ANTIBODIES SPECIFIC FOR STRUCTURALLY DISORDERED SEQUENCES

Abstract

The present invention relates to a method for generating and/or obtaining specific binding moieties against intrinsically disordered proteins (IDPs) and/or intrinsically disordered protein domains which tend to be immunologically inert and lack immunogenicity in animals, in particular in mammals. The present invention also relates to such specific binding moieties, in particular to antibodies and/or to antigen binding fragments thereof, specifically binding to structurally disordered and/or intrinsically disordered sequences, in particular to Pro/Ala-rich sequences (PAS). These binding moieties, antibodies, antigen binding fragments are first in class since they bind to/recognize disordered peptides or polypeptide fragments as also comprised in such intrinsically disordered proteins, in particular PAS polypeptides. The inventive binding moieties, antibodies, antigen binding fragments are, without being limiting, particularly useful in diagnostic settings as well as research tools. The present invention relates to a method for generating and/or obtaining specific binding moieties against intrinsically disordered proteins (IDPs) and/or intrinsically disordered protein domains which tend to be immunologically inert and lack immunogenicity in animals, in particular in mammals. The present invention also relates to such specific binding moieties, in particular to antibodies and/or to antigen binding fragments thereof, specifically binding to structurally disordered and/or intrinsically disordered sequences, in particular to Pro/Ala-rich sequences (PAS). These binding moieties, antibodies, antigen binding fragments are first in class since they bind to/recognize disordered peptides or polypeptide fragments as also comprised in such intrinsically disordered proteins, in particular PAS polypeptides. The inventive binding moieties, antibodies, antigen binding fragments are, without being limiting, particularly useful in diagnostic settings as well as research tools.

Claims

1. A method for generating an antigen binding molecule, preferably an antibody or an antigen-binding fragment thereof, directed against intrinsically disordered peptides/proteins and/or intrinsically disordered peptide/protein domains, said method comprising the step of immunizing a non-human mammal with an antigen wherein said antigen is a conjugate of an immunoadjuvant and one or more P/A peptides, wherein each P/A peptide is independently a peptide consisting of about 5 to about 100 amino acid residues, wherein at least 60% of the amino acid residues of said peptide are independently selected from proline and alanine, and wherein a protecting group R.sup.N is attached to the N-terminal amino group of said peptide.

2. The method of claim 1, wherein each P/A peptide is independently a peptide
R.sup.N(P/A)-R.sup.C, wherein (P/A) is an amino acid sequence consisting of about 8 to about 90 amino acid residues, wherein at least 70% of the number of amino acid residues in (P/A) are independently selected from proline and alanine, wherein (P/A) includes at least one proline residue and at least one alanine residue, wherein R.sup.N is a protecting group which is attached to the N-terminal amino group of (P/A), wherein R.sup.C is an amino acid residue which is bound via its amino group to the C-terminal carboxy group of (P/A) and which comprises at least one carbon atom between its amino group and its carboxy group, and wherein each P/A peptide is conjugated to the immunoadjuvant via an amide linkage formed from the carboxy group of the C-terminal amino acid residue R.sup.C of the P/A peptide and a free amino group of the immunoadjuvant.

3. The method of claim 2, wherein (P/A) in said antigen is an amino acid sequence consisting of about 10 to about 80 amino acid residues, wherein at least 70% of the number of amino acid residues in (P/A) are independently selected from proline and alanine, wherein at least 95% of the number of amino acid residues in (P/A) are independently selected from proline, alanine and serine, and wherein (P/A) includes at least one proline residue and at least one alanine residue.

4. The method of claim 2, wherein (P/A) in said antigen is an amino acid sequence consisting of 20 to 40 amino acid residues independently selected from proline, alanine and serine, wherein at least 70% of the number of amino acid residues in (P/A) are independently selected from proline and alanine, and wherein (P/A) includes at least one proline residue and at least one alanine residue.

5. The method of claim 2, wherein the proportion of the number of proline residues comprised in (P/A) to the total number of amino acid residues comprised in (P/A) is ?10% and ?70%, preferably ?20% and ?50%, more preferably ?25% and ?40%.

6. The method of claim 2, wherein (P/A) in said antigen consists of (i) five or more partial sequences independently selected from ASPA (SEQ ID NO: 86), APAP (SEQ ID NO: 87), SAPA (SEQ ID NO: 88), AAPA (SEQ ID NO: 89) and APSA (SEQ ID NO: 84), and (ii) optionally one, two or three further amino acid residues independently selected from proline, alanine and serine.

7. The method of claim 2, wherein (P/A) consists of (i) the sequence ASPA-APAP-ASPA-APAP-SAPA, (ii) the sequence AAPA-APAP-AAPA-APAP-AAPA, (iii) the sequence APSA-APSA-APSA-APSA-APSA, (iv) a duplication of any of the aforementioned sequences, or (v) a combination of two of the aforementioned sequences.

8. The method of claim 1, wherein R.sup.N is selected from pyroglutamoyl (Pga), homopyroglutamoyl, formyl, acetyl, hydroxyacetyl, methoxyacetyl, ethoxyacetyl, propoxyacetyl, propionyl, 2-hydroxypropionyl, 3-hydroxypropionyl, 2-methoxypropionyl, 3-methoxypropionyl, 2-ethoxypropionyl, 3-ethoxypropionyl, butyryl, 2-hydroxybutyryl, 3-hydroxybutyryl, 4-hydroxybutyryl, 2-methoxybutyryl, 3-methoxybutyryl, 4-methoxybutyryl, glycine betainyl, o-aminobenzoyl, NH(C.sub.1-6 alkyl), N,N(C.sub.1-8 alkyl).sub.2, N,N,N-tri(C.sub.1-6-alkyl).sub.3, N,N-tetramethylene, and N,N-pentamethylene.

9. The method of claim 2, wherein R.sup.C is H.sub.2N(C.sub.1-12 hydrocarbyl)-COOH, wherein it is preferred that R.sup.C is selected from H.sub.2N(CH.sub.2).sub.1-10COOH, H.sub.2N-phenyl-COOH, and H.sub.2N-cyclohexyl-COOH, wherein it is more preferred that R.sup.C is selected from H.sub.2NCH.sub.2COOH (Gly), H.sub.2N(CH.sub.2).sub.2COOH (D-Ala), H.sub.2N(CH.sub.2).sub.3COOH, H.sub.2N(CH.sub.2).sub.4COOH, H.sub.2N(CH.sub.2).sub.5COOH, H.sub.2N(CH.sub.2).sub.6COOH, H.sub.2N(CH.sub.2).sub.7COOH, H.sub.2N(CH.sub.2).sub.8COOH, p-aminobenzoic acid, and 4-aminocyclohexanecarboxylic acid, and wherein it is even more preferred that R.sup.C is H.sub.2N(CH.sub.2).sub.5COOH.

10. The method of claim 1, wherein the P/A peptide(s) comprised in said antigen adopt(s) a random coil conformation and/or wherein the P/A peptide(s) comprised in said antigen is/are devoid of charged residues.

11. The method of claim 1, wherein the immunoadjuvant is selected from keyhole limpet hemocyanin (KLH), ovalbumin (OVA), and bovine serum albumin (BSA), preferably wherein the immunoadjuvant is keyhole limpet hemocyanin (KLH).

12. An antigen as defined in claim 1.

13. (canceled)

14. The method of claim 1, wherein said intrinsically disordered peptides/proteins and/or intrinsically disordered peptide/protein domains are Pro/Ala-rich sequences, preferably wherein said Pro/Ala-rich sequences are amino acid sequences consisting of at least 20 amino acid residues forming random coil conformation and whereby said amino acid residues forming said random coil conformation are selected from Pro (P), Ala (A) and Ser (S), preferably from Pro (P) and Ala (A).

15. The method of claim 14, wherein said Pro/Ala-rich sequences comprise at least one epitope of the structure (P/S)A(A/S)P; and/or PA(A/S)P; preferably wherein said epitope comprises an epitope stretch selected from the group consisting of PAAP, PASP, PAPASP, PAPAAP, PASPAAP, and APSA.

16. An antigen binding molecule, preferably an antibody or an antigen-binding fragment thereof, directed against intrinsically disordered peptides/proteins and/or intrinsically disordered peptide/protein domains, which is obtainable by the method of claim 1.

17. An antigen-binding molecule, wherein said antigen-binding molecule is selected from the group consisting of: a) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 35 [anti-PA(S) MAb 1.1], the CDR-H2 as defined in SEQ ID NO: 36 [anti-PA(S) MAb 1.1], and the CDR-H3 as defined in SEQ ID NO: 37 [anti-PA(S) MAb 1.1]; and/or a variable light (VL) chain comprising the CDR-L1 as defined in SEQ ID NO: 38 [anti-PA(S) MAb 1.1], the CDR-L2 as defined in SEQ ID NO: 39 [anti-PA(S) MAb 1.1], and the CDR-L3 as defined in SEQ ID NO: 40 [anti-PA(S) MAb 1.1]; or is an antibody or an antigen-binding fragment thereof binding to the same epitope as an antibody comprising any one or more of the CDRs of (a); b) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 41 [anti-PA(S) MAb 1.2], the CDR-H2 as defined in SEQ ID NO: 42 [anti-PA(S) MAb 1.2], and the CDR-H3 as defined in SEQ ID NO: 43 [anti-PA(S) MAb 1.2]; and/or a variable light (VL) chain comprising the CDR-L1 as defined in SEQ ID NO: 44 [anti-PA(S) MAb 1.2], the CDR-L2 as defined in SEQ ID NO: 45 [anti-PA(S) MAb 1.2], and the CDR-L3 as defined in SEQ ID NO: 46 [anti-PA(S) MAb 1.2]; or is an antibody or an antigen-binding fragment thereof binding to the same epitope as an antibody comprising any one or more of the CDRs of (b); c) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 47 [anti-PA(S) MAb 2.1], the CDR-H2 as defined in SEQ ID NO: 48 [anti-PA(S) MAb 2.1], and the CDR-H3 as defined in SEQ ID NO: 49 [anti-PA(S) MAb 2.1]; and/or a variable light (VL) chain comprising the CDR-L1 as defined in SEQ ID NO: 50 [anti-PA(S) MAb 2.1], the CDR-L2 as defined in SEQ ID NO: 51 [anti-PA(S) MAb 2.1], and the CDR-L3 as defined in SEQ ID NO: 52 [anti-PA(S) MAb 2.1]; or is an antibody or an antigen-binding fragment thereof binding to the same epitope as an antibody comprising any one or more of the CDRs of (c); d) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 53 [anti-PA(S) MAb 2.2], the CDR-H2 as defined in SEQ ID NO: 54 [anti-PA(S) MAb 2.2], and the CDR-H3 as defined in SEQ ID NO: 55 [anti-PA(S) MAb 2.2]; and/or a variable light (VL) chain comprising the CDR-L1 as defined in SEQ ID NO: 56 [anti-PA(S) MAb 2.2], the CDR-L2 as defined in SEQ ID NO: 57 [anti-PA(S) MAb 2.2], and the CDR-L3 as defined in SEQ ID NO: 58 [anti-PA(S) MAb 2.2]; or is an antibody or an antigen-binding fragment thereof binding to the same epitope as an antibody comprising any one or more of the CDRs of (d); e) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 59 [anti-PA(S) MAb 3.1], the CDR-H2 as defined in SEQ ID NO: 60 [anti-PA(S) MAb 3.1], and the CDR-H3 comprising or consisting of the amino acid sequence Trp-Gly-Arg [anti-PA(S) MAb 3.1]; and/or a variable light (VL) chain comprising the CDR1-L as defined in SEQ ID NO: 62 [anti-PA(S) MAb 3.1], the CDR2-L as defined in SEQ ID NO: 63 [anti-PA(S) MAb 3.1], and the CDR3-L as defined in SEQ ID NO: 64 [anti-PA(S) MAb 3.1]; or is an antibody or an antigen-binding fragment thereof binding to the same epitope as an antibody comprising any one or more of the CDRs of (e); and f) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 65 [anti-PA(S) MAb 3.2], the CDR-H2 as defined in SEQ ID NO: 66 [anti-PA(S) MAb 3.2], and the CDR-H3 as defined in SEQ ID NO: 67 [anti-PA(S) MAb 3.2]; and/or a variable light (VL) chain comprising the CDR-L1 as defined in SEQ ID NO: 68 [anti-PA(S) MAb 3.2], the CDR-L2 as defined in SEQ ID NO: 69 [anti-PA(S) MAb 3.2], and the CDR-L3 as defined in SEQ ID NO: 70 [anti-PA(S) MAb 3.2]; or is an antibody or an antigen-binding fragment thereof binding to the same epitope as an antibody comprising any one or more of the CDRs of (f)

18. The antigen-binding molecule of claim 17, wherein said antigen-binding molecule is an antibody or an antigen-binding fragment thereof, which: a) comprises a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 [anti-PA(S) MAb 1.1], SEQ ID NO: 13 [anti-PA(S) MAb 1.2], SEQ ID NO: 15 [anti-PA(S) MAb 2.1], SEQ ID NO: 17 [anti-PA(S) MAb 2.2], SEQ ID NO: 19 [anti-PA(S) MAb 3.1] or SEQ ID NO: 21 [anti-PA(S) MAb 3.2], or a sequence having 85%, preferably 87%, more preferably at least 90% sequence identity to SEQ ID NO: 11, 13, 15, 17, 19 or 21; and/or comprises a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 12 [anti-PA(S) MAb 1.1], SEQ ID NO: 14 [anti-PA(S) MAb 1.2], SEQ ID NO: 16 [anti-PA(S) MAb 2.1], SEQ ID NO: 18 [anti-PA(S) MAb 2.2], SEQ ID NO: 20 [anti-PA(S) MAb 3.1] or SEQ ID NO: 22 [anti-PA(S) MAb 3.2], or a sequence having 85%, preferably 87%, more preferably at least 90% sequence identity to SEQ ID NO: 12, 14, 16, 18, 20 or 22; or b) is an antibody or an antigen-binding fragment thereof binding to the same epitope as an antibody of (a).

19. The antigen-binding molecule of claim 17, wherein the antigen-binding molecule is an antigen-binding fragment selected from a Fab fragment, a F(ab).sub.2 fragment, a Fv fragment or a scFv fragment.

20. The antigen-binding molecule of claim 17, wherein the antigen-binding molecule is conjugated or fused to a reporter molecule and/or a label.

21. The antigen-binding molecule of claim 17, wherein the antigen-binding molecule is employed in matrix-based protein/peptide purification or immobilization.

22. The antigen-binding molecule of claim 17, wherein said antigen-binding molecule is selected from the group consisting of: a) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 35 [anti-PA(S) MAb 1.1], the CDR-H2 as defined in SEQ ID NO: 36 [anti-PA(S) MAb 1.1], and the CDR-H3 as defined in SEQ ID NO: 37 [anti-PA(S) MAb 1.1]; and/or a variable light (VL) chain comprising the CDR-L1 as defined in SEQ ID NO: 38 [anti-PA(S) MAb 1.1], the CDR-L2 as defined in SEQ ID NO: 39 [anti-PA(S) MAb 1.1], and the CDR-L3 as defined in SEQ ID NO: 40 [anti-PA(S) MAb 1.1]; b) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 41 [anti-PA(S) MAb 1.2], the CDR-H2 as defined in SEQ ID NO: 42 [anti-PA(S) MAb 1.2], and the CDR-H3 as defined in SEQ ID NO: 43 [anti-PA(S) MAb 1.2]; and/or a variable light (VL) chain comprising the CDR-L1 as defined in SEQ ID NO: 44 [anti-PA(S) MAb 1.2], the CDR-L2 as defined in SEQ ID NO: 45 [anti-PA(S) MAb 1.2], and the CDR-L3 as defined in SEQ ID NO: 46 [anti-PA(S) MAb 1.2]; c) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 47 [anti-PA(S) MAb 2.1], the CDR-H2 as defined in SEQ ID NO: 48 [anti-PA(S) MAb 2.1], and the CDR-H3 as defined in SEQ ID NO: 49 [anti-PA(S) MAb 2.1]; and/or a variable light (VL) chain comprising the CDR-L1 as defined in SEQ ID NO: 50 [anti-PA(S) MAb 2.1], the CDR-L2 as defined in SEQ ID NO: 51 [anti-PA(S) MAb 2.1], and the CDR-L3 as defined in SEQ ID NO: 52 [anti-PA(S) MAb 2.1]; d) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 53 [anti-PA(S) MAb 2.2], the CDR-H2 as defined in SEQ ID NO: 54 [anti-PA(S) MAb 2.2], and the CDR-H3 as defined in SEQ ID NO: 55 [anti-PA(S) MAb 2.2]; and/or a variable light (VL) chain comprising the CDR-L1 as defined in SEQ ID NO: 56 [anti-PA(S) MAb 2.2], the CDR-L2 as defined in SEQ ID NO: 57 [anti-PA(S) MAb 2.2], and the CDR-L3 as defined in SEQ ID NO: 58 [anti-PA(S) MAb 2.2]; e) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 59 [anti-PA(S) MAb 3.1], the CDR-H2 as defined in SEQ ID NO: 60 [anti-PA(S) MAb 3.1], and the CDR-H3 comprising or consisting of the amino acid sequence Trp-Gly-Arg [anti-PA(S) MAb 3.1]; and/or a variable light (VL) chain comprising the CDR1-L as defined in SEQ ID NO: 62 [anti-PA(S) MAb 3.1], the CDR2-L as defined in SEQ ID NO: 63 [anti-PA(S) MAb 3.1], and the CDR3-L as defined in SEQ ID NO: 64 [anti-PA(S) MAb 3.1]; or and f) an antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain comprising the CDR-H1 as defined in SEQ ID NO: 65 [anti-PA(S) MAb 3.2], the CDR-H2 as defined in SEQ ID NO: 66 [anti-PA(S) MAb 3.2], and the CDR-H3 as defined in SEQ ID NO: 67 [anti-PA(S) MAb 3.2]; and/or a variable light (VL) chain comprising the CDR-L1 as defined in SEQ ID NO: 68 [anti-PA(S) MAb 3.2], the CDR-L2 as defined in SEQ ID NO: 69 [anti-PA(S) MAb 3.2], and the CDR-L3 as defined in SEQ ID NO: 70 [anti-PA(S) MAb 3.2]; or

23. The antigen-binding molecule of claim 17 wherein said antigen-binding molecule is an antibody or an antigen-binding fragment thereof, which: comprises a variable heavy (VH) chain sequence comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 11 [anti-PA(S) MAb 1.1], SEQ ID NO: 13 [anti-PA(S) MAb 1.2], SEQ ID NO: 15 [anti-PA(S) MAb 2.1], SEQ ID NO: 17 [anti-PA(S) MAb 2.2], SEQ ID NO: 19 [anti-PA(S) MAb 3.1] and SEQ ID NO: 21 [anti-PA(S) MAb 3.2], or comprises a variable light (VL) chain sequence comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 12 [anti-PA(S) MAb 1.1], SEQ ID NO: 14 [anti-PA(S) MAb 1.2], SEQ ID NO: 16 [anti-PA(S) MAb 2.1], SEQ ID NO: 18 [anti-PA(S) MAb 2.2], SEQ ID NO: 20 [anti-PA(S) MAb 3.1] or SEQ ID NO: 22 [anti-PA(S) MAb 3.2].

Description

FIGURES

[0215] FIG. 1: Amino acid sequence alignment for the V.sub.H (A) and V.sub.L (B) domains of the anti-PAS antibodies of this invention (prior to the introduction of flanking restriction sites for subcloning). CDRs are labelled with a black outline. Identical amino acid positions with regard to the V.sub.H and V.sub.L sequences of Anti-PA(S) MAb 1.1 shown at the top of each alignment are depicted as ., gaps in the amino acid sequence alignment are indicated by -.

[0216] FIG. 2: Exemplary western blot analysis demonstrating that the different anti-PAS antibodies of this invention bind specifically to the corresponding PASylated fusion proteins. Western blots incubated with cell culture supernatant from hybridoma clones (A) Anti-PA(S) MAb 2.2, (B) Anti-PA(S) MAb 2.1, (C) purified Anti-PA(S) MAb 2.1, (D) Anti-PA(S) MAb 3.1, (E) Anti-PA(S) MAb 3.2, (F) Anti-PA(S) MAb 1.1, (G) Anti-PA(S) MAb 1.2 and (H) the anti-mouse IgG Fc-specific alkaline phosphatase (produced in goat, Sigma-Aldrich) secondary antibody as control. The following samples were applied to SDS-PAGE for western blotting: MPageRuler Prestained Protein Ladder, 10 to 180 kDa (Thermo Fisher Scientific); 1PAS #1(200)-IL1Ra (SEQ ID NO: 72); 2P/A #1(200)-IL1Ra (SEQ ID NO: 73); 3APSA(200)-IL1Ra (SEQ ID NO: 74); 4pooled human serum (SEQENS IVD/H2B), 1:200 diluted in ddH.sub.2O and spiked with 1 ?g IL1Ra (Kineret/Anakinra, SOBI); 5E. coli BL21 whole cell protein, lysed in SDS sample buffer.

[0217] FIG. 3: Exemplary results of ELISA experiments to detect PAS sequences or PASylated fusion proteins with the anti-PAS antibodies of this invention: (A) ELISA with the Fab fragment of Anti-PA(S) MAb 2.1 and IL1Ra-PAS #1(800), PAS #1(600)-Leptin and P/A #1(600)-GMCSF as test substances as well as BSA as control. (B) ELISA with the Fab fragment of Anti-PA(S) MAb 2.2 and P/A #1(600) as test substance. (C) ELISA with the Fab fragment of Anti-PA(S) MAb 1.2 and IL1Ra-PAS #1(800), PAS #1(600)-Leptin and P/A #1(600)-GMCSF test substances as well as BSA as control. (D) ELISA with hybridoma supernatant of Anti-PA(S) MAb 1.2, captured using an anti-mouse IgG Fc specific from goat pre-adsorbed to the microtiter plate, and hu4D5-PAS #1(200) as test substance.

[0218] FIG. 4: Exemplary results of ELISA experiments to detect PAS sequences or PASylated fusion proteins with the anti-PAS antibodies of this invention: (A) ELISA with the Fab fragment of Anti-PA(S) MAb 3.1 and APSA(200)-IL1Ra as test substance. (B) ELISA with the Fab fragment of Anti-PA(S) MAb 3.2 and APSA(200)-IL1Ra as test substance. (C) ELISA with the Fab fragment of Anti-PA(S) MAb 1.1 and PAS #1(600)-Leptin as test substance. (D) MAb capture ELISA (see FIG. 3D) with hybridoma supernatant of Anti-PA(S) MAb 2.1 and hu4D5-P/A #1(200) as test substance.

[0219] FIG. 5: SPOT assay results for the hybridoma culture supernatant of Anti-PA(S) MAb 1.1 and Anti-PA(S) MAb 1.2. Consecutive 12-mer peptides from the PAS #1 and P/A #1 sequences, each shifted by one residue in SEQ ID NOs: 5 and 6, respectively, were synthesized C-terminally anchored on a hydrophilic membrane. After color development of the membrane, the spot intensities were scanned and quantified with the software CLIQS ver. 1.2.044 (TotalLab) and are displayed as bar graphs. Epitope sequences are highlighted in bold.

[0220] FIG. 6: SPOT assay results for the Fab fragment of Anti-PA(S) MAb 2.1 and the hybridoma culture supernatant of Anti-PA(S) MAb 2.2. For explanation, see FIG. 5.

[0221] FIG. 7: SPOT assay results for the hybridoma culture supernatants of Anti-PA(S) MAb 3.1 and Anti-PA(S) MAb 3.2. A 10-mer peptide comprising the sequence AAPSAAPSAA was synthesized C-terminally anchored on a hydrophilic membrane whereby positions 3 to 8 were consecutively substituted by all twenty proteinogenic amino acids. After color development of the membrane, the spot intensities were scanned and quantified with the software CLIQS ver. 1.2.044 (TotalLab) and are displayed as bar graphs. Bars corresponding to the residue in the original sequence AAPSAAPSAA are filled.

[0222] FIG. 8: Exemplary SPR sensorgrams for Anti-PA(S) MAb 3.1 (APSA(200)-IL1Ra as analyte) as well as Anti-PA(S) MAb 1.1 (PAS #1(200)-IL1Ra as analyte) and Anti-PA(S) MAb 1.2 (PAS #1(200)-IL1Ra as analyte measured on a Biacore X 100 instrument). Hybridoma supernatants were applied to a CM3 sensorchip (GE Healthcare) coated with an anti-mouse antibody (Mouse Antibody Capture Kit; GE Healthcare). Injection phases are labeled with black bars, together with the corresponding concentration of injected analyte.

[0223] FIG. 9: Principle of the affinity purification of PASylated proteins using a column with immobilised anti-PAS Fab 1.2. (A) Schematic illustration of the one-step purification of a PASylated protein: (i) application of the cell extract containing the PASylated protein of interest, (ii) column washing with running buffer and (iii) elution of the PASylated protein of interest by applying a 1 M solution of L-prolinamide in running buffer. (B) Crystal structure (PDB ID: 7031) of Fab 1.2 (cartoon diagram) in complex with its PAS #1 epitope peptide (shown as stick model at the top). (C) Relevant part of the PAS #1 peptide epitope recognized by Fab 1.2. (D) Chemical structure of L-prolinamide.

[0224] FIG. 10: Exemplary chromatograms of the affinity purification of a PASylated protein using an immobilized anti-PAS antibody of this invention. The Fab fragment of Anti-PA(S) MAb 1.2 was covalently immobilized to a 1 ml HiTrap HP column (GE Healthcare) to serve as affinity matrix. The StrepII-eGFP-PAS #1(200) fusion protein (SEQ ID NO: 71) was applied as a test protein for purification from (A, C) a previously purified protein solution and (B, D) a whole cell extract of BL21 E. coli cells expressing StrepII-eGFP-PAS #1(200). The protein elution from the chromatography column was monitored by measuring the general absorbance at 280 nm and also the specific absorbance of the eGFP chromophore at 488 nm in parallel (A, B). After application of the protein sample and washing with buffer, the bound PAS fusion protein was eluted with a 1 M solution of L-prolinamide, which also showed strong absorbance at 280 nm (possibly due to contamination with aromatic amino acids). SDS-PAGE analysis (C, D) revealed the presence of StrepII-eGFP-PAS #1(200) in these elution fractions. Samples: MPierce Unstained Protein MW Marker (Thermo Fisher Scientific); 0pure StrepII-eGFP-PAS #1(200); 1fractions 7-7.5 ml; 2fractions 7.5-8 ml; 3fractions 8-8.5 ml; 4fractions 8.5-9 ml; 5fraction 9-9.5 ml; 6fractions 9.5-10 ml; 7fractions 10-10.5 ml. 1*whole cell lysate of BL21 E. coli cells expressing StrepII-eGFP-PAS #1(200); 2*fractions 2-4 ml; 3*fractions 7-7.5 ml; 4*fractions 7.5-8 ml; 5*fractions 8-8.5 ml; 6*fractions 8.5-9 ml; 7*fractions 9-9.5 ml; 8*fractions 9.5-10 ml; 9*fractions 10-10.5 ml. This experiment demonstrates that the anti-PAS affinity column specifically binds the PASylated test protein, StrepII-eGFP-PAS #1(200), which can be eluted under mild conditions by applying a solution of L-prolinamide.

[0225] FIG. 11: Exemplary chromatograms and SDS PAGE analysis documenting the one-step PAS affinity purification of therapeutically relevant PASylated proteins using an immobilized anti-PAS antibody of this invention. The Fab fragment of Anti-PA(S) Mab 1.2 was covalently immobilized to a 1 ml HiTrap HP column (GE Healthcare) to serve as affinity matrix. The C-terminally PASylated Anticalin H1GA-PAS #1(200)-His.sub.6 (SEQ ID NO: 90) (A, B) and the N-terminally PASylated cytokine PAS #1(800)-IL1 Ra (SEQ ID NO: 91) (C, D) were purified from either the periplasmic or from the cell fraction of E. coli BL21 respectively. Protein elution from the chromatography column was monitored by measuring the absorbance at 280 nm. After application of the protein sample and washing with buffer, the bound PAS fusion proteins were eluted with a 1 M solution of L-prolinamide in running buffer. SDS-PAGE analysis (B, D) revealed the presence of the PASylated proteins in the elution fraction. Samples: Unstained Protein MW Marker (Thermo Fisher Scientific)-PE/CE (periplasmic extract/whole cell extract of E. coli BL21 cells expressing the respective proteins)-FT (flow through)-Wash-Elution. Arrows indicate the protein bands corresponding to H1GA-PAS #1(200)-His.sub.6 and PAS #1(800)-IL1Ra respectively. This experiment demonstrates that N- and C-terminally PASylated proteins carrying PAS tags comprising 200-800 residues can be purified with high selectivity.

[0226] FIG. 12: Exemplary chromatogram of the affinity purification of a PASylated protein using an immobilized anti-PAS antibody of this invention. The Fab fragment of Anti-PA(S) MAb 1.2 was covalently immobilized to a 1 ml HiTrap HP column (GE Healthcare) to serve as affinity matrix. The pre-purified StrepII-eGFP-PAS #1(200) fusion protein (SEQ ID NO: 71) was applied as a test protein. Protein elution from the chromatography column was monitored by measuring the specific absorbance of the eGFP chromophore at 488 nm. After application of the protein sample and washing with buffer, the bound PAS fusion protein was eluted under particularly mild conditions (1 M L-prolinamide, 100 mM Tris, 150 mM NaCl, 1 mM EDTA, pH adjusted to 8.0 with HCl). This experiment demonstrates that the anti-PAS affinity column quantitatively binds the PASylated test protein, StrepII-eGFP-PAS #1(200), which can be eluted under mild buffer conditions by applying L-prolinamide.

[0227] FIG. 13: Crystal structures of synthetic PAS epitope peptides in complex with recombinant Fab fragments of anti-PAS antibodies of the invention: (A) P/A #1-epitope peptide bound to Anti-PA(S) MAb 2.2, (B) PAS #1-epitope peptide bound to Anti-PA(S) MAb 1.1, (C) Anti-PA(S) MAb 1.2 and (D) Pga-(APSA).sub.3 peptide bound to Anti-PA(S) MAb 3.1. Epitope peptides are shown as sticks (dark gray), Fab heavy (middle grey) and light (light grey) chains are shown as wires.

[0228] FIG. 14: Pharmacokinetic (PK) study of PASylated Thymosin alpha 1 in Wistar rats. (A) Linear range of the standard curve used for quantification of PASylated Thymosin alpha 1 in rat plasma samples via ELISA setup A schematically illustrated in FIG. 15. (B) PASylated Thymosin alpha 1 was subcutaneously injected at a dose of 3.4 mg/kg body weight into female Wistar rats (N=5). The concentration of the fusion protein in plasma was quantified by a sandwich ELISA using Anti-PA(S) MAb 2.1 as capture antibody and the alkaline phosphatase conjugated Anti-PA(S) MAb 1.2 as detection reagent. Data were plotted against sampling time post injection and fitted using a one-compartment model. The PK profile shows distinct absorption and elimination phases of the PASylated peptide drug (for PK parameters see Table 1).

[0229] FIG. 15: Exemplary ELISA setups for detection of PASylated molecules. (A) Sandwich ELISA using an anti-PAS antibody adsorbed to a microtiter plate in order to capture a PASylated molecule and a second anti-PAS antibody conjugated to a reporter enzyme as detection reagent. (B) Sandwich ELISA using an anti-PAS antibody adsorbed to a microtiter plate in order to capture a PASylated molecule and a second enzyme-coupled antibody directed against the biological active moiety of the PASylated drug. (C) ELISA using a binding partner of the protein, peptide or small molecule drug (e.g. a receptor, here designated target) adsorbed to a microtiter plate in order to capture the PASylated molecule and an enzyme-coupled anti-PAS antibody of this invention to detect the PASylated drug. (D) Schematic illustration of a competitive ELISA showing a PASylated analyte molecule competing with a PASylated and biotinylated molecule for the binding site(s) of an anti-PA(S) MAb which is adsorbed to a microtiter plate. PASylated and biotinylated molecules bound to the MAb are subsequently detected by a streptavidin-enzyme conjugate.

[0230] FIG. 16: Fluorescence titration of the Fab fragment of Anti-PA(S) Mab 2.2, applied at 1 ?M in 100 mM Tris/HCl pH 7.5, with the synthetic epitope peptide Abz-APAPAAPA. RFUrelative fluorescence units (fluorescence excitation at 280 nm, signal detection at 340 nm).

[0231] FIG. 17: Surface plasmon resonance (SPR) spectroscopy on a Biacore X100 instrument (Cytiva, Freiburg, Germany) using anti-PA(S) Mab 1.1. to capture a PASylated anti-Galectin Fab fragment and to determine the PAS-Fab binding kinetics towards its antigen Galectin-3. (A) Sensorgram showing the immobilization of a PASylated antibody fragment on a CM5 surface plasmon resonance (SPR) sensor chip (Cytiva). (1) The carboxylate groups on the dextran hydrogel surface of the chip were converted to reactive N-hydroxysuccinimide ester (NHS) groups using EDC/NHS chemistry. (2) Anti-PA(S) Mab 1.1 was covalently conjugated to the chip surface via the activated NHS esters, and (3) unreacted NHS-esters were saturated using 0.1 M ethanolamine. (B) Single cycle kinetic experiment showing (1) the non-covalent immobilization of a PASylated anti-Galectin Fab, followed by (2) five consecutive injections from a 1:2 dilution series (0.1 nM to 1.6 nM) of Galectin-3 as well as two acidic regeneration steps. (C) The baseline of the reference-corrected sensorgram of the single cycle kinetic experiment from (B) was set to zero as well as a start Time=0 s and fitted to a global 1:1 Langmuir binding model using the Biacore X100 evaluation software.

EXAMPLES

Example 1: Methods Employed in the Present Invention

A. Preparation of PAS Peptide Conjugates for Immunization

[0232] Three different peptides were obtained by solid phase synthesis (Pga-PAS #1(40)-Ahx and Pga-P/A #1(40)-Ahx: Peptide Specialty Laboratories-PSL, Heidelberg, Germany; Pga-APSA(40)-Ahx: Almac Sciences, Edinburgh, Scotland), each with a blocked N-terminus:

TABLE-US-00002 Pga-PAS#1(40)-Ahx(Pga-ASPAAPAPASPAAPAPSAPA- ASPAAPAPASPAAPAPSAPA-Ahx;SEQIDNO:5); Pga-P/A#1(40)-Ahx(Pga-AAPAAPAPAAPAAPAPAAPA- AAPAAPAPAAPAAPAPAAPA-Ahx;SEQIDNO:6); Pga-APSA(40)-Ahx(Pga-APSAAPSAAPSAAPSAAPSA- APSAAPSAAPSAAPSAAPSA-Ahx;SEQIDNO:7).

[0233] Pga means a pyroglutamyl residue (also known as 2-pyrrolidone-5-carboxylic acid or 5-oxoproline) and Ahx means aminohexanoic acid; all other residues are standard proteinogenic L-amino acids denoted by their single-letter abbreviations. The 40mer PAS peptides were designed with sufficient length in order to encompass at least two copies of the corresponding PAS sequence repeat, in some embodiments comprising 20 residues, thus also including at least one instance of the junction between two adjacent sequence repeats. Of note, such junctions would also constitute potential epitopes in longer recombinant PAS polypeptides. As all peptides contained chemically inert side chains only and had a blocked N-terminus, their single C-terminal carboxylate group (in fact, the one of the Ahx linker residue) was activated selectively and used for directed chemical conjugation to the ?-amino groups of Lys side chains of KLH, which was employed as a highly immunogenic carrier protein (Swaminathan et al., 2014). To this end, 50 mg of each peptide was dissolved in 1450 ?l dimethylsulfoxide (DMSO) and activated with a 10 fold molar amount of each 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU; Iris Biotech, Marktredwitz, Germany) and N,N-diisopropylethylamine (DIPEA; Sigma-Aldrich, Taufkirchen, Germany). 10 mg KLH (Thermo Scientific, Waltham, MA) was dissolved in water, dialyzed against PBS (4 mM KH.sub.2PO.sub.4, 16 mM Na.sub.2HPO.sub.4, 115 mM NaCl), adjusted to a concentration of 2.3 mg/ml in a volume of 4.35 ml and mixed with the activated peptide solution. After incubation on ice for 30 min, the solution was dialyzed against 25 mM Na-borate pH 9.0 and the conjugate was purified by anion exchange chromatography on a Source 15Q column (GE Healthcare, Munich, Germany) equilibrated with the same buffer. The conjugate was eluted in a linear concentration gradient of 0-500 mM NaCl applied in running buffer, monitored at 280 nm. Eluate fractions of the main peak were pooled, dialyzed against PBS, concentrated to 2 mg/ml, sterile-filtered through a 0.22 ?m Millex-GV PVDF filter (Merck, Darmstadt, Germany) and flash-frozen in liquid nitrogen.

B. Immunization of Mice and Generation of Hybridoma Cells

[0234] Using the PAS peptide-KLH conjugates described above as antigen, Balb/c mice were immunized and hybridomas were prepared according to standard procedures (ProMab Biotechnologies, Richmond, CA). For each antigen, five Balb/c mice were immunized subcutaneously with 50 ?g antigen together with Freund's complete adjuvant (CFA). Three weeks after priming, three booster injections (five for APSA(40)-KLH), each with 25 ?g antigen and Freund's incomplete adjuvant (IFA), were applied at intervals of two weeks. A final boost with 50 ?g of antigen without adjuvant was administered intraperitoneally two weeks after the last boost. Spleen cells were harvested from animals and fused with Sp2/0 myeloma cells for hybridoma clone generation using standard procedures well known in the art.

[0235] Promising hybridoma clones were propagated in cell culture using DMEM (Biochrom, Berlin, Germany) containing 10% v/v FCS (Ultra low IgG One Shot, Life Technologies, NY), 6 mM L-alanyl-L-glutamine (Biochrom), 1:100 penicillin/streptomycin (Biochrom) and supplemented with 10% v/v Hybridoma Premium Medium (ProMab Biotechnologies). Secreted anti-PAS MAbs in the cell culture supernatants were characterized by real-time surface plasmon resonance (SPR) spectroscopy and enzyme-linked immunosorbent assay (ELISA).

[0236] For some studies, Anti-PA(S) MAbs were purified from the hybridoma supernatants using a 1 ml HiTrap Protein G HP column (GE Healthcare) operated at a flow rate of 1 ml/min using an Akta Explorer 10 chromatography workstation (GE Healthcare). The hybridoma supernatant was diluted with binding buffer (20 mM NaP.sub.i pH 7.0) at a 1:1 ratio and applied to the column, which had been pre-equillibrated with 10 column volumes of binding buffer. After washing with 10 column volumes of binding buffer, the antibody was eluted with 2 column volumes of elution buffer (0.1 M glycine/HCl pH 2.7). To preserve the activity of acid-labile IgGs, 200 ?l of 1 M Tris/HCl pH 9.0 per 1 ml collection volume were added to each collection tube prior to the fractionation. Fractions containing the affinity-purified MAb were subsequently dialyzed against 200 volumes of storage buffer (20 mM KPi, 125 mM NaCl, 50% glycerol, pH 7.2) and frozen at ?21? C. Protein concentration was determined by measuring the absorbance at 280 nm (A.sub.280=1.4 equalling a concentration of 1.0 mg/ml IgG).

C. Characterization of Hybridoma MAbs by ELISA and SPR

[0237] Characterization of hybridoma MAbs by ELISA was performed using NUNC Maxisorp F 96-well plates (Thermo Fisher Scientific, Munich, Germany) coated with 50 ?l of a 5 ?g/ml solution of anti-mouse IgG Fc-specific goat antibody (Sigma-Aldrich) in PBS for 1 h, followed by twice washing with PBS and blocking with 3% w/v bovine serum albumin (BSA) in PBS/T (PBS+0.1% v/v Tween 20) for 1 h. After washing with PBS/T, the wells were incubated for 1 h with 50 ?l of each hybridoma supernatant diluted 1:100 in PBS/T and washed again. Then, 50 ?l solutions of the following PASylated proteins (each 8 nM) were applied in 1:2 dilution series with PBS and incubated for 1 h: hu4D5-PAS #1(200) (Schlapschy et al., 2013), hu4D5-P/A #1(200) (WO 2011/144756 A1) or APSA(200)-IL1Ra (SEQ ID NO: 74), which had been labeled with DIG-NHS (Santa Cruz Biotechnology, Dallas, TX) according to the manufacturer's instructions. After washing with PBS/T, 50 ?l of a 1:1000 dilution of anti-human kappa light chain antibody alkaline phosphatase conjugate (Sigma-Aldrich) or anti-DIG-Fab alkaline phosphatase conjugate (Roche Diagnostics) was applied to each well and incubated for 1 h. After final washing with PBS, 50 ?l of 0.5 mg/ml p-nitrophenyl phosphate in AP buffer (100 mM Tris/HCl pH 8.8, 100 mM NaCl, 5 mM MgCl.sub.2) was added and signal development was recorded at 405 nm for 15 min at 1 min intervals using a Synergy 2 photometer (BioTek Instruments, Bad Friedrichshall, Germany). The concentration-dependent signals (?A/?t) were evaluated following a published procedure (Voss & Skerra, 1997) using the formula:

[MAb.Math.Ag]=[MAb].sub.t.Math.[Ag].sub.t/(K.sub.D+[Ag].sub.t)

[0238] [MAb.Math.Ag] is the detectable amount of antibody/antigen complex, which is proportional to the ?A/?t signal measured for each well; [MAb].sub.t is the total amount of immobilized antibody, which corresponds to the asymptotic maximal signal of the binding curve; [Ag].sub.t is the (variable) total concentration of PAS antigen applied to each well and K.sub.D is the dissociation constant of the antibody/antigen complex resulting from the curve fit, which was evaluated with KaleidaGraph (Synergy Software, Reading, PA).

[0239] SPR measurements were performed at 25? C. either on a Biacore X 100 or Biacore T 200 instrument (GE Healthcare) using a mouse antibody capture kit and CM3 sensor chips (both from GE Healthcare). Culture supernatants were diluted 1:5 in HBS-ET buffer (0.01 M HEPES/NaOH pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Tween20), and a 30 ?l sample was injected at a flow rate of 10 ?l/min. A concentration series of the following test antigens, as appropriate, was injected onto the sensor ship using single cycle kinetics (Karlsson et al., 2006) at a flow rate of 30 ?l/min: PAS #1(200)-IL1Ra (SEQ ID NO: 72), P/A #1(200)-IL1Ra (SEQ ID NO: 73), P/A #1(600)-GMCSF (SEQ ID NO: 75), APSA(200)-IL1Ra (SEQ ID NO: 74) and hu4D5-P/A #1(200) WO 2011/144756 A1. The sensor chip was regenerated with 10 mM glycine/HCl pH 1.7 for 100 s. After subtraction of signals from a reference channel and a blank baseline measured with HBS-ET buffer, data were fitted using the Biacore X100 evaluation software ver. 2.0.1 (GE Healthcare) and a bivalent analyte model. The rate equations used by the fitting algorithm are as follows:

[00001]$\mathrm{Bivalent}\mathrm{analyte}\left(A\right)\mathrm{binds}\mathrm{to}\mathrm{ligand}\left(B\right).$$A\left(\mathrm{solution}\right)=\mathrm{Conc}$$A\left[0\right]=0$$\mathrm{dA}/\mathrm{dt}=\left(\mathrm{tc}*f^\left(1/3\right)\right)*\left(\mathrm{Conc}-A\right)-\left(\left(2*\mathrm{ka}1\right)*A*B-\mathrm{kd}1*\mathrm{AB}\right)$$B\left[0\right]=\mathrm{RMax}$$\mathrm{dB}/\mathrm{dt}=-\left(\left(2*\mathrm{ka}1\right)*A*B-\mathrm{kd}1*\mathrm{AB}\right)-\left(\mathrm{ka}2*\mathrm{AB}*B-\left(2*\mathrm{kd}2\right)*\mathrm{AB}2\right)$$\mathrm{AB}\left[0\right]=0$$\mathrm{dAB}/\mathrm{dt}=\left(\left(2*\mathrm{ka}1\right)*A*B-\mathrm{kd}1*\mathrm{AB}\right)-\left(\mathrm{ka}2*\mathrm{AB}*B-\left(2*\mathrm{kd}2\right)*\mathrm{AB}2\right)$$\mathrm{AB}2\left[0\right]=0$$\mathrm{dAB}2/\mathrm{dt}=\left(\mathrm{ka}2*\mathrm{AB}*B-\left(2*\mathrm{kd}2\right)*\mathrm{AB}2\right)$$\mathrm{Total}\mathrm{response}:$$\mathrm{AB}+\mathrm{AB}2+\mathrm{RI}$

[0240] Parameters: Conc, analyte concentration [M]; tc, mass transfer constant; f, volume flow rate of solution through the flow cell [m.sup.3.Math.s.sup.?1]; RMax, binding capacity; RI, refractive index.

D. Cloning of V-Genes from Hybridoma Cells

[0241] Hybridoma cells were mechanically lysed and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), followed by cDNA synthesis using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) with an oligo(dT).sub.18 primer. Ig V-gene regions were PCR-amplified from this cDNA with Q5 DNA polymerase (New England Biolabs, Frankfurt/M. Germany) using a set of 63 forward primers covering all mouse germline V.sub.L/V.sub.H gene segments (Chardes et al., 1999) together with the reverse primers RMK (5-GAC CTC CAC GGA GTC AGC-3; SEQ ID NO: 77) for the light chain and RMG (5-AGG TCG CCA CAC GTG TGG-3; SEQ ID NO: 78) for the heavy chain (Loers et al., 2014). Forward primers were initially applied in pools of 5-15 in order to reduce the required number of PCR reactions and, after a PCR product was identified for such a pool, individually to generate a single PCR product. After that, suitable PCR products were isolated by agarose gel electrophoresis using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI) and subjected to double-stranded DNA sequencing using the Mix2Seq Kit (Eurofins Genomics, Ebersberg, Germany).

E. Construction of Bacterial Expression Plasmids for Fab Fragments

[0242] For cloning of the V-genes on the bacterial expression vector pASK88 (Schiweck & Skerra, 1995), the products from the V-gene amplification described above were PCR-amplified with primer pairs that were designed to introduce suitable flanking restriction sites following a previously published routine procedure (Loers et al., 2014; Peplau et al., 2020). The resulting PCR products were cut with the corresponding restriction enzymes, isolated by agarose gel electrophoresis, and the V.sub.H and V.sub.L genes, respectively, were inserted into pASK88, which had been cut with the corresponding restriction enzymes, in two consecutive ligations. The coding regions for the Anti-PA(S) MAb 2.1, Anti-PA(S) MAb 1.2 and Anti-PA(S) MAb 3.1 were obtained by gene synthesis with suitable flanking restriction sites (GeneArt, Regensburg, Germany) based on V-gene sequences determined for these hybridomas by ProMab Biotechnologies.

F. E. coli Production and Purification of Fab Fragments

[0243] pASK88 derivatives harboring the V-genes of Anti-PA(S) MAb 2.1, Anti-PA(S) MAb 2.2, Anti-PA(S) MAb 1.1, Anti-PA(S) MAb 1.2, Anti-PA(S) MAb 3.1 and Anti-PA(S) MAb 3.2 were used to express the chimeric Fab fragments (murine variable domains from the hybridomas fused to human constant domains) either in 6?2 l shake flask culture using E. coli strain JM83 (Yanisch-Perron et al., 1985) or via 8 l bench top fermentation using the strain KS272 (Meerman & Georgiou, 1994) and following published procedures (Schiweck & Skerra, 1995; Skerra, 1994). The recombinant proteins were purified from the periplasmic cell extract via immobilized metal ion affinity chromatography (IMAC), followed by cation exchange chromatography (CEX) on a Resource S 6 ml column and size exclusion chromatography (SEC) on a HiLoad 16/60 Superdex75 prep grade column (both from GE Healthcare). Protein concentrations were determined by measuring the absorbance at 280 nm using calculated extinction coefficients (Gasteiger et al., 2003) of 88405 M.sup.?1 cm.sup.?1, 89895 M.sup.?1 cm.sup.?1, 77405 M.sup.?1 cm.sup.?1, 66405 M.sup.?1 cm.sup.?1, 69955 M.sup.?1 cm.sup.?1 or 57465 M.sup.?1 cm.sup.?1 for the chimeric Fab fragments of Anti-PA(S) MAb 2.1, Anti-PA(S) MAb 2.2, Anti-PA(S) MAb 1.1, Anti-PA(S) MAb 1.2, Anti-PA(S) MAb 3.1 or Anti-PA(S) MAb 3.2, respectively. Protein integrity and purity were checked by SDS-PAGE (Fling & Gregerson, 1986) and electrospray ionization mass spectrometry (ESI-MS) on a maXis Q-TOF instrument (Bruker Daltonics, Bremen).

G. Antigen Affinity Measurement of Fabs by ELISA, Fluorescence Titration and SPR

[0244] A NUNC Maxisorp F 96-well plate was coated with either 50 ?l of 10 ?g/ml P/A #1(600) polypeptide (Breibeck & Skerra, 2018) in PBS for the recombinant Fab fragments of Anti-PA(S) MAb 2.1 and Anti-PA(S) MAb 2.2, 50 ?l of 10 ?g/ml PAS #1(600)-Ieptin (Morath et al., 2015) in PBS for the Fab fragments of Anti-PA(S) MAb 1.1 and Anti-PA(S) MAb 1.2, or 50 ?l of 10 ?g/ml APSA(200)-IL1Ra (SEQ ID NO: 74) for the Fab fragments of Anti-PA(S) MAb 3.1 and Anti-PA(S) MAb 3.2, and incubated at 4? C. overnight. After a single washing step with PBS/T, the wells were blocked with 3% w/v BSA (NeoFROXX, Einhausen, Germany) in PBS/T for 1 h, followed by washing and 1 h incubation with 50 ?l of an appropriate dilution series of each purified Fab fragment in PBS/T. The wells were washed again with PBS/T followed by incubation with 50 ?l of a 1:1000 dilution of anti-human kappa light chain goat antibody conjugated to alkaline phosphatase (Sigma-Aldrich) in PBS/T for 1 h. After final washing twice each with PBS/T and PBS, signals were developed with p-nitrophenyl phosphate and measured and evaluated as described herein above.

[0245] Fluorescence titration was performed as previously described (Voss & Skerra, 1997) using a LS-50B luminescence spectrometer (Perkin Elmer, Norwalk, CT) equipped with a 2 ml quartz cuvette thermostated at 25? C. with wavelengths of 280 nm for excitation and 340 nm for detection (integrating the signal over 5 s). 2 ml of a 1 ?M solution of the purified Fab fragment of the Anti-PA(S) MAb 2.2 in 100 mM Tris/HCl pH 7.5 was titrated with a 5 mM solution of the Abz-APAPAAPA peptide (Peptide Specialty Laboratories-PSL, Heidelberg, Germany) (Abz means ortho-aminobenzoyl) in aliquots of 1 ?l up to a total volume of 22 ?l. Data were normalized to an initial fluorescence of 100% and fitted by non-linear least-squares regression with KaleidaGraph (Synergy Software, Reading, PA) as described (Edwardraja et al., 2017) including correction of the inner filter effect by titration of N-acetyl-tryptophanamide with the same peptide.

[0246] SPR measurements with the Fab fragments of the corresponding Anti-PA(S) MAbs were performed at 25? C. on a Biacore X 100 instrument (GE Healthcare). PAS #1(200)-IL1Ra, P/A #1(200)-IL1Ra or thioredoxinA-APSA(200) were biotinylated with a 20-fold molar amount of succinimidyl-6-(biotinamido)hexaonate (Sigma Aldrich) according to the manufacturer's instructions and individually immobilized as ligands on a biotin CAPture chip (GE Healthcare) following the manufacturer's protocol. Before immobilisation of each ligand, the sensorchip was regenerated with two consecutive injections of 30% v/v acetonitrile, 0.25 M NaOH for 120 s as well as 6 M guanidine/HCl, 0.25 M NaOH for 120 s. A concentration series of the recombinant Fab fragment was injected onto the sensorchip using single cycle kinetics and a flow rate of 30 ?l/min. After subtraction of signals from both a reference channel and a blank baseline measured with HBS-ET buffer, data were fitted using the Biacore X100 evaluation software ver. 2.0.1 (GE Healthcare) with a 1:1 binding model. The rate equations used by the fitting algorithm are as follows:

[00002]$A\left(\mathrm{solution}\right)=\mathrm{Conc}$$A\left[0\right]=0$$\mathrm{dA}/\mathrm{dt}=\left(\mathrm{tc}*f^\left(1/3\right)\right)*\left(\mathrm{Conc}-A\right)-\left(\mathrm{ka}*A*B-\mathrm{kd}*\mathrm{AB}\right)$$B\left[0\right]=\mathrm{RMax}$$\mathrm{dB}/\mathrm{dt}=-\left(\mathrm{ka}*A*B-\mathrm{kd}*\mathrm{AB}\right)$$\mathrm{AB}\left[0\right]=0$$\mathrm{dAB}/\mathrm{dt}=\left(\mathrm{ka}*A*B-\mathrm{kd}*\mathrm{AB}\right)$$\mathrm{Total}\mathrm{response}:$$\mathrm{AB}+\mathrm{RI}$

[0247] Parameters: Conc, analyte concentration [M]; tc, mass transfer constant; f, volume flow rate of solution through the flow cell [m.sup.3.Math.s.sup.?1]; RMax, binding capacity; RI, refractive index.

H. SPOT Synthesis of Immobilized Peptide Arrays and Epitope Mapping Arrays of 20 overlapping 12mer peptides covering the entire amino acid sequence of the PAS #1 or P/A #1 amino acid sequence repeat, or a 10mer peptide comprising the sequence AAPSAAPSAA, consecutively substituted to all twenty proteinogenic amino acids at positions 3 to 8, were synthesized on a hydrophilic membrane according to a standard protocol (Frank, 2002) using a MultiPep SPOT synthesizer (Intavis, K?ln, Germany). Detection of binding activity on the membranes was performed according to a published procedure (Zander et al., 2007) after incubating with either the purified Fab fragment or the hybridoma cell culture supernatant containing the secreted MAb, followed by anti-human kappa light chain antibody alkaline phosphatase conjugate (Sigma-Aldrich) or anti-mouse IgG Fc specific antibody alkaline phosphatase conjugate (Sigma-Aldrich), respectively.

I. Detection of PASylated Proteins by Western Blotting

[0248] Anti-PA(S) MAbs from hybridoma supernatants were tested for detection of PASylated proteins on western blots. A set of different PASylated proteins (PAS #1(200)-IL1Ra (SEQ ID NO: 72), P/A #1(200)-IL1Ra (SEQ ID NO: 73), APSA(200)-IL1Ra (SEQ ID NO: 74) as well as, for control, human serum (human serum (PL), pooled; SEQENS IVD/H2B, Limoges, France) diluted 1:200 in water and spiked with 1 ?g IL1Ra (Kineret/Anakinra; Swedish Orphan Biovitrum, Stockholm, Sweden) and E. coli BL21 whole cell lysate were subjected to SDS-PAGE followed by semi-dry electrotransfer on a nitrocellulose membrane. After washing with PBS/T, the membrane was incubated with a 1:2000 dilution in PBS/T of anti-PAS MAbs as hybridoma supernatants or a 1:200000 dilution in case of the purified Anti-PA(S) MAb 2.1. Bound MAbs were detected using a 1:50.000 dilution of an anti-mouse IgG Fc-specific goat antibody conjugated with alkaline phosphatase (Sigma-Aldrich) in PBS/T followed by chromogenic reaction with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT) (both from Carl Roth, Karlsruhe).

J. Pharmacokinetic Analysis in Rats

[0249] A pharmacokinetic (PK) study in female Wistar rats, at 8-9 weeks age, was conducted at the Aurigon Toxicological Research Center (ATRC, Dunakeszi, Hungary) in compliance with applicable animal welfare regulations. Up to 3 animals per cage were housed in a controlled environment at 22?3? C. with a relative humidity of 50?20%, 12 h light and 12 h dark. Purified PASylated Thymosin alpha 1 (SEQ ID NO. 76) (3.4 mg/kg) was administered subcutaneously via a single injection into the rat dorsal area. Blood samples (100 ?l) were taken from 5 animals each at various time points. Following collection in K3-EDTA tubes (Greiner Bio-One, Frickenhausen, Germany), samples were centrifuged at room temperature for 10 min (3000?g) and the resulting plasma was stored at ?15 to ?30? C. PASylated Thymosin alpha 1 in these samples was quantified using a sandwich ELISA (see Method K & FIG. 15A). Potential alternative ELISA setups suitable for the quantification of PASylated peptides or proteins in blood or plasma/serum samples of animals or human patients are illustrated in FIG. 15, panels B to D.

K. Quantification of PASylated Thymosin Alpha 1 in Rat Plasma by ELISA

[0250] Female Wistar rats (n=5), at 8-9 week age (Aurigon Toxicological Research Center, Dunakeszi, Hungary) were subcutaneously injected with PASylated Thymosin alpha 1 (SEQ ID NO. 76) (3.4 mg/kg) and blood samples (100 ?l) were collected in K3-EDTA tubes (Greiner Bio-One, Frickenhausen, Germany) at various time points. For the quantification of PASylated Thymosin alpha 1 administered in the rat PK study (Method J) Nunc Maxisorb ELISA 96 well plates (Thermo Fisher Scientific) were coated with 100 ?g/ml of the Anti-PA(S) MAb 2.1 in PBS at 4? C. overnight. After washing twice with PBS/T, free binding sites were blocked with 3% w/v BSA in PBS/T at room temperature for 1 h.

[0251] Then, the plate was washed 3 times with PBS/T and the rat plasma samples were applied, each in a 1:2 dilution series, in PBS/T, which had been supplemented with 0.5% (v/v) plasma from an untreated animal in order to maintain a constant proportion of rat plasma constituents. In the same manner, a standard curve was prepared using dilution series of the purified PASylated Thymosin alpha 1 at defined concentrations in PBS/T containing the same amount of rat plasma as the test samples. After incubation for 1 h at room temperature, wells were washed 3 times with PBS/T. To detect bound PASylated Thymosin alpha 1, wells were incubated for 1 h with 50 ?l of a 1 ?g/ml PBS/T solution of the Anti-PA(S) MAb 1.2, which had been conjugated with alkaline phosphatase using the Lightning-Link alkaline phosphatase antibody labeling kit (BioTechne, Wiesbaden, Germany). After washing twice with PBS/T and twice with PBS, the enzymatic activity was detected using p-nitrophenyl phosphate (0.5 mg/ml). To this end, the plate was incubated for 20 min at 30? C., the absorbance was measured at 405 nm using a SpectraMax M5e microtiter plate reader (Molecular Devices, Sunnyvale, CA), and the PASylated Thymosin alpha 1 concentrations were quantified by comparison with the standard curve (FIG. 14A).

[0252] Data were plotted against the sampling time post injection and fitted using a one-compartment model using Phoenix WinNonlin 6.3 software. The resulting PK parameters (Table 1) and PK profile (FIG. 14B) are typical for a long-acting peptide drug and prove that the anti-PAS antibodies of this invention are suited to quantify PASylated drug concentrations in mammalian plasma. Of note, the ELISA setup applied in this method (FIG. 15A) uses an anti-PAS antibody as capture antibody and, thus, avoids the detection of endogenous rat Thymosin alpha 1, which shares 100% sequence identity with the human peptide. Alternative ELISA setups are illustrated, for example, in FIG. 15 and are well known in the art (Vashist & Luong, 2018).

TABLE-US-00003 TABLE 1 Pharmacokinetic parameters of PASylated Thymosin alpha 1 (T?1) in rats. Listed are the maximum serum concentration of the drug (Cmax), the time to reach Cmax (Tmax), the area under the curve (AUC), the distribution half-life (t.sub.1/2?), the elimination half-life (t.sub.1/2?) and clearance (CL). Parameter PASylated T?1 Cmax (mg/l) 25.6 ? 4.4 tmax (h) 22.7 ? 1.1 AUC.sub.0-? (h ?g/ml) 1586.7 ? 295.1 t.sub.1/2? (h) 15.7 ? 0.8 t.sub.1/2? (h) 15.9 ? 0.9 CL (ml/h/kg) 2.2 ? 0.4
L. Co-Crystallization of Anti-PAS Fab Fragments with PAS Peptides, X-Ray Data Collection and Molecular Model Building

[0253] The purified recombinant Fab fragments of Anti-PA(S) MAb 2.2, Anti-PA(S) MAb 1.1 and Anti-PA(S) MAb 3.1 were directly co-crystallized with their cognate PAS peptides, whereas in the case of the Fab of Anti-PA(S) MAb 1.2 a complex with an anti-human kappa V.sub.HH domain described in (Ereno-Orbea et al., 2018) was initially prepared. To this end, the purified Fab was incubated for 1 h at 4? C. with a three-fold molar amount of the V.sub.HH domain (Thermo Fisher Scientific). The protein mixture was subjected to SEC on a HiLoad 16/60 Superdex75 prep grade column and the Fab.Math.V.sub.HH complex was separated from excess anti-human kappa V.sub.HH domain and isolated in one peak using 10 mM HEPES/NaOH pH 6.5, 70 mM NaCl as running buffer.

[0254] The different protein solutions were concentrated using Amicon Ultracel centrifugal filter units (MWCO 10 kDa; Millipore, Billerica, MA) as follows: Anti-PA(S) MAb 2.2 to 9.6 mg/ml in 20 mM HEPES/NaOH pH 6.5, 80 mM NaCl; Anti-PA(S) MAb 3.1 to 9.2 mg/ml in 10 mM HEPES, pH 6.5, 100 mM NaCl; Anti-PA(S) MAb 1.1 to 8.4 mg/ml and Anti-PA(S) MAb 1.2, as Fab.Math.V.sub.HH, to 13.7 mg/ml, both in 10 mM HEPES/NaOH pH 6.5, 70 mM NaCl. For co-crystallization, each concentrated protein solution was mixed with the appropriate peptide from a >50 mM stock solution in water at a molar ratio of 1:3 (Fab:peptide) and incubated for 1 h at 4? C. Then, protein crystallization screens were performed via the sitting drop vapor diffusion method and equivolume mixtures of protein and reservoir solutions, leading to a total drop volume in the range of 300-1000 nl. For refinement of promising crystallization conditions, further screens were set up using the hanging drop vapor diffusion method with a reservoir volume of 1 ml and droplets composed of 1 ?l protein and 1 ?l reservoir solution. Crystals appeared within one week at 20? C. under the conditions listed in Table 3. Protein crystals were harvested, transferred into the precipitant buffer supplemented with 20% w/v PEG200 for Anti-PA(S) MAb 2.2, 20% w/v ethyleneglycol for Anti-PA(S) MAb 1.1 and Anti-PA(S) MAb 1.2 or 20% w/v glycerol for Anti-PA(S) MAb 3.1 and immediately frozen in liquid nitrogen.

[0255] A single-wavelength X-ray synchrotron data set was collected at 100 K from each crystal at the MX beamline BL14.2 of BESSY II operated by the Helmholtz-Zentrum Berlin, Germany or, for the Fab fragment of Anti-PA(S) MAb 3.1, at the protein crystallography beamline X06SA-PXI of the Swiss Light Source (SLS), Villigen-PSI, Switzerland. The diffraction data (Table 3) were reduced with the XDS program package (Kabsch, 2010) and molecular replacement was carried out with Phaser (McCoy et al., 2007) using the constant and variable domains of the Fab 101F (PDB ID: 3QQ9) as search models to solve the structure of the Anti-PA(S) MAb 2.2 Fab.Math.P/A #1 complex. The structures of Anti-PA(S) MAb 1.1 Fab.Math.PAS #1 and Anti-PA(S) MAb 1.2 Fab.Math.PAS #1 were solved by molecular replacement with the refined structure of the Fab of Anti-PA(S) MAb 2.2 as search model, also including the anti-human kappa V.sub.HH domain (PDB ID: 6ANA) in the latter case. Structure of anti-PA(S) MAb 3.1 Fab.Math.APSA was solved by molecular replacement with the refined structure of the anti-PA(S) MAb 1.2 Fab as search model, not including the anti-human kappa V.sub.HH domain. The protein model was manually adjusted with Coot (Emsley et al., 2010) and refined with Refmac5 (Murshudov et al., 2011). The peptide and water molecules were manually built in Coot in the course of the refinement process. The final structural models were validated using the MolProbity server (Williams et al., 2018). Crystal contact sites as well as accessible and buried surface areas (ASA and BSA, respectively) were analysed with PISA (Krissinel & Henrick, 2007) (calculated with the light and heavy chains connected as a continuous uninterrupted amino acid chain in the input file). Molecular graphics were prepared with PyMOL (Schr?dinger, Cambridge, MA) using the APBS module (Baker et al., 2001) for calculation of electrostatics. Atomic distances were calculated with CONTACT (Winn et al., 2011).

[0256] Polypeptides were denoted L for the Ig light chain, H for the Ig heavy chain and P for each bound PAS peptide whereas the anti-human kappa V.sub.HH domain was assigned the chain identifier X. In case of Anti-PA(S) MAb 1.1, with two Fab.Math.peptide complexes in the asymmetric unit, the one with the higher average crystallographic B-factor was assigned chain identifiers A, B and Q, respectively.

M. Affinity Purification of StrepII-eGFP-PAS #1(200), H1GA-PAS #1(200)-His.SUB.6 .and PAS #1(800)-IL1Ra Using an Anti-PAS Fab Immobilized on a Sepharose Column

[0257] In total 5 mg of the purified Fab fragment of Anti-PA(S) MAb 1.2 was covalently immobilized on a 1 ml HiTrap NHS-activated HP column (GE Healthcare) according to the manufacturer's protocol. In brief, the column was washed with ice-cold 1 mM HCl prior to injection of the Fab in 1 ml coupling buffer (0.2 M NaHCO.sub.3, 0.5 M NaCl, pH 8.3) and incubation for 30 min at 25? C. Washing and deactivation of excess reactive groups was performed by repeated alternating injections of 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3 and 0.1 M Na-acetate, 0.5 M NaCl, pH 4.

[0258] Purification of the PASylated test proteins StrepII-eGFP-PAS #1(200), H1GA-PAS #1(200)-His.sub.6 and PAS #1(800)-IL1Ra on this column was performed using an AKTA Pure 25 chromatography system operated at a flow rate of 1 ml/min. The column was first equilibrated with 2 ml of running buffer (100 mM Tris/HCl pH 8, 150 mM NaCl, 1 mM EDTA), followed by injection of either (i) pure StrepII-eGFP-PAS #1(200) (SEQ ID NO: 71) or (ii) a whole cell lysate of E. coli BL21 cells expressing StrepII-eGFP-PAS #1(200) or (iii) a periplasmic extract of E. coli BL21 cells expressing H1GA-PAS #1(200)-His.sub.6 (SEQ ID NO: 90), or (iv) a whole cell lysate of E. coli BL21 cells expressing PAS #1(800)-IL1Ra (SEQ ID NO: 91). Unbound proteins were washed off the column with 2 ml running buffer, then bound protein was eluted by applying 2-3 ml of a 1 M solution of L-prolinamide (Sigma Aldrich) in running buffer or, alternatively, 1 M L-prolinamide, 100 mM Tris, 150 mM NaCl, 1 mM EDTA, pH adjusted to 8.0 with HCl, followed by regeneration of the column with running buffer. In order to monitor both the presence of proteins in general and the specific presence of StrepII-eGFP-PAS #1(200), UV absorbance was detected at 280 nm and 488 nm, respectively (FIG. 10A, B). SDS-PAGE analysis confirmed the specific elution of the pure PASylated protein (FIG. 10C, D). Due to an apparent impurity in the commercial L-prolinamide substance that led to a background absorption at 280 nm, a blank chromatogram (without application of a PASylated protein) was recorded and used for subtraction to obtain the corrected chromatogram for H1GA-PAS #1(200)-His.sub.6 and PAS #1(800)-IL1Ra (FIGS. 11A, C). Specific elution of the pure PASylated proteins was confirmed by SDS-PAGE analysis (FIGS. 11B, D).

N. Preparation of PASylated Test Proteins

[0259] All test proteins and peptides fused to PAS sequences with different compositions and lengths used in the methods herein described were produced in E. coli either via cytoplasmic expression or via periplasmic secretion from conventional expression vectors harbouring corresponding synthetic genes according to routine procedures well described in the art, e.g. in WO 2008/155134 A1, WO 2011/144756 A1, WO 2017/109087 A1, WO 2018/234455 A1 or in (Binder & Skerra, 2017; Breibeck & Skerra, 2018; Morath et al., 2015; Schlapschy et al., 2013).

O. Deposits

[0260] The following MAbs of this invention were deposited by XL-protein GmbH, Lise-Meitner-Strasse 30, 85354 Freising, Germany as cell cultures at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstrasse 7B, 38124 Braunschweig, Germany, which was recognized by the World Intellectual Property Organization as an International Depositary Authority according to the Budapest Treaty for the deposit of animal and human cell cultures on 28 Feb. 1991: [0261] Anti-PA(S)Mab 1.1=DSM ACC3365 [0262] Anti-PA(S)Mab 2.1=DSM ACC3366 [0263] Anti-PA(S)Mab 3.1=DSM ACC3367

Example 2: Generation of Monoclonal Anti-PAS Antibodies and their Specific Detection of PAS Sequences

[0264] Antibodies against three different PAS peptide sequences, PAS #1 (SEQ ID NO: 1), P/A #1 (SEQ ID NO: 2) and APSA (SEQ ID NO: 3), were raised in mice. To this end, the animals were immunized with corresponding synthetic N-terminally protected 40mer peptides as described in Example 1 herein above that were chemically coupled via their C-terminal carboxylate groups to mariculture keyhole limpet hemocyanin (KLH) as a highly immunogenic T-cell dependent carrier antigen/immunoadjuvant (Swaminathan et al., 2014). In the case of PAS #1 and P/A #1 the 40mer covered exactly two copies of the designed 20mer sequence repeat (Breibeck & Skerra, 2018; Schlapschy et al., 2013), whereas the APSA peptide comprised 10 copies of the 4-residue motif, which may be considered as a kind of simplified Pro/Ala-rich sequence pattern.

[0265] These 2?20mer and/or 40mer PAS peptides were designed in order to encompass at least two copies of the corresponding PAS sequence repeat, thus including at least one copy of the junction between two adjacent sequence repeats, which also constitutes a potential epitope in longer recombinant PAS polypeptides. After four to six rounds of immunization as well as a final boost, each with 25-50 ?g antigen, spleen cells were isolated from five mice per antigen and fused with Sp2/0 myeloma cells to generate hybridomas. For each immunization campaign, antibodies from 40 hybridoma clones were characterized by ELISA using recombinant fusion proteins comprising the corresponding PAS polypeptides (200-600 residues), with the goal to screen for (i) sequence-specific and context-independent recognition of PAS sequences and (ii) identification of antibodies showing potential cross-reactivity between the different PAS sequences. MAb capture ELISAs with hybridoma culture supernatants were performed, applying the PAS fusion protein in a concentration-dependent fashion, to determine the dissociation constants (K.sub.D). Hybridoma culture supernatants of promising candidates were characterized with regard to antigen affinity and binding kinetics by real-time surface plasmon resonance (SPR) spectroscopy. Corresponding methods are described in Example 1.

[0266] Based on their K.sub.D values resulting from the ELISA and SPR measurements, also considering the absorption amplitudes in the concentration-dependent ELISAs, eight clones with distinct properties were selected each from the PAS #1(40)-KLH and P/A #1(40)-KLH immunization and tested for linear epitope recognition on a synthetic peptide array using the Synthetic Peptides On Transfer membranes (SPOT) technique (Frank, 2002). This assay revealed PAPAAP (SEQ ID NO: 8) and PAPASP (SEQ ID NO: 9) as epitope sequences for the Anti-PA(S) MAbs 2.1 and 2.2, while the Anti-PA(S) MAbs 1.1 and 1.2 predominantly recognized the peptide motif PASPAAP (SEQ ID NO: 10) (see FIGS. 5 and 6). Due to the simpler repetitive nature of the APSA sequence, a SPOT substitution analysis was performed for this antigen (FIG. 7). Therefore, a 10-mer peptide comprising the sequence AAPSAAPSAA, consecutively substituted to all twenty essential amino acids at positions 3 to 8, was synthesized C-terminally anchored on a hydrophilic membrane. Interestingly, antibodies generated by immunization with PAS #1(40)-KLH only recognized the PAS #1 polypeptide sequence (Anti-PA(S) MAbs 1.1 and 1.2), while some MAbs from the P/A #1(40)-KLH immunization also showed cross-reactivity with PAS #1 (Anti-PA(S) MAb 2.1) beyond the P/A #1 epitope. Surprisingly, the generalized sequence motif recognized by all Anti-PA(S) MAbs of the invention emerged as (P/S)A(A/S)P or as PA(A/S)P.

[0267] To verify the applicability of the monoclonal antibodies of the invention in the detection of PASylated fusion proteins, the Anti-PA(S) MAbs from the hybridoma supernatants were tested in western blotting experiments wherein specific detection of PASylated fusion proteins was confirmed. Furthermore, no cross-reactivity to the non-PASylated protein version, human serum proteins or proteins in an E. coli whole cell lysate was detected (FIG. 2).

Example 3: Cloning of V-Gene Sequences of MAbs and Fab Production

[0268] For each antigen, the two most promising hybridoma clones were selected for further analysis, based on their affinities to the target sequences as well as cross-reactivity to other PAS sequences: Anti-PA(S) MAb 2.1 and Anti-PA(S) MAb 2.2 for P/A #1; Anti-PA(S) MAb 1.1 and Anti-PA(S) MAb 1.2 for PAS #1, Anti-PA(S) MAb 3.1 and Anti-PA(S) MAb 3.2 for APSA.

[0269] To determine their V-gene sequences from the mRNA/cDNA, the coding regions for each V.sub.H and V.sub.L domain were reverse-transcribed and amplified by polymerase chain reaction (PCR) using suitable oligodeoxynucleotide primers as described in Example 1 herein above. The cloned V-gene sequences (for Anti-PA(S) MAb 2.2, Anti-PA(S) MAb 1.1 and Anti-PA(S) MAb 3.2) or, alternatively, corresponding synthetic DNA fragments (for Anti-PA(S) MAb 2.1, Anti-PA(S) MAb 1.2 and Anti-PA(S) MAb 3.1) were then inserted into a bacterial expression vector encoding the first human IgG1 heavy chain and ? light chain constant regions to allow expression of the corresponding chimeric Fab fragments (Schiweck & Skerra, 1995; Skerra, 1994). The Fab fragments were produced in a functional state by periplasmic secretion in E. coli both at the shake flask and at the bench top fermenter scale and purified to homogeneity by IMAC, CEX and SEC (see Example 1).

[0270] The following amino acid sequences were obtained (see FIG. 1; CDRs according to the definition by (Kabat et al., 1991) are labelled with a black frame): [0271] VH Anti-PA(S) MAb 1.1 (SEQ ID NO: 23): [0272] VL Anti-PA(S) MAb 1.1 (SEQ ID NO: 24): [0273] VH Anti-PA(S) MAb 1.2 (SEQ ID NO: 25): [0274] VL Anti-PA(S) MAb 1.2 (SEQ ID NO: 26 [0275] VH Anti-PA(S) MAb 2.1 (SEQ ID NO: 27): [0276] VL Anti-PA(S) MAb 2.1 (SEQ ID NO: 28): [0277] VH Anti-PA(S) MAb 2.2 (SEQ ID NO: 29): [0278] VL Anti-PA(S) MAb 2.2 (SEQ ID NO: 30): [0279] VH Anti-PA(S) MAb 3.1 (SEQ ID NO: 31): [0280] VL Anti-PA(S) MAb 3.1 (SEQ ID NO: 32): [0281] VH Anti-PA(S) MAb 3.2 (SEQ ID NO: 33): [0282] VL Anti-PA(S) MAb 3.2 (SEQ ID NO: 34):

[0283] Of note, apart from the method of (Kabat et al., 1991) for determining CDRs, which is largely based on cross-species sequence variability there is at least one other approach well known in the art, which is based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997; Chothia et al., 1989). As used herein, a CDR preferentially refers to the definition by Kabat (supra) but may also refer to CDRs defined by the other said approach or by a combination of both approaches. Amino acids were numbered using sequential numbering.

Example 4: Characterization of Binding Affinities (Anti-PAS Monoclonals and Anti-PAS Fabs

[0284] The monoclonal antibodies (MAbs) of the invention and as obtained by the methods and Examples provided herein as well as the corresponding recombinant anti-PAS Fabs were investigated in quantitative ELISAs and real-time SPR measurements in order to precisely determine their K.sub.D values towards the different PAS polypeptides (see also Table 2). These measurements essentially confirmed the findings from the preliminary hybridoma screening.

[0285] At least one MAb with particularly high affinity was identified for each type of PAS antigen, here evident from a K.sub.D value in the one-digit nanomolar range measured for the Fab: 2 nM towards P/A #1 for Anti-PA(S) MAb 2.1; 23 nM towards PAS #1 for Anti-PA(S) MAb 1.1; 2 nM towards APSA for Anti-PA(S) MAb 3.1. Compared with the previously investigated intact MAbs, the affinities measured for the Fabs were usually by 1-2 orders weaker, which is most likely due to the avidity effect that arises when the bivalent MAb interacts with a long PAS polypeptide that harbors multiple copies of the epitope (for example, 30 copies of the repetitive 20 PAS #1 amino acid stretch in a 600-residue PAS polypeptide). Anti-P/A #1 Fabs were either specific for the P/A #1 sequence or cross-reactive with the PAS #1 sequence as well, while the anti-PAS #1 Fabs showed specificity towards the PAS #1 sequence only. Anti-APSA antibody fragments were either specific for the APSA sequence or cross-reactive with the PAS #1 and P/A #1 polypeptides (see the following Table 2).

TABLE-US-00004 TABLE 2 Affinities of the MAbs and corresponding Fabs towards their cognate PAS (poly)peptides measured by ELISA, SPR and fluorescence titration (FT). Antigen PAS#1 P/A#1 APSA Anti-PA(S) MAb Hybridoma clone Format Assay Target 1.1 1.2 2.1 2.2 3.1 3.2 MAb SPR P/A#1(200)-IL1Ra n.q. n.q. 429 pM 1.1 nM n.q. 20 nM MAb ELISA hu4D5- n.q. n.q. 170 pM 69 nM n.q. 400 ? 100 P/A#1(200) pM Fab SPR P/A#1(200)-IL1Ra n.d. n.d. 648 nM 7 ?M n.d. 446 nM Fab ELISA P/A#1(600) n.q. n.q. 2 ? 0.2 nM 8 ? 1 nM n.q. 48 ? 4 nM MAb SPR PAS#1(200)- 1 nM 44 nM 1.7 nM 22 nM n.q. 80 nM IL1Ra MAb ELISA hu4D5- 37 ? 10 162 ? 11 113 pM n.q. n.q. 294 ? 92 nM PAS#1(200) pM pM Fab SPR PAS#1(200)- 8.8 ?M 3.7 ?M 4.4 nM n.d. n.d. n.q. IL1Ra Fab ELISA PAS#1(600)- 23 ? 5 123 ? 20 2.2 ? 0.1 n.q. n.q. 311 ? 16 nM Leptin nM nM nM MAb SPR APSA(200)-IL1Ra n.q. n.q. n.q. n.q. 19 pM 108 pM MAb ELISA APSA(200)-IL1Ra n.q. n.q. n.q. n.q. 118 ? 2 pM 251 ? 5 pM Fab SPR APSA(200)-TrxA n.d. n.d. n.d. n.d. 2.5 nM 137 nM Fab ELISA APSA(200)-IL1Ra n.d. n.d. n.d. n.d. 1.9 ? 0.01 1.6 ? 0.1 nM nM Fab FT Abz-APAPAAPA n.d. n.d. >100 ?M 9.2 ? 0.1 ?M n.d. n.d. n.q.: not quantifiable n.d.: not determined

[0286] To determine the monovalent affinity of Anti-PA(S) MAb 2.1 and Anti-PA(S) MAb 2.2 towards their epitope sequence, fluorescence titration (FT) experiments were performed with the corresponding recombinant Fab fragments and the synthetic peptide Abz-APAPAAPA (SEQ ID NO: 4) (carrying an N-terminal o-aminobenzoyl group as fluorescence resonance energy transfer probe). While no reliable K.sub.D value could be deduced for the Fab of the Anti-PA(S) MAb 2.1, a K.sub.D=9.2?0.1 ?M was determined for the Fab of the Anti-PA(S) MAb 2.2 (FIG. 16). This value has to be compared with K.sub.D=8?1 nM measured for the pure P/A #1(600) antigen in the ELISA, which was ?1000 fold higher (see Table 2). This difference must even be higher for the Fab of the Anti-PA(S) MAb 2.1 and indicates that not only the avidity effect but also more peculiar aspects, such as the precise molecular neighborhood of the epitope peptide or the epitope density within a polypeptide with many sequence repeats, can considerably influence the apparent affinity.

Example 5: Structural Characterization of PAS Peptide Binding by Co-Crystallization with Anti-PAS Fabs

[0287] The structural mechanism of antigen recognition by some of the Anti-PA(S) MAbs of this invention was analyzed using X-ray crystallography. Accordingly, recombinant anti-PA(S) Fab fragments as prepared using the methods described in Example 1 herein above were subjected to co-crystallization experiments with their cognate synthetic peptides, whose sequences were either based on the epitope sequences determined by the SPOT assay as described above or, in case of the simple APSA motif, comprised a twelve amino acid stretch with three APSA repeats. To avoid charges at the N-termini, which would be absent in longer (poly)peptide stretches, these were blocked with pyroglutamic acid (Pga) or by acetylation. Diffraction quality crystals were obtained for the Fab.Math.peptide complexes of Anti-PA(S) MAb 2.2 Fab.Math.P/A #1 and Anti-PA(S) MAb 1.1 Fab.Math.PAS #1 (see appended Table 3). In case of Anti-PA(S) MAb 2.1 and Anti-PA(S) MAb 1.2 we applied a recently published strategy that utilizes an anti-human kappa light chain V.sub.HH domain to facilitate (co)-crystallization of (our chimeric) Fab fragments (Ereno-Orbea et al., 2018). Indeed, this approach led to crystals for the Fab of the Anti-PA(S) MAb 1.2 in complex with the PAS #1 epitope peptide, which diffracted to a high resolution of 1.55 ? at a synchrotron X-ray source. The structure of the Anti-PA(S) MAb 2.2 Fab.Math.P/A #1 complex was solved by molecular replacement using the constant and variable domains of the functionally unrelated anti-human RSV Fab 101F (PDB ID: 3QQ9) as search models. Subsequently, the structures of the complexes Anti-PA(S) MAb 1.1 Fab.Math.PAS #1 and Anti-PA(S) MAb 1.2 Fab.Math.PAS #1.Math.V.sub.HH were solved by molecular replacement with the refined structure of the Fab 3F3E2Anti-PA(S) MAb 2.2 as search model, as well as the anti-human kappa light chain V.sub.HH domain (PDB ID: 6ANA) in the latter case. The structure of Anti-PA(S) MAb 3.1 Fab.Math.(APSA).sub.3 was solved by molecular replacement with the refined structure of the Anti-PA(S) MAb 1.2 Fab as search model, not including the anti-human kappa VHH domain in this case. After manual positioning of the PAS #1, P/A #1 and (APSA).sub.3 peptides, crystallographic refinement was completed, leading to R.sub.free values of 23-27% (Table 3).

TABLE-US-00005 TABLE 3 X-ray diffraction and refinement statistics for recombinant Fab fragments of Anti-PAS MAbs crystallized in complex with PAS peptide epitopes. Anti-PA(S) MAb Anti-PA(S) MAb Anti-PA(S) MAb Anti-PA(S) MAb 2.2 1.1 1.2 + VHH 3.1 Fab Peptide ligand Ac-APAPAAPA Pga-APASPAAPA Pga-APASPAAPA Pga-APSAAPSAAPSA Crystallization condition 11% w/v 18% w/v 22% w/v 20% w/v PEG6000 PEG3350 PEG3350 PEG3350 100 mM 100 mM HEPES 300 mM K- 200 mM Li- Tris/HCl PH 8 pH 7.5 acetate Nitrate 200 mM MgCl.sub.2 200 mM MgCl.sub.2 Data collection statistics Wavelength (?) 0.9184 0.9184 0.9184 1 Resolution range (?) 35-2.55 (2.65-2.55)* 35-2.65 (2.75-2.65) 35-1.55 (1.65-1.55) 30-1.85 (1.95-1.85) Space group I422 P4.sub.22.sub.12 C2 C2 Unit cell a, b, c (?) 122.0, 122.0, 138.4 102.6, 102.6, 199.9 141.3, 44.4, 93.0 94.9, 61.3, 78.6 ?, ?, ? (?) 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 109.1, 90.0 90.0, 106.3, 90 assemblies per asym. unit 1 2 1 1 Unique reflections 17370 (1866) 30855 (3054) 77314 (12963) 36237 (5351) Multiplicity 26.2 (25.2) 22.2 (18.3) 6.9 (6.8) 3.0 (3.1) Completeness (%) 99.9 (100) 96.9 (93.3) 97.2 (95.8) 97.6 (99.2) Mean I/?l 17.0 (5.1) 24.3 (2.1) 15.0 (1.6) 9.2 (2.1) Wilson B-factor (?.sup.2) 28.3 63.3 30.7 36.5 R.sub.meas (%) 21.3 (75.1) 11.2 (167.5) 6.3 (107.0) 8.3 (74.6) Refinement statistics Resolution (?) 34.63-2.55 (2.62-2.55) 34.13-2.65 (2.72-2.65) 33.39-1.55 (1.59-1.55) 45.6-1.85 (1.898-1.85) R.sub.work (%) 19.5 (26.2) 22.0 (39.4) 19.4 (34.1) 21.3 (35.9) R.sub.free (%) 23.8 (32.0) 26.9 (37.8) 23.2 (35.4) 26.8 (37.8) Number of non-H atoms: Protein/peptide 3379/50 6662/122 4374/61 3490/77 Solvent 125 53 388 176 Average B value (?.sup.2) Protein/peptide 29.6/42.8 73.8/58.5 28.7/25.7 44.6/58.7 Solvent 24.7 46.2 34.5 41.2 RMS (bonds/angles) 0.004/1.327 0.004/1.357 0.011/1.685 0.009/1.532 Ramachandran statistics.sup.# Favored (%) 96.8 94.1 97.7 96.6 Outliers (%) 0 0.2 0 0 Rotamer outliers (%) 1.5 2.9 0 1 Clash score.sup.# 1.63 2.47 1.6 2.09 *Values for the highest resolution shell are shown in brackets. .sup.#Calculated with MolProbity (Williams et al., 2018).

[0288] Further analysis of these crystal structures showed that the PAS peptides were bound to all four Fabs in a more or less relaxed conformation, covering a wide area of the antigen-binding site with at least four of the six complementarity-determining regions (CDRs) involved. Due to the lack of polar side chainsexcept for one Ser residue in the PAS #1 epitope peptide and three Ser residues in the (APSA).sub.3 peptidethe interactions are predominantly mediated through hydrogen bonds with peptide main-chain atoms (see appended Table 4) and Van-der-Waals contacts (see appended Table 5) including some local hydrophobic interactions, whereas salt bridges are completely absent, as expected. Interestingly, in each case at least one Ala residue of the PAS peptide is involved in relevant interactions with the anti-PAS Fab; hence, Ala can be considered as a hot spot for antibody interactions in PAS epitopes. Up to now, Ala, the amino acid with the smallest side chain, has been regarded to play a negligible role in protein-protein/peptide recognition. In fact, the strategy of alanine-scanning mutagenesis (Cunningham & Wells, 1989) has found wide application to dissect critical residues for receptor-ligand or antibody-antigen binding, assuming a quasi inert role of the Ala methyl side chain for molecular interactions. Unexpectedly, this invention reveals that Ala actually can adopt a central role in antigen recognition, as exemplified in particular with two crystal structures, the Anti-PA(S) MAb 2.2 Fab.Math.P/A #1 and the Anti-PA(S) MAb 1.1 Fab.Math.PAS #1. Indeed, being completely buried in the binding pocket, and with its carbonyl oxygen involved in two hydrogen bonds, Ala.sup.P5 acts as a hot spot residue (Clackson & Wells, 1995) in the antibody-peptide interface of the complex Anti-PA(S) MAb 2.2 Fab.Math.P/A #1. Likewise, the structure of the Anti-PA(S) MAb 1.1 Fab reveals a hole in the middle of the antigen-binding site which is perfectly molded to accommodate the methyl group of Ala.sup.P7, thereby allowing high shape complementarity and a densely packed interface.

[0289] The structure of anti-PA(S) MAb 3.1 Fab in complex with the (APSA).sub.3 peptide reveals a distinct groove in the paratope between V.sub.H and V.sub.L chains in which the peptide is bound in an elongated shape. Binding involves residues from all three APSA repeats in the peptide and is primarily mediated by hydrogen bonds with the peptide main chain atoms or peptide Ser side chains, as well as hydrophobic interactions of peptide Pro and Ala side chains. Similar to the structures of the Anti-PA(S) MAb 2.2 Fab.Math.P/A #1 and the Anti-PA(S) MAb 1.1 Fab.Math.PAS #1, Ala residues in the epitope play an important role in mediating hydrogen bonds and Van-der-Waals contacts (Tables 4 and 5).

[0290] Unexpectedly, in the case of the Anti-PA(S) MAb 1.2 Fab in complex with the PAS #1 epitope peptide the N-terminal pyroglutamyl residue of the peptide also contributes to the complex formation with three hydrogen bonds. These hydrogen bonds would not be possible in a complex with a longer PAS #1 (poly)peptide where the position of the Pga residue would be occupied by Pro. While a Pro residue would fit perfectly at this position in the crystal structure, the further N-terminal course of a longer polypeptide chain would lead to a steric clash with the Fab.

[0291] In order to elucidate any conformational similarities between the bound PAS #1 peptides in the complexes with the anti-PA(S) MAb 1.1 Fab or anti-PA(S) MAb 1.2 Fab, a superposition between their structures was performed. Indeed, the four residues Ser.sup.P5 to Ala.sup.P8 showed an excellent match of their C.sub.? positions, with a root mean square deviation (RMSD) of only 0.15 ?. Secondary structure analysis with STRIDE (Frishman & Argos, 1995) identified a type I ?-turn for this four-residue stretch. Apart from the intramolecular hydrogen bond between the Ser.sup.P5 carbonyl oxygen and the Ala.sup.P8 amide hydrogen, this turn is stabilized by a hydrogen bond between the Ser.sup.P5 hydroxyl group and the Ala.sup.P7 amide hydrogen. This type of ?-turn is classified as SPXX turn and occurs in gene regulatory proteins where it acts as DNA-binding motif (Suzuki & Yagi, 1991). However, despite their mutual similarity in the two Fab complexes, these turns are bound in different orientations: in the complex of anti-PA(S) MAb 1.1 Fab.Math.PAS #1 this turn nestles into the binding pocket whereas it is exposed to the solvent in the complex with the anti-PA(S) MAb 1.2 Fab.Math.PAS #1. Of note, a similar analysis with STRIDE identified no secondary structure features neither for the P/A #1 epitope peptide in the complex with the anti-P/A #1 MAb 2.2 Fab, nor for the (APSA).sub.3 peptide in complex with anti-PA(S) MAb 3.1 Fab.

[0292] In the context of this invention, MAbs that specifically recognize linear epitopes in structurally disordered Pro/Ala-rich (poly)peptides with three different sequences; i.e. sequences as provided in SEQ ID Nos: 1, 2 and 3 are generated by means and methods as provided herein. The inventive anti-PA(S) MAbs, or their recombinant versions and fragments, offer valuable bioanalytical and diagnostic tools for the biochemical study as well as biopharmaceutical development of PASylated drug candidates (Binder & Skerra, 2017; Gebauer & Skerra, 2018; Richter et al., 2020), including suitable assays for clinical studies.

TABLE-US-00006 TABLE 4 Hydrogen-bonding interactions between Anti- PA(S) MAbs and PAS epitope peptides. Peptide Fab Distance Structure atom atom [?] Anti-PA(S) MAb Ace.sup.P1-O Tyr.sup.L98-O.sup.? 2.47 2.2 FabP/A#1 Ala.sup.P3-N Tyr.sup.L98-O 2.85 Ala.sup.P5-O Asn.sup.H52-N.sup.?2 3.17 Ala.sup.P5-O Trp.sup.H54-N.sup.?1 2.94 Pro.sup.P7-O Asp.sup.H104-O.sup.?2 2.67 Anti-PA(S) MAb Pro.sup.P3-O Tyr.sup.L36-O.sup.? 2.61 1.1 FabPAS#1 Ser.sup.P5-N Arg.sup.H103-O 2.87 Ser.sup.P5-O.sup.? Ser.sup.L95-O 2.46 Pro.sup.P9-O Arg.sup.H103-N.sup.? 2.42 Anti-PA(S) MAb Pga.sup.P1-N Asp.sup.H107-O 2.80 1.2 FabPAS#1 Pga.sup.P1-N Tyr.sup.L54-O.sup.? 2.89 Pga.sup.P1-O.sup.? Ala.sup.H109-N 2.96 Ala.sup.P2-N Asp.sup.H107-O 3.05 Ala.sup.P2-O Asp.sup.H107-N 2.79 Pro.sup.P3-O Tyr.sup.L36-O.sup.? 2.59 Ala.sup.P4-N Gly.sup.H105-O 2.99 Ser.sup.P5-O.sup.? Trp.sup.L96-O 2.50 Ala.sup.P10-O.sup.oxt Asp.sup.H104-N 3.09 Anti-PA(S) MAb Pga.sup.P1-O Ile.sup.H28-N 3.05 3.1 FabAPSA Ser.sup.P4-O.sup.? Trp.sup.H99-O 2.69 Ala.sup.P6-O ArgL51-N.sup.? 2.74 Ser.sup.P8-O.sup.? ArgL51-N.sup.? 2.95 Ser.sup.P8-O Asn.sup.L39-N.sup.?2 2.84 Ala.sup.P10-N Gly.sup.L96-O 3.08 Ala.sup.P13-N Glu.sup.H33-O.sup.?2 3.04

TABLE-US-00007 TABLE 5 Van-der-Waals atom contacts between Anti-PAS MAbs and the PAS epitope peptides (?4.0 ?). Peptide Number of Structure residue contacts Fab residues Anti-PA(S) MAb Ace.sup.P1 5 Tyr.sup.L98 2.2 FabP/A#1 Ala.sup.P1 8 Tyr.sup.L31, Tyr.sup.L98 Pro.sup.P2 25 Tyr.sup.L31, Tyr.sup.L38, Tyr.sup.L98 Ala.sup.P3 10 Tyr.sup.L98, Asn.sup.L99, Tyr.sup.L100 Pro.sup.P4 5 Tyr.sup.L97, Tyr.sup.L98, Tyr.sup.L100, Pro.sup.H106 Ala.sup.P5 18 Tyr.sup.L100, Pro.sup.L101, Trp.sup.H49 Asn.sup.H52, Trp.sup.H54, Tyr.sup.H60 Ala.sup.P6 17 Trp.sup.H54, Tyr.sup.H60, Asp.sup.H104 Pro.sup.P7 22 Trp.sup.H54, Trp.sup.H55, Thr.sup.H56, Asp.sup.H58 Tyr.sup.H60, Asp.sup.H104 Ala.sup.P8 Anti-PA(S) MAb Pga.sup.P1 4 Tyr.sup.L34 1.1 FabPAS#1 Ala.sup.P2 10 Tyr.sup.L34, Tyr.sup.H104 Pro.sup.P3 11 Ser.sup.L32, Tyr.sup.L34, Tyr.sup.L36, Tyr.sup.H104 Ala.sup.P4 7 Tyr.sup.L36, Arg.sup.H103 Ser.sup.P5 21 Tyr.sup.L36, Ser.sup.L95, Arg.sup.H103, Tyr.sup.H104, Tyr.sup.H105 Pro.sup.P6 7 Ser.sup.L31, Tyr.sup.L36, Arg.sup.L96 Ala.sup.P7 11 Ser.sup.L95, Arg.sup.L96, Glu.sup.L97, Leu.sup.L98, Tyr.sup.H105 Ala.sup.P8 Pro.sup.P9 10 Trp.sup.H52, Ile.sup.H56, Arg.sup.H103 Ala.sup.P10 Anti-PA(S) MAb Pga.sup.P1 30 Lys.sup.L53, Tyr.sup.L54, Tyr.sup.H106, Asp.sup.H107, Tyr.sup.H108, Ala.sup.H109 1.2 FabPAS#1 Ala.sup.P2 15 Tyr.sup.L34, Tyr.sup.L54, Tyr.sup.H106, Asp.sup.H107 Pro.sup.P3 13 Tyr.sup.L34, Tyr.sup.L36, Gly.sup.H105 Ala.sup.P4 13 Tyr.sup.L36, Ile.sup.H102, Gly.sup.H105, Tyr.sup.H106, Asp.sup.H107 Ser.sup.P5 16 Tyr.sup.L36, Trp.sup.L96 Pro.sup.P6 3 Thr.sup.L31, Trp.sup.L96 Ala.sup.P7 7 Trp.sup.L96, Glu.sup.L97 Ala.sup.P8 2 Trp.sup.L96, Ile.sup.H102 Pro.sup.P9 7 Ile.sup.L98, Ile.sup.H102, Tyr.sup.H103 Ala.sup.P10 7 Tyr.sup.H103, Asp.sup.H104 Anti-PA(S) MAb Pga.sup.P1 9 Tyr.sup.H27, Ile.sup.H28, Tyr.sup.H32 3.1 FabAPSA Ala.sup.P2 9 Tyr.sup.H27, Tyr.sup.H32 Pro.sup.P3 22 Val.sup.H2, Tyr.sup.H27, Tyr.sup.H32, Leu.sup.H98, Trp.sup.H99, Arg.sup.H101 Ser.sup.P4 12 Arg.sup.L51, Trp.sup.H99, Arg.sup.H101 Ala.sup.P5 3 Tyr.sup.L54, Trp.sup.H99 Ala.sup.P6 22 Arg.sup.L51, Tyr.sup.L54, Leu.sup.L55, Trp.sup.H99 Pro.sup.P7 22 Arg.sup.L51, Trp.sup.H99 Ser.sup.P8 29 Tyr.sup.L37, Asn.sup.L39, Arg.sup.L51, Gly.sup.L96, Trp.sup.L94, Trp.sup.H99, Gly.sup.H100 Ala.sup.P9 17 Tyr.sup.L37, Gly.sup.L96, His.sup.L101 Ala.sup.P10 16 His.sup.L31, Tyr.sup.L37, Gly.sup.L96 Pro.sup.P11 6 Glu.sup.H33 Ser.sup.P12 9 Leu.sup.L99, Glu.sup.H33, Val.sup.H59 Ala.sup.P13 16 Glu.sup.H33, Ile.sup.H51, His.sup.H52, Asn.sup.H57

[0293] In all crystallized Fab complexes provided herein, a high abundance of Tyr residues in the paratope is evident. These residues are responsible for the majority of hydrophobic contacts (appended Table 5), thus creating a surface well suited to bind antigens poor in charge or polar side chains. In fact, 62%, 49% 43% and 23% of all contacts ?4.0 ? are mediated by Tyr in the Anti-PA(S) MAb 2.2, Anti-PA(S) MAb 1.2, Anti-PA(S) MAb 1.1 and Anti-PA(S) MAb 3.1, respectively. Interestingly, the Anti-PA(S) MAb 2.2 revealed a high Tyr content and also has a high affinity among the crystallized complexes. This is in line with previous analyses, which indicate that a high content of Tyr in antibody paratopes generally contributes to enhanced antigen specificity and affinity (Birtalan et al., 2008; Birtalan et al., 2010).

[0294] The data provided herein shed light on the mechanism of molecular recognition of disordered epitopes by antibodies. With no salt bridges and no pronounced side chain interactions arising from the PAS epitope peptides in all assessed Fab structures, complex formation is mainly driven by hydrogen bonds involving the peptide backbone (appended Table 4) as well as Van-der-Waals contacts (appended Table 5) including some local hydrophobic interactions. Due to the feature-less nature of the PAS peptides, the few atom groups capable of polar interactions have to be capitalized efficiently. This is nicely demonstrated with the structure of Anti-PA(S) MAb 1.2, for example, where a short segment of the backbone hydrogen bond network with the PAS #1 peptide resembles an antiparallel ?-sheet. In the two Fab complexes with the PAS #1 epitope peptide, which comprises one Ser residue, both antibodies engage the only available polar side chain for formation of hydrogen bonds.

[0295] The same is the case in the structure of Anti-PA(S) MAb 3.1, where two of the three Ser side chains are involved in hydrogen bonding. Nevertheless, in line with the limited energy gain of such hydrogen bonds in a competing aqueous environment (Gao et al., 2009). In fact, it seems that the Anti-PA(S) MAbs 1.1 and 1.2 do not much benefit from this interaction considering their significantly lower affinity compared with the best MAbs raised against anti-P/A #1 (see Table 2). The observation that at least four of the six CDRs are involved in the peptide-antibody interactions in all assessed Fab complexes (see Table 6) highlights the need to involve an extended interface to more or less tightly bind structurally flexible antigens.

TABLE-US-00008 TABLE 6 CDRs involved in atom contacts between Anti-PA(S) MAbs and the PAS epitope peptides (?4.0 ?). Sequential numbering is used for the amino acid sequences of all antibodies; hence, the numbers of the CDRs may be individually different. CDR L1 CDR L2 CDR L3 CDR H1 CDR H2 CDR H3 Anti-PA(S) L24-38 L54-60 L93-101 H26-35 H50-65 H98-109 MAb 1.1 Ser.sup.L32 Ser.sup.L95 Trp.sup.H52 Arg.sup.H103 Tyr.sup.L34 Arg.sup.L96 Ile.sup.H56 Tyr.sup.H104 Tyr.sup.L36 Glu.sup.L97 Tyr.sup.H105 Leu.sup.L98 Anti-PA(S) L24-38 L54-60 L93-101 H26-35 H50-66 H99-112 MAb 1.2 Tyr.sup.L31 Lys.sup.L53 Trp.sup.L96 Ile.sup.H102 Tyr.sup.L34 Tyr.sup.L54 Glu.sup.L97 Tyr.sup.H103 Tyr.sup.L36 Ile.sup.L98 Asp.sup.H104 Gly.sup.H105 Tyr.sup.H106 Asp.sup.H107 Tyr.sup.H108 Ala.sup.H109 Anti-PA(S) L24-40 L56-62 L95-102 H26-37 H52-67 H100-110 MAb 2.2 Tyr.sup.L31 Tyr.sup.L97 Asn.sup.H52 Asp.sup.H104 Tyr.sup.L38 Tyr.sup.L98 Trp.sup.H54 Pro.sup.H106 Asn.sup.199 Trp.sup.H55 Tyr.sup.L00 Thr.sup.H56 Pro.sup.L101 Asp.sup.H58 Tyr.sup.H60 Anti-PA(S) L24-39 L55-61 L94-102 H26-35 H50-66 H99-101 MAb 3.1 His.sup.L31 Leu.sup.L55 Gly.sup.L96 Tyr.sup.H27 Ile.sup.H51 Trp.sup.H99 Tyr.sup.L37 Ile.sup.H28 His.sup.H52 Arg.sup.H101 Asn.sup.L39 Tyr.sup.H32 Asn.sup.H57 Glu.sup.H33 Val.sup.H59

Example 6: SPR Spectroscopy Using Anti-PA(S) Mab 1.1 to Capture a PASylated Anti-Galectin Fab Fragment and to Determine the PAS-Fab Binding Kinetics Towards its Antigen Galectin-3

[0296] The anti-PA(S) Mab 1.1 antibody of this invention was used as a tool for the stable non-covalent capturing of a PASylated humanized anti-Galectin Fab fragment (Peplau et al., 2021) on a surface plasmon resonance (SPR) sensor chip to determine the affinity of this Fab to its antigen Galectin-3. A Biacore X100 instrument (Cytiva, Freiburg, Germany), operated with HBS/T (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% v/v Tween 20) as running buffer at a flow rate of 30 ?l/min, was charged with a carboxymethyl dextran-coated CM5 sensor chip (Cytiva). The carboxylate groups of the dextran hydrogel in both flow channels were converted to reactive N-hydroxysuccinimide ester groups using an amine coupling kit (Cytiva) by injecting a 1:1 mixture of 483 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 100 mM N-hydroxysuccinimide (NHS) for 430 s at a flow rate of 5 ?l/min. Next, the protein A affinity purified recombinant anti-PA(S) Mab 1.1 obtained from Genscript (Piscataway, NJ, USA) was covalently immobilized onto the chip surface by injection of a 100 ?g/ml anti-PA(S) Mab 1.1 solution in 10 mM Na-acetate pH 4.5 for 600 s at a flow rate of 5 ?l/min. Unreacted NHS ester groups were finally blocked by injection of an 0.1 M ethanolamine solution for 430 s at a flow rate of 5 ?l/min. This procedure (FIG. 17) resulted in an anti-PA(S) Mab 1.1 surface density of approximately 13500 resonance units (?RU).

[0297] To investigate the binding kinetics of the anti-Galectin-PAS(200) Fab fragment (SEQ ID NO: 92 and SEQ ID NO: 93) towards recombinant Galectin-3 (Uniprot Identifier P17931) carrying a C173T mutation and a C-terminal Strep-tag II (SEQ ID NO: 94), the purified PASylated Fab fragment was diluted in HBS/T to 3.57 ?g/ml and injected into flow channel 2 for 40 s at a flow rate of 5 ?l/min, followed by buffer flow for 600 s. This resulted in a PAS-Fab surface density of approximately 580 resonance units (ARU), which remained stable within ?7% (FIG. 17B). Subsequently, a single cycle kinetic experiment was performed using five consecutive injections from a 1:2 dilution series (1.6 nM to 0.1 nM) of the antigen Galectin-3 at a flow rate of 30 ?L/min, each for 60 s, finally followed by a 3600 s dissociation period. After that, the chip was regenerated by two subsequent injections of 10 mM glycine/HCl pH 2.4 for 60 s at a flow rate of 30 ?l/min. This regeneration procedure allowed reuse of the same MAb-functionalized sensor surface for further measurements with PASylated proteins over more than two month.

[0298] The reference-corrected sensorgram (FIG. 17C) showed binding curves typical for a bimolecular reaction between the anti-Galectin-PAS(200) Fab fragment and its antigen Galectin-3. These data were fitted to a global 1:1 Langmuir binding model using Biacore X100 evaluation software (Cytiva), resulting in an association rate of 7.9?10.sup.6 M.sup.?1 s.sup.?1, a dissociation rate of 3.0?10.sup.?5 s.sup.?1 and an equilibrium dissociation constant (K.sub.D value) of 3.8 pM.

[0299] These findings demonstrate that the highly specific anti-PAS 1.1 MAb of this invention offers a valuable tool for the stable non-covalent capturing of a PASylated protein on an SPR sensor chip. Furthermore, mild acidic regeneration using 10 mM glycine/HCl pH 2.4 completely removed the PASylated protein, together with its ligand, offering the ability to reuse the same MAb-functionalized sensor surface for further measurements with PASylated proteins.

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