Techniques for predicting, detecting and reducing aspecific protein interference in assays involving immunoglobulin single variable domains

Abstract

This invention provides, and in certain specific but non-limiting aspects relates to: assays that can be used to predict whether a given ISV will be subject to protein interference as described herein and/or give rise to an (aspecific) signal in such an assay (such as for example in an ADA immunoassay). Such predictive assays could for example be used to test whether a given ISV could have a tendency to give rise to such protein interference and/or such a signal; to select ISV's that are not or less prone to such protein interference or to giving such a signal; as an assay or test that can be used to test whether certain modification(s) to an ISV will (fully or partially) reduce its tendency to give rise to such interference or such a signal; and/or as an assay or test that can be used to guide modification or improvement of an ISV so as to reduce its tendency to give rise to such protein interference or signal; methods for modifying and/or improving ISV's to as to remove or reduce their tendency to give rise to such protein interference or such a signal; modifications that can be introduced into an ISV that remove or reduce its tendency to give rise to such protein interference or such a signal; ISV's that have been specifically selected (for example, using the assay(s) described herein) to have no or low(er)/reduced tendency to give rise to such protein interference or such a signal; modified and/or improved ISV's that have no or a low(er)/reduced tendency to give rise to such protein interference or such a signal.

Claims

1. A fusion protein comprising: at least two heavy chain variable domains (VH or VHH), wherein at least one of the at least two heavy chain variable domains (VH or VHH) binds serum albumin and the remaining of the at least two heavy chain variable domains (VH or VHH) bind(s) a therapeutic target; and one of the at least two heavy chain variable domains (VH or VHH) is at the C-terminal end of the fusion protein, wherein the heavy chain variable domain (VH or VHH) at the C-terminal end of the fusion protein binds serum albumin and terminates in the sequence VTVSS(X).sub.n (SEQ ID NO: 34, wherein: (i) n=1 or 2 in which each X is independently chosen from glycine (G), valine (V), leucine (L) or isoleucine (I); or (ii) n=3, 4 or 5 in which each X is independently chosen from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I).

2. The fusion protein according to claim 1, in which: (a) n=1, 2 or 3 in which each X=Gly; or (b) n=3 in which each X=Ala.

3. The fusion protein according to claim 1, in which said VH is a camelized VH or wherein said VHH is a humanized VHH.

4. Pharmaceutical composition that comprises a fusion protein according to claim 1, and at least one suitable carrier, diluent or excipient.

5. A fusion protein comprising: at least two heavy chain variable domains (VH or VHH), wherein at least one of the at least two heavy chain variable domains (VH or VHH) binds serum albumin and the remaining of the at least two heavy chain variable domains (VH or VHH) bind(s) a therapeutic target; and one of the at least two heavy chain variable domains (VH or VHH) is at the C-terminal end of the fusion protein, wherein the heavy chain variable domain (VH or VHH) at the C-terminal end of the fusion protein binds serum albumin and terminates in the sequence VTVSS(X).sub.n (SEQ ID NO: 34, in which: (a) n=1 or 2 and each X is chosen from any naturally occurring amino acid except cysteine (C) or alanine (A); or (b) n=3, 4 or 5 and each X is independently chosen from any naturally occurring amino acid except cysteine (C).

6. The fusion protein according to claim 5, wherein n=3, 4 or 5 in which each X is independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) and isoleucine (I).

7. The fusion protein according to claim 5, in which said VH is a camelized VH or wherein said VHH is a humanized VHH.

8. The fusion protein according to claim 5, wherein n=1 or 2 in which each X is independently chosen from glycine (G), valine (V), leucine (L) or isoleucine (I).

9. Pharmaceutical composition that comprises a fusion protein according to claim 5, and at least one suitable carrier, diluent or excipient.

Description

(1) The invention will now be further described by means of the following non-limiting preferred aspects, examples and figures, in which:

(2) FIG. 1A to 1C schematically shows some non-limiting examples of ADA assay formats. Some representative but non-limiting protocols for performing these assays are mentioned in Example 4.

(3) FIG. 2 schematically shows a representative 3D structure of an ISV, such as a Nanobody.

(4) FIG. 3 is a binding curve (obtained using the BIACORE assay described in Example 3) showing the binding of NB's 3.4 to 3.9 (SEQ ID NO's. 5 to 10) to the immobilized polyclonal antibody obtained in Example 2.

(5) FIG. 4 is a binding curve (obtained using the BIACORE assay described in Example 3) showing the binding of NB's 3.4, 3.11, 3.12 and 3.13 (SEQ ID NO's: 5, 12, 13 and 14) to the immobilized polyclonal antibody obtained in Example 2.

(6) FIG. 5 is a binding curve (obtained using the BIACORE assay described in Example 3) showing the binding of NB's 3.4, 3.14 and 3.15 (SEQ ID NO's: 5, 15 and 16) to the immobilized polyclonal antibody obtained in Example 2.

(7) FIG. 6 is a binding curve (obtained using the BIACORE assay described in Example 3) showing the binding of NB's 3.1, 3.2 and 3.4 (SEQ ID NO's: 3, 4 and 5) to the immobilized polyclonal antibody obtained in Example 2.

(8) FIG. 7 is a binding curve (obtained using the BIACORE assay described in Example 3) showing the binding of NB's 4.1 and 4.2 (SEQ ID NO's: 17 and 18) to the immobilized polyclonal antibody obtained in Example 2.

(9) FIG. 8 is a binding curve (obtained using the BIACORE assay described in Example 3) showing the binding of NB's 6.1, 6.2, 6.4 and 6.5 (SEQ ID NO's 19 to 22) to the immobilized polyclonal antibody obtained in Example 2.

(10) The sequences referred to in the present description and claims are listed in Table A below (SEQ ID NO's: 1 to 36) and in Table B below (SEQ ID NO's: 37 to 89, showing the sequences used in Example 8 and setting out the corresponding reference sequence).

(11) TABLE-US-00001 TABLE A  SEQ ID Name NO: Sequence ISV Ex. 1/2- 1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK GLEWVSGIKSSGDSTRYAGSVKGRFTISRDNAKNTLYLQMNSL RPEDTAVYYCAKSRVSRTGLYTYDNRGQGTLVTVSSGGGGSG GGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTF NNYAMGWFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDR FTISRDNAKNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFE YDYSGQGTLVTVSS Alt. ISV 2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGK GREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLR PEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGG GSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYL QMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGK GREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLR PEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS >Nb3.1 3 EVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRTG EPRELVATITGGSSINYGDFVKGRFTISIDNAKNTVYLQMNNLK PEDTAVYYCNFNKYVTSRDTWGQGTQVTVSS >Nb3.2 4 EVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRTG EPRELVATITGGSSINYGDFVKGRFTISIDNAKNTVYLQMNNLK PEDTAVYYCNFNKYVTSRDTWGQGTQVTVSSAAAEQKLISEED LNGAAHHHHHH >Nb3.4 5 EVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGE PRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPE DTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb3.5 6 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGW YRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQ MNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSAA >Nb3.6 7 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGW YRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQ MNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSA >Nb3.7 8 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGW YRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQ MNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSG >Nb3.8 9 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGW YRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQ MNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSGG >Nb3.9 10 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGW YRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQ MNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSGGG >Nb3.10 11 HHHHHHEVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGW YRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQ MNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb3.11 12 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGW YRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQ MNSLKPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb3.12 13 HHHHHHEVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGW YRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQ MNSLRPEDTAVYYCNFNKYVTSRDTWGQGTQVTVSS >Nb3.13 14 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGW YRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQ MNSLRPEDTAVYYCNFNKYVTSRDTWGQGTQVTVSS >Nb3.14 15 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGW YRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQ MNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVQVSS >Nb3.15 16 HHHHHHEVQLVESGGGSVQPGGSLRLSCAASRSIGRLDRMGW YRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQ MNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb4.1 17 EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPG KGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLR PEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSS >Nb4.2 18 EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPG KGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLR PEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGG GSRDWDFDVFGGGTPVGG >Nb6.1 19 EVQLVESGGGLVQPGGSLRLSCIASGLPFSTKSMGWFRQAPGK EREFVARISPGGTSRYYGDFVKGRFAISRDNAKNTTWLQMNSL KAEDTAVYYCASGERSTYIGSNYYRTNEYDYWGTGTQVTVSS AAAEQKLISEEDLNGAAHHHHHH >Nb6.2 20 EVQLVESGGGLVQPGGSLRLSCIASGLPFSTKSMGWFRQAPGK EREFVARISPGGTSRYYGDFVKGRFAISRDNAKNTTWLQMNSL KAEDTAVYYCASGERSTYIGSNYYRTNEYDYWGTGTQVTVSS >Nb6.4 21 EVQLLESGGGLVQPGGSLRLSCAASGLPFSTKSMGWFRQAPGK GREFVSRISPGGTSRYYGDFVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCASGERSTYIGSNYYRTNEYDYWGQGTLVTVSSA AAEQKLISEEDLNGAAHHHHHH >Nb6.5 22 EVQLLESGGGLVQPGGSLRLSCAASGLPFSTKSMGWFRQAPGK GREFVSRISPGGTSRYYGDFVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCASGERSTYIGSNYYRTNEYDYWGQGTLVTVSS Example 1C: 23 HHHHHHEVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMG wildtype WFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA KNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGT QVTVSS Example 1C: 24 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMG (A14P) WFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA KNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGT QVTVSS Example 1C:  25 HHHHHHEVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMG (K83R) WFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA KNTLYLQMNSLRPEDTAVYTCAASAIGSGALRRFEYDYSGQGT QVTVSS Example 1C:  26 HHHHHHEVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMG (Q108L) WFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA KNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGT LVTVSS Example 1C: 27 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMG (A14P, K83R, WFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA Q108L) KNTLYLQMNSLRPEDTAVYTCAASAIGSGALRRFEYDYSGQGT LVTVSS Example 1c:  28 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMG (A14P, R39Q, WFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA K83R, T91Y, KNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGT Q108L) LVTVSS Example 1C:  29 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMG (A14P, R39Q, WFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA K83R, T91Y, KNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGT Q108L)-1A LVTVSSA Example 1C:  30 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMG (A14P, R39Q, WFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA K83R, T91Y, KNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGT Q108L)-3A LVTVSSAAA Nb3.16 31 DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPREL VATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYC NFNKYVTSRDTWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPG NSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRM GWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQM NSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS Nb3.17 32 DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPREL VATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYC NFNKYVTSRDTWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPG NSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRM GWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQM NSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSA C-terminal 33 VTVSS sequence C-terminal 34 VTVSS(X).sub.n sequence 21-4-3, IGH 35 QIQLVQSGPELKKPGETVKISCKASGYTFTAYSMHWVKQAPG consensus KGLKWMGWINTVTGEPAYADDFKGRFAFSLETSASTAYLQIS SLKNEDTATYFCTRGLIHFYYWGQGTTLTVSSAKTTPPSVYPL APGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTF PAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKK IVPRDC 21-4-3-IGK 36 DIQMTQTPSSLSASLGGRVTITCKASQDIHNFISWYQHKPGKV consensus PRLIIHDTSTLQPGIPSRFSGSGSGRDYSFSITNLEPEDIATYYCL HYDNLLRSFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASV VCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTY SMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

(12) TABLE-US-00002 TABLE B  Mutations SEQ C-terminal in the  ID amino C-terminal Reference NO acid(s) region Sequence sequence 37 A none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 37 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL added C- YADSVKGRFTISRDNAKTTLYL terminal amino QMNSLRPEDTAVYYCTIGGSLS acid residues RSSQGTLVTVSSA 38 A none EVQLVESGGGLVQPGGSLRLSC SEQ ID NO: 38 AASGFTFSSYAMSWVRQAPGK without the GLEWVSGIKSSGDSTRYAGSV added C- KGRFTISRDNAKNTLYLQMNS terminal amino LRPEDTAVYYCAKSRVSRTGL acid residues YTYDNRGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSEVQLVE SGGGLVQPGGSLRLSCAASGRT FNNYAMGWFRQAPGKEREFV AAITRSGVRSGVSAIYGDSVKD RFTISRDNAKNTLYLQMNSLRP EDTAVYYCAASAIGSGALRRFE YDYSGQGTLVTVSSA 39 A none EVQLVESGGGLVQPGGSLRLSC SEQ ID NO: 39 AASGFTFSSYPMGWFRQAPGK without the GREFVSSITGSGGSTYYADSVK added C- GRFTISRDNAKNTLYLQMNSLR terminal amino PEDTAVYYCAAYIRPDTYLSRD acid residues YRKYDYWGQGTLVTVSSGGG GSGGGSEVQLVESGGGLVQPG NSLRLSCAASGFTFSSFGMSWV RQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYL QMNSLRPEDTAVYYCTIGGSLS RSSQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQPGGSLRLSC AASGFTFSSYPMGWFRQAPGK GREFVSSITGSGGSTYYADSVK GRFTISRDNAKNTLYLQMNSLR PEDTAVYYCAAYIRPDTYLSRD YRKYDYWGQGTLVTVSSA 40 A none EVQLVESGGGLVQPGGSLRLSC SEQ ID NO: 40 AASGFTFSDYWMYWVRQAPG without the KGLEWVSEINTNGLITKYPDSV added C- KGRFTISRDNAKNTLYLQMNS terminal amino LRPEDTAVYYCARSPSGFNRG acid residues QGTLVTVSSGGGGSGGGSEVQ LVESGGGLVQPGNSLRLSCAAS GFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPED TAVYYCTIGGSLSRSSQGTLVT VSSGGGGSGGGSEVQLVESGG GLVQPGGSLRLSCAASGFTFSD YWMYWVRQAPGKGLEWVSEI NTNGLITKYPDSVKGRFTISRD NAKNTLYLQMNSLRPEDTAVY YCARSPSGFNRGQGTLVTVSSA 41 A none EVQLVESGGGLVQPGGSLRLSC SEQ ID NO: 41 AASGSVFKINVMAWYRQAPGK without the GRELVAGIISGGSTSYADSVKG added C- RFTISRDNAKNTLYLQMNSLRP terminal amino EDTAVYYCAFITTESDYDLGRR acid residues YWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSC AASGFTFSSFGMSWVRQAPGK GLEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLR PEDTAVYYCTIGGSLSRSSQGT LVTVSSA 42 A none EVQLVESGGGLVQPGGSLRLSC SEQ ID NO: 42 AASGRTFSYNPMGWFRQAPGK without the GRELVAAISRTGGSTYYPDSVE added C- GRFTISRDNAKRMVYLQMNSL terminal amino RAEDTAVYYCAAAGVRAEDG acid residues RVRTLPSEYTFWGQGTQVTVS SAAAEVQLVESGGGLVQPGGS LRLSCAASGRTFSYNPMGWFR QAPGKGRELVAAISRTGGSTYY PDSVEGRFTISRDNAKRMVYL QMNSLRAEDTAVYYCAAAGV RAEDGRVRTLPSEYTFWGQGT QVTVSSA 43 A none DVQLVESGGGLVQPGGSLRLS SEQ ID NO: 43 CAASGFILDYYAIGWFRQAPGK without the EREGVLCIDASDDITYYADSVK added C- GRFTISRDNSKNTVYLQMNSLR terminal amino PEDTAVYYCATPIGLSSSCLLE acid residues YDYDYWGQGTLVTVSSGGGG SGGGSEVQLLESGGGLVQPGG SLRLSCAASGFTFRSFGMSWVR QAPGKGPEWVSSISGSGSDTLY ADSVKGRFTISRDNSKNTLYLQ MNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSA 44 A none DVQLVESGGGLVQPGGSLRLS SEQ ID NO: 44 CAASRSIGRLDRMGWYRHRPG without the EPRELVATITGGSSINYGDSVK added C- GRFTISIDNSKNTVYLQMNSLR terminal amino PEDTAVYYCNFNKYVTSRDTW acid residues GQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCA ASGFTFSSFGMSWVRQAPGKG LEWVSSISGSGSDTLYADSVKG RFTISRDNAKTTLYLQMNSLRP EDTAVYYCTIGGSLSRSSQGTL VTVSSGGGGSGGGSEVQLVES GGGLVQPGGSLRLSCAASRSIG RLDRMGWYRHRPGEPRELVAT ITGGSSINYGDSVKGRFTISIDN SKNTVYLQMNSLRPEDTAVYY CNFNKYVTSRDTWGQGTLVTV SSA 45 none P14A, EVQLVESGGGLVQAGGSLRLS SEQ ID NO: 45 P41T, S62F, CAASRSIGRLDRMGWYRHRTG without the S74A, EPRELVATITGGSSINYGDFVK added C- S82bN, GRFTISIDNAKNTVYLQMNNLK terminal amino R83K, PEDTAVYYCNFNKYVTSRDTW acid residues L108Q GQGTQVTVSS and without the mentioned mutations in the C-terminal region 46 AAEQKLI A14P, EVQLVESGGGLVQAGGSLRLS SEQ ID NO: 46 SEEDLNG T41P, F62S, CAASRSIGRLDRMGWYRHRTG without the AAHHHH A74S, EPRELVATITGGSSINYGDFVK added C- HH N82bS, GRFTISIDNAKNTVYLQMNNLK terminal amino K83R, PEDTAVYYCNFNKYVTSRDTW acid residues Q108L GQGTQVTVSSAAAHHHHHHG and without the AAEQKLISEEDLNGAA mentioned mutations in the C-terminal region 47 GGGGSG none EVQLVESGGGLVQPGGSLRLSC SEQ ID NO: 47 GGSRDW AASGSVFKINVMAWYRQAPGK without the DFDVFGG GRELVAGIISGGSTSYADSVKG added C- GTPVGG RFTISRDNAKNTLYLQMNSLRP terminal amino EDTAVYYCAFITTESDYDLGRR acid residues YWGQGTLVTVSSGGGGSGGGS RDWDFDVFGGGTPVGG 48 AAEQKLI none EVQLVESGGGLVQPGGSLRLSC SEQ ID NO: 48 SEEDLNG IASGLPFSTKSMGWFRQAPGKE without the AAHHHH REFVARISPGGTSRYYGDFVKG added C- HH RFAISRDNAKNTTWLQMNSLK terminal amino AEDTAVYYCASGERSTYIGSNY acid residues YRTNEYDYWGTGTQVTVSSAA AEQKLISEEDLNGAAHHHHHH 49 AAEQKLI V5L, I23A, EVQLLESGGGLVQPGGSLRLSC SEQ ID NO: 49 SEEDLNG E44G, AASGLPFSTKSMGWFRQAPGK without the AAHHHH A49S, GREFVSRISPGGTSRYYGDFVK added C- HH A68T, GRFTISRDNSKNTLYLQMNSLR terminal amino A74S, AEDTAVYYCASGERSTYIGSNY acid residues T78L, YRTNEYDYWGQGTLVTVSSAA and without the W79Y, AEQKLISEEDLNGAAHHHHHH mentioned K83R, mutations in the T110Q, C-terminal Q108L region 50 none L11S HHHHHHEVQLVESGGGSVQPG SEQ ID NO: 50 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL mentioned YADSVKGRFTISRDNAKTTLYL mutations in the QMNSLRPEDTAVYYCTIGGSLS C-terminal RSSQGTLVTVSS region 51 none T110Q HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 51 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL mentioned YADSVKGRFTISRDNAKTTLYL mutations in the QMNSLRPEDTAVYYCTIGGSLS C-terminal RSSQGTLVQVSS region 52 none S112G HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 52 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL mentioned YADSVKGRFTISRDNAKTTLYL mutations in the QMNSLRPEDTAVYYCTIGGSLS C-terminal RSSQGTLVTVGS region 53 none S113G HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 53 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL mentioned YADSVKGRFTISRDNAKTTLYL mutations in the QMNSLRPEDTAVYYCTIGGSLS C-terminal RSSQGTLVTVSG region 54 none L11S, HHHHHHEVQLVESGGGSVQPG SEQ ID NO: 54 T110Q, NSLRLSCAASGFTFSSFGMSWV without the S112G, RQAPGKGLEWVSSISGSGSDTL mentioned S113G YADSVKGRFTISRDNAKTTLYL mutations in the QMNSLRPEDTAVYYCTIGGSLS C-terminal RSSQGTLVQVGG region 55 A none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 55 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL added C- YADSVKGRFTISRDNAKTTLYL terminal amino QMNSLRPEDTAVYYCTIGGSLS acid residues RSSQGTLVTVSSA 56 G S113G HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 56 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL added C- YADSVKGRFTISRDNAKTTLYL terminal amino QMNSLRPEDTAVYYCTIGGSLS acid residues RSSQGTLVTVSSGG and without the mentioned mutations in the C-terminal region 57 AS none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 57 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL added C- YADSVKGRFTISRDNAKTTLYL terminal amino QMNSLRPEDTAVYYCTIGGSLS acid residues RSSQGTLVTVSSAS 58 AST none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 58 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL added C- YADSVKGRFTISRDNAKTTLYL terminal amino QMNSLRPEDTAVYYCTIGGSLS acid residues RSSQGTLVTVSSAST 59 ASTK none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 59 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL added C- YADSVKGRFTISRDNAKTTLYL terminal amino QMNSLRPEDTAVYYCTIGGSLS acid residues RSSQGTLVTVSSASTK 60 ASP none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 60 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL added C- YADSVKGRFTISRDNAKTTLYL terminal amino QMNSLRPEDTAVYYCTIGGSLS acid residues RSSQGTLVTVSSASP 61 AP none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 61 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL added C- YADSVKGRFTISRDNAKTTLYL terminal amino QMNSLRPEDTAVYYCTIGGSLS acid residues RSSQGTLVTVSSAP 62 APT none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 62 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL added C- YADSVKGRFTISRDNAKTTLYL terminal amino QMNSLRPEDTAVYYCTIGGSLS acid residues RSSQGTLVTVSSAPT 63 W none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 63 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL added C- YADSVKGRFTISRDNAKTTLYL terminal amino QMNSLRPEDTAVYYCTIGGSLS acid residues RSSQGTLVTVSSW 64 L none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 64 NSLRLSCAASGFTFSSFGMSWV without the RQAPGKGLEWVSSISGSGSDTL added C- YADSVKGRFTISRDNAKTTLYL terminal amino QMNSLRPEDTAVYYCTIGGSLS acid residues RSSQGTLVTVSSL 65 none P14A HHHHHHEVQLVESGGGLVQA SEQ ID NO: 65 GGSLRLSCAASRSIGRLDRMG without the WYRHRPGEPRELVATITGGSSI mentioned NYGDSVKGRFTISIDNSKNTVY mutations in the LQMNSLRPEDTAVYYCNFNKY C-terminal VTSRDTWGQGTLVTVSS region 66 none L11S HHHHHHEVQLVESGGGSVQPG SEQ ID NO: 66 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVTVSS region 67 none R83K HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 67 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLKPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVTVSS region 68 none P14A, HHHHHHEVQLVESGGGLVQA SEQ ID NO: 68 L108Q GGSLRLSCAASRSIGRLDRMG without the WYRHRPGEPRELVATITGGSSI mentioned NYGDSVKGRFTISIDNSKNTVY mutations in the LQMNSLRPEDTAVYYCNFNKY C-terminal VTSRDTWGQGTQVTVSS region 69 none L108Q HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 69 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTQVTVSS region 70 none T110Q HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 70 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVQVSS region 71 none S113G HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 71 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVTVSG region 72 none S112G, HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 72 S113G GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVTVGG region 73 G S112G, HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 73 S113G GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY added C- GDSVKGRFTISIDNSKNTVYLQ terminal amino MNSLRPEDTAVYYCNFNKYVT acid residues SRDTWGQGTLVTVGGG and without the mentioned mutations in the C-terminal region 74 G none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 74 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY added C- GDSVKGRFTISIDNSKNTVYLQ terminal amino MNSLRPEDTAVYYCNFNKYVT acid residues SRDTWGQGTLVTVSSG 75 AA none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 75 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY added C- GDSVKGRFTISIDNSKNTVYLQ terminal amino MNSLRPEDTAVYYCNFNKYVT acid residues SRDTWGQGTLVTVSSAA 76 GGG none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 76 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY added C- GDSVKGRFTISIDNSKNTVYLQ terminal amino MNSLRPEDTAVYYCNFNKYVT acid residues SRDTWGQGTLVTVSSGGG 77 A none HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 77 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY added C- GDSVKGRFTISIDNSKNTVYLQ terminal amino MNSLRPEDTAVYYCNFNKYVT acid residues SRDTWGQGTLVTVSSA 78 none Q13R HHHHHHEVQLVESGGGLVRPG SEQ ID NO: 78 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVTVSS region 79 GG none HHHHHEVQLVESGGGLVQPGG SEQ ID NO: 79 SLRLSCAASRSIGRLDRMGWY without the RHRPGEPRELVATITGGSSINYG added C- DSVKGRFTISIDNSKNTVYLQM terminal amino NSLRPEDTAVYYCNFNKYVTS acid residues RDTWGQGTLVTVSSGG 80 none T110Q, HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 80 S112G, GSLRLSCAASRSIGRLDRMGW without the S113G YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVQVGG region 81 none L11V HHHHHHEVQLVESGGGVVQP SEQ ID NO: 81 GGSLRLSCAASRSIGRLDRMG without the WYRHRPGEPRELVATITGGSSI mentioned NYGDSVKGRFTISIDNSKNTVY mutations in the LQMNSLRPEDTAVYYCNFNKY C-terminal VTSRDTWGQGTLVTVSS region 82 none P84A HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 82 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRAEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVTVSS region 83 none T87A HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 83 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDAAVYYCNFNKYVT C-terminal SRDTWGQGTLVTVSS region 84 none S112G HHHHHHEVQLVESGGGLVQPG SEQ ID NO: 84 GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVTVGS region 85 none L11S, HHHHHHEVQLVESGGGSVQPG SEQ ID NO: 85 T110Q, GSLRLSCAASRSIGRLDRMGW without the S112G, YRHRPGEPRELVATITGGSSINY mentioned S113G GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVQVGG region 86 none L11S, HHHHHHEVQLVESGGGSVQPG SEQ ID NO: 86 T110Q GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVQVSS region 87 none L11S, HHHHHHEVQLVESGGGSVQPG SEQ ID NO: 87 S112G, GSLRLSCAASRSIGRLDRMGW without the S113G YRHRPGEPRELVATITGGSSINY mentioned GDSVKGRFTISIDNSKNTVYLQ mutations in the MNSLRPEDTAVYYCNFNKYVT C-terminal SRDTWGQGTLVTVGG region 88 A L11S, HHHHHHEVQLVESGGGSVQPG SEQ ID NO: 88 T110Q GSLRLSCAASRSIGRLDRMGW without the YRHRPGEPRELVATITGGSSINY added C- GDSVKGRFTISIDNSKNTVYLQ terminal amino MNSLRPEDTAVYYCNFNKYVT acid residues SRDTWGQGTLVQVSSA and without the mentioned mutations in the C-terminal region 89 none L11S, HHHHHHEVQLVESGGGSVQA SEQ ID NO: 89 P14A, GGSLRLSCAASRSIGRLDRMG without the T110Q, WYRHRPGEPRELVATITGGSSI mentioned S112G, NYGDSVKGRFTISIDNSKNTVY mutations in the S113G LQMNSLRPEDTAVYYCNFNKY C-terminal VTSRDTWGQGTLVQVGG region
Experimental Part:

Example 1: Generation of a Polyclonal Analytical Antibody

(13) A polyclonal antibody (IgG fraction) that can be used as the “analytical antibody” was generated as follows:

(14) A. Identification of Suitable Plasma Samples for Isolating the Polyclonal Antibody

(15) Twenty plasma samples from healthy individuals that were never treated with an ISV were evaluated for presence of antibodies against ISV that can be used as the analytical antibody in the invention.

(16) The ISV that was initially used in this Example was SEQ ID NO: 1. Subsequently, to confirm that the interaction is not specific for this particular ISV, but is an aspecific protein-protein interaction that may occur with a number of ISV's, the assays below were repeated with other ISV's (see paragraph C) below). As an alternative for SEQ ID NO:1, for example SEQ ID NO:2 may also be used.

(17) The assay used was an ECL (Electrochemiluminescence) based bridging assay that used biotinylated ISV (a biotinylated variant of SEQ ID NO:1) to capture and sulfo-tagged ISV to detect anti-drug antibodies. A similar format is also used for performing ADA assays. Biotinylation and sulfo-tagging of the ISV was done using standard coupling chemistry on primary amines using Sulfo-NHS-LC-Biotin (Pierce) and Sulfo-tag NHS-Ester (MSD), respectively according to the manufacturer's instructions. The plasma samples were diluted 1/5 in PBS/0.1% casein and were incubated for 30 minutes at 37° C., 600 RPM in 96 well polypropylene plates. The samples (50 μL) were then diluted 1/3 in 1:1 mixture (100 μL) of 2 μg/ml biotinylated and 2 μg/ml sulfo-tagged ISV (SEQ ID NO:1) and incubated for 1 hour at RT, 600 RPM. MSD MA®96-well Standard Streptavidin plates were blocked with 150 μL/well Superblock® T20 for 1 hour at RT, then washed 3 times with PBS/0.05% Tween20 (=wash buffer). Sample/1:1 mix (biotinylated and sulfo-tagged ISV (SEQ ID NO:1) (50.0 μL) was transferred from the polypropylene plate to the MSD plate and incubated for 1 hour at RT, 600 rpm. Plates were washed three times prior to addition of 2× Read Buffer (MSD) (150 μL/well) and reading the ECL units (ECLU) on an MSD instrument (Sector Imager 2400 reader). Samples were screened as positive or negative using the screening cut-point determined during method validation. The screening cut-point was calculated based on the background values of 118 individual plasma samples from healthy individuals that were never treated with an ISV, using appropriate statistical analysis as recommended by the guidelines for ADA assay development (Shankar, 2008). A non-parametric assessment was used and the cut-off value was calculated based on the 95.sup.th percentile, after exclusion of outliers.

(18) Six plasma samples were clearly scored as positive: IHuP #002-001-ABL-01, IHuP #002-001-ABL-08, IHuP #002-001-ABL-10, IHuP #002-001-ABL-15. IHuP #002-001-ABL-19 and IHuP #002-001-ABL-20 (Table I).

(19) These samples were further analyzed in a drug displacement set-up (confirmatory assay) to confirm the specificity of the positive screening outcome (Table II). Therefore, the samples were diluted 1/5 in PBS/0.1% casein containing 12.5 μg/mL ISV (SEQ ID NO:1) and were incubated for 30 minutes at 37° C., 600 RPM in 96 well polypropylene plates. The samples (50 μL) are then diluted 1/3 in 1:1 mixture (100 μL) of 2 μg/ml biotinylated and 2 μg/ml sulfo-tagged ISV (SEQ ID NO:1) and incubated for 1 hour at RT, 600 RPM. Subsequently, sample/1:1 mix (biotinylated and sulfo-tagged ISV) (50.0 μL) was transferred from the polypropylene plate to the blocked MSD MA®96-well Standard Streptavidin plate as described above for the screening assay and incubated for 1 hour at RT, 600 rpm. Plates were washed three times prior to addition of 2× Read Buffer (MSD) (150 μt/well) and measuring ECL units (ECLU) on an MSD instrument (Sector Imager 2400 reader). Samples were confirmed as true positives using the confirmatory cut-point determined during method validation and was calculated on the ECL response of 118 individual plasma samples from healthy individuals that were never treated with ISV, that were spiked with 50 μg/ml ISV (SEQ ID NO:1) using appropriate statistical analysis as recommended by the guidelines for ADA assay development (Shankar, 2008). A minimal signal reduction of 50% was calculated based on the 99% confidence interval.

(20) Samples that were positive in the ECL based bridging assay and that were confirmed as positive in the drug displacement set-up assay were selected as a source for generating the polyclonal antibody using affinity chromatography.

(21) TABLE-US-00003 TABLE I screening results of 20 plasma samples in the ADA ISV assay. Sample ID ECLU screening assay IHuP#002-001-ABL-01 13081 IHuP#002-001-ABL-02 56 IHuP#002-001-ABL-03 272 IHuP#002-001-ABL-04 125 IHuP#002-001-ABL-05 70 IHuP#002-001-ABL-06 99 IHuP#002-001-ABL-07 170 IHuP#002-001-ABL-08 659358 IHuP#002-001-ABL-09 798 IHuP#002-001-ABL-10 1101 IHuP#002-001-ABL-11 83 IHuP#002-001-ABL-12 72 IHuP#002-001-ABL-13 403 IHuP#002-001-ABL-14 62 IHuP#002-001-ABL-15 1141 IHuP#002-001-ABL-16 159 IHuP#002-001-ABL-17 72 IHuP#002-001-ABL-18 170 IHuP#002-001-ABL-19 4503 IHuP#002-001-ABL-20 8243

(22) TABLE-US-00004 TABLE II Confirmation of positively screened plasma samples in the confirmatory assay. A confirmatory cut-point of 50% was used for evaluation of the results. One sample was not confirmed as a true positive sample ECLU screening ECLU confirmatory % signal Plasma sample ID assay: plasma assay: plasma inhibition IHuP#002-001- 13081 685 95 ABL-01 IHuP#002-001- 659358 169410 74 ABL-08 IHuP#002-001- 1101 582 47 ABL-10 IHuP#002-001- 1141 467 59 ABL-15 IHuP#002-001- 4503 1531 66 ABL-19 IHuP#002-001- 8243 1450 82 ABL-20

(23) A further three serum samples from individuals that not have been treated with an ISV were also evaluated using the ECL based bridging assay described above and confirmed using the drug displacement set-up assay.

(24) Two serum samples were clearly scored as positive in the ECL based bridging assay: IHUS #B09032311A3 and IHUS #B09032311A20 (Table III). The 2 positively screened samples were further analyzed in the drug displacement set-up to confirm the specificity of the positive screening outcome.

(25) TABLE-US-00005 TABLE III Screening and confirmatory results of 3 serum samples and corresponding IgG purified fraction ECL ECL signal ECL signal signal ECL signal screening confirmatory screening confirmatory assay: assay: % signal assay: assay: % signal Serum sample ID serum serum inhibition IgG IgG inhibition IHUS#B09032311A3 2388 286 88% 3716 370 90% IHUS#B09032311A20 19272 915 95% 31309 1160 96% IHUS#B09032311A1 62
B. Generation of Purified Polyclonal IgG Fraction.

(26) A polyclonal IgG was purified from the samples IHUS #B09032311A3 and IHUS #B09032311A20 (see above) using Protein G HP Spin Trap Columns (GE Healthcare) according to the manufacturer's instructions. In short, after removal of the storage solution form the column by centrifugation (30 s at 100×g), the column was equilibrated by adding binding buffer (20 mM sodium phosphate, pH 7.0). After centrifugation, the solution containing the desired polyclonal was added (max 1 mg in 600 μl) and column was incubated for 4 min while gently mixing. The column was then centrifuged and washed 2× by successive addition of binding buffer (600 μl) and centrifugation. After addition of 400 μl elution buffer (0, 1 M glycine-HCL, pH 2.7) and mixing by inversion, the antibody was eluted by centrifugation in 30 μl neutralization buffer (1M Tris-HCL, pH 9.0).

(27) In order to confirm that the IgG fraction thus obtained was involved in aspecific binding to the ISV(s), the purified IgG antibody was analyzed in the ECL based bridging assay described above and confirmed using the drug displacement set-up assay used under A) above. In both samples (IHUS #B09032311A3 and IHUS #B09032311A20), purified IgG antibody was confirmed to be involved in the aspecific binding leading to a positive signal in the assays (Table III). This confirmed that the purified polyclonal IgG could be used as an “analytical antibody”, and it was used as such in (the assays of) Examples 3 and 5.

(28) C. Aspecific Binding to Other ISV's.

(29) In order to determine whether the protein interference observed is specific for a single ISV, and/or is specific for a particular region, epitope or antigenic determinant on ISV's, and/or for certain mutations made to wildtype ISV's (such as one or more humanizing mutations), the ECL based bridging assay and the drug displacement set-up assay (both as described under A) above, with SEQ ID NO: 1 being used as the sulfo-tagged ISV) were repeated using the plasma samples IHUS #B09032311A3, IHUS #B09032311A20 and IHUS #B09032311A1. As these plasma samples contain the polyclonal “analytical” antibody isolated under B) above, this also provides information on the specificity, selectivity and epitope recognition of the polyclonal analytical antibody.

(30) 8 ISV's were tested (SEQ ID NO's 23 to 30, respectively—see Table A above), of which one was a wildtype VHH (SEQ ID NO: 23) and the other 7 ISV's were humanized versions of the wildtype sequence with different humanizing substitutions. Two ISV's (SEQ ID NO's: 29 and 30) also contained additional amino acid residues at the C-terminus (1 and 3 additional alanine residues, respectively).

(31) The data are shown in Table IV. Without being limited to any explanation or hypothesis, it can be seen that changes to the C-terminal region (as defined herein) can apparently strongly influence the extent to which the plasma samples used can give rise to protein interference. For example, it can be seen that adding one or three amino acid residues to the C-terminus can strongly reduce the tendency for protein interference to arise (for example, only 18 and 13% reduction in the ECLU assay with sample IHUS #B09032311A3 for SEQ ID NO's: 29 and 30, compared to 90% reduction for SEQ ID NO: 28, the corresponding humanized variant without any amino acid residues added to the C-terminus). Similarly, introducing a proline residue at position 14 of the wildtype sequence can apparently also strongly influence the extent to which the plasma samples used can give rise to protein interference (for example, only 20% reduction in the ECLU assay with sample IHUS #B09032311A3 for the wildtype sequence of SEQ ID NO's: 23, compared to 91% reduction for SEQ ID NO: 24, the wildtype sequence with an A14P substitution). K83R and Q108L, which are also substitutions close to the C-terminal region, also lead to some increase in the tendency to give rise to protein interference, but not as much as the A14P substitution, and the total combined effect of the A14P+K83R+Q108L substitutions can be negated by adding one or more amino acid residues to the C-terminus (compare again the data for SEQ ID NO's: 29 and 30 with the data for the other humanized variants).

(32) Based on this data, it was also concluded that apparently, the polyclonal analytical antibody recognized the C-terminal region (as defined herein) of ISV's generally. As can be seen from FIG. 2, position 14 (and to a lesser degree positions 83 and 108) also form parts of the C-terminal region of an ISV (when the three-dimensional ternary structure of an ISV is taken into account).

(33) TABLE-US-00006 TABLE IV Evaluation of different Nanobody variants as competitor in the ISV ADA assay using the analytical antibody. Serum sample ID IHUS#B09032311A3 IHUS#B09032311A20 IHUS#B09032311A1 ECLU in screening Nanobody Variant (right hand assay (using SEQ ID NO: 1) column mentions the humanizing 2217 18494 62 substitutions and C-terminal ECLU ECLU ECLU additions made compared to the confirmatory % confirmatory % confirmatory % wildtype sequence of SEQ ID NO: 23 assay reduction assay reduction assay reduction SEQ ID NO: Wildtype VHH 1778 20 8682 53 60 4 23 SEQ ID NO: Wildtype VHH + 205 91 668 96 56 10 24 A14P SEQ ID NO: Wildtype VHH + 1403 37 6912 63 62 1 25 K83R SEQ ID Wildtype VHH + 1533 31 6991 62 59 5 NO: 26 Q108L SEQ ID NO: Wildtype VHH + 156 93 628 97 57 8 27 A14P + K83R + Q108L SEQ ID NO: Wildtype VHH + 228 90 570 97 58 6 28 A14P + R39Q + K83R + T91Y + Q108L SEQ ID NO: Wildtype VHH + 1814 18 15087 18 60 3 29 A14P + R39Q + K83R + T91Y + Q108L + 1 additional A at C-terminus (A114) SEQ ID NO: Wildtype VHH + 1933 13 15244 18 62 0 30 A14P + R39Q + K83R + T91Y + Q108L + 3 A's at C- terminus (A114 + A115 + A116)

Example 2: Affinity Purification of Analytical Antibody

(34) This Example describes two methods that can be used to isolate from a biological fluid from a human subject an analytical antibody that is able to recognize and/or bind the C-terminal end of an ISV. The antibody is isolated from 4 different serum samples that were characterized in that these induced a positive signal in an ADA assay according to the test as described in Example 1.

(35) Starting from serum samples, each of these protocols provide a purified preparation of interference factor(s) that can be used as the analytical antibody in the methods described herein. These methods can also more generally be used to purify the interference factor(s) for other purposes (for example, the interference factor(s) purified using the protocols below were also used experimentally in Example 8 in order to show that binding to an ISV or ISV-construct by monoclonal 21-4 is predictive for binding of the same ISV or ISV-construct by interference factors, and thus by the tendency of said ISV or ISV-construct to undergo aspecific protein interference in an ADA assay).

Example 2A: Purification Using Protein a and Affinity Chromatography

(36) In a first step, the IgG antibody fraction was enriched from the serum samples using protein A affinity chromatography. Typical columns that were used for this enrichment included HiTrap MabselectSure and MabSelectXtra (GE Healthcare); PorosMabCapture A (Applied Biosystems). Purification of the IgG antibodies from the serum samples was performed in an automated and similar manner over all experiments. Chromatographic runs were performed on the AKTA purifier systems (GE Healthcare) and logged in real-time using UNICORN protein purification software (GE Healthcare). Briefly, the serum sample was diluted 1:1 with D-PBS (Dulbecco's Phosphate-Buffered Saline) and 0.22 μm filtered before uploading on the column at a fixed flow rate of 0.5 mL/min. The column was washed to remove non-specific binding components over 5 column volumes using D-PBS at a flow rate of 0.5 mL/min. The IgG fraction was eluted by acidic elution, using 100 mM Glycine pH 2.6 buffer, and a flow rate of 0.5 mL/min. After elution, the fractions were neutralized using 1.5 M Tris buffer pH 8.8. SDS-PAGE was run to confirm the isolation of IgG antibodies in the elution.

(37) In a second step, the interfering IgGs were further enriched by applying the protein A purified IgG fraction from the 4 different sera onto ISV coupled affinity columns. More specifically, the interfering IgG were further enriched by binding to a column containing an ISV with sequence of SEQ ID NO: 1. To this, the ISV was covalently linked to Sepharose 4 fast flow (GE Healthcare) using the CNBr (Cyanogen bromide)-coupling method according to the manufacturer's procedure. The affinity purification was performed in an automated and similar manner over all experiments. Chromatographic runs were performed on the AKTA purifier systems and logged in UNICORN. Briefly, the IgG enriched sample (up to 10 mL loading volume) was uploaded on the column at a fixed flow rate of 0.5 mL/min. The column was washed to remove non-specific binding-components over 5 column volumes using D-PBS at a flow rate of 0.5 mL/min. The ISV-binding components were eluted by acidic elution, using 100 mM Glycine pH 2.6 buffer, and a flow rate of 0.5 mL/min. After elution, the fractions were neutralized using 1.5 M Tris buffer pH 8.8. The fractions were analyzed using SDS-PAGE which confirmed the isolation of IgG antibodies in the elution (data not shown).

(38) These fractions were pooled and used for further analyses such as those described in Example 3.

Example 2B: Purification Suing CaptureSelect™ Chromatography

(39) Alternatively, interference factor(s) were recovered from plasma and purified using the commercially available IgA binding affinity resin CaptureSelect hIgA™ (BAC BV), which is based on camelid-derived heavy-chain only variable domains (VHH). The collected ‘IgA fraction’ containing IgA together with interfering IgG was subsequently loaded onto a protein A column to remove the IgA fraction. The protein A column was processed according to generic IgG purification conditions (running buffer: PBS; elution buffer: 100 mM glycine pH=2.7; post elution neutralization via 1M Tris). The interference factor was recovered from the Prot A elution in >95% yield.

(40) In a variation to this method, another CaptureSelect affinity resin (CaptureSelect Alpha-1 Antitrypsin resin, a VHH based commercially available affinity resin, not targeting any antibody related proteins) was be used. This resin provided a high interference factor binding efficacy and allowed for a selective 2 step elution: antitrypsin via neutral pH elution using 2.0 M MgCl2, followed by the interference factor elution via an acidic step (0.1 M Glycine pH3.0, similar to protein A/G elution conditions; neutralisation using 1.5M Tris). This one step purification yielded up to 15 μg interfering IgG1 per mL high interference plasma, which is approximately 0.3% of the total IgG present. Optionally, the neutralised interference fraction can be desalted and further purified via a Size Exclusion Column equilibrated in D-PBS.

Example 3: Influence of Different ISV Substitutions on the Tendency of ISV to Give Rise to Protein Interference

(41) As mentioned in the description above, the present invention makes available certain assays and techniques which make it possible to make an assessment of whether or not a given ISV has a tendency to give rise to protein interference. These include the ECL based bridging assay and the drug displacement set-up assay used in Example 1, as well as the BIACORE assay described in this Example 3 and the bridging/competition ADA assay described in the further Examples below.

(42) As also mentioned in the description above, these assays can also be used to determine whether specific changes (such as amino acid deletions, substitutions or additions) can influence (and preferably reduce) the tendency of a given ISV to give rise to protein interference. Some of these changes will be or become clear to the skilled person based on the disclosure herein and on the experimental data presented in Example 1 and this Example 3.

(43) As already indicated by the data generated in Example 1, it appears that certain mutations in or close to the C-terminal region (as defined herein) of an ISV can (strongly) influence its tendency to give rise to protein interference. For example, adding a few amino acid residues to the C-terminus (such as 1 or 3 alanine residues) appears to strongly reduce the tendency of an ISV to give rise to protein interference, and appears even to be able to negate the presence of other substitutions (for example, in or close to the C-terminal region) which appear to increase the tendency to give rise to protein interference (for example, an A14P substitution).

(44) In this Example 3, both the effect of other substitutions as well as the effect of adding additional amino acids to the C-terminus was investigated by comparing related ISV's with different substitutions, using the analytical polyclonal antibody generated in Example 2. The analysis was done by measuring the kinetics of interaction between each of the ISV's investigated and the analytical polyclonal by means of surface Plasmon resonance (SPR) using the Biacore™ T100 biosensor from GE Healthcare. The ISV tested in this Example 3 were those of SEQ ID NO's 3 to 22 (see Table A above and Table V below).

(45) In a typical experiment, a polyclonal antibody solution was prepared at 10 μg/ml in 10 mM NaOAc pH5.0. This polyclonal antibody was then immobilized on a CM5 sensorchip using amine coupling by the EDC/NHS method (EDC═N-ethyl-N′43-diethylamino-propyTh carbodiimide; NHS═N-hydroxysuccinimide) according to the manufacturer's procedure. The amount immobilized gave approximately 2700 response units (RU). A fixed concentration of 500 nM of ISV was then injected onto the surface for 120 seconds at a flow rate of 45 μl per minute. Because no efficient regeneration buffer could be identified, the dissociation time was elongated to 2400 seconds. The signal obtained by injecting the ISV onto a blank flow cell was subtracted from the signal obtained by injecting the ISV onto the polyclonal antibody bound flow cell. The blank flow cell was activated/deactivated in a similar way as the flow cell for the polyclonal antibody, but without adding protein. Also, a blank injection (HBS-EP+running buffer (HBS=Hepes Buffered Saline: GE Healthcare) was subtracted to correct for possible baseline drift.

(46) To examine the effect of adding amino acid residues to the C-terminus, the influence of adding 1 or 2 alanines and 1, 2 or 3 glycines was investigated by comparing the binding of ISV with the different additions, using an analytical polyclonal antibody generated as described in example 2. The ISV's generated and tested for this purpose were NB's 3.4 to 3.9 (SEQ ID NO's: 5 to 10).

(47) As representative examples of the kind of data obtained, FIG. 3 shows the binding of NB's 3.4 to 3.9 to the immobilized polyclonal antibody. Table V summarizes the results obtained.

(48) TABLE-US-00007 TABLE V SEQ ID Position Position Position Position Binding** Clone ID NO 113.sup.(1) 114.sup.(1) 115.sup.(1) 116.sup.(1) (RU) NB 3.4 5 S 75 NB 3.5 6 S A 9 NB 3.6 7 S A 8 A NB 3.7 8 S G 31 NB 3.8 9 S G G 13 NB 3.9 10 S G G G 13 **Binding signal obtained at the end of injection (=maximal RU signal) .sup.(1)In this numbering, position 113 is the last “S” of the C-terminal VTVSS motif, and positions 114, 115 and 116 are the positions immediately following (downstream) of said position 113.

(49) To examine the effect of (other) substitutions in the C-terminal region, the influence of different substitutions was investigated by comparing related ISV's containing these substitutions, using the same analytical polyclonal antibody as described above. The analysis was done as described above.

(50) The ISVs containing said substitutions that were tested were NB's 3.1, 3.2 and 3.4 (SEQ ID NO's 3, 4 and 5); NB's 3.10 to 3.15 (SEQ ID NO's 11 to 16), which were compared with NB 3.4; NB's 4.1 and 4.2 (SEQ ID NO's 17 and 18) and NB's 6.1, 6.2, 6.4 and 6.5 (SEQ ID NO's 19 to 22).

(51) As representative examples of the kind of data obtained: FIG. 4 shows the binding of NB's 3.4, 3.11, 3.12 and 3.13 to the immobilized polyclonal antibody; FIG. 5 shows the binding of NB's 3.4, 3.14 and 3.15 to the immobilized polyclonal antibody; FIG. 6 shows the binding of NB's 3.1, 3.2 and 3.4 to the immobilized polyclonal antibody; FIG. 7 shows the binding of NB's 4.1 and 4.2 to the immobilized polyclonal antibody; FIG. 8 shows the binding of NB's 6.1, 6.2, 6.4 and 6.5 to the immobilized polyclonal antibody.

(52) Tables VI, VII and VIII summarize the results obtained.

(53) TABLE-US-00008 TABLE VI SEQ ID Position Position Position Binding** Clone ID NO 14.sup.(1) 83.sup.(1) 108.sup.(1) (RU) NB 3.4 5 P R L 75 NB 3.10 11 A R L 91 NB 3.11 12 P K L 88 NB 3.12 13 A R Q 86 NB 3.13 14 P R Q 90 **Binding signal obtained at the end of injection (=maximal RU signal) .sup.(1)numbering according to Kabat.

(54) TABLE-US-00009 TABLE VII SEQ ID Position Position Binding** Clone ID NO 11.sup.(1) 110.sup.(1) (RU) NB 3.4 5 L T 75 NB 3.14 15 L Q 79 NB 3.15 16 S T 22 **Binding signal obtained at the end of injection (=maximal RU signal) .sup.(1)numbering according to Kabat.

(55) TABLE-US-00010 TABLE VIII SEQ ID Position Position Binding** Clone ID NO: Position 14 83.sup.(1) 108.sup.(2) Tag* (RU) NB 3.1 3 A K Q − 2 NB 3.2 4 A K Q + 0 NB 3.4 5 P R L − 59 Position Position Binding** Clone ID Position 14 83.sup.(3) 108.sup.(4) Tag* (RU) NB 4.1 17 P R L − 51 NB 4.2 18 P R L + 0 Position Position Binding** Clone ID Position 14 83.sup.(5) 108.sup.(6) Tag* (RU) NB 6.1 19 P K Q + 0 NB 6.2 20 P K Q − 39 NB 6.4 21 P R L + 0 NB 6.5 22 P R L − 66 *if “+”, this ISV contains additional amino acids at the C-terminal VTVSS end **Binding signal obtained at the end of injection (=maximal RU signal) .sup.(1)numbering acc. to Kabat (corresponds to the a.a. at position 87 in SEQ ID NO's 3 to 5). .sup.(2)numbering acc. to Kabat (corresponds to the a.a. at position 123 in SEQ ID NO's 3 to 5). .sup.(3)numbering acc. to Kabat (corresponds to the a.a. at position 86 in SEQ ID NO's 17 and 18). .sup.(4)numbering acc. to Kabat (corresponds to the a.a. at position 116 in SEQ ID NO's 17 and 18). .sup.(5)numbering acc. to Kabat (corresponds to the a.a. at position 86 in SEQ ID NO's 19 to 22). .sup.(6)numbering acc. to Kabat (corresponds to the a.a. at position 112 in SEQ ID NO's 19 to 22).

(56) Again, without being limited to any specific hypothesis or explanation, the data presented above shows that (various) substitutions to the C-terminal region (as defined herein) of an ISV can alter/improve its tendency to give rise to protein interference.

Example 4: Representative Protocols for Performing the ADA Assays of FIG. 1

(57) This Example gives some representative but non-limiting conditions that could be used for performing the competitive/bridging ADA assays schematically shown in FIG. 1: ADA assay of FIG. 1A in solution: Samples 100% matrix, 30′, 37° C., Acid treatment using acetic acid in 10 matrix, 5′, RT, Preincubation/acid neutralisation sample: ISV-Sulfo (:Tris) 1:1:1 (1:0.9:0.9:0.1), 1 h, RT; On plate 1 h, RT; Wash 3×, Readbuffer 4× ADA assay of FIG. 1B in solution: Samples 20% matrix, 30′, 37° C., Preincubation sample: ISV-Sulfo 1:1:1, lh, RT, On plate 1 h, RT, Wash 3×, Readbuffer 2× Sequential ADA assay of FIG. 1C: Capture ISV-Bio, 1 h, RT, Wash 3×, Samples 20% matrix, 15′, RT, On plate: 2h, RT, Wash 3×, Detection ALX-0141-Sulfo, lh, RT, Wash 3×. Readbuffer 4×

Example 5: Predicting Sensitivity of the ISV to Aspecific Protein Interference Using the Analytical Antibody

(58) This example describes a bridging/competition ADA assay using the analytical antibody that can be used to predict sensitivity of an ISV to aspecific protein interference.

(59) The ISV to be tested is diluted at a concentration of 10 μg/ml and incubated with the analytical antibody at 400 ng/ml, purified according to Example 2, and incubated at 37° C. at 600 rpm in 96 well polypropylene plates. The sample (50 μL) is then diluted 1/3 in 1:1 mixture (100 μL) of 2 μg/ml biotinylated and 2 μg/ml sulfo-tagged ISV and incubated for 1 hour at RT, 600 RPM. MSD MA®96-well Standard Streptavidin plates are blocked with 150 μL/well Superblock® T20 for 1 hour at RT, then washed 3 times with PBS/0.05% Tween20 (=wash buffer). Sample/1:1 mix (biotinylated and sulfo-tagged ISV) (50.0 μL) is transferred from the polypropylene plate to the MSD plate and incubated for 1 hour at RT, 600 rpm. Plates are washed three times prior to addition of 2× Read Buffer (MSD) (150 μL/well) and reading the ECL units (ECLU) on an MSD instrument (Sector Imager 2400 reader).

(60) Using this assay, the ISVs of SEQ ID NO's 23 to 30 were tested and compared. The data are shown in Table IX. These data not only show that the assay described in this Example can be used to predict the tendency of an ISV to give rise to protein interference, but the data generated also confirm the findings from the earlier Examples on the effect of substitutions in the C-terminal region. As can be seen, addition of 3 (and to lesser extent 1) Alanine residues at the C-terminus of the fully humanized ISV abolished its capacity to compete with binding of the analytical antibody. Mutating position 14 on the wild type ISV variant from Alanine to Proline clearly increased its capacity as competitor in the assay, (=making the ISV variant more prone to aspecific protein interference), whereas mutating position 83 and 108 did not clearly influenced the sensitivity of the ISV to aspecific protein interference.

(61) TABLE-US-00011 TABLE IX ID affinity purified antibody IHuP#002-001-ABL-08 ECLU in screening assay (using SEQ ID NO: 1) 2919 Nanobody Variant (right hand column mentions ECLU the humanizing substitutions and C-terminal confirm- % additions made compared to the wildtype atory reduc- sequence of SEQ ID NO: 23) assay tion SEQ ID NO: 23 Wildtype VHH 2706 7.3 SEQ ID NO: 24 Wildtype VHH + A14P 268 90.8 SEQ ID NO: 25 Wildtype VHH + K83R 2460 15.71 SEQ ID NO: 26 Wildtype VHH + Q108L 2533 13.23 SEQ ID NO: 27 Wildtype VHH + 319 89.1 A14P + K83R + Q108L SEQ ID NO: 28 Wildtype VHH + A14P + 251 91.4 R39Q + K83R + T91Y + Q108L SEQ ID NO: 29 Wildtype VHH + A14P + 1207 58.64 R39Q + K83R + T91Y + Q108L + 1 additional A at C-terminus (A114) SEQ ID NO: 30 Wildtype VHH + A14P + 3301 −13.09 R39Q + K83R + T91Y + Q108L + 3 A's at C-terminus (A114 + A115 + A116)

Example 6: Influence of the Addition of Amino Acids to the C-Terminus of Anti-OX40L Nanobodies on their OX40L Blocking Potency

(62) This example demonstrates that the C-terminal extension has no influence on activity or blocking potency of the Nanobodies.

(63) The in vitro potency of the trivalent bispecific sequence optimized anti-OX40L Nanobody Nb 3.16 (SEQ ID NO: 31) was compared with the potency of the corresponding Nanobody containing one additional Ala at its C-terminus Nb 3.17 (SEQ ID NO: 32).

(64) A first assay, the T-cell activation assay, was performed as follows. PBMCs were isolated from buffy coats (Red Cross, Ghent. Belgium) from healthy donors using Ficoll Paque Plus reagent (GE Healthcare) and washed using RPMI 1640 complete medium (RPMI1640+GlutaMAX+25 mM HEPES+10% fetal bovine serum+1% Penicillin/Streptomycin; Invitrogen). The PBMC's (1×10.sup.5 cells/well) were stimulated with phytohaemagglutinin (PHA-L; final concentration 0.6 μg/ml) before the addition to 1×10.sup.4 hOX40L expressing CHO cells (irradiated with gamma scintillator at 3000 RAD; UZ Gent, Belgium) and dilution series of anti-OX40L Nanobodies RPMI 1640 complete medium and incubated for 22 hours at 37° C. in CO, incubator. Production of IL2 by the PBMCs was measured in ELISA. Wells of a Maxisorp plate were coated overnight at 4° C. with anti-human IL2 monoclonal antibody (BD Biosciences). After washing and blocking of the coated wells, a 1/2 dilution of cell supernatant was added. As a standard, 1/2 serial dilutions of recombinant human IL2 (BD Biosciences) starting from 2000 μg/ml were included. Detection was done using biotinylated anti-human IL2 monoclonal antibody (BD Biosciences) and HRP conjugated streptavidin (Thermo Scientific) and esTMB (SDT Reagents). The reaction was stopped with 1N HCl and the OD was read at 450 nm. As expected, the potency of the trivalent bispecific sequence optimized Nanobody Nb 3.17 (IC50=0.13 nM, 95% CI=0.098-0.17 nM) was comparable to that of Nb 3.16 (IC50=0.10 nM, 95% CI=0.071-0.15 nM).

(65) In a second ELISA-based competition assay, a dilution series (from 1.5 μM to 0.083 μM) of the Nanobodies were pre-incubated overnight at room temperature with 100 ng/ml human OX40/Fc (R&D Systems) and 10 ng/mlbiotinylated human OX40L (R&D Systems; in-house biotinylated as described in Example 1) in PBS+0.1% BSA+0.01% Tween-20. Next, the samples were incubated on Maxisorp plates coated with 10 ug/ml anti-human Fc Nanobody (in-house generated) and blocked with PBS+1% BSA+0.1% Tween-20. Bound human OX40/Fc was detected using HRP conjugated streptavidin (Thermo Scientific) and sTMB (SDT Reagents). The reaction was stopped with 1N HCl and the OD was read at 450 nm. In accordance with the cell-based assay, the potency of the trivalent bispecific sequence optimized Nanobodies Nb 3.17 (IC50=0.178 nM, 95% CI=0.152-0.200 nM) was comparable to that of Nb 3.16 (IC50=0.179 nM, 95% CI=0.149-0.215 nM).

Example 7: Generation of Monoclonal Antibody 21-4-3

(66) Two groups of different mice strains (BALB/c and NMRI—three mice each) were intraperitoneally immunized with the Nanobody construct of SEQ ID NO:98 in WO 2006/122825, in a water-in-oil emulsion of equal volumes of antigen and Freund's complete or incomplete adjuvant) over a period of 39 days, with boosting until suitable antiserum titers were obtained.

(67) After asphyxiation of the stimulated mice in CO2, the spleens were aseptically removed and a single cell suspension of pooled spleens was prepared. Spleen cells and myeloma cells were washed several times with DMEM and fused in the presence of 1 ml 50% (w/v) PEG 3350 (ratio spleen cells to SP2/0 3:1). For fusion was used the myeloma cell line SP2/0-Ag14 from German Collection of Microorganisms and Cell Cultures (DSMZ GmbH, Braunschweig). This cell line is a hybrid between BALB/c spleen cells and the myeloma cell line P3x63Ag8. The so produced hybridomas were resuspended in CGM containing 20% FCS and aminopterin (HAT medium) and plated out (140 μl/well) into eight 96-well tissue culture flat-bottom plates (Corning-Costar) containing 140 μl/well CGM (20% FCS) with peritoneal excudate cells as feeder cells. The plates were incubated for 10 days in a complete growth medium (CGM) containing DMEM with supplements 2-mercaptoethanol, L-Glutamin, Stable Glutamin, HT and non essential amino acids (in concentrations recommended by the supplier) and FCS at different concentrations (10%, 15% or 20%). During this period cells were fed two times with HAT medium. The cell culture supernatants from hybridoma cells usually contained 1 to 20 μg/ml antibody, which were tested in a binding ELISA to confirm binding to the Nanobody construct of SEQ ID NO:98 in WO 2006/122825.

(68) Cells from positive IgG producing wells were transferred into wells of 48 well plates and cultivated for 2-4 days (depending on growth characteristic of cells). Binding ELISA's on ALX081 and human/cynomolgus IgG were carried out in order to exclude the unspecific binders. Hybridoma cells expressing binders specific for the Nanobody construct of SEQ ID NO:98 in WO 2006/122825 were twice cloned using limited dilution. After fusion and rescreening 7 primary cultures producing antibodies against ALX-081 were identified. All these primary cultures produced antibodies not cross-reacting with human or cynomolgus IgG. The primary cultures were recloned (twice).

(69) Clone 21-4 (one of the clones that stably produced antibodies against ALX-081 after the second cloning) was given the designation “ABH0015” and was deposited with the Belgian Coordinated Collections of Micro-organisms (BCCM) in Ghent, Belgium on Jun. 4, 2012 under accession number LMBP-9680-CB. The mouse monoclonal produced by ABH0015 was called 21-4-3: isotype determination for 21-4-3 showed an IgG1 heavy chain and a kappa light chain, which were sequenced (see SEQ ID NO's: 35 and 36, respectively). 21-4-3 was shown to bind to the C-terminal region of the Nanobody construct of SEQ ID NO:98 in WO 2006/122825 (data not shown).

Example 8: Binding of 21-4 to an ISV is Predictive of the Tendency of an ISV to Undergo Aspecific Protein Interference

(70) This Example together with the following Example 9 demonstrates that binding of the monoclonal 21-4 to an ISV can be used to predict (within the degrees of certainty indicated in this Example) of whether a given ISV will have a tendency to undergo aspecific protein interference (e.g. in an ADA assay).

(71) This Example 8 in particular shows that 21-4 can be used to predict whether certain proposed modifications to a given ISV (such as adding one or more amino acid residues to the C-terminus of an ISV and/or substituting one or more amino acid substitutions within the C-terminal region of an ISV) will lead to a reduction of the tendency of said ISV to undergo aspecific protein interference.

(72) In short, a set of 53 different Nanobodies and Nanobody constructs (see Table B and SEQ ID NO's: 38 to 89) were tested for binding by monoclonal 21-4-3. The same Nanobodies and Nanobody constructs were also tested for binding by purified preparations of interference factor(s) obtained from three different human donors (referred to herein as “Donor 8”, “Donor 19” and “Donor 30”), to see if there was any correlation between binding by 21-4 and by the purified interference factors.

(73) It was established that binding of an ISV by 21-4 can indeed be used to predict binding of the same ISV's by the interference factor(s) (within the overall degree of confidence provided by the data set out herein).

(74) To demonstrate this, as detailed by the experimental data set out below, the binding of the 53 Nanobodies or Nanobody constructs (as listed in Table B; see SEQ ID NO's: 38 to 89) by 21-4 was measured using a Biacore T100 (according to the protocol set out below) and was compared to binding of a reference Nanobody or construct (also listed in Table B), as measured using the same Biacore instrument and the same protocol. The results are shown in Table X below.

(75) TABLE-US-00012 TABLE X  More than More than 70% 90% reduction reduction reduction reduction reduction reduction in binding in binding in of inter- of inter- of inter- of Nanobody of Nanobody binding ference in ference in ference in to 21-4-3 to 21-4-3 of 21-4-3 serum from serum from serum from predicts  predicts  vs Donor A Donor B Donor C >50% >50% binding compared compared compared reduction reduction mutations of to to to in binding in binding SEQ C-terminal to the Reference Reference Reference Reference of nanobody of nanobody ID amino C-terminal Sequence Sequence Sequence Sequence to inter- to inter- NO: acid(s) region (=100%) (=100%) (=100%) (=100%) ference ference 37 A none   7%   3%  21%  31% ok ok 38 A none   0%   9%  25%   7% ok ok 39 A none   0%  10%  43%  35% ok ok 40 A none   1%   6% #N/A #N/A ok ok 41 A none   7%   6%   9% #N/A ok ok 42 A none   0%   1%   4% #N/A ok ok 43 A none   3%   3%  20% #N/A ok ok 44 A none   1%   5% #N/A #N/A ok ok 45 none P14A, P41T,  22%   0% #N/A #N/A ok S62F, S74A, S82bN, R83K, L108Q 46 AAEQKLI A14P, T41P,   2%   0% #N/A #N/A ok ok SEEDLN F62S, A74S, GAAHHH N82bS, K83R, HHH Q108L 47 GGGGSG none   4%   1% #N/A #N/A ok ok GGSRDW DFDVFG GGTPVG G 48 AAEQKLI none   3%   0% #N/A #N/A ok ok SEEDLN GAAHHH HHH 49 AAEQKLI V5L, I23A,   4%   0% #N/A #N/A OK OK SEEDLN E44G, A49S, GAAHHH A68T, A74S, HHH T78L, W79Y, K83R, T110Q, Q108L 50 none L11S  44%  77%  33% #N/A (<70% (<90% reduction) reduction) 51 none T110Q  88%  85%  84% #N/A (<70% (<90% reduction) reduction) 52 none S112G 100%  84%  58% #N/A (<70% (<90% reduction) reduction) 53 none S113G  13%  85%  88% #N/A NOK (<90% reduction) 54 none L11S, T110Q,  16%  39%  16% #N/A OK OK S112G, S113G 55 A none   6%   2%  21%  31% OK OK 56 G S113G   3%   2%  25%   0% OK OK 57 AS none   6%   1%   2% #N/A OK OK 58 AST none   6%   2%   2% #N/A OK OK 59 ASTK none   6%   2%   1% #N/A OK OK 60 ASP none   6%   2%   1% #N/A OK OK 61 AP none   6%   2%   2% #N/A OK OK 62 APT none   6%   2%   1% #N/A OK OK 63 W none   3%   4%   8% #N/A OK OK 64 L none   6%   3%   4% #N/A OK OK 65 none P14A  23%  73% 121% 64% NOK (<90% reduction) 66 none L11S  48%  81%  29%  84% (<70% (<90% reduction) reduction) 67 none R83K 101% 102% 117%  96% (<70% (<90% reduction) reduction) 68 none P14A, L108Q  26%  38% 115%  49% NOK (<90% reduction) 69 none L108Q 106%  80% 120%  84% (<70% (<90% reduction) reduction) 70 none T110Q 106%  90% 105%  98% (<70% (<90% reduction) reduction) 71 none S113G  44%  88% 105%  87% (<70% (<90% reduction) reduction) 72 none S112G, S113G  45%  70%  47%  56% (<70% (<90% reduction) reduction) 73 G S112G, S113G   1%   6%   8%   9% OK OK 74 G none   1%   4%  41%  33% OK OK 75 AA none   1%   1%  11%  16% OK OK 76 GGG none   2%   2%  17%  20% OK OK 77 A none   1%   2%  12%  13% OK OK 78 none Q13R   1%  96%  96% 100% NOK NOK 79 GG none   2%   1%  17%  13% OK OK 80 none T110Q, S112G,  51%  65%   5%  58% (<70% (<90% S113G reduction) reduction) 81 none L11V  75%  94%  50%  89% (<70% (<90% reduction) reduction) 82 none P84A  56%  96%  80% 100% (<70% (<90% reduction) reduction) 83 none T87A  79%  56%  73%  61% (<70% (<90% reduction) reduction) 84 none S112G  91%  84%  50%  83% (<70% (<90% reduction) reduction) 85 none L11S, T110Q,  32%  46%   5%  42% (<70% (<90% S112G, S113G reduction) reduction) 86 none L11S, T110Q  64%  76%   6%  86% (<70% (<90% reduction) reduction) 87 none L11S, S112G,  41%  51%   5%  39% (<70% (<90% S113G reduction) reduction) 88 A L11S, T110Q   1%   1%   5%  11% OK OK 89 none L11S, Pl4A,   2%  14%   5%  17% OK OK T110Q, S112G, S113G

(76) For each of the 53 Nanobodies or Nanobody constructs tested, the reference was chosen such that compared to the reference, the tested Nanobodies or Nanobody constructs either had one or more additional amino acid residues at the C-terminal end (which were added in order to test the effect of such addition on protein interference, and in particular in order to reduce said interference) and/or one or more mutations within the C-terminal region (for example, as a result of humanization compared to the reference).

(77) The results were expressed as a percentage reduction in binding (measured as RU units) for the given Nanobody versus the binding of the reference (also measured in RU units—for example, if the measured binding level (RU) of the reference Nanobody was 276 and the binding level of the given Nanobody (also in RU) was 9, then the reduction in binding level was to a level of [9 RU/276 RU]×100%=3%), which means a reduction of 97% compared to the reference (100%).

(78) Similarly, binding of the purified interference factor(s) from each of the three donors to each of the 53 Nanobodies or Nanobody constructs was measured using the same Biacore instrument and compared to binding of the purified interference factor(s) to the same reference Nanobody or construct. The results were similarly expressed as a percentage reduction in binding of the interference factor to the given Nanobody or Nanobody construct vs the reference.

(79) It was found that for essentially all Nanobody or Nanobody construct in which one or more amino acid residues had been added to the C-terminal end compared to the reference, that the binding of the interference factor(s) was dramatically reduced. This again confirms that adding one or more amino acid residues to the C-terminal end of an ISV (VTVSS) can reduce aspecific protein interference in an ADA assay. It was also found that in the majority of cases, only making substitutions within the C-terminal region (i.e. without adding one or more amino acid residues to the C-terminus) compared to the reference often did not have a similar dramatic impact on the binding of the interference factor(s).

(80) The data was then further analysed to determine whether a reduction in binding by 21-4 compared to the reference was in any way correlated with a reduction in binding by each of the three different preparations of purified interference factor compared to the reference. Such correlations were found.

(81) For example, it was found that of the 54 Nanobodies or Nanobody constructs tested, 36 showed a reduction in binding by 21-4 of more than 70% compared to their respective reference sequence (with most of these 36 having one or more additional amino acid residues at the C-terminus, in some cases in combination with substitutions within the C-terminal region). Of these 36, 32 also showed reduction in binding by the interference factor(s) compared to the reference of more than 50% (and in a large number of cases, in particular for Nanobodies or Nanobody constructs with one or more amino acid residues added at the C-terminus, the reduction was far greater than 50%, such as more than 70% or even more than 90%, see the data given in the Table X). This demonstrates that in 32 out of 36 cases (i.e. 89%), a reduction in binding by 21-4 of more than 70% (compared to the reference=100%) is predictive for a reduction in binding by the interference factors of more than 50% (compared to the same reference). For clarity, in each case, the reduction was calculated as 100%−[the percentage given in the Tables below for the level of reduction achieved with the Nanobody tested].

(82) Similarly, it was found that of the 53 Nanobodies or Nanobody constructs tested, 33 showed a reduction in binding by 21-4 of more than 90% compared to their respective reference sequence (again, with most of these 33 having one or more additional amino acid residues at the C-terminus, in some cases in combination with substitutions within the C-terminal region). Of these 33, 32 also showed reduction in binding by the interference factor(s) compared to their respective reference sequence of more than 50%. This demonstrates that in 32 out of 33 cases (i.e. 97%), a reduction in binding by 21-4 of more than 90% (compared to the reference) is predictive for a reduction in binding by the interference factors of more than 50% (compared to the same reference).

(83) It should also be noted that such a reduction in binding of the interference factor(s) by more than 50% (as evidenced by a reduction of binding by 21-4 of more than 70%) means that such interference factor(s) essentially no longer interfere(s) with an ADA assay for the ISV in question: experimental confirmation using an ADA assay showed that when the binding by the interference factor(s) is reduced by more than 45%, that no significant influence of the presence of the interference factor(s) on the ADA assay could be observed. In this respect, it will be also be clear to the skilled person that this will even more so be the case when the binding by interference factor(s) is reduced to an extent far greater than 50% (such as by more than 70% or even more than 90%), as is observed in some cases (see again the data presented herein).

(84) In fact, it has been found that a reduction of more than 45% of binding by 21-4 is indicative of a reduction of binding by interference factors of more than 45%, which as mentioned means that the interference factor(s) no longer interfere with the ADA assay.

(85) Moreover, the data presented herein on the correlation between (reduction in) binding by 21-4 and (reduction in) binding by interference factor also allowed the present inventors to set an absolute value for the binding by 21-4 below which it can be expected (within the confidence provided by the data set out in this Example 8) that an ISV or ISV-based construct will not be susceptible to binding by interference factor(s) in a way that could interfere with an ADA assay. As set out in the following Example 9, this value is 500 RU (determined and calculated as set out in Example 9).

(86) Monoclonal 21-4 was purified from the culture medium of the hybridoma obtained in Example 7 above, as follows: Hybridoma cells secreting the monoclonal antibody 21-4-3 were cultured in spinner flasks in serum free medium (CD Hybridoma, Gibco, supplemented with 8 mM L-glutamine (Invitrogen) and 1× cholesterol (250× cholersterol lipid concentrate, Gibco)) at a volume of 100 mL or 500 mL. The cleared supernatant was filtered, and the murine IgG1 captured on a ProteinA column (HiTrap MabSelect SuRe, 5 mL, GE Healthcare) at a reduced flow rate of 2 mL/min. Bound antibody was eluted in 0.1M citrate buffer pH3.0, and elution fractions (of 5 mL) directly neutralized with 1 mL of 1M TRIS pH9. Purity of the antibody was verified by reducing and non-reducing SDS-PAGE.

(87) The purified preparations of interference factor(s) from Donors 8 and 19 were obtained from serum samples from said donors by means of affinity purification, essentially as described in Example 2A. The interference factor(s) from Donor 30 were obtained from a serum sample of Donor 30, essentially as described in Example 2B.

(88) To determine the binding of 21-4 to each of the Nanobodies or Nanobody constructs, the protocol described in Example 9 was used.

(89) The binding of the interference factors from the three donors to each of the Nanobodies or Nanobody constructs was determined using a Biacore T100 essentially as described in Example 3, using the interference factor from each of the donors 8, 19 and 30, directly immobilized on a CM5 sensor chip.

Example 9: Protocol for Predicting Whether an ISV Will have a Tendency to Undergo Aspecific Protein Interference (Using Monoclonal 21-4

(90) Binding measurements were performed using a Biacore T100 using a CM5 T120416 sensor chip, with running buffer HBS-EP+, 25° C. 21-4 was captured via immobilized rabbit anti-mouse IgG, as it was found that directly immobilized mAb 21-4-3 surface could not efficiently be regenerated. The anti-mouse IgG used was a polyclonal rabbit anti-mouse IgG antibodies reacting with all IgG subclasses, IgA and IgM (GE Healthcare; Cat #BR-1008-38; Lot #10056316). Immobilisation of the anti-mouse IgG was performed using manual amine coupling using a 7 minute injection of EDC/NHS for activation and a 7 minute injection of 1M ethanolamine HCl pH 8.5 for deactivation (Biacore, amine coupling kit). Binding conditions are listed in Table XI. Based on the immobilization level and MW of the proteins, the theoretical R.sub.max for mAb21-4-3 binding to the immobilized anti-mouse IgG was ˜13000RU (when one mAb21-4-3 molecule is binding to one anti-mouse IgG molecule).

(91) TABLE-US-00013 TABLE XI Immobili- Conc. Contact Flow rate Immobilization zation Protein (μg/ml) time (s) (μl/min) buffer level (RU) Anti-mouse 30 420 5 10 mM acetate 13028 IgG pH 5.0 Anti-mouse 30 420 5 10 mM acetate 13318 IgG 24 pH 5.0

(92) The conditions used for the binding experiment (Biacore T100) using 21-4 immobilized in the manner are given in Table XII. The anti-mouse IgG surface could successfully be regenerated after capture of mAb21-4-3 and injection of all samples (with a limited increase for baseline level after each regeneration).

(93) TABLE-US-00014 TABLE XII Capture Flow path Flow rate (μl/min) 10 Contact time (s) 180 Concentration (μg/ml) 10 Binding and dissociation Flow path 3, 4 Flow rate (μl/min) Sample contact time (s) 120 Sample concentration (nM) 500 Dissociation time (s) 600 Regeneration1 Flow path 3, 4 Flow rate (μl/min) 10 Regeneration contact time (s) 180 Regeneration buffer 10 mM Glycine-HCl pH 1.7 Stabilization time (s) 120 If . . . Then . . . Else If after regeneration 1 >20RU on Fc4 Else exit cycle Regeneration2 Flow path 3, 4 Flow rate (jol/min) 10 Regeneration contact time (s) 120 Regeneration buffer 10 mM Glycine-HCl pH 1.7 Stabilization time (s) 120

(94) The above protocol was used to generate the 21-4 binding data set out in Table X. When the absolute values for RU were considered (after adjusting the measured RU value for the molecular weight of the ISV, protein or polypeptide according to the formula ([RU measured]/[MW of the protein]×10.sup.6), it was found that the Nanobodies and Nanobody constructs mentioned in Table X that had an added alanine residue and that showed >90% reduction in binding to both 21-4 as well as interference factors, generally provided RU values of between 30RU and 400RU (with the corresponding reference Nanobodies or polypeptides—as listed in Table B—having RU values of more than 1000, usually more than 1500, and often more than 2000).

(95) Based on this, it was considered that an (adjusted) RU value of less than 500 in this assay would be clearly indicative of an ISV (or a protein or polypeptide that comprises as least one IS, as described herein) that will (essentially) not be bound by interference factors in a manner that would interfere with an ADA assay.

Example 10

(96) A sub-selection of anti-c-MET Nanobodies were fused at their C-terminus to an anti-human serum albumin (HSA) binding Nanobody (ALB11 or ALB23), separated by a 9GS-linker. Constructs had an additional C-terminal alanine residue. See Table XIII below, showing SEQ ID NOs: 90-99.

(97) TABLE-US-00015 TABLE XIII  SEQ ID Name of clone NO: Amino acid sequences A007901255 90 DVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIG WFRQAPGEEREGVSSISSTYGLTYYADSVKGRFTISS DNSKNTVYLQMNSLRPEDTAVYYCAATPIGLIGLDA YEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQ MNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA A007901256 91 DVQLVESGGGLVQPGGSLRLSCAASGFAFDDYAIG WFRQAPGEEREGVSSISSTYGLTYYADSVKGRFTISS DNSKNTVYLQMNSLRPEDTAVYYCAATPIGLIGLDA YEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQ MNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA A007901257 92 DVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIG WFRQAPGKEREGVSSISSTYGLTYYADSVKGRFTISS DNSKNTVYLQMNSLRPEDTAVYYCAATPIGLIGLDA YEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQ MNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA A007901258 93 DVQLVESGGGLVQPGGSLRLSCAASGFAFDDYAIG WFRQAPGKEREGVSSISSTYGLTYYADSVKGRFTISS DNSKNTVYLQMNSLRPEDTAVYYCAATPIGLIGLDA YEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQ MNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA A007901259 94 DVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIG WFRQAPGEERLGVSSISSTYGLTYYADSVKGRFTISS DNSKNTVYLQMNSLRPEDTAVYYCAATPIGLIGLDA YEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQ MNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA A007901260 95 DVQLVESGGGLVQPGGSLRLSCAASGFAFDDYAIG WFRQAPGEERLGVSSISSTYGLTYYADSVKGRFTISS DNSKNTVYLQMNSLRPEDTAVYYCAATPIGLIGLDA YEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQ MNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA A007901261 96 DVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIG WFRQAPGKERLGVSSISSTYGLTYYADSVKGRFTISS DNSKNTVYLQMNSLRPEDTAVYYCAATPIGLIGLDA YEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQ MNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA A007901262 97 DVQLVESGGGLVQPGGSLRLSCAASGFAFDDYAIG WFRQAPGKERLGVSSISSTYGLTYYADSVKGRFTISS DNSKNTVYLQMNSLRPEDTAVYYCAATPIGLIGLDA YEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQ MNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA A007901263 98 DVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIG WFRQAPGEEREGVSSISSTYGLTYYADPVKGRFTISS DNSKNTVYLQMNSLRPEDTAVYYCAATPIGLIGLDA YEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQ MNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA A007901222 99 DVQLVESGGGLVQPGGSLRLSCAASGFILDYYAIGW FRQAPGKEREGVLCIDASDDITYYADSVKGRFTISRD NSKNTVYLQMNSLRPEDTAVYYCATPIGLSSSCLLE YDYDYWGQGTLVTVSSGGGGSGGGSEVQLLESGG GLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKG PEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYL QMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA

(98) Nanobodies were expressed in P. pastoris in a culture volume of 5 mL. Nanobody expression was induced by addition of methanol and allowed to continue for 48 hours at 30° C. The cleared supernatants were used as starting material for purification by ProteinA affinity chromatography (MabCap A POROS, Applied Biosystems).

(99) The Nanobodies were tested in a thermal shift assay (as described in Examples 21 and 22 of U.S. Ser. No. 13/435,567), by analytical sizing (as described in Examples 21 and 22 of U.S. Ser. No. 13/435,567), and in the HGF-competition Alphascreen (as described in Examples 2 and 22 of U.S. Ser. No. 13/435,567). The HGF-competition Alphascreen was modified (as compared to the initially described set-up) for higher sensitivity by increasing the concentration of biotinylated HGF from 0.1 nM to 0.4 nM, and lowering the concentration of cMet/Fc from 0.1 nM to 0.016 nM (see Table XIV).

(100) The mobility of the clones A007901255 to A007901260 in analytic SEC on Phenomenex matrix was similar: The Nanobodies eluted at the expected molecular weight and no significant aggregation was observed.

(101) TABLE-US-00016 TABLE XIV Alphascreen [in pM] Tm at monovalent 9GS-Alb11-Ala SEQ ID pH 7.5 form fusion Clone number NO [in ° C.] IC.sub.50 95% CI IC.sub.50 95% CI A007901255 142 60.2 91  82-100 93 66-130 A007901256 143 59.8 48 40-58 85 64-110 A007901259 146 59.4 43 35-53 59 46-75  A007901260 147 59.4 35 27-47 64 46-89 

Example 11

(102) Some non-limiting examples of some Alb-23 like sequences directed against (human) serum albumin are given in SEQ ID NOs: 101 to 106. The variants of SEQ ID NOs: 101 to 106 (or other Alb-23 variants with 1 to 3 amino acid residues at the C-terminus, which may each be independently chosen from naturally occurring amino acid residues and may for example be independently chosen from A. G, V, L and I) may in particular be used when the albumin-binding Nanobody is provided at the C-terminal end of the polypeptide or protein construct.

(103) TABLE-US-00017 Alb-23:  [SEQ ID NO: 100] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSS  Alb-23D:  [SEQ ID NO: 101] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSA  Alb-23E:  [SEQ ID NO: 102] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSAA Alb-23F:  [SEQ ID NO: 103] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSAAA  Alb-23G:  [SEQ ID NO: 104] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSG  Alb-23H:  [SEQ ID NO: 105] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSGG  Alb-23I:  [SEQ ID NO: 106] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSGGG 

(104) The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced herein.