RU2798971C2 - Methods for prediction, detection and reduction of non-specific protein interference in analysis methods using single variable domain of immunoglobulins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a new method of reducing non-specific protein interference. The invention discloses a single variable domain (ISV) of an immunoglobulin heavy chain, in the C-terminal region of the amino acid sequence of which a number of amino acid substitutions have been introduced in order to reduce the tendency of the protein to non-specific interference.

EFFECT: proteins and protein constructs containing such modified ISVs with improved properties may be useful in therapy and diagnostics.

21 cl, 9 ex, 12 tbl, 9 dwg

Description

The present invention relates to the region of single variable domains of immunoglobulins.

In general, an immunoglobulin single variable domain or "ISV" is defined in this document as an amino acid sequence that:

- has an immunoglobulin-specific chain fold, or which, under suitable conditions (e.g., physiological conditions), is capable of forming an immunoglobulin-specific chain fold (i.e., by folding (folding)) in order to form an immunoglobulin variable domain (such as, e.g. VH, VL or VHH domain);

and which

- forms (or under suitable conditions is able to form) an immunoglobulin variable domain containing a functional antigen-binding site (in the sense that it does not require interaction with another immunoglobulin variable domain (such as VH-VL interaction) to form a functional antigen-binding site).

Some examples of immunoglobulin single variable domains currently known in the art are VHH and/or (other) Nanobodies, dAbs and (single) domain antibodies. Of these, as of the filing of this application, several Nanotels are in phase I and phase II clinical trials. It is important to have available reliable assays for the examination of biological samples obtained from people who are treated with ISVs (eg, participants in clinical trials, and after the introduction of such ISVs into the market, patients treated with such ISVs).

This is important not only for regulatory purposes, but also for the treatment of patients with biologics, as prescribing clinicians would also like to have reliable assays available to monitor various aspects of treatment.

For example, in clinical studies of biological drug molecules, it is important to evaluate their immunogenic potential, and in particular the level to which they are able to detect so-called “anti-drug antibodies (anti-drug antibodies)” or “ADA”. This is determined using the so-called “anti-drug antibody assay” or “ADA (immuno) assay” (see, for example, review by Shankar et al., Journal of Pharmaceutical and Biomedical Analysis, 48 (2008), 1267-1281; and also Mire- Sluis et al., J. Immunol. Meth. 289 (2004), 1-16; Peng et al., Journal of Pharmaceutical and Biomedical Analysis, 54 (2011), 629-635; and Loyet et al., J. Immunol Meth 345 (2009), 17-28 Such an ADA assay and its methods are known in the art of pharmacology and are standard and commonly used in clinical trials of biological drugs (and also required by many regulatory authorities around the world). ).

For example, as described on pages 3 and 4 of the Mire-Sluis article and as, for example, also schematically illustrated with the Figures in the Peng article, quite a few different kinds of ADA assays are known, such as bridge ELISA, direct immunofluorescence (direct ELISA), indirect ELISA format, radioimmunoprecipitation analysis (RIP), surface plasmon resonance and electrochemiluminescent bridge analysis. Other formats for conducting an ADA immunoassay will be apparent to those skilled in the art.

It has been proven that a number of different commercially available technology platforms, also well known to the person skilled in the art, are suitable for preparing and conducting an ADA analysis. These include, but are not limited to, the MSD platform (Mesoscale), Gyrolab (Gyros), and the octet platform (Fortebio).

In addition, some non-limiting examples of types of ADA analysis are schematically shown in Figures 1A - 1C.

In general, it should be noted that in such an ADA assay to detect or measure ADA to ISV, ISV is used as an “analytical agent” (i.e., as a compound used to detect the presence of any ADA in the test sample) , and ADA is an “antigen” (i.e., a compound that should be detected in the test sample). Thus, in these assays, ISV is typically/often bound to a carrier (such as an ELISA plate) while ADA (if any) is present in the sample being assayed.

In order to better understand the invention described in this document, it should first be noted that in the methods used in this specification to predict whether an ISV will cause protein interference, the ISV will typically be used as an "antigen" (i.e., as a compound, to be detected), and the antibody (described later in this document) is used as an “analyzing agent” (i.e., as a means to detect whether a given ISV binds or not, respectively; and thus, whether there is a high or increased risk of protein interference or not, respectively). Thus, in this method of the invention, the antibody used as the assay agent (which is also referred to in this document as “assay antibody”) will typically be associated with a carrier (i.e., an ELISA plate) and the ISV will be in the test sample. However, it should be noted that the invention is not limited to assay methods in which the "analytical antibody" is bound to a carrier. For example, in an alternative way to perform the assay according to the invention (as shown, for example, in Figure 1 and described in the Examples), the analytical antibody is instead used as a bridging agent and thus will be in solution, and will not be associated with the tablet ( although indirectly it is related to the tablet via the ISV that covers the tablet). At the same time, in the specific bridged assay method described in the Examples (which is a competitive assay method), the assay antibody is still used as the assay agent (i.e., to determine whether the ISV of interest binds or not, respectively ; and thus whether there is a high or increased risk of protein interference or not, respectively). Also, based on the further disclosure, this document provides that one skilled in the art will be able to develop other assays in which an analytical antibody can be used as an analytical agent in order to determine whether a given ISV can bind or not, respectively; and thus has a high or increased risk of protein interference or not).

As a result of research into single chain Fvs or "ScFvs" (which are constructs containing immunoglobulin single variable domains that, like ISVs, are not associated with constant domains), it has been described in the art that the C-terminus of an immunoglobulin variable domain forms a hydrophobic region that, in an antibody, is "immersed" in the interaction (contact) between the variable domain and the constant domain, but which becomes accessible to the solvent when the variable domain is not associated with the constant domain (Nieba et al., Protein Engineering, 10, 435-444 (1997)). It has also been described that the accessible C-terminus can form B-cell epitopes that can mediate and/or interact with (emerging and/or existing) drug antibodies (WO 11/07586), the presence of which can then be detected. using the aforementioned ADA analysis. For this reason, it has been proposed to mutate certain amino acid residues forming part of the C-terminus of variable domains to reduce said hydrophobicity and/or remove said epitopes. For example, Nieba et al. propose to mutate positions 11, 14, 41, 84, 87 and/or 89 of the VH region (Kabat numbering), whereas WO 11/07586 proposes to mutate positions 99, 101 and/or 148 (AHo numbering) of the VL- domain or positions 12, 97, 98, 99, 103 and/or 144 of the VH domain (again AHo numbering - these positions correspond to positions 11, 83, 84, 85, 89 and 103 according to Kabat).

However, none of these sources conclude that certain proteins present in the subject's blood or serum can interfere with ADA analysis using ISV, and therefore these sources do not address the issue of how to avoid aspecific protein interference. in such ADA assays in order to enable the use of the ADA assay to detect the actual presence/extent of (emerging or existing) anti-drug antibodies in the sample to be tested.

In contrast, the present invention provides assay methods and methods that allow one of skill in the art to readily predict whether or not a single variable domain of an immunoglobulin will be prone to undergo aspecific protein interference in an ADA assay. In addition, the assay methods and methods described herein enable one skilled in the art, when it is found that a variable domain may have a propensity or risk for such protein interference in an ADA assay, to easily check for (putative) modifications of the variable domain in order to predict whether such (presumed) modifications will reduce or almost completely prevent such protein interference.

The present invention also describes a number of modifications that can be made to the variable domains to reduce or actually prevent such protein interference. In accordance with one non-limiting aspect, this modification involves the introduction of a limited number (as further described in this document) amino acid residues (as further described in this document) to the C-terminus of the variable domain. Surprisingly, it has been found that for many different variable domains or constructs based on them, the addition of even a single amino acid residue at the C-terminus (for example, a single alanine residue) can largely or even almost completely eliminate the problem of protein interference in ADA analysis, even despite the fact that the addition of one such amino acid alone is not sufficient to "cover" or "sink" the hydrophobic region, which, according to Nieba et al., is present at the C-terminus of ISV. Similarly, but without wishing to limit the invention in any way by any mechanism or explanation, it is also contemplated that the addition of such a single amino acid would not be sufficient to "cover" or "sink" any B-cell epitopes that, according to WO 11 /07586 may be present at the C-terminus of the variable domain. It should also be noted that although according to this specific aspect of the present invention, the addition of a limited number or even a single amino acid to the C-terminus of the variable domain (i.e., without any substitutions within the C-terminus itself, as proposed Nieba et al and WO 11/07586) can, and in many cases will, greatly reduce or even virtually eliminate the problem of aspecific protein interference, it is also within the scope of this aspect of the invention that such additions at the C-terminus are combined with mutations at the C- end section. In this regard, however, it should also be noted that the invention is not limited to the rationale behind the creation of such mutations. For example, it is well known to mutate amino acid residues within the C-terminus (including those positions clearly indicated by Nieba et al. and in WO 11/07586) in order to humanize a variable domain (including, without limitation, the V _HH domain) or in order to "camelize" the V _H domain (for example, reference may be made to WO 08/020079 and several other Ablynx NV applications mentioned in this specification).

It is contemplated that the methods, assays, and modifications provided herein may be applied to any variable domain that does not bind to or otherwise interact with a constant domain (or other moiety or peptide fragment that functions to "screen", "covering" or "sinking" the C-terminal region of the variable domain) and, more generally, any variable domain that has solvent-accessible C-terminal regions. However, according to one preferred but non-limiting aspect of the invention, methods, methods of analysis and modification can be applied in particular to heavy chain variable domains (V _H domains), and according to one particular aspect of the invention, to V _HH domains.

It is also contemplated that the techniques, methods of analysis and modification described herein may be appropriately applied to protein constructs containing one or more variable domains, and in particular to those constructs in which the variable domain forms the C-terminal portion of the construct or , in the case of the methods and methods of analysis described in this document, in which the C-terminal region of the variable domain is otherwise available to the solvent. In addition, in accordance with one preferred but non-limiting aspect of the invention, methods, methods of analysis and modification are applied to constructs in which the V _H domain (and in particular the V _HH domain) forms the C-terminal part of the construct or, in the case of methods and methods of analysis of this invention, for other reasons available to the solvent.

Some non-limiting examples of such constructs are multivalent, multispecific (such as bispecific), or multiparatopic (such as biparatopic) constructs that contain two or more ISVs linked directly or through one or more suitable linkers (and again in accordance with one specific aspect, V _H or V _HH domain) forming the C-terminal portion of such a construct. For example, and without limitation, such a construct may consist entirely of V _H domains, and in particular of Nanobodies (i.e., V _HH domains, humanized V _HH domains, or camelized V _H domains), again linked directly or through one or more suitable linkers. For some non-limiting examples of such constructs and a general idea of how such constructs can be made (in particular based on Nanobodies), reference may be made to Conrath et al., JBC 276, 10(9), 7346 (2001) and to a review article by Muyldermans. Reviews in Mol. Biotechnol., 74:27 (2001).

However, it is also contemplated that the invention may be applied to other constructs that have a solvent-accessible variable domain and in particular have a variable domain at their C-terminus, such as, for example, single-chain Fvs, and in particular ScFvs, which have a heavy chain variable domain at the C-terminus.

In the present detailed description and claims, terms like “ISV”, “analytical agent”, and “protein interference” have the meanings set forth below.

In particular, the ISV described herein may be a Nanobody or (other) ISV (ie other than Nanobody) that is a VH domain or that contains a VH domain; and preferably is Nanotel.

Also, any protein or polypeptide containing an ISV (such as an ISV-based drug) preferably has said (or at least one) such ISV at its C-terminus. Further, said ISV may specifically be a Nanobody or (other) ISV (ie other than Nanobody) which is a VH domain or contains a VH domain; and preferably is Nanotel.

The invention described in this document is particularly intended and suitable for the use of ISVs that contain, are based on, and/or are derived from heavy chain variable domains such as VH domains (including human VH domains), and Nanobodies such as VHH domains (including humanized and sequence optimized VHH domains) or camelized VH domains. They can be synthetic (eg, derived from a synthesized library and/or based on fixed framework regions), semi-synthetic (eg, humanized, camelized, or sequence-optimized, or obtained by affinity maturation or CDR grafting starting from natural VH or VHH domain) or VH or VHH domains of completely natural origin. The invention will thus be described hereinafter in relation to ISVs that are, are based on, and/or are derived from VH or VHH domains.

In developing the present invention, it has been found that when using certain assay methods (such as, for example, ADA immunoassay) to examine biological samples (such as blood samples, including whole blood, serum and plasma, intraocular fluid, bronchoalveolar fluid/BALF, cerebrospinal fluid or other samples of biological fluids) protein interference may be observed, and that such protein interference may generate a non-specific signal in some of these assays and/or in some of these samples. It has also been found that such protein interference may occur not only in samples that have been obtained from subjects who have been treated with ISVs (and in particular with Nanobodies; or proteins, polypeptides, or other biologics that contain at least one such ISV or Nanotelo) and/or who have been administered ISV (e.g., patients or participants in clinical trials), but also in samples from subjects who have never received ISV (showing that such interference is likely due to non-specific protein-protein interactions with pre-existing proteins, and not any new ADA that has just appeared).

While it has been found that such protein interference and/or such signal in such assays is not associated with any alteration or reduction in the pharmacological properties (such as pharmacokinetic/PK or pharmacodynamic/PD properties) of the ISV, it would be desirable to have methods suitable for predicting, detecting, reducing and/or, if possible, avoiding such non-specific protein interference. This is the main purpose of the present invention.

In particular, the invention provides and, in certain specific but non-limiting aspects, relates to:

- methods of analysis that can be used to predict whether a given ISV will be subject to such protein interference and/or will result in such a (non-specific) signal in such an assay (such as, for example, an ADA immunoassay). Such predictive analysis methods can be used, for example, to test whether a given ISV tends to generate such protein interference and/or such signal; to select ISVs that are not or less susceptible to such protein interference or signaling; as an analysis method or test that can be used to test whether certain modification(s) of an ISV will (in whole or in part) reduce its propensity to generate such interference or such signal; and/or as an assay or test that can be used to direct modification or improvement of the ISV so as to reduce its propensity to generate such protein interference or signal;

- methods for modifying and/or improving ISVs with respect to eliminating or reducing their tendency to generate such interference or such a signal;

- modified or improved ISVs that do not have or have a reduced propensity to generate such protein interference or such signal;

- ISVs that have been specifically selected in such a way (eg, using the assay method(s) described herein) that they do not or have a lower/reduced propensity to generate such protein interference or such signal;

- modified and/or improved ISVs that do not have or have a low/lower/reduced propensity to generate such protein interference or such signal.

For example, in a first non-limiting aspect, the invention relates to a method that can be used to predict whether a given ISV or Nanobody (or ISV or Nanobody drug) will generate (or have a high or increased risk of causing) protein interference in an immunoassay. (i.e., after administration to a subject, a biological fluid sample is obtained from said subject, and said sample is subjected to an immunoassay described later in this document), said method comprising conducting an immunoassay that at least comprises the steps of:

(i) contacting said ISV or Nanobody (or ISV or Nanobody drug) with an antibody that has been derived from a human and that has been selected, engineered and/or isolated based on its ability to recognize and/or bind to C -terminus of ISV or Nanotel ("analytical antibody"); And

(ii) determining whether said ISV or Nanobody (or ISV or Nanobody drug) is bound by said antibody in said immunoassay.

In this method, when an ISV, Nanobody, ISV-based or Nanobody-based drug is bound by said analytical antibody, said ISV, Nanobody, ISV-based or Nanobody-based drug can be expected to generate (or have a high or increased risk of generating) such protein interference (as defined later in this document). Based on this, for example, said ISV, Nanobody, ISV-based drug or Nanobody-based drug can be modified or improved so as to reduce or eliminate their tendency to generate such protein interference (which again can be determined using the above method assay), and this document will describe some strategies that can be used to modify said ISV, Nanobody, ISV drug, or Nanobody (which include, for example, adding a small amount of amino acid residues to the C-terminus and/or introducing one or more specific amino acid substitutions).

Thus, in a broad sense, the invention makes available to the skilled person assay methods and techniques that can be used to predict the propensity of an ISV, Nanobody, ISV-based or Nanobody-based drug to generate protein interference and/or as a tool to improve ISV in such a way to reduce or avoid their tendency to generate protein interference. To this end, the invention also provides the skilled person with the means to select ISVs, Nanotels, ISV drugs, or Nanotels based drugs based on their low or reduced ability (or lack of ability) to generate protein interference. Thus, the invention provides the skilled person with an important analysis method and tool that can be used in the optimization and development of ISVs, Nanotels, ISV-based drugs or Nanotel-based drugs.

Also, as described hereinafter, the invention provides the skilled artisan with a number of ways in which an ISV, Nanobody, ISV drug, or Nanobody drug can be modified or improved in such a manner as to reduce or avoid its propensity to generate protein interference. Thus, the invention generally makes available modified and/or improved ISVs, Nanobodies, ISV drugs or Nanobodies with reduced, low and without any propensity to generate protein interference.

As described later in this document, the invention can particularly be used to predict whether a given ISV or Nanobody (or ISV or Nanobody drug) will generate protein interference (as described later in this document) in an immunoassay, and in particular, in ADA analysis. For example, said ADA assay may be an ADA assay designed to detect or measure ADA against ISV in a general sense, and in particular may be an ADA assay designed to detect or measure ADA against ISV used in steps (i) and (ii) above.

In addition, as mentioned above, the ISV described here may, in particular, be a Nanobody or (other) ISV (ie other than Nanobody) that is a VH domain or that contains a VH domain; and preferably is Nanotel.

Also, any protein or polypeptide containing an ISV (such as an ISV drug) preferably has said (or at least one) such ISV at its C-terminus. In addition, said ISV may in particular be either a Nanobody or (another) ISV (ie other than a Nanobody) which is a VH domain or which contains a VH domain; and preferably is Nanotel.

The sample tested using the specified analysis method or ADA analysis is also referred to in this document as the “test sample” or “analyzed sample”. To avoid confusion, “test sample” or “analyte sample” should not be confused with the biological sample that is used here as starting material for the preparation of the (polyclonal or monoclonal) “analytical antibody” used in the invention.

In one particular preferred but non-limiting aspect, the invention can be used to predict whether a given ISV or Nanobody (or ISV or Nanobody drug) will generate protein interference (as described later in this document) in an immunoassay (and in particular , in ADA analysis), which implies the use of such an ISV. Also, said ADA assay may, for example, be an ADA assay designed to detect or measure ADA against ISV in a broad sense, and may specifically be an ADA assay to detect or measure ADA against ISV used in the steps ( i) and (ii) above.

In an even more specific but non-limiting aspect, the invention can be used to predict whether a given ISV or Nanobody (or ISV or Nanobody drug) will generate protein interference (as described later in this document) in an immunoassay (and in particular , in ADA-analysis), which is intended to detect or measure the content in the sample of any ADA to ISV. For example, such an immunoassay may be one of the well-known types of ADA assay (for which reference is made in the description to the prior art ADA assays), which is performed to detect or measure the "presence" of any ADA to the specified ISV in the "test sample ”, where the specified “study sample” is a sample of biological fluid (as described here), obtained from the subject, which was injected with the specified ISV (as described later in this document).

As described later in this document, in all of these aspects (and additional aspects of the invention described herein), the invention can also be used to select ISVs that are not or less susceptible to such protein interference in such an immunoassay or ADA assay; as an assay method or test that can be used to test whether a particular ISV modification(s) will (in whole or in part) reduce its propensity to generate such interference in such an immunoassay or ADA assay; and/or as an assay method or test that can be used to direct modification or improvement of the ISV so as to reduce its tendency to generate protein interference in such an immunoassay or ADA assay.

Other aspects, embodiments, advantages and uses of the invention will become apparent from the following description.

In this detailed description, whenever the term “ISV” is used, it should be understood that:

- such ISV is preferably a Nanobody, where the term "Nanobody" in most cases is as defined in WO 08/020079 or WO 09/138519, and in a specific aspect, as a rule, means VHH, humanized VHH or camelized VH (such as a camelized human VH) or, in most cases, a VHH with an optimized sequence (eg, optimized for chemical stability and/or solubility, maximum overlap with known human framework regions, and maximum expression). It should be noted that the terms Nanobody or Nanobody are registered trademarks of Ablynx N.V. and thus may also be referred to as Nanobody® and/or Nanobody®);

- the term “ISV” in its broadest sense also includes “ISV-based biologicals” and, in cases where the ISV is a Nanobody, “Nanobody-based biologicals”. An “ISV-based biological product” is defined in this specification as a protein, polypeptide, or other biological drug that contains or actually consists of at least one (eg, one, two, or three) ISVs. Similarly, a “Nanobody-based biological product” is defined as a protein, polypeptide, or other biological drug that contains or actually consists of at least one (eg, one, two, or three) Nanobodies. As with the term “ISV”, whenever the term “ISV-based biologic” is used, it should be understood that such ISV-based biological is preferably a Nanotel-based biologic. In the context of the present invention, both “ISV-based biologics” and “Nanobody-based biologics” may, for example, be monovalent, bivalent (or multivalent), bispecific (or multispecific), and biparatopic (or multiparatopic) ISV constructs, or Nanobody-construction, respectively. In addition, any ISV-based or Nanobodies-based biological preparation may, for example, in addition to one or more (for example, one, two or three) ISVs or Nanobodies optionally additionally contain one or more (for example, one or two) other additional therapeutic agents and/or one or more (eg one or two) other agents that affect the pharmacokinetic or pharmacodynamic properties of the ISV-based or Nanobody-based biological product (such as half-life). Suitable examples of such additional therapeutic or other agents are known to the skilled person and in most cases may include, for example, any therapeutically active protein, polypeptide or other binding domain or binding unit, as well as modifications, for example, described on pages 149 - 152 of WO 09 /138159. The ISV based biological or the Nanotel based biological is preferably therapeutic or intended for use as a therapeutic agent (which includes prophylaxis and diagnosis) and for this purpose preferably contains at least one ISV to a therapeutically important target (such as, eg RANK-L, vWF, IgE, RSV, CXCR4, IL-23 or other interleukins, etc.). As separate, but non-limiting examples of such ISV-based or Nanotel-based biological preparations, various applications of Ablynx N.V. (such as, for example, and without limitation WO 2004/062551, WO 2006/122825, WO 2008/020079 and WO 2009/068627), as well as, for example (and without limitation) applications such as WO 06/038027, WO 06 /059108, WO 07/063308, WO 07/063311, WO 07/066016 and WO 07/085814. In addition, in this detailed description, unless this document expressly states otherwise, all terms mentioned herein have the meaning given in WO 09/138519 (or the prior art given in WO 09/138519) or WO 08/020079 ( or the prior art given in WO 08/020079). Also, in cases where a method or procedure is not described in detail herein, it can be carried out as described in WO 09/138519 (or the prior art given in WO 09/138519) or WO 08/020079 (or the prior art given in WO 08/020079).

In particular, the following terms have their original meanings and/or, where applicable, may be defined in the form described on pages 62-75 of WO 09/138519: “agonist”, “antagonist”, “inverse agonist”, “nonpolar uncharged amino acid residue”, “polar uncharged amino acid residue”, “polar charged amino acid residue”, “sequence identity”, “absolutely the same” and “amino acid difference” (when referring to the sequence comparison of two amino acid sequences), “mostly (in ) isolated (form)”, “domain”, “binding domain”, “antigenic determinant”, “epitope”, “to” or “against” (antigen), “specificity” and “half-life”. In addition, the terms “modulate” and “modulate”, “interaction site”, “specific to”, “cross-block”, “cross-block” and “cross-block” and “basically independent of pH” have values as defined on (and/or may be defined as described on) pages 74-79 of Applicant's WO 10/130832. Also, when referring to a construct, compound, protein, or polypeptide of the invention, terms like “univalent”, “bivalent” (or “multivalent”), “bispecific” (or “multispecific”), and “biparatopic” (or “multiparatopic”) may have the meaning given in WO 09/138.519, WO 10/130832 or WO 08/020079.

The term "half-life" as used herein in relation to an ISV, Nanotel, ISV biologic, Nanotel biologic, or any other amino acid sequence, compound, or polypeptide can in most cases be defined as described in paragraph o) on page 57 of WO 08/020079, and as indicated there, refers to the time required for a 50% decrease in serum concentration of an amino acid sequence, compound or polypeptide in vivo, for example, due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms. The in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined by any known method, for example, by pharmacokinetic analysis. Suitable techniques will be apparent to one skilled in the art and in most cases they can be as described in paragraph o) on page 57 of WO 08/020079. In addition, as indicated in paragraph o) on page 57 of WO 08/020079, the half-life can be expressed using parameters such as t1/2-alpha, t1/2-beta and area under the curve (AUC). In this regard, it should be noted that the term "half-life" as used herein specifically refers to t1/2-beta or final half-life (where t1/2-alpha and/or AUC or both may be disregarded). For example, reference may be made to the Experimental Section below, as well as standard guidelines such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinete analysis: A Practical Approach (1996). Reference may also be made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). Similarly, the terms “increased half-life” or “increased half-life”, which are also defined in paragraph o) on page 57 of WO 08/020079, refer in particular to an increase in t1/2-beta, with or without an increase in t1/ 2-alpha and/or AUC or both.

When a term is not specifically defined in this specification, it has the meaning generally accepted in the art and understood by those skilled in the art. Reference may be made to standard manuals such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd. Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987); Lewin, "Genes II", John Wiley & Sons, New York, N.Y., (1985); Old et al., “Principles of Gene Manipulation: An Introduction to Genetic Engineering”, 2nd edition, University of California Press, Berkeley, CA (1981); Roitt et al., “Immunology” (6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential Immunology, 10th Ed. Blackwell Publishing, UK (2001); and Janeway et al., “Immunobiology” (6th Ed.), Garland Science Publishing/Churchill Livingstone, New York (2005), as well as the general prior art cited therein.

Also in this specification, the amino acid residues of the Nanobody are numbered according to the conventional VH domain numbering given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91) for VHH domains of the Camelids family in Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195; or in those mentioned in this description. In accordance with this numbering, FR1 of Nanobody contains amino acid residues at positions 1-30, CDR1 of Nanobody contains amino acid residues at positions 31-35, FR2 of Nanobody contains amino acids at positions 36-49, CDR2 of Nanobody contains amino acid residues at positions 50-65, FR3 of Nanobody contains amino acid residues at positions 66-94, Nanobody CDR3 contains amino acid residues at positions 95-102, and Nanobody FR4 contains amino acid residues at positions 103-113. [In this regard, it should be noted that (as is well known in the art for VH domains and for VHH domains) the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated in the Kabat numbering (available meaning that one or more positions according to the Kabat numbering may not be occupied in the available (in this) sequence, or the given sequence may contain more amino acid residues than the number allowed by the Kabat numbering). Generally, this means that the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the given sequence. However, in most cases, it can be argued that, according to Kabat numbering and regardless of the number of amino acid residues in the CDR, position 1, according to Kabat numbering, corresponds to the beginning of FR1 and vice versa, position 36, according to Kabat numbering, corresponds to the beginning of FR2 and vice versa, position 66 according to Kabat numbering corresponds to the beginning of FR3 and vice versa, and position 103 according to Kabat numbering corresponds to the beginning of FR4 and vice versa].

Alternative methods for numbering amino acid residues of VH domains, which can also be applied to Camelids VHH domains and Nanobodies, are those described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in the present description, aspects and figures, unless otherwise indicated, the numbering according to Kabat in relation to VHH domains according to Riechmann and Muyldermans will be used.

It should also be noted that the Figures, any Sequence Listings and Experimental Part/Examples are provided to further illustrate the invention and should not be interpreted or construed as limiting the scope of the invention and/or the appended claims in any way, unless this document clearly states otherwise.

Additionally, it should be noted that the present invention is not specifically limited to any causal relationship, explanation, hypothesis, or mechanism of protein interference (and/or immunoassay-derived signals) that is observed in, and which can be reduced according to the present invention. However, it is recognized that the blood or serum (or other body fluids referred to in this document) of certain individuals or groups of individuals may contain distinct (pre-existing) proteins that, under certain conditions, may (non-specifically) bind to ISV, resulting in an interfering signal in some of the assay methods used to examine blood or serum samples obtained from such individuals. This is based on the observation made during the development of the present invention that non-specific protein interference, which is considered by the present invention, occurs not only when analyzing samples obtained from subjects who have previously been administered ISV, but also when analyzing samples obtained from subjects who have not previously been administered ISV. received ISV.

In particular, based on observations made during the development of the present invention, and although the invention is not limited thereto, it is believed that such pre-existing proteins may, in particular, bind to the C-terminus of such ISVs (which, in the full-length conventional 4-strand monoclonal antibody, as well as in the “heavy chain only” antibodies found in the Camelidae family, are associated with the rest of the antibody, i.e. with the CH1 region in conventional monoclonal antibodies and the hinge region in the heavy chain of Camelidae antibodies, respectively - and thus in such full length antibodies can be protected from such protein interference).

This is supported by the findings obtained by the present inventors during the development of the present invention (which are described later in this document) that certain (simple) modifications of ISVs at their C-terminus can significantly reduce or actually prevent protein interference. Accordingly, methods for modifying ISVs in this way, as well as ISVs that have been modified in this way, constitute additional aspects of the invention, as described later in this document.

In particular, the present invention can be used to reduce or avoid protein interference and/or signals due to non-specific binding in immunoassays that are performed on biological samples (such as blood or serum samples) obtained from a subject who has been administered a (biological) drug ( in which case such samples are referred to herein as “test samples” or “test samples”). Examples are immunoassays used to determine the parameters of drug distribution and antibody formation after administration of a biological agent to a subject, such as those mentioned in the “Guideline on the Clinical Investigation of the Pharmacokinetics Therapeutic Proteins” (document CHMP/EWP/89249/2004, dated January 27, 2007 ) issued by the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMEA). As stated on pages 4 and 5 of this document:

“Several possible deficiencies have been identified that can lead to erroneous determination of drug distribution and antibody production parameters. The following questions should be considered […]:

immunoassay

Drug analysis:

[…]

(iii) Interference with endogenous substances.

(iv) Interference with plasma components or anti-drug antibodies that bind to the test substance and inhibit complementary binding to the immobilized antibody.”

In particular, the invention can be used to predict, reduce or prevent this type of interference in immunoassays that are used in the analysis of test samples / test samples of biological fluids taken from subjects who have been administered ISV (and in particular Nanobodies; or a biological preparation based on ISV or Nanotel Biological as defined later in this document).

The invention can particularly be used to predict, reduce or prevent this type of (non-specific) protein interference in immunoassays that are used to determine drug distribution parameters and/or to determine the production of any ADA (anti-drug) antibodies. In this regard, it should be noted that, in most cases, in this Detailed Description and in the appended claims, when using the phrase "predicting, reducing or preventing protein interference" includes not only predicting, reducing or preventing such protein interference as such, but also predicting, reducing or preventing the occurrence of non-specific signals in immunoassays (such as those in which (non-specific) signals associated with protein interference may occur, such as in ADA assays), and in particular predicting, reducing or preventing the occurrence of non-specific signals in such immunoassays, which, when observed in such an assay, are typically explained, associated with, and/or considered to be indicative of (non-specific) protein interference. In this regard, it should be noted, and as indicated in this document, the present invention is not limited to any causation, explanation, hypothesis or mechanism.

In one specific, but non-limiting aspect, the invention can be used to predict, prevent, or reduce protein interference in an "anti-drug antibody" or "ADA" assay that is performed on samples (i.e., "test samples") of body fluids taken from subjects. , which were introduced ISV (and, in particular, Nanotel; or a biological product based on ISV or a biological product based on Nanotel).

In another specific but non-limiting aspect, the invention can be used to predict, prevent, or reduce protein interference (and/or non-specific signals generally associated with the same) in an “anti-drug antibody” or “ADA” assay, which is used to detect, measuring and/or describing the presence of (any) anti-drug antibodies to one or more ISVs (and in particular to Nanobodies; or an ISV-based biological or Nanobodies-based biological, as defined hereinafter). In particular, the invention can be used to predict, prevent, or reduce such protein interference in “anti-drug antibodies” or “ADA” assays that are performed on samples (i.e., “test samples”) of body fluids, and more specifically on samples of body fluids. obtained from subjects who have been administered one or more of these ISVs or Nanotel (ISV-based biologic or Nanotel-based biologic, as defined hereinafter). For example, the invention can be used to predict, prevent, or reduce protein interference in such an “anti-drug antibody” or “ADA” assay, which is used to detect, measure, and/or characterize the presence of (any) anti-drug antibodies to an ISV or Nanobody (or biological ISV-based preparation or Nanotel-based biological preparation, as defined hereinafter) that were administered to the subject from which the sample was obtained (either as part of a clinical study or during therapy).

Thus, in one single, but non-limiting, aspect, the invention can be used to predict, prevent, or reduce protein interference (and/or non-specific signals usually associated with the same) in biological samples (i.e., "test samples") obtained from a subject who has been administered one or more of these ISVs or Nanotel (or an ISV-based biologic or a Nanotel-based biological, as defined hereinafter), which samples are suitable for and/or intended for use in immunological analysis such as ADA analysis. As indicated, such a biological sample may be blood (including whole blood, serum, or plasma), intraocular fluid, bronchoalveolar fluid/BALF, cerebrospinal fluid, or any other suitable biological fluid or sample suitable for use in an immunoassay, and in particular, ADA analysis.

In one specific, but non-limiting aspect, such test sample may be obtained from a subject who has undergone multiple administrations (e.g., at least 1 to 3 separate administrations over a period of at least 10 days, such as at least , one month or more) and/or long-term treatment (i.e., treatment for at least 10 days, such as at least one month) of ISV, Nanotel, ISV-based biologic (as defined below in this document) or a Nanotel-based biological product (as defined later in this document). Such an ISV, Nanotelo, ISV-based biologic, or Nanotel-based biologic may be administered to said subject, for example, as part of therapy or as part of a clinical study.

In one specific, but non-limiting aspect, the test sample may be obtained from a subject administered with an ISV, Nanobody, ISV-based biologic, or Nanobody-based biologic that has (and/or is provided with) an extended half-life (as described in this document). , and compared to a monovalent ISV), e.g., a half-life of at least 1 day, preferably at least 3 days, more preferably at least 7 days, e.g. at least 10 days, subject to whom the same is/was administered.

For example, and without limitation, such an ISV, Nanobody, ISV-based biological, or Nanobody-based biological can acquire an increased half-life by functionalization and/or by incorporating a moiety or binding unit into the construct that increases the half-life of the construct. Examples of such functionalization, fragments or binding units are clear to the skilled person and may include pegylation, fusion to serum albumin, or fusion to a peptide or binding unit that can bind to a serum protein, such as serum albumin. Such a serum albumin-binding peptide or binding domain may be any suitable serum albumin-binding peptide or binding domain capable of increasing the half-life of the construct (compared to a similar construct without a serum albumin-binding peptide or binding domain), and may in particular be a binding a serum albumin peptide as described by the applicant in WO 2008/068280 (and in particular WO 2009/127691 and unpublished provisional application US 61/301,819), or a serum albumin binding ISV (such as a serum albumin binding Nanobody; e.g. Alb-1 or a humanized variant of Alb-1, such as Alb-8, which is mentioned in WO 06/122787).

Thus, in one specific, but non-limiting aspect, such a biological sample can be obtained from a subject who has been administered an ISV, Nanobody, ISV-based biological, or Nanobody containing a (human) serum albumin-binding peptide or binding domain.

As mentioned above, in one non-limiting aspect, the invention broadly relates to a method that can be used to predict whether a given ISV or Nanobody (or ISV or Nanobody drug) will produce (or have a high or increased propensity to to generation) protein interference (as described later in this document) in immunoassay (i.e., after administration of said ISV to a subject, a body fluid sample is obtained from said subject, and said body fluid is subjected to immunoassay as described later in this document), wherein said method includes conducting an immunoassay comprising at least the steps of:

(i) contacting said ISV or Nanobody (or ISV or Nanobody drug) with an antibody that has been obtained from a subject (human) and has been selected, engineered and/or isolated based on its ability to recognize and/or bind with the C-terminus of ISV or Nanotel (“analytical antibody”); And

(ii) establishing whether said ISV or Nanobody (or ISV or Nanobody drug) is bound by said antibody in said immunoassay.

And in this case, the ISV described in this document may, in particular, be a Nanobody or (other) ISV (ie other than Nanobody) which is a VH domain or which contains a VH domain; and preferably is Nanotel.

Also, any protein or polypeptide containing an ISV (such as an ISV drug) preferably has said (or at least one) such ISV at its C-terminus. In addition, said ISV may in particular be a Nanobody or (other) ISV (ie other than Nanobody) which is a VH domain or which contains a VH domain; and preferably is Nanotel.

In an alternative embodiment, also described later in this document, instead of the aforementioned human-derived antibody, a monoclonal antibody referred to in this document as "21-4-3" (or "21-4" for short, see SEQ ID NO. 35 and 36 for VH and VL sequences). 21-4 was generated using hybridoma technology, starting with a mouse immunized with Nanobody construct SEQ ID NO:98 in WO 2006/122825 as described further in Example 7, and a hybridoma cell line (named “ABH0015”) expressing 21-4 was deposited on June 4, 2012 at the BCCM Microorganism Collection, Ghent, Belgium, under accession number LMBP-9680-CB. The monoclonal 21-4 has been shown to recognize the C-terminus of the Nanobody construct SEQ ID NO:98 in WO 2006/122825, the C-terminus of which consists of Nanobody (humanized V _HH ) derived to von Willebrand factor (vWF). Initially, 21-4 was induced as an analytical reagent for use in the detection of Nanotel protein (in particular Nanobody construct SEQ ID NO:98 in WO 2006/122825) in samples (sera); unexpectedly, it has now been found that 21-4 can also be used to predict whether an ISV has a propensity to undergo non-specific protein interference (more than some other comparable (mouse) monoclonal antibodies produced to the Nanobody construct of SEQ ID NO:98 in WO 2006/122825 or another Nanobody).

In particular, it has been found that if measuring the binding of 21-4 to an ISV (or to a protein or polypeptide containing an ISV at its C-terminus, or a similar protein or polypeptide as defined herein) gives an RU value of less than 500 ( after adjusting the measured RU value for the molecular weight of the protein, according to the formula [measured RU]/[MW of protein] x 10 ⁶ ) when determined according to the protocol outlined in Example 9, in this case, the specified ISV or protein will probably not have a tendency to be subject to protein interference (within the accuracy of the data set forth in the Examples below). For use in the above formula, MW (molecular weight) can be calculated as the sum of all MW of all amino acid residues present in the ISV.

Accordingly, any ISV, protein or polypeptide described herein preferably has a 21-4 binding RU value of less than 500 (set according to the protocol set forth in Example 9 and after adjusting the measured RU value for the molecular weight of the used ISV or protein, according to the above formula).

Thus, this aspect of the invention mainly relates to a method that can be used to predict whether a given ISV or Nanobody (or ISV or Nanobody drug) will generate (or has a high or increased propensity to generate) a protein interference (as described later in this document) in immunoassay (i.e., after said ISV is administered to a subject, a body fluid sample is obtained from said subject and said body fluid is subjected to immunoassay as described later in this document), wherein said method, includes conducting an immunoassay comprising at least the steps of:

(i) contacting said ISV or Nanobody (or ISV or Nanobody drug) with a 21-4 monoclonal antibody (i.e., used as an “analyte antibody”); And

(ii) establishing whether said ISV or Nanobody (or ISV or Nanobody drug) binds to monoclonal antibody 21-4 in said immunoassay.

Said method can in particular be carried out using BiaCore or similar technical equipment, and more specifically using the protocol set forth in Example 9. As indicated in this document, when binding of an ISV or an ISV-based drug in this protocol shows an RU value of less than than 500 (after adjusting the measured RU value for the molecular weight of the protein, according to the formula [measured RU]/[MW of protein] x 10 ⁶ ), the indicated ISV or ISV-based protein is not likely to be bound by any factor (s) interferences present in human blood or serum and/or will likely not be prone to non-specific protein interference in the ADA assay (i.e., within the confidence limits set forth in the experimental section below).

And in this case, as indicated in this document, the described ISV may, in particular, be a Nanobody or (other) ISV (ie other than Nanobody) which is a VH domain or which contains a VH domain; and preferably is Nanotel.

Also, any protein or polypeptide containing an ISV (such as an ISV drug) preferably has said (or at least one) such ISV at its C-terminus. Moreover, said ISV may in particular be a Nanobody or (other) ISV (ie other than Nanobody) which is a VH domain or which contains a VH domain; and preferably is Nanotel.

As already stated in this document, the above method using 21-4 can also be used to determine whether an ISV or a protein or polypeptide containing ISV binds (or tends to bind) to an interference factor(s) that are present in blood or serum. person.

Also, as stated in this document, it is contemplated that said method using 21-4 can also be used to predict whether any protein or polypeptide (such as an antibody fragment or ScFv) having a VH domain on its C- Finally, bind (or tend to bind) to an interference factor(s) that are present in human blood or serum and/or have a propensity for protein interference in an ADA assay.

In addition to 21-4, it is contemplated that an antibody or antibody fragment (e.g., a suitable Fab fragment) containing the heavy chain and light chain variable domains of 21-4 (see SEQ ID NOS: 35 and 36, respectively), or even just the CDR -sequences 21-4 (suitably grafted into other suitable VH and VK scaffolds) can also be used in the methods described here.

As described later in this document, the invention can particularly be used to predict whether a given ISV or Nanobody (or ISV or Nanobody drug) will generate protein interference (as described later in this document) in an immunoassay that is an ADA analysis. Said ADA assay, for example, may be an ADA assay to detect or measure ADA to ISVs, and in particular may be an ADA assay to detect or measure ADA to ISVs used in steps (i) and (ii) above. .

In one particular preferred but non-limiting aspect, the invention can be used to predict whether a given ISV or Nanobody (or ISV or Nanobody drug) will generate protein interference (as described later in this document) in an immunoassay (and in particular , in ADA analysis), which implies the use of such an ISV. Again, said ADA assay may, for example, be an ADA assay to detect or measure ADA to ISV, and may in particular be an ADA assay to detect or measure ADA to ISV used in steps (i) and ( ii) above.

In an even more specific but non-limiting aspect, the invention can be used to predict whether a given ISV or Nanobody (or ISV or Nanobody drug) will generate protein interference (as described below) in an immunoassay (and in particular in an ADA). -analysis) which implies the use of such an ISV. For example, such an immunoassay may be an ADA assay (i.e., involving an ISV) that is performed to detect or measure the presence of any ADA to a specified ISV in a test sample, where said sample is a body fluid sample (as described in this document) that is received from the subject to whom the specified ISV has been administered (as described later in this document). For example, as indicated later in this document, said sample (i.e., “test sample”) may be a sample of fluid (including whole blood, serum or plasma), intraocular fluid, bronchoalveolar fluid/BALF, cerebrospinal fluid, or any other suitable biological fluid, and may in particular be a biological sample suitable and/or intended for use in an immunological assay, such as an ADA assay.

As described later in this document, in all of these aspects (and additional aspects of the invention described in this document), the invention can also be used to select ISVs that do not or are less susceptible to protein interference in such immunoassays or ADA assays; as an assay or test that can be used to test whether certain ISV modification(s) will (in whole or in part) reduce its propensity to generate such interference in immunoassays or ADA assays; and/or as an assay or assay that can be used to direct modification or improvement of the ISV so as to reduce its propensity to generate protein interference in such immunoassays or ADA assays.

As already indicated, step (i) of the method of the invention comprises contacting said ISV or Nanotel (or ISV or Nanotel based drug) with an antibody that has been obtained from a subject (human) and that has been isolated based on its ability to recognize and/or bind to the C-terminus of the ISV or Nanotel (as described later in this document). In said step (i) of the method described herein, “specified ISV or Nanobody (or ISV or Nanobody drug)” is used as the antigen in the immunoassay (i.e., as the substance to be detected). Also, in said step (i), “an antibody that was obtained from a subject (human) and that was selected/isolated based on its ability to recognize and/or bind to the C-terminus of an ISV or Nanobody” is used as the analytical reagent ( i.e. in the same manner as other antibodies are used in an immunoassay to detect the presence of the antigen for which they are specific).

As already indicated, and for a better understanding of the invention described here, it should be noted that, in step (i) ISV will in most cases be used as an “antigen” (i.e., as a compound intended to be detected), and “ "analytic antibody" will be used as an assay agent (i.e., as a means to determine whether a given ISV binds or not, respectively; and thus has a high or increased risk of causing protein interference or not, respectively). For example, when step (i) is carried out in an ELISA format, the "antibody/assay agent" will typically be bound to the carrier (ie, ELISA plate) and the ISV will be in the test sample.

It should be noted that, in contrast, ADA analysis to detect or measure ADA to ISV uses ISV as the “analytical agent” (i.e., as the compound used to detect the presence of any ADA) and ADA are “antigen” (i.e., intended to be detected by the compound). Thus, in such assays, ISV will usually/often be associated with the carrier (eg, ELISA plate), while ADA (if any) will be present in the sample being assayed.

However, as already indicated, it should be noted in general terms that the invention is not limited to assay methods in which the "analytical antibody" binds to a carrier. For example, in an alternative way of performing the assay according to the invention (as shown in Example 5), the assay antibody is instead used as a bridging agent and thus will not be bound to the plate, but will be in solution (although it is indirectly bound to the plate via ISV that covers the tablet). However, also in the separate ("bridged") assay method described in Example 5 (which is a competitive assay), the assay antibody is still used as the assay agent (i.e., to determine if the ISV of interest binds or not , respectively; and thus has a high or increased risk of causing protein interference or not, respectively). Furthermore, it is contemplated that, based on the further disclosure, one skilled in the art may be able to develop other assay formats in which an assay antibody can be used as an assay agent to determine if a given ISV can bind and thus has a high or increased risk of generating protein interference. or not).

Used in stage (i) "analytical antibody" may be a polyclonal or monoclonal antibody.

Where the analyte antibody is a polyclonal antibody, it may, for example, be a polyclonal antibody (preparation) that has been prepared/purified/isolated from a biological sample obtained from a subject (human) (such as blood, plasma, B cells or other suitable biological sample or fluid from which the polyclonal antibodies can be appropriately isolated). This may be, for example, a suitable biological sample that is obtained from a person who has received at least one ISV (such as the ISV used in step (i), however this is not mandatory or necessary), but it can also be (and preferably is) a suitable biological sample from a person who has never received or has never been treated with ISV. More importantly, the polyclonal antibody is obtained from said biological sample in a manner that includes at least one affinity step using an affinity matrix or column carrying ISV as the affinity group (and one or more additional steps to obtain/purify/ isolation of polyclonal antibodies known per se). For example, a polyclonal antibody can be generated from such a biological sample by affinity chromatography using an ISV-bearing affinity column as described in Example 2, for example. using an affinity matrix carrying ISV as an antigen. Typically, such methods are known in the art, and suitable examples of them will be clear to the person skilled in the art, based on the disclosure in this document.

In particular, such a polyclonal antibody (preparation) may be an IgG (or IgG fraction).

For example, it may be a polyclonal antibody obtained by a method involving (immuno)affinity chromatography performed on a sample of a biological fluid obtained from a human, using as an antigen associated with an ISV affinity matrix (and, in particular, a Nanotel, such as VHH, a humanized VHH and/or a sequence-optimized VHH, or a camelized VH, such as a camelized human VH) that does not contain a C-terminal marker (i.e., the C-terminus of which ends with the amino acid sequence VTVSS (SEQ ID NO:33)). In particular, the ISV used as the affinity template-bound antigen may be a humanized VHH or a sequence-optimized VHH (or alternatively, a corresponding camelized human VH) whose C-terminus ends in the amino acid sequence VTVSS (SEQ ID NO:33) . In one separate, but non-limiting aspect, the affinity template-bound ISV used as antigen can be a humanized VHH or a sequence-optimized VHH that, as a result of such humanization or sequence optimization, contains a proline (P) residue at position 14, while the corresponding intact ( “naive”) VHH contains an alanine (A) at position 14 (in other words, the ISV used as antigen is a humanized variant of VHH that naturally contains an alanine at position 14, with the alanine residue as a result of humanization or sequence optimization was replaced by a proline residue (P)). The ISV used as an antigen may also contain one or more amino acid substitutions due to such humanization or sequence optimization, for example as generally described in WO 08/020079 or WO 09/138519.

Some specific examples of ISVs that can be used as an antigen to create/isolate an "analyte antibody" are given in SEQ ID NOs: 1 and 2.

Further, the method used to obtain a polyclonal antibody, in addition to the (immuno)affinity steps, may also include one or more additional steps for isolating/purifying the polyclonal antibody from a biological sample (before or after the affinity steps). And in this case, such stages and methods for their implementation will be clear to the specialist.

Thus, in one aspect, the invention includes a method that comprises steps (i) and (ii) described herein, wherein the “analytical antibody” (i.e., an antibody that has been obtained from a subject (human) and that has been selected/ isolated based on its ability to recognize and/or bind to the C-terminus of ISV or Nanotel) was obtained from a biological sample obtained from a person (in this case, the specified biological sample is a sample suitable for use in the method of creating/isolation of an antibody from the specified sample) using a method comprising at least one affinity step (such as an affinity chromatography step, e.g., immunoaffinity chromatography) in which an ISV (and preferably a Nanobody) is used as the antigen, and preferably an ISV is used as the antigen which contains the amino acid sequence VTVSS (SEQ ID NO:33) as the C-terminal sequence, and more preferably, a humanized Nanobody or a sequence-optimized Nanobody is used as an antigen which contains the amino acid sequence VTVSS (SEQ ID NO:33) as A C-terminal sequence, and even more preferably, a humanized Nanobody or a sequence-optimized Nanobody is used as an antigen which contains the amino acid sequence VTVSS (SEQ ID NO:33) as the C-terminal sequence, and which contains a proline residue at position 14, such as a Nanobody containing the amino acid sequence VTVSS (SEQ ID NO:33) as the C-terminal sequence and containing a proline residue at position 14 that has been introduced into the Nanobody as a result of said sequence humanization or optimization (e.g. to replace an alanine residue that naturally located at the indicated position of VHH that has been humanized and/or whose sequence has been optimized).

The above ISVs can also be used in methods for isolating monoclonal antibodies (again starting from a suitable biological sample obtained from a human), which are suitable for use in the invention as an “analytical antibody”.

For example, such a monoclonal antibody may be derived from blood, B cells, or other suitable sample or material for antibody isolation, may be selected based on its ability to recognize or bind to the (C-terminus) of an ISV or Nanobody (in which the ISV used in as an antigen during screening and/or selection is preferably as described in the preceding paragraphs, including preferences set for such an ISV/antigen). Such screening and selection may be carried out in any convenient manner, for example using B-cell selection and/or propagation methods substantially the same or similar to the B-cell selection methods described in EP 0 488 470, WO 92/02551, EP 1 633 787, WO 01/55216, WO 02/26829, WO 04/051268, WO 04/102198 or WO 04/106377, or by methods similar to the Nanoclone technique described in WO 06/079372 (but using human B cells, not camel B cells).

Once one or more B cells expressing a suitable antibody have been established/isolated, said antibody can be isolated, expressed and/or produced by any suitable method. For example, said B cell(s) can be immortalized as a hybridoma producing the desired antibody/antibodies (using techniques well known per se to create hybridomas starting from selected B cells) and said antibody/antibodies can then be isolated from the (culture supernatant) of said hybridoma(house), again using suitable techniques generally recognized in the art and described in various manuals, as well as described and/or indicated patent publications mentioned in the previous paragraph.

Alternatively, said B cell(s) can be expanded using known B cell expansion techniques and the antibody/antibodies can be isolated from the (culture supernatant) of said expanded B cell(s). And in this case, this can be done using suitable methods generally recognized in the art and described in various manuals, as well as described and/or indicated in the patent publications mentioned in the previous paragraphs.

In yet another alternative, DNA encoding the antibody/antibodies of interest can be obtained (e.g., by amplification) from the indicated B cell(s) or other suitable cells, or directly (e.g., using suitable single cell PCR cloning techniques). ) or after appropriate expansion of the desired B cell(s). Said DNA can then be appropriately expressed in a suitable host cell or host organism to provide the desired antibody/antibodies. And in this case, this can be done using suitable methods generally recognized in the art and described in various manuals, as well as described and/or indicated in the patent publications mentioned in the previous paragraphs.

It is also possible to generate monoclonal antibodies suitable for use as an "analyte antibody" in a manner that involves "repertory cloning" (starting with a suitable sample obtained from a subject (human)) and selecting the cloned "repertoire" to find antibodies that bind with the ISV used as the antigen (and in this case, the ISV(s) used as the antigen during screening and/or selection is preferably as described in the previous paragraphs, including the preferences established for such ISV/antigen) . Repertoire cloning techniques and various methods for visualizing cloned repertoires for selection and screening (such as phage display, ribosomal display, and yeast display) are well understood by those skilled in the art and are described, for example, in EP 0 589877, EP 0 774 511, WO 90/ 14430 and EP 0368 684), as well as various guides on the subject.

In general, the biological sample that is used as the starting point for the production of a (polyclonal or monoclonal) analytical antibody can be any suitable sample (i.e., suitable as starting material for the production of a polyclonal or monoclonal antibody, respectively) derived from any suitable subject (person). For example, in one separate but non-limiting aspect, such a sample may be obtained from a woman, and in particular a post-menopausal woman. Thus, in one separate but non-limiting aspect, the analytical antibody used in steps (i) and (ii) above was derived from a biological sample taken/obtained from a post-menopausal woman (or was derived from an antibody taken/ from a post-menopausal woman).

Also, the biological sample used as a starting point for obtaining a polyclonal or monoclonal analytical antibody can be obtained from a subject who has previously received ISV (for example, as part of a clinical trial or for therapeutic purposes), but preferably it is obtained from a subject to whom ISV has not been previously administered.

However, it should be noted that the invention is not limited, in particular, to the source of the analytical antibody/antibodies used, and in some cases, using the methods described in this document, the possibility of obtaining (creating, isolating) other suitable analytical antibodies from other sources, including commercially available human blood or plasma (and even blood, plasma or B cells from other mammalian or primate species such as the baboon or cynomolgus macaque).

As mentioned above, the (polyclonal or monoclonal) analytical antibody used in steps (i) and (ii) must be capable of recognizing or binding to the C-terminus of the ISV or Nanotel, and most preferably is selected and/or isolated based on its ability to bind to the C-terminus of ISV or Nanotel.

As can be seen from Figure 2, when the ISV is based on or derived from the VH or VHH domain, the C-terminus of the ISV contains the amino acid sequence VTVSS (SEQ ID NO:33), and accordingly, the analytical antibody should be able to recognize any ISV having the amino acid sequence VTVSS (SEQ ID NO:33) at its C-terminus. In addition, as seen in Figure 2, (at least some of the amino acid residues in) the VTVSS sequence (SEQ ID NO:33) is part of a putative epitope on ISV that also includes, among other residues, the amino acid residue at position 14 (and amino acid residues bordering/close to the same position in the amino acid sequence, such as positions 11, 13 and 15), and may also contain an amino acid residue at position 83 (and amino acid residues bordering/close to the same position in the amino acid sequence, such as positions 82, 82a, 82b and 84) and/or the amino acid residue at position 108 (and amino acid residues bordering/close to the same position in the amino acid sequence, such as position 107). For example, position 109 is the first V C-terminal VTVSS (SEQ ID NO:33) sequence, and it has been shown that position 110 can also affect protein interference). In addition, referred to in this document in a generalized sense "C-terminal region" means that this C-terminal region at least contains the C-terminal sequence of VTVSS (SEQ ID NO:33) and the amino acid residue at position 14, and may also contain amino acid residues at positions 83 and 108, and possibly also amino acid residues at positions 13, 15, 82b, 83, 84 and 107.

Without limitation to any hypothesis or explanation, and as already stated, in a full length 4-chain antibody or a full length heavy chain only antibody, such as camelids, the C-terminus of the VH or VHH domain is linked to the rest of the antibodies (i.e. with the CH1 region of a conventional monoclonal antibody or the hinge region of heavy chain-only Camelid antibodies, respectively), and thus, in such full-length antibodies, can be protected from protein interference and/or "hidden" by interaction VH/VL (in conventional 4-chain antibodies) such that this “C-terminal region” is generally not hydrophilic and/or accessible as an interaction site for proteins present in the blood, serum, or body of the subject being administered such an ISV. However, if the ISV or Nanobody is used per se (i.e., not bound to any other part of the antibody), or if an ISV drug or Nanobody drug that carries an ISV or Nanobody at its C-terminus, this C-terminal epitope is available for (non-specific) interaction with other proteins, and again without being limited by any hypothesis or explanation, it is assumed that this C-terminal region may now be available for (non-specific) interaction with one or more proteins preexisting in the “sample of interest” (e.g., one or more IgGs) and that this may cause protein interference and/or non-specific signals in immunoassays (and in particular in ADA assays).

As indicated, the methods described in this document can be used to predict, reduce or prevent protein interaction, and can also be used as a tool to direct the modification of an ISV, Nanobody, ISV drug or Nanobody drug in such a way as to provide the listed together with a (partially or preferably almost completely) reduced propensity to generate protein interference.

In particular, as can be seen from the previous paragraph, and again without being limited by any hypothesis or explanation, it is believed (and is part of the idea of the present invention) that (certain) modifications of the "C-terminal region" will change (and preferably reduce) the propensity ISV to non-specific protein interaction, which is also observed experimentally (see, for example, the experimental results presented in Examples 1C and 3 below).

On this basis, and again without being limited by any hypothesis or explanation, the present invention also proposes certain modifications that can be introduced for this purpose in the C-terminal region of the ISV, Nanotel, ISV-based drug or Nanotel-based drug (( potential) the effectiveness of which can be analyzed using the methods described in this document). Also, based on the ideas presented in the document, it is envisaged that the person skilled in the art will be able to select, create or propose other (possible) modifications of the C-terminal region that could be introduced for this purpose (and the (potential) effectiveness of which, in this case, could be analyzed using the methods described in this document).

Returning to the analytical antibody used in the invention, it is preferably a (polyclonal or monoclonal) antibody that recognizes the C-terminal region (as defined above) of ISV, and in particular, but without limitation, the C-terminal region of the Nanotel.

For example, in one separate, but non-limiting aspect, an "analyst antibody" can be a polyclonal or monoclonal antibody that recognizes (and/or is able to bind to and, in particular, specifically bind to) the C-terminal region of an ISV or Nanobody, C-terminus which ends in VTVSS (SEQ ID NO:33) but does not recognize (and/or is not able to specifically bind to) the C-terminal region of an ISV or Nanotel (which may be a different ISV, but preferably is the same ISV) in that case when there is one or more additional amino acid residues (eg, 1 to 5 amino acid residues, or alternatively, a small peptide sequence or even another polypeptide or protein) associated with the C-terminal VTVSS (SEQ ID NO:33).

In another, more specific, but still non-limiting aspect, an “analyst antibody” can be a polyclonal or monoclonal antibody that recognizes (and/or is able to bind to and, in particular, specifically bind to) the C-terminal region of an ISV or Nanobody. , whose C-terminus sequence ends in VTVSS (SEQ ID NO:33), and in which position 14 is occupied by an amino acid that does not occur naturally at position 14 and/or is modified from an amino acid that occurs at position 14 in the natural sequence (e.g., as a result of humanization, camelization, and/or sequence optimization), but which does not recognize (and/or is not able to specifically bind to) the C-terminal region of an ISV or Nanotel (which may be a different ISV, but is preferably the same ISV) which has one or more additional amino acid residues (eg 1 to 5 amino acid residues, or alternatively a small peptide sequence or even another polypeptide or protein) linked to the C-terminal VTVSS (SEQ ID NO:33); and/or in which position 14 is occupied by an amino acid naturally found at position 14 (eg, alanine or, when the ISV naturally contains proline at position 14, proline).

For example, an “analyst antibody” can also be a polyclonal or monoclonal antibody that recognizes (and/or is able to bind to and, in particular, specifically bind to) the C-terminal region of an ISV or Nanotel whose C-terminal sequence ends in VTVSS (SEQ ID NO:33) and in which position 14 is occupied by a proline (and in particular where position 14 has been changed to a proline, e.g. as a result of humanization, camelidization and/or sequence optimization) but does not recognize the C-terminal a region of an ISV or Nanobody (which may be a different ISV, but is preferably the same ISV) that has one or more additional amino acid residues (e.g., 1 to 5 amino acid residues, or alternatively a small peptide sequence or even a different polypeptide or protein) associated with the C-terminal VTVSS (SEQ ID NO:33); and/or in which position 14 is occupied by alanine.

An “analytical antibody” can also be a polyclonal or monoclonal antibody that recognizes (and/or is able to bind to and, in particular, specifically bind to) the C-terminal region of an ISV or Nanotel whose C-terminal sequence ends in VTVSS (SEQ ID NO :33) and whose position 14 is occupied by a proline (in particular, if the proline residue is naturally located at the indicated position in the indicated ISV), but does not recognize the C-terminal region of the ISV or Nanotel (which may be another ISV, but is preferably is the same ISV) that has one or more additional amino acid residues (e.g., 1 to 5 amino acid residues, or alternatively a small peptide sequence or even a different polypeptide or protein) linked to the C-terminal VTVSS (SEQ ID NO:33) in which position 14 is still occupied by (natural or unmodified) proline.

“Analytical antibody” can also, for example, be a polyclonal or monoclonal antibody that recognizes (C-terminal region) the sequence of ISV, referred to in this document as “Nb 3.4” (SEQ ID NO: 5), but does not recognize (C-terminal plot) sequence of ISV, referred to in this document as “Nb 3.1” (SEQ ID NO: 3), and/or (preferably, and) does not recognize the sequence of ISV, referred to in this document as “Nb 3.2” (SEQ ID NO: 4).

It can be determined whether the “analyte antibody” (or does not recognize) ISV or Nanobody (and/or is or is not able (specifically) to bind to ISV or Nanobody) using any suitable binding assay method (such as Biacore) as well as using the BIACORE assay described in Example 3 or an ADA assay such as the bridged/competitive ADA assay described in Example 5 (see also Figures 1A-1C and Figure 1B in particular).

Suitable formats/methods for conducting such analysis will be apparent to those skilled in the art based on the present disclosure, and include, for example, (without limitation):

- Colorimetric analysis, such as ELISA using a plate directly or indirectly coated with an analytical antibody, and detection (detection) of bound ISV using a monoclonal or polyclonal anti-ISV antibody. Other convenient alternative techniques include, but are not limited to, electrochemiluminescence (MSD platform), fluorescence (DELFIA, GYROS) and other methods based on secondary detection of bound ISV.

- Surface plasmon resonance (such as BIACORE) or other real-time biosensor method (i.e., different from that of SPR) with directly or indirectly immobilized analyte antibody and control of later-introduced ISV binding. These methods do not require further discovery of the associated ISV. A typical method for conducting this type of assay is described in Example 3.

-Study of the competitive behavior of ISVs in the bridging assay (ADA-assay) using an analytical antibody instead of an ADA-containing biological fluid. For bridge analysis, various technologies can be used, such as ELISA, MSD platform. Typical methods for performing this type of assay are shown schematically in Figures 1A-1C, and one particular example of this type of assay is also described in Example 5.

- Any chromatographic method in which the analytical antibody is immobilized on a chromatographic matrix and the specific capture/isolation of ISV occurs from solution.

When a suitable analytical antibody has been prepared using one of the methods described herein or one of the examples (or a method substantially equivalent thereto), said analytical antibody can be used to determine whether a given ISV or Nanobody (or ISV-based drug or based on Nanobody) generate (or have a high or increased propensity to generate) protein interference (as defined in this document), i.e. by carrying out steps (i) and (ii) described above. As already described herein, this typically involves contacting said ISV, Nanobody, ISV drug, or Nanobody drug with an assay antibody and determining whether said ISV, Nanobody, ISV drug, or drug based on the Nanobody (and/or binds and, in particular, specifically binds) by the specified analytical antibody (and, in particular, whether the C-terminal region of the specified ISV or Nanobody or any ISV or Nanobody forming the C-terminus of the specified drug is recognized based on ISV or drug based on Nanotel, specified by the analytical antibody).

In most cases, this can be done using any suitable technique to determine whether the antigen (in the case of ISV, Nanotel, ISV-based drug or Nanotel-based drug) is bound by an antibody, and suitable methods of (immuno)assay are known to the person skilled in the art. Some non-limiting examples are suitable ELISA techniques (including, for example, sandwich ELISA); wherein, depending on the ELISA format used (as one skilled in the art will understand), the plate may be coated with either the assay antibody or ISV and the assay antibody or ISV may be labeled for detection. For example, other approaches involve the use of a BIAcore instrument (in which case, an analytical antibody or ISV can be applied to a chip, see, for example, Example 3). Another alternative would be a competitive bridge assay (as, for example, illustrated in Example 5) that examines the ability of an ISV to compete with another ISV, Nanobody, ISV-based drug, or Nanobody-based drug known to bind by an assay antibody. (or vice versa). These and other suitable techniques for determining whether a given ISV, Nanobody, ISV-based drug, or Nanobody-based drug is bound (specifically) or recognized by an analyte antibody will be understood by those skilled in the art from the disclosure herein.

In addition, based on the disclosure, it is clear that the present invention (and, in particular, the analytical antibody used in the present invention) can be used to determine whether or not a given ISV, Nanobody, ISV-based drug, or Nanobody-based drug has an interaction site. (such as an interaction site present at or within the C-terminal region and/or part of which is formed by the C-terminal region) capable of undergoing (non-specific) protein interactions with one or more proteins or other compounds that may be present in the biological sample (i.e., “test sample”) obtained from a subject that is subjected to an immunoassay, such as an ADA assay (in particular, an ADA assay to determine the presence of any ISV ADA, Nanobody, ISV-based drug or drug based on Nanotele). Thus, in the case where an ISV, Nanobody, ISV drug, or Nanobody drug is recognized by an assay antibody used in the invention, it is highly likely that said ISV, Nanobody, ISV drug, or Nanobody drug is The nanobodies contain such an (accessible or unprotected) site of interaction and thus will tend to generate protein interference (as defined herein) when used in such an immunoassay or ADA assay to test a test sample. As one skilled in the art will understand, this should be avoided preferably either by selecting/using another ISV, Nanobody, ISV drug, or Nanobody drug, or by modifying the ISV, Nanobody, ISV drug, or Nanobody drug, resulting in whereby its propensity for such protein interference will be greatly reduced or virtually eliminated (in which case this can be tested using the method and assay antibody disclosed herein).

As also understood by one of skill in the art based on the disclosure, such modification may, for example, involve making one or more modifications (such as amino acid insertions, additions, deletions, or substitutions) to an interaction site on an ISV, Nanotel, ISV drug, or Nanobody, whereby its ability to undergo a (non-specific) protein interaction with one or more proteins or other compounds that may be present in the test sample will be reduced or eliminated. Further, this can be done with a limited amount of "trial and error" by introducing one or more modifications, and then checking whether this ability has been eliminated or not, in this case using the method and analytical antibody disclosed in this document. For example, one or more such modifications can be introduced and then the ability of the modified ISV to bind to the assay antibody can be compared to that of the original/unmodified ISV. Alternatively, the ability of the modified ISV to still compete with the original ISV for binding to the assay antibody can be established using a competitive bridge format (as illustrated in Example 5, for example) or using a BIAcore (see, for example, Example 3).

Further, and while the invention is not limited to any hypothesis or explanation, based on the experimental evidence set forth in the examples below, the inventors have found that this interaction site is likely to be located at/near the C-terminal region (as determined in this document) or said interaction site forms part of the C-terminal region (or the C-terminal region forms part of the interaction site). This is based, for example, at least in part on the observation that if an ISV has a propensity to generate protein interference and has VTVSS (SEQ ID NO:33) as amino acid residues at its C-terminus, then the addition of a limited number of amino acid residues ( e.g., 1 to 10, e.g., 1 to 5, e.g., 1, 2, 3, 4, or 5) or alternatively, a label or other peptide, protein, or other fragment towards this C-terminus will generally significantly reduce or practically eliminate the indicated tendency. In some cases, it has been found that even adding 1, 2, or 3 amino acid residues to the C-terminal VTVSS (SEQ ID NO:33) (which can be any suitable amino acid(s) or combination of amino acids, each of which can be independently selected from any naturally occurring amino acids such as those listed in Table A-2 on page 64 of WO 09/138519, for example and without limitation from alanine, glycine, valine, leucine and isoleucine) can already significantly reduce or virtually eliminate this tendency. This is also partly based on the observation that in some cases where VHH naturally contains an alanine residue at position 14 (which is said to form part of the C-terminal region; see Figure 2), naturally occurring VHH often has no (or low ) a propensity to generate protein interference, whereas a corresponding VHH in which said alanine at position 14 is substituted for a proline residue (e.g. for the purpose of humanization or sequence optimization) may result in an increased propensity to generate protein interference (i.e., compared to VHH with alanine at position 14).

In one aspect, the invention relates to VHH, Nanobody (as referred to in this document, and in particular humanized VHH or camelized VH, such as camelized human VH) or another ISV (or ISV drug or Nanobody drug with VHH). , Nanobody, or other ISV at its C-terminus) that has been modified (for example, by introducing one or more amino acid substitutions, additions, or deletions) and, in particular, modified at the C-terminal regions (for example, by one or more amino acid substitutions or inserts in the C-terminal region) such that (i) it significantly reduced its propensity (eg, at least statistically significantly reduced propensity) to generate protein interference (as defined herein); and/or for example, (ii) it has in the method of the invention described herein (for example, as in the separate assay method described in Example 3 or 5) a significantly reduced ability to bind to the analytical antibody described herein (such as a polyclonal antibody, described in Example 2 and used in Examples 3 and 5) in both cases is preferred over the same VHH, Nanotel or ISV, but without modifications.

Thus, in one aspect, the invention relates to a VHH, a Nanobody (as referred to in this document, and in particular a humanized VHH or a camelized VH, such as a camelized human VH) or other ISV (an ISV-based drug or a VH-based drug). Nanobodies with a VHH, Nanobody, or other ISV at its C-terminus) that is a VHH or a VH domain (i.e., an ISV that is a VH domain or derived from a VH domain) and/or that has been based or derived from the (amino acid sequence) of a VHH or VH domain, wherein the VHH, Nanotbody, or ISV contains the amino acid sequence VTVSS(X) _n (SEQ ID NO:34) at its C-terminus, where n is a number from 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4, or 5 (and preferably 1 or 2, such as 1), and where each X is a (preferably natural) amino acid residue that is independently selected (and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I); however, as can be seen from the data below, other (preferably natural) amino acid residues or combinations of the above preferred amino acid residues with other amino acid residues (such as serine, proline, threonine and/or lysine) can also be used. Preferably, said VHH, Nanobody, or ISV with the amino acid sequence VTVSS(X) _n (SEQ ID NO:34) at its C-terminus is one that (i) has a significantly reduced propensity (e.g., at least a statistically significant reduced propensity ) causing protein interference (as defined in this document); and/or (ii) has, in the method of the invention described herein (such as the specific assay described in Example 3 or 5), a significantly reduced ability to bind to the analyte antibody described herein (such as the polyclonal antibody described in Example 2 ), in both cases, preferably over the same VHH, Nanotel or ISV, but with the amino acid sequence VTVSS (SEQ ID NO:33) at its C-terminus. For example, you can refer to the analysis and data presented in Example 3.

The above VHHs, Nanobodies or (other) ISVs preferably have a 21-4 binding RU value of less than 500 (determined according to the protocol set forth in Example 9 and after correcting the measured RU value for molecular weight). It should also be noted that at any time reference is made in the description or claims to any C-terminal sequence of VTVSS(X) _n (including any of aspects (a) to (p) below), which corresponds to one single aspect of the invention, none of the amino acids X is a cysteine residue.

For example, in some preferred aspects, the C-terminus of an ISV or an ISV-containing construct (in the event that the C-terminus is an ISV derived from VH, VHH, or Nanotel) may be:

- VTVSS(X) _n , where n = 1 and X = Ala;

- VTVSS(X) _n , where n = 2 and each X = Ala;

- VTVSS(X) _n , where n = 3 and each X = Ala;

- VTVSS(X) _n , where n = 2 and at least one X = Ala (while the remaining amino acid residues X are independently selected from any natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- VTVSS(X) _n , where n = 3 and at least one X = Ala (while the remaining amino acid residues X are independently selected from any natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- VTVSS(X) _n , where n = 3 and at least two X = Ala (while the remaining amino acid residues X are independently selected from any natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- VTVSS(X) _n , where n = 1 and X = Gly;

- VTVSS(X) _n , where n = 2 and each X = Gly;

- VTVSS(X) _n , where n = 3 and each X = Gly;

- VTVSS(X) _n , where n = 2 and at least one X = Gly (while the remaining amino acid residues X are independently selected from any natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- VTVSS(X) _n , where n = 3 and at least one X = Gly (while the remaining amino acid residues X are independently selected from any natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- VTVSS(X) _n , where n = 3 and at least two X = Gly (while the remaining amino acid residues X are independently selected from any natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- VTVSS(X) _n , where n = 2 and each X = Ala or Gly;

- VTVSS(X) _n , where n = 3 and each X = Ala or Gly;

- VTVSS(X) _n where n = 3 and at least one X = Ala or Gly (with the remaining amino acid residues X being independently selected from any natural amino acids, but preferably independently selected from Val, Leu and/or Ile ); or

- VTVSS(X) _n , where n = 3 and at least two X = Ala or Gly (while the remaining amino acid residues X are independently selected from any natural amino acids, but preferably independently selected from Val, Leu and/or Ile );

while aspects (a), (b), (c), (g), (h), (i), (m) and (n) are particularly preferred, with aspects where n = 1 or 2 are preferred , and aspects where n = 1 are particularly preferred.

It should also be noted that at any time reference is made in the description or claims to any C-terminal sequence of VTVSS(X) _n (including any of aspects (a) to (p) above), which corresponds to one single aspect of the invention, none of the amino acids X is a cysteine residue.

Thus, in one preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanobody being preferred), which has on C-terminally the sequence VTVSS(X) _n , where n=1 and X=Ala (or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 2 and each X = Ala (or a protein or polypeptide containing such an ISV (and preferably such a Nanobody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 2 and at least one X = Ala (with the remaining amino acid residues of X being independently selected from any naturally occurring amino acids, but preferably being independently selected from Val, Leu and/or Ile) (or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanobody being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 3 and at least one X = Ala (with the remaining amino acid residues X being independently selected from any naturally occurring amino acids, but preferably being independently selected from Val, Leu and/or Ile) (or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus sequence VTVSS(X) _n where n = 3 and at least two X = Ala (with the remaining X amino acid residues being independently selected from any naturally occurring amino acids, but preferably being independently selected from Val, Leu and/or Ile) (or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 3 and each X = Ala (or a protein or polypeptide containing such an ISV (and preferably such a Nanobody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n , where n=1 and X=Gly (or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 2 and each X = Gly (or a protein or polypeptide containing such an ISV (and preferably such a Nanobody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 3 and each X = Gly (or a protein or polypeptide containing such an ISV (and preferably such a Nanobody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 2 and at least one X = Gly (with the remaining amino acid residues of X being independently selected from any naturally occurring amino acids, but preferably independently selected from Val, Leu and/or Ile) ( or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 3 and at least one X = Gly (with the remaining amino acid residues of X being independently selected from any naturally occurring amino acids, but preferably independently selected from Val, Leu and/or Ile) ( or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus sequence VTVSS(X) _n where n = 3 and at least two X = Gly (with the remaining X amino acid residues being independently selected from any naturally occurring amino acids, but preferably independently selected from Val, Leu and/or Ile) ( or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 2 and each X = Ala or Gly (or a protein or polypeptide containing such an ISV (and preferably such a Nanobody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 3 and each X = Ala or Gly (or a protein or polypeptide containing such an ISV (and preferably such a Nanobody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 3 and at least one X = Ala or Gly (with the remaining amino acid residues X being independently selected from any natural amino acids, but preferably independently selected from Val, Leu and/or Ile ) (or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n where n = 3 and at least two X = Ala or Gly (with the remaining amino acid residues X being independently selected from any naturally occurring amino acids, but preferably independently selected from Val, Leu and/or Ile ) (or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus the sequence VTVSS(X) _n , where n = 1, 2, or 3, where each X = Ala or Gly.

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanotelo being preferred), which has at the C-terminus sequence VTVSS(X) _n , where:

- n = 1, 2 or 3, where each X = Ala or Gly; or

- n = 2 or 3, where all but one X = Ala or Gly (while the remaining amino acid residues X are independently selected from any naturally occurring amino acids, but preferably independently selected from Val, Leu and/or Ile)

or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain (ISV) which is a Nanobody or (other) ISV containing a VH sequence or derived from a VH sequence (with Nanobody being preferred), which has a C-terminus sequences VTVSS(X) _n , where:

- n = 1, 2 or 3, where each X = Ala or Gly; or

- n = 2 or 3, where at least one X = Ala or Gly (while the remaining amino acid residues X are independently selected from any naturally occurring amino acids, but preferably independently selected from Val, Leu and/or Ile);

- n = 2 or 3, where all but one X = Ala or Gly (while the remaining amino acid residues X are independently selected from any natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

or a protein or polypeptide containing such an ISV (and preferably such a Nanotbody) at its C-terminus.

In the above aspects, in the case of the specified (other) “ISV that contains a VH sequence or is derived from a VH sequence”, it means any ISV that contains a VH sequence or is derived from a VH sequence and is not a Nanobody (i.e., not being VHH, humanized VHH, or camelized VH). For example, such (another) ISV may, for example, be a VH-based (single) domain antibody, a VH-dAb™ based or a VH-based microbody (see WO 00/29004).

And in this case, it should be noted that at any time any of the ISVs mentioned in the description has the C-terminal sequence VTVSS(X) _n (including, without limitation, the ISVs mentioned in the previous aspects), which corresponds to one single aspect of the invention, wherein none of the X amino acid residues in the sequence VTVSS(X) _n is a cysteine residue.

As further described herein, any such protein or polypeptide, for example, may be a construct that contains two or more ISVs (eg, two or more Nanobodies), optionally linked via one or more suitable linkers. Thus, such a construct may be, for example, a divalent, trivalent, tetravalent, or pentavalent construct (such as a divalent, trivalent, tetravalent, or pentavalent Nanobody construct), and may, for example, be a divalent, trivalent, tetravalent, or pentavalent construct (such as a divalent , trivalent, tetravalent, or pentavalent Nanobody construct) that is a bispecific, trispecific, or biparatopic construct (including, for example, monospecific, bispecific, or biparatopic constructs that can also bind to serum albumin (preferred) or other serum protein to increase half-life).

Again, the Nanobodies, ISVs, and proteins/polypeptides according to each of the aspects described above preferably have an RU value for binding 21-4 of less than 500 (determined according to the protocol set forth in Example 9, and after correcting for the measured RU values based on the molecular weight of the ISV or protein used in accordance with the above formula).

As stated in this document, it is also contemplated that the invention can also be applied to other proteins or polypeptides (and in particular antibody fragments such as Fab fragments or other proteins or polypeptides based on antibody fragments such as ScFv) having VH domain at their C-terminus. Thus, in another aspect, the invention relates to a protein or polypeptide (such as ScFv) which has a VH domain at its C-terminus with the amino acid sequence VTVSS(X) _n (SEQ ID NO:34) at its C-terminus. where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5, and where each X is a (preferably natural) amino acid residue independently selected (and preferably independently selected from a group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) Further, in certain specific aspects, said C-terminus may be any of (a) - (p) above, and preferably in accordance with one of (a), (b), (c), (g), (h), (i), (m) or (n), while n is 1, 2 or 3, and preferably 1 or 2.

Again, such proteins or polypeptides preferably have a 21-4 binding RU value of less than 500 (determined according to the protocol set forth in Example 9 and after adjusting the measured RU value for the ISV or protein molecular weight, used in accordance with the above formula). Also, further, in accordance with one particular aspect of the invention, none of the X amino acid residues in the C-terminal sequence of VTVSS(X) _n is a cysteine residue.

The invention further relates to a pharmaceutical composition comprising an ISV (and preferably a therapeutic ISV) or a protein or polypeptide comprising at least one ISV (and preferably at least one therapeutic ISV) wherein said ISV, protein or polypeptide is as previously described (i.e., an ISV, protein or polypeptide in accordance with one or more of the aspects described herein and in particular in accordance with one or more of the aspects described on the previous pages; and more specifically ISV, protein or polypeptide having a C-terminus/sequence that corresponds to one or more of the aspects described here), and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use), and optionally one or more additional active substances. Such compositions, carriers, diluents and excipients, for example, may be as described in WO 08/020079 in relation to pharmaceutical compositions that contain a Nanobody or a protein or polypeptide that contains at least one Nanobody (and as already mentioned, according to the present invention, ISV is also preferably Nanotel).

The invention further relates to an ISV, or a protein or polypeptide containing at least one ISV, for use in the treatment of a disease in a human (e.g., a patient in need of such treatment), said ISV, protein, or polypeptide being as it was previously described (i.e. is an ISV, a protein, or a polypeptide according to one or more of the aspects described herein and, in particular, according to one or more of the aspects described on the previous pages; and more specifically, an ISV, a protein or a polypeptide having a C-terminus/sequence that corresponds to one or more of the aspects described here).

The invention further relates to the use of an ISV or a protein or polypeptide containing at least one ISV in the preparation of a pharmaceutical composition, wherein said ISV, protein or polypeptide is as previously described (i.e. is an ISV, a protein or a polypeptide according to one or more of the aspects described herein and, in particular, according to one or more of the aspects described on the previous pages; and more specifically, an ISV, protein or polypeptide having a C-terminus/sequence that corresponds to one or more of the aspects described here).

The invention further relates to a method of treatment comprising administering to a subject (human) (e.g., a patient in need of such treatment) an ISV or a protein or polypeptide containing at least one ISV in a pharmaceutical composition, wherein said ISV, protein or polypeptide is is, as described herein (i.e., is an ISV, protein, or polypeptide according to one or more of the aspects described herein and, in particular, according to one or more of the aspects described on the preceding pages; and more specifically an ISV, a protein or polypeptide having a C-terminus/sequence that corresponds to one or more of the aspects described herein); or a pharmaceutical composition (described above) containing at least one such ISV, protein or polypeptide.

With respect to the foregoing, it is clear that the therapeutic use of the ISVs, proteins and polypeptides described herein is a very important aspect of the invention, since therapeutic use (or clinical studies of such ISVs, proteins and polypeptides for therapeutic use) may include the use of an ADA assay to determine whether said ISV, protein, or polypeptide is immunogenic (i.e., can produce ADA when administered to a human). In this respect, it is also understood that questions regarding possible immunogenicity must be considered, in particular when the therapeutic agent is used for longer periods of time (weeks, months or years) and/or has a half-life (preferably expressed as t1/ 2-beta) in a human for at least 3 days, such as at least one week, and up to 10 days or more.

Thus, in accordance with one particular aspect of the invention, the ISV, protein, polypeptide, or pharmaceutical composition described herein is for the treatment of a chronic disease in a human, and/or such ISV, protein, polypeptide, described herein, is for presence in the bloodstream of the subject (i.e., at a pharmacologically active level) to which it is administered (i.e., at a therapeutically active dose) for at least one week, preferably at least two weeks, e.g. at least a month; and/or such ISV, protein, polypeptide described herein has a half-life (preferably expressed as t1/2-beta) in humans of at least 3 days, e.g., at least one week, and up to 10 days or more; and/or such an ISV, protein, polypeptide, or pharmaceutical composition described herein is intended to be administered to a human in two or more doses administered over at least 3 days, such as at least one week, for example, at least two weeks, or at least one month, or even longer (i.e., at least 3 months, at least 6 months, or at least one year), or even entered constantly.

The invention further relates to a method for (significantly) reducing or substantially eliminating the propensity of an ISV, Nanobody or ISV drug or Nanobody drug to cause protein interference, said method at least comprising the steps of:

- optionally determining the propensity of an ISV, Nanobody, ISV drug, or Nanobody drug to generate protein interference using a method that at least comprises steps (i) and (ii) specified herein;

- modification of the specified ISV, Nanobody, ISV-based drug or Nanobody-based drug by introducing one or more amino acid substitutions, additions or deletions to the specified ISV or Nanobody, or to the C-terminus of the ISV or Nanobody (if any) of the drug on based on ISV or drug based on Nanotel; and, in particular, by introducing one or more amino acid substitutions, insertions into the C-terminal region of the specified ISV or Nanobody or into the C-terminal region of the C-terminal ISV or Nanobody (if any) of the ISV-based drug or Nanobody-based drug , for example, by adding to the C-terminus a sequence of from 1 to 10, for example, from 1 to 5, for example, 1, 2, 3, 4, or 5 amino acid residues, each of which is independently selected from any naturally occurring amino acids (such as those listed in Table A-2 on page 64 of WO 09/138519, for example and without limitation, from alanine, glycine, valine, leucine or isoleucine);

- determining the propensity of the thus modified ISV, Nanobody, ISV-based drug or Nanobody-based drug to generate protein interference using a method that at least includes steps (i) and (ii) specified in this document; optionally in a manner that compares the propensity of the ISV, Nanobody, ISV-based drug, or Nanobody-based drug modified in this way to generate protein interference with the propensity of the original ISV, Nanobody, ISV-based drug, or Nanobody-based drug to generate protein interference. interferences (including, without limitation, their comparison in a competitive assay for binding by an analytical antibody, as described herein). Alternatively, the method described herein using 21-4 may be used.

The invention will now be described by means of the following non-limiting preferred aspects, examples and Figures, in which:

- Figures 1A - 1C schematically show some non-limiting examples of ADA analysis formats. Some exemplary, but non-limiting, protocols for performing these assays are provided in Example 4.

- Figure 2 schematically shows a typical 3D structure of an ISV such as a Nanobody.

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

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

- Figure 5 is a binding curve (obtained using the BIACORE assay described in Example 3) showing the binding of NB 3.4, 3.14 and 3.15 (SEQ ID NOs: 5, 15 and 16) to the immobilized polyclonal antibody prepared in Example 2.

- Figure 6 is a binding curve (obtained using the BIACORE assay described in Example 3) showing the binding of NB 3.1, 3.2 and 3.4 (SEQ ID NOs: 3, 4 and 5) to the immobilized polyclonal antibody obtained in Example 2.

- Figure 7 is a binding curve (obtained using the BIACORE assay described in Example 3) showing the binding of NB 4.1 and 4.2 (SEQ ID NOs 17 and 18) to the immobilized polyclonal antibody prepared in Example 2.

- Figure 8 is a binding curve (obtained using the BIACORE assay described in Example 3) showing the binding of NB 6.1, 6.2, 6.4 and 6.5 (SEQ ID NOs 19 to 22) to the immobilized polyclonal antibody prepared in Example 2.

- Figure 9 is a table showing the sequences used in Example 8 (SEQ ID NOs 37 to 89) and the corresponding reference sequence.

The sequences referred to in the present specification and claims are listed in Table A below (SEQ ID NOs: 1 to 37) and in Figure 9 (SEQ ID NOs: 38 to 89).

Table A

Name SEQID NO: Subsequence ISV Ex. 1/2- 1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIKSSGDSTRYAGSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAKSRVSRTGLYTYDNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMGWFRQAPGKEREFVAAITR SGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGTLVTVSS alt. ISV 2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYAD SVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS >Nb3.1 3 EVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELVATITGGSSINYGDFVKGRFTISIDNAKNTVYLQMNNLKPEDTAVYYCNFNKYVTSRDTWGQGTQVTVSS >Nb3.2 4 EVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELVATITGGSSINYGDFVKGRFTISIDNAKNTVYLQMNNLKPEDTAVYYCNFNKYVTSRDTWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH >Nb3.4 5 EVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb3.5 6 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSAA >Nb3.6 7 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSA >Nb3.7 8 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSG >Nb3.8 9 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSGG >Nb3.9 10 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSGGG >Nb3.10 eleven HHHHHHEVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb3.11 12 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLKPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb3.12 13 HHHHHHEVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTQVTVSS >Nb3.13 14 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTQVTVSS >Nb3.14 15 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVQVSS >Nb3.15 16 HHHHHHEVQLVESGGGSVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb4.1 17 EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSS >Nb4.2 18 EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSRDWDFDVFGGGTPVGG >Nb6.1 19 EVQLVESGGGLVQPGGSLRLSCIASGLPFSTKSMGWFRQAPGKEREFVARISPGGTSRYYGDFVKGRFAISRDNAKNTTWLQMNSLKAEDTAVYYCASGERSTYIGSNYYRTNEYDYWGTGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH >Nb6.2 20 EVQLVESGGGLVQPGGSLRLSCIASGLPFSTKSMGWFRQAPGKEREFVARISPGGTSRYYGDFVKGRFAISRDNAKNTTWLQMNSLKAEDTAVYYCASGERSTYIGSNYYRTNEYDYWGTGTQVTVSS >Nb6.4 21 EVQLLESGGGLVQPGGSLRLSCAASGLPFSTKSMGWFRQAPGKGREFVSRISPGGTSRYYGDFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASGERSTYIGSNYYRTNEYDYWGQGTLVTVSSAAAEQKLISEEDLNGAAHHHHHH >Nb6.5 22 EVQLLESGGGLVQPGGSLRLSCAASGLPFSTKSMGWFRQAPGKGREFVSRISPGGTSRYYGDFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASGERSTYIGSNYYRTNEYDYWGQGTLVTVSS Example 1C: wild type 23 HHHHHHEVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGTQVTVSS Example 1C: (A14P) 24 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMGWFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGTQVTVSS Example 1C: (K83R) 25 HHHHHHEVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLRPEDTAVYTCAASAIGSGALRRFEYDYSGQGTQVTVSS Example 1C: (Q108L) 26 HHHHHHEVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGTLVTVSS Example 1C: (A14P,K83R,Q108L) 27 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMGWFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLRPEDTAVYTCAASAIGSGALRRFEYDYSGQGTLVTVSS Example 1c: (A14P,R39Q,K83R,T91Y,Q108L) 28 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMGWFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGTLVTVSS Example 1C: (A14P,R39Q,K83R,T91Y,Q108L)-1A 29 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMGWFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGTLVTVSSA Example 1C: (A14P,R39Q,K83R,T91Y,Q108L)-3A thirty HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMGWFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGTLVTVSSAAA Nb3.16 31 DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTIS RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS Nb3.17 32 DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTIS RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSA C-terminal sequence 33 VTVSS C-terminal sequence 34 VTVSS(X) _n 21-4-3, IGH consensus 35 QIQLVQSGPELKKPGETVKISCKASGYTFTAYSMHWVKQAPGKGLKWMGWINTVTGEPAYADDFKGRFAFSLETSASTAYLQISSLKNEDTATYFCTRGLIHFYYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDTLSSSSVTVPSSTWPSETVTCNVA HPASSTKVDKKIVPRDC 21-4-3-IGK consensus 36 DIQMTQTPSSLSASLGGRVTITCKASQDIHNFISWYQHKPGKVPRLIIHDTSTLQPGIPSRFSGSGSGRDYSFSITNLEPEDIATYYCLHYDNLLRSFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS TSPIVKSFNRNEC

Experimental part:

Example 1: Creation of a polyclonal analytical antibody.

A polyclonal antibody (IgG fraction) that can be used as an “analyte antibody” was prepared as follows:

- Identification of plasma samples suitable for polyclonal antibody isolation

Twenty plasma samples from healthy individuals who had never been treated with ISV were evaluated for the presence of antibodies to ISV, which can be used as analytical antibodies in the invention.

In this Example, ISV SEQ ID NO: 1 was used initially. Then, to confirm that the interaction is not specific to that individual ISV, but is a non-specific protein-protein interaction that can occur with a range of ISVs, the following analysis was repeated with other ISV (see point C) below). Alternatively, SEQ ID NO:1 can also be used, for example, SEQ ID NO:2.

The assay method used was an ECL (electrochemiluminescence) bridging assay using biotinylated ISV (biotinylated variant of SEQ ID NO:1) for capture and sulfo-labeled ISV for detection of antidrug antibodies. A similar format was also used for the ADA analysis. Biotinylation and sulfo-labeling of ISV was carried out using conventional primary amine coupling chemistry using Sulfo-NHS-LC-Biotin (Pierce) and Sulfo-label NHS-Ester (MSD), respectively, according to the manufacturer's instructions. Plasma samples were diluted 1/5 in PBS/0.1% casein and incubated for 30 minutes at 37°C, 600 rpm in 96-well polypropylene plates. Samples (50 µl) were then diluted 1/3 in a 1:1 mixture (100 µl) of 2 µg/ml biotinylated and 2 µg/ml sulfo-labeled ISV (SEQ ID NO:1) and incubated for 1 hour at room temperature (RT ), 600 rpm. MSD ^MA® 96-well standard streptavidin plates were blocked with 150 μl/well ^Superblock® T20 for 1 hour at RT, then washed 3 times with PBS/0.05% Tween20 (=wash buffer). Sample/1:1 mixture (biotinylated and sulfo labeled ISV (SEQ ID NO:1) (50.0 µl) was transferred from a polypropylene plate to an MSD plate and incubated for 1 hour at RT, 600 rpm. Plates were washed three times before adding 2x Reading Buffer (MSD) (150 µl/well) and readings were taken in ECL units (ECLU) on an MSD instrument (Sector Imager 2400 Plate Spectrophotometer) Samples were sorted as positive or negative using a sorting split point determined by The cut-off point was calculated from the baseline values of 118 individual plasma samples from healthy individuals who had never been treated with ISV using an appropriate statistical analysis as recommended in the ADA analysis design guide (Shankar, 2008). non-parametric estimate, and the split point value was calculated based on the ^95th percentile after outliers were excluded.

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

These samples were further analyzed in a drug displacement set-up (confirmatory analysis) to confirm the specificity of a positive screening result (Table II). Accordingly, samples were diluted 1/5 in PBS/0.1% casein containing 12.5 μg/ml ISV (SEQ ID NO:1) and incubated for 30 minutes at 37°C, 600 rpm at 96- well polypropylene plates. Samples (50 µl) were then diluted 1/3 in a 1:1 mixture (100 µl) of 2 µg/ml biotinylated and 2 µg/ml sulfo-labeled ISV (SEQ ID NO:1) incubated for 1 hour at RT, 600 rpm /min Next, a sample/1:1 mixture (biotinylated and sulfo-labeled ISV) (50.0 μl) was transferred from the polypropylene plate to an MSD ^MA® blocked 96-well streptavidin standard plate as described above for screening analysis and incubated for 1 hours at RT, 600 rpm. The plates were then washed 3 times before adding 2x read buffer (MSD) (150 μl/well) and measured ECL units (ECLU) using an MSD instrument (Sector Imager 2400 Plate Spectrophotometer). Samples were confirmed as truly positive using the confirmatory cut-off point determined during method validation, and ECL responses were calculated from 118 individual plasma samples from healthy, never-treated ISV individuals administered 50 μg/mL ISV (SEQ ID NO: 1) using an appropriate statistical analysis as recommended in the ADA Analysis Development Guide (Shankar, 2008). The minimum 50% signal reduction was calculated based on the 99% confidence interval.

Samples that were positive in the ECL-based bridge assay and that were confirmed positive in the drug displacement set-up assay were selected as a source for polyclonal antibody generation using affinity chromatography.

Table I: Screening results of 20 plasma samples in ADA ISV analysis

Sample ID ECLU screening assay IHuP#002-001-ABL-01 13081 IHuP#002-001-ABL-02 56 IHuP#002-001-ABL-03 272 IHuP#002-001-ABL-04 125 IHuP#002-001-ABL-05 70 IHuP#002-001-ABL-06 99 IHuP#002-001-ABL-07 170 IHuP#002-001-ABL-08 659358 IHuP#002-001-ABL-09 798 IHuP#002-001-ABL-10 1101 IHuP#002-001-ABL-11 83 IHuP#002-001-ABL-12 72 IHuP#002-001-ABL-13 403 IHuP#002-001-ABL-14 62 IHuP#002-001-ABL-15 1141 IHuP#002-001-ABL-16 159 IHuP#002-001-ABL-17 72 IHuP#002-001-ABL-18 170 IHuP#002-001-ABL-19 4503 IHuP#002-001-ABL-20 8243

Table II: Confirmation of plasma samples collected as positive by confirmatory assay. A confirmatory cutoff point of 50% was used to evaluate the results. One sample was not confirmed as a true positive

Plasma Sample ID ECLU Screening Assay: Plasma ECLU confirmatory assay: plasma % signal suppression IHuP#002-001-ABL-01 13081 685 95 IHuP#002-001-ABL-08 659358 169410 74 IHuP#002-001-ABL-10 1101 582 47 IHuP#002-001-ABL-15 1141 467 59 IHuP#002-001-ABL-19 4503 1531 66 IHuP#002-001-ABL-20 8243 1450 82

Three more serum samples from individuals who had never been treated with ISV were also evaluated using the ECL-based bridging assay described above and validated using drug displacement set-up analysis.

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

Table III: Results of screening and confirmatory analysis of 3 serum samples and the corresponding purified IgG fraction

Plasma Sample ID ECL signal in screening assay: serum ECL signal in confirmatory assay: serum % signal suppression ECL signal in screening analysis: IgG ECL signal in confirmatory assay: IgG % signal suppression IHUS#B09032311A3 2388 286 88% 3716 370 90% IHUS#B09032311A20 19272 915 95% 31309 1160 96% IHUS#B09032311A1 62

- Obtaining a purified fraction of polyclonal IgG.

Polyclonal IgG was purified from samples IHUS#B09032311A3 and IHUS#B09032311A20 (see above) using Protein G HP Spin Trap columns (GE Healthcare) according to the manufacturer's instructions. Briefly, after removing the storage solution from the column by centrifugation (30 s at 100 x g), the column was equilibrated by adding binding buffer (20 mM sodium phosphate, pH 7.0). After centrifugation, a solution containing the desired polyclonal antibody (maximum 1 mg in 600 μl) was added and the column was incubated for 4 minutes with gentle agitation. The column was then centrifuged and washed 2x with sufficient binding buffer (600 µl). After adding 400 μl of elution buffer (0.1 M glycine-HCL, pH 2.7) and mixing by inversion, the antibody was eluted by centrifugation with 30 ml of neutralizing buffer (1 M Tris-HCL, pH 9.0).

To confirm that the IgG fraction thus obtained was involved in non-specific binding to ISV(s), the purified IgG antibody was analyzed using the ECL-based bridge assay described above and confirmed using the drug displacement set-up assay used in A) above. . For both samples (IHUS#B09032311A3 and IHUS#B09032311A20), the participation of the purified IgG antibody in non-specific binding leading to a positive signal in the assay was confirmed (Table III). Thus, it was confirmed that the purified polyclonal IgG can be used as an "analyte antibody", and it was used as such (in the assay) in Examples 3 and 5.

- Non-specific binding to other ISVs.

In order to establish whether the observed protein interference is specific to an individual ISV and/or specific to an individual site, epitope or antigenic determinant of the ISV and/or to certain mutations introduced into the wild-type ISV (such as one or more humanizing mutations), the above ECL-based bridging assay and drug displacement set-up assay (both as described under A, using SEQ ID NO: 1 as sulfo-labeled ISV) were repeated using plasma samples IHUS#B09032311A3, IHUS#B09032311A20 and IHUS#B09032311A1. Because these plasma samples contained the polyclonal “assay” antibody isolated in B) above, this also provided information regarding the specificity, selectivity, and epitope recognition of the polyclonal assay antibody.

8 samples of ISVs were tested (SEQ ID NOs 23-30, respectively, see Table A above), of which one sample was wild-type VHH (SEQ ID NO: 23) and the remaining 7 ISVs were humanized variants of the wild-type sequence with various humanizing substitutions. . Two ISVs (SEQ ID NO's: 29 and 30) also contained additional amino acid residues at the C-terminus (1 and 3 additional alanine residues, respectively).

Data are shown in Table IV. Without being limited by any explanation or hypothesis, it can be seen that changes in the C-terminal region (as indicated in this document) can obviously greatly influence the degree to which the plasma samples used generate protein interference. For example, it can be seen that the introduction of one or three amino acid residues at the C-terminus can greatly reduce the tendency for protein interference (for example, a decrease of only 18 and 13% in ECLU analysis of sample IHUS#B09032311A3 with SEQ ID NOS: 29 and 30 compared with a 90% reduction for SEQ ID NO: 28 corresponding to the humanized variant without adding any amino acid residues at the C-terminus). Similarly, the introduction of a proline residue at position 14 of the wild-type sequence can obviously also greatly affect the extent to which plasma samples generate protein interference (eg, only a 20% reduction in ECLU analysis for wild-type sample IHUS#B09032311A3 of SEQ ID NO : 23, compared to a 91% decrease for SEQ ID NO: 24, wild-type sequence with A14P substitution). K83R and Q108L, which are also substitutions located close to the C-terminal region, also lead to some increase in the propensity to generate protein interference, but not as strong as the A14P substitution, and the overall net effect of the A14P+K83R+Q108L substitutions can be leveled. by adding one or more amino acid residues to the C-terminus (compare data for SEQ ID NOs: 29 and 30 again with data for other humanized variants).

Based on these data, it was also concluded that, apparently, the polyclonal analytical antibody in most cases recognized the C-terminal region (as defined in this document) of ISV. As can be seen from Figure 2, position 14 (and to a lesser extent positions 83 and 108) also form part of the C-terminal region of the ISV (when the three-dimensional tertiary structure of the ISV is taken into account).

Table IV: Evaluation of Different Variants of Nanotel as Competitor in ISV ADA Analysis Using Analytical Antibody

Plasma Sample ID IHUS#B09032311A3 IHUS#B09032311A20 IHUS#B09032311A1 ECLU in screening assay (using SEQ ID NO: 1) 2217 18494 62 Nanotel variant
(columns to the right indicate made
humanizing substitutions and C-terminal additions, according to
compared to
sequence of SEQ ID NO:23 wild type) ECLU confirmatory analysis % reduction ECLU confirmatory analysis % reduction ECLU confirmatory analysis % reduction SEQ ID NO: 23 VHH wild type 1778 20 8682 53 60 4 SEQ ID NO: 24 VHH wild type +A14P 205 91 668 96 56 10 SEQ ID NO: 25 VHH wild type +K83R 1403 37 6912 63 62 1 SEQ ID NO: 26 VHH wild type +Q108L 1533 31 6991 62 59 5 Plasma Sample ID IHUS#B09032311A3 IHUS#B09032311A20 IHUS#B09032311A1 ECLU in screening assay (using SEQ ID NO: 1) 2217 18494 62 Nanobody variant (columns on the right indicate humanizing substitutions made and C-terminal
additions, compared to wild-type SEQ ID NO:23) ECLU confirmatory analysis % reduction ECLU confirmatory analysis % reduction ECLU confirmatory analysis % reduction SEQ ID
NO: 27 VHH wild type +A14P+K83R+Q108L 156 93 628 97 57 8 SEQ ID
NO: 28 VHH wild type + A14P + R39Q + K83R + T91Y + Q108L 228 90 570 97 58 6 SEQ ID
NO: 29 VHH wild type + A14P + R39Q + K83R + T91Y + Q108L + 1
additional A at C-terminus (A114) 1814 18 15087 18 60 3 SEQ ID
NO: 30 VHH wt + A14P + R39Q + K83R + T91Y + Q108L + 3 A’s at C-terminus (A114+A115+A116) 1933 13 15244 18 62 0

Example 2: Affinity Purification of an Analytical Antibody

This Example describes two methods that can be used to isolate from human body fluid an analytical antibody that is able to recognize and/or bind to the C-terminus of ISV. The antibody was isolated from 4 different sera samples, which tended to elicit a positive signal in the ADA assay according to the test described in Example 1 above.

Starting with serum samples, each of these protocols provides a purified preparation of interference factor(s) that can be used as an analytical antibody in the methods described in this document. These methods can also be used more generally to purify interference factor(s) for other purposes (e.g., interference factor(s) purified using the protocols described below were also used in the experiments in Example 8 in order to show that ISV binding or ISV construct of monoclonal antibody 21-4 is predictive of binding of the same ISV or ISV construct to interference factors, and thus the propensity of said ISV or ISV construct to undergo non-specific protein interference in ADA analysis).

Example 2A: Purification using Protein A and affinity chromatography.

In the first step, the IgG antibody fraction was enriched from serum samples using protein A affinity chromatography. Representative columns used for this enrichment contained HiTrap MabselectSure and MabSelectXtra (GE Healthcare); PorosMabCapture A (Applied Biosystems). Purification of IgG antibodies from serum samples was carried out in an automated way, the same for all experiments. Chromatography was performed on an AKTA purification system (GE Healthcare) and readings were recorded in real time using UNICORN software (GE Healthcare). Briefly, a serum sample was diluted 1:1 with D-PBS (Dulbecco's Phosphate Buffer) and filtered through a 0.22 µm filter before being loaded onto the column at a set rate of 0.5 ml/min. The column was washed to remove non-specifically bound components with 5 column volumes using D-PBS at a fixed flow rate of 0.5 ml/min. The IgG fraction was eluted by acid elution using a buffer containing 100 mM glycine pH 2.6 at a flow rate of 0.5 ml/min. After elution, the fractions were neutralized with 1.5 M Tris buffer pH 8.8. SDS-PAGE was performed in the eluate to confirm the isolation of the IgG antibody.

In a second step, the interfering IgGs were further enriched with protein A purified IgG fractions from 4 different sera on ISV bound affinity columns. More specifically, interfering IgGs were further enriched by coupling to a column containing ISVs with the sequence of SEQ ID NO: 1. To do this, ISVs were covalently coupled to Sepharose 4 fast flow (GE Healthcare) using the CNBr (cyanogen bromide) coupling method according to the procedure manufacturer. Affinity purification was carried out in an automated way, the same for all experiments. Chromatography was carried out on an AKTA purification system and the results were recorded using UNICORN. Briefly, an IgG enriched sample (up to 10 ml loading volume) was loaded onto the column at a set flow rate of 0.5 ml/min. The column was washed to remove non-specifically bound components with 5 column volumes using D-PBS at a flow rate of 0.5 ml/min. ISV binding components were eluted by acid elution using a buffer containing 100 mM glycine pH 2.6 at a flow rate of 0.5 ml/min. After elution, the fractions were neutralized with 1.5 M Tris buffer, pH 8.8. Fractions were analyzed using SDS-PAGE, which confirmed the isolation of IgG antibodies in the eluate (data not shown).

These fractions were pooled and used for further analyzes such as those described in Example 3.

Example 2B: Purification Using CaptureSelect™ Chromatography.

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

A variation of this method used a different CaptureSelect affinity resin (CaptureSelect Alpha-1 Antitrypsin resin, a commercially available VHH-based affinity resin not targeting any antibody-related proteins). This resin provides high binding efficiency of the interference factor and allows selective two-step elution of antitrypsin by elution at neutral pH using 2.0 M MgCl ₂ , followed by elution of the interference factor by acid step (0.1 M glycine pH3.0, similar to elution conditions for protein A/G; neutralization with 1.5M Tris). The yield of one-step purification was up to 15 μg of interfering IgG1 per ml of plasma with high interference, which is approximately 0.3% of the total IgG present. The neutralized interfering fraction can optionally be desalted and further purified using a size exclusion column equilibrated with D-PBS.

Example 3: Effect of Different Substitutions on the Propensity of ISVs to Generate Protein Interference

As stated in the description above, the present invention provides certain analysis methods and techniques that make it possible to assess whether a given ISV has a propensity to generate protein interference or not. These include the ECL-based bridging and drug displacement set-up analyzes used in Example 1, as well as the BIACORE analysis described in Example 3 and the bridging/competitive ADA analysis described further in the Examples below.

As previously mentioned, these assays can also be used to determine if certain changes (eg, amino acid deletions, substitutions, or additions) can affect (and preferably reduce) the propensity of a given ISV to generate protein interference. Some of these changes will become apparent to those skilled in the art based on the disclosure and experimental data presented in Example 1 and in this Example 3.

As already evident from the data obtained in Example 1, certain mutations in or near the C-terminal region (as defined in this document) of ISV can (strongly) influence its propensity to generate protein interference. For example, adding a few amino acid residues to the C-terminus (for example, 1 or 3 alanine residues) seems to greatly reduce the tendency of ISV to generate protein interference, and, apparently, is even able to neutralize the presence of other substitutions (for example, in or nearby from the C-terminal region) that seem to increase the propensity to generate protein interference (eg, A14P substitution).

In this Example 3, the effect of other substitutions, as well as the effect of adding additional amino acids to the C-terminus, was examined by comparing related ISVs with different substitutions using the analytical polyclonal antibody prepared in Example 2. The analysis was performed by measuring the interaction kinetics between each of the ISVs under study and analytical polyclonal antibody using surface plasmon resonance (SPR) using a Biacore ^TM T100 GE Healthcare biosensor. In this Example 3, ISV SEQ ID NO 3 - 22 were tested (see Table A above and Table V below).

In a typical experiment, a 10 μg/ml solution of the polyclonal antibody in 10 mM NaOAc pH5.0 was prepared. This polyclonal antibody was then immobilized on a CM5 sensor chip using an amine coupling using the EDC/NHS method (EDC=N-ethyl-N'-[3-diethylamino-propyl]-carbodiimide; NHS=N-hydroxysuccinimide) in according to the manufacturer's instructions. The immobilized amount provided approximately 2700 response units (RU). ISV was then applied to the surface at a set concentration of 500 nM for 120 seconds at a flow rate of 45 μl per minute. Since no efficient buffer regeneration was found, the dissociation time was increased to 2400 seconds. The signal obtained when ISV was introduced into an empty flow cell was subtracted from the signal obtained when ISV was introduced into a flow cell with bound polyclonal antibody. The empty flow cell was activated/deactivated in the same manner as the polyclonal antibody flow cell, but without the addition of protein. The result of a "blank" injection (HBS-EP + running buffer (HBS = saline buffered with Hepes: GE Healthcare) was also subtracted to correct for possible baseline bias.

To study the effect of adding amino acid residues at the C-terminus, the effect of adding 1 or 2 alanine residues and 1, 2 or 3 glycine residues was investigated by comparing ISV binding with different additions using an analytical polyclonal antibody prepared as described in Example 2. For this purpose were created and tested ISV representing NB from 3.4 to 3.9 (SEQ ID NO: 5 to 10).

Since typical examples of this type of data are obtained, Figure 3 shows the binding of NB 3.4 to 3.9 with an immobilized polyclonal antibody. The results obtained are summarized in Table V.

Table V

clone ID SEQID NO Position
113 ⁽¹⁾ Position
114 ⁽¹⁾ Position
115 ⁽¹⁾ Position
116 ⁽¹⁾ Binding **
(RU) NB 3.4
NB 3.5
NB 3.6 5
6
7 S
S
S A
A A 75
9
8 NB 3.7
NB 3.8
NB 3.9 8
9
10 S
S
S G
G
G G
G G 31
13
13

**: binding signal obtained at the end of administration (= maximum RU signal)

⁽¹⁾ in this numbering, position 113 is the last "S" of the VTVSS C-terminal motif, and positions 114, 115, and 116 are the positions immediately (towards the C-terminus) following the indicated position 113.

To study the effect of (other) substitutions in the C-terminal region, the effect of different substitutions was investigated by comparing related ISVs containing these substitutions using the same analytical polyclonal antibody described above. The analysis was carried out as described above.

ISVs containing these tested substitutions are NB 3.1, 3.2 and 3.4 (SEQ ID NOS 3, 4 and 5); NB 3.10 - 3.15 (SEQ ID NOs 11 to 16), which were compared with NB 3.4; NB 4.1 and 4.2 (SEQ ID NOS 17 and 18) and NB 6.1, 6.2, 6.4 and 6.5 (SEQ ID NOS 19-22).

Typical examples of this type of data have been obtained:

- Figure 4 shows the binding of NB 3.4, 3.11, 3.12 and 3.13 with immobilized polyclonal antibody;

- Figure 5 shows the binding of NB 3.4, 3.14 and 3.15 with immobilized polyclonal antibody;

- Figure 6 shows the binding of NB 3.1, 3.2 and 3.4 with immobilized polyclonal antibody;

- Figure 7 shows the binding of NB 4.1 and 4.2 with immobilized polyclonal antibody;

- The figure 8 shows the binding of NB 6.1, 6.2, 6.4 and 6.5 with immobilized polyclonal antibody.

The results obtained are summarized in Tables VI, VII and VIII.

Table VI

clone ID SEQID NO Position
14 ⁽¹⁾ Position
83 ⁽¹⁾ Position
108 ⁽¹⁾ Binding **
(RU) NB 3.4
NB 3.10
NB 3.11
NB 3.12
NB 3.13 5
eleven
12
13
14 P
A
P
A
P R
R
K
R
R L
L
L
Q
Q 75
91
88
86
90

**: binding signal obtained at the end of administration (= maximum RU signal)

⁽¹⁾ numbering according to Kabat.

Table VII

clone ID SEQID NO Position
11 ⁽¹⁾ Regulation 110 ⁽¹⁾ Binding**
(RU) NB 3.4
NB 3.14
NB 3.15 5
15
16 L
L
S T
Q
T 75
79
22

**: binding signal obtained at the end of administration (= maximum RU signal)

⁽¹⁾ numbering according to Kabat.

Table VIII

clone ID SEQID NO: Regulation 14 Regulation 83 ⁽¹⁾ Regulation 108 ⁽²⁾ Label* Binding **
(RU) NB 3.1
NB 3.2
NB 3.4 3
4
5 A
A
P K
K
R Q
Q
L -
+
- 2
0
59 clone ID Regulation 14 Regulation 83 ⁽³⁾ Regulation 108 ⁽⁴⁾ Label * Binding **
(RU) NB 4.1
NB 4.2 17
18 P
P R
R L
L -
+ 51
0 clone ID Regulation 14 Regulation 83 ⁽⁵⁾ Regulation 108 ⁽⁶⁾ Label * Binding **
(RU) NB 6.1
NB 6.2
NB 6.4
NB 6.5 19
20
21
22 P
P
P
P K
K
R
R Q
Q
L
L +
-
+
- 0
39
0
66

*: in case of “+”, this ISV contains additional amino acids at the C-terminus of VTVSS

**: binding signal obtained at the end of administration (= maximum RU signal)

⁽¹⁾ : numbering acc. with Kabat (corresponding to aa at position 87 in SEQ ID NOs 3 to 5).

⁽²⁾ : numbering acc. with Kabat (corresponding to aa at position 123 in SEQ ID NOs 3 to 5).

⁽³⁾ : numbering acc. with Kabat (corresponds to aa at position 86 in SEQ ID NOs 17 and 18).

⁽⁴⁾ : numbering acc. with Kabat (corresponding to aa at position 116 in SEQ ID NOs 17 and 18).

⁽⁵⁾ : numbering acc. with Kabat (corresponding to aa at position 86 in SEQ ID NOs 19 to 22).

⁽⁶⁾ : numbering acc. with Kabat (corresponding to aa at position 112 in SEQ ID NOs 19 to 22).

Further, without being limited to any particular hypothesis or explanation, the data presented above shows that (various) substitutions in the C-terminal region (as defined herein) of ISV can alter/improve its propensity to generate protein interference.

Example 4: Exemplary protocols for conducting ADA assays shown in Figure 1

This Example provides some exemplary, but non-limiting, conditions that can be used to perform competitive/bridging ADA assays, shown schematically in Figure 1:

- ADA analysis in Figure 1A in solution: Samples 100% matrix, 30', 37°C, acid treatment using acetic acid in 10 matrix, 5', RT, sample pre-incubation/neutralization with acid: Sulfo-ISV (:Tris ) 1:1:1 (1:0.9:0.9:0.1), 1h, RT; on plate 1 h, RT; wash 3x, read buffer 4X

- ADA analysis in Figure 1B in solution: Samples 20% matrix, 30', 37°C, sample pre-incubation: ISV--Sulfo 1:1:1, 1h, RT, on plate 1h, RT, wash 3x, read buffer 2x

- Serial ADA analysis in Figure 1C: Capture ISV-Bio, 1 h, RT, wash 3X, Samples 20% matrix, 15', RT, on plate 2 h, RT, wash 3X, detection of Sulfo-ALX-0141, 1 h , RT, wash 3x, read buffer 4X

Example 5 Prediction of ISV susceptibility to non-specific protein interference using an analytical antibody

This example describes a bridged/competitive ADA assay using an analytical antibody that can be used to predict the sensitivity of ISVs to non-specific protein interference.

Test ISV was diluted to a concentration of 10 μg/ml and incubated with 400 ng/ml of analytical antibody purified according to Example 2 and incubated at 37° C. at 600 rpm in 96-well polypropylene plates. The sample (50 µl) was then diluted 1/3 in a 1:1 mixture (100 µl) of 2 µg/ml biotinylated and 2 µg/ml sulfo-labeled ISV and incubated for 1 hour at RT, 600 rpm. MSD ^MA® 96-well standard streptavidin plates were blocked with 150 μl/well ^Superblock® T20 for 1 hour at RT, then washed 3 times with PBS/0.05% Tween20 (=wash buffer). Sample and 1:1 mixture (biotinylated and sulfo-labelled ISV) (50.0 μl) were transferred from polypropylene plate to MSD plate and incubated for 1 hour at RT, 600 rpm. The plates were washed three times before adding 2x Reading Buffer (MSD) (150 μl/well) and ECL units (ECLU) were read on an MSD instrument (Sector Imager 2400 Plate Spectrophotometer).

With this analysis was tested and compared ISV SEQ ID NO 23 - 30. The data are shown in Table IX. These data not only show that the assay method described in this Example can be used to predict the propensity of ISVs to generate protein interference, but also confirm the data from previous Examples on the effect of substitutions in the C-terminal region. As can be seen, the addition of 3 (and, to a lesser extent, 1) alanine residues to the C-terminus of fully humanized ISV eliminated its ability to compete with analytical antibody binding. Altering position 14 in the wild-type ISV variant from alanine to proline apparently increased its ability as a competitor in the assay (= making the ISV variant more prone to non-specific protein interference), while altering positions 83 and 108 did not appear to affect the sensitivity of ISV to non-specific protein interference.

Table IX

Affinity Purified Antibody ID IHuP#002-001-ABL-08 ECLU in screening assay (using SEQ ID NO:1) 2919 Nanobody variant (bars on the right indicate humanizing substitutions and C-terminal additions made, compared to wild-type SEQ ID NO:23) ECLU confirmatory analysis % reduction SEQ ID NO: 23 VHH wild type 2706 7.3 SEQ ID NO: 24 VHH wild type +A14P 268 90.8 SEQ ID NO: 25 VHH wild type +K83R 2460 15.71 SEQ ID NO: 26 VHH wild type +Q108L 2533 13.23 SEQ ID NO: 27 VHH wild type +A14P+K83R+Q108L 319 89.1 SEQ ID NO: 28 VHH wild type + A14P + R39Q + K83R + T91Y + Q108L 251 91.4 SEQ ID NO: 29 VHH wt + A14P + R39Q + K83R + T91Y + Q108L + 1 extra A at C-terminus (A114) 1207 58.64 SEQ ID NO: 30 VHH wt + A14P + R39Q + K83R + T91Y + Q108L + 3 A at C-terminus (A114+A115+A116) 3301 -13.09

Example 6 Effect of adding amino acids to the C-terminus of anti-OX40L Nanotel on their OX40L blocking activity

This example demonstrates that C-terminal extension does not affect the activity or blocking activity of Nanotel.

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

The first assay, a T cell activation assay, was performed as described below. PBMC were isolated from buffy coats (Red Cross, Ghent, Belgium) of healthy donors using Ficoll Paque Plus reagent (GE Healthcare), washed with complete RPMI 1640 medium (RPMI1640 + GlutaMAX + 25 mM HEPES + 10% fetal bovine serum + 1% penicillin/ streptomycin, Invitrogen). PBMC (1x10 ⁵ cells/well) were stimulated with phytohemagglutinin (PHA-L; final concentration 0.6 μg/ml) before the addition of 1x10 ⁴ hOX40L CHO expressing cells (irradiated with a gamma scintillator at 3000 rad; UZ Gent, Belgium) and a dilution series of anti- OX40L Nanobody RPMI 1640 in complete medium and incubated for 22 hours at 37°C in a CO ₂ incubator. The production of IL2 by PBMCs was measured by ELISA. The wells of a Maxisorp plate were coated overnight at 4° C. with an anti-human IL2 monoclonal antibody (BD Biosciences). After washing and blocking the coated wells, a ½ dilution of cell supernatant was added. A ½ dilution series of recombinant human IL2 (BD Biosciences) starting at 2000 pg/mL was included as a standard. Detection was performed with a biotinylated anti-human IL2 monoclonal antibody (BD Biosciences) and HRP conjugated streptavidin (Thermo Scientific) and esTMB (SDT Reagents). The reaction was stopped with 1N HCl and OD readings were taken at 450 nm. As expected, the activity of Nanotel Nb 3.17, optimized with the trivalent bispecific sequence (IC50 = 0.13 nM, 95% CI = 0.098-0.17 nM), was comparable to that of Nb 3.16 (IC50 = 0.10 nM, 95% CI = 0.071- 0.15 nM).

In a second competitive ELISA-based assay, a dilution series (from 1.5 μM to 0.083 pM) of Nanotel was pre-incubated overnight at room temperature with 100 ng/ml human OX40/Fc (R&D Systems) and 10 ng/ml biotinylated human OX40L (R&D Systems; biotinylated in situ as described in Example 1) in PBS+0.1% BSA+0.01% Tween-20. Then, samples were incubated in Maxisorp plates coated with 10 μg/ml anti-human Fc Nanotel (produced in situ) and blocked with PBS + 1% BSA + 0.1% Tween-20. Bound human OX40/Fc was detected using HRP-conjugated streptavidin (Thermo Scientific) and sTMB (SDT Reagents). The reaction was stopped with 1N HCl and OD readings were taken at 450 nm. According to cellular analysis, the activity of Nb 3.17, an optimized trivalent bispecific sequence, Nanotel (IC50=0.178nM, 95% CI=0.152-0.200nM) was comparable to that of Nb 3.16 (IC50=0.179nM, 95% CI=0.149-0.215nM).

Example 7: Preparation of monoclonal antibody 21-4-3

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

After asphyxation of stimulated mice in CO2, the spleens were removed under sterile conditions and a single cell suspension was prepared from the pooled spleens. Spleen cells and myeloma cells were washed several times with DMEM and combined in the presence of 1 ml of 50% (w/v) PEG 3350 (ratio of spleen cells to SP2/0 3:1). For fusion, the myeloma cell line SP2/0-Ag14 from the Collection of Microorganisms and Cell Cultures of Germany (DSMZ GmbH, Braunschweig) was used. This cell line is a hybrid between BALB/c spleen cells and P3x63Ag8 myeloma cell line. The hybridomas thus obtained were resuspended in CGM containing 20% FCS and aminopterin (HAT medium) and plated (140 μl/well) in eight 96-well flat bottom tissue culture plates (Corning-Costar) containing 140 μl/well CGM (20 % FCS) together with peritoneal exudate cells as feeding cells. The plates were incubated for 10 days in complete growth medium (CGM) containing DMEM supplemented with 2-mercaptoethanol, L-glutamine, stable glutamine (Stable Glutamine), HT and non-essential amino acids (at concentrations recommended by the supplier) and FCS at various concentrations ( 10%, 15% or 20%). During this period, HAT medium was added to the cells twice. Supernatants (culture media) from hybridoma cell cultures typically contained 1 to 20 μg/ml antibody, which was tested in a binding ELISA to confirm binding to the Nanobody construct of SEQ ID NO:98 in WO 2006/122825.

Cells from wells producing positive IgG were transferred to wells of 48-well plates and cultured for 2-4 days (depending on cell growth rates). An ELISA binding assay was performed for ALX081 and human/cynomolgus IgG in order to exclude non-specific binders. Hybridoma cells expressing binders specific for the Nanobody construct SEQ ID NO:98 in WO 2006/122825 were cloned twice using serial dilutions. After fusion and re-screening, 7 primary cultures producing antibodies to ALX-081 were established. All of these primary cultures produced antibodies that did not cross-react with human or cynomolgus IgG. Primary cultures were cloned (twice).

Clone 21-4 (one of the clones that stably produced antibodies to ALX-081 after the second cloning) was designated "ABH0015" and was deposited with the Belgian Coordinated Collections of Microorganisms (BCCM) in Ghent, Belgium on June 4, 2012 under accession number LMBP- 9680-CB. The mouse monoclonal antibody produced by ABH0015 was named 21-4-3: the isotype determined for 21-4-3 showed an IgG1 heavy chain and a kappa light chain that were sequenced (see SEQ ID NOS: 35 and 36, respectively). 21-4-3 has been shown to bind to the C-terminal portion of the Nanobody construct of SEQ ID NO:98 in WO 2006/122825 (data not shown).

Example 8 Binding of 21-4 to ISV predicts ISV propensity to undergo non-specific protein interference

This Example, together with the following Example 9, demonstrates the binding of monoclonal 21-4 to an ISV that is used to predict (within the accuracy specified in this Example) whether a given ISV will be prone to non-specific protein interference (eg, in an ADA assay).

In particular, this Example 8 shows that 21-4 can be used to predict whether certain proposed modifications for a given ISV (such as adding one or more amino acid residues to the C-terminus of the ISV and/or substituting one or more amino acids within the C- terminal region of the ISV) lead to a decrease in the propensity of said ISV to non-specific protein interference.

Briefly, a set of 53 different Nanobodies or Nanobody constructs (see Figure 9 and SEQ ID NO's: 38-89) was tested for binding to monoclonal 21-4-3. The same Nanobodies and Nanobody constructs were also tested for binding with purified interference factor(s) preparations from three different human donors (referred to in the document as "Donor 8", "Donor 19" and "Donor 30") to see if there is a correlation between the 21-4 binding and the purified interference factors.

It has been found that ISV binding by antibody 21-4 can indeed be used to predict binding of the same ISV by interference factor(s) (within the overall confidence provided by the data presented in the description).

To demonstrate this, as detailed in the experimental data below, binding of 53 Nanobodies or Nanobody constructs (as listed in Figure 9; see SEQ ID NOs: 38-89) 21-4 was measured using Biacore T100 (as shown below). protocol) and compared with the binding of a reference Nanobody or construct (also listed in Figure 9) also measured on the Biacore instrument and using the same protocol. The results are shown in Table X below.

For each of the 53 Nanobodies or Nanobody constructs tested, a reference sample (reference) was selected for comparison, the tested Nanobody or Nanobody constructs had one or more additional amino acid residues at the C-terminus (which were added in order to test the effect of such an addition on protein interference , and in particular in order to reduce said interference) and/or one or more mutations within the C-terminal region (eg due to hymanization compared to a reference).

Results were expressed as percentage reduction in binding (measured in RU units) of a given Nanobody versus reference binding (also measured in RU units – e.g. if the measured binding level (RU) of a reference Nanobody was 276, and the binding level of a given Nanobody (also in RU) was 9, then the decrease in the level of binding occurred to the level of [9 RU/276 RU] x 100% = 3%), which means a decrease of 97% compared to the reference (100%).

Similarly, the binding of the purified interference factor(s) obtained from each of the three donors to each of 53 Nanobodies or Nanobody constructs was measured using the same Biacore instrument and compared with the binding of the purified interference factor(s) to the same reference Nanobody or construction. The results were expressed as a percentage of the decrease in the binding of the interference factor with a given Nanobody or Nanobody design against the reference one.

It was found that virtually all Nanobodies or Nanobody constructs in which one or more amino acid residues were added to the C-terminus compared to the reference showed a significant decrease in the binding of the interference factor(s). This again confirms that adding one or more amino acid residues to the C-terminus of the ISV (VTVSS) can reduce non-specific protein interference in the ADA assay. It has also been found that, in most cases, only substitution of amino acid residues within the C-terminal region (i.e., without adding one or more amino acid residues to the C-terminus) compared to the reference does not have such a significant effect on the binding of the factor(s) interference.

The results were then further analyzed to determine if the decrease in 21-4 binding correlated in any way with the decrease in binding of each of the three different purified interference factor preparations compared to the reference. Such correlations have been found.

For example, of the 54 Nanotels or Nanotel constructs tested, 36 showed a greater than 70% reduction in antibody 21-4 binding compared to their corresponding reference sequence (with most of these 36 having one or more additional amino acid residues per C-terminus, in some cases in combination with substitutions within the C-terminal region). Of these 36, 32 also showed a decrease in binding by interference factor(s) compared to the reference by more than 50% (and in a large number of cases, in particular for Nanotel or Nanotel constructs with the addition of one or more amino acid residues at the C-terminus , the reduction was much more than 50%, for example, more than 70% or even more than 90%, see data presented in Table X). This shows that in 32 out of 36 cases (i.e. 89%), a decrease in 21-4 binding of more than 70% (versus reference = 100%) predicts a decrease in interference factor binding of more than 50% ( compared to the same benchmark). For the sake of clarity, in each case the reduction was calculated as 100% - [the percentage given in the tables below for the level of reduction achieved using the tested Nanobody].

Similarly, of the 53 Nanotels or Nanotel constructs tested, 33 showed a greater than 90% reduction in antibody 21-4 binding compared to their respective reference sequence (and in this case, most of these 36 had one or more additional amino acid residues at the C-terminus, in some cases in combination with substitutions within the C-terminus). Of these 33, 32 also showed a reduction in binding by the interference factor(s) compared to their respective reference sequence by more than 50%. This shows that in 32 out of 33 cases (i.e. 97%), a decrease in antibody 21-4 binding of more than 90% (compared to reference) predicts a decrease in interference factor binding of more than 50% (compared to with a standard).

It should be noted that this decrease in the binding of the interference factor(s) by more than 50% (confirmed by a decrease in the binding of 21-4 by more than 70%) means that such an interference factor(s) does not actually interfere with the ADA analysis anymore. With respect to the discussed ISV: Experimental confirmation using the ADA assay has shown that when binding by the interference factor(s) is reduced by more than 45%, then there is no significant effect of the presence of the interference factor(s) on the ADA assay. In this regard, those skilled in the art will also appreciate that this will be all the more the case when the binding interference factor(s) is reduced to a much greater extent than 50% (e.g., more than 70% or even more than 90%) as observed in some cases (see again the data presented here).

In fact, a greater than 45% decrease in 21-4 binding was found to be indicative of a greater than 45% decrease in interference factor binding, which, as already discussed, means that the interference factor(s) no longer interferes with ADA- analysis.

Moreover, the data presented here regarding the correlation between (decrease in) 21-4 binding and (decrease in) interference factor binding also allows the present inventors to establish an absolute value for 21-4 binding below which, as can be expected (within the confidence provided by the data presented in this Example 8) that the ISV or ISV-based construct will not be susceptible to binding by interference factor(s) to interfere with the ADA analysis. As set in the following Example 9, this value is 500 RU (determined and calculated as set in Example 9).

Monoclonal 21-4 was purified from the hybridoma culture medium prepared in Example 7 above as follows: Hybridoma cells secreting monoclonal antibody 21-4-3 were cultured in spinner flasks in serum-free medium (CD Hybridoma, Gibco, supplemented with 8mM L -glutamine (Invitrogen) and 1×cholesterol (250×lipid cholesterol concentrate, Gibco)) in a volume of 100ml or 500ml. The purified supernatant was filtered and mouse IgG1 was captured on a ProteinA column (HiTrap MabSelect SuRe, 5ml, GE Healthcare) at a reduced flow rate of 2ml/min. The bound antibody was eluted in 0.1M citrate buffer pH 3.0 and the elution fractions (5ml) were directly neutralized with 1ml 1M TRIS pH9. The purity of the antibody was confirmed by reducing and non-reducing SDS-PAGE analysis.

Purified preparations of interference factor(s) from Donors 8 and 19 were obtained from serum samples from the indicated donors by affinity purification, essentially as described in Example 2A. Interference factor(s) from Donor 30 were obtained from a serum sample of Donor 30, in principle as described in Example 2B.

The protocol described in Example 9 was used to determine binding of 21-4 to each of the Nanobodies or Nanobody constructs.

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

Example 9: Protocol for predicting whether an ISV will be prone to non-specific protein interference (using monoclonal 21-4)

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

Table XI

Protein Conc. (µg/ml) Contact time (sec) Flow rate (µl/min) Buffer for immobilization Level of immobilization Anti-mouse IgG thirty 420 5 10mM acetate pH5 13028 Anti-mouse IgG thirty 420
24 5 10mM acetate pH5 13318

Conditions used in binding experiments (Biacore T100) using immobilized 21-4 are also shown in Table XII. The anti-mouse IgG surface could be successfully regenerated after mAb21-4-3 capture and injection of all samples (with a limited increase from baseline after each regeneration).

Table XII

Capture (catching) flow path 4 Flow rate (µl/min) 10 Contact time (sec) 180 Concentration (µg/ml) 10 Binding and dissociation flow path 3.4 Flow rate (µl/min) 45 Sample contact time (sec) 120 Sample Concentration (nM) 500 Dissociation time (sec) 600 Regeneration 1 flow path 3.4 Flow rate (µl/min) 10 Contact regeneration time (sec) 180 regeneration buffer 10mM Glycine-HCl pH1.7 Stabilization time (sec) 120 if…then…more If after regeneration 1 >20RU on Fc4 Another exit cycle Regeneration 2 flow path 3.4 Flow rate (µl/min) 10 Contact regeneration time (sec) 120 regeneration buffer 10mM Glycine-HCl pH1.7 Stabilization time (s) 120

The above protocol was used to obtain the binding results of 21-4 shown in Table X. When absolute RU values were considered (after adjusting the measured RU value according to the molecular weight of the ISV, protein, or polypeptide according to the formula ([RU measured]/[MW protein] x 10 ⁶ ), it was found that the Nanobodies and the Nanobody constructs mentioned in Table X that had an additional alanine residue and that showed a >90% reduction in binding in relation to 21-4 as well as in relation to interference factors, as a rule, provided RU values between 30RU and 400RU (with the corresponding reference Nanobodies or polypeptides - as listed in Figure 9 - having RU values greater than 1000, typically greater than 1500, and often greater than 2000).

Based on this, it is believed that a (corrected) RU value of less than 500 in this assay method can clearly show an ISV (or a protein or polypeptide containing at least one IS, as described here) that will not (actually) be bound by interference factors in such a way as to interfere with ADA analysis.

The entire contents of all references (including bibliography, issued patents, published patent applications and concurrent patent applications) cited throughout this application are expressly incorporated herein by reference, in particular to the parent reference that is mentioned in the specification.

SEQUENCE LIST

<110> Ablynx N.V.

<120> TECHNIQUES FOR PREDICTING, DETECTING AND REDUCING ASPECIFIC

PROTEIN INTERFERENCE IN ASSAYS INVOLVING IMMUNOGLOBULIN SINGLE

VARIABLE DOMAINS

<130> P011-08-PCT

<160> 89

<170>PatentIn version 3.5

<210> 1

<211> 270

<212> PRT

<213> -

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Lys Ser Ser Gly Asp Ser Thr Arg Tyr Ala Gly Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Arg Val Ser Arg Thr Gly Leu Tyr Thr Tyr Asp Asn Arg

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

130 135 140

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

145 150 155 160

Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Tyr Ala Met

165 170 175

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala

180 185 190

Ile Thr Arg Ser Gly Val Arg Ser Gly Val Ser Ala Ile Tyr Gly Asp

195 200 205

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

210 215 220

Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr

225 230 235 240

Tyr Cys Ala Ala Ser Ala Ile Gly Ser Gly Ala Leu Arg Arg Phe Glu

245 250 255

Tyr Asp Tyr Ser Gly Gln Gly Thr Leu Val Thr Val Ser Ser

260 265 270

<210> 2

<211> 385

<212> PRT

<213> -

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val

35 40 45

Ser Ser Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Tyr Ile Arg Pro Asp Thr Tyr Leu Ser Arg Asp Tyr Arg Lys

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

130 135 140

Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser

145 150 155 160

Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro

165 170 175

Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp

180 185 190

Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

195 200 205

Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu

210 215 220

Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser

225 230 235 240

Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

245 250 255

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

260 265 270

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

275 280 285

Ser Ser Tyr Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg

290 295 300

Glu Phe Val Ser Ser Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala

305 310 315 320

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

325 330 335

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val

340 345 350

Tyr Tyr Cys Ala Ala Tyr Ile Arg Pro Asp Thr Tyr Leu Ser Arg Asp

355 360 365

Tyr Arg Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

370 375 380

Ser

385

<210> 3

<211> 117

<212> PRT

<213> -

<400> 3

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Gly Arg Leu Asp

20 25 30

Arg Met Gly Trp Tyr Arg His Arg Thr Gly Glu Pro Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Phe Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 4

<211> 140

<212> PRT

<213> -

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Gly Arg Leu Asp

20 25 30

Arg Met Gly Trp Tyr Arg His Arg Thr Gly Glu Pro Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Phe Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu

115 120 125

Asp Leu Asn Gly Ala Ala His His His His His His His

130 135 140

<210> 5

<211> 117

<212> PRT

<213> -

<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Gly Arg Leu Asp

20 25 30

Arg Met Gly Trp Tyr Arg His Arg Pro Gly Glu Pro Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 6

<211> 125

<212> PRT

<213> -

<400> 6

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala

115 120 125

<210> 7

<211> 124

<212> PRT

<213> -

<400> 7

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 8

<211> 124

<212> PRT

<213> -

<400> 8

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

115 120

<210> 9

<211> 125

<212> PRT

<213> -

<400> 9

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly

115 120 125

<210> 10

<211> 126

<212> PRT

<213> -

<400> 10

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

<210> 11

<211> 123

<212> PRT

<213> -

<400> 11

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 12

<211> 123

<212> PRT

<213> -

<400> 12

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 13

<211> 123

<212> PRT

<213> -

<400> 13

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 14

<211> 123

<212> PRT

<213> -

<400> 14

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 15

<211> 123

<212> PRT

<213> -

<400> 15

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Gln Val Ser Ser

115 120

<210> 16

<211> 123

<212> PRT

<213> -

<400> 16

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Ser Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 17

<211> 121

<212> PRT

<213> -

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val Phe Lys Ile Asn

20 25 30

Val Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val

35 40 45

Ala Gly Ile Ile Ser Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Ile Thr Thr Glu Ser Asp Tyr Asp Leu Gly Arg Arg Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 146

<212> PRT

<213> -

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val Phe Lys Ile Asn

20 25 30

Val Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val

35 40 45

Ala Gly Ile Ile Ser Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Ile Thr Thr Glu Ser Asp Tyr Asp Leu Gly Arg Arg Tyr Trp Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Ser Arg Asp Trp Asp Phe Asp Val Phe Gly Gly Gly Thr Pro Val

130 135 140

Gly Gly

145

<210> 19

<211> 151

<212> PRT

<213> -

<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Leu Pro Phe Ser Thr Lys

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Arg Ile Ser Pro Gly Gly Thr Ser Arg Tyr Tyr Gly Asp Phe Val

50 55 60

Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Thr Trp

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Glu Arg Ser Thr Tyr Ile Gly Ser Asn Tyr Tyr Arg Thr

100 105 110

Asn Glu Tyr Asp Tyr Trp Gly Thr Gly Thr Gln Val Thr Val Ser Ser

115 120 125

Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala

130 135 140

Ala His His His His His His

145 150

<210> 20

<211> 128

<212> PRT

<213> -

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Leu Pro Phe Ser Thr Lys

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Arg Ile Ser Pro Gly Gly Thr Ser Arg Tyr Tyr Gly Asp Phe Val

50 55 60

Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Thr Trp

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Glu Arg Ser Thr Tyr Ile Gly Ser Asn Tyr Tyr Arg Thr

100 105 110

Asn Glu Tyr Asp Tyr Trp Gly Thr Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 21

<211> 151

<212> PRT

<213> -

<400> 21

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Pro Phe Ser Thr Lys

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val

35 40 45

Ser Arg Ile Ser Pro Gly Gly Thr Ser Arg Tyr Tyr Gly Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Glu Arg Ser Thr Tyr Ile Gly Ser Asn Tyr Tyr Arg Thr

100 105 110

Asn Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala

130 135 140

Ala His His His His His His

145 150

<210> 22

<211> 128

<212> PRT

<213> -

<400> 22

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Pro Phe Ser Thr Lys

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val

35 40 45

Ser Arg Ile Ser Pro Gly Gly Thr Ser Arg Tyr Tyr Gly Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Glu Arg Ser Thr Tyr Ile Gly Ser Asn Tyr Tyr Arg Thr

100 105 110

Asn Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 23

<211> 134

<212> PRT

<213> -

<400> 23

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp Phe Arg Arg Ala Pro Gly

35 40 45

Lys Glu Arg Glu Phe Val Ala Ala Ile Thr Arg Ser Gly Val Arg Ser

50 55 60

Gly Val Ser Ala Ile Tyr Gly Asp Ser Val Lys Asp Arg Phe Thr Ile

65 70 75 80

Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

85 90 95

Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys Ala Ala Ser Ala Ile Gly

100 105 110

Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr

115 120 125

Gln Val Thr Val Ser Ser

130

<210> 24

<211> 134

<212> PRT

<213> -

<400> 24

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp Phe Arg Arg Ala Pro Gly

35 40 45

Lys Glu Arg Glu Phe Val Ala Ala Ile Thr Arg Ser Gly Val Arg Ser

50 55 60

Gly Val Ser Ala Ile Tyr Gly Asp Ser Val Lys Asp Arg Phe Thr Ile

65 70 75 80

Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

85 90 95

Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys Ala Ala Ser Ala Ile Gly

100 105 110

Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr

115 120 125

Gln Val Thr Val Ser Ser

130

<210> 25

<211> 134

<212> PRT

<213> -

<400> 25

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp Phe Arg Arg Ala Pro Gly

35 40 45

Lys Glu Arg Glu Phe Val Ala Ala Ile Thr Arg Ser Gly Val Arg Ser

50 55 60

Gly Val Ser Ala Ile Tyr Gly Asp Ser Val Lys Asp Arg Phe Thr Ile

65 70 75 80

Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

85 90 95

Arg Pro Glu Asp Thr Ala Val Tyr Thr Cys Ala Ala Ser Ala Ile Gly

100 105 110

Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr

115 120 125

Gln Val Thr Val Ser Ser

130

<210> 26

<211> 134

<212> PRT

<213> -

<400> 26

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp Phe Arg Arg Ala Pro Gly

35 40 45

Lys Glu Arg Glu Phe Val Ala Ala Ile Thr Arg Ser Gly Val Arg Ser

50 55 60

Gly Val Ser Ala Ile Tyr Gly Asp Ser Val Lys Asp Arg Phe Thr Ile

65 70 75 80

Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

85 90 95

Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys Ala Ala Ser Ala Ile Gly

100 105 110

Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser

130

<210> 27

<211> 134

<212> PRT

<213> -

<400> 27

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp Phe Arg Arg Ala Pro Gly

35 40 45

Lys Glu Arg Glu Phe Val Ala Ala Ile Thr Arg Ser Gly Val Arg Ser

50 55 60

Gly Val Ser Ala Ile Tyr Gly Asp Ser Val Lys Asp Arg Phe Thr Ile

65 70 75 80

Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

85 90 95

Arg Pro Glu Asp Thr Ala Val Tyr Thr Cys Ala Ala Ser Ala Ile Gly

100 105 110

Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser

130

<210> 28

<211> 134

<212> PRT

<213> -

<400> 28

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly

35 40 45

Lys Glu Arg Glu Phe Val Ala Ala Ile Thr Arg Ser Gly Val Arg Ser

50 55 60

Gly Val Ser Ala Ile Tyr Gly Asp Ser Val Lys Asp Arg Phe Thr Ile

65 70 75 80

Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

85 90 95

Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ser Ala Ile Gly

100 105 110

Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser

130

<210> 29

<211> 137

<212> PRT

<213> -

<400> 29

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly

35 40 45

Lys Glu Arg Glu Phe Val Ala Ala Ile Thr Arg Ser Gly Val Arg Ser

50 55 60

Gly Val Ser Ala Ile Tyr Gly Asp Ser Val Lys Asp Arg Phe Thr Ile

65 70 75 80

Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

85 90 95

Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ser Ala Ile Gly

100 105 110

Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ala Ala

130 135

<210> 30

<211> 137

<212> PRT

<213> -

<400> 30

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly

35 40 45

Lys Glu Arg Glu Phe Val Ala Ala Ile Thr Arg Ser Gly Val Arg Ser

50 55 60

Gly Val Ser Ala Ile Tyr Gly Asp Ser Val Lys Asp Arg Phe Thr Ile

65 70 75 80

Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

85 90 95

Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ser Ala Ile Gly

100 105 110

Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ala Ala

130 135

<210> 31

<211> 367

<212> PRT

<213> -

<400> 31

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Gly Arg Leu Asp

20 25 30

Arg Met Gly Trp Tyr Arg His Arg Pro Gly Glu Pro Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val

115 120 125

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu

130 135 140

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met

145 150 155 160

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

165 170 175

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln

195 200 205

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile

210 215 220

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu

245 250 255

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

260 265 270

Ala Ala Ser Arg Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg

275 280 285

His Arg Pro Gly Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly

290 295 300

Ser Ser Ile Asn Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

305 310 315 320

Ile Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg

325 330 335

Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr

340 345 350

Ser Arg Asp Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

355 360 365

<210> 32

<211> 368

<212> PRT

<213> -

<400> 32

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Gly Arg Leu Asp

20 25 30

Arg Met Gly Trp Tyr Arg His Arg Pro Gly Glu Pro Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val

115 120 125

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu

130 135 140

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met

145 150 155 160

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

165 170 175

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln

195 200 205

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile

210 215 220

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu

245 250 255

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

260 265 270

Ala Ala Ser Arg Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg

275 280 285

His Arg Pro Gly Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly

290 295 300

Ser Ser Ile Asn Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

305 310 315 320

Ile Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg

325 330 335

Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr

340 345 350

Ser Arg Asp Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

355 360 365

<210> 33

<211> 5

<212> PRT

<213> -

<400> 33

Val Thr Val Ser Ser

15

<210> 34

<211> 6

<212> PRT

<213> -

<220>

<221> MISC_FEATURE

<222>(6)..(6)

<223> X = a group (X)n, in which N = 1-10, preferably 1-5 and X is any

amino acids.

<400> 34

Val Thr Val Ser Ser Xaa

15

<210> 35

<211> 218

<212> PRT

<213> -

<400> 35

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr

20 25 30

Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Val Thr Gly Glu Pro Ala Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Thr Arg Gly Leu Ile His Phe Tyr Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala

115 120 125

Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu

130 135 140

Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly

145 150 155 160

Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp

165 170 175

Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro

180 185 190

Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Ser Thr Lys

195 200 205

Val Asp Lys Lys Ile Val Pro Arg Asp Cys

210 215

<210> 36

<211> 214

<212> PRT

<213> -

<400> 36

Asp Ile Gln Met Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile His Asn Phe

20 25 30

Ile Ser Trp Tyr Gln His Lys Pro Gly Lys Val Pro Arg Leu Ile Ile

35 40 45

His Asp Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Thr Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu His Tyr Asp Asn Leu Leu Arg

85 90 95

Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Ser Ser Glu Gln Leu Thr Ser Gly

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 37

<211> 122

<212> PRT

<213> -

<400> 37

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 38

<211> 271

<212> PRT

<213> -

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Lys Ser Ser Gly Asp Ser Thr Arg Tyr Ala Gly Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Arg Val Ser Arg Thr Gly Leu Tyr Thr Tyr Asp Asn Arg

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

130 135 140

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

145 150 155 160

Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Tyr Ala Met

165 170 175

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala

180 185 190

Ile Thr Arg Ser Gly Val Arg Ser Gly Val Ser Ala Ile Tyr Gly Asp

195 200 205

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

210 215 220

Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr

225 230 235 240

Tyr Cys Ala Ala Ser Ala Ile Gly Ser Gly Ala Leu Arg Arg Phe Glu

245 250 255

Tyr Asp Tyr Ser Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

260 265 270

<210> 39

<211> 386

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody or Nanobody construct

<400> 39

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val

35 40 45

Ser Ser Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Tyr Ile Arg Pro Asp Thr Tyr Leu Ser Arg Asp Tyr Arg Lys

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

130 135 140

Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser

145 150 155 160

Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro

165 170 175

Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp

180 185 190

Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

195 200 205

Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu

210 215 220

Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser

225 230 235 240

Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

245 250 255

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

260 265 270

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

275 280 285

Ser Ser Tyr Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg

290 295 300

Glu Phe Val Ser Ser Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala

305 310 315 320

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

325 330 335

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val

340 345 350

Tyr Tyr Cys Ala Ala Tyr Ile Arg Pro Asp Thr Tyr Leu Ser Arg Asp

355 360 365

Tyr Arg Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

370 375 380

Ser Ala

385

<210> 40

<211> 364

<212> PRT

<213> -

<400> 40

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Ser Gly Phe Asn Arg Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu

115 120 125

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu

130 135 140

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp

145 150 155 160

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser

165 170 175

Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn

195 200 205

Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly

210 215 220

Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

245 250 255

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

260 265 270

Ser Gly Phe Thr Phe Ser Asp Tyr Trp Met Tyr Trp Val Arg Gln Ala

275 280 285

Pro Gly Lys Gly Leu Glu Trp Val Ser Glu Ile Asn Thr Asn Gly Leu

290 295 300

Ile Thr Lys Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

305 310 315 320

Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro

325 330 335

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Pro Ser Gly Phe Asn

340 345 350

Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

355 360

<210> 41

<211> 246

<212> PRT

<213> -

<400> 41

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val Phe Lys Ile Asn

20 25 30

Val Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val

35 40 45

Ala Gly Ile Ile Ser Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Ile Thr Thr Glu Ser Asp Tyr Asp Leu Gly Arg Arg Tyr Trp Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

130 135 140

Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

145 150 155 160

Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

165 170 175

Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp

180 185 190

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr

195 200 205

Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr

210 215 220

Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu

225 230 235 240

Val Thr Val Ser Ser Ala

245

<210> 42

<211> 260

<212> PRT

<213> -

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn

20 25 30

Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro

100 105 110

Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

Ala Ala Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

130 135 140

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe

145 150 155 160

Ser Tyr Asn Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg

165 170 175

Glu Leu Val Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro

180 185 190

Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg

195 200 205

Met Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

210 215 220

Tyr Tyr Cys Ala Ala Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg

225 230 235 240

Thr Leu Pro Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr

245 250 255

Val Ser Ser Ala

260

<210> 43

<211> 250

<212> PRT

<213> -

<400> 43

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly

130 135 140

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

145 150 155 160

Phe Thr Phe Arg Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

165 170 175

Lys Gly Pro Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

180 185 190

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

195 200 205

Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

210 215 220

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

245 250

<210> 44

<211> 368

<212> PRT

<213> -

<400> 44

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Gly Arg Leu Asp

20 25 30

Arg Met Gly Trp Tyr Arg His Arg Pro Gly Glu Pro Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val

115 120 125

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu

130 135 140

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met

145 150 155 160

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

165 170 175

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln

195 200 205

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile

210 215 220

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu

245 250 255

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

260 265 270

Ala Ala Ser Arg Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg

275 280 285

His Arg Pro Gly Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly

290 295 300

Ser Ser Ile Asn Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

305 310 315 320

Ile Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg

325 330 335

Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr

340 345 350

Ser Arg Asp Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

355 360 365

<210> 45

<211> 117

<212> PRT

<213> -

<400> 45

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Gly Arg Leu Asp

20 25 30

Arg Met Gly Trp Tyr Arg His Arg Thr Gly Glu Pro Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Phe Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 46

<211> 143

<212> PRT

<213> -

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Gly Arg Leu Asp

20 25 30

Arg Met Gly Trp Tyr Arg His Arg Thr Gly Glu Pro Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Phe Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser Ala Ala Ala His His His His His His His Gly Ala

115 120 125

Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala

130 135 140

<210> 47

<211> 146

<212> PRT

<213> -

<400> 47

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val Phe Lys Ile Asn

20 25 30

Val Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val

35 40 45

Ala Gly Ile Ile Ser Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Ile Thr Thr Glu Ser Asp Tyr Asp Leu Gly Arg Arg Tyr Trp Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Ser Arg Asp Trp Asp Phe Asp Val Phe Gly Gly Gly Thr Pro Val

130 135 140

Gly Gly

145

<210> 48

<211> 151

<212> PRT

<213> -

<400> 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Leu Pro Phe Ser Thr Lys

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Arg Ile Ser Pro Gly Gly Thr Ser Arg Tyr Tyr Gly Asp Phe Val

50 55 60

Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Thr Trp

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Glu Arg Ser Thr Tyr Ile Gly Ser Asn Tyr Tyr Arg Thr

100 105 110

Asn Glu Tyr Asp Tyr Trp Gly Thr Gly Thr Gln Val Thr Val Ser Ser

115 120 125

Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala

130 135 140

Ala His His His His His His

145 150

<210> 49

<211> 151

<212> PRT

<213> -

<400> 49

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Pro Phe Ser Thr Lys

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val

35 40 45

Ser Arg Ile Ser Pro Gly Gly Thr Ser Arg Tyr Tyr Gly Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Glu Arg Ser Thr Tyr Ile Gly Ser Asn Tyr Tyr Arg Thr

100 105 110

Asn Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala

130 135 140

Ala His His His His His His

145 150

<210> 50

<211> 121

<212> PRT

<213> -

<400> 50

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Ser Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 51

<211> 121

<212> PRT

<213> -

<400> 51

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Gln Val Ser Ser

115 120

<210> 52

<211> 121

<212> PRT

<213> -

<400> 52

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Gly Ser

115 120

<210> 53

<211> 121

<212> PRT

<213> -

<400> 53

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Gly

115 120

<210> 54

<211> 121

<212> PRT

<213> -

<400> 54

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Ser Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Gln Val Gly Gly

115 120

<210> 55

<211> 122

<212> PRT

<213> -

<400> 55

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 56

<211> 123

<212> PRT

<213> -

<400> 56

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly

115 120

<210> 57

<211> 123

<212> PRT

<213> -

<400> 57

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

115 120

<210> 58

<211> 124

<212> PRT

<213> -

<400> 58

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120

<210> 59

<211> 125

<212> PRT

<213> -

<400> 59

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

<210> 60

<211> 124

<212> PRT

<213> -

<400> 60

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Pro

115 120

<210> 61

<211> 123

<212> PRT

<213> -

<400> 61

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Pro

115 120

<210> 62

<211> 124

<212> PRT

<213> -

<400> 62

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Pro Thr

115 120

<210> 63

<211> 122

<212> PRT

<213> -

<400> 63

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Trp

115 120

<210> 64

<211> 122

<212> PRT

<213> -

<400> 64

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

20 25 30

Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly

35 40 45

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

50 55 60

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75 80

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

85 90 95

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Leu

115 120

<210> 65

<211> 123

<212> PRT

<213> -

<400> 65

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 66

<211> 123

<212> PRT

<213> -

<400> 66

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Ser Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 67

<211> 123

<212> PRT

<213> -

<400> 67

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 68

<211> 123

<212> PRT

<213> -

<400> 68

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 69

<211> 123

<212> PRT

<213> -

<400> 69

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 70

<211> 123

<212> PRT

<213> -

<400> 70

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Gln Val Ser Ser

115 120

<210> 71

<211> 123

<212> PRT

<213> -

<400> 71

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Gly

115 120

<210> 72

<211> 123

<212> PRT

<213> -

<400> 72

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Glyn Gly Thr Leu Val Thr Val Gly Gly

115 120

<210> 73

<211> 124

<212> PRT

<213> -

<400> 73

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Glyn Gly Thr Leu Val Thr Val Gly Gly Gly

115 120

<210> 74

<211> 124

<212> PRT

<213> -

<400> 74

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

115 120

<210> 75

<211> 125

<212> PRT

<213> -

<400> 75

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala

115 120 125

<210> 76

<211> 126

<212> PRT

<213> -

<400> 76

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

<210> 77

<211> 124

<212> PRT

<213> -

<400> 77

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 78

<211> 123

<212> PRT

<213> -

<400> 78

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 79

<211> 124

<212> PRT

<213> -

<400> 79

His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

1 5 10 15

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser

20 25 30

Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly Glu

35 40 45

Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr

50 55 60

Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser Lys

65 70 75 80

Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

115 120

<210> 80

<211> 123

<212> PRT

<213> -

<400> 80

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Glyn Gly Thr Leu Val Gln Val Gly Gly

115 120

<210> 81

<211> 123

<212> PRT

<213> -

<400> 81

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 82

<211> 123

<212> PRT

<213> -

<400> 82

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 83

<211> 123

<212> PRT

<213> -

<400> 83

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Ala

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 84

<211> 123

<212> PRT

<213> -

<400> 84

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Gly Ser

115 120

<210> 85

<211> 123

<212> PRT

<213> -

<400> 85

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Ser Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Glyn Gly Thr Leu Val Gln Val Gly Gly

115 120

<210> 86

<211> 123

<212> PRT

<213> -

<400> 86

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Ser Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Gln Val Ser Ser

115 120

<210> 87

<211> 123

<212> PRT

<213> -

<400> 87

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Ser Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Glyn Gly Thr Leu Val Thr Val Gly Gly

115 120

<210> 88

<211> 124

<212> PRT

<213> -

<400> 88

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Ser Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Gln Gly Thr Leu Val Gln Val Ser Ser Ala

115 120

<210> 89

<211> 123

<212> PRT

<213> -

<400> 89

His His His His His His His Glu Val Gln Leu Val Glu Ser Gly Gly Gly

1 5 10 15

Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg

20 25 30

Ser Ile Gly Arg Leu Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly

35 40 45

Glu Pro Arg Glu Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn

50 55 60

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser

65 70 75 80

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Asn Phe Asn Lys Tyr Val Thr Ser Arg Asp Thr

100 105 110

Trp Gly Glyn Gly Thr Leu Val Gln Val Gly Gly

115 120

Claims (45)

1. Heavy chain immunoglobulin single variable domain with reduced propensity for non-specific protein interference for use in therapy where the heavy chain single variable domain of an immunoglobulin is modified at the C-terminus of its sequence by (i) introducing one or more C-terminal amino acid substitutions selected from valine (V) at position 11, glutamine (Q) at position 110, glycine (G) at position 112 and glycine (G) at position 113, where amino acid residues are numbered according to Kabat; And (ii) adding to the C-terminus of the sequence 1-10 amino acid residues, each independently selected from any naturally occurring amino acid. 2. The immunoglobulin heavy chain single variable domain of claim 1, which has been modified at the C-terminus of its sequence by (i) introducing one or more C-terminal amino acid substitutions selected from valine (V) at position 11, glutamine (Q) at position 110, glycine (G) at position 112 and glycine (G) at position 113, where amino acid residues are numbered according to Kabat; And (ii) adding to the C-terminal sequence 1, 2, 3, 4 or 5 amino acid residues, each independently selected from any naturally occurring amino acid. 3. Heavy chain single immunoglobulin variable domain according to any one of paragraphs. 1 or 2, wherein said natural amino acids are independently selected from alanine, glycine, valine, leucine, or isoleucine. 4. Heavy chain single immunoglobulin variable domain according to any one of paragraphs. 1-3, which has the sequence VTVSS(X)n at its C-terminus, where a) n=1, 2 or 3, where each X=Ala or Gly; or b) n=1, 2 or 3, where each X=Ala; or c) n=1, 2 or 3, where each X=Gly; or d) n=2 or 3, where at least one X=Ala or Gly; or e) n=2 or 3, where all but one X=Ala or Gly. 5. Heavy chain single immunoglobulin variable domain according to any one of paragraphs. 1-4, which has the sequence VTVSS(X)n at its C-terminus, where: - X is Ala and n is 1, 2 or 3; or - X is Gly and n is 2 or 3. 6. Heavy chain single immunoglobulin variable domain according to any one of paragraphs. 1-5 which has the sequence VTVSS(X)n at its C-terminus, where X is Ala and n is 1. 7. Heavy chain single immunoglobulin variable domain according to any one of paragraphs. 1-6, in which the C-terminal part of its sequence was modified by introducing a valine(V) residue at position 11. 8. Heavy chain single immunoglobulin variable domain according to any one of paragraphs. 1-7 which has a significantly reduced propensity, such as at least a statistically significantly reduced propensity, to generate protein interference, compared to the same immunoglobulin single variable domain but without modifications. 9. Heavy chain single immunoglobulin variable domain according to any one of paragraphs. 1-8, which has, in the assay described in Example 3, a significantly lower propensity to bind to the polyclonal antibody described in Example 2 compared to the same immunoglobulin single variable domain but without modifications. 10. Heavy chain single immunoglobulin variable domain according to any one of paragraphs. 1-8, which has, in the assay described in Example 5, a significantly lower propensity to bind to the polyclonal antibody described in Example 2 compared to the same immunoglobulin single variable domain but without modifications. 11. Heavy chain single immunoglobulin variable domain according to any one of paragraphs. 1-10, which is a humanized VHH or camelized VH, such as human camelized VH. 12. A polypeptide for use in therapy, where the polypeptide contains a heavy chain single immunoglobulin variable domain according to any one of paragraphs. 1-11 at its C-terminus. 13. A polypeptide according to claim 12 which is a bivalent, multivalent, bispecific, multispecific, biparatopic, or multiparatopic immunoglobulin heavy chain single variable domain construct. 14. A polypeptide for use in therapy, containing one or more heavy chain single immunoglobulin variable domains according to any one of paragraphs. 1-11 at its C-terminus and additionally containing one or more other therapeutic agents and/or one or more other agents that affect the pharmacokinetic or pharmacodynamic properties of the polypeptide. 15. The polypeptide of claim 14, wherein one or more other agents affect the half-life of the polypeptide. 16. The polypeptide of claim 15, wherein one or more other agents increase the half-life of the polypeptide. 17. The polypeptide according to any one of paragraphs. 14-16 wherein one or more other agents comprise a single immunoglobulin variable domain that binds serum albumin. 18. The use of a single variable domain of an immunoglobulin according to any one of paragraphs. 1-11, in the method of treatment. 19. The use of a polypeptide according to any one of paragraphs. 12-17, in the method of treatment. 20. Pharmaceutical composition which contains a single immunoglobulin variable domain according to any one of paragraphs. 1-11 or a polypeptide according to any one of paragraphs. 12-17 in a therapeutically active dose and at least one suitable carrier, diluent or excipient. 21. A biological drug, such as a protein or polypeptide, for use in therapy, where the biological drug contains at least one immunoglobulin single variable domain according to any one of paragraphs. 1-11 at its C-terminus.

Images