Antibodies specific for structurally disordered sequences

Abstract

The present invention relates to a method for generating and / or obtaining specific binding moieties for intrinsically disordered proteins (IDPs) and / or domains of intrinsically disordered proteins that tend to be immunologically inactive and lack immunogenicity in animals, particularly in mammals. The present invention also relates to such specific binding moieties, particularly antibodies and / or antigen-binding fragments thereof, that specifically bind to structurally disordered and / or intrinsically disordered sequences, particularly Pro / Ala rich sequences (PAS). These binding moieties, antibodies, antigen-binding fragments are first-in-class, as they bind to / recognize such "intrinsically disordered proteins", particularly disordered peptides or polypeptide fragments, such as also included in PAS polypeptides. The binding moieties, antibodies, antigen-binding fragments of the present invention are particularly useful, but not limited to, in diagnostic settings as well as in research tools. The present invention relates to a method for generating and / or obtaining specific binding moieties for intrinsically disordered proteins (IDPs) and / or domains of intrinsically disordered proteins that tend to be immunologically inactive and lack immunogenicity in animals, particularly in mammals. The present invention also relates to such specific binding moieties, in particular antibodies and / or antigen-binding fragments thereof, that specifically bind 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 because they bind to / recognize such "intrinsically disordered proteins", in particular disordered peptides or polypeptide fragments, such as also included in PAS polypeptides. The binding moieties, antibodies, antigen-binding fragments of the present invention are particularly useful in, but not limited to, diagnostic settings as well as research tools.

Description

[Technical field]

[0001] The present invention relates to methods for generating and / or obtaining specific binding moieties for intrinsically disordered proteins (IDPs) and / or domains of intrinsically disordered proteins that tend to be immunologically inactive and lack immunogenicity in animals, particularly in mammals. The present invention also relates to such specific binding moieties, particularly antibodies and / or antigen-binding fragments thereof, that specifically bind to structurally disordered and / or intrinsically disordered sequences, particularly Pro / Ala rich sequences (PAS). These binding moieties, antibodies, antigen-binding fragments are first-in-class, as they bind to / recognize such "intrinsically disordered proteins", particularly disordered peptides or polypeptide fragments, such as also included in PAS polypeptides. The binding moieties, antibodies, antigen-binding fragments of the present invention are particularly useful, but not limited to, in diagnostic settings as well as in research tools.

[0002] The present invention specifically and in one particular embodiment relates to a method for generating antigen-binding molecules, preferably antibodies or antigen-binding fragments thereof, directed against intrinsically unfolded peptides / proteins and / or domains of intrinsically unfolded peptides / proteins, comprising the step of immunizing a non-human mammal with an antigen, said antigen being a conjugate of an immune adjuvant and one or more P / A peptides, each P / A peptide being independently a peptide of about 5 to about 100 amino acid residues, at least 60% of the amino acid residues of said peptides being independently selected from proline and alanine, and each P / A peptide being independently selected from a protecting group R N is attached to the N-terminal amino group of the peptide.

[0003] The present invention also relates to well-defined specific hybridomas comprising a nucleic acid sequence encoding a specific binding moiety / antibody / antibody fragment of the invention and / or a nucleic acid sequence encoding a variable region (such as a variable heavy and / or variable light chain sequence) and / or a complementarity determining region (CDR) of said specific binding moiety / antibody / antibody fragment of the invention. The present invention further relates to a nucleic acid molecule encoding the CDR and / or the light or heavy chain variable region of an antibody of the invention, as well as to a vector comprising said nucleic acid molecule. The present invention also relates to a host cell comprising a vector of the invention, as well as to a method for producing a binding moiety / antibody / antibody fragment of the invention, comprising the steps of culturing a host cell of the invention and / or a hybridoma of the invention under suitable conditions and isolating the binding moiety / antibody / antibody fragment produced. Thus, the present invention also relates to a hybridoma and / or a host cell expressing a binding moiety / antibody / antibody fragment of the invention.

[0004] Furthermore, the present invention relates to binding moieties / antibodies / antibody fragments obtainable by the method of the invention, to compositions comprising at least one binding moiety, antibody or antigen-binding fragment of the invention, to hybridomas and / or host cells of the invention, to nucleic acid molecules of the invention, to vectors of the invention, to hybridomas / host cells of the invention, or to binding moieties, antibodies or antigen-binding fragments produced by the method of the invention. The present invention also relates to the use of binding moieties, antibodies or antigen-binding fragments of the invention for detecting, quantifying and / or identifying intrinsically disordered proteins and / or domains of intrinsically disordered proteins, in particular PAS sequences and / or molecules comprising PAS sequences. Such detection, quantification or identification may also be performed on biological samples according to the invention, for example on blood or plasma samples, or on samples of cerebrospinal fluid, vitreous of the eye, tissue sections, etc. The present invention also provides research tools and / or diagnostic reagents for the preclinical and clinical development of PASed biologics. Also provided herein are means and methods for screening biological samples obtained from subjects, particularly human patients, treated with such PASylated biologics, i.e. drug conjugates comprising a biologically active (protein) drug and a Pro / Ala rich sequence (PAS) containing, for example, only small residues Pro, Ala and Ser, or Pro and Ala. Said drug conjugates are not limited to protein drugs or biologics, but can also include "small molecule" drugs and chemical drugs, as well as carbohydrate drugs and nucleic acid drugs.

[0005] The present invention also relates to the use of the binding moieties, antibodies or antigen-binding fragments of the present invention for the preparation of tools, such as in diagnostic settings, for laboratory use, in research and / or development, including preclinical or clinical development, in purification methods, etc. For example, the binding moieties, antibodies or antigen-binding fragments of the present invention can be used in matrix-based protein / peptide purification or immobilization. In particular, affinity matrices for the purification of intrinsically disordered proteins and / or domains of intrinsically disordered proteins, in particular PAS sequences and / or molecules containing PAS sequences, can be prepared using the compounds of the present invention provided herein, such as the antibodies of the present invention or their antigen-binding fragments.

[0006] In this specification, several documents, including patent applications and manufacturer's manuals, are cited. The disclosures of these documents are not considered relevant to the patentability of this invention, but are incorporated herein by reference in their entirety. More specifically, all references are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. [Background technology]

[0007] Intrinsically disordered proteins or protein domains (IDPs) are common in nature and play important roles in signal transduction and protein trafficking, as is the case for example with the transcriptional activation domains of synaptojanin or RelA (Snead & Eliezer, 2019; Tantos et al., 2012; Wright & Dyson, 2015). Such IDPs are also abundant in a wide range of pathogens and are therefore potential targets for combating infections (Feng et al., 2006). In contrast to specific interactions with structured proteins, the properties of disordered peptide or polypeptide segments (as also found in IDPs) are features that are exploited by pathogens to evade immune responses, often posing a challenge to the immune system in generating cognate antibodies (Giri et al., 2016; Goh et al., 2016).

[0008] Antigens are mostly proteins or peptides, and these surface epitopes serve as interaction points for specific antibody recognition. Epitopes are generally classified into two categories: (i) linear epitopes, defined by their primary structure, and (ii) conformational epitopes, in which key amino acids are discontinuous in the amino acid sequence but are closely spaced within a (structure-defined) three-dimensional fold (Barlow et al., 1986). It has long been assumed that epitopes are overwhelmingly discontinuous (Barlow et al., 1986); in fact, more recent analyses suggest that conformational epitopes account for approximately 90% of all B-cell epitopes present in natural proteins (Huang & Honda, 2006).

[0009] Although interactions between disordered proteins have been analyzed in detail (Fong et al., 2009; Meszaros et al., 2007; Uversky, 2019), only a few studies have addressed the structural aspects of complex formation between disordered protein antigens and well-defined binding partners such as antibodies (Fassolari et al., 2013; MacRaild et al., 2016). In general, peptides tend to bind in a planar manner than proteins, forming interfaces with antibodies that often involve the peptide backbone but are dominated by hydrogen bonds (London et al., 2010). Furthermore, IDPs present smaller epitopes than folded antigens and appear to be more efficient in terms of free energy gain per contact residue (MacRaild et al., 2016). Structural analysis of protein antigens has shown that residues in disordered epitopes are more likely to participate in hydrogen bonds and salt bridges than residues in conformational epitopes (MacRaild et al., 2016). More specifically, as the prior art postulates, interfaces with peptides are typically rich in large hydrophobic side chains, such as Phe, Leu, Trp, Tyr, and Ile, which act as hot spots for binding ( London et al., 2010 ).

[0010] In the past decade, conformationally disordered artificial polypeptides have attracted attention in the field of pharmaceutical biotechnology, where they are used as functional replacements for the highly hydrophilic chemical polymer polyethylene glycol (PEG) to tailor the in vivo properties of protein fusion partners or drug conjugates (Schellenberger et al., 2009; Schlapschy et al., 2013). For example, the conjugation of pharmacologically active proteins or peptides or ("small") molecules with long polypeptides containing the three small amino acids Pro, Ala, and / or Ser, known as PASylation®, dramatically increases the hydrodynamic volume and extends the plasma half-life by delaying renal filtration (Binder & Skerra, 2017). Thus, Pro / Ala-rich sequence (PAS) polypeptides have been developed as biological replacements for polyethylene glycol (PEG) to generate biopharmaceuticals with extended plasma half-lives. As with the chemical macromolecule PEG, recombinant PAS polypeptides are conformationally disordered and highly soluble in water. Indeed, these biosynthetic polymers, which lack any charged or apparent hydrophobic side chains, are extreme examples of IDPs.

[0011] As discussed above, the PASylation® approach relies on Pro / Ala-rich sequences (PAS), i.e. conformationally disordered polypeptide chains with increased hydrodynamic volume that contain only the small residues Pro, Ala and Ser or Pro and Ala. These PAS sequences are hydrophilic, uncharged biopolymers with very similar biophysical properties to PEG, whose chemical conjugation to drugs is an established method for plasma half-life extension. In contrast to PEG, PAS polypeptides are described as allowing simple fusion to therapeutic proteins or peptides at the genetic level, thus allowing the production of fully active therapeutic proteins in Escherichia coli or other host cells, obviating the need for in vitro coupling or modification steps (Binder & Skerra, 2017). Moreover, Pro / Ala-rich sequences (PAS) / PAS polypeptides are biodegradable, thus avoiding organ accumulation, while exhibiting stability in serum and being non-toxic. One further advantage of the PASylation® technology is that it provides PAS polypeptides that are in fact immunologically inert, which is therefore of great advantage for medical and therapeutic applications. However, the lack of immunogenicity is also the reason why no antibodies against such intrinsically disordered proteins (IDPs) and / or domains of intrinsically disordered proteins have been described in the art. However, such specific antibodies are desired, especially as research tools and in diagnostic situations, including patient stratification and / or monitoring of treatment responses. Summary of the Invention [Problem to be solved by the invention]

[0012] The technical problem underlying the present invention is therefore to provide means and methods for the preparation of binding moieties, in particular antibodies and / or antibody fragments, which specifically bind intrinsically disordered proteins or protein domains, in particular disordered polypeptide chains with increased hydrodynamic volume containing the amino acid residues Pro and Ala and / or Pro, Ala and Ser (PAS). [Means for solving the problem]

[0013] This technical problem is solved by the embodiments provided herein and in the appended claims. In a first embodiment, the present invention relates to a method for generating an antigen-binding molecule, preferably an antibody or an antigen-binding fragment thereof, directed against an intrinsically unnatural peptide / protein and / or a domain of an intrinsically unnatural peptide / protein, comprising: immunizing a non-human mammal with an antigen, The antigen is a conjugate of an immune adjuvant and one or more P / A peptides, each P / A peptide being independently a peptide of about 5 to about 100 amino acid residues, at least 60% of the amino acid residues of the peptides being independently selected from proline and alanine, and each P / A peptide being independently selected from the group consisting of a protecting group R N is attached to the N-terminal amino group of the peptide.

[0014] In the context of the present invention and as illustrated in the examples, the inventors have surprisingly discovered that when conjugated to an immune adjuvant / highly immunogenic carrier protein such as KLH in combination with an immunization scheme as disclosed herein, which preferably forms a protein complex with a molecular mass of more than about 5 megadaltons (5MDa), the PAS polypeptide can induce a PAS-directed antibody response in non-human mammals, particularly in mice. This is unexpected and is made possible by the means and methods as disclosed herein. In general, conjugation to an immune adjuvant is believed to provide additional T cell epitopes and thus enhance the immunogenicity of the peptide portion of the conjugate. Thus, it is believed that the ability of the immune system is enhanced to generate antibodies that specifically bind to such peptides. However, it has been shown previously in the same BALB / c mouse strain used in the present invention (Schlapschy et al., 2013) that conjugation of the PAS moiety to a protein moiety such as human IFNα2b and repeated immunization of the mice did not result in the generation of any PAS-specific antibodies, but rather in the generation of IFNα2b-specific antibodies, even with the use of Freund's adjuvant as an immune booster. Similarly, in plasma samples from mice that had been repeatedly treated with PAS-hGH, IgG reactive to the human growth hormone (hGH) moiety was detectable by Western blot, but there was no cross-reactivity with the PAS sequence fused to other proteins, indicating that the PAS polypeptide itself is not immunogenic (Schlapschy et al., 2013). Indeed, this complete lack of PAS-directed immunogenicity, even when fused to a protein moiety, is a key feature of the PASylation technology, which can be attributed directly to the nature of the small, biochemically inert amino acids Pro, Ala and Ser that constitute such PA(S) peptides.

[0015] The prior art postulates that interfaces with peptides are usually rich in large hydrophobic side chains such as Phe, Leu, Trp, Tyr and Ile (contained in peptides used for conjugation to carrier proteins) that serve as hot spots for binding (London et al., 2010). Conversely, and therefore surprisingly, the attached crystal structure of the binding moiety of the present invention reveals a particularly relevant and unprecedented role of Ala in the recognition of PA(S) peptides. This is particularly surprising because all of the amino acids contained in PA(S) peptides have immunologically / chemically inert side chains and lack any charged and / or obvious hydrophobic side chains that could form strong hydrophobic and / or electrostatic interactions. In addition, the random coil behavior of PA(S) (poly)peptides under physiological conditions induces a significant entropic cost for the disorder-order transition upon complexation with binding proteins such as antibodies. This has also been demonstrated by numerous in vivo imaging studies using PASylated antibody fragments as well as in preclinical animal experiments with repeated dosing of the protein, where neither any non-specific binding to non-target tissues or organs nor PAS-specific immune responses were detectable (Bolze et al., 2016; Harari et al., 2014; Mendler et al., 2015; Richter et al., 2020). Taken together, these PAS-specific sequence / structural features therefore pose unique and significant challenges for generating PA(S)-specific antibodies, which have been overcome by the methods and peptides / antigens of the invention as disclosed herein. Corresponding exemplary P / A peptides / antigens are also described herein below.

[0016] The present invention relates in certain embodiments to a method for generating specific binding moieties, in particular antigen-binding molecules, directed against intrinsically disordered proteins and / or domains or peptides of intrinsically disordered proteins, comprising the steps of immunizing a non-human mammal with an antigen, Thereby, the antigen is a conjugate of an immune adjuvant and one or more P / A peptides, Here, each P / A peptide is independently peptide R N -(P / A)-R C and (P / A) is an amino acid sequence consisting of about 5 to about 100 amino acids, at least 60% of the number of amino acid residues in (P / A) are independently selected from proline and alanine, (P / A) contains at least one proline residue and at least one alanine residue, R N is a protecting group attached to the N-terminal amino group of (P / A), R C is an amino acid residue that is linked via its amino group to the C-terminal carboxy group of (P / A) and that contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6 carbon atoms between its amino group and its carboxy group; Each P / A peptide is composed of the C-terminal amino acid residue R C is conjugated to the immune adjuvant via an amide bond formed between the carboxy group of and a free amino group of the immune adjuvant.

[0017] In a preferred embodiment, the method for generating said antigen binding molecule, preferably said antibody or said antigen binding fragment thereof (directed against an intrinsically unnatural peptide / protein and / or a domain of an intrinsically unnatural peptide / protein) as described herein is a method of immunization of a non-human with said P / A peptide, where P / A peptides are independently peptide R N -(P / A)-R C and is a peptide consisting of about 8 to about 90 amino acid residues, At least 70% of the number of amino acid residues in (P / A) are independently selected from proline and alanine, and (P / A) contains at least one proline residue and at least one alanine residue; R Nis a protecting group attached to the N-terminal amino group of (P / A), R C is an amino acid residue that is linked via its amino group to the C-terminal carboxy group of (P / A) and that includes at least one carbon atom between its amino group and its carboxy group; Each P / A peptide is composed of the C-terminal amino acid residue R C It is conjugated to the immunoadjuvant via an amide bond formed between the carboxy group of and a free amino group of the immunoadjuvant.

[0018] As used herein, the term "specific binding moiety(s)" includes in particular the described antigen-binding molecules, antibodies and antigen-binding fragments thereof discussed herein. However, the term also includes other molecules that are capable of specifically binding said naturally unfolded peptides / proteins, but are not of the usual antibody format. Such "binding moieties" may include in particular molecules such as fusion proteins or (protein) constructs that contain a binding moiety based on or derived from an antibody obtainable using the means and methods provided herein. Such constructs may in particular comprise at least one, at least two or three complementarity determining regions (CDRs) of an antibody / antibody fragment provided herein and / or obtainable using the methods of the invention.

[0019] In a particular embodiment, the present invention provides means and methods for generating antigen-binding molecules, preferably antibodies or antigen-binding fragments thereof, directed against intrinsically unfolded peptides / proteins and / or domains of intrinsically unfolded peptides / proteins. In a preferred embodiment, the present invention provides means and methods for generating antibodies and / or antigen-binding fragments thereof, directed against and / or specifically binding to intrinsically unfolded peptides / proteins (IDPs) and / or domains of intrinsically unfolded proteins, in particular antibodies and / or antigen-binding fragments thereof, that specifically bind to Pro / Ala rich sequences ("PAS"), "PAS" sequences / "PAS" portions.

[0020] Pro / Ala-rich sequences ("PAS"), "PAS" sequences / "PAS" moieties are defined herein and are also described in WO2008 / 155134 and WO2011 / 144756. These "PAS" moieties also relate to peptides consisting of at least 7 amino acid residues that form a random coil conformation, as further described in (Schlapschy et al., 2013) or (Binder & Skerra, 2017), whereby the amino acid residues that form the random coil conformation are selected from Pro(P), Ala(A) and Ser(S), or from Pro(P) and Ala(A). Pro / Ala-rich sequences, such as those contained in proteinaceous drug conjugates, among others, are also described as "(P / A)" sequences, for example in WO2018 / 234455. Such a (P / A) sequence, i.e., herein, a Pro / Ala-rich sequence, can consist of about 7 to about 1200 amino acid residues, where at least 80% of the number of amino acid residues in (P / A) are independently selected from proline and alanine, and (P / A) comprises at least one proline residue and at least one alanine residue. Furthermore, as will be apparent from the disclosure herein and known in the art, the term "Pro / Ala-rich sequence (PAS)", "PAS", "PAS portion", or "PAS sequence" should not be construed as being limited to intrinsically disordered proteins / peptides (IDPs) and / or proteins / peptides that form a random coil conformation that contains only Pro and Ala. The above terms also encompass the corresponding proteins / peptides that are composed primarily of Pro, Ala, and Ser. Further amino acids may also be included to a minor extent, as disclosed, inter alia, in WO2008 / 155134, WO2011 / 144756 or WO2018 / 234455, all of which are incorporated by reference above.

[0021] As discussed herein, conjugation of drugs with such (P / A) sequences and / or Pro / Ala-rich sequences (PAS), also known as PASylation®, dramatically increases hydrodynamic volume and extends plasma half-life by slowing renal filtration in vivo (Griffiths et al., 2019; Langin et al., 2018; Richter et al., 2020).

[0022] Thus, the present invention provides means and methods for obtaining specific binding moieties, in particular antibodies and / or antigen-binding fragments thereof, that specifically bind to structurally disordered and / or naturally unordered sequences, in particular Pro / Ala-rich sequences (PAS). The prior art does not provide or describe any antibodies and / or antigen-binding fragments directed against structurally disordered and / or naturally unordered sequences, in particular Pro / Ala-rich sequences. Moreover, it has been previously described that recombinant polypeptides composed of Pro, Ala and Ser, or even only Pro and Ala, are highly hydrophilic and structurally unordered, regardless of the exact amino acid sequence - provided that certain repeat patterns or long homoamino acid stretches are avoided (Breibeck & Skerra, 2018; Schlapschy et al., 2013). In particular, PAS polypeptides, whose amino acid sequence is precisely defined at the genetic level, are completely neutral, while their side chains - in particular in the case of Ser-free P / A sequences - lack obvious polar groups. Thus, the strong hydrophilicity of Pro / Ala-rich sequences (PAS) is explained by the exposure of peptide groups to aqueous solvent in the absence of rigid secondary structures (Breibeck & Skerra, 2018). Last but not least, due to the lack of obvious polar groups, Pro / Ala-rich sequences / (PAS) sequences lack immunogenicity in animals, especially in mammals. This low immunogenicity is indeed one of the hallmarks of PASylation® technology in the pharmaceutical and medical fields.

[0023] PAS polypeptides as used in the PASylation® technology are known to lack immunogenicity (be "immunologically inert") in mammals, especially in rodents such as mice, rats or rabbits, i.e. animals routinely used for the preparation of (monoclonal) antibodies. Indeed, as a result of their restricted amino acid composition, PAS polypeptides lack both charged and bulky hydrophobic side chains that normally play a role in molecular recognition, especially in the immune response. In addition, their random coil behavior under physiological conditions causes an enormous entropic cost for the disorder-order transition upon complex formation with binding proteins such as antibodies. This has also been demonstrated by numerous in vivo imaging studies using PASylated antibody fragments as well as in preclinical animal experiments involving repeated dosing of the protein, where neither any non-specific binding to non-target tissues or organs nor a PAS-specific immune response was detectable (Bolze et al., 2016; Griffiths et al., 2019; Harari et al., 2014; Mendler et al., 2015; Richter et al., 2020).

[0024] However, despite these lack of immunogenicity of Pro / Ala rich sequences (PAS) in previous animal studies, the inventors have succeeded in generating binding moieties that specifically bind different Pro / Ala rich sequences (PAS), in particular (monoclonal) antibodies. This success is based on the novel and inventive approach of specific antigens to be used in the immunization of non-human animals. Said inventive antigens are herein referred to as (P / A) sequences [wherein (P / A) is an amino acid sequence] or (P / A) antigens [format R as defined herein]. N -(P / A)-R C In the present specification, the term "immunoadjuvant" is used to refer to a (P / A) sequence or (P / A) antigen, which are characterized by their conjugation with a highly immunogenic carrier protein ("immunoadjuvant"), such as keyhole limpet hemocyanin (KLH). The (P / A) sequence or (P / A) antigen is represented by the carboxy group of the C-terminal amino acid or linker residue of the (P / A) sequence / antigen (referred to herein as "R C") to one or more free amino groups of the immunoadjuvant. Furthermore, the (P / A) sequence / antigen used in the context of the present invention may be N-terminally blocked, i.e., linked to the N-terminal amino group of the (P / A) sequence / antigen and designated "R N " This also obviates the need for the formation of N-terminal specific antibodies.

[0025] The method of the present invention comprises the steps of: N -(P / A)-R C The present invention includes immunization of non-human mammals (particularly mouse(s)) with (P / A) peptides / sequences / antigens, such as those provided in the form of a peptide / sequence / antigen (P / A) having a protecting group "R" at the N-terminus. N " and an immune adjuvant linked to the C-terminus. However, it is also envisaged that the antigen used for immunization comprises multiple of said (P / A) peptides / sequences / antigens. Thus, the antigen used in the context of the present invention is a conjugate of an immune adjuvant and one or more (P / A) peptides / sequences / antigens as disclosed herein.

[0026] Preferred (P / A) peptides / sequences / antigens are: an amino acid sequence / antigen consisting of about 5 to about 100 amino acid residues, more preferably about 8 to about 90 amino acid residues, wherein at least 60%, at least 70% of the number of amino acid residues in (P / A) are independently selected from proline and alanine, and (P / A) comprises at least one proline residue and at least one alanine residue; an amino acid sequence / antigen consisting of about 5 to about 100 amino acid residues, more preferably 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, and at least 95% of the number of amino acid residues in (P / A) are independently selected from proline, alanine and serine, and (P / A) comprises at least one proline residue and at least one alanine residue; and / or An amino acid sequence / antigen consisting of about 20 to about 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 (P / A) contains at least one proline residue and at least one alanine residue. may include.

[0027] In one embodiment of the present invention, the (P / A) peptide / sequence / antigen used in the methods provided herein for immunization of a non-human mammal may be a (P / A) sequence / antigen, in which the ratio of the number of proline residues contained in said (P / A) to the total number of amino acid residues contained in (P / A) is ≧about 10% and ≦about 70%, preferably ≧about 20% and ≦about 50%, more preferably ≧about 25% and ≦about 40%.

[0028] In accordance with the above, a (P / A) peptide / sequence / antigen as used in the context of the present invention may be a (P / A) peptide / sequence / antigen consisting of (i) five or more subsequences independently selected from "ASPA", "APAP", "SAPA", "AAPA" and "APSA", and (ii) optionally one, two or three additional amino acid residues independently selected from proline (P), alanine (A) and serine (S). The (P / A) peptide / sequence / antigen may also include multimers and combinations of these subsequences independently selected from "ASPA", "APAP", "SAPA", "AAPA" and "APSA". In one embodiment, said (P / A) peptide / sequence / antigen consists of (i) the sequence ASPA-APAP-ASPA-APAP-SAPA (SEQ ID NO:1), (ii) the sequence AAPA-APAP-AAPA-APAP-AAPA (SEQ ID NO:2), (iii) the sequence APSA-APSA-APSA-APSA-APSA (SEQ ID NO:3), (iv) a duplication of any of the foregoing sequences, or (v) a combination of at least two of the foregoing sequences.

[0029] Non-limiting examples of such peptides / sequences / antigens are (i) ASPA-APAP-ASPA-APAP-SAPA-ASPA-APAP-ASPA-APAP-SAPA, (ii) AAPA-APAP-AAPA-APAP-AAPA-AAPA-APAP-AAPA-APAP-AAPA, or (iii) (P / A) consisting of the sequence APSA-APSA-APSA-APSA-APSA-APSA-APSA-APSA-APSA-APSA-APSA-APSA. Multimers of these sequences are also included in the scope of the invention and may be used in the non-human animal immunization methods provided herein. Non-limiting examples of embodiments are also provided in the experimental section as "PAS#1", "P / A#1", or "APSA". In the experimental section, such 20-mer peptides (or multimers thereof, such as 40-mers exemplified in SEQ ID NOs: 5, 6 or 7) conjugated to ("immune adjuvants") and N-terminally blocked were used as illustrative examples. Surprisingly, several monoclonal antibodies (MAbs) with high avidity and high specificity for the PAS sequence motif have been obtained using the novel and inventive methods disclosed herein.

[0030] Thus, in a preferred embodiment of the invention, the method for producing said antigen-binding molecules, said antibodies and / or said antigen-binding fragments comprises immunization of a non-human animal with an antigen comprising one or more P / A peptides, wherein the P / A peptide is independently a peptide R N -(P / A)-R C and The P / A peptide is a peptide consisting of about 5 to about 100 amino acid residues, and at least 70% of the number of amino acid residues in (P / A) are independently selected from proline and alanine, and (P / A) contains at least one proline residue and at least one alanine residue; R N is a protecting group attached to the N-terminal amino group of (P / A), R Cis an amino acid residue that is linked via its amino group to the C-terminal carboxy group of (P / A) and that includes at least one carbon atom between its amino group and its carboxy group; Each P / A peptide is composed of the C-terminal amino acid residue R C It is conjugated to the immunoadjuvant via an amide bond formed between the carboxy group of and a free amino group of the immunoadjuvant.

[0031] Also, in the context of this preferred embodiment of the invention, the explanations for P / A peptides / antigens and / or (P / A) provided herein above apply here too. As discussed herein above, the inventors have surprisingly achieved success, by the means and methods provided herein, in the generation of (non-human) monoclonal antibodies (MAbs), especially directed against intrinsically unfolded proteins and / or domains or peptides of intrinsically unfolded proteins, in particular against PAS sequences, as used in the known PASylation® approach / technology. Such intrinsically unfolded proteins and / or domains or peptides of intrinsically unfolded proteins or PAS polypeptides as used in the PASylation® technology are known to lack immunogenicity (be "immunologically inactive") in mammals, in particular in rodents such as mice, rats or rabbits, i.e. animals routinely used for the preparation of (monoclonal) antibodies. This success is the result of the use of antigens for immunization consisting of and / or comprising the P / A peptides / antigens or (P / A) (or multimers thereof) defined herein, as illustrated herein and in the accompanying experimental part, in the examples and in the accompanying figures. As discussed herein, said P / A peptides / antigens can adopt a random coil conformation. Furthermore, an antigen can comprise two or more "P / A peptides" as defined herein. The P / A peptides comprised in said antigen can be multiple copies of the same P / A sequence as defined herein, such as non-limiting sequences independently selected from "ASPA", "APAP", "SAPA", "AAPA" and "APSA". Examples are provided herein and are also exemplified in sequences such as SEQ ID NO: 5, 6 or 7. Again, an antigen as used in the context of the present invention is an antigen conjugate of an immune adjuvant and one or more P / A peptides, where each P / A peptide is independently structured. R N -(P / A)-R C It is a peptide.

[0032] In the context of the present invention, R Nis a protecting group attached to the N-terminal amino group of the (P / A) amino acid sequence as defined herein. N are pyroglutamoyl (Pga; also known as 2-pyrrolidone-5-carboxylic acid or 5-oxoproline), 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 1~6 alkyl), -N, N(C 1~8 Alkyl)2, N,N,N-tri(C 1~6 alkyl), N,N-tetramethylene, and N,N-pentamethylene.

[0033] R N But N-(C 1~6 alkyl) group, N,N-di(C 1~6 alkyl) group, or N,N,N-tri(C 1~6 alkyl) group, 1, 2 or 3 C bonded to the nitrogen atom of the amino group of the (P / A) part to be protected 1~6 It is understood that an alkyl group will be present. In the case of two or three alkyl groups attached to the nitrogen atom, each alkyl group is independently C 1~6 It is an alkyl group and may therefore be the same or different. In the case of three alkyl groups, the nitrogen atom is an ammonium group.

[0034] In addition, R NIt will be understood that when is an N,N-tetramethylene or N,N-pentamethylene group, both ends of the tetramethylene or pentamethylene carbon chain are bonded to the nitrogen atom of the same protected amino group and thus, together with the nitrogen atom to which they are attached, form a saturated five- or six-membered ring (i.e., a pyrrolidine or piperidine ring).

[0035] As used herein, "R C " is an amino acid residue that is linked via its amino group to the C-terminal carboxy group of the (P / A) amino acid sequence defined herein, C " contains at least 1, at least 2, at least 3, at least 4, at least 5 or 6 carbon atoms between its amino group and its carboxy group. In a preferred embodiment, the "R C " is H2N-(C 1~12 In another preferred embodiment, "R C " is H2N-(CH2) 1~10 -COOH, HN-phenyl-COOH, and HN-cyclohexyl-COOH. Even more preferred is "R C is selected from the group consisting of HN-CH-COOH(Gly), HN-(CH)-COOH(β-Ala), HN-(CH)-COOH, HN-(CH)-COOH, HN-(CH)-COOH, HN-(CH)-COOH, HN-(CH)-COOH, HN-(CH)-COOH, p-aminobenzoic acid, and 4-aminocyclohexanecarboxylic acid. Most preferably, R C is H2N-(CH2)5-COOH (aminohexanoic acid).

[0036] In a preferred embodiment, and as illustrated in the accompanying examples, the P / A peptides contained in the antigens used in the means and methods of the present invention adopt a random coil conformation. Moreover, the P / A peptides contained in the antigens lack charged residues. As discussed herein, and also as disclosed in WO2008 / 155134, WO2011 / 144756 or WO2018 / 234455, among others, above, to a minor extent, further amino acids may be included. Moreover, these further amino acids preferably lack any charged residues and / or any obvious hydrophobic side chains. An exemplary non-limiting amino acid may be glycine.

[0037] An antigen as used in the context of the present invention and as provided herein is a conjugate of an immune adjuvant with one or more P / A peptides as defined herein. Such "immune adjuvants" are known in the art and described as highly immunogenic carrier proteins, which are preferably, but not exclusively, foreign (i.e., derived from a different species) to the subject to which they are administered. Preferably, and as illustrated in the examples, such immune adjuvants form protein complexes with a molecular mass of more than about 5 megadaltons (5 000 000 Da). A preferred example of such an immune adjuvant is KLH. Likewise, such highly immunogenic carrier proteins induce detectable antibody titers (i.e., antibodies that bind to them) directed against the "immune adjuvant" / highly immunogenic carrier protein itself. In the context of the present invention, the P / A peptide or (P / A) amino acid sequence as defined herein is conjugated to the ε-amino group of a lysine residue or to the free N-terminal amino group of said immune adjuvant. The immune adjuvant may be selected from the group consisting of, but not limited to, keyhole limpet hemocyanin (KLH), ovalbumin (OVA), and bovine serum albumin (BSA).Preferably, the immune adjuvant is keyhole limpet hemocyanin (KLH).

[0038] In accordance with the above, non-limiting examples of antigens of the present invention and for use in the context of the means and methods provided herein are: Pga-PAS#1(40)-Ahx: (Pga-ASPA-APAP-ASPA-APAP-SAPA-ASPA-APAP-ASPA-APAP-SAPA-Ahx; SEQ ID NO:5); Pga-P / A#1(40)-Ahx: (Pga-AAPA-APAP-AAPA-APAP-AAPA-AAPA-APAP-AAPA-APAP-AAPA-Ahx; SEQ ID NO: 6); Pga-APSA(40)-Ahx:(Pga-APSA-APSA-APSA-APSA-APSA-APSA-APSA-APSA-APSA-APSA-Ahx; SEQ ID NO: 7) It is.

[0039] Pga stands for pyroglutamyl residue (also known as 2-pyrrolidone-5-carboxylic acid or 5-oxoproline) and Ahx stands for aminohexanoic acid. These exemplary 40-mer "P / A" peptides are therefore designed to include at least two copies of the corresponding "PAS repeat sequence". The peptides contain only chemically inert side chains, have a blocked N-terminus, and their single C-terminal carboxylate group (indeed, one of the Ahx linker residues) is selectively activated and used for specific chemical conjugation to the ε-amino group of the Lys side chain of an immune adjuvant, i.e., KLH in the attached example.

[0040] Thus, the present invention provides in one embodiment a novel and inventive antigen, which may be used in particular and without exception in the method of the present invention for generating antigen-binding molecules (particularly antibodies) directed against intrinsically unnatural proteins and / or domains or peptides of intrinsically unnatural proteins in non-human animals, in particular in rodents such as mice and rats, but also in other mammals, including but not limited to horses, sheep, goats, camelids, etc. Thus, the present invention also relates to the antigens as defined and provided herein. The subject matter of the present invention is also the use of such antigen(s) in the method of the present invention. Thus, the present invention also relates to the non-therapeutic use of antigens as provided herein for generating antigen-binding molecules, preferably antibodies or antigen-binding fragments thereof, directed against intrinsically unnatural peptides / proteins and / or domains of intrinsically unnatural peptides / proteins, including immunization of non-human mammals.

[0041] The binding moieties, particularly antigen-binding molecules, most particularly antibodies or antigen-binding fragments thereof, as obtainable and obtainable by the present invention, are directed against intrinsically disordered proteins and / or domains or peptides of intrinsically disordered proteins.These binding moieties, antigen-binding molecules, antibodies or antigen-binding fragments thereof are also part of the present invention, and they preferably and specifically bind to structurally disordered and / or intrinsically disordered sequences, particularly Pro / Ala rich sequences (PAS), as also known in the art.Such Pro / Ala rich sequences (PAS) are defined herein and also described in WO2008 / 155134 and WO2011 / 144756. As discussed above, these "PAS" moieties also relate to peptides consisting of at least 7 amino acid residues that form a random coil conformation and up to about 2000 amino acid residues, as further described in (Schlapschy et al., 2013) or (Binder & Skerra, 2017), whereby said amino acid residues that form said random coil conformation are selected from Pro(P), Ala(A) and Ser(S), or Pro(P) and Ala(A).The "binding targets" of the antigen-binding molecules provided herein, most particularly antibodies or antigen-binding fragments thereof, are therefore in preferred embodiments intrinsically disordered proteins and / or domains of intrinsically disordered proteins, which are Pro / Ala-rich sequences (PAS) and / or form a random coil conformation, and / or have at least 10, at least 20, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 180, at least 190, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 800, at least 1000, at least 1200, at least 1400, at least 1600, at least 1800, at least 1900, at least 2500, at least 3500, at least 4000, at least 5000, at least 6000, at least 8000, at least 9000, at least 10000, at least 12000, at least 14000, at least 16000, at least 18000, at least 10 ... , at least 190, at least 200, or about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1500 or about 2000 amino acid residues, whereby the amino acid residues forming the random coil conformation are selected from Pro(P), Ala(A) and Ser(S), or are Pro(P) and Ala(A). Further definitions and explanations regarding Pro / Ala rich sequences (PAS) that form random coil conformations are provided, inter alia, in WO2008 / 155134 and WO2011 / 144756, both of which are incorporated herein by reference.

[0042] In a further embodiment of the invention, the binding moiety, particularly the antigen-binding molecule, most particularly an antibody or antigen-binding fragment thereof, can be bound to a Pro / Ala-rich sequence (PAS molecule; "PAS"), wherein the PAS can be an amino acid sequence consisting of about 7 to about 2000, preferably about 7 to about 1200, amino acid residues, wherein at least 80% of the number of amino acid residues in the "PAS" are independently selected from proline and alanine, and wherein the "PAS" comprises at least one proline residue and at least one alanine residue. The PAS can also be an amino acid sequence consisting of about 8 to about 400 amino acid residues, wherein at least 85% of the number of amino acid residues in the "PAS" are independently selected from proline and alanine, and wherein at least 95% of the number of amino acid residues in the "PAS" are independently selected from proline, alanine, glycine and serine, and wherein the "PAS" comprises at least one proline residue and at least one alanine residue. The binding moieties of the invention can also specifically bind to Pro / Ala-rich sequences (PAS molecules; "PAS"), where "PAS" is an amino acid sequence consisting of 10 to 60 amino acid residues independently selected from proline, alanine, glycine and serine, where at least 95% of the number of amino acid residues in the "PAS" are independently selected from proline and alanine, and where the "PAS" comprises at least one proline residue and at least one alanine residue. Corresponding "PAS molecules" are also described in WO2018 / 234455, which is also incorporated by reference.

[0043] Thus, in certain embodiments, the binding moieties, antigen-binding molecules or antibodies (or antigen-binding fragments thereof) of the invention specifically bind to Pro / Ala-rich sequences (PAS) and / or to amino acid sequences consisting of at least 20, preferably at least 40, preferably at least 60, preferably at least 80, more preferably at least 100, more preferably at least 120, more preferably at least 140, more preferably at least 160, more preferably at least 180, more preferably at least 200, more preferably at least 300 to about 1200 amino acid residues that form a random coil conformation, whereby the amino acid residues that form the random coil conformation are selected from Pro(P), Ala(A) and Ser(S), or are Pro(P) and Ala(A). Thus, the preferred target Pro / Ala-rich sequences (PAS molecules; "PAS") of the binding moieties of the invention comprise or consist of alanine, serine and proline, or comprise alanine and proline.

[0044] In one embodiment of the present invention, the binding moiety of the present invention, particularly the antigen-binding molecule, most particularly the antibody or antigen-binding fragment thereof, is capable of binding to and / or detecting at least one epitope on said PAS target sequence. The epitope may be a linear epitope, but also an epitope caused by a three-dimensional structure. The accompanying non-limiting examples provide sufficient evidence for corresponding binding studies, including epitope mapping, SPOT epitope analysis, antigen affinity measurements (e.g. by ELISA), surface plasmon resonance (SPR) real-time measurements, Western blotting, and also by co-crystallization of antigen-binding fragments (particularly Fab fragments). In one embodiment of the present invention, but not limited thereto, the antigen-binding molecule of the present invention, most particularly the antibody or antigen-binding fragment thereof, is capable of binding to and / or detecting at least one epitope on said PAS target sequence. (P / S)A(A / S)P; and / or PA(A / S)P It is capable of binding a Pro / Ala-rich sequence that contains at least one epitope of

[0045] The epitope may be or may comprise an epitope stretch selected from the group consisting of PAPAAP (SEQ ID NO: 8), PAPASP (SEQ ID NO: 9), PASPAAP (SEQ ID NO: 10), PSAAPS (SEQ ID NO: 79), ASPAAP (SEQ ID NO: 80), PASPAA (SEQ ID NO: 81), PAAP (SEQ ID NO: 82), PASP (SEQ ID NO: 83), APSA (SEQ ID NO: 84) and PSAA (SEQ ID NO: 85).

[0046] As illustrated herein in the accompanying Examples and below, the present invention provides several novel and inventive antibodies or antigen-binding fragments thereof. Again, without limitation and without being bound by theory, Epitope detection of “PAAP” is predicted for anti-PA(S) MAb 2.2, anti-PA(S) MAb 2.1, anti-PA(S) MAb 1.1, and anti-PA(S) MAb 1.2; Epitope detection of “PASP” is presumed for anti-PA(S) MAb 2.2, anti-PA(S) MAb 2.1, anti-PA(S) MAb 1.1, and anti-PA(S) MAb 1.2; Epitope detection of “PAPASP” is predicted for anti-PA(S) MAb 2.2 and anti-PA(S) MAb 2.1; Epitope detection of “PAPAAP” is predicted for anti-PA(S) MAb 2.2 and anti-PA(S) MAb 2.1; Epitope detection of “PASPAAP” is predicted for anti-PA(S) MAb 1.1 and anti-PA(S) MAb 1.2; Epitope detection of “PASPAA” is predicted for anti-PA(S) MAb 1.1; Epitope detection of “ASPAAP” is predicted for anti-PA(S) MAb 1.1 and anti-PA(S) MAb 1.2; Epitope detection of “APSA” is predicted for anti-PA(S) MAb 3.1 and anti-PA(S) MAb 3.2; Epitope detection of “PSAA” is predicted for anti-PA(S) MAb 3.1 and anti-PA(S) MAb 3.2; Epitope detection of "PSAAPS" is predicted for anti-PA(S) MAb 3.2.

[0047] Also, as per the attached non-limiting but highly exemplary (co)crystallization data of the antigen-binding molecules (Fab fragments) provided herein, further epitope studies in the Examples illustrate that the antigen-binding molecules of the present invention (i.e., antibodies / their antigen-binding fragments) bind to epitopes that contain alanine residues (A, Ala). Interestingly, at least one Ala residue in the Pro / Ala-rich sequence is involved in proper interaction with anti-PAS Fab; thus, without being limiting, alanine can be considered as a "hot spot" for the interaction of the antibodies of the present invention with the PAS epitope in the Pro / Ala-rich sequence. Until the present invention, Ala, the amino acid with the smallest side chain, has been ascribed a minor role in protein-protein / peptide recognition. In fact, the strategy of alanine scanning mutagenesis (Cunningham & Wells, 1989) has found wide application to probe residues critical for receptor-ligand binding or antibody-antigen binding, which resulted in the assumption of a semi-inactive role of the Ala methyl side chain for molecular interactions. Unexpectedly, the present invention reveals that Ala can indeed play a central role in antigen recognition, particularly as illustrated by the crystal structure studies provided in the context of the present invention.

[0048] In this regard, the present invention also provides complexes between the binding moieties, antigen-binding molecules, antibodies / antigen-binding fragments thereof of the present invention and Pro / Ala-rich sequence (PAS) molecules and epitopes as provided herein, in particular epitopes containing alanine. These epitopes can include structures such as (P / S)A(A / S)P and / or PA(A / S)P. Examples are provided herein.

[0049] In a further embodiment of the present invention, a complex between a specific binding moiety, particularly an antigen-binding molecule of the present invention (such as an antibody and an antigen-binding fragment thereof) and a Pro / Ala-rich sequence / (PAS) molecule, as obtainable by the means and methods provided herein, is provided and claimed herein. Also, a complex between a binding moiety or an antigen-binding molecule of the present invention (such as an antibody and an antigen-binding fragment thereof) and a fusion protein and / or a drug conjugate comprising a Pro / Ala-rich sequence ((PAS) molecule) is part of the present invention. Such an "anti-"PAS" complex" of the present invention is particularly useful, but not limited to, in methods of diagnosis, screening, and as a research tool as provided herein.

[0050] As discussed above and illustrated in the accompanying examples, the inventors provide for the first time binding moieties, antigen-binding molecules, antibodies / antigen-binding fragments thereof that specifically bind intrinsically disordered proteins and / or domains or peptides of intrinsically disordered proteins, in particular Pro / Ala-rich sequence (PAS) molecules and / or epitopes constituted by or formed by such Pro / Ala-rich sequence (PAS) molecules.

[0051] The present invention therefore comprises binding moieties, antigen-binding molecules, antibodies / antigen-binding fragments thereof as obtainable and / or obtained by the means, in particular the methods, provided herein. The present invention therefore also provides specific binding moieties, preferably antigen-binding molecules, more preferably antibodies, obtainable by the methods provided herein, and / or specific binding moieties, preferably antigen-binding molecules, more preferably antibodies, which specifically bind to intrinsically disordered proteins and / or domains of intrinsically disordered proteins or to antigenic parts of said intrinsically disordered proteins and / or domains of intrinsically disordered proteins, (i) wherein said intrinsically disordered protein and / or domain of an intrinsically disordered protein is a Pro / Ala rich sequence (PAS) and / or an amino acid sequence of at least 20 amino acid residues that form a random coil conformation, whereby said amino acid residues that form a random coil conformation are selected from Pro(P), Ala(A) and Ser(S) or are Pro(P) and Ala(A), and / or (ii) The specific binding moiety, preferably the antigen-binding molecule, binds to an epitope of the structure: (P / S)A(A / S)P and / or PA(A / S)P. Such an epitope may be selected from the group consisting of PAPAAP (SEQ ID NO: 8), PAPASP (SEQ ID NO: 9), PASPAAP (SEQ ID NO: 10), PSAAPS (SEQ ID NO: 79), ASPAAP (SEQ ID NO: 80), PASPAA (SEQ ID NO: 81), PAAP (SEQ ID NO: 82), PASP (SEQ ID NO: 83), APSA (SEQ ID NO: 84) and PSAA (SEQ ID NO: 85).

[0052] The binding moieties of the present invention can be antigen-binding molecules as well as antibodies (MAbs) or antigen-binding fragments thereof (e.g., Fabs). The antigen-binding molecules can be immunoglobulins (Igs), antibodies, antigen-binding fragments thereof, bispecific antibodies, IgG antibodies, camel / llama heavy chain antibodies (camelid antibodies), immunoglobulin novel antigen receptors (IgNARs) or antibody mimetics. The present invention also includes antibodies, antibody constructs thereof, antigen-binding fragments thereof, engineered via recombinant means based on the binding moieties, antigen-binding molecules of the present invention as well as antibodies or antigen-binding fragments thereof, and obtainable by the means and methods provided herein. For example, the corresponding sequence information of antigen-binding molecules can be used in the construction of such engineered / recombinant binding moieties / antigen-binding molecules. Such engineered / recombinant binding moieties / antigen-binding molecules can be based, inter alia, on the CDR sequences of antibodies obtained by the methods of the present invention or as exemplarily provided herein.

[0053] The present invention also provides an antigen-binding molecule / antibody that may be selected from the group consisting of monoclonal antibodies, chimeric antibodies, recombinant antibodies, and antigen-binding fragments of recombinant or chimeric antibodies. The antigen-binding fragments according to the invention may be fragments such as, but not limited to, Fab fragments, Fab' fragments, (Fab')2 fragments, single chain variable fragments (scFv), single domain antibodies, or VHH domains or nanobodies. The term "antibody" as used herein also includes humanized antibodies, or antibodies displayed on the surface of phages, yeast cells, bacterial cells, or mammalian cells. The antibodies of the present invention may be IgG1, IgG2, IgG2a or IgG2b, IgG3, or IgG4 antibodies.

[0054] The PAS binding moieties / antibodies of the invention (anti-PA(S) MAbs) show substantially no or very low cross-reactivity with proteins lacking structurally disordered PAS sequences, sequence stretches, (poly)peptide segments or domains of proteins. In particular, said anti-PA(S) MAbs show no or very low cross-reactivity with human plasma proteins and / or plasma proteins from primates, mammals, rodents, in particular monkeys, macaques, baboons, mice, rats, rabbits, dogs, pigs, cows, sheep. Furthermore, in another embodiment, the anti-PA(S) MAbs show no or very low cross-reactivity with host cell proteins from production organisms commonly used in the fields of recombinant protein production, genetic engineering or biotechnology, e.g., bacteria such as E. coli, Corynebacterium glutamicum or Pseudomonas fluorescens, or yeasts such as Saccharomyces cerevisiae or Pichia pastoris, or mammalian cells such as CHO, HEK, NS0 or COS cells.

[0055] The PAS binding moieties / antibodies (anti-PA(S) MAbs) according to the invention have high affinity / low dissociation constants (K D Such a K D Values ​​can be determined using many techniques well known in the art, for example using ELISA or SPR measurements as illustrated in the examples further disclosed herein below. Of note, such measurements can be performed on intact antibodies (MAbs) or on antigen-binding fragments thereof, for example Fab, Fv or scFv fragments, and the corresponding K DValues ​​can vary depending on the type of antibody protein (intact or fragment) and the exact assay used (ELISA, SPR, fluorescence titration, etc.). In general, preferred K D The values ​​are less than 500 μM, less than 200 μM, less than 100 μM, less than 50 μM, less than 10 μM, more preferably less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, even more preferably less than 1 nM, less than 500 pM, less than 200 pM or less than 100 pM. In the case of the use of the anti-PA(S) MAbs according to the invention in bioanalytical or diagnostic assays, particularly low K D Values ​​are preferred, for example less than 10 nM, less than 5 nM or less than 2 nM, even more preferably less than 1 nM, less than 500 pM, less than 200 pM or less than 100 pM.

[0056] Without being bound by theory, and as shown in the accompanying examples, the apparent affinity of the binding moieties / antibodies of the invention is influenced by avidity effects, which appear to be most evident for bivalent MAbs when interacting with long PAS repeat sequences containing multiple epitopes. X-ray structural analysis of recombinant Fab fragments of the antibodies of the invention complexed with their cognate PAS epitope peptides reveals that the interaction is dominated by a hydrogen bond network with the peptide backbone as well as multiple van der Waals interactions resulting from close shape complementarity. As discussed above, and most surprisingly, Ala, the amino acid with the smallest side chain (apart from Gly, which lacks a side chain), emerged as a crucial feature for antigen recognition of the binding moieties / antibodies of the invention. Said Ala provides a major contribution at the center of the paratope of the various "anti-PAS complexes".

[0057] The present invention also provides specific but non-limiting examples of binding moieties / antigen-binding molecules / antibodies of the invention and / or antigen-binding fragments of these antibodies of the invention. In this context, the term "antigen-binding molecule" also includes antigen-binding fragments, while said term also preferably includes antigen-binding fragments of antibodies of the invention, which are in particular directed against intrinsically unnatural peptides / proteins and / or domains of intrinsically unnatural peptides / proteins as provided herein and described herein and / or obtainable by the method of the present invention.

[0058] Thus, the present invention provides a) CDR-H1 [anti-PA(S) MAb 1.1] as defined in SEQ ID NO: 35; CDR-H2 [anti-PA(S) MAb 1.1] as defined in SEQ ID NO: 36; CDR-H3 [anti-PA(S) MAb 1.1] defined in SEQ ID NO: 37; and / or CDR-L1 [anti-PA(S) MAb 1.1] as defined in SEQ ID NO: 38; CDR-L2 [anti-PA(S) MAb 1.1] as defined in SEQ ID NO: 39; CDR-L3 [anti-PA(S) MAb 1.1] as defined in SEQ ID NO: 40 or an antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs of (a); b) CDR-H1 [anti-PA(S) MAb 1.2] as defined in SEQ ID NO: 41; CDR-H2 [anti-PA(S) MAb 1.2] as defined in SEQ ID NO: 42; CDR-H3 [anti-PA(S) MAb 1.2] defined in SEQ ID NO: 43; and / or CDR-L1 [anti-PA(S) MAb 1.2] as defined in SEQ ID NO: 44; CDR-L2 [anti-PA(S) MAb 1.2] as defined in SEQ ID NO: 45; CDR-L3 [anti-PA(S) MAb 1.2] as defined in SEQ ID NO: 46; or an antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs of (b); c) CDR-H1 [anti-PA(S) MAb 2.1] as defined in SEQ ID NO: 47; CDR-H2 [anti-PA(S) MAb 2.1] as defined in SEQ ID NO: 48; CDR-H3 [anti-PA(S) MAb 2.1] defined in SEQ ID NO: 49; and / or CDR-L1 [anti-PA(S) MAb 2.1] as defined in SEQ ID NO: 50; CDR-L2 [anti-PA(S) MAb 2.1] as defined in SEQ ID NO: 51; CDR-L3 [anti-PA(S) MAb 2.1] as defined in SEQ ID NO:52; or an antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs of (c); d) CDR-H1 [anti-PA(S) MAb 2.2] as defined in SEQ ID NO: 53; CDR-H2 [anti-PA(S) MAb 2.2] as defined in SEQ ID NO: 54; CDR-H3 [anti-PA(S) MAb 2.2] defined in SEQ ID NO: 55; and / or CDR-L1 [anti-PA(S) MAb 2.2] as defined in SEQ ID NO: 56; CDR-L2 [anti-PA(S) MAb 2.2] as defined in SEQ ID NO: 57; CDR-L3 [anti-PA(S) MAb 2.2] as defined in SEQ ID NO: 58; or an antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs of (d); e) CDR-H1 [anti-PA(S) MAb 3.1] as defined in SEQ ID NO: 59; CDR-H2 [anti-PA(S) MAb 3.1] as defined in SEQ ID NO: 60; a CDR-H3 comprising or consisting of the amino acids Trp-Gly-Arg; and / or CDR-L1 [anti-PA(S) MAb 3.1] as defined in SEQ ID NO: 62; CDR-L2 [anti-PA(S) MAb 3.1] as defined in SEQ ID NO: 63; CDR-L3 [anti-PA(S) MAb 3.1] as defined in SEQ ID NO: 64; or an antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs of (e); And f) CDR-H1 [anti-PA(S) MAb 3.2] as defined in SEQ ID NO: 65; CDR-H2 [anti-PA(S) MAb 3.2] as defined in SEQ ID NO: 66; CDR-H3 [anti-PA(S) MAb 3.2] defined in SEQ ID NO: 67; and / or CDR-L1 [anti-PA(S) MAb 3.2] as defined in SEQ ID NO: 68; CDR-L2 [anti-PA(S) MAb 3.2] as defined in SEQ ID NO: 69; CDR-L3 [anti-PA(S) MAb 3.2] as defined in SEQ ID NO: 70; or (f) an antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs of Also provided is an antigen-binding domain selected from the group consisting of:

[0059] The sequence "Trp-Gly-Arg" as constituting anti-PA(S)Mab3.1 is herein designated as SEQ ID NO: 61. However, in the attached sequence listing, this SEQ ID NO is represented as "000", since this sequence consists of only three amino acids, and it should be understood that BiSSAP does not allow the inclusion of sequences with only three amino acid residues. Also, the ST.25 standard dictates that only sequences with a length of four or more amino acid residues shall be included in the corresponding sequence listing.

[0060] In one embodiment, the present invention relates to an antigen-binding molecule that binds to the same epitope as any of the antibodies or antigen-binding fragments of the present invention or that is obtainable by the means and methods of the present invention. In one particular embodiment of the present invention, said antigen-binding molecule binds to the same epitope as any of the antibodies or antigen-binding fragments defined herein above by (a) to (f).

[0061] As discussed herein above, the present invention also includes antigen-binding molecules that are antigen-binding fragments of the antibodies of the present invention. These antigen-binding fragments may be selected from the group consisting of Fab fragments, F(ab')2 fragments, Fv fragments or scFv fragments. Such antigen-binding fragments are illustrated in the accompanying examples, including data from protein crystallography and data on epitope binding. Other means and methods for epitope elucidation as well as epitope binding are also fully provided in the accompanying experimental section. Corresponding techniques include immunological assays such as ELISA and Western blot, as well as SPOT assays for epitope mapping, and also more complex techniques such as X-ray structure analysis of the complex between recombinant Fab fragments and PAS epitope peptides. However, the skilled artisan is in a position to easily deduce the epitope binding of a given antigen-binding molecule, including antibodies and / or antigen-binding fragments thereof.

[0062] In the context of the present invention, the term "binding to the same epitope" is not limited to linear epitopes, the term can also include binding to the same three-dimensional conformation or to a "conformational" epitope.

[0063] In a further embodiment, the present invention relates to an antigen-binding molecule, in particular an antibody or an antigen-binding fragment thereof, wherein said antigen-binding molecule, antibody or antigen-binding fragment thereof comprises: (a) 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 with SEQ ID NO: 11, 13, 15, 17, 19 or 21. a variable heavy (VH) 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 with SEQ ID NO: 12, 14, 16, 18, 20 or 22. or, (b) An antibody that binds to the same epitope as the antibody (a).

[0064] In this regard, SEQ ID NO: 11, 13, 15, 17, 19 or 21 provide the heavy chain variable region / variable heavy (VH) chain sequences of exemplary antibodies, while SEQ ID NO: 12, 14, 16, 18, 20 or 22 provide the light chain variable region / light heavy (VL) chain sequences of exemplary antibodies. Of note, the CDR-H3 of the heavy chain SEQ ID NO: 19 [anti-PA(S) MAb 3.1] as shown in SEQ ID NO: 19 is relatively short and contains only three amino acids, namely the amino acids Trp-Gly-Arg (SEQ ID NO: 61; characterized by the "000" sequence as a placeholder in the attached sequence protocol for "anti-PA(S) MAb 3.1").

[0065] A particularly preferred antigen binding molecule or antibody of the present invention, i.e., the antibody designated herein as anti-PA(S) MAb 1.1, a) a variable heavy (VH) chain comprising CDR-H1 as defined in SEQ ID NO: 35, CDR-H2 as defined in SEQ ID NO: 36, and CDR-H3 as defined in SEQ ID NO: 37, and a variable light (VL) chain sequence comprising CDR-L1 as defined in SEQ ID NO: 38, CDR-L2 as defined in SEQ ID NO: 39, and CDR-L3 as defined in SEQ ID NO: 40, or b) an antibody that binds to the same epitope as the antibody of (a); It is an antigen-binding molecule or an antibody.

[0066] A further preferred antigen binding molecule or antibody of the present invention, i.e., the antibody designated herein as anti-PA(S) MAb 1.2, comprises: a) comprising a variable heavy (VH) chain comprising CDR-H1 as defined in SEQ ID NO: 41, CDR-H2 as defined in SEQ ID NO: 42 and CDR-H3 as defined in SEQ ID NO: 43, and a variable light (VL) chain sequence comprising CDR-L1 as defined in SEQ ID NO: 44, CDR-L2 as defined in SEQ ID NO: 45 and CDR-L3 as defined in SEQ ID NO: 46, or b) an antibody that binds to the same epitope as the antibody of (a); It is an antigen-binding molecule or an antibody.

[0067] A further preferred antigen binding molecule or antibody of the present invention, i.e., the antibody designated herein as anti-PA(S) MAb 2.1, comprises: a) comprising a variable heavy (VH) chain comprising CDR-H1 as defined in SEQ ID NO: 47, CDR-H2 as defined in SEQ ID NO: 48 and CDR-H3 as defined in SEQ ID NO: 49, and a variable light (VL) chain sequence comprising CDR-L1 as defined in SEQ ID NO: 50, CDR-L2 as defined in SEQ ID NO: 51 and CDR-L3 as defined in SEQ ID NO: 52; or b) an antibody that binds to the same epitope as the antibody of (a); It is an antigen-binding molecule or an antibody.

[0068] A further preferred antigen binding molecule or antibody of the present invention, i.e., the antibody designated herein as anti-PA(S) MAb 3.1, comprises: a) comprising a variable heavy (VH) chain comprising CDR-H1 as defined in SEQ ID NO: 59, CDR-H2 as defined in SEQ ID NO: 60 and CDR-H3 comprising or consisting of the amino acid sequence Trp-Gly-Arg, and a variable light (VL) chain sequence comprising CDR-L1 as defined in SEQ ID NO: 62, CDR-L2 as defined in SEQ ID NO: 63 and CDR-L3 as defined in SEQ ID NO: 64, or b) an antibody that binds to the same epitope as the antibody of (a); It is an antigen-binding molecule or an antibody.

[0069] The binding moieties / antibodies of the present invention provide valuable insight into how antibodies against antigens known to be "immunologically inert" (such as PAS sequences) can be obtained via immunization approaches as provided herein. Furthermore, the present invention also provides means and methods by which binding moieties / antibodies can be obtained that specifically bind to and / or recognize "uncharacterized peptides" that lack obvious hydrophobic or charged side chains and / or have no defined secondary structure and / or contain a random coil conformation or configuration. The binding moieties / antibodies as provided in the context of the present invention and characterized herein also provide valuable tools for the preclinical and clinical development of drug conjugates, such as PASylated biologics or PASylated (small molecule) drugs - "PASylated" meaning conjugated to a PAS molecule / sequence (poly)peptide.

[0070] Exemplary PASylated proteins or peptides include, but are not limited to, adenosine deaminase, agalsidase alpha, alpha-human atrial natriuretic peptide, amylin or analogs, anti-HIV fusion inhibitors (such as enfluvitide), asparaginase (such as calaspargase), B-domain deleted factor VIII (such as veroctocog alpha or octofactor), bactericins including endolysins and ectolysins, bicyclic peptides (such as TG-758), bradykinin antagonists (such as icatiban), Other agents that may be used include: brain natriuretic peptide (BNP or B-type natriuretic peptide), calcitonin, CD19 antagonists, CD20 antagonists (such as Rituxan), CD3 receptor antagonists, CD40 antagonists, CD40L antagonists (such as dapirorizumab or antova), cerebroside sulfatase, chorionic gonadotropin, coagulation factor IV, coagulation factor IX, coagulation factor VIIa (such as eptacog alfa), coagulation factor VIII (such as susoctocog alfa), coagulation factor Xa, coagulation factor XIII (catride cacog), complement component 5a antagonists, complement factor C3 inhibitors, C-peptide, crisantaspase, CTLA-4 antagonists, C-type natriuretic peptide, deoxyribonuclease I (such as dornase alfa), EGFR receptor antagonists, erythropoietin (such as erythropoietin alpha or erythropoietin zeta), exendin-4, exendin-4 analogs (such as exendin 9-39), FcγIIB receptor antagonists, fibroblast growth factor 1 (human acidic fibroblast growth factor), fibroblast growth factor 18, fibroblast growth factor 2 (human basic fibroblast growth factor), fibroblast growth factor 21, fibroblast growth factor receptor 2 antagonists (such as FPA144), follicle-stimulating hormones (such as follitropin alfa or follitropin beta), gastric inhibitory polypeptide (GIP), GIP analogs, GLP-1, GLP-1 analogs (such as lixisenatide, liraglutide or semiglutide), GLP-2, GLP-2 analogs (such as teduglutide), glucagon or analogs, glucocerebrosidase (such as imiglucerase), gonadorelin,Gonadotropin-releasing hormone agonists (such as goserelin, buserelin, triptorelin, leuprolide, protirelin, resiliencelin, fertirelin or desiorelin), gonadotropin-releasing hormone antagonists (such as abarelix, cetrorelix, degarelix, ganirelix or teverelix), gp120, gp160, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), ghrelin, growth hormone (such as human, feline, bovine or porcine growth hormone), hematoxylin (HE), erythropoietin (EGF ... , hepatocyte growth factor, hepcidin antagonist, hsp70 antagonist, human chorionic gonadotropin (such as chorionic gonadotropin alpha), human parathyroid hormone, hyalosidase or bob hyaluronidase, hyaluronidase (such as human hyaluronidase PH-20), glucocerebrosidase, iduronate-2-sulfatase, insulin, insulin analogs, insulin-like growth factor 1, insulin-like growth factor 2, integrin α4β1 antagonist, interferon tau, interferon-alpha, Interferon-alpha antagonists, interferon-alpha superagonists, interferon-alpha-n3 (such as Alferon N injection), interferon-beta, interferon-gamma, interferon-lambda, interleukins, interleukin-2 fusion proteins (such as DAB(389)IL-2), interleukin receptor antagonists (such as interleukin-1 receptor antagonist, EBI-005 or anakinra), interleukin-11 (such as oplerevukin), Interleukin-12, interleukin-17 receptor antagonist, interleukin-18 binding protein, interleukin-2, interleukin-22, interleukin-22 receptor subunit alpha (IL-22ra) antagonist, interleukin-38 (IL-38), interleukin-4, interleukin-6 receptor antagonist, interleukin-7, kynureninase, L-arginine degrading enzymes (such as arginase or arginine deiminase), leptin, L-iduronidase,L-phenylalanine degrading enzymes (such as phenylalanine hydroxylase or phenylalanine ammonia-lyase), N-acetylgalactosamine-6-sulfatase (such as erosulfase alpha), nanophytin, neutrophil gelatinase-associated lipocalin, anticalin, octreotide, Ornithodoros moubata ( moubata) Complement inhibitors (OmCI / Covasin), parathormone (PTH), PD1 antagonists, PD1L antagonists, PDGF antagonists, (PYY3-36), phenylalanine ammonia lyase (variase, etc.), phylomers, platelet-derived growth factor, relaxin, relaxin analogs, secretin, RGD peptides, serine protease inhibitors (conestat alpha, etc.), soluble CD64, soluble DCC (deleted in colorectal cancer) receptor, soluble Fc receptors (CD16, CD32, CD64, etc.), soluble tumor necrosis factor I receptor (sTNF-RI), soluble tumor necrosis factor II receptor (sTNF-RII), soluble VEGF receptor, somatostatin, somatostatin stimulant analogs (such as pasireotide or CAP-232), stresscopins, T cell receptor ligands, teriparatide (PTH1-34), thymosin alpha 1, thymosin beta 4, thymosin beta 15, tumor necrosis factor (TNF alpha), tumor necrosis factor alpha antagonists, uricase (such as rasburicase or pegadolicase), urocortin, vasoactive intestinal peptide, vasopressin, vasopressin analogs (such as desmopressin, felypressin or teripressin), VEGF antagonists (such as rambitumab or bevacizumab), VEGF antagonists, adnectins, PDGF antagonists, DARPins, von Willebrand factor (such as vonicog alpha).

[0071] Exemplary PAS-modified small molecule drugs include, but are not limited to, amanitin, auristatin, calicheamicin, camptothecin, digoxigenin, fluorescein, doxorubicin, fumagillin, dexamethasone, geldanamycin, paclitaxel, docetaxel, irinotecan, cyclosporine, buprenorphine, naltrexone, naloxone, vindesine, vancomycin, risperidone, aripiprazole, palonosetron, granisetron, cytarabine, nucleic acids (such as antisense nucleic acids), small interfering RNA (siRNA), microRNA (miR) inhibitors, and microRNA inhibitors. RNA mimics, DNA aptamers, RNA aptamers, LNA (locked nucleic acids), RNA vaccines, DNA vaccines, carbohydrates suitable for the preparation of vaccines, such as tumor-associated carbohydrate antigens (TACA, α-GalNAc-O-Ser / Thr), sialyl-Tn antigens (e.g. NeuAcα(2,6)-GalNAcα-O-Ser) / Thr), Thomsen-Friedenreich antigens (Galβ1-3GalNAcα1), Lewis Y (e.g. Fucα(1,2)-Galβ(1,4)-[Fucα(1,3)]-GalNAc), sialyl-Lewis X or sialyl-Lewis A.

[0072] In certain contexts, it may be useful for example for research purposes, in screening methods, including as a diagnostic tool, patient stratification, etc., that the antigen-binding molecules of the present invention, in particular antibodies or antigen-binding fragments thereof, comprise a tag and / or label. Thus, the present invention also relates to antigen-binding molecules / antibodies / antigen-binding fragments thereof as obtainable by the means and methods of the present invention and / or as provided herein, wherein said antigen-binding molecules / antibodies / antigen-binding fragments thereof are conjugated or fused to a reporter molecule(s), tag(s), and / or label(s). Such reporter molecules, tags and / or labels are very well known in the art and include, inter alia, small molecule fluorescent dyes (including N-hydroxysuccinimide esters, isothiocyanates, iodoacetates or maleimides) applied, for example, in a chemically activated manner, such as xanthene derivatives, e.g. fluorescein, rhodamine, Alexa dyes such as Alexa488, cyanine derivatives such as Cy3 or Cy5, organoboron compounds such as 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), small molecules (haptens) such as biotin or digoxigenin, fluorescent proteins such as green fluorescent protein or a derivative thereof, red fluorescent protein or a derivative thereof or allophycocyanin, enzymes such as alkaline phosphatase, horseradish peroxidase, or enzymes catalyzing visible light emission (bioluminescence), such as luciferase.

[0073] The present invention also provides a polynucleotide encoding at least one of the variable heavy (VH) and / or variable light (VL) chain sequences of the antigen-binding molecule of the present invention, in particular an antibody or antigen-binding fragment thereof. In a preferred embodiment of the present invention, the polynucleotide encodes at least one of the following: a variable heavy (VH) and / or variable light (VL) chain sequence of an antigen-binding molecule, in particular an antibody or antigen-binding fragment thereof, capable of specifically binding to a Pro / Ala-rich sequence (PAS) and / or an amino acid sequence consisting of at least 4 or at least 10 or at least 20 amino acid residues forming a random coil conformation, whereby the amino acid residues forming the random coil conformation are selected from Pro(P), Ala(A) and Ser(S), or are Pro(P) and Ala(A), or capable of specifically binding to an antigenic portion thereof. The polynucleotide of the present invention has the structure: (P / S)A(A / S)P; and / or PA(A / S)P The antigen-binding molecule (or fragment thereof) can be encoded that is capable of binding to an epitope of the polypeptide.

[0074] The polynucleotide of the present invention preferably encodes at least one of the variable heavy (VH) chain sequences and / or at least one of the variable light (VL) chain sequences of an antigen-binding molecule, particularly an antibody or antigen-binding fragment, as provided herein. Preferably, said antigen-binding molecule, particularly an antibody or antigen-binding fragment, binds an epitope on an intrinsically disordered protein and / or on a domain or peptide of an intrinsically disordered protein. Preferably, said intrinsically disordered protein and / or domain or peptide of an intrinsically disordered protein comprises or consists of a Pro / Ala-rich sequence (PAS). The epitope may comprise an epitope / epitope stretch as disclosed herein and may be selected from the group consisting of PAPAAP (SEQ ID NO: 8), PAPASP (SEQ ID NO: 9), PASPAAP (SEQ ID NO: 10), PSAAPS (SEQ ID NO: 79), ASPAAP (SEQ ID NO: 80), PASPAA (SEQ ID NO: 81), PAAP (SEQ ID NO: 82), PASP (SEQ ID NO: 83), APSA (SEQ ID NO: 84) and PSAA (SEQ ID NO: 85).

[0075] Corresponding polynucleotide / nucleic acid molecules, including DNA or RNA, can be easily obtained through routine sequencing methods known to those skilled in the art and as illustrated in the accompanying examples. As a source for such sequencing techniques, B cells from non-human animals immunized according to the methods of the present invention can be used. Such cells include "hybridoma cells" that can be produced without exception, as illustrated in manuals on the production of monoclonal antibodies, such as (Harlow & Lane, 1988). Examples of such polynucleotide / nucleic acid molecules of the present invention, including DNA or RNA, are the above-mentioned polynucleotide / nucleic acid molecules, including the DNA contained in the deposited clones DSM ACC3365, DSM ACC3366 or DSM ACC3367. These deposited clones are hybridomas that contain polynucleotides that can code for exemplary monoclonal antibodies (anti-PA(S) MAbs) of the present invention, anti-PA(S) Mab 1.1, anti-PA(S) Mab 2.1 and anti-PA(S) Mab 3.1, respectively.

[0076] As is evident from the enclosed deposit receipt, these three hybridomas were deposited on November 13, 2020 (2020-11-13) at "DSMZ" (Leibnitz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) in accordance with the provisions of the Budapest Treaty and received the following accession numbers from the International Depository Authority: DSM ACC3365 (anti-PA(S)Mab 1.1); DSM ACC3366 (anti-PA(S)Mab 2.1) and DSM ACC3367 (anti-PA(S)Mab 3.1).

[0077] The present invention also relates to these deposits and thus to the hybridomas DSM ACC3365, DSM ACC3366 and DSM ACC3367. The present invention also relates to a host cell comprising a polynucleotide of the present invention, i.e., a polynucleotide encoding at least one of the variable heavy (VH) chain sequence and / or variable light (VL) chain sequence of an antigen-binding molecule, particularly an antibody or antigen-binding fragment, of the present invention. The (host) cell of the present invention may also be a cell expressing a polynucleotide contained in a hybridoma as provided herein, such as DSM ACC3365, DSM ACC3366 or DSM ACC3367. The hybridoma may also be a host cell of the present invention.

[0078] Also provided herein is a method for producing an antigen-binding molecule of the present invention, in particular an antibody or an antigen-binding fragment, comprising culturing a hybridoma of the present invention and / or culturing a host cell of the present invention, such as a bacterial cell or a mammalian cell. The production of the antigen-binding molecule of the present invention may comprise a routine culturing step of a host cell and / or a hybridoma of the present invention. Further hybridomas producing an antigen-binding molecule of the present invention, in particular an antibody or an antigen-binding fragment, can be obtained without exception by the means and methods provided herein for producing binding moieties, in particular antigen-binding molecules, directed against and / or specifically binding to an intrinsically disordered protein and / or a domain or peptide of an intrinsically disordered protein as defined herein. The method for producing an antigen-binding molecule of the present invention may also include isolation or purification of the antigen-binding molecule from the culture system, for example from the culture broth of a host cell / hybridoma.

[0079] In one embodiment, the present invention also provides a method for producing an antibody that specifically binds to a Pro / Ala rich sequence (PAS) as defined herein or to an antigenic portion thereof, said method comprising administering to a non-human mammal the Pro / Ala rich sequence (PAS), and / or administering an amino acid sequence of at least 20, preferably 40, amino acid residues which form a random coil conformation, whereby said amino acid residues which form a random coil conformation are selected from Pro(P), Ala(A) and Ser(S), or are Pro(P) and Ala(A), or to an antigenic portion thereof; (i) the Pro / Ala-rich sequence (PAS) and / or the amino acid sequence consisting of at least 20, preferably 40, amino acid residues forming a random coil conformation, structure: (P / S)A(A / S)P and / or PA(A / S)P and comprising at least one epitope / epitope stretch of (ii) the Pro / Ala-rich sequence (PAS) and / or the amino acid sequence of at least 20, preferably 40, amino acid residues that form a random coil conformation comprises a protecting group attached to the N-terminus; (iii) An immune adjuvant is linked to the C-terminus of said Pro / Ala-rich sequence (PAS) and / or said amino acid sequence consisting of at least 20, preferably 40, amino acid residues that form a random coil conformation.

[0080] In one embodiment, in said method for producing an antibody that specifically binds to a Pro / Ala rich sequence (PAS) as described above, said epitope in (i) comprises an epitope / epitope stretch selected from the group consisting of PAPAAP (SEQ ID NO: 8), PAPASP (SEQ ID NO: 9), PASPAAP (SEQ ID NO: 10), PSAAPS (SEQ ID NO: 79), ASPAAP (SEQ ID NO: 80), PASPAA (SEQ ID NO: 81), PAAP (SEQ ID NO: 82), PASP (SEQ ID NO: 83), APSA (SEQ ID NO: 84) and PSAA (SEQ ID NO: 85). In a further embodiment, the epitope / epitope stretch is N -(P / A)-R C The peptide is included in the peptide defined herein above as

[0081] The present invention also relates to compositions comprising binding moieties, in particular antigen-binding molecules, as produced by the means and methods of the present invention, capable of specifically binding domains or peptides of intrinsically disordered proteins. The claimed compositions can also comprise binding moieties, in particular antigen-binding molecules, as obtainable by the methods of the present invention, as well as binding moieties to binding moieties, in particular antigen-binding molecules, produced by the methods provided herein above.

[0082] The present invention also includes compositions comprising a specific antigen as defined herein, which is a conjugate of an immune adjuvant and one or more P / A peptides as defined above, wherein each of said P / A peptides independently has the structure R N -(P / A)-R C The peptide may be:

[0083] The binding moieties of the present invention, in particular the antigen-binding molecules / antibodies or antigen-binding fragments thereof, are particularly useful as research tools and as bioanalytical tools. They may also be used for in vitro screening of patient samples, such as blood samples obtained from individuals treated with PASed drugs and / or proteins. On the other hand, samples obtained from individuals who have never received PASed drugs and / or proteins may also be tested in vitro with the binding moieties of the present invention, in particular the antigen-binding molecules / antibodies or antigen-binding fragments thereof. This may be considered as a "negative control" and may be useful to evaluate or avoid false positive reactions of the antibodies of the present invention. Thus, the compositions of the present invention, in particular those comprising antigen-binding molecules / antibodies or antigen-binding fragments thereof, may be useful in screening (patients) and / or for tracking the time course of (concomitant) treatment of said patients / individuals with PASed (small molecule) drugs and / or protein / peptide drugs. Thus, the present invention also relates to diagnostic compositions.

[0084] In accordance with the above, the present invention also relates to a method for detecting a marker in a biological sample, (i) Pro / Ala-rich sequence (PAS), (ii) a conjugate of a protein, peptide or small molecule drug with a Pro / Ala rich sequence (PAS); and / or (iil) a conjugate of a protein, peptide or small molecule drug with an amino acid sequence consisting of at least 20 amino acid residues that form a random coil conformation, whereby the amino acid residues that form the random coil conformation are selected from Pro(P), Ala(A) and Ser(S), or are Pro(P) and Ala(A). A method for detecting the

[0085] The claimed method may therefore be an in vitro method using a biological sample obtained from an individual, in particular from a mammal, preferably from a human, who has been or is to be treated with a PASylated drug and / or protein / peptide.

[0086] The in vitro method may include contacting the biological sample with the antigen-binding molecule and / or antibody of the present invention under conditions permissive for the binding of the antigen-binding molecule and / or antibody to the Pro / Ala-rich sequence (PAS) (i) or (ii) and / or to the amino acid residues forming the random coil conformation (iii). The method may also include, as a further step, detection of whether a complex is formed between the antigen-binding molecule and / or the antibody and the Pro / Ala-rich sequence (PAS) and / or the amino acid residues forming the random coil conformation. The (positive) detection of the Pro / Ala-rich sequence (PAS) and / or the amino acid residues forming the random coil conformation in the biological sample may indicate, for example, whether a drug / protein containing a Pro / Ala-rich sequence (PAS), i.e., a "PASylated (small molecule) drug and / or a protein drug or peptide drug", is still present in the body of the individual. This is considered to be a qualitative assay. However, the time course and / or quantification of drugs / proteins containing Pro / Ala rich sequences (PAS) in such biological samples is also contemplated. Such assays also include "screening assays" of individual biological samples.

[0087] The in vitro detection of complexes formed between the binding moieties of the present invention, in particular antigen-binding molecules / antibodies or antigen-binding fragments thereof, and said conjugates of said Pro / Ala rich sequences (PAS) and / or protein / peptide drugs / small drugs containing such Pro / Ala rich sequences (PAS) is a routine task for the skilled person. Such detection of the formed complexes may include known techniques such as immunohistochemistry, immunofluorescence imaging, enzyme-linked immunosorbent assay (ELISA), Western blotting, electrochemiluminescence (ECL) immunoassays (ECLIA), surface plasmon resonance (SPR, Biacore), lateral flow immunoassays, paper-based immunoassays, acoustic wave-based immunoassays, interferometry-based immunoassays, nano- and micromaterial-based immunoassays, microcantilever-based sensors, quartz crystal microbalance-based sensors, electrochemical immunosensors, Lab-on-a-Chip (LOC) immunoassays, smartphone-based immunoassays, mass spectrometry-based immunoassays (MSIA, Immuno-MALDI, Immuno-MRM, SISCAPA) or immunoprecipitation. X-ray examination and imaging are also envisaged, for example after corresponding labelling of the binding moieties of the invention with radioactive substances, as is well known in the art.

[0088] It is also envisioned that the binding moieties, particularly antigen-binding molecules / antibodies or antigen-binding fragments thereof, may be used in vivo on individuals, e.g., in research settings, whereby non-human animals may be tested and screened with these compounds and compositions of the invention.

[0089] In accordance with the above, the present invention also relates to a method for monitoring the response of a subject or animal to treatment with a PASed drug conjugate, said method comprising: (a) a protein or peptide or small molecule drug conjugate with a Pro / Ala rich sequence (PAS); and / or (b) a conjugate of a protein drug or peptide or small molecule with an amino acid sequence consisting of at least 20 amino acid residues that form a random coil conformation, whereby the amino acid residues that form the random coil conformation are selected from Pro(P), Ala(A) and Ser(S), or are Pro(P) and Ala(A). The present invention also includes the use of an antigen-binding molecule and / or antibody or composition of the present invention for and / or in measuring levels of circulating Pro / Ala-rich sequence (PAS) molecules and / or fusion proteins and / or drug conjugates comprising Pro / Ala-rich sequence (PAS) molecules in blood samples, preferably plasma or serum samples, at one or more time points before and at one or more time points after treatment of a subject / patient or non-human test individual with PAS.

[0090] The method may also involve the detection of time courses and / or time-dosage relationships, particularly when samples taken at various time points following said treatment of said subject / patient or said non-human test individual with any of the conjugates defined in (a) or (b) above are screened.

[0091] Furthermore, detection of the complex formed between the binding moiety of the present invention, in particular the antigen-binding molecule / antibody or antigen-binding fragment thereof, and said Pro / Ala-rich sequence (PAS) and / or said conjugate of a protein drug / small drug comprising such a Pro / Ala-rich sequence (PAS) is routine, and the embodiments provided herein above also apply, mutatis mutandis, to this "monitoring method".

[0092] The invention is further illustrated by the following non-limiting figures and examples. [Brief description of the drawings]

[0093] [Figure 1]FIG. 1 shows an amino acid sequence alignment of the VH(A) and VL(B) domains of the anti-PAS antibodies of the invention (prior to the introduction of flanking restriction sites for subcloning). The CDRs are labeled with black outlines. Identical amino acid positions with respect to the VH and VL sequences of anti-PA(S) MAb 1.1 shown above each alignment are depicted as "·" and gaps in the amino acid sequence alignment are indicated as "-". [Diagram 2] Figure 1 shows exemplary Western blot analyses demonstrating that various anti-PAS antibodies of the invention specifically bind to the corresponding PASylated fusion proteins. Western blots incubated with cell culture supernatants from (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 hybridoma clones, and (H) anti-mouse IgG Fc-specific alkaline phosphatase (produced in goat, Sigma-Aldrich) secondary antibody as a control. The following samples were applied to SDS-PAGE for Western blotting: M - PageRuler Prestained Protein Ladder, 10-180 kDa (Thermo Fisher Scientific); 1 - PAS#1(200)-IL1Ra (SEQ ID NO: 72); 2 - P / A#1(200)-IL1Ra (SEQ ID NO: 73); 3 - APSA(200)-IL1Ra (SEQ ID NO: 74); 4 - Pooled human serum (SEQENS IVD / H2B), diluted 1:200 in ddH2O and spiked with 1 μg IL1Ra (Kineret / Anakinra, SOBI); 5 - E. coli BL21 whole cell protein, dissolved in SDS sample buffer. [Diagram 3]Figure 1 shows exemplary results of ELISA experiments for detecting PAS sequences or PASylated fusion proteins using anti-PAS antibodies of the invention: (A) ELISA with Fab fragments of anti-PA(S) MAb 2.1 and IL1Ra-PAS#1(800)(800), PAS#1(600)-leptin and P / A#1(600)-GMCSF as test substances and BSA as control. (B) ELISA with Fab fragments of anti-PA(S) MAb 2.2 and P / A#1(600) as test substance. (C) ELISA with Fab fragments of anti-PA(S) MAb 1.2 and IL1Ra-PAS#1(800), PAS#1(600)-leptin and P / A#1(600)-GMCSF as test substances and BSA as control. (D) ELISA with hybridoma supernatants of anti-PA(S) MAb 1.2 and hu4D5-PAS#1(200) as the test substance, captured using anti-mouse IgG Fc specific from goat preadsorbed to microtiter plates. [Figure 4] Figure 3 shows exemplary results of ELISA experiments for detecting PAS sequences or PASylated fusion proteins using anti-PAS antibodies of the invention: (A) ELISA using Fab fragments of anti-PA(S) MAb 3.1 and APSA(200)-IL1Ra as test substances; (B) ELISA using Fab fragments of anti-PA(S) MAb 3.2 and APSA(200)-IL1Ra as test substances; (C) ELISA using Fab fragments of anti-PA(S) MAb 1.1 and PAS#1(600)-leptin as test substances; (D) MAb capture ELISA using hybridoma supernatants of anti-PA(S) MAb 2.1 and hu4D5-P / A#1(200) as test substances (see Figure 3D). [Diagram 5]Figure 1 shows the results of SPOT assay of hybridoma culture supernatants of anti-PA(S) MAb 1.1 and anti-PA(S) MAb 1.2. Consecutive 12-mer peptides from PAS#1 and P / A#1 sequences, shifted by one residue in SEQ ID NO: 5 and 6, respectively, were synthesized with C-terminus anchoring on hydrophilic membranes. After chromogenic development of the membranes, spot intensities were scanned and quantified using the software CLIQS ver.1.2.044 (TotalLab), and are displayed as bar graphs. Epitope sequences are highlighted in bold type. [Figure 6] FIG. 5 shows the results of a SPOT assay of hybridoma culture supernatants of anti-PA(S) MAb 2.1 Fab fragment and anti-PA(S) MAb 2.2. For explanation, see FIG. [Figure 7] Figure 1: SPOT assay results of hybridoma culture supernatants of anti-PA(S) MAb 3.1 and anti-PA(S) MAb 3.2. A 10-mer peptide containing the sequence AAPSAAPSAA was synthesized with C-terminus anchoring on a hydrophilic membrane, whereby positions 3-8 were successively replaced by all 20 proteinogenic amino acids. After chromogenic development of the membrane, the spot intensities were scanned and quantified using the software CLIQS ver.1.2.044 (TotalLab), which is displayed as a bar graph. The bars corresponding to the residues of the original sequence AAPSAAPSAAA are filled. [Figure 8] Exemplary SPR sensorgrams of anti-PA(S) MAb 3.1 (APSA(200)-IL1Ra as analyte) and 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 X100 instrument). Hybridoma supernatants were applied to a CM3 sensor chip (GE Healthcare) coated with anti-mouse antibody (Mouse Antibody Capture Kit; GE Healthcare). Injection phases are labeled with black bars along with the corresponding concentrations of injected analytes. [Figure 9] Diagram of the principle of affinity purification of PASylated proteins using a column with immobilized anti-PAS Fab 1.2. (A) Schematic of one-step purification of PASylated proteins: (i) application of a 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 1 M of L-prolinamide in running buffer. (B) Crystal structure of Fab 1.2 (schematic diagram) in complex with the PAS#1 epitope peptide of Fab 1.2 (shown as a stick model at the top) (PDB ID: 7O31). (C) Relevant portion of the PAS#1 peptide epitope recognized by Fab 1.2. (D) Chemical structure of L-prolinamide. [Figure 10]Illustrative chromatograms of affinity purification of PASylated proteins using immobilized anti-PAS antibodies of the present invention. The Fab fragment of anti-PA(S) MAb 1.2 was covalently immobilized on a 1 ml HiTrap HP column (GE Healthcare) to serve as an affinity matrix. StrepII-eGFP-PAS#1(200) fusion protein (SEQ ID NO: 71) was applied as a test protein for purification from (A, C) pre-purified protein solutions and (B, D) whole cell extracts of BL21 E. coli cells expressing StrepII-eGFP-PAS#1(200). Elution of the protein from the chromatography column was monitored by measuring the general absorbance at 280 nm and 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 eluted fractions. Samples: M - Pierce Unstained Protein MW Marker (Thermo Fisher Scientific); 0 - pure StrepII-eGFP-PAS#1(200); 1 - fraction 7-7.5 ml; 2 - fraction 7.5-8 ml; 3 - fraction 8-8.5 ml; 4 - fraction 8.5-9 ml; 5 - fraction 9-9.5 ml; 6 - fraction 9.5-10 ml; 7 - fraction 10-10.5 ml. 1* - whole cell lysate of BL21 E. coli cells expressing StrepII-eGFP-PAS#1(200);2* - fraction 2-4 ml;3* - fraction 7-7.5 ml;4* - fraction 7.5-8 ml;5* - fraction 8-8.5 ml;6* - fraction 8.5-9 ml;7* - fraction 9-9.5 ml;8* - fraction 9.5-10 ml;9* - fraction 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 application of an L-prolinamide solution. [Figure 11] Exemplary chromatograms and SDS PAGE analyses documenting one-step PAS affinity purification of therapeutically relevant PASylated proteins using immobilized anti-PAS antibodies of the invention. Fab fragments of anti-PA(S) MAb 1.2 were covalently immobilized on a 1 ml HiTrap HP column (GE Healthcare) to serve as an affinity matrix. C-terminally PASylated anticalin H1GA-PAS#1(200)-His6 (SEQ ID NO: 90) (A, B) and N-terminally PASylated cytokine PAS#1(800)-IL1Ra (SEQ ID NO: 91) (C, D) were purified from either periplasmic or subcellular fractions of E. coli BL21, respectively. Elution of proteins from the chromatography column was monitored by measuring absorbance at 280 nm. After application of protein samples and washing with buffer, bound PAS fusion proteins were eluted with 1 M running buffer of L-prolinamide. SDS-PAGE analysis (B, D) revealed the presence of PAS-ylated proteins in the eluted fractions. Samples: Unstained Protein MW Marker (Thermo Fisher Scientific) - PE / CE (periplasmic / whole cell extracts 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)-His6 and PAS#1(800)-IL1Ra, respectively. This experiment demonstrates that N-terminally and C-terminally PAS-ylated proteins bearing PAS tags containing 200-800 residues can be purified with high selectivity. [Figure 12]Illustrative chromatograms of affinity purification of PASylated proteins using immobilized anti-PAS antibodies of the invention. Fab fragments of anti-PA(S) MAb 1.2 were covalently immobilized on a 1 ml HiTrap HP column (GE Healthcare) to serve as an affinity matrix. Prepurified StrepII-eGFP-PAS#1(200) fusion protein (SEQ ID NO: 71) was applied as a test protein. Elution of the protein from the chromatographic 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 (1M 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 application of L-prolinamide solution. [Figure 13] Crystal structures of synthetic PAS epitope peptides complexed 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) Pga-(APSA)3 peptide bound to anti-PA(S) MAb 1.2 and (D) anti-PA(S) MAb 3.1. The epitope peptides are shown as sticks (dark grey) and the Fab heavy (medium grey) and light (light grey) chains are shown as wires. [Figure 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, as illustrated in FIG. 15. (B) PASylated thymosin alpha 1 was subcutaneously injected into female Wistar rats (N=5) at a dose of 3.4 mg / kg body weight. The concentration of the fusion protein in plasma was quantified by sandwich ELISA using anti-PA(S) MAb 2.1 as capture antibody and alkaline phosphatase-conjugated anti-PA(S) MAb 1.2 as detection reagent. Data were plotted against sampling time after injection and fitted using a 1-compartment model. The PK profile shows distinct absorption and elimination phases of the PASylated peptide drug (see Table 1 for PK parameters). [Figure 15] Illustrative ELISA setups for the detection of PASylated molecules. (A) Sandwich ELISA using an anti-PAS antibody adsorbed to a microtiter plate to capture PASylated molecules and a second anti-PAS antibody conjugated to a reporter enzyme as a detection reagent. (B) Sandwich ELISA using an anti-PAS antibody adsorbed to a microtiter plate to capture PASylated molecules and a second enzyme-coupled antibody directed against the biologically active portion of the PASylated drug. (C) ELISA using a binding partner (e.g., receptor, referred to here as target) of a protein, peptide or small molecule drug adsorbed to a microtiter plate to capture PASylated molecules and an enzyme-coupled anti-PAS antibody of the present invention to detect the PASylated drug. (D) Schematic of a competitive ELISA showing PASylated test substance molecules competing with PASylated and biotinylated molecules for the binding sites of anti-PA(S) MAb adsorbed to a microtiter plate. PASylated and biotinylated molecules bound to MAb are then detected by a streptavidin enzyme conjugate. [Figure 16]Fluorescence titration of Fab fragment of anti-PA(S) Mab2.2 with synthetic epitope peptide Abz-APAPAAPA, applied at 1 μM in 100 mM Tris / HCl pH 7.5. RFU-relative fluorescence units (fluorescence excitation at 280 nm, signal detection at 340 nm). [Figure 17] Surface plasmon resonance (SPR) spectroscopy on a Biacore X100 instrument (Cytiva, Freiburg, Germany) using anti-PA(S)Mab1.1 to capture PASylated anti-galectin Fab fragments and determine the PAS-Fab binding kinetics to its antigen galectin-3. (A) Sensorgram showing immobilization of PASylated antibody fragments on a CM5 surface plasmon resonance (SPR) sensor chip (Cytiva). (1) 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)Mab1.1 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) (1) Noncovalent immobilization of PASylated anti-galectin Fab followed by (2) five consecutive injections from a 1:2 dilution series of galectin-3 (0.1 nM to 1.6 nM) as well as two acidic regeneration steps. (C) The baseline-corrected sensorgram of the single cycle kinetic experiment from (B) was set to zero with a start time of 0 s and fitted to a global 1:1 Langmuir binding model using Biacore X100 evaluation software. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0094] Example 1: Methods used in the present invention A. Preparation of PAS-peptide conjugates for immunization 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: Pga-PAS#1(40)-Ahx (Pga-ASPAAPAPASPAAPAPSAPA-ASPAAPAPASPAAPAPSAPA-Ahx; SEQ ID NO:5); Pga-P / A#1(40)-Ahx (Pga-AAPAAPAPAAPAAPAPAAPA-AAPAAPAPAAPAAPAPAAPA-Ahx; SEQ ID NO:6); Pga-APSA(40)-Ahx (Pga-APSAAPSAAPSAAPSAAPSA-APSAAPSAAPSAAPSAAPSA-Ahx; SEQ ID NO:7).

[0095] Pga means 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 described by their one-letter abbreviations. 40-mer PAS peptides were designed with sufficient length to encompass at least two copies of the corresponding PAS repeats, but in some embodiments, resulted in 20 residues, including at least one instance of a junction between two adjacent repeats. Of note, such junctions would also constitute potential epitopes in longer recombinant PAS polypeptides. Since all peptides contained only chemically inert side chains and had blocked N-termini, their single C-terminal carboxylate group (indeed, one of the Ahx linker residues) was selectively activated and used for direct chemical conjugation to the ε-amino group of the Lys side chain of KLH, which was used as a highly immunogenic carrier protein (Swaminathan et al., 2014). For this purpose, 50 mg of each peptide was dissolved in 1450 μl of dimethyl sulfoxide (DMSO) and activated with 10-fold molar amounts of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU; Iris Biotech, Marktredwitz, Germany) and N,N-diisopropylethylamine (DIPEA; Sigma-Aldrich, Taufkirchen, Germany). 10 mg of KLH (Thermo Scientific, Waltham, MA) was dissolved in water, dialyzed against PBS (4 mM KH2PO4, 16 mM Na2HPO4, 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 with a linear gradient of 0 to 500 mM NaCl applied in running buffer and monitored at 280 nm. Elution fractions of the main peak were pooled, dialyzed against PBS, concentrated to 2 mg / ml, filter-sterilized through a 0.22 μm Millex-GV PVDF filter (Merck, Darmstadt, Germany), and flash-frozen in liquid nitrogen. B. Immunization of Mice and Generation of Hybridoma Cells Balb / c mice were immunized using the above PAS peptide-KLH conjugate as antigen, and hybridomas were prepared according to standard procedures (ProMab Biotechnologies, Richmond, CA). For each antigen, five Balb / c mice were subcutaneously immunized with 50 μg of antigen together with Freund's complete adjuvant (CFA). Three weeks after priming, three booster injections (five for APSA(40)-KLH) were administered at two-week intervals, each with 25 μg of antigen and Freund's incomplete adjuvant (IFA). Two weeks after the last boost, a final boost with 50 μg of antigen without adjuvant was administered intraperitoneally. Spleen cells were harvested from the animals and fused with Sp2 / 0 myeloma cells for hybridoma clone generation using standard procedures well known in the art.

[0096] Promising hybridoma clones were expanded in cell culture using DMEM (Biochrom, Berlin, Germany) supplemented with 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 10% v / v Hybridoma Premium Medium (ProMab Biotechnologies). Secreted anti-PAS MAbs in cell culture supernatants were characterized by real-time surface plasmon resonance (SPR) spectroscopy and enzyme-linked immunosorbent assay (ELISA).

[0097] For some studies, anti-PA(S) MAbs were purified from 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). Hybridoma supernatants were purified using binding buffer (20 mM NaP i The antibody was diluted 1:1 with 100 mM Tris / HCl pH 7.0 and applied to a column pre-equilibrated 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 IgG, 200 μl of 1 M Tris / HCl pH 9.0 per ml of collection volume was added to each collection tube before fractionation. The fractions containing the affinity-purified MAb were then 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 280 = 1.4 is equivalent to a concentration of 1.0 mg / ml IgG). C. Characterization of Hybridoma MAbs by ELISA and SPR Characterization of hybridoma MAbs by ELISA was performed using NUNC Maxisorp F 96-well plates (Thermo Fisher Scientific, Munich, Germany) coated with 50 μl of 5 μg / ml of anti-mouse IgG Fc-specific goat antibody (Sigma-Aldrich) in PBS, followed by washing twice with PBS and blocking for 1 h with 3% w / v bovine serum albumin (BSA) in PBS / T (PBS + 0.1% v / v Tween 20). After washing with PBS / T, wells were incubated for 1 h with 50 μl of each hybridoma supernatant diluted 1:100 in PBS / T and washed again. Then, 50 μl of the following PASylated proteins (8 nM each) were applied in a 1:2 dilution series in PBS and incubated for 1 h: hu4D5-PAS#1(200) (Schlapschy et al., 2013), hu4D5-P / A#1(200) (WO2011 / 144756A1) 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 μl 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 a final wash with PBS, 50 μl of 0.5 mg / ml p-nitrophenyl phosphate in AP buffer (100 mM Tris / HCl pH 8.8, 100 mM NaCl, 5 mM MgCl2) was added and signal development was recorded at 405 nm at 1 min intervals for 15 min using a Synergy 2 photometer (BioTek Instruments, Bad Friedrichshall, Germany). The concentration-dependent signal (ΔA / Δt) was calculated using the equation: [MAb Ag] = [MAb] t [Ag] t / (KD+Ag) t ) The antibody / antigen complex was assessed according to published procedures ( Voss & Skerra, 1997 ) using [MAb·Ag], which is the detectable amount of antibody / antigen complex, which is proportional to the ΔA / Δt signal measured for each well; [MAb] t is the total amount of immobilized antibody, which corresponds to the asymptotic maximum signal of the binding curve; [Ag] t is the (variable) total concentration of PAS antigen applied to each well, and K D is the dissociation constant of the antibody / antigen complex resulting from curve fitting, evaluated with KaleidaGraph (Synergy Software, Reading, PA).

[0098] SPR measurements were performed on either a Biacore X100 or Biacore T200 instrument (GE Healthcare) using a mouse antibody capture kit and a CM3 sensor chip (both from GE Healthcare) at 25° C. 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 Tween 20) and 30 μl of sample was injected at a flow rate of 10 μl / min. Where appropriate, a concentration series of the following test antigens was injected onto the sensor chip at a flow rate of 30 μl / min using single cycle kinetics (Karlsson et al., 2006): 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) WO2011 / 144756A1. The sensor chip was regenerated with 10 mM glycine / HCl pH 1.7 for 100 s. After subtraction of the signal from the reference channel and the blank baseline measured in HBS-ET buffer, the data were fitted using the Biacore X100 evaluation software ver. 2.0.1 (GE Healthcare) and the bivalent analyte model. The rate equation used by the fitting algorithm is: A divalent test substance (A) binds to a ligand (B).

[0099] A(solution)=concentration A[0]=0 dA / dt=(tc*f^(1 / 3))*(concentration A)-((2*ka1)*A*B-kd1*AB) B[0]=RMax dB / dt=-((2*ka1)*A*B-kd1*AB)-(ka2*AB*B-(2*kd2)*AB2) AB[0]=0 dAB / dt=((2*ka1)*A*B-kd1*AB)-(ka2*AB*B-(2*kd2)*AB2) AB2[0]=0 dAB2 / dt=(ka2*AB*B-(2*kd2)*AB2) Total Response: AB+AB2+RI Parameters: concentration, analyte concentration [M]; tc, mass transfer constant; f, volumetric flow rate of solution through the flow cell [m 3 ·s -1 ]; RMax, binding capacity; RI, refractive index. D. Cloning of V genes from hybridoma cells Hybridoma cells were mechanically lysed and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), followed by oligo(dT) 18 cDNA synthesis was performed using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) with primers: all mouse germline V, V(Gamma) and V(Gamma) together with 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). L / V HIgV gene regions were PCR amplified from their cDNA with Q5 DNA polymerase (New England Biolabs, Frankfurt / M. Germany) using a set of 63 forward primers covering gene segments (Chardes et al., 1999). Forward primers were first applied in pools of 5–15 to reduce the required number of PCR reactions and to generate single PCR products individually after identifying PCR products for such pools. Appropriate PCR products were then 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 For cloning of V genes on the bacterial expression vector pASK88 (Schiweck & Skerra, 1995), products from the V gene amplifications described above were PCR amplified using primer pairs designed to introduce appropriate flanking restriction sites according to previously published routine procedures (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 H Genes and V L Each gene was inserted into pASK88, which had been cut with the corresponding restriction enzyme in two successive ligations. The coding regions for anti-PA(S) MAb 2.1, anti-PA(S) MAb 1.2, and anti-PA(S) MAb 3.1 were obtained by gene synthesis using appropriate flanking restriction sites based on the V gene sequences determined for these hybridomas by ProMab Biotechnologies (GeneArt, Regensburg, Germany). F. E. coli production and purification of Fab fragments 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 chimeric Fab fragments (hybridoma-derived mouse variable domains fused to human constant domains) either in 6 × 2 liter shake flask cultures using E. coli strain JM83 (Yanisch-Perron et al., 1985) or via 8 liter benchtop fermentation using strain KS272 (Meerman & Georgiou, 1994) and following published procedures (Schiweck & Skerra, 1995; Skerra, 1994). Recombinant proteins were purified from periplasmic cell extracts 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). 88405M for 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. -1 cm -1 , 89895M -1 cm -1 , 77405M -1 cm -1 , 66405M -1 cm -1 , 6995M -1 cm -1 or 57465M -1 cm -1Protein concentrations were determined by measuring absorbance at 280 nm using a calculated extinction coefficient of (Gasteiger et al., 2003). 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 Fab by ELISA, fluorescence titration and SPR NUNC Maxisorp F 96-well plates were coated with either P / A#1(600)polypeptide (Breibeck & Skerra, 2018) at 10 μg / ml in 50 μl PBS for the recombinant Fab fragments of anti-PA(S)MAb 2.1 and anti-PA(S)MAb 2.2, PAS#1(600)-leptin (Morath et al., 2015) at 10 μg / ml in 50 μl PBS for the Fab fragments of anti-PA(S)MAb 1.1 and anti-PA(S)MAb 1.2, or APSA(200)-IL1Ra (SEQ ID NO: 74) at 10 μg / ml in 50 μl PBS for the Fab fragments of MAb 3.1 and anti-PA(S)MAb 3.2, and incubated overnight at 4°C. After one 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 incubation with 50 μl of the appropriate dilution series of each purified Fab fragment in PBS / T for 1 h. The wells were washed again with PBS / T, followed by incubation with 50 μl 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 two final washes with PBS / T and PBS, respectively, the signal was developed with p-nitrophenyl phosphate and measured and evaluated as described herein above.

[0100] Fluorescence titrations were performed as previously described (Voss & Skerra, 1997) using a LS-50B luminescence spectrometer (Perkin Elmer, Norwalk, CT) with a 2 ml quartz cuvette thermostated at 25°C at wavelengths of 280 nm for excitation and 340 nm for detection (signal integrated over 5 s). 2 ml of a solution containing 1 μM of purified Fab fragment of anti-PA(S) MAb 2.2 in 100 mM Tris / HCl pH 7.5 was titrated with a 5 mM solution of Abz-APAPAAPA peptide (Peptide Specialty Laboratories-PSL, Heidelberg, Germany) (Abz stands for ortho-aminobenzoyl) in aliquots ranging from 1 μl up to a total volume of 22 μl. Data were normalized to 100% initial fluorescence and fitted by nonlinear least-squares regression using KaleidaGraph (Synergy Software, Reading, PA) as described ( Edwardraja et al., 2017 ), including correction for inner filter effects by titration of N-acetyl-tryptophanamide with the same peptide.

[0101] SPR measurements with the Fab fragments of the corresponding anti-PA(S) MAbs were performed on a Biacore X100 instrument (GE Healthcare) at 25°C. PAS#1(200)-IL1Ra, P / A#1(200)-IL1Ra, or thioredoxin A-APSA(200) were biotinylated with 20-fold molar amount of succinimidyl-6-(biotinamide)hexanoate (Sigma Aldrich) according to the manufacturer's instructions and immobilized individually as ligands on a BiotinCAPture chip (GE Healthcare) according to the manufacturer's protocol. Prior to immobilization of each ligand, the sensor chip was regenerated with two consecutive injections of 30% v / v acetonitrile, 0.25 M NaOH for 120 s and 6 M guanidine / HCl, 0.25 M NaOH for 120 s. A concentration series of recombinant Fab fragments was injected over the sensor chip using single cycle kinetics and a flow rate of 30 μl / min. After subtraction of the signal from both the reference channel and the blank baseline measured in HBS-ET buffer, the data were fitted using Biacore X100 evaluation software ver. 2.0.1 (GE Healthcare) using a 1:1 binding model. The rate equation used by the fitting algorithm is: A(solution)=concentration A[0]=0 dA / dt=(tc*f^(1 / 3))*(concentration A)-(ka*A*B-kd*AB) B[0]=RMax dB / dt=-(ka*A*B-kd*AB) AB[0]=0 dAB / dt=(ka*A*B-kd*AB) Total Response: AB+RI Parameters: concentration, analyte concentration [M]; tc, mass transfer constant; f, volumetric flow rate of solution through the flow cell [m 3 ·s -1 ]; RMax, binding capacity; RI, refractive index. H. SPOT synthesis and epitope mapping of immobilized peptide arrays Arrays of 20 overlapping 12-mer peptides covering the entire amino acid sequence of the PAS#1 or P / A#1 amino acid repeats, or 10-mer peptides containing the sequence AAPSAAPSAA consecutively substituted at positions 3-8 by all 20 proteinogenic amino acids, were synthesized on hydrophilic membranes using a MultiPep SPOT synthesizer (Intavis, Koln, Germany) according to standard protocols (Frank, 2002). Detection of binding activity on the membranes was performed following published procedures (Zander et al., 2007) after incubation with either purified Fab fragments or hybridoma cell culture supernatants containing secreted MAbs, 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 PAS-conjugated proteins by Western blotting Anti-PA(S) MAbs from hybridoma supernatants were tested for the 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)) diluted 1:200 in water and spiked with 1 μg IL1Ra (Kineret / Anakinra; Swedish Orphan Biovitrum, Stockholm, Sweden) and E. coli BL21 whole cell lysate as well as human serum (human serum (PL), pooled, SEQENS IVD / H2B, Limoges, France) in the case of controls were subjected to SDS-PAGE followed by semi-dry electrotransfer onto nitrocellulose membranes. After washing with PBS / T, the membranes were incubated with a 1:2000 dilution in PBS / T of anti-PAS MAb as hybridoma supernatant or a 1:200000 dilution in PBS / T of purified anti-PA(S) MAb 2.1. Bound MAb was detected using a 1:50,000 dilution in PBS / T of an anti-mouse IgGFc-specific goat antibody conjugated with alkaline phosphatase (Sigma-Aldrich), followed by a color reaction with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitroblue tetrazolium (NBT) (both from Carl Roth, Karlsruhe). J. Pharmacokinetic analysis in rats Pharmacokinetic (PK) studies were performed in female Wistar rats at 8-9 weeks of age at the Aurigon Toxicological Research Center (ATRC, Dunakeszi, Hungary) in accordance with applicable animal welfare regulations. Up to three animals per cage were housed in a controlled environment at 22±3°C, with a relative humidity of 50±20% and 12 h light / 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 dorsal region of the rats. Blood samples (100 μl) were taken from each of the five animals at various time points. Following collection into K3-EDTA tubes (Greiner Bio-One, Frickenhausen, Germany), samples were centrifuged (3000×g) for 10 min at room temperature 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 and FIG. 15A). A potential alternative ELISA setup suitable for quantification of PASylated peptides or proteins in blood or plasma / serum samples of animals or human patients is illustrated in FIG. 15, panels B-D. K. Quantification of PAS-conjugated thymosin alpha 1 in rat plasma by ELISA Female Wistar rats (n=5) at 8-9 weeks of age (Aurigon Toxicological Research Center, Dunakeszi, Hungary) were subcutaneously injected with PAS-Thymosin alpha 1 (SEQ ID NO: 76) (3.4 mg / kg) and blood samples (100 μl) were collected at different time points into K3-EDTA tubes (GreinerBio-One, Frickenhausen, Germany). For quantification of administered PAS-Thymosin alpha 1 in rat PK studies (Method J), Nunc Maxisorb ELISA 96-well plates (Thermo Fisher Scientific) were coated with anti-PA(S) MAb 2.1 in PBS at 100 μg / ml overnight at 4°C. After two washes with PBS / T, free binding sites were blocked with 3% w / v BSA in PBS / T for 1 h at room temperature.

[0102] The plates were then washed three times with PBS / T, and rat plasma samples were applied, each at a 1:2 dilution series, in PBS / T, which had been supplemented with 0.5% (v / v) plasma from untreated animals to maintain a constant proportion of rat plasma components. Similarly, a standard curve was generated using a dilution series of purified PAS-tyrosine thymosin alpha 1 at a defined concentration in PBS / T containing the same amount of rat plasma as the test samples. After 1 h of incubation at room temperature, the wells were washed three times with PBS / T. To detect bound PAS-tyrosine thymosin alpha 1, the wells were incubated for 1 h with 50 μl of a 1 μg / ml solution of anti-PA(S) MAb 1.2 in PBS / T, which had been conjugated to alkaline phosphatase using the Lightning-Link Alkaline Phosphatase Antibody Labeling Kit (BioTechne, Wiesbaden, Germany). After washing twice with PBS / T and twice with PBS, enzyme activity was detected using p-nitrophenyl phosphate (0.5 mg / ml). For this purpose, plates were incubated for 20 min at 30° C., absorbance was measured at 405 nm using a SpectraMax M5e microtiter plate reader (Molecular Devices, Sunnyvale, CA), and PAS-ylated thymosin alpha 1 concentrations were quantified by comparison with a standard curve (FIG. 14A).

[0103] Data were plotted against sampling time after injection and fitted using a 1-compartment model using Phoenix WinNonlin 6.3 software. The resulting PK parameters (Table 1) and PK profile (Figure 14B) are typical for long-acting peptide drugs, proving that the anti-PAS antibodies of the present invention are suitable for quantifying the concentration of PASylated drugs in mammalian plasma. Of note, the ELISA setup applied in the present method (Figure 15A) uses an anti-PAS antibody as a capture antibody, thus avoiding 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 Figure 15 and are well known in the art (Vashist & Luong, 2018).

[0104] [Table 1]

[0105] Cocrystallization of anti-PAS Fab fragment with L.PAS peptide, X-ray data collection, and construction of molecular model 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 cocrystallized with their cognate PAS peptides, whereas in the case of the Fab of anti-PA(S) MAb 1.2, the anti-human kappa V peptide was cocrystallized with the cognate PAS peptides described in (Ereno-Orbea et al., 2018). H The complex with the H domain was first prepared. For this purpose, purified Fab was diluted with 3x molar amount of V H The protein mixture was subjected to SEC on a HiLoad 16 / 60 Superdex75 prep-grade column, and Fab V H The H complex was then purified using excess anti-human kappa V H The H domain was separated and isolated in one peak using 10 mM HEPES / NaOH pH 6.5, 70 mM NaCl as the running buffer.

[0106] The various protein solutions were concentrated using Amicon Ultracel centrifugal filter units (MWCO 10 kDa; Miiiipore, 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 to Fab·V H H, 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 at 4 °C for 1 h. Protein crystallization screening was then performed via sitting drop vapor diffusion and equal mixtures of protein and reservoir solution, resulting in total drop volumes ranging from 300 to 1000 nl. Upon refining promising crystallization conditions, further screening was set up using hanging drop vapor diffusion with a reservoir volume of 1 ml and droplets composed of 1 μl protein and 1 μl reservoir solution. Crystals appeared within 1 week at 20 °C under the conditions listed in Table 3. Protein crystals were harvested, transferred to precipitation buffer supplemented with 20% w / v PEG200 for anti-PA(S) MAb 2.2, 20% w / v ethylene glycol 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.

[0107] Single-wavelength X-ray synchrotron data sets were collected at 100 K from each crystal at the MX beamline BL14.2 of the 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 at the Swiss Light Source (SLS), Villigen-PSI, Switzerland. Diffraction data (Table 3) were organized with the XDS program package (Kabsch, 2010) and molecular replacement was performed with Phaser (McCoy et al., 2007) using the constant and variable domains of Fab 101F (PDB ID: 3QQ9) as a search model to solve the structure of the anti-PA(S) MAb 2.2 Fab·P / A#1 complex. The structures of anti-PA(S) MAb 1.1 Fab·PAS#1 and anti-PA(S) MAb 1.2 Fab·PAS#1 are shown in Fig. 1. The latter is anti-human kappa V H The H domain (PDB ID: 6ANA) was also analyzed by molecular replacement with the refined structure of the Fab of anti-PA(S) MAb 2.2 as a search model. The structure of anti-PA(S) MAb 3.1Fab·APSA was analyzed by molecular replacement with the refined structure of the Fab of anti-human kappa V HThe H-domain was not included and the structure was analyzed by molecular replacement with the refined structure of anti-PA(S) MAb 1.2 Fab as a search model. The protein model was manually adjusted in Coot (Emsley et al., 2010) and refined with Refmac5 (Murshudov et al., 2011). Peptide and water molecules were manually constructed in Coot during the purification process. The final structural model was validated using the MolProbity server (Wiiiiams et al., 2018). The contact sites and exposed and buried surface areas (ASA and BSA, respectively) of the crystals were analyzed 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 generated with PyMOL (Schrodinger, Cambridge, MA) using the APBS module (Baker et al., 2001) for electrostatic calculations. Interatomic distances were calculated using CONTACT (Winn et al., 2011).

[0108] The polypeptides are designated L for Ig light chain, H for Ig heavy chain, and P for each of the bound PAS peptides, while anti-human kappa V H The H domain was assigned the chain identifier X. For anti-PA(S) MAb 1.1, when there were two Fab·peptide complexes in the asymmetric unit, the one with the higher average crystallographic B factor was assigned the chain identifiers A, B, and Q, respectively. Affinity purification of StrepII-eGFP-PAS#1(200), H1GA-PAS#1(200)-His6, and PAS#1(800)-IL1Ra using anti-PAS Fab immobilized on M-Sepharose column A total of 5 mg of purified Fab fragments of anti-PA(S) MAb 1.2 were covalently immobilized on a 1 ml HiTrap NHS-activated HP column (GE Healthcare) according to the manufacturer's protocol. Briefly, the column was washed with ice-cold 1 mM HCl, followed by injection of 1 ml of Fab coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) and incubation at 25°C for 30 min. Washing and inactivation 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.

[0109] Purification of the PASylated test proteins, StrepII-eGFP-PAS#1(200), H1GA-PAS#1(200)-His6 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)-His6 (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 of running buffer, and then bound proteins were eluted by applying 2-3 ml of 1 M running buffer of L-prolinamide (Sigma Aldrich) or alternatively 1 M L-prolinamide, 100 mM Tris, 150 mM NaCl, 1 mM EDTA, pH adjusted to 8.0 with HCl, followed by column regeneration with running buffer. 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 (Figure 10A,B). SDS-PAGE analysis confirmed the specific elution of pure PASylated proteins (Figure 10C,D). Due to apparent impurities in the commercial L-prolinamide material that resulted in background absorption at 280 nm, blank chromatograms (without application of PASylated protein) were recorded and used for subtraction to obtain corrected chromatograms of H1GA-PAS#1(200)-His6 and PAS#1(800)-IL1Ra (Figure 11A,C). Specific elution of pure PASylated proteins was confirmed by SDS-PAGE analysis (Figure 11B,D). N. Preparation of PAS-conjugated test proteins All test proteins and peptides fused to PAS sequences of various compositions and lengths used in the methods described herein were produced in E. coli via cytoplasmic expression from conventional expression vectors harboring the corresponding synthetic genes or via periplasmic secretion according to routine procedures well described in the art, e.g., in WO2008 / 155134A1, WO2011 / 144756A1, WO2017 / 109087A1, WO2018 / 234455A1 or in (Binder & Skerra, 2017; Breibeck & Skerra, 2018; Morath et al., 2015; Schlapschy et al., 2013). O.Deposit The following MAbs of the invention have been 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 is recognized by the World Intellectual Property Organization as an International Depositary Authority pursuant to the Budapest Treaty on the Deposit of Animal and Human Cell Cultures, on February 28, 1991.

[0110] ·Anti-PA(S)Mab 1.1=DSM ACC3365 ·Anti-PA(S)Mab 2.1=DSM ACC3366 ·Anti-PA(S)Mab 3.1=DSM ACC3367 Example 2: Generation of monoclonal anti-PAS antibodies and their specific detection of PAS sequences 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. For this purpose, animals were immunized with the corresponding synthetic N-terminally protected 40-mer peptides, as described herein above in Example 1, which were chemically coupled via the C-terminal carboxylate group to marine-farmed 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 40-mers exactly covered two copies of the designed 20-mer repeat sequence (Breibeck & Skerra, 2018; Schlapschy et al., 2013), whereas the "APSA" peptide constituted 10 copies of the four-residue motif, which can be considered as a kind of simplified Pro / Ala-rich sequence pattern.

[0111] These 2 × 20-mer and / or 40-mer PAS peptides were designed to encompass at least two copies of the corresponding PAS repeat sequence, including at least one copy of the junction between two adjacent repeat sequences, which would also constitute a potential epitope in the longer recombinant PAS polypeptide. After 4-6 rounds of immunization with 25-50 μg of antigen, respectively, and a final boost, spleen cells were isolated from 5 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 containing the corresponding PAS polypeptide (200-600 residues), with the goal of screening for (i) sequence-specific and context-independent recognition of the PAS sequence, and (ii) identification of antibodies showing possible cross-reactivity between different PAS sequences. MAb capture ELISA with hybridoma culture supernatants was performed, applying the PAS fusion proteins in a concentration-dependent manner to determine the dissociation constants (K D) was determined. Hybridoma culture supernatants of promising candidates were characterized with respect to antigen affinity and binding kinetics by real-time surface plasmon resonance (SPR) spectroscopy. The corresponding method is described in Example 1.

[0112] Its K measured by ELISA and SPR D Based on the values ​​and also taking into account the absorbance amplitudes of the concentration-dependent ELISA, eight clones with clearly different properties were selected from the immunization of PAS#1(40)-KLH and P / A#1(40)-KLH, respectively, and tested for recognition of linear epitopes on synthetic peptide arrays using the Synthetic Peptides On Transfer Membrane (SPOT) technique (Frank, 2002). The assay revealed that "PAPAAP" (SEQ ID NO: 8) and "PAPASP" (SEQ ID NO: 9) as epitope sequences for anti-PA(S) MAbs 2.1 and 2.2, whereas anti-PA(S) MAbs 1.1 and 1.2 predominantly recognized the peptide motif "PASPAAP" (SEQ ID NO: 10) (see Figures 5 and 6). Due to the simpler repetitive nature of the APSA sequence, a SPOT displacement analysis was performed on this antigen (Figure 7). Therefore, a 10-mer peptide containing the sequence AAPSAAPSAA consecutively substituted at positions 3-8 with all 20 essential amino acids was synthesized with C-terminal anchoring on a hydrophilic membrane. Interestingly, antibodies generated by immunization with PAS#1(40)-KLH recognized only the PAS#1 polypeptide sequence (anti-PA(S) MAb 1.1 and 1.2), whereas some MAbs derived from 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, a generalized sequence motif recognized by all anti-PA(S) MAbs of the present invention emerged as "(P / S)A(A / S)P" or as PA(A / S)P.

[0113] To verify the applicability of the monoclonal antibodies of the present invention in detecting PASylated fusion proteins, anti-PA(S) MAbs from hybridoma supernatants were tested in Western blotting experiments, where specific detection of PASylated fusion proteins was confirmed, and no cross-reactivity was detected with non-PASylated protein versions, human serum proteins, or proteins in E. coli whole cell lysates (Figure 2).

[0114] Example 3: Cloning of MAb V gene sequences and Fab production For each antigen, the two most promising hybridoma clones (anti-PA(S) MAb 2.1 and anti-PA(S)) were selected for further analysis based on their affinity to the target sequence and 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, and anti-PA(S) MAb 3.1 and anti-PA(S) MAb 3.2 for APSA. To determine their V gene sequences from mRNA / cDNA, each V was analyzed using the same method as described herein above in Example 1. H Domains and V L The coding regions of the domains were reverse transcribed and amplified by polymerase chain reaction (PCR) using appropriate oligodeoxynucleotide primers. 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, the 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 bacterial expression vectors encoding the first human IgG1 heavy chain and kappa light chain constant regions to allow expression of the corresponding chimeric Fab fragments (Schiweck & Skerra, 1995; Skerra, 1994). The Fab fragments were produced functionally by periplasmic secretion in E. coli at both shake flask and benchtop fermenter scale and purified to homogeneity by IMAC, CEX, and SEC (see Example 1).

[0115] The following amino acid sequences were obtained (see FIG. 1; CDRs according to the definition by (Kabat et al., 1991) are marked with black boxes): VH Anti-PA(S) MAb 1.1 (SEQ ID NO: 23): VL Anti-PA(S) MAb 1.1 (SEQ ID NO: 24): VH Anti-PA(S) MAb 1.2 (SEQ ID NO:25): VL Anti-PA(S) MAb 1.2 (SEQ ID NO:26): VH Anti-PA(S) MAb 2.1 (SEQ ID NO:27): VL Anti-PA(S) MAb 2.1 (SEQ ID NO:28): VH Anti-PA(S) MAb 2.2 (SEQ ID NO:29): VL Anti-PA(S) MAb 2.2 (SEQ ID NO:30): VH Anti-PA(S) MAb 3.1 (SEQ ID NO:31): VL Anti-PA(S) MAb 3.1 (SEQ ID NO:32): VH Anti-PA(S) MAb 3.2 (SEQ ID NO:33): VL Anti-PA(S) MAb 3.2 (SEQ ID NO:34): It is noteworthy that apart from the method for determining CDRs, which is based mostly on interspecies sequence diversity (Kabat et al., 1991), 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, CDR refers preferentially to the definition by Kabat (supra), but may also refer to CDRs defined by other of the above approaches or by a combination of both approaches. Consecutive numbers are used to number the amino acids.

[0116] Example 4: Characterization of binding affinity (anti-PAS monoclonal antibodies and anti-PAS Fab) 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 analyzed for their K D To precisely determine K values, quantitative ELISA and real-time SPR measurements were investigated (see also Table 2). These measurements essentially confirmed the findings from the preliminary hybridoma screening. For each type of PAS antigen, at least one MAb with particularly high affinity was identified, with K values ​​in the single-digit nanomolar range measured for the Fab. D It is evident from the values: 2 nM for P / A#1 for anti-PA(S) MAb 2.1; 23 nM for PAS#1 for anti-PA(S) MAb 1.1; 2 nM for APSA for anti-PA(S) MAb 3.1. Compared to the intact MAbs previously investigated, the affinities measured for the Fabs were usually 1-2 orders of magnitude weaker, most likely due to avidity effects that occur when bivalent Mabs interact with long PAS polypeptides that harbor multiple copies of the epitope (e.g., 30 copies of the repetitive 20 amino acid stretch of PAS#1 in a 600 residue PAS polypeptide). The anti-P / A#1 Fabs were either specific for the P / A#1 sequence or cross-reactive with the PAS#1 sequence, whereas the anti-PAS#1 Fabs showed specificity only for the PAS#1 sequence. Anti-APSA antibody fragments were either specific for the APSA sequence or cross-reactive with the PAS#1 and P / A#1 polypeptides (see Table 2 below).

[0117] [Table 2]

[0118] To determine the monovalent affinity of anti-PA(S) MAb 2.1 and anti-PA(S) MAb 2.2 for their epitope sequences, fluorescence titration (FT) experiments were performed using the corresponding recombinant Fab fragments and the synthetic peptide Abz-APAPAAPA (SEQ ID NO: 4), which carries an N-terminal o-aminobenzoyl group as a fluorescence resonance energy transfer probe. A reliable K D Although it was not possible to derive a K value for the Fab of anti-PA(S) MAb 2.2, D = 9.2 ± 0.1 μM (Figure 16). This value is the same as the K measured for the pure P / A#1(600) antigen by ELISA. D =8±1 nM, which is approximately 1000-fold higher (see Table 2). The difference must be even higher for the Fab of anti-PA(S) MAb 2.1, indicating that not only avidity effects but also more specific aspects such as the exact molecular vicinity of the epitope peptide or epitope density within a polypeptide with multiple repeat sequences can significantly affect the apparent affinity.

[0119] Example 5: Structural characterization of PAS peptide binding by co-crystallization with anti-PAS Fab The structural mechanism of antigen recognition by some of the anti-PA(S) MAbs of the invention was analyzed using X-ray crystallography. Thus, recombinant anti-PA(S) Fab fragments prepared using the method described herein above in Example 1 were subjected to co-crystallization experiments with their cognate synthetic peptides, the sequences of which were based on the epitope sequences determined by SPOT assays as described above or, in the case of simple APSA motifs, included a stretch of 12 amino acids with three APSA repeats. To avoid charges at the N-terminus, which may 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·peptide complexes of anti-PA(S) MAb 2.2 Fab·P / A#1 and anti-PA(S) MAb 1.1 Fab·PAS#1 (see attached Table 3). In the case of anti-PA(S) MAb 2.1 and anti-PA(S) MAb 1.2, we used anti-human kappa light chain V H We applied a recently published strategy that utilizes H domains to facilitate (co)crystallization of (our chimeric) Fab fragments (Ereno-Orbea et al., 2018). Indeed, this approach led to crystals of the Fab of anti-PA(S) MAb 1.2 in complex with the PAS#1 epitope peptide, which diffracted at a high resolution of 1.55 Å at a synchrotron X-ray source. The structure of the anti-PA(S) MAb 2.2 Fab·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 a search model. The complexes, anti-PA(S) MAb 1.1 Fab·PAS#1 and anti-PA(S) MAb 1.2 Fab·PAS#1·V HThe structure of H was solved by molecular replacement using the refined structure of Fab 3F3E2 anti-PA(S) MAb 2.2 as a search model, and in the latter case the refined structure of the anti-human kappa light chain VHH domain (PDB ID: 6ANA). The structure of anti-PA(S) MAb 3.1 Fab·(APSA)3 was solved by molecular replacement using the refined structure of anti-PA(S) MAb 1.2 Fab as a search model, in this case without including the anti-human kappa VHH domain. After manual positioning of the PAS#1, P / A#1 and (APSA)3 peptides, crystallographic refinement was completed, resulting in R free The values ​​ranged from 23 to 27% (Table 3).

[0120] [Table 3]

[0121] Further analysis of these crystal structures showed that the PAS peptide is bound to all four Fabs in a more or less relaxed conformation, thus 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 chains - except for one Ser residue in the PAS#1 epitope peptide and three Ser residues in the (APSA)3 peptide - interactions are predominantly mediated through hydrogen bonds with atoms of the peptide backbone (see attached Table 4) and van der Waals contacts (see attached Table 5), including some local hydrophobic interactions, while salt bridges are completely absent, as expected. Interestingly, in each case at least one Ala residue of the PAS peptide is involved in the proper interaction with the anti-PAS Fab; therefore, Ala can be considered as a hotspot for antibody interactions in the PAS epitope. So far, Ala, the amino acid with the smallest side chain, has been ascribed a negligible role in protein-protein / peptide recognition. In fact, the strategy of alanine scanning mutagenesis (Cunningham & Wells, 1989) has found wide application to probe residues critical for receptor-ligand or antibody-antigen binding, resulting in the assumption of a semi-inert role of the Ala methyl side chain for molecular interactions. Unexpectedly, the present invention reveals that Ala can indeed play a pivotal role in antigen recognition, as exemplified in particular by two crystal structures, anti-PA(S) MAb 2.2 Fab·P / A#1 and anti-PA(S) MAb 1.1 Fab·PAS#1. Indeed, Ala P5 is completely buried in the binding pocket, its carbonyl oxygen is involved in two hydrogen bonds, and serves as a "hot spot" residue in the antibody-peptide interface of the complex PA(S) MAb 2.2 Fab P / A#1 (Clackson & Wells, 1995). Similarly, the structure of anti-PA(S) MAb 1.1 Fab reveals a hole in the center of the antigen-binding site that is occupied by Ala. P7The methyl groups of the carboxyl groups are perfectly shaped to accommodate the methyl groups of the carboxyl groups, thereby allowing for high shape complementarity and a closely packed interface.

[0122] The structure of anti-PA(S) MAb 3.1 Fab in complex with the (APSA)3 peptide reveals that V H Chain and V L It is a characteristic groove in the paratope between the chains where the peptide binds in an extended conformation. Binding involves residues from all three APSA repeats in the peptide and is mediated primarily by hydrogen bonds with atoms in the peptide backbone or with Ser side chains of the peptide, as well as hydrophobic interactions of Pro and Ala side chains of the peptide. As in the structures of anti-PA(S) MAb 2.2 Fab·P / A#1 and anti-PA(S) MAb 1.1 Fab·PAS#1, Ala residues in the epitope play an important role in mediating hydrogen bonds and van der Waals contacts (Tables 4 and 5).

[0123] Unexpectedly, in the case of anti-PA(S) MAb 1.2 Fab complexed with the PAS#1 epitope peptide, the N-terminal pyroglutamyl residue of the peptide also contributes to the complex formation by three hydrogen bonds. Such hydrogen bonds seem not to be possible in complexes with longer PAS#1 (poly)peptides, where the position of the Pga residue would be occupied by Pro. The Pro residue would fit perfectly in this position in the crystal structure, whereas the further N-terminal course of the longer polypeptide chain would result in steric hindrance with the Fab.

[0124] To elucidate any conformational similarities between the bound PAS#1 peptide in complex with anti-PA(S) MAb 1.1 Fab or anti-PA(S) MAb 1.2 Fab, superpositions between the structures were performed. P5 ~Ala P8 The four residues of these C αThe positions showed an excellent match, in this case with a root mean square deviation (RMSD) of only 0.15 Å. Secondary structure analysis with STRIDE (Frishman & Argos, 1995) identified a type I β-turn within this four-residue stretch. P5 Carbonyl oxygen of Ala P8 Apart from the intramolecular hydrogen bond between the amide hydrogens, this turn is P5 Hydroxyl group and Ala P7 It is stabilized by hydrogen bonds between the amide hydrogens. This type of β-turn, classified as an SPXX turn, occurs in gene regulatory proteins, where it serves as a DNA-binding motif (Suzaki & 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·PAS#1, the turn is close to the binding pocket, whereas in the complex with anti-PA(S) MAb 1.2 Fab·PAS#1, it is exposed to the solvent. Of note, a similar analysis using STRIDE did not identify secondary structural features for the P / A#1 epitope peptide in the complex with anti-P / A#1 MAb 2.2 Fab, nor for the (APSA)3 peptide complexed with anti-PA(S) MAb 3.1 Fab.

[0125] In the context of the present invention, MAbs are generated by means and methods as provided herein that specifically recognize linear epitopes in structurally disordered Pro / Ala-rich (poly)peptides having three distinct sequences: i.e., sequences as provided in SEQ ID NOs: 1, 2 and 3. The anti-PA(S) MAbs of the present invention, or recombinant versions and fragments thereof, provide valuable bioanalytical and diagnostic tools for biochemical studies of PASylated drug candidates as well as biopharmaceutical development, including assays suitable for clinical studies (Binder & Skerra, 2017; Gebauer & Skerra, 2018; Richter et al., 2020).

[0126] [Table 4]

[0127] [Table 5]

[0128] In all crystallized Fab complexes provided herein, high abundance Tyr residues in the paratope are evident. These residues are responsible for the majority of hydrophobic contacts (see attached Table 5), thus creating a surface that is well suited to bind antigens lacking charged or polar side chains. In fact, 62%, 49%, 43%, and 23% of all contacts of ≦4.0 Å are mediated by Tyr in 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, anti-PA(S) MAb 2.2 reveals high Tyr content and also has high affinity among the crystallized complexes. This is consistent with previous analyses, which indicate that high Tyr content in antibody paratopes contributes overall to enhanced antigen specificity and affinity (Birtalan et al., 2008; Birtalan et al., 2010).

[0129] The data provided herein shed light on the mechanism of molecular recognition of disordered epitopes by antibodies. In the absence of salt bridges and obvious side chain interactions arising from the PAS epitope peptide in all Fab structures evaluated, complex formation is mainly driven by hydrogen bonds involving the peptide backbone (Appendix Table 4) as well as van der Waals contacts (Appendix Table 5), including some local hydrophobic interactions. The monotonicity of the PAS peptide requires that a small number of atomic groups capable of polar interactions are efficiently exploited. This is nicely demonstrated by the structure of anti-PA(S) MAb 1.2, where, for example, a short segment of the backbone hydrogen bond network with the PAS#1 peptide resembles an antiparallel β-sheet. In the two Fab complexes with the PAS#1 epitope peptide containing one Ser residue, both antibodies engage the only polar side chain available for hydrogen bond formation. The same is true for the structure of anti-PA(S) MAb 3.1, where two of the three Ser side chains are involved in hydrogen bonds. Nevertheless, consistent with the limited energy gain of such hydrogen bonds in a competitive aqueous environment (Gao et al., 2009). Indeed, anti-PA(S) MAbs 1.1 and 1.2 do not appear to benefit significantly from this interaction, given their significantly lower affinity compared to the best MAb raised against anti-P / A#1 (see Table 2). The observation that at least four of the six CDRs are involved in peptide-antibody interactions in all Fab complexes evaluated (see Table 6) highlights the need to engage extended interfaces to bind conformationally flexible antigens more or less tightly.

[0130] [Table 6]

[0131] Example 6: Anti-PA(S)Mab1.1 is used to capture PASylated anti-galectin Fab fragments and determine PAS-Fab binding kinetics to its antigen Galectin-3, SPR spectroscopy The anti-PA(S)Mab1.1 antibody of the present invention was used as a tool for the non-covalent and stable capture 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 the Fab to its antigen, galectin-3. A carboxymethyldextran-coated CM5 sensor chip (Cytiva) was loaded onto 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 μl / min. 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 μl / min. Protein A affinity purified recombinant anti-PA(S)Mab1.1 obtained from Genscript (Piscataway, NJ, USA) was then covalently immobilized onto the chip surface by injection of a solution of 100 μg / ml of anti-PA(S)Mab1.1 in 10 mM Na acetate pH 4.5 at a flow rate of 5 μl / min for 600 s. Unreacted NHS ester groups were finally blocked by injection of a 0.1 M ethanolamine solution at a flow rate of 5 μl / min for 430 s. This procedure (Figure 17) resulted in an anti-PA(S) Mab1.1 surface density of approximately 13500 resonance units (ΔRU).

[0132] To investigate the binding kinetics of anti-galectin-PAS(200) Fab fragments (SEQ ID NO: 92 and SEQ ID NO: 93) to recombinant galectin-3 (Uniprot Identifier P17931) carrying a C173T mutation and a C-terminal Strep-tag II (SEQ ID NO: 94), purified PASylated Fab fragments were diluted to 3.57 μg / ml in HBS / T and injected into flow channel 2 at a flow rate of 5 μl / min for 40 s, followed by 600 s of buffer flow. This resulted in a PAS-Fab surface density of approximately 580 resonance units (ΔRU), which remained stable within ±7% (Figure 17B). A single cycle kinetic experiment was then performed using five consecutive injections from a 1:2 dilution series of the antigen galectin-3 (1.6 nM to 0.1 nM) for 60 s each at a flow rate of 30 μL / min, followed finally by a dissociation period of 3600 s. The chip was then regenerated with two successive injections of 10 mM glycine / HCl, pH 2.4 for 60 s at a flow rate of 30 μl / min. This regeneration procedure allowed the reuse of the same MAb-functionalized sensor surface for further measurements with PAS-ylated proteins for more than two months.

[0133] The reference-corrected sensorgram (Figure 17C) shows a typical binding curve for the 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), yielding an association rate of 7.9 × 10 6 M -1 s -1 , dissociation rate 3.0×10 -5 s -1 , equilibrium dissociation constant (K D The result was 3.8 pM.

[0134] These findings demonstrate that the highly specific anti-PAS1.1 MAb of the present invention provides a valuable tool for the non-covalent and stable capture of PASylated proteins on SPR sensor chips. Moreover, mild acid regeneration using 10 mM glycine / HCl pH 2.4 completely removed the PASylated proteins together with their ligands, thus providing the ability to reuse the same MAb-functionalized sensor surface for further measurements of PASylated proteins. References Al-Lazikani, B., et al. (1997) J Mol Biol 273 , 927-948. Baker, NA, et al. (2001) Proc Natl Acad Sci USA 98 , 10037-10041. Barlow, DJ, et al. (1986) Nature 322 , 747-748. Binder, U. & Skerra, A. (2017) Curr Opin Colloid Interface Sci 31 , 10-17. Birtalan, S., et al. (2008) J Mol Biol 377 , 1518-1528. Birtalan, S., et al. (2010) Mol Biosyst 6 , 1186-1194. Bolze, F., et al. (2016) Endocrinology 157 , 233-244. Breibeck, J. & Skerra, A. (2018) Biopolymers 109 . Chardes, T., et al. (1999) FEBS Lett 452 , 386-394. Chothia, C., et al. (1989) Nature 342 , 877-883. Clackson, T. & Wells, J.A. (1995) Science 267 , 383-386. Cunningham, B.C. & Wells, J.A. (1989) Science 244 , 1081-1085. Edwardraja, S., et al. (2017) ACS Synth Biol 6 , 2241-2247. Emsley, P., et al. (2010) Acta Crystallogr D Biol Crystallogr 66 , 486-501. Ereno-Orbea, J., et al. (2018) J Mol Biol 430 , 322-336. Fassolari, M., et al. (2013) J Biol Chem 288 , 13110-13123. Feng, Z.P., et al. (2006) Mol Biochem Parasitol 150 , 256-267. Fling, S.P. & Gregerson, D.S. (1986) Anal Biochem 155 , 83-88. Fong, J.H., et al. (2009) PLoS Comput Biol 5 , e1000316. Frank, R. (2002) J Immunol Methods 267 , 13-26. Frishman, D. & Argos, P. (1995) Proteins 23 , 566-579. Gao, J., et al. (2009) Nat Struct Mol Biol 16 , 684-690. Gasteiger, E., et al. (2003) Nucleic Acids Res 31 , 3784-3788. Gebauer, M. & Skerra, A. (2018) Bioorg Med Chem 26 , 2882-2887. Giri, R., et al. (2016) Front Cell Infect Microbiol 6 , 144. Goh, G.K., et al. (2016) Mol Biosyst 12 , 1881-1891. Griffiths, K., et al. (2019) MAbs 11 , 1331-1340. Harari, D., et al. (2014) J Biol Chem 289 , 29014-29029. Harlow, E. & Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Huang, J. & Honda, W. (2006) BMC Immunol 7 , 7. Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest. 5th ed. National Institute of Health, Bethesda, MD. Kabsch, W. (2010) Acta Crystallogr D Biol Crystallogr 66 , 125-132. Karlsson, R., et al. (2006) Anal Biochem 349 , 136-147. Krissinel , E. & Henrick , K. ( 2007 ) J Mol Biol 372 , 774-797. Langin, M., et al. (2018) Nature 564 , 430-433. Loers, G., et al. (2014) Biochem J 460 , 437-446. London, N., et al. (2010) Structure 18 , 188-199. MacRaild, CA, et al. (2016) Structure 24 , 148-157. McCoy, AJ, et al. (2007) J Appl Crystallogr 40 , 658-674. Meerman , HJ & Georgiou , G. ( 1994 ) Bio / Technology 12 , 1107-1110. Mendler, CT, et al. (2015) MAbs 7 , 96-109. Meszaros, B., et al. (2007) J Mol Biol 372 , 549-561. Morath, V., et al. (2015) Mol Pharm 12 , 1431-1442. Murshudov, GN, et al. (2011) Acta Crystallogr D Biol Crystallogr 67 , 355-367. Peplau, E., et al. (2020) Thyroid 30 , 1314-1326. Peplau, E., et al. (2021) Sci Rep 11 , 7358. Richter, A., et al. (2020) Nucl Med Mol Imaging 54 , 114-119. Schellenberger, V., et al.(2009) Nat Biotechnol 27 , 1186-1190. Schiweck, W. & Skerra, A. (1995) Proteins 23 , 561-565. Schlapschy, M., et al. (2013) Protein Eng Des Sel 26 , 489-501. Skerra, A. (1994) Gene 141 , 79-84. Snead, D. & Eliezer, D. (2019) J Biol Chem 294 , 3325-3342. Suzuki, M. & Yagi, N. (1991) Proc Biol Sci 246 , 231-235. Swaminathan, A., et al. (2014) Br J Clin Pharmacol 78 , 1135-1142. Tantos, A., et al. (2012) Mol Cell Endocrinol 348 , 457-465. Uversky, V.N. (2019) Front Phys 7 , 10. Vashist, S.K. & Luong, J.H.T. (2018) Handbook of Immunoassay Technologies - Approaches, Performances, and Applications. 1st ed. Academic Press, London, UK. Voss, S. & Skerra, A. (1997) Protein Eng 10, 975-982. Williams, C.J., et al. (2018) Protein Sci 27 , 293-315. Winn, M.D., et al. (2011) Acta Crystallogr D Biol Crystallogr 67 , 235-242. Wright, P.E. & Dyson, H.J. (2015) Nat Rev Mol Cell Biol 16 , 18-29. Yanisch-Perron, C., et al.(1985) Gene 33 , 103-119. Zander, H., et al. (2007) J Mol Recognit 20 , 185-196.

[0135]

Chem.

[0136]

Chem.

[0137]

Chem.

Claims

1. 1. An antigen-binding molecule directed against an intrinsically unnatural peptide / protein and / or a domain of an intrinsically unnatural peptide / protein, a) CDR-H1 as defined in SEQ ID NO: 35, CDR-H2 as defined in SEQ ID NO: 36; CDR-H3 as defined in SEQ ID NO: 37; and a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:38; CDR-L2 as defined in SEQ ID NO:39; CDR-L3 as defined in SEQ ID NO: 40; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising An antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody comprising the CDR described in a); b) CDR-H1 as defined in SEQ ID NO: 41; and CDR-H2 as defined in SEQ ID NO: 42; CDR-H3 as defined in SEQ ID NO: 43; and a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:44; CDR-L2 as defined in SEQ ID NO:45; CDR-L3 as defined in SEQ ID NO: 46; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising an antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody comprising the CDR described in b); c) CDR-H1 as defined in SEQ ID NO: 47; and CDR-H2 as defined in SEQ ID NO: 48; CDR-H3 as defined in SEQ ID NO: 49; and a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:50; CDR-L2 as defined in SEQ ID NO:51; CDR-L3 as defined in SEQ ID NO:52; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising an antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody comprising the CDR described in c); d) CDR-H1 as defined in SEQ ID NO:53; and CDR-H2 as defined in SEQ ID NO:54; CDR-H3 as defined in SEQ ID NO:55; and a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:56; CDR-L2 as defined in SEQ ID NO:57; CDR-L3 as defined in SEQ ID NO:58; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising An antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody comprising the CDR described in d); e) CDR-H1 as defined in SEQ ID NO:59; and CDR-H2 as defined in SEQ ID NO: 60; a CDR-H3 that comprises or consists of the amino acid sequence Trp-Gly-Arg; and a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:62; CDR-L2 as defined in SEQ ID NO:63; CDR-L3 as defined in SEQ ID NO:64; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising An antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody comprising the CDR described in e) above; And f) CDR-H1 as defined in SEQ ID NO: 65; and CDR-H2 as defined in SEQ ID NO:66; CDR-H3 as defined in SEQ ID NO:67; and a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:68; CDR-L2 as defined in SEQ ID NO:69; CDR-L3 as defined in SEQ ID NO: 70; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising An antibody or an antigen-binding fragment thereof that binds to the same epitope as the antibody comprising the CDR described in f). The antigen-binding molecule is selected from the group consisting of:

2. A method for producing an antigen-binding molecule, preferably an antibody or an antigen-binding fragment thereof, directed against an intrinsically unnatural peptide / protein and / or a domain of an intrinsically unnatural peptide / protein, comprising the steps of immunizing a non-human mammal with an antigen, the antigen is a conjugate of an immune adjuvant and one or more P / A peptides; Each P / A peptide is independently a peptide of about 5 to about 100 amino acid residues, at least 60% of the amino acid residues of said peptides are independently selected from proline and alanine, and each P / A peptide is independently selected from the group consisting of a protecting group R Ｎ is attached to the N-terminal amino group of said peptide.

3. Each P / A peptide is independently Ｎ -(P / A)-R Ｃ and wherein (P / A) is an amino acid sequence consisting of about 8 to about 90 amino acids, at least 70% of the number of amino acid residues in (P / A) are independently selected from proline and alanine, and (P / A) contains at least one proline residue and at least one alanine residue; R Ｎ is a protecting group attached to the N-terminal amino group of (P / A), R Ｃ is an amino acid residue that is attached via its amino group to the C-terminal carboxy group of (P / A) and that contains at least one carbon atom between its amino group and its carboxy group; Each P / A peptide is represented by the C-terminal amino acid residue R Ｃ The method of claim 2, wherein the immunoadjuvant is conjugated to the immunoadjuvant via an amide bond formed between the carboxy group of and a free amino group of the immunoadjuvant.

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

5. The method according to claim 3 or 4, wherein (P / A) in the antigen is an amino acid sequence consisting of 20 to 40 amino acid residues independently selected from proline, alanine and serine, at least 70% of the number of amino acid residues in (P / A) are independently selected from proline and alanine, and (P / A) contains at least one proline residue and at least one alanine residue.

6. The method according to any one of claims 3 to 5, wherein the ratio of the number of proline residues contained in (P / A) to the total number of amino acid residues contained in (P / A) is ≧10% and ≦70%, preferably ≧20% and ≦50%, more preferably ≧25% and ≦40%.

7. 7. The method of any one of claims 3 to 6, wherein (P / A) in the antigen consists of (i) five or more subsequences 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 additional amino acid residues independently selected from proline, alanine and serine.

8. 8. The method of any one of claims 3 to 7, 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-APSA, (iv) a duplication of any of the foregoing sequences, or (v) a combination of two of the foregoing sequences.

9. R Ｎ are 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 １～６ alkyl), -N, N(C １～８ Alkyl) ２ , N,N,N-tri(C １～６ -alkyl) ３ 9. The method of claim 2, wherein the alkyl group is selected from N,N-tetramethylene and N,N-pentamethylene.

10. R Ｃ Is H ２ N-(C １～１２ hydrocarbyl)-COOH, R Ｃ H ２ N-(CH ２ ) １～１０ -COOH, H ２ N-phenyl-COOH, and H ２ N-cyclohexyl-COOH, R Ｃ H ２ N-CH ２ -COOH(Gly), H ２ N-(CH ２ ) ２ -COOH(β-Ala), H ２ N-(CH ２ ) ３ -COOH, H ２ N-(CH ２ ) ４ -COOH, H ２ N-(CH ２ ) ５ -COOH, H ２ N-(CH ２ ) ６ -COOH, H ２ N-(CH ２ ) ７ -COOH, H ２ N-(CH2) ８ More preferably, R is selected from —COOH, p-aminobenzoic acid, and 4-aminocyclohexanecarboxylic acid; Ｃ Is H ２ N-(CH ２ ) ５ The method according to any one of claims 3 to 9, wherein -COOH is even more preferred.

11. 11. The method of claim 2, wherein the P / A peptide contained in the antigen adopts a random coil conformation and / or the P / A peptide contained in the antigen lacks charged residues.

12. 12. The method according to any one of claims 2 to 11, wherein the immune adjuvant is selected from keyhole limpet hemocyanin (KLH), ovalbumin (OVA), and bovine serum albumin (BSA), preferably the immune adjuvant is keyhole limpet hemocyanin (KLH).

13. An antigen according to any one of claims 2 to 12.

14. Non-therapeutic use of the antigen according to claim 13 for the generation of antigen-binding molecules, preferably antibodies or antigen-binding fragments thereof, directed against naturally unnatural peptides / proteins and / or domains of naturally unnatural peptides / proteins, including immunization of non-human mammals.

15. the intrinsically unfolded peptide / protein and / or domain of the intrinsically unfolded peptide / protein is a Pro / Ala rich sequence, Preferably, the Pro / Ala-rich sequence is an amino acid sequence consisting of at least 20 amino acid residues that form a random coil conformation, whereby the amino acid residues that form the random coil conformation are selected from Pro(P), Ala(A) and Ser(S), preferably from Pro(P) and Ala(A). The antigen-binding molecule of claim 1, the method of any one of claims 2 to 12, or the non-therapeutic use of claim 14.

16. The Pro / Ala rich sequence has the structure (P / S)A(A / S)P; and / or PA(A / S)P and Preferably, the epitope comprises an epitope stretch selected from the group consisting of PAAP, PASP, PAPASP, PAPAAP, PASPAAP, and APSA.

17. 17. An antigen-binding molecule, preferably an antibody or an antigen-binding fragment thereof, directed against an intrinsically unfolded peptide / protein and / or a domain of an intrinsically unfolded peptide / protein, obtainable by a method according to any one of claims 2 to 12, 15 or 16.

18. a) CDR-H1 as defined in SEQ ID NO: 35, CDR-H2 as defined in SEQ ID NO: 36; CDR-H3 as defined in SEQ ID NO: 37; and / or a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:38; CDR-L2 as defined in SEQ ID NO:39; CDR-L3 as defined in SEQ ID NO: 40; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising An antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs described in a); b) CDR-H1 as defined in SEQ ID NO: 41; and CDR-H2 as defined in SEQ ID NO: 42; CDR-H3 as defined in SEQ ID NO: 43; and / or a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:44; CDR-L2 as defined in SEQ ID NO:45; CDR-L3 as defined in SEQ ID NO: 46; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising An antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs described in b); c) CDR-H1 as defined in SEQ ID NO: 47; and CDR-H2 as defined in SEQ ID NO: 48; CDR-H3 as defined in SEQ ID NO: 49; and / or a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:50; CDR-L2 as defined in SEQ ID NO:51; CDR-L3 as defined in SEQ ID NO:52; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising An antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs described in c); d) CDR-H1 as defined in SEQ ID NO:53; and CDR-H2 as defined in SEQ ID NO:54; CDR-H3 as defined in SEQ ID NO:55; and / or a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:56; CDR-L2 as defined in SEQ ID NO:57; CDR-L3 as defined in SEQ ID NO:58; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising An antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs described in d); e) CDR-H1 as defined in SEQ ID NO:59; and CDR-H2 as defined in SEQ ID NO: 60; a CDR-H3 that comprises or consists of the amino acid sequence Trp-Gly-Arg; and / or a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:62; CDR-L2 as defined in SEQ ID NO:63; CDR-L3 as defined in SEQ ID NO:64; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising An antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs described in e); And f) CDR-H1 as defined in SEQ ID NO: 65; and CDR-H2 as defined in SEQ ID NO:66; CDR-H3 as defined in SEQ ID NO:67; and / or a variable heavy (VH) chain comprising CDR-L1 as defined in SEQ ID NO:68; CDR-L2 as defined in SEQ ID NO:69; CDR-L3 as defined in SEQ ID NO: 70; or an antibody or antigen-binding fragment thereof comprising a variable light (VL) chain comprising An antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising any one or more of the CDRs described in f).

18. The antibody or antigen-binding fragment thereof of claim 17, selected from the group consisting of:

19. An antibody or antigen-binding fragment thereof, (a) the amino acid sequence of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21; or a sequence having 85%, preferably 87%, more preferably at least 90% sequence identity with SEQ ID NO: 11, 13, 15, 17, 19 or 21. and / or the amino acid sequence of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22; or a sequence having 85%, preferably 87%, more preferably at least 90% sequence identity with SEQ ID NO: 12, 14, 16, 18, 20 or 22. A variable light (VL) chain sequence comprising: or (b) an antibody or an antigen-binding fragment thereof that binds to the same epitope as the antibody described in (a); An antigen-binding molecule according to any one of claims 1 and 15 to 18.

20. Fab fragment, F(ab') ２ The antigen-binding molecule of any one of claims 1 and 15 to 19, which is an antigen-binding fragment selected from a fragment, an Fv fragment, or an scFv fragment.

21. 21. The antigen-binding molecule of any one of claims 1 and 15 to 20, which is conjugated or fused to a reporter molecule and / or a label.

22. 22. The antigen-binding molecule of any one of claims 1 and 15 to 21, for use in matrix-based protein / peptide purification or immobilization.

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