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

Abstract

This invention provides a method to avoid protein interference and / or nonspecific signaling in assays (e.g., ADA immunoassays) when an immunoglobulin single variable domain (ISV) is affected. [Solution] The ISV is modified and / or improved by adding a limited number of amino acid residues to the C-terminus of the variable domain. Even if it is not sufficient to "cover" or "fill" the hydrophobic patch present at the C-terminus of the ISV, the problem of protein interference in the ADA assay can be substantially or even essentially completely eliminated.

Description

[Technical Field]

[0001] This invention relates to the field of immunoglobulin monovariable domains.

[0002] An immunoglobulin monovariate domain or "ISV" is generally used herein. - It contains an immunoglobulin fold, or can form an immunoglobulin fold under appropriate conditions (e.g., physiological conditions) (i.e., by folding) to form an immunoglobulin variable domain (e.g., a VH, VL, or VHH domain); and - Defined as an amino acid sequence that forms an immunoglobulin variable domain containing a functional antigen-binding site (or is capable of forming such an immunoglobulin variable domain under suitable conditions) (in the sense that it does not require interaction with another immunoglobulin variable domain (e.g., VH-VL interaction) to form a functional antigen-binding site).

[0003] Some examples of immunoglobulin monovariable domains currently known in the art are VHH and / or (other) nanobodies, dAbs, and (mono)domain antibodies. Among these, various nanobodies are in Phase I and Phase II clinical trials as of the filing date of this application. Therefore, it is important to make available reliable assays for analyzing biological samples from people treated with ISVs (e.g., subjects in clinical trials and patients treated with such ISVs after they are marketed).

[0004] Clinicians prescribing treatments also want reliable assays available to monitor various treatment methods, which is important not only for control purposes but also for patient treatment with biological drugs.

[0005] For example, in the clinical development of biological drug molecules, it is important to evaluate their immunogenicity, particularly the extent to which they can induce so-called "anti-drug antibodies" or "ADA". This is determined using so-called "anti-drug antibody" or "ADA (immuno) assays" (see, for example, the review by Shankar et al., Journal of Pharmaceutical and Biomedical Analysis, 48 (2008), 1267-1281; and 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 ADA assays, and the methods for performing them, are standard knowledge in the field of pharmacology and are commonly used in the clinical development of biological drug products (and are required by various regulatory authorities around the world).

[0006] As described, for example, on pages 3 and 4 of the Mire-Sluis paper and schematically illustrated, for example, in the figures of the Peng paper, a number of different ADA assay formats are known, such as "ELISA-bridging format", "ELISA-direct format", "indirect format", radioimmunoprecipitation assay (RIP), "surface plasmon resonance", and "electrochemiluminescence-bridging format". Other formats for performing ADA immunoassays will be apparent to those skilled in the art.

[0007] Those skilled in the art will also be familiar with a number of different commercially available technical platforms that have been shown to be suitable for preparing and performing ADA assays. These include, but are not limited to, the MSD platform (Mesoscale), the Gyrolab (Gyros), and the octet platform (Fortebio).

[0008] Some non-limiting examples of the ADA assay format are also schematically shown in FIGS. 1A-1C.

[0009] Generally, in such ADA assays for detecting or measuring ADA against an ISV, it should be noted that the ISV is used as the "analyte" (i.e., the compound used to detect whether ADA is present in the sample being tested), and the ADA is the "antigen" (i.e., the compound to be detected in the sample being tested). Thus, in these assays, the ISV is typically / often bound to a carrier (e.g., an ELISA plate), whereas the ADA is present (if any) in the sample being assayed.

[0010] To better understand the present invention as described herein, it should be noted that, in contrast, in the methods used herein to predict whether an ISV will cause protein interference, the ISV is typically used as an "antigen" (i.e., as the compound to be detected), and the antibody (which will be further described herein) is used as an "analyte" (i.e., as a means to detect whether a given ISV binds to it and whether there is a high or increased risk of causing protein interference, respectively). Thus, in the methods of the present invention, the antibody used as the analyte (which will also be referred to herein as the "analytical antibody") is typically bound to a carrier (i.e., to an ELISA plate), and the ISV is the sample to be tested (which is present in the sample). However, it should be noted that, in general, the present invention is not limited to assays in which the "analytical antibody" is bound to a carrier. For example, in alternative methods of carrying out the assay of the present invention (as shown in Figure 1 and described in the Examples), the analytical antibody is used instead as a bridging agent, so it is present in solution rather than bound to a plate (although it is indirectly bound to the plate via the ISV coated on the plate). However, even in the specific bridging assays described in the examples (which are competitive assays), analytical antibodies are still used as analytical agents (i.e., to determine whether each ISV of interest binds and whether there is a high or increased risk of causing protein interference). Based on further disclosures herein, it is conceivable that those skilled in the art could design other assay formats in which analytical antibodies may be used as analytical agents to determine whether each given ISV can bind and whether there is a high or increased risk of causing protein interference.

[0011] As a result of studies on single-stranded Fv or "ScFv" (these are constructs containing a single immunoglobulin variable domain that is not bound to a constant domain, similar to ISV), it has been explained in the art that the C-terminus of the single immunoglobulin variable domain forms a hydrophobic patch that is embedded at the interface between the variable domain and the constant domain in antibodies, but becomes exposed to the solvent when the variable domain is not bound to the constant domain (Nieba et al., Protein Engineering, 10, 435-444 (1997)). The exposed C-terminus can give rise to (novel and / or existing) anti-drug antibodies and / or form a B-cell epitope that can interact with (novel and / or existing) anti-drug antibodies (International Publication No. 11 / 07586), and it has also been explained that the presence of these can be determined using the ADA assay described above. For this reason, it has been proposed to make mutations in some of the amino acid residues that form part of the C-terminus of the variable domain to reduce the hydrophobicity and / or remove the epitope. For example, Nieba et al. propose mutating positions 11, 14, 41, 84, 87 and / or 89 (Kabat numbering) in the VH domain, whereas International Publication No. 11 / 07586 proposes mutating positions 99, 101 and / or 148 (AHo numbering) in the VL domain, or positions 12, 97, 98, 99, 103 and / or 144 (AHo numbering, as before - these positions correspond to positions 11, 83, 84, 85, 89 and 103 in Kabat).

[0012] However, none of these references acknowledge that certain proteins present in the subject's blood or serum may interfere with ADA assays using ISVs. Consequently, these references do not address (or provide solutions to) the challenge of how to avoid such nonspecific protein interference in ADA assays so that they can be used to determine the true presence / absence of (novel or existing) anti-drug antibodies in the sample being tested.

[0013] In contrast, the present invention provides methods and assays that enable those skilled in the art to easily predict whether an immunoglobulin monovariate domain is prone to nonspecific protein interference in an ADA assay. The methods and assays described herein also enable those skilled in the art to easily test a candidate modification to a variable domain in order to predict whether the modification will reduce or essentially completely avoid such protein interference if the variable domain appears to be prone to or at risk of such protein interference in an ADA assay.

[0014] The present invention also describes numerous modifications that can be made to a variable domain to reduce or essentially avoid such protein interference. In one non-limiting embodiment, this modification involves adding a limited number of amino acid residues (more described herein) to the C-terminus of the variable domain. Surprisingly, it has been found that for a number of different variable domains or constructs based thereon, adding even just one amino acid residue (e.g., one alanine residue) to the C-terminus can substantially or more essentially eliminate the problem of protein interference in the ADA assay, even if such a single amino acid addition itself is not sufficient to "cover" or "fill" the hydrophobic patch present at the C-terminus of the ISV, according to Nieba et al. Similarly, without limiting the present invention to any or any mechanism or description, it is also conceivable that such a single amino acid addition is not sufficient to "cover" or "fill" the B cell epitope that may be present at the C-terminus of the variable domain, according to International Publication No. 11 / 07586. According to this specific aspect of the present invention, by adding a limited number or even one amino acid to the C-terminus of a variable domain (i.e., without creating a substitution within the C-terminal region itself, as proposed by Nieba et al. and International Publication No. 11 / 07586), the problem of nonspecific protein interference can be significantly reduced, or even essentially eliminated in many cases. It should be noted, however, that combining such additions to the C-terminus with mutations within the C-terminal region is also within the scope of this aspect of the present invention. However, it should be noted that in this regard, the present invention is not particularly limited in terms of the rationale for creating such mutations. For example, a variable domain (but not limited to V) HH To humanize (including the domain), or V HIt is well known that mutations are made in C-terminal amino acid residues (including those explicitly mentioned by Nieba et al. and in International Publication 11 / 07586) in order to “camelize” the domain (see, for example, International Publication No. 08 / 020079 and some other applications by Ablynx NV referenced herein).

[0015] The methods, assays, and modifications taught herein may be applied to a variety of variable domains that are not linked to, or are otherwise linked to, a constant domain (or another group or peptide moiety that "protects," covers, or "fills" the C-terminal region of the variable domain), and more generally, to variable domains having a C-terminal region exposed to a solvent. However, according to one preferred but non-limiting aspect of the present invention, these methods, assays, and modifications may be applied in particular to heavy chain variable domains (V H ) may be applied, and according to one specific aspect of the present invention, V HH Applicable to domains.

[0016] The methods, assays, and modifications described herein may be appropriately applied to protein constructs containing one or more variable domains, in particular to such constructs in which the variable domains form the C-terminal portion of the construct, or otherwise, in the case of the methods and assays described herein, the C-terminal region of the variable domains is exposed to a solvent. As before, according to one preferred but non-limiting aspect of the present invention, these methods, assays, and modifications may be applied to V H Domain (especially V) HH The domain) forms the C-terminal portion of the construct, or, in the case of the methods and assays of the present invention, is applied to the construct that is exposed to the solvent.

[0017] Some non-limiting examples of such structures include two or more ISVs (as before, according to one specific embodiment, V) connected directly or via one or more suitable linkers. H or V HHa domain) and is a multivalent, multispecific (e.g., bispecific) or multiparatopic (e.g., biparatopic) construct containing two or more ISVs that form the C-terminal portion of such a construct. For example, without limitation, such a construct may be a V domain, particularly a nanobody (i.e., V domain), a humanized V domain, or a camelized V domain, directly or linked via one or more suitable linkers as described above. H domain, particularly a nanobody (i.e., V HH domain, a humanized V HH domain or a camelized V H domain) and may be composed entirely. For some non-limiting examples of such constructs and general teachings on how such constructs can be made (particularly based on nanobodies), see, for example, Conrath et al., JBC 276, 10(9), 7346 (2001) and the review article by Muyldermans in Reviews in Mol. Biotechnol., 74: 27 (2001).

[0018] However, for example, the present invention is also contemplated to be applicable to other constructs having a variable domain exposed to a solvent, particularly those having a variable domain at their C-terminus, such as a single-chain Fv having its heavy-chain variable domain at the C-terminus, particularly ScFv.

[0019] In this specification and the claims, terms such as "ISV", "analytical agent" and "protein interference" have meanings further defined herein.

[0020] In particular, the ISV described herein may be particularly a nanobody, or a VH domain or an (other) ISV (i.e., other than a nanobody) containing a VH domain, preferably a nanobody.

[0021] Furthermore, any protein or polypeptide containing an ISV (e.g., an ISV-based drug) preferably has the above (or at least one) such ISV at its C-terminus. As before, the ISV may be a nanobody, a VH domain, or an ISV (other than a nanobody) containing a VH domain, and is preferably a nanobody.

[0022] The present invention as described herein includes, in particular, 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, and is intended to be applied to ISVs based on and / or derived therefrom, and is suitable for application to such ISVs. These may be synthetic (e.g., obtained starting from a synthetic library and / or based on a certain framework region), semi-synthetic (e.g., obtained starting from a natural VH or VHH domain by humanization, camelization or sequence optimization, or affinity maturation or CDR transplantation), or entirely naturally occurring VH or VHH domains. Accordingly, the present invention will be further described herein with reference to VH or VHH domains and ISVs based on and / or derived from VH or VHH domains.

[0023] In establishing the present invention, it was found that protein interference can occur in several assays (e.g., ADA immunoassays) used to analyze biological samples (e.g., blood samples including whole blood, serum or plasma, ocular fluid, bronchoalveolar lavage fluid / BALF, cerebrospinal fluid, or other biological fluid samples), and that such protein interference can produce nonspecific signals in some of these assays or some of these samples. It was also found that such protein interference can occur not only in samples obtained from subjects (e.g., patients or clinical trial subjects) treated with and / or administered with ISVs (particularly nanobodies; or proteins, polypeptides, or other biological drugs containing at least one such ISV or nanobody), but also in samples derived from subjects that have never been administered ISVs (this suggests that such interference may be due to nonspecific protein-protein interactions with existing proteins rather than novel ADAs).

[0024] Although such protein interference and / or signals in such assays have been found not to be related to changes or reductions in the pharmacological properties of ISVs (e.g., pharmacokinetic / PK properties or pharmacodynamic / PD properties), it is desirable that techniques be available to predict, detect, reduce, and / or avoid such nonspecific protein interferences. This is a general objective of the present invention.

[0025] In particular, the present invention provides the following, and in certain specific but non-limiting embodiments, the following: -An assay that can be used to predict whether a given ISV will be subject to such protein interference and / or produce such (nonspecific) signals in such assays (e.g., ADA immunoassays). Such predictive assays can be used, for example, to test whether a given ISV is likely to be prone to producing such protein interference and / or such signals; to select ISVs that are not prone to or have little tendency to produce such protein interference or such signals; to test whether certain modifications to an ISV reduce (all or partly) its tendency to produce such interference or such signals; and / or to guide modifications or improvements to ISVs to reduce their tendency to produce such protein interference or signals; - Methods for modifying and / or improving ISVs to eliminate or reduce the tendency to produce such protein interference or such signals; - Modifications that may be introduced into ISVs to eliminate or reduce the tendency to produce such protein interference or such signals; - ISVs specifically selected (for example, using assays described herein) that do not have a tendency to produce such protein interference or such signals, or have a lower / reduced tendency to do so; - Modified and / or improved ISVs that do not have a tendency to produce such protein interference or such signals, or have a lower / reduced tendency to do so.

[0026] For example, in a first non-limiting embodiment, the present invention may be used to predict whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) causes protein interference (or has a high or increased risk of causing protein interference) in an immunoassay (i.e., as further described herein, administering it to a subject and then obtaining a sample of biological fluid from the subject and subjecting the sample to an immunoassay), (i) A step of contacting an antibody obtained from a human subject, selected, prepared and / or isolated based on its ability to recognize the C-terminus of an ISV or nanobody and / or its ability to bind to the C-terminus of an ISV or nanobody ("analytical antibody"), with the ISV or nanobody (or an ISV-based drug or a nanobody-based drug); and (ii) A step of determining whether the ISV or nanobody (or ISV-based drug or nanobody-based drug) is bound by the antibody in the immunoassay. The present invention relates to a method for performing an immunoassay that includes at least the following:

[0027] In this method, when an ISV, nanobody, ISV-based drug, or nanobody-based drug is bound by the analytical antibody, it may be expected that the ISV, nanobody, ISV-based drug, or nanobody-based drug will cause (or have a high or increased risk of causing) such protein interference (as further defined herein). Based on this, for example, the ISV, nanobody, ISV-based drug, or nanobody-based drug may be modified or improved to reduce or eliminate the tendency to cause such protein interference (which can be determined again using the above assay), and several measures that may be used to modify the ISV, nanobody, ISV-based drug, or nanobody-based drug are described herein (e.g., adding a few amino acid residues to the C-terminus and / or introducing one or more specific amino acid substitutions).

[0028] Accordingly, the present invention generally provides those skilled in the art with assays and methods / techniques that can be used to predict the tendency of ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs to cause protein interference, and / or as tools to improve ISVs to reduce or avoid the tendency to cause protein interference. By doing so, the present invention also provides those skilled in the art with means for selecting ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs based on their low or reduced ability (or lack thereof) to cause protein interference. Accordingly, the present invention provides those skilled in the art with important assays and tools that can be used in the optimization and development of ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs.

[0029] As will be further described herein, the present invention teaches those skilled in the art a number of ways in which ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs can be modified or improved to reduce or avoid the tendency to cause protein interference. Accordingly, the present invention also generally makes available modified and / or improved ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs that have a reduced tendency, a low tendency, or no tendency to cause protein interference.

[0030] As further described herein, the present invention can be particularly used to predict whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) will cause protein interference (as further described herein) in an immunoassay, in particular an ADA assay. The ADA assay may be, for example, an ADA assay for detecting or measuring ADA against an ISV in general, or in particular an ADA assay for detecting or measuring ADA against an ISV used in steps (i) and (ii) above.

[0031] As previously stated herein, the ISV described herein may be a nanobody, or it may be a VH domain or an ISV containing a VH domain (i.e., other than a nanobody), and is preferably a nanobody.

[0032] Furthermore, any protein or polypeptide containing an ISV (e.g., an ISV-based drug) preferably has the above-mentioned (or at least one) such ISV at its C-terminus. As before, the ISV may be a nanobody, or it may be a VH domain or an ISV containing a VH domain (i.e., other than a nanobody), and is preferably a nanobody.

[0033] The sample tested in the immunoassay or ADA assay described herein is also referred to herein as the “test sample” or “assay sample.” To avoid confusion, such “test samples” or “assay samples” should not be confused with the biological samples used herein as starting materials for obtaining the (polyclonal or monoclonal) “analytical antibodies” used in the present invention.

[0034] In one particularly preferred but non-limiting embodiment, the present invention may be used to predict whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) will cause protein interference (as further described herein) in an immunoassay (in particular an ADA assay) involving the use of such ISV. As before, the ADA assay may be, for example, an ADA assay for detecting or measuring ADA against an ISV in general, and in particular an ADA assay for detecting or measuring ADA against an ISV used in steps (i) and (ii) above.

[0035] In a more specific but non-limiting embodiment, the present invention may be used to predict whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) will cause protein interference (as further described herein) in an immunoassay (in particular an ADA assay) aimed at determining or measuring whether a sample contains ADA against the ISV. As before, for example, such an immunoassay may be one of the known types of ADA assays (see, for example, the prior art of ADA assays cited herein) performed to determine or measure whether ADA against the ISV is present in a “test sample,” the test sample being a sample of biological fluid (as described herein) obtained from a subject (as further described herein) to which the ISV has been administered.

[0036] As further described herein, in all these embodiments (and further embodiments of the invention as described herein), the invention may also be used to select ISVs that have little or no tendency to cause such protein interference in such immunoassays or ADA assays; it may be used as an assay or test to test whether certain modifications to an ISV reduce (all or partly) the tendency to cause such interference in such immunoassays or ADA assays; and / or it may be used as an assay or test to guide modifications or improvements to an ISV to reduce the tendency to cause such protein interference in such immunoassays or ADA assays.

[0037] Other aspects, embodiments, advantages, and uses of the present invention will become apparent from further descriptions herein.

[0038] Whenever the term "ISV" is used in this specification, the following should be understood: - Such ISVs are preferably nanobodies (the term "nanobody" is generally defined in International Publication No. 08 / 020079 or International Publication No. 09 / 138519), and in specific embodiments, generally mean VHH, humanized VHH or camelized VH (e.g., camelized human VH), or generally sequence-optimized VHH (e.g., optimized for chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression). Note that the terms Nanobody or Nanobodies are registered trademarks of Ablynx NV and may also be referred to as Nanobody® and / or Nanobodies®; - The term "ISV" in its broadest sense also includes "ISV-based biological formulations," and if the ISV is a nanobody, it also includes "nanobody-based biological formulations." "ISV-based biological formulation" is defined herein as a protein, polypeptide, or other biological drug containing at least one (e.g., one, two, or three) ISVs, or essentially consisting of such ISVs. Similarly, "nanobody-based biological formulation" is defined as a protein, polypeptide, or other biological drug containing at least one (e.g., one, two, or three) nanobodies, or essentially consisting of such nanobodies. As with the term "ISV," whenever the term "ISV-based biological formulation" is used, it should be understood that such an ISV-based biological formulation is preferably a nanobody-based biological formulation. Within the context of the present invention, both "ISV-based biological formulation" and "nanobody-based biological formulation" may be, for example, monovalent, divalent (or polyvalent), bispecific (or multiplespecific), and diparatope (or multiparatope) ISV constructs or nanobody constructs, respectively. Furthermore, any ISV-based bioagent or nanobody-based bioagent may optionally include, in addition to one or more (e.g., one, two, or three) ISVs or nanobodies, one or more (e.g., one or two) other further therapeutic parts, and / or one or more (e.g., one or two) other parts that affect the pharmacokinetic or pharmacodynamic properties (e.g., its half-life) of the ISV-based bioagent or nanobody-based bioagent. Suitable examples of such further therapeutic parts or other parts will be apparent to those skilled in the art, and may generally include any therapeutically active protein, polypeptide, or other binding domain or binding unit, as well as modifications such as those described on pages 149-152 of International Publication No. 09 / 138159.ISV-based biological agents or nanobody-based biological agents are preferably therapeutic agents or intended for therapeutic uses (including prevention and diagnosis), and for this purpose preferably contain at least one ISV against a therapeutically relevant target (e.g., RANK-L, vWF, IgE, RSV, CXCR4, IL-23, or other interleukins). For some specific but non-limiting examples of such ISV-based biological agents or nanobody-based biological agents, see, for example, various applications by Ablynx NV (e.g., but not limited to International Publication Nos. 2004 / 062551, 2006 / 122825, 2008 / 020079 and 2009 / 068627), and applications such as (e.g., but not limited to) International Publication Nos. 06 / 038027, 06 / 059108, 07 / 063308, 07 / 063311, 07 / 066016 and 07 / 085814. Furthermore, unless otherwise explicitly stated herein, all terms used herein have the meanings set forth in International Publication No. 09 / 138519 (or the prior art cited in International Publication No. 09 / 138519) or International Publication No. 08 / 020079 (or the prior art cited in International Publication No. 08 / 020079). Also, if a method or technique is not specifically described herein, it may be implemented as described in International Publication No. 09 / 138519 (or the prior art cited in International Publication No. 09 / 138519) or International Publication No. 08 / 020079 (or the prior art cited in International Publication No. 08 / 020079).

[0039] In particular, the following terms have the same meaning as set out on pages 62-75 of International Publication No. 09 / 138519, and / or may be determined as set out on those pages where applicable: “agonist,” “antagonist,” “reverse agonist,” “nonpolar uncharged amino acid residue,” “polar uncharged amino acid residue,” “polar charged amino acid residue,” “sequence identity,” “exactly the same,” and “amino acid difference” (when referring to a sequence comparison of two amino acid sequences), “essentially isolated (form),” “domain,” “binding domain,” “antigenic determinant,” “epitope,” “against” or “directed against” (antigen), “specificity,” and “half-life.” In addition, the terms “modulating,” “to modulate,” “interaction site,” “specific to,” “cross-block,” “cross-blocked,” and “cross-blocking,” as well as “essentially pH-independent,” are defined (and / or may be determined as described on pages 74-79) of the applicant’s International Publication No. 10 / 130832. Furthermore, when referring to the constructs, compounds, proteins, or polypeptides of the present invention, terms such as “monovalent,” “divalent” (or “polyvalent”), “bispecificity” (or “multispecificity”) and “diparatope” (or “multiparatope”) may have the meanings set forth in International Publication No. 09 / 138.519, International Publication No. 10 / 130832, or International Publication No. 08 / 020079.

[0040] The term “half-life” as used herein with respect to ISVs, nanobodies, ISV-based biological agents or nanobody-based biological agents or any other amino acid sequences, compounds or polypeptides may generally be defined as described in paragraph o) on page 57 of International Publication No. 08 / 020079, as stated therein, and refers to the time required for the serum concentration of an amino acid sequence, compound or polypeptide to decrease by 50% in vivo, for example, by the degradation of the sequence or compound by natural mechanisms and / or elimination or sequestration of the sequence or compound. The in vivo half-life of the amino acid sequences, compounds or polypeptides of the present invention may be determined by any method known in itself, for example, pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art, and may generally be as described in paragraph o) on page 57 of International Publication No. 08 / 020079. As also stated in paragraph o) on page 57 of International Publication No. 08 / 020079, the half-life may be expressed using parameters such as t1 / 2-α, t1 / 2-β and area under the concentration curve (AUC). In this regard, it should be noted that the term “half-life” as used herein refers in particular to t1 / 2-β or final half-life (without considering t1 / 2-α and / or AUC or both). See, for example, the experimental section below and standard handbooks such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinetic analysis: A Practical Approach (1996). See also “Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). Similarly, the terms “increased half-life” or “increased half-life” are defined in paragraph o) on page 57 of International Publication No. 08 / 020079, and refer in particular to an increase in t1 / 2-β, with or without an increase in t1 / 2-α and / or AUC or both.

[0041] Unless otherwise specified herein, a term shall have its ordinary meaning in the art, which is obvious to those skilled in the art. For example, 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, NY, (1985); Old et al. 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. al., “Immunobiology” See standard handbooks such as (6th Ed.), Garland Science Publishing / Churchill Livingstone, New York (2005), as well as the general background technologies cited herein.

[0042] Furthermore, in this specification, the amino acid residues of the nanobodies are numbered according to the general numbering of VH domains shown by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to the VHH domain derived from camels in the paper Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195, or as referenced herein. According to this numbering, nanobody FR1 contains amino acid residues at positions 1-30, nanobody CDR1 contains amino acid residues at positions 31-35, nanobody FR2 contains amino acid residues at positions 36-49, nanobody CDR2 contains amino acid residues at positions 50-65, nanobody FR3 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 with respect to the VH domain and VHH domain, 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 by Kabat numbering (i.e., the actual sequence may not be present at one or more positions indicated by Kabat numbering, and the actual sequence may contain more amino acid residues than the number permitted by Kabat numbering). This generally means that Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence.However, generally speaking, according to Kabat numbering, regardless of the number of amino acid residues in the CDR, position 1 in Kabat numbering corresponds to the start of FR1 and vice versa; position 36 in Kabat numbering corresponds to the start of FR2 and vice versa; position 66 in Kabat numbering corresponds to the start of FR3 and vice versa; and position 103 in Kabat numbering corresponds to the start of FR4 and vice versa.

[0043] An alternative method for numbering amino acid residues in the VH domain is the method described by Chothia et al. (Nature 342, 877-883 (1989)) (the so-called "AbM definition" and the so-called "contact definition"), and this method can be applied similarly to camel-derived VHH domains and nanobodies. However, in this specification, embodiments and drawings, unless otherwise indicated, we follow Kabat numbering as applied to the VHH domain by Riechmann and Muyldermans.

[0044] It should also be noted that the drawings, sequence lists, and experimental sections / examples are provided solely for the purpose of further illustrating the present invention and should not be construed or understood to limit the scope of the present invention and / or the appended claims unless otherwise explicitly indicated herein.

[0045] It should be further noted that the present invention does not specifically limit any causes, explanations, hypotheses, or mechanisms of protein interference (and / or signals occurring in immunoassays) observed and reduced in accordance with the present invention. However, it is assumed that the blood or serum (or other biological fluids such as those described herein) of a particular individual or population may contain certain (existing) proteins that, under certain conditions, can bind (non-specifically) to ISVs and result in interference signals in certain assays used to analyze blood or serum samples obtained from such individuals. This is particularly based on observations made in establishing the present invention that non-specific protein interference addressed by the present invention occurs not only when assaying samples obtained from subjects who have previously been administered ISVs, but also when assaying samples obtained from subjects who have not previously been administered ISVs.

[0046] In particular, based on observations made in establishing the present invention, it is thought that such (existing) proteins may be particularly capable of binding to the C-terminus of such ISVs, but the present invention is not limited thereto (in conventional full quadruple-chain monoclonal antibodies and “heavy chain alone” antibodies found in camels, the C-terminus is linked to the rest of the antibody—i.e., the CH1 region of conventional monoclonal antibodies and the hinge region of camel heavy chain antibodies, respectively—so such complete antibodies may be protected from such protein interference).

[0047] This is supported by the findings made by the inventors in establishing the present invention (which are further described herein) that certain (simple) modifications at the C-terminus of ISVs can substantially reduce or essentially suppress such protein interference. Accordingly, methods for such modification of ISVs, and such modified ISVs, form further embodiments of the present invention, as further described herein.

[0048] The present invention may be particularly used to reduce or avoid protein interference and / or signaling due to nonspecific binding in immunoassays performed on biological samples (e.g., blood or serum samples) obtained from subjects administered with (biological) drugs (as before, such samples are also referred to herein as “test samples” or “assay samples”). Some examples of this are immunoassays used to characterize pharmacokinetics and antibody formation when biological drugs are administered to subjects, as mentioned in the “Guideline on the Clinical Investigation of the Pharmacokinetics of Therapeutic Proteins” (document dated January 27, 2007, CHMP / EWP / 89249 / 2004) 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 potential drawbacks have been identified, which could lead to erroneous characterization regarding pharmacokinetics and antibody formation. The following issues should be considered [...]: Immunoassay Drug assay: [...] (iii) Interference by endogenous substances. (iv) Interference by anti-drug antibodies that bind to plasma components or the sample and inhibit complementary binding to the capture antibody.

[0049] The present invention may be particularly used to predict, reduce, or avoid this type of interference in immunoassays used to analyze test samples / assay samples of biological fluids taken from subjects administered with ISVs (in particular, nanobodies; or, as further defined herein, ISV-based biological agents or nanobody-based biological agents).

[0050] The present invention may be particularly used to predict, reduce, or avoid this type of (non-specific) protein interference in immunoassays used to characterize drug pharmacokinetics and / or determine the formation of ADA (anti-drug) antibodies. In this regard, it should be noted that where expressions such as “predict, reduce, or avoid protein interference” are used in this specification and the appended claims, this means not only predicting, reducing, or avoiding such protein interference itself, but also generally predicting, reducing, or avoiding the occurrence of non-specific signals in immunoassays (e.g., assays in which (non-specific) signals related to protein interference may occur, e.g., ADA assays), and in particular predicting, reducing, or avoiding the occurrence of non-specific signals in such immunoassays that, when observed in such assays, are usually attributable to, related to, and / or considered as signs of (non-specific) protein interference. In this regard, it should be noted that, in general, as stated herein, the present invention does not specifically limit any cause, explanation, hypothesis, or mechanism.

[0051] In one specific but non-limiting embodiment, the present invention may be used to predict, avoid, or reduce such protein interference in “anti-drug antibody” or “ADA” assays performed on a sample of biological fluid (i.e., “test sample”) taken from a subject administered with an ISV (in particular, nanobody; or, as further defined herein, an ISV-based biological agent or nanobody-based biological agent).

[0052] In a specific but non-limiting alternative embodiment, the present invention may be used to predict, avoid, or reduce such protein interference (and / or nonspecific signals typically associated with such protein interference) in “anti-drug antibody” or “ADA” assays used to detect, measure, and / or characterize the presence of (any) anti-drug antibodies against one or more ISVs (in particular, nanobodies; or ISV-based biological agents or nanobody-based biological agents as defined herein). In particular, the present invention may be used to predict, avoid, or reduce such protein interference in such “anti-drug antibody” or “ADA” assays performed on samples of biological fluids (i.e., “test samples”), more specifically, on samples of biological fluids obtained from subjects administered with one or more such ISVs or nanobodies (or ISV-based biological agents or nanobody-based biological agents as defined herein). For example, the present invention may be used to predict, avoid, or reduce such protein interference in such “anti-drug antibody” or “ADA” assays used to detect, measure and / or characterize the presence of (any) anti-drug antibodies against ISVs or nanobodies (or ISV-based biological agents or nanobody-based biological agents as further defined herein) administered to a subject sample from which a sample has been obtained (in connection with a clinical trial and / or treatment).

[0053] Accordingly, in one specific but non-limiting embodiment, the present invention relates to a biological sample (i.e., a "test sample") obtained from a subject to which one or more such ISVs or nanobodies (or ISV-based biological agents or nanobody-based biological agents as defined herein) have been administered, which is suitable for an immunoassay such as the ADA assay and / or can be used to predict, avoid or reduce such protein interference (and / or nonspecific signals usually associated with such protein interference) in a sample intended for use in said immunoassay. As stated above, such a biological sample may be blood (including whole blood, serum or plasma), ocular fluid, bronchoalveolar lavage fluid / BALF, cerebrospinal fluid, or any other suitable biological fluid or sample suitable for use in an immunoassay, particularly the ADA assay.

[0054] In one specific but non-limiting embodiment, such test samples may be obtained from subjects subjected to multiple administrations (e.g., at least one to three individual administrations over a period of at least 10 days, e.g., at least one month or longer) and / or chronic treatment (i.e., treatment for at least 10 days, e.g., at least one month) with an ISV, nanobody, ISV-based biological agent (as further defined herein) or nanobody-based biological agent (as further defined herein). Such an ISV, nanobody, ISV-based biological agent or nanobody-based biological agent may be administered to the subject, for example, in connection with treatment or a clinical trial.

[0055] In one specific but non-limiting embodiment, such test samples may be obtained from subjects to whom an ISV, nanobody, ISV-based biological agent, or nanobody-based biological agent having an increased half-life (as defined herein and compared to monovalent ISVs), for example, at least 1 day, preferably at least 3 days, more preferably at least 7 days, for example at least 10 days (and / or given the increased half-life).

[0056] For example, but not limited to, such ISVs, nanobodies, ISV-based biological agents, or nanobody-based biological agents may be given an extended half-life by functionalization and / or by including a moiety or binding unit in the construct that increases the half-life of the construct. Examples of such functionalizations, moieties or binding units will be obvious to those skilled in the art and may be, for example, those described herein, such as pegylation, fusion with serum albumin, or fusion with a peptide or binding unit that can bind to a serum protein such as serum albumin. Such serum albumin-binding peptides or binding domains may be any suitable serum albumin-binding peptides or binding domains that can increase the half-life of the construct (compared to the same construct without a serum albumin-binding peptide or binding domain), in particular the serum albumin-binding peptides described in the applicant's International Publication 2008 / 068280 (and in particular, both the applicant's International Publication 2009 / 127691 and unpublished U.S. application 61 / 301,819), or serum albumin-binding ISVs (e.g., serum albumin-binding nanobodies; e.g., Alb-1 or a humanized form of Alb-1, e.g., Alb-8 (see, for example, International Publication 06 / 122787)).

[0057] Therefore, in one specific but non-limiting embodiment, such biological samples may be obtained from subjects who have been administered an ISV, nanobody, ISV-based biological agent, or nanobody-based biological agent containing a (human) serum albumin-binding peptide or binding domain.

[0058] As already described above, in one non-limiting embodiment, the present invention can generally be used to predict whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) will cause protein interference (as further described herein) (or have a high tendency or increased tendency to cause protein interference) in an immunoassay (i.e., as further described herein, administering the ISV to a subject, obtaining a sample of biological fluid from the subject, and subjecting the biological fluid to an immunoassay), (i) A step of contacting an antibody obtained from a human subject, selected, prepared and / or isolated based on its ability to recognize the C-terminus of an ISV or nanobody and / or its ability to bind to the C-terminus of an ISV or nanobody ("analytical antibody"), with the ISV or nanobody (or an ISV-based drug or a nanobody-based drug); and (ii) A step of determining whether the ISV or nanobody (or ISV-based drug or nanobody-based drug) is bound by the antibody in the immunoassay. The present invention relates to a method for performing an immunoassay that includes at least the following:

[0059] To reiterate, as previously stated, as described herein, the ISV described herein may be a nanobody, a VH domain, or an ISV (other than a nanobody) containing a VH domain, and is preferably a nanobody.

[0060] Furthermore, any protein or polypeptide containing an ISV (e.g., an ISV-based drug) preferably has the above (or at least one) such ISV at its C-terminus. As before, the ISV may be a nanobody, a VH domain, or an ISV (other than a nanobody) containing a VH domain, and is preferably a nanobody.

[0061] In alternative embodiments (which will also be further described herein), a monoclonal antibody referred to herein as "21-4-3" (or abbreviated as "21-4"; see SEQ ID NOs. 35 and 36 for the sequences of VH and VL) may be used instead of the above-mentioned antibody obtained from human subjects. 21-4 was prepared using hybridoma technology, starting from mice immunized with the nanobody construct of SEQ ID NO. 98 in International Publication No. 2006 / 122825, as further described in Example 7. A hybridoma cell line expressing 21-4 (referred to as "ABH0015") was deposited with BCCM, Ghent, Belgium on June 4, 2012, under accession number LMBP-9680-CB. Monoclonal 21-4 is the C-terminus of the nanobody construct of Sequence ID No. 98 in International Publication No. 2006 / 122825, and is a nanobody (humanized V) created in relation to von Willebrand factor (vWF). HH It was shown to recognize the C-terminus consisting of ). 21-4 was originally prepared as an analytical reagent for use in detecting protein nanobodies in (serum) samples (particularly the nanobody construct of SEQ ID NO. 98 in International Publication No. 2006 / 122825); surprisingly, it was found that 21-4 can also be used to predict whether ISVs are prone to nonspecific protein interference (more so than several other comparable (mouse) monoclonals prepared for the nanobody construct of SEQ ID NO. 98 in International Publication No. 2006 / 122825, or for other nanobodies).

[0062] In particular, the measurement of the binding of 21-4 to ISV (or a protein or polypeptide containing ISV at its C-terminus, or a similar protein or polypeptide as described herein) is determined according to the protocol shown in Example 9 (the measured RU value for the protein molecular weight is given by the formula [measured RU] / [protein molecular weight (MW)] x 10 6If the RU value is less than 500 (after adjustment according to the formula), it has been found that the ISV or protein may not be prone to protein interference (within the limits of the reliability provided by the data shown in the following examples). For the above formula, the molecular weight (MW) can be calculated as the sum of the total molecular weights (MW) of all amino acid residues present in the ISV.

[0063] Accordingly, the ISVs, proteins, or polypeptides described herein preferably have such RU values ​​of less than 500 for binding by 21-4 (determined according to the protocol shown in Example 9 and after adjusting the measured RU values ​​for the molecular weight of the ISV or protein used according to the formula shown above).

[0064] Accordingly, this aspect of the present invention can generally be used to predict whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) will cause protein interference (as further described herein) (or have a high tendency or increased tendency to cause protein interference) in an immunoassay (i.e., as further described herein, administering the ISV to a subject and then obtaining a sample of the biological fluid from the subject and subjecting the biological fluid to an immunoassay), (i) a step of contacting the monoclonal antibody 21-4 (i.e., used as an "analytical antibody") with the ISV or nanobody (or an ISV-based drug or a nanobody-based drug); and (ii) A step of determining whether the ISV or nanobody (or ISV-based drug or nanobody-based drug) is bound by the monoclonal antibody 21-4 in the immunoassay. The present invention relates to a method for performing an immunoassay that includes at least the following:

[0065] The above method may be carried out, in particular, using BiaCore or a similar technique, and more specifically, using the protocol shown in Example 9. As described herein, the binding of the ISV or ISV-based drug in this protocol is (measured RU value for the molecular weight of the protein is given by the formula [measured RU] / [molecular weight of the protein (MW)] x 10 6 If the RU value is less than 500 (after adjustment according to the above), the ISV or ISV-related protein may not be bound by interfering factors present in human blood or serum, and / or may not be prone to nonspecific protein interference in the ADA assay (i.e., within the confidence range shown in the experimental section below).

[0066] To reiterate, as previously stated, as described herein, the ISV described herein may be a nanobody, a VH domain, or an ISV (other than a nanobody) containing a VH domain, and is preferably a nanobody.

[0067] Furthermore, any protein or polypeptide containing an ISV (e.g., an ISV-based drug) preferably has the above (or at least one) such ISV at its C-terminus. As before, the ISV may be a nanobody, a VH domain, or an ISV (other than a nanobody) containing a VH domain, and is preferably a nanobody.

[0068] Furthermore, as described herein, the above method using 21-4 may also be used to determine whether an ISV or an ISV-containing protein or polypeptide is bound (or tends to be bound) by an interfering factor present in human blood or serum.

[0069] Furthermore, as described herein, the method using 21-4 may also be used to predict whether any protein or polypeptide having a VH domain at its C-terminus (e.g., an antibody fragment or ScFv) is bound (or tends to be bound) by interfering factors present in human blood or serum, and / or is prone to protein interference in the ADA assay.

[0070] In addition to 21-4, antibodies or antibody fragments (e.g., appropriate Fab fragments) containing the heavy chain variable domain and light chain variable domain of 21-4 (see SEQ ID NOs. 35 and 36, respectively), or containing only the CDR sequence of 21-4 (appropriately transplanted into other appropriate VH and VK frameworks), may also be used in the methods described herein.

[0071] As further described herein, the present invention may be particularly used to predict whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) will cause protein interference (as further described herein) in an immunoassay, which is an ADA assay. The ADA assay may be, for example, an ADA assay for detecting or measuring ADA against an ISV in general, and in particular an ADA assay for detecting or measuring ADA against an ISV used in steps (i) and (ii) above.

[0072] In one particularly preferred but non-limiting embodiment, the present invention may be used to predict whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) will cause protein interference (as further described herein) in an immunoassay (in particular an ADA assay) involving the use of such ISV. As before, the ADA assay may be, for example, an ADA assay for detecting or measuring ADA against an ISV in general, and in particular an ADA assay for detecting or measuring ADA against an ISV used in steps (i) and (ii) above.

[0073] In a more specific but non-limiting embodiment, the present invention may be used to predict whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) will cause protein interference (as further described herein) in an immunoassay (in particular an ADA assay) involving the use of such ISV. For example, such an immunoassay may be an ADA assay (i.e., including an ISV) performed to determine or measure whether the ADA against the ISV is present in a test sample, which is a sample of biological fluid (as described herein) obtained from a subject (as further described herein) to which the ISV has been administered. For example, as further described herein, the sample (i.e., “test sample”) may be a sample (including whole blood, serum or plasma), ocular fluid, bronchoalveolar lavage fluid / BALF, cerebrospinal fluid, or any other suitable biological fluid, and in particular a biological sample that is suitable for an immunoassay such as an ADA assay and / or intended for use in such an immunoassay.

[0074] As further described herein, in all these embodiments (and further embodiments of the invention as described herein), the invention may also be used to select ISVs that have little or no tendency to cause such protein interference in such immunoassays or ADA assays; it may be used as an assay or test that can be used to test whether certain modifications to an ISV reduce (all or partly) the tendency to cause such interference in such immunoassays or ADA assays; and / or it may be used as an assay or test that can be used to guide modifications or improvements to an ISV to reduce the tendency to cause such protein interference in such immunoassays or ADA assays.

[0075] As described above, step (i) of the method of the present invention comprises contacting an ISV or nanobody (or an ISV-based drug or nanobody-based drug) with an antibody obtained from a human subject, selected / isolated based on its ability to recognize the C-terminus of an ISV or nanobody and / or its ability to bind to the C-terminus of an ISV or nanobody (as further described herein). In step (i) of the method described herein, the "ISV or nanobody (or an ISV-based drug or nanobody-based drug)" is used as an antigen (i.e., as the substance to be detected) in the immunoassay. Also in step (i), the "antibody obtained from a human subject, selected / isolated based on its ability to recognize the C-terminus of an ISV or nanobody and / or its ability to bind to the C-terminus of an ISV or nanobody" is used as an analytical reagent (i.e., in the same way as other antibodies used in an immunoassay to detect the presence of the antigen they are targeting).

[0076] As already stated, in order to better understand the present invention as described herein, it should be noted that in step (i), the ISV is typically used as the “antigen” (i.e., as the compound to be detected) and the “analytical antibody” is used as the “analyte” (i.e., as a means to detect whether a given ISV binds to it, and thereby whether there is a high risk or increased risk of causing protein interference, respectively). For example, if step (i) is performed in ELISA format, the “antibody / analyte” is typically bound to a carrier (i.e., an ELISA plate), and the ISV is the sample to be tested (present in it).

[0077] In contrast, it should be noted that in ADA assays for detecting or measuring ADA against ISV, ISV is used as the “analyte” (i.e., the compound used to detect the presence of ADA), while ADA is the “antigen” (i.e., the compound to be detected). Therefore, in these assays, ISV is usually / often conjugated to a carrier (e.g., an ELISA plate), while ADA (if any) is present in the sample subjected to the assay.

[0078] However, as already stated, it should be noted that the present invention is not generally limited to assays in which the “analytical antibody” is bound to a carrier. For example, in alternative methods of carrying out the assay of the present invention (as shown in Example 5), the analytical antibody is used instead as a bridging agent, so it is present in solution rather than bound to the plate (although it is indirectly bound to the plate via the ISV coated on the plate). However, even in the particular (bridging) assay described in Example 5 (which is a competing assay), the analytical antibody is still used as an analytical agent (i.e., to determine whether each ISV of interest will bind and whether there is a high or increased risk of causing protein interference). Based on further disclosures herein, it is also conceivable that those skilled in the art could design other forms of assays in which the analytical antibody may be used as an analytical agent to determine whether each given ISV can bind and whether there is a high or increased risk of causing protein interference.

[0079] The "analytical antibody" used in step (i) may be a polyclonal antibody or a monoclonal antibody.

[0080] If the analytical antibody is a polyclonal antibody, it may be, for example, a polyclonal antibody (preparation) obtained / purified / isolated from a obtained biological sample of a human subject (e.g., blood, plasma, B cells, or another suitable biological sample or biological fluid from which a polyclonal antibody can be appropriately isolated). This may be, for example, a suitable biological sample obtained from a human subject administered with at least one ISV (e.g., the ISV used in step (i), but which is not required or essential), or a suitable biological sample derived from a human subject that has never been administered with an ISV or treated with an ISV (preferred). More importantly, the polyclonal antibody is obtained from the biological sample by a method comprising at least one affinity step (and one or more further steps to obtain / purify / isolate a polyclonal antibody, which is known in itself) using an affinity matrix or affinity column that holds the ISV as the affinity portion. For example, polyclonal antibodies may be obtained from such biological samples by affinity chromatography using an affinity column that retains ISV, as described, for example, in Example 2. This can be carried out using, for example, well-known techniques of immunoaffinity chromatography for isolating antibodies from biological samples using an affinity matrix that retains ISV as an antigen. Such techniques are generally known in the art, and suitable examples thereof will be apparent to those skilled in the art based on the disclosure herein.

[0081] Such polyclonal antibodies (preparations) may, in particular, be IgG (or an IgG fraction).

[0082] For example, it could be a polyclonal antibody obtained by a method involving (immuno)affinity chromatography performed on a sample of biological fluid obtained from a human subject, using an ISV (in particular, a nanobody, e.g., VHH, humanized and / or sequence-optimized VHH, or camelized VH, e.g., camelized human VH) that does not contain a C-terminal tag (i.e., its C-terminus terminates with the amino acid sequence VTVSS (SEQ ID NO: 33)) as an antigen bound to an affinity matrix. In particular, the ISV used as the antigen bound to the affinity matrix may be a humanized or sequence-optimized VHH (or the corresponding camelized human VH) whose C-terminus terminates with the amino acid sequence VTVSS (SEQ ID NO: 33). In one specific but non-limiting embodiment, an ISV used as an antigen bound to an affinity matrix may be a humanized or sequence-optimized VHH containing a proline (P) residue at position 14 (the corresponding “native” VHH contains alanine (A) at position 14) as a result of such humanization or sequence optimization (in other words, an ISV used as an antigen is a humanized form of a VHH that originally contains alanine (A) at position 14, and this alanine residue is replaced with a proline (P) residue as a result of humanization or sequence optimization). An ISV used as an antigen may also include one or more other amino acid substitutions as a result of such humanization or sequence optimization, as generally described in, for example, International Publication No. 08 / 020079 or International Publication No. 09 / 138519.

[0083] Some specific examples of ISVs that can be used as antigens to produce / isolate the "analytical antibodies" used in the present invention are shown in SEQ ID NOs: 1 and 2.

[0084] As before, the methods used to obtain polyclonal antibodies may include, in addition to the (immuno)affinity step, one or more further steps (performed before or after the affinity step) for isolating / purifying the polyclonal antibodies from a biological sample. As before, such steps and techniques for carrying them out will be obvious to those skilled in the art.

[0085] Accordingly, in one embodiment, the present invention is a method further described herein, comprising steps (i) and (ii) described herein, wherein the “analytical antibody” (i.e., an antibody obtained from a human subject, selected / isolated based on its ability to recognize the C-terminus of an ISV or nanobody and / or its ability to bind to the C-terminus of an ISV or nanobody) is used, wherein an ISV (preferably a nanobody) is used as the antigen, preferably an ISV containing the amino acid sequence VTVSS (Sequence ID 33) as its C-terminal sequence is used as the antigen, more preferably a humanized and / or sequence-optimized nanobody containing the amino acid sequence VTVSS (Sequence ID 33) as its C-terminal sequence is used as the antigen, and even more preferably the C-terminal sequence is the amino acid sequence VTVSS (Sequence ID 33) The method includes obtaining a biological sample obtained from a human subject (the biological sample is a sample suitable for use in a method for producing / isolating antibodies from a sample) using a method which includes a humanized and / or sequence-optimized nanobody, for example, one containing the amino acid sequence VTVSS (SEQ ID NO: 33) as its C-terminal sequence, and the nanobody containing a proline residue at position 14 introduced into the nanobody as a result of the humanized and / or sequence-optimized (e.g., substitution of an alanine residue originally present at the position of the humanized and / or sequence-optimized VHH), and which is obtained from a biological sample obtained from a human subject (the biological sample is a sample suitable for use in a method for producing / isolating antibodies from a sample).

[0086] The above ISV can also be used in a method for isolating a monoclonal antibody suitable for use in the present invention as an "analytical antibody" (starting from a suitable biological sample obtained from a human, as before).

[0087] For example, such monoclonal antibodies may be obtained starting from blood, B cells, or another sample or material suitable for isolating antibodies, or they may be selected based on their ability to recognize ISVs or nanobodies (or their C-terminus) and / or to bind to ISVs or nanobodies (or their C-terminus) (as before, the ISVs used as antigens in screening and / or selection are preferably those described in the previous paragraph, including the preferred ISVs / antigens mentioned above). Such screening and selection may be carried out by any suitable method, for example, by using B cell selection and / or amplification techniques that are essentially the same as or appropriately similar to the B cell selection techniques described in European Patent Publication No. 0488470, International Publication No. 92 / 02551, European Patent Publication No. 1633787, International Publication No. 01 / 55216, International Publication No. 02 / 26829, International Publication No. 04 / 051268, International Publication No. 04 / 102198 or International Publication No. 04 / 106377, or by using techniques similar to the nanocloning techniques described in International Publication No. 06 / 079372 (except for using human B cells rather than camel B cells).

[0088] Once one or more B cells expressing a suitable antibody have been identified / isolated, the antibody may be isolated, expressed and / or produced by any suitable method. For example, the B cells may be immortalized as a hybridoma producing the desired antibody / antibodies (using techniques that are themselves well known for constructing hybridomas starting from selected B cells), and the antibody / antibodies may then be isolated from the hybridoma (or its culture supernatant) again using suitable techniques that are well established in the art, described in various handbooks and manuals, and also described in and / or referenced in the patent publications mentioned in the preceding paragraph.

[0089] Alternatively, the B cells may be amplified using B cell amplification techniques that are themselves known, and antibodies / antibodies may be isolated from the amplified B cells (or their culture supernatant). As before, this can be carried out using appropriate techniques that are well established in the art, described in various handbooks and manuals, as well as described in and / or referenced in the patent publications mentioned in the preceding paragraph.

[0090] In yet another alternative method, the DNA encoding the antibody / antibodies of interest may be obtained directly from the B cells or other suitable cells (e.g., using a suitable single-cell PCR cloning technique) or after the desired B cells have been appropriately amplified (e.g., by amplification). The DNA can then be appropriately expressed in a suitable host cell or host organism to provide the desired antibody / antibodies. As before, this can be carried out using suitable techniques that are well established in the art, described in various handbooks and manuals, and also described in and / or referenced in the patent publications mentioned in the preceding paragraph.

[0091] It is also possible to produce monoclonal antibodies suitable for use as “analytical antibodies” by a method that includes repertory cloning (starting from a suitable sample obtained from a human subject) and screening the cloned repertory for antibodies that bind to ISVs used as antigens (as before, the ISVs used as antigens in screening and / or selection are preferably those described in the preceding paragraph, and the preferred ones described for such ISVs / antigens). Methods for repertory cloning and various techniques for presenting the cloned repertory for selection and screening (e.g., phage display, ribosome display, and yeast display) are apparent to those skilled in the art and are described, for example, in European Patent Application Publication No. 0589877, European Patent Application Publication No. 0774511, International Publication No. 90 / 14430, and European Patent Application Publication No. 0368684, as well as in various handbooks on this subject.

[0092] In general, the biological sample used as a starting point for obtaining (polyclonal or monoclonal) analytical antibodies can be any suitable sample obtained from any suitable human subject (i.e., suitable as a starting material for obtaining polyclonal or monoclonal antibodies, respectively). In one specific but non-limiting embodiment, such a sample may be obtained, for example, from a woman, particularly a post-clonal woman. Thus, in one specific but non-limiting embodiment, the analytical antibodies used in steps (i) and (ii) above are obtained starting from a biological sample obtained from / derived from a post-clonal woman (or derived from antibodies obtained from / derived from a post-clonal woman).

[0093] Furthermore, the biological sample used as a starting point for obtaining the (polyclonal or monoclonal) analytical antibody may be obtained from a subject who has previously received ISV (for example, as part of a clinical trial or therapeutically), but preferably from a subject who has not previously received ISV.

[0094] However, it should be noted that the present invention is not particularly limited to the origin of the analytical antibody / antibodies used, and in some cases it has been proven that the techniques described herein can be used to obtain (prepare, isolate) other suitable analytical antibodies from other sources, such as commercially available human blood or plasma (and even blood, plasma, or B cells from other mammalian or primate species, e.g., baboons or cynomolgus monkeys).

[0095] As described above, the (polyclonal or monoclonal) analytical antibodies used in steps (i) and (ii) should be able to recognize or bind to the C-terminus of the ISV or nanobody, and most preferably be selected and / or isolated based on this ability to bind to the C-terminus of the ISV or nanobody.

[0096] As can be seen from Figure 2, when an ISV is based on or derived from a VH or VHH domain, the C-terminus of the ISV contains the amino acid sequence VTVSS (SEQ ID NO: 33). Accordingly, the analytical antibody should be able to recognize any ISV that has the amino acid sequence VTVSS (SEQ ID NO: 33) at its C-terminus. As can be seen further from Figure 2, the sequence VTVSS (SEQ ID NO: 33) (at least some of its amino acid residues) is part of a putative epitope on the ISV that may include, among other residues, the amino acid residue at position 14 (and the amino acid residues adjacent to / near the amino acid residue at position 14 of the amino acid sequence, e.g., positions 11, 13, and 15), the amino acid residue at position 83 (and the amino acid residues adjacent to / near the amino acid residue at position 83 of the amino acid sequence, e.g., positions 82, 82a, 82b, and 84), and / or the amino acid residue at position 108 (and the amino acid residue adjacent to / near the amino acid residue at position 108 of the amino acid sequence, e.g., position 107. Position 109 is the first V in the C-terminal VTVSS (SEQ ID NO: 33) sequence, and for example, position 110 has also been shown to affect protein interference). This is collectively referred to as the "C-terminal region" in this specification, and this C-terminal region is understood to include at least the C-terminal sequence VTVSS (SEQ ID NO: 33) and the amino acid residue at position 14, and may also include the amino acid residues at positions 83 and 108, and possibly positions 13, 15, 82b, 83, 84 and 107.

[0097] As already stated, and as before, not limited to any hypothesis or explanation, in complete quadruple-chain monoclonal antibodies or complete heavy-chain monoantibodies (e.g., those found in camels), the C-terminus of the VH or VHH domain is linked to the rest of the antibody—i.e., the CH1 region of a normal monoclonal or the hinge region of a camel heavy-chain antibody, respectively—so such complete antibodies are protected from such protein interference and / or may be protected by the VH / VL interaction (of a normal quadruple-chain antibody), and therefore this "C-terminal region" is not normally exposed to solvents and / or is not accessible as an interaction site for proteins present in the blood, plasma or body of a human to whom such an ISV is administered. However, when the ISV or nanobody itself is used (i.e., not linked to any other part of an antibody), or when an ISV-based drug or nanobody-based drug having an ISV or nanobody at its C-terminus is used, this C-terminal epitope is available for (non-specific) interactions with other proteins, and, as before, is not limited to any hypothesis or explanation, the C-terminal region may now be accessible to (non-specific) protein interactions with one or more proteins already present in the “test sample” to be tested (e.g., one or more IgGs), which is assumed to cause protein interference and / or non-specific signaling in immunoassays (particularly ADA assays).

[0098] As described above, the methods described herein can be used to predict, reduce, or avoid such protein interactions. They can also be used as tools to lead to modifications of ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs to provide ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs in which the tendency to produce such protein interference is suppressed (partially or preferably essentially completely).

[0099] As is clear from the preceding paragraph, and as before, not limited to any hypothesis or explanation, it is particularly expected (and is part of the teachings of the present invention) that (certain) modifications to the “C-terminal region” will alter (preferably reduce) the ISV’s tendency to undergo such nonspecific protein interactions, and this is also experimentally observable (see, for example, the experimental results shown in Examples 1C and 3 below).

[0100] Based on this, and without being limited to any hypothesis or explanation as set forth herein, the present invention also teaches certain modifications that can be introduced into the C-terminal region of ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs for this purpose (the (potential) efficacy of these modifications may be tested using the methods described herein). Furthermore, based on the teachings herein, it is assumed that those skilled in the art can select, design, or propose other modifications (candidates) to the C-terminal region that can be introduced for this purpose (the (potential) efficacy of these modifications may be tested using the methods described herein, as set forth herein).

[0101] Returning to the analytical antibody used in the present invention, it is preferably an antibody (polyclonal or monoclonal) that recognizes the C-terminal region of ISV (as defined above), but not limited to, the C-terminal region of nanobodies.

[0102] For example, in one specific but non-limiting embodiment, the “analytical antibody” may be polyclonal or monoclonal, which recognizes (and / or binds to, particularly specifically to) the C-terminal region of an ISV or nanobody whose sequence ends with VTVSS (SEQ ID NO: 33), but does not recognize (and / or cannot specifically bind to) the C-terminal region of an ISV or nanobody (these may be different ISVs, but preferably the same ISV) if there are one or more further amino acid residues (e.g., one to five amino acid residues, or a small peptide sequence, or yet another polypeptide or protein) linked to the C-terminal VTVSS (SEQ ID NO: 33).

[0103] In another, more specific but still non-limiting embodiment, the “analytical antibody” may be polyclonal or monoclonal, which recognizes (and / or can bind to, particularly specifically to) the C-terminal region of an ISV or nanobody that has been modified compared to the amino acid originally present at position 14 (e.g., as a result of humanization, camelidization and / or sequence optimization), but has one or more further amino acid residues (e.g., one to five amino acid residues, or a small peptide sequence, or yet another polypeptide or protein) linked to the C-terminal VTVSS (SEQ ID NO: 33); and / or does not recognize (and / or cannot specifically bind to) the C-terminal region of an ISV or nanobody (these may be different ISVs, but preferably the same ISV) whose C-terminal region is the amino acid originally present at position 14 (e.g., alanine, or proline if the ISV originally contains proline at position 14).

[0104] For example, an "analytical antibody" may also recognize (and / or bind to, particularly specifically bind to) the C-terminal region of an ISV or nanobody whose sequence ends at VTVSS (SEQ ID NO: 33) and whose 14th position is proline (especially if the 14th position is modified to proline, for example, as a result of humanization, camelization, and / or sequence optimization), but which has one or more further amino acid residues (e.g., one to five amino acid residues, or a small peptide sequence, or yet another polypeptide or protein) linked to the C-terminal VTVSS (SEQ ID NO: 33); and / or may be polyclonal or monoclonal, which does not recognize the C-terminal region of an ISV or nanobody whose 14th position is alanine (these may be different ISVs, but preferably the same ISV).

[0105] The “analytical antibody” may also be polyclonal or monoclonal, which recognizes (and / or binds to, particularly specifically binds to, the C-terminal region of an ISV or nanobody) whose sequence terminates at VTVSS (SEQ ID NO: 33) and whose 14th position is proline (especially if a proline residue is originally present at the position of the ISV), but does not recognize the C-terminal region of an ISV or nanobody (these may be different ISVs, but preferably the same ISV) which has one or more further amino acid residues (e.g., one to five amino acid residues, or a small peptide sequence, or yet another polypeptide or protein) linked to the C-terminal VTVSS (SEQ ID NO: 33) and whose 14th position is still (originally present or unmodified) proline.

[0106] The “analytical antibody” may also be polyclonal or monoclonal, for example, that recognizes the C-terminal region of the ISV sequence (SEQ ID NO: 5) referred to herein as “Nb3.4”, but does not recognize the C-terminal region of the ISV sequence (SEQ ID NO: 3) referred to herein as “Nb3.1”, and / or (preferably) does not recognize the ISV sequence (SEQ ID NO: 4) referred to herein as “Nb3.2”.

[0107] For the purposes described above, whether the “analytical antibody” recognizes (or does not recognize) ISVs or nanobodies (and / or can bind (specifically) to ISVs or nanobodies) can be determined using any suitable binding assay (e.g., Biacore), or it may be determined using an ADA assay such as the BIACORE assay described in Example 3 or the ADA bridging / competition assay described in Example 5 (see Figures 1A-1C, and especially Figure 1B).

[0108] Appropriate forms / techniques for conducting such assays will be apparent to those skilled in the art based on the disclosures herein, and include, for example (but not limited to), the following: - A colorimetric assay, such as ELISA, in which an analytical antibody is directly or indirectly coated onto a plate, and the bound ISV is detected with a monoclonal or polyclonal anti-ISV antibody. Other useful alternative techniques for this setup include, but are not limited to, electrochemiluminescence (MSD platform), fluorescence emission (DELFIA, GYROS), and other methods that rely on secondary detection of bound ISV. - Surface plasmon resonance (e.g., BIACORE), or other real-time biosensor methods (i.e., those not using SPR) that directly or indirectly immobilize an analytical antibody and subsequently monitor the binding of injected / administered ISVs. These methods do not require further detection of bound ISVs. A typical method for performing this type of assay is described in Example 3. - Analytical antibodies are used instead of ADA containing biological fluid to analyze the competitive behavior of ISVs in a bridging assay (ADA assay). Various techniques such as ELISA and MSD platforms can be used for the bridging assay. Typical methods for performing this type of assay are schematically shown in Figures 1A to 1C, and a specific example of this type of assay is also described in Example 5. - Any chromatographic method for immobilizing analytical antibodies on a chromatographic matrix to specifically capture / isolate ISVs from solution.

[0109] Once a suitable analytical antibody is obtained using one of the methods described herein or in one of the examples (or a method essentially equivalent thereto), the analytical antibody may be used to determine whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) causes protein interference (as defined herein) (or has a high tendency or increased tendency to cause protein interference) by performing steps (i) and (ii) above. As already described herein, this generally involves contacting the analytical antibody with the ISV, nanobody, ISV-based drug or nanobody-based drug, and determining whether the ISV, nanobody, ISV-based drug or nanobody-based drug is recognized by the analytical antibody (and / or bound, in particular specifically, by the analytical antibody) (in particular whether the C-terminal region of the ISV or nanobody, or the C-terminal region of the ISV or nanobody forming the C-terminus of the ISV-based drug or nanobody-based drug, is recognized by the analytical antibody).

[0110] This can generally be carried out using any suitable technique for determining whether an antibody binds to an antigen (in this case, an ISV, nanobody, ISV-based drug, or nanobody-based drug). Suitable (immuno)assay techniques will be obvious to those skilled in the art. Some non-limiting examples are suitable ELISA techniques (e.g., including sandwich ELISA); in this case, depending on the ELISA format used (obvious to those skilled in the art), the analytical antibody or ISV may be coated onto a plate or the analytical antibody or ISV may be detectably labeled. Other techniques may include, for example, the use of a BIAcore instrument (in this case, as before, the analytical antibody or ISV may be coated onto a chip; see, for example, Example 3). Another alternative method may be a competitive bridging assay (e.g., illustrated in Example 5) that tests the ability of an ISV to compete (or vice versa) with another ISV, nanobody, ISV-based drug, or nanobody-based drug known to be bound by an analytical antibody. These and other suitable techniques for determining whether a given ISV, nanobody, ISV-based drug, or nanobody-based drug is (specifically) bound to or recognized by an analytical antibody will be apparent to those skilled in the art based on the disclosure herein.

[0111] Based on the disclosures herein, it is also clear that the present invention (in particular the analytical antibodies used herein) can be used to determine whether a given ISV, nanobody, ISV-based drug, or nanobody-based drug contains an interaction site (for example, an interaction site located in or within the C-terminal region, and / or formed in part by the C-terminal region) that can undergo (nonspecific) protein interactions with one or more proteins or other components that may be present in a biological sample (i.e., the "test sample") obtained from a subject to be subjected to an immunoassay such as an ADA assay (in particular an ADA assay for determining the presence of ADA against an ISV, nanobody, ISV-based drug, or nanobody-based drug). Therefore, when an ISV, nanobody, ISV-based drug, or nanobody-based drug is recognized by the analytical antibody used herein, the ISV, nanobody, ISV-based drug, or nanobody-based drug is very likely to contain such an (accessible or exposed) interaction site, and thereby is likely to cause such protein interference (as defined herein) when used in such an immunoassay or ADA assay for testing a test sample. As will be apparent to those skilled in the art, this should preferably be avoided by selecting / using a different ISV, nanobody, ISV-based drug, or nanobody-based drug, if possible, or by modifying the ISV, nanobody, ISV-based drug, or nanobody system so that the tendency for such protein interference is substantially reduced or essentially eliminated (as before, this can be tested using the methods and analytical antibodies disclosed herein).

[0112] Furthermore, as will be apparent to those skilled in the art based on the disclosures herein, such modifications may include making one or more modifications (e.g., insertion, addition, deletion, or substitution of amino acids) to interaction sites on ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs such that the ability to undergo (nonspecific) protein interactions with one or more proteins or other components that may be present in the test sample is suppressed or eliminated. As before, this can be carried out with little trial and error by introducing one or more modifications and then testing whether this ability has been suppressed using the methods and analytical antibodies disclosed herein again. For example, one or more such modifications may be introduced and then the ability of the modified ISV to bind to the analytical antibody may be compared to the ability of the original / unmodified ISV. Alternatively, the ability of the modified ISV to (still) compete with the original ISV for binding to the analytical antibody may be determined using a competitive bridging format (e.g., illustrated in Example 5) or using BIAcore (e.g., see Example 3).

[0113] As before, the present invention is not limited to any hypothesis or description, but based on the experimental evidence shown in the following examples, the inventors have found that the interaction site may be located in / near the C-terminal region (as defined herein), or that the interaction site may form part of the C-terminal region (or that the C-terminal region may form part of this interaction site). This is at least in part based on the observation that, for example, ISVs tend to cause such protein interference and have VTVSS (SEQ ID NO: 33) as an amino acid residue at their C-terminus, the tendency is usually substantially reduced or essentially eliminated by adding 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 a tag or another peptide, protein, or other part to this C-terminus. In some cases, it has been found that the aforementioned tendency can be immediately substantially reduced or essentially eliminated by adding one, two, or three amino acid residues (which may be any suitable amino acid or combination of amino acids that can be independently selected from, for example, but not limited to, alanine, glycine, valine, leucine, or isoleucine, such as those listed in Table A-2 on page 64 of International Publication No. 09 / 138519) to the C-terminal VTVSS (SEQ ID NO: 33). This is partly based on the observation that in some cases where VHHs naturally contain an alanine residue at position 14 (as mentioned above, this forms part of the C-terminal region; see Figure 2), naturally occurring VHHs often do not tend to cause such protein interference (or have a low tendency to do so), whereas corresponding VHHs in which the alanine at position 14 is replaced with a proline residue (for example, for humanization or sequence optimization purposes) may consequently have an increased tendency to cause such protein interference (i.e., compared to VHHs with alanine at position 14).

[0114] In one embodiment, the present invention relates to a VHH, nanobody (as defined herein, particularly humanized VHH or camelid VH, e.g., camelid human VH) or another ISV (or an ISV-based drug or nanobody-based drug having a VHH, nanobody or other ISV at its C-terminus), which in any of the following cases is preferably modified (e.g., by introducing the substitution, addition or deletion of one or more amino acids) to have a substantially reduced tendency to produce protein interference (as defined herein) (e.g., a statistically relevant reduced tendency) compared to the same VHH, nanobody or ISV that is otherwise unmodified, and / or (ii) to have a substantially reduced ability to be bound by the analytical antibody described herein (e.g., the polyclonal antibody described in Example 2 and used in Examples 3 and 5) in the methods of the present invention described herein (e.g., the specific assay described in Example 3 or Example 5), particularly a VHH, nanobody or another ISV whose C-terminal region is modified (e.g., by substituting or adding one or more amino acids within the C-terminal region).

[0115] Accordingly, in one embodiment, the present invention is a VHH or VH domain (i.e., an ISV that is a VH domain or derived from a VH domain), and / or a VHH, nanobody (as defined herein, particularly humanized VHH or camelid VH, e.g., camelid human VH) or another ISV (or an ISV-based drug or nanobody-based drug having VHH, a nanobody or other ISV at its C-terminus), wherein the amino acid sequence is VTVSS(X) nThe present invention relates to a VHH, nanobody, or other ISV having (SEQ ID NO: 34) (wherein n is 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 (preferably 1 or 2, e.g., 1), and each X is an amino acid residue (preferably naturally occurring) 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 shown below, other (preferably naturally occurring) amino acid residues, or combinations of the above preferred amino acid residues with other amino acid residues (e.g., serine, proline, threonine, and / or lysine)) at its C-terminus. Preferably, the amino acid sequence VTVSS(X) n The VHH, nanobody, or ISV having (SEQ ID NO: 34) at its C-terminus is preferably in any of the following cases compared to the same VHH, nanobody, or ISV having the amino acid sequence VTVSS (SEQ ID NO: 33) at its C-terminus, except that it has (i) a substantially reduced tendency to cause protein interference (as defined herein) (e.g., at least a statistically significant reduced tendency); and / or (ii) a substantially reduced ability to be bound by the analytical antibody described herein (e.g., the polyclonal antibody described in Example 2) in the methods of the present invention described herein (e.g., the specific assay described in Example 3 or Example 5). See, for example, the assay and data shown in Example 3.

[0116] The above VHH, nanobody, or (other) ISV is preferably such that it has an RU value of less than 500 for binding by 21-4 (determined according to the protocol shown in Example 9 and after adjusting the measured RU value for the molecule (molecu)). In the description or claims herein, the C-terminal sequence VTVSS(X) n Whenever referring to any of the above embodiments (a) to (p), it should also be noted that, according to one specific embodiment of the present invention, none of the amino acids X are cysteine ​​residues.

[0117] For example, in some preferred embodiments, the C-terminus of an ISV or ISV-containing structure (where this C-terminus is an ISV, VHH, or nanobody derived from VH) may be: (a) VTVSS(X) n (In the equation, n=1 and X=Ala); (b) VTVSS(X) n (In the equation, n=2 and each X=Ala); (c)VTVSS(X) n (In the formula, n=3 and each X=Ala); (d)VTVSS(X) n (wherein n=2 and at least one X=Ala (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)); (e)VTVSS(X) n (wherein n=3 and at least one X=Ala (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)); (f)VTVSS(X) n (wherein n=3 and at least two X=Ala (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)); (g)VTVSS(X) n (In the equation, n=1 and X=Gly); (h)VTVSS(X) n (In the formula, n=2 and each X=Gly); (i) VTVSS(X) n (In the formula, n=3 and each X=Gly); (j)VTVSS(X) n (wherein n=2 and at least one X=Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)); (k)VTVSS(X) n(wherein n=3 and at least one X=Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)); (l)VTVSS(X) n (wherein n=3 and at least two X=Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)); (m)VTVSS(X) n (In the formula, n=2 and each X=Ala or Gly); (n)VTVSS(X) n (In the formula, n=3 and each X=Ala or Gly); (o)VTVSS(X) n (wherein n=3 and at least one X=Ala or Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)); or (p)VTVSS(X) n (wherein n=3 and at least two X=Ala or Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile); Embodiments (a), (b), (c), (g), (h), (i), (m), and (n) are particularly preferred, embodiments where n=1 or n=2 are preferred, and embodiments where n=1 are particularly preferred.

[0118] In the description or claims herein, the C-terminal sequence VTVSS(X) n Whenever referring to any of the above embodiments (a) to (p), it should also be noted that, according to one specific embodiment of the present invention, none of the amino acids X are cysteine ​​residues.

[0119] Therefore, in a preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody is preferred), and the sequence VTVSS(X) n This invention relates to an immunoglobulin single variable domain (ISV) having a C-terminus of (wherein n=1 and X=Ala) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus).

[0120] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n This invention relates to an immunoglobulin single variable domain (ISV) having a C-terminus of (wherein n=2 and each X=Ala) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus).

[0121] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n The present invention relates to an immunoglobulin single variable domain (ISV) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus) (wherein n=2 and at least one X=Ala (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)).

[0122] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) nThe present invention relates to an immunoglobulin single variable domain (ISV) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus) (wherein n=3 and at least one X=Ala (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)).

[0123] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n The present invention relates to an immunoglobulin single variable domain (ISV) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus) (wherein n=3 and at least two X=Ala (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)).

[0124] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n This relates to an immunoglobulin single variable domain (ISV) having a C-terminus of (wherein n=3 and each X=Ala) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus).

[0125] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) nThis invention relates to an immunoglobulin single variable domain (ISV) having a C-terminus of (wherein n=1 and X=Gly) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus).

[0126] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n This invention relates to an immunoglobulin single variable domain (ISV) having a C-terminus of (wherein n=2 and each X=Gly) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus).

[0127] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n This relates to an immunoglobulin single variable domain (ISV) having a C-terminus of (wherein n=3 and each X=Gly) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus).

[0128] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n The present invention relates to an immunoglobulin single variable domain (ISV) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus) (wherein n=2 and at least one X=Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)).

[0129] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n The present invention relates to an immunoglobulin single variable domain (ISV) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus) (wherein n=3 and at least one X=Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)).

[0130] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n The present invention relates to an immunoglobulin single variable domain (ISV) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus) (wherein n=3 and at least two X=Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile)).

[0131] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n This relates to an immunoglobulin single variable domain (ISV) having a C-terminus of (wherein n=2 and each X=Ala or Gly) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus).

[0132] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n This relates to an immunoglobulin single variable domain (ISV) having a C-terminus of (wherein n=3 and each X=Ala or Gly) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus).

[0133] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n The present invention relates to an immunoglobulin single variable domain (ISV) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus) (wherein n=3 and at least one X=Ala or Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile)).

[0134] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n The present invention relates to an immunoglobulin single variable domain (ISV) (or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus) (wherein n=3 and at least two X=Ala or Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile)).

[0135] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n This relates to a single variable immunoglobulin domain (ISV) having a C-terminus of (wherein n=1, 2, or 3, each X=Ala or Gly).

[0136] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n (In the formula: - n=1, 2, or 3, each X=Ala or Gly; or - A single variable immunoglobulin domain (ISV) having a C-terminus with n=2 or 3, where all but one X=Ala or Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile) Or relating to a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus.

[0137] In another preferred embodiment, the present invention relates to an immunoglobulin single variable domain (ISV) which is a nanobody or an ISV comprising or derived from a VH sequence (nanobody preferred), and the sequence VTVSS(X) n (In the formula: - n=1, 2, or 3, each X=Ala or Gly; or - n=2 or 3, at least one X=Ala or Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile); - A single variable immunoglobulin domain (ISV) having a C-terminus with n=2 or 3, where all but one X=Ala or Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile) Or relating to a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus.

[0138] In the above embodiments, the “(other) ISV comprising or derived from a VH sequence” means an ISV comprising or derived from a VH sequence that is not a nanobody (i.e., not VHH, humanized VHH, or camelized VH). For example, such an (other) ISV could be, for example, a (single) domain antibody based on VH, a dAb™ based on VH, or a microbody based on VH (see International Publication No. 00 / 29004).

[0139] As before, one of the ISVs mentioned herein is the C-terminal sequence VTVSS(X) n Whenever the sequence VTVSS(X) has (including, but not limited to, the ISV referred to in the preceding embodiment), according to one specific aspect of the present invention, n It should be noted that none of the amino acid X components are cysteine ​​residues.

[0140] As will be further described herein, such proteins or polypeptides may be constructs containing two or more ISVs (e.g., two or more nanobodies) linked together, for example, via one or more suitable linkers. Thus, for example, such constructs may be divalent, trivalent, tetravalent, or pentavalent constructs (e.g., divalent, trivalent, tetravalent, or pentavalent nanobodies), or divalent, trivalent, tetravalent, or pentavalent constructs (e.g., divalent, trivalent, tetravalent, or pentavalent nanobodies) that are, for example, bispecific, triplicate, or diparatope constructs (e.g., monospecific, bispecific, or diparatope constructs that can also bind to serum albumin (preferably) or another serum protein for extension of half-life).

[0141] As before, the nanobodies, ISVs, and proteins / polypeptides of each of the above embodiments are preferably such that they have an RU value of less than 500 for binding by 21-4 (determined according to the protocol shown in Example 9 and after adjusting the measured RU value for the molecular weight of the ISV or protein used according to the formula shown above).

[0142] As described herein, the present invention may also be applied to other proteins or polypeptides having a VH domain at its C-terminus (in particular antibody fragments such as Fab fragments, or other proteins or polypeptides based on antibody fragments, e.g., ScFv). Accordingly, in another embodiment, the present invention may apply to the C-terminal amino acid sequence VTVSS(X) nThe present invention relates to a protein or polypeptide (e.g., ScFv) having a VH domain having (SEQ ID NO: 34) (wherein n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5, and each X is an amino acid residue independently selected (preferably independently selected) (preferably naturally occurring) from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)) at its C-terminus. As before, according to some specific embodiments, the C-terminus may be any of (a) to (p) above, preferably one of (a), (b), (c), (g), (h), (i), (m), or (n), where n is 1, 2, or 3, preferably 1 or 2.

[0143] As before, such proteins or polypeptides are preferably those having an RU value of less than 500 for binding by 21-4 (determined according to the protocol shown in Example 9 and after adjusting the measured RU value for the molecular weight of the ISV or protein used according to the formula shown above). Also as before, according to a specific embodiment of this aspect of the present invention, the C-terminal sequence VTVSS(X) n None of the amino acid X components are cysteine ​​residues.

[0144] The present invention further relates to a pharmaceutical composition comprising an ISV (preferably a therapeutic ISV) or a protein or polypeptide containing at least one ISV (preferably at least one therapeutic ISV), such as an ISV, protein or polypeptide as further described herein (i.e., one or more embodiments described herein, particularly one or more embodiments described on earlier pages; more specifically, an ISV, protein or polypeptide having one or more C-terminuses / sequences of embodiments described herein), at least one suitable (i.e., suitable for pharmaceutical use) carrier, diluent or excipient, and optionally one or more further active substances. Such a composition, carrier, diluent or excipient may be, for example, as described in International Publication No. 08 / 020079 for a pharmaceutical composition comprising a nanobody or a protein or polypeptide containing at least one nanobody (as already stated, according to the present invention, the ISV is preferably also a nanobody).

[0145] The present invention further relates to ISVs or proteins or polypeptides comprising at least one ISV for use in the treatment of diseases in humans (e.g., patients requiring such treatment), as ISVs, proteins or polypeptides as further described herein (i.e., one or more embodiments described herein, in particular one or more embodiments described on earlier pages; more specifically, ISVs, proteins or polypeptides having one or more C-terminuses / sequences of embodiments described herein).

[0146] The present invention further relates to the use of an ISV or a protein or polypeptide comprising at least one ISV in the preparation of a pharmaceutical composition, wherein the ISV, protein or polypeptide is as further described herein (i.e., one or more of the embodiments described herein, in particular one or more of the embodiments described on earlier pages; more specifically, an ISV, protein or polypeptide having one or more C-terminuses / sequences of the embodiments described herein).

[0147] The present invention further relates to a method of treatment comprising administering a pharmaceutical composition (above) to a human subject (e.g., a patient requiring such treatment) in the form of a pharmaceutical composition preparation, comprising an ISV or a protein or polypeptide comprising at least one ISV, as further described herein (i.e., one or more embodiments described herein, particularly one or more embodiments described on earlier pages; more specifically, an ISV, protein or polypeptide having one or more C-terminuses / sequences described herein); or a pharmaceutical composition comprising at least one such ISV, protein or polypeptide (above).

[0148] In relation to the above, the therapeutic uses of ISVs, proteins, and polypeptides described herein (or the clinical development of such ISVs, proteins, and polypeptides for such therapeutic uses) may involve the use of an ADA assay to determine whether the ISV, protein, or polypeptide is immunogenic (i.e., capable of producing ADA when administered to a human subject), so it is clear that the therapeutic uses of ISVs, proteins, and polypeptides described herein are very important embodiments of the present invention. In this regard, it is also clear that concerns about possible immunogenicity must be addressed in particular when therapeutic agents are used over longer periods (over weeks, months, or years) and / or have a half-life of at least 3 days, for example, at least 1 week, up to 10 days or more (preferably expressed as t1 / 2-β) in human subjects.

[0149] Accordingly, according to a specific embodiment of the present invention, the ISV, protein, polypeptide, or pharmaceutical composition described herein is intended to treat chronic diseases in humans, and / or, such ISV, protein, polypeptide described herein is intended to be present in the circulatory system of a subject to which it is administered (i.e., at a therapeutically active dose) for a period of at least one week, preferably at least two weeks, for example, at least one month; and / or, such ISV, protein, polypeptide described herein has a half-life (preferably expressed as t1 / 2-β) in a human subject of at least three days, for example, at least one week, up to 10 days or more; and / or, such ISV, protein, polypeptide, or pharmaceutical composition described herein is intended to be administered to humans over a period of at least three days, for example, at least one week, for example, at least two weeks or at least one month or longer (i.e., at least three months, at least six months or at least one year), or in two or more doses administered chronically.

[0150] The present invention further relates to a method for (substantially) reducing or essentially eliminating the tendency of ISVs, nanobodies, or ISV-based drugs or nanobody-based drugs to cause protein interference, - A step of determining the tendency of an ISV, nanobody, ISV-based drug, or nanobody-based drug to cause protein interference, using a method that optionally includes at least steps (i) and (ii) as referred to herein; - Modifying the ISV, nanobody, ISV-based drug, or nanobody-based drug by introducing substitution, addition, or deletion of one or more amino acids into the ISV or nanobody, or into the C-terminal ISV or nanobody of an ISV-based drug or nanobody-based drug (if any); in particular, by introducing substitution or addition of one or more amino acids into the C-terminal region of the ISV or nanobody, or into the C-terminal region of the C-terminal ISV or nanobody of an ISV-based drug or nanobody-based drug (if any), for example, by adding one to ten amino acid residues, for example one to five, for example one, two, three, four, or five amino acid residues, independently selected from any naturally occurring amino acids (for example, the amino acids listed in Table A-2 on page 64 of International Publication No. 09 / 138519, for example, alanine, glycine, valine, leucine, or isoleucine), to the C-terminus of the sequence; - A method comprising at least steps (i) and (ii) as referred to herein; optionally, a method that allows for the determination of the tendency of the thus modified ISV, nanobody, ISV-based drug, or nanobody-based drug to induce protein interference, by comparing the tendency of the thus modified ISV, nanobody, ISV-based drug, or nanobody-based drug to induce protein interference with the tendency of the original ISV, nanobody, ISV-based drug, or nanobody-based drug to induce protein interference (including, but not limited to, comparing them in a competitive assay for binding to analytical antibodies as described herein). The present invention relates to a method comprising at least , or the method described herein with the use of 21-4.

[0151] Next, the present invention will be further described by the following non-limiting preferred embodiments, examples, and drawings. [Brief explanation of the drawing]

[0152] [Figure 1A]Figure 1A schematically shows a non-limiting example of the ADA assay format. Several representative but non-limiting protocols for performing this assay are described in Example 4. [Figure 1B] Figure 1B schematically shows a non-limiting example of the ADA assay format. Several representative but non-limiting protocols for performing this assay are described in Example 4. [Figure 1C] Figure 1C schematically shows a non-limiting example of the ADA assay format. Several representative but non-limiting protocols for performing this assay are described in Example 4. [Figure 2] Figure 2 schematically shows a typical three-dimensional (3D) structure of ISVs such as nanobodies. [Figure 3] Figure 3 shows the binding curves of NB3.4-3.9 (SEQ ID NOs. 5-10) to the immobilized polyclonal antibodies obtained in Example 2 (obtained using the BIACORE assay described in Example 3). [Figure 4] Figure 4 shows the binding curves (obtained using the BIACORE assay described in Example 3) of NB3.4, 3.11, 3.12, and 3.13 (SEQ ID NOs. 5, 12, 13, and 14) to the immobilized polyclonal antibodies obtained in Example 2. [Figure 5] Figure 5 shows the binding curves (obtained using the BIACORE assay described in Example 3) of NB3.4, 3.14, and 3.15 (SEQ ID NOs. 5, 15, and 16) to the immobilized polyclonal antibodies obtained in Example 2. [Figure 6] Figure 6 shows the binding curves (obtained using the BIACORE assay described in Example 3) of NB3.1, 3.2, and 3.4 (SEQ ID NOs: 3, 4, and 5) to the immobilized polyclonal antibodies obtained in Example 2. [Figure 7] Figure 7 shows the binding curves of NB4.1 and 4.2 (SEQ ID NOs. 17 and 18) to the immobilized polyclonal antibodies obtained in Example 2 (obtained using the BIACORE assay described in Example 3). [Figure 8] Figure 8 shows the binding curves of NB6.1, 6.2, 6.4, and 6.5 (SEQ ID NOs. 19-22) to the immobilized polyclonal antibodies obtained in Example 2 (obtained using the BIACORE assay described in Example 3). [Figure 9-1] Figure 9-1 shows the sequences used in Example 8 (sequence numbers 37-39) and a table showing the corresponding reference sequences. [Figure 9-2] Figure 9-2 shows the sequences used in Example 8 (sequences 40-42) and a table showing the corresponding reference sequences. [Figure 9-3] Figure 9-3 shows the sequences used in Example 8 (sequences 43-46) and a table showing the corresponding reference sequences. [Figure 9-4] Figure 9-4 shows the sequences used in Example 8 (sequence numbers 47-51) and a table showing the corresponding reference sequences. [Figure 9-5] Figure 9-5 shows the sequences (sequence numbers 52-58) used in Example 8, and a table showing the corresponding reference sequences. [Figure 9-6] Figure 9-6 shows the sequences (sequence numbers 59-65) used in Example 8, and a table showing the corresponding reference sequences. [Figure 9-7] Figure 9-7 shows the sequences (sequence numbers 66-72) used in Example 8, and a table showing the corresponding reference sequences. [Figure 9-8] Figure 9-8 shows the sequences (sequence numbers 73-79) used in Example 8, and a table showing the corresponding reference sequences. [Figure 9-9] Figure 9-9 shows the sequences used in Example 8 (sequences 80-86) and a table showing the corresponding reference sequences. [Figure 9-10] Figure 9-10 shows the sequences used in Example 8 (sequence numbers 87-89) and a table showing the corresponding reference sequences.

[0153] The sequences referred to in this specification and in the claims are listed in Table A (Sequence IDs 1-37) and Figure 9 (Sequence IDs 38-89).

[0154] [Table 1] TIFF2026067951000002.tif211170 TIFF2026067951000003.tif225170 TIFF2026067951000004.tif236170 TIFF2026067951000005.tif207170 TIFF2026067951000006.tif113170 [Examples]

[0155] Example 1: Preparation of polyclonal analytical antibodies. Polyclonal antibodies (IgG fraction) that can be used as "analytical antibodies" were prepared as follows:

[0156] A. Identification of suitable plasma samples for the isolation of polyclonal antibodies. In 20 plasma samples derived from healthy individuals who had never been treated with ISV, the presence of antibodies against ISV that can be used as analytical antibodies in this invention was evaluated.

[0157] The ISV initially used in this embodiment was SEQ ID NO: 1. Subsequently, the assay was repeated using other ISVs to confirm that the interaction was not specific to this particular ISV but was a nonspecific protein-protein interaction that could occur with many ISVs (see paragraph C below). Instead of SEQ ID NO: 1, for example, SEQ ID NO: 2 may be used.

[0158] The assay used was an ECL (electrochemiluminescence) bridging assay, using biotinylated ISV (biotinylated variant of SEQ ID NO: 1) for capture and sulfotaged ISV for detection of anti-drug antibodies. A similar format was used for the ADA assay. Biotinylation and sulfotagling of ISV were performed using standard coupling chemical reactions to primary amines, with Sulfo-NHS-LC-Biotin (Pierce) and Sulfo-tag NHS-Ester (MSD) respectively, according to the manufacturer's instructions. Plasma samples were diluted 1 / 5 with PBS / 0.1% casein and incubated in 96-well polypropylene plates at 37°C and 600 RPM for 30 minutes. Next, the sample (50 μL) was diluted by 1 / 3 with a 1:1 mixture (100 μL) of 2 μg / ml biotinylated ISV and 2 μg / ml sulfotagled ISV (SEQ ID NO: 1), and incubated at room temperature (RT) and 600 RPM for 1 hour. An MSD MA® 96-well standard streptavidin plate was blocked at room temperature (RT) for 1 hour using 150 μL / well of Superblock® T20, and then washed three times with PBS / 0.05% Tween20 (=wash buffer). The sample / 1:1 mixture (biotinylated ISV and sulfotagled ISV (SEQ ID NO: 1)) (50.0 μL) was transferred from the polypropylene plate to an MSD plate and incubated at room temperature (RT) and 600 rpm for 1 hour. The plates were washed three times, then 2 x Read Buffer (MSD) (150 μL / well) was added, and ECL units (ECLU) were read using an MSD instrument (Sector Imager 2400 reader). Samples were screened as positive or negative using the screening cutpoint determined during method validation. The screening cutpoint was calculated using appropriate statistical analysis as recommended by the ADA assay development guidelines (Shankar, 2008), based on background values ​​from 118 individual plasma samples from healthy individuals who had never been treated with ISV. Nonparametric assessments were used, and the cutoff value was calculated based on the 95th percentile after removing outliers.

[0159] Six plasma samples were clearly scored as positive: IHuP#002-001-ABL-01, IHuP#002-001-ABL-08, IHuP#002-001-ABL-10, IHuP#002-001-ABL-15, IHuP#002-001-ABL-19, and IHuP#002-001-ABL-20 (Table I).

[0160] These samples were further analyzed using a drug substitution setup (confirmatory assay) to confirm the specificity of the positive screening results (Table II). Therefore, the samples were diluted 1 / 5 with PBS / 0.1% casein containing 12.5 μg / mL of ISV (SEQ ID NO: 1) and incubated in a 96-well polypropylene plate at 37°C and 600 RPM for 30 minutes. Next, the sample (50 μL) was diluted 1 / 3 with a 1:1 mixture (100 μL) of 2 μg / mL of biotinylated ISV and 2 μg / mL of sulfotagled ISV (SEQ ID NO: 1) and incubated at room temperature (RT) and 600 RPM for 1 hour. Next, for the screening assay, as described above, the sample / 1:1 mixture (biotinylated ISV and sulfotaged ISV) (50.0 μL) was transferred from a polypropylene plate to a blocked MSD MA® 96-well standard streptavidin plate and incubated at room temperature (RT) at 600 rpm for 1 hour. After washing the plate three times, 2 x Read Buffer (MSD) (150 μL / well) was added, and ECL units (ECLU) were measured using an MSD instrument (Sector Imager 2400 reader). Samples were confirmed as true positive using the confirmation cutpoint determined during method validation, and ECL responses were calculated using appropriate statistical analysis as recommended by the ADA assay development guidelines (Shankar, 2008) based on the ECL response of 118 individual plasma samples (spiked with 50 μg / ml ISV (SEQ ID NO: 1)) from healthy individuals who had never been treated with ISV. The minimum signal decline of 50% was calculated based on the 99% confidence interval.

[0161] Samples that were positive in the ECL bridging assay and confirmed positive in the drug substitution setup assay were selected as materials for the production of polyclonal antibodies using affinity chromatography.

[0162] [Table 2]

[0163] [Table 3]

[0164] Three additional serum samples from individuals not treated with ISV were also evaluated using the ECL bridging assay described above and confirmed using the drug substitution setup assay.

[0165] Two serum samples were clearly scored as positive in the ECL bridging assay: IHUS#B09032311A3 and IHUS#B09032311A20 (Table III). The two samples screened as positive were further analyzed in a drug substitution setup to confirm the specificity of the positive screening results.

[0166] [Table 4]

[0167] B. Preparation of purified polyclonal IgG fraction. 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, the column was equilibrated by removing the storage solution from the column by centrifugation (100xg for 30 seconds) and then adding binding buffer (20mM sodium phosphate, pH 7.0). After centrifugation, the column was incubated for 4 minutes with the addition of the solution containing the desired polyclonal (up to 1 mg in 600 μl) and gentle mixing. The column was then centrifuged and washed twice by successively adding binding buffer (600 μl) and centrifuged again. The antibody was eluted by adding 400 μl of eluent (0.1 M glycine-HCl, pH 2.7), inverting and mixing, and then centrifugation in 30 μl of neutralizing buffer (1 M Tris-HCl, pH 9.0).

[0168] To confirm that the IgG fraction thus obtained was involved in nonspecific binding to ISV, the purified IgG antibody was analyzed using the ECL bridging assay described above and confirmed using the drug substitution setup assay used in A) above. In both samples (IHUS#B09032311A3 and IHUS#B09032311A20), the purified IgG antibody was found to be involved in nonspecific binding, resulting in a positive signal in the assay (Table III). This confirmed that purified polyclonal IgG could be used as an "analytical antibody," and it was used as such in Examples 3 and 5 (the assays).

[0169] C. Non-specific binding to other ISVs. To determine whether the observed protein interference was specific to a single ISV and / or specific to a particular region, epitope, or antigenic determinant on the ISV and / or to certain mutations made against the wild-type ISV (e.g., one or more humanization mutations), the ECL bridging assay and drug substitution setup assay (both using SEQ ID NO: 1 as the sulfotagged ISV, as described in A) above) were repeated using plasma samples IHUS#B09032311A3, IHUS#B09032311A20, and IHUS#B09032311A1. Since these plasma samples contain the polyclonal "analysis" antibody isolated in B) above, this also provides information about the specificity, selectivity, and epitope recognition of the polyclonal analysis antibody.

[0170] Eight ISVs were tested (SEQ ID NOs. 23-30, respectively - see Table A above). Of these, one was wild-type VHH (SEQ ID NOs. 23), while the other seven ISVs were humanized versions of the wild-type sequence with different humanization substitutions. Two ISVs (SEQ ID NOs. 29 and 30) also contained additional amino acid residues at the C-terminus (one and three additional alanine residues, respectively).

[0171] The data is shown in Table IV. While not limited to any explanation or hypothesis, it can be seen that mutations in the C-terminal region (as defined herein) can significantly affect the degree to which the plasma sample used can produce protein interference. For example, the addition of one or three amino acid residues to the C-terminus can significantly reduce the tendency for protein interference to occur (e.g., for SEQ ID NO: 28 (the corresponding humanized variant without added amino acid residues at the C-terminus), the reduction in the ECLU assay using sample IHUS#B09032311A3 was 90%, compared to only 18% and 13% for SEQ ID NOs: 29 and 30). Similarly, the introduction of a proline residue at position 14 of the wild-type sequence can also significantly affect the degree to which the plasma sample used can produce protein interference (e.g., for SEQ ID NO: 24 (the wild-type sequence with substitution A14P), the reduction in the ECLU assay using sample IHUS#B09032311A3 was 91%, compared to only 20% for the wild-type sequence of SEQ ID NO: 23). K83R and Q108L (which are also substitutions near the C-terminus) also increase the tendency to cause protein interference to some extent, but not to the same extent as the A14P substitution. The overall effect of the combined A14P + K83R + Q108L substitutions can be neutralized by adding one or more amino acid residues to the C-terminus (data from SEQ ID NOs. 29 and 30 were compared again with data from other humanized mutants).

[0172] Based on this data, we concluded that the polyclonal analytical antibody clearly recognized the C-terminal region of ISV (as defined herein) in general. 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 ISV (when considering the three-dimensional structure of ISV).

[0173] [Table 5]

[0174] [Table 6]

[0175] Example 2: Affinity Purification of Analytical Antibodies This example describes two methods that can be used to isolate analytical antibodies capable of recognizing and / or binding to the C-terminus of ISV from biological fluids derived from human subjects. Antibodies are isolated from four different serum samples characterized by inducing a positive signal in the ADA assay of the test described in Example 1.

[0176] Starting from serum samples, each of these protocols provides purified preparations of interfering factors that can be used as analytical antibodies in the methods described herein. These methods can also be used more generally to purify interfering factors for other purposes (for example, in Example 8, purified interfering factors were experimentally used using the following protocols to show that binding to an ISV or ISV construct by monoclonal 21-4 predicts binding to the same ISV or ISV construct by the interfering factor, thereby predicting the tendency of the ISV or ISV construct to undergo nonspecific protein interference in the ADA assay).

[0177] Example 2A: Purification using Protein A and affinity chromatography In the first step, the IgG antibody fraction was concentrated from serum samples using protein A affinity chromatography. The standard columns used for this concentration included HiTrap MabselectSure and MabSelectXtra (GE Healthcare); and PorosMabCapture A (Applied Biosystems). Purification of IgG antibodies from serum samples was performed automatically and similarly in all experiments. Chromatography runs were performed using AKTA purifier systems (GE Healthcare) and logged in in real time using UNICORN protein purification software (GE Healthcare). In short, serum samples were diluted 1:1 with D-PBS (Dulbecco phosphate-buffered saline), filtered through 0.22 μm, and uploaded to the column at a constant flow rate of 0.5 mL / min. The column was washed with more than five times the column volume of D-PBS at a flow rate of 0.5 mL / min to remove nonspecific binding components. The IgG fraction was eluted by acid elution using 100 mM glycine buffer (pH 2.6) at a flow rate of 0.5 mL / min. After elution, the fraction was neutralized using 1.5 M Tris buffer (pH 8.8). SDS-PAGE was performed to confirm the isolation of IgG antibodies in the eluate.

[0178] In the second step, interfering IgG was further enriched by applying protein A purified IgG fractions from four different serum samples to an ISV-binding affinity column. More specifically, interfering IgG was further enriched by binding to a column containing an ISV with the sequence of SEQ ID NO: 1. For this purpose, the ISV was covalently bound to Sepharose 4 fast flow (GE Healthcare) using a CNBr (cyanide bromide) coupling method according to the manufacturer's procedure. Affinity purification was performed automatically and similarly in all experiments. Chromatography runs were performed on AKTA purifier systems and logged in with UNICORN. Briefly, the IgG enriched sample (maximum loading volume of 10 mL) was uploaded to the column at a constant flow rate of 0.5 mL / min. The column was washed with more than five times the column volume of D-PBS at a flow rate of 0.5 mL / min to remove nonspecific binding components. ISV-binding components were eluted by acid elution using 100 mM glycine buffer (pH 2.6) at a flow rate of 0.5 mL / min. After elution, the fraction was neutralized using 1.5 M Tris buffer (pH 8.8). This fraction was analyzed using SDS-PAGE to confirm the isolation of IgG antibodies in the eluate (data not shown).

[0179] These fractions were pooled and used for further analysis, for example, the analysis described in Example 3.

[0180] Example 2B: Purification using CaptureSelect™ chromatography. Alternatively, interfering factors were recovered from plasma and purified using the commercially available IgA-binding affinity resin CaptureSelect hIgA® (BAC BV) (based on a single variable heavy chain domain (VHH) derived from camel). Subsequently, the recovered "IgA fraction" containing IgA along with interfering IgG was loaded onto a Protein A column, and the IgA fraction was removed. The Protein A column was processed according to typical IgG purification conditions (running buffer: PBS; elution buffer: 100 mM glycine (pH=2.7); neutralization after elution with 1 M Tris). Interfering factors were recovered from the Protein A (Prot A) eluate in yield >95%.

[0181] A variation of this method used a different CaptureSelect affinity resin (a commercially available affinity resin based on VHH, CaptureSelect Alpha-1 antitrypsin resin that does not target antibody-related proteins). This resin provided a high interference factor binding effect and enabled a two-step selective elution: antitrypsin treatment by neutral pH elution using 2.0 M MgCl2, followed by elution of the interference factors in an acidic step (neutralization using 0.1 M glycine (pH 3.0); 1.5 M Tris, similar to the elution conditions for protein A / G). This one-step purification yielded up to 15 μg of interference IgG1 per 1 mL of highly interference plasma, which is approximately 0.3% of the total IgG present. If necessary, the neutralized interference fraction can be desalted and further purified using a size exclusion column equilibrated with D-PBS.

[0182] Example 3: The effect of various ISV substitutions on the tendency of ISVs to cause protein interference. As described above, the present invention makes available certain assays and techniques that enable the evaluation of whether a given ISV tends to cause protein interference. These include the ECL bridging assay and drug substitution setup assay used in Example 1, as well as the BIACORE assay described in this Example 3, and the bridging / competition ADA assay described in the following further examples.

[0183] Furthermore, as described above, these assays can also be used to determine whether specific mutations (e.g., amino acid deletions, substitutions, or additions) can affect (preferably reduce) the tendency of a given ISV to cause protein interference. Some of these mutations are or will become apparent to those skilled in the art based on the disclosures herein and the experimental data presented in Example 1 and this Example 3.

[0184] As already demonstrated by the data prepared in Example 1, certain mutations within or near the C-terminal region (as defined herein) of an ISV appear to be able to (strongly) influence its tendency to cause protein interference. For example, the addition of several amino acid residues to the C-terminus (e.g., one or three alanine residues) appears to strongly reduce the ISV's tendency to cause protein interference and may even neutralize the presence of other substitutions (e.g., substitution A14P) (e.g., within or near the C-terminal region) that appear to increase the tendency to cause protein interference.

[0185] In this Example 3, the effects of other substitutions and the effect of adding additional amino acids to the C-terminus were investigated by comparing related ISVs with different substitutions using the analytical polyclonal antibody prepared in Example 2. The analysis was performed by measuring the dynamics of the interaction between each investigated ISV and the analytical polyclonal antibody by surface plasmon resonance (SPR) using a Biacore® T100 biosensor (GE Healthcare). The ISVs tested in this Example 3 were ISVs SEQ ID NOs: 3 to 22 (see, for example, Table A above and Table V below).

[0186] In a standard experiment, a polyclonal antibody solution was prepared at a concentration of 10 μg / ml in 10 mM NaOAc (pH 5.0). This polyclonal antibody was then immobilized onto a CM5 sensor chip using amine coupling via the EDC / NHS method (EDC = N-ethyl-N'-[3-diethylaminopropyl]-carbodiimide; NHS = N-hydroxysuccinimide) according to the manufacturer's procedure. The immobilized amount was approximately 2700 reaction units (RU). A constant concentration of 500 nM ISV was then injected onto the surface at a flow rate of 45 μl / min over 120 seconds. Since no effective regeneration buffer could be identified, the dissociation time was extended to 2400 seconds. The signal obtained by injecting ISV into a blank flow cell was subtracted from the signal obtained by injecting ISV into a flow cell bound to the polyclonal antibody. The blank flow cell was activated / deactivated in the same manner as the polyclonal antibody flow cell, except that no protein was added. Additionally, the blank (HBS-EP + running buffer; HBS = Hepes-buffered saline: GE Healthcare) was subtracted to correct for possible baseline variability.

[0187] To investigate the effect of adding amino acid residues to the C-terminus, the effects of adding one or two alanine residues and one, two, or three glycine residues were investigated by comparing the binding of ISVs with different additions using analytical polyclonal antibodies prepared as described in Example 2. The ISVs prepared and tested for this purpose were NB3.4-3.9 (SEQ ID NOs. 5-10).

[0188] As a representative example of the data types obtained, Figure 3 shows the binding of NB3.4-3.9 to immobilized polyclonal antibodies. The results obtained are summarized in Table V.

[0189] [Table 7]

[0190] To investigate the effects of (other) substitutions within the C-terminal region, the effects of different substitutions were examined by comparing related ISVs containing three substitutions using the same analytical polyclonal antibody as described above. The analysis was performed as described above.

[0191] The ISVs containing the aforementioned substitutions that were tested were NB3.1, 3.2, and 3.4 (SEQ ID NOs: 3, 4, and 5); NB3.10 to 3.15 (SEQ ID NOs: 11 to 16), which were compared with NB3.4; NB4.1 and 4.2 (SEQ ID NOs: 17 and 18) and NB6.1, 6.2, 6.4, and 6.5 (SEQ ID NOs: 19 to 22).

[0192] As representative examples of the data types obtained: - Figure 4 shows the binding of NB3.4, 3.11, 3.12, and 3.13 to immobilized polyclonal antibodies; - Figure 5 shows the binding of NB3.4, 3.14, and 3.15 to immobilized polyclonal antibodies; - Figure 6 shows the binding of NB3.1, 3.2, and 3.4 to immobilized polyclonal antibodies; - Figure 7 shows the binding of NB4.1 and 4.2 to immobilized polyclonal antibodies; - Figure 8 shows the binding of NB6.1, 6.2, 6.4, and 6.5 to immobilized polyclonal antibodies.

[0193] The results obtained are summarized in Tables VI, VII, and VIII.

[0194] [Table 8]

[0195] [Table 9]

[0196] [Table 10]

[0197] As before, we are not limited to any specific hypothesis or explanation, but the data presented above shows that (various) substitutions of the C-terminal region of ISV (as defined herein) can alter / improve the tendency to cause protein interference.

[0198] Example 4: A typical protocol for performing the ADA assay shown in Figure 1. This example presents some representative but non-limiting conditions that can be used to perform the competitive / bridging ADA assay schematically shown in Figure 1: - ADA assay in Figure 1A (in solution): 100% sample matrix, 30 minutes (30'), 37°C, acid treatment (acetic acid used for 10 matrix), 5 minutes (5'), room temperature (RT), pre-incubation / acid neutralization. Sample: ISV-sulfo(:Tris)1:1:1 (1:0,9:0,9:0,1), 1 hour, room temperature (RT); 1 hour on plate, room temperature (RT); 3 washes, Readbuffer 4X - ADA assay in Figure 1B (in solution): Sample 20% matrix, 30 minutes (30'), 37°C, pre-incubation. Sample: ISV--Sulfo 1:1:1, 1 hour, room temperature (RT), 1 hour on plate, room temperature (RT), 3 washes, 2x Readbuffer - Sequential ADA assay in Figure 1C: Capture ISV-biotinylation (Bio), 1 hour, room temperature (RT), 3 washes, sample 20% matrix, 15 minutes (15'), room temperature (RT), on plate; 2 hours, room temperature (RT), 3 washes, detection ALX-0141-sulfo, 1 hour, room temperature (RT), 3 washes, Readbuffer 4X

[0199] Example 5: Predict the sensitivity of ISV to nonspecific protein interference using analytical antibodies. This embodiment describes a bridging / competitive ADA assay using an analytical antibody that can be used to predict the sensitivity of ISV to nonspecific protein interference.

[0200] The ISV to be tested is diluted to a concentration of 10 μg / ml, incubated with 400 ng / ml analytical antibody, purified according to Example 2, and incubated in a 96-well polypropylene plate at 37°C and 600 rpm. Then, the sample (50 μL) is diluted 1 / 3 with a 1:1 mixture (100 μL) of 2 μg / ml biotinylated ISV and 2 μg / ml sulfotagged ISV, and incubated at room temperature (RT) and 600 RPM for 1 hour. The MSD MA® 96-well standard streptavidin plate is blocked at room temperature (RT) for 1 hour with 150 μL / well of Superblock® T20, and then washed three times with PBS / 0.05% Tween20 (=wash buffer). Transfer the sample / 1:1 mixture (biotinylated ISV and sulfotagged ISV) (50.0 μL) from a polypropylene plate to an MSD plate and incubate at room temperature (RT) at 600 rpm for 1 hour. Wash the plate three times, then add 2 x Read Buffer (MSD) (150 μL / well) and read the ECL units (ECLU) using an MSD instrument (Sector Imager 2400 reader).

[0201] This assay was used to test and compare ISVs of SEQ ID NOs. 23–30. The data are shown in Table IX. These data not only demonstrate that the assay described in this embodiment can be used to predict the tendency of ISVs to cause protein interference, but the data produced also support findings from previous embodiments regarding the effect of substitutions within the C-terminal region. As can be seen, the addition of three (and to a lesser extent, one) alanine residues to the C-terminus of fully humanized ISVs neutralized their ability to compete with the binding of the analytical antibody. The alanine-to-proline mutation at position 14 in wild-type ISV mutants clearly increased their ability as competitors in this assay (= making ISV mutants more prone to nonspecific protein interference), whereas mutations at positions 83 and 108 had no apparent effect on the sensitivity of ISVs to nonspecific protein interference.

[0202] [Table 11]

[0203] Example 6: Effect of amino acid addition to the C-terminus of anti-OX40L nanobodies on their OX40L inhibitory efficacy. This embodiment demonstrates that C-terminal extension does not affect the activity or inhibitory effect of the nanobody.

[0204] The in vitro efficacy of the trivalent, bispecific, sequence-optimized anti-OX40L nanobody Nb3.16 (SEQ ID NO: 31) was compared to that of the corresponding nanobody Nb3.17 (SEQ ID NO: 32), which contains one additional Ala at its C-terminus.

[0205] The initial assay (T cell activation assay) was performed as follows: PBMCs were isolated from buffy coat (Red Cross, Ghent, Belgium) derived from healthy donors using Ficoll Paque Plus reagent (GE Healthcare), and washed with RPMI1640 complete medium (RPMI1640 + GlutaMAX + 25 mM HEPES + 10% fetal bovine serum + 1% penicillin / streptomycin; Invitrogen). PBMCs (1 x 10⁻¹⁶ cells) 5 Stimulate the cells (1 / well) with phytohemagglutinin (PHA-L; final concentration 0.6 μg / ml), then 1 x 10 4 Individual hOX40L-expressing CHO cells (irradiated with 3000 RAD using a γ scintillator; UZ Gent, Belgium) and anti-OX40L nanobodies were added to a dilution series in RPMI1640 complete medium and incubated in a CO2 incubator at 37°C for 22 hours. IL2 production by PBMCs was measured by ELISA. Maxisorp plate wells were coated overnight at 4°C with an anti-human IL2 monoclonal antibody (BD Biosciences). After washing and blocking the coated wells, the cell supernatant was... 1 A 2 / 2 dilution was added. Recombinant human IL-2 (BD Biosciences) was used as the standard. 1The study included a 2 / 2 dilution series (starting at 2000 pg / ml). Detection was performed using biotinylated anti-human IL2 monoclonal antibody (BD Biosciences), HRP-labeled streptavidin (Thermo Scientific), and esTMB (SDT Reagents). The reaction was stopped with 1N HCl, and the OD was measured at 450 nm. As expected, the potency of the trivalent, bispecific sequence-optimized nanobody Nb3.17 (IC50 = 0.13 nM, 95% confidence interval (CI) = 0.098~0.17 nM) was comparable to that of Nb3.16 (IC50 = 0.10 nM, 95% confidence interval (CI) = 0.071~0.15 nM).

[0206] In the second ELISA competitive assay, a dilution series of nanobodies (1.5 μM to 0.083 pM) were pre-incubated overnight at room temperature in PBS + 0.1% BSA + 0.01% Tween-20 with 100 ng / ml human OX40 / Fc (R&D Systems) and 10 ng / ml biotinylated human OX40L (R&D Systems; biotinylated in-house as described in Example 1). Next, samples were incubated on Maxisorp plates coated with 10 ug / ml anti-human Fc nanobodies (prepared in-house) and blocked with PBS + 1% BSA + 0.1% Tween-20. Binding human OX40 / Fc was detected using HRP-labeled streptavidin (Thermo Scientific) and sTMB (SDT Reagents). The reaction was stopped with 1N HCl, and the OD was measured at 450 nm. Consistent with cell assays, the potency of the trivalent, bispecific, sequence-optimized nanobody Nb3.17 (IC50 = 0.178 nM, 95% confidence interval (CI) = 0.152 to 0.200 nM) was comparable to that of Nb3.16 (IC50 = 0.179 nM, 95% confidence interval (CI) = 0.149 to 0.215 nM).

[0207] Example 7: Preparation of monoclonal antibody 21-4-3. Two different mouse strains (BALB / c and NMRI-, 3 mice each) were immunized intraperitoneally over a 39-day period using the nanobody construct of Sequence ID No. 98 (equal volume of antigen and Freund's complete or incomplete adjuvant in water-in-oil emulsion) from International Publication No. 2006 / 122825, with additional immunizations until an appropriate antiserum titer was obtained.

[0208] After suffocating stimulated mice with CO2, the spleens were removed under sterile conditions, and a single-cell suspension of pooled spleens was prepared. Spleen cells and myeloma cells were washed several times with DMEM and fused in the presence of 1 ml of 50% (w / v) PEG3350 (spleen cell to SP2 / 0 ratio of 3:1). For fusion, the myeloma cell line SP2 / 0-Ag14 from the German Collection of Microorganisms and Cell Cultures (DSMZ GmbH, Braunschweig) was used. This cell line is a hybrid between BALB / c spleen cells and the myeloma cell line P3x63Ag8. The hybridomas produced in this manner were resuspended in CGM (HAT medium) containing 20% ​​FCS and aminopterin, and seeded (140 μl / well) into eight 96-well flat-bottom tissue culture plates (Corning-Costar) containing 140 μl / well of CGM (20% FCS) along with peritoneal excudate cells as feeder cells. The plates were incubated for 10 days in complete growth medium (CGM) containing DMEM supplemented with 2-mercaptoethanol, L-glutamine, stable glutamine, HT, and non-essential amino acids (at concentrations recommended by the supplier), as well as FCS at different concentrations (10%, 15%, or 20%). During this period, the cells were fed twice with HAT medium. Cell culture supernatants from hybridoma cells typically contain antibodies in concentrations of 1 μg / ml to 20 μg / ml. These were tested by binding ELISA to confirm binding to the nanobody construct of Sequence ID No. 98 (International Publication No. 2006 / 122825).

[0209] Cells from IgG-positive wells were transferred to wells in a 48-well plate and cultured for 2 to 4 days (depending on cell growth characteristics). ELISA for ALX081 and human / cynomolgus monkey IgG was performed to eliminate nonspecific conjugates. Hybridoma cells expressing a conjugate specific to the nanobody construct of Sequence ID No. 98 (International Publication No. 2006 / 122825) were cloned twice using limited dilutions. After fusion and rescreening, seven primary cultures producing antibodies against ALX-081 were identified. All of these primary cultures produced antibodies that did not cross-react with human or cynomolgus monkey IgG. The primary cultures were re-cloned (twice).

[0210] Clone 21-4 (one of the clones that stably produced antibodies against ALX-081 after the second cloning) was named "ABH0015" and deposited with the Belgian Coordinated Collections of Micro-organisms (BCCM) in Ghent, Belgium on June 4, 2012, under accession number LMBP-9680-CB. The mouse monoclonal produced by ABH0015 was named 21-4-3. Isotype determination of 21-4-3 revealed that it consisted of IgG1 heavy chain and κ light chain, which were then sequenced (see SEQ ID NOs. 35 and 36, respectively). 21-4-3 was shown to bind to the C-terminal region of the nanobody construct of SEQ ID NO. 98 in International Publication No. 2006 / 122825 (data not shown).

[0211] Example 8: Binding of 21-4 to ISV predicts that ISV is prone to nonspecific protein interference. In this example, in conjunction with Example 9 below, we demonstrate that the binding of monoclonal 21-4 to an ISV can be used to predict (within the confidence shown in this example) whether a given ISV is prone to nonspecific protein interference (e.g., in an ADA assay).

[0212] This Example 8 specifically shows that a certain modification candidate for a given ISV (e.g., addition of one or more amino acid residues to the C-terminus of the ISV, and / or substitution of one or more amino acid substitutions within the C-terminal region of the ISV) can use 21-4 to predict whether it will result in a decrease in the tendency of the ISV to undergo non-specific protein interference.

[0213] Briefly, a set of 53 different nanobodies and nanobody constructs (see FIGS. 9 and SEQ ID NOs: 38-89) were tested for binding by monoclonal 21-4-3. To confirm whether there is a correlation between the binding by 21-4 and the binding by the purified interfering factor, the same nanobodies and nanobody constructs were also tested for binding by purified preparations of interfering factors obtained from three different human donors (referred to herein as "Donor 8", "Donor 19", and "Donor 30").

[0214] It was demonstrated that the binding of 21-4 to the ISV can actually be used to predict the binding of the interfering factor to the same ISV (within the overall reliability range provided by the data shown here).

[0215] To demonstrate this, as detailed by the experimental data shown below, using a Biacore T100 (of the protocol shown below), the binding of 53 nanobodies or nanobody constructs (listed in FIG. 9; see SEQ ID NOs: 38-89) by 21-4 was measured and compared to the binding of a reference nanobody or construct (similarly listed in FIG. 9) when measured using the same Biacore instrument and the same protocol. The results are shown in Table X below.

[0216]

Table 12

[0217] For each of the 53 tested nanobodies or nanobody constructs, a reference was selected such that, compared to the reference, the tested nanobody or nanobody construct had one or more additional amino acid residues (added specifically to reduce said interference in order to test the effect of such addition on protein interference) at the C-terminus and / or (e.g., as a result of humanization compared to the reference) one or more mutations within the C-terminal region.

[0218] The results were expressed as the rate of decrease of the binding of a given nanobody (measured in RU units) compared to the binding of the reference (also measured in RU units) - for example, if the measured binding level (RU) of the reference nanobody is 276 and the binding level (also RU) of a given nanobody is 9, the decrease in the binding level is at a level of [9RU / 276RU] x 100% = 3%, which means a 97% decrease compared to the reference (100%).

[0219] Similarly, the binding of the purified interfering factor derived from each of three donors to each of the 53 nanobodies or nanobody constructs was measured using the same Biacore instrument and compared to the binding of the same purified interfering factor to the same reference nanobody or construct. The results were likewise expressed as the rate of decrease of the binding of the interfering factor to a given nanobody or nanobody construct compared to the reference.

[0220] For all nanobodies or nanobody constructs with one or more amino acid residues added to the C-terminus compared to the reference, a dramatic reduction in interference factor binding was found. This further supports the idea that adding one or more amino acid residues to the C-terminus (VTVSS) of ISVs can reduce nonspecific protein interference in the ADA assay. It was also found that in most cases, simply making substitutions within the C-terminal region compared to the reference (i.e., not adding one or more amino acid residues to the C-terminus) does not have a similarly dramatic effect on interference factor binding.

[0221] Next, the data was further analyzed to determine whether the decrease in binding by 21-4 compared to the reference correlated in any way with the decrease in binding by each of the three different preparations of the purification interferant compared to the reference. Such a correlation was found.

[0222] For example, 36 of the 54 nanobodies or nanobody constructs tested showed a reduction of over 70% in binding by 21-4 compared to their respective reference sequences (most of these 36 had one or more additional amino acid residues at the C-terminus, and in some cases in combination with substitutions within the C-terminal region). 32 of these 36 also showed a reduction of over 50% in binding by interfering factors compared to the reference (and in many cases, particularly for nanobodies or nanobody constructs with one or more added amino acid residues at the C-terminus, the reduction was much greater than 50% (e.g., over 70% or even over 90%). See data shown in Table X). This demonstrates that in 32 of the 36 cases (i.e., 89%), a reduction of over 70% in binding by 21-4 (compared to the reference = 100%) predicts a reduction of over 50% in binding by interfering factors (compared to the same reference). For clarity, in each case, the reduction was calculated as 100% - [the percentage shown in the table below for the level of reduction achieved in the tested nanobody].

[0223] Similarly, 33 of the 53 nanobodies or nanobody constructs tested were found to show a reduction of over 90% in binding by 21-4 compared to their respective reference sequences. (As before, most of these 33 had one or more additional amino acid residues at the C-terminus, and in some cases in combination with substitutions within the C-terminal region.) 32 of these 33 also showed a reduction of over 50% in binding by interfering factors compared to their respective reference sequences. This demonstrates that in 32 out of 33 cases (i.e., 97%), a reduction of over 90% in binding by 21-4 (compared to the reference) predicts a reduction of over 50% in binding by interfering factors (compared to the same reference).

[0224] It should also be noted that such a reduction of more than 50% in the binding of interfering factors (evidence of which is a reduction of more than 70% in binding by 21-4) means that such interfering factors no longer essentially interfere with the ADA assay of the ISV in question: experimental confirmation using the ADA assay has shown that when the binding by interfering factors is reduced by more than 45%, no significant effect of the presence of the interfering factor on the ADA assay is observed. In this regard, it is obvious to those skilled in the art that this situation is even more pronounced when the binding by interfering factors is reduced by much more than 50% (e.g., more than 70% or even more than 90%), as is observed in some cases (see again the data presented here).

[0225] In fact, the more than 45% reduction in binding by 21-4 was found to correspond to a more than 45% reduction in binding of the interfering factor, which, as mentioned above, means that the interfering factor no longer interferes with the ADA assay.

[0226] Furthermore, based on the data presented here regarding the correlation between binding (reduction) by 21-4 and binding (reduction) by interfering factors, the inventors were able to set an absolute value for binding by 21-4 below which it can be predicted (within the reliability range provided by the data presented in this Example 8) that ISV or ISV-system constructs will not be easily affected by binding by interfering factors in a manner that may interfere with the ADA assay. This value is 500 RU (determined and calculated as shown in Example 9 below).

[0227] Monoclonal 21-4 was purified from the culture medium of the hybridomas obtained in Example 7 as follows: Hybridoma cells secreting monoclonal antibody 21-4-3 were cultured in a spinner flask containing 100 mL or 500 mL of serum-free medium (CD Hybridoma, Gibco supplemented with 8 mM L-glutamine (Invitrogen) and 1 × cholesterol (250 × cholesterol lipid concentrate, Gibco)). The clear supernatant was filtered, and mouse IgG1 was captured on a protein A column (HiTrap MabSelect SuRe, 5 mL, GE Healthcare) at a low flow rate of 2 mL / min. The bound antibody was eluted with 0.1 M citrate buffer (pH 3.0), and the eluted fraction (5 mL) was directly neutralized with 1 mL of 1 M TRIS (pH 9). The purity of the antibody was confirmed by reduced or unreduced SDS-PAGE.

[0228] Purified preparations of interfering factors derived from donors 8 and 19 were obtained essentially by affinity purification from serum samples derived from the donors, as described in Example 2A. Interfering factors derived from donor 30 were obtained essentially from a serum sample of donor 30, as described in Example 2B.

[0229] To determine the binding of 21-4 to each nanobody or nanobody construct, the protocol described in Example 9 was used.

[0230] The binding of interference factors from the three donors to each nanobody or nanobody construct was determined using a Biacore T100, as essentially described in Example 3, with CM5 sensor chips directly immobilized with interference factors from donors 8, 19, and 30, respectively.

[0231] Example 9: A protocol for predicting whether ISVs tend to be subject to nonspecific protein interference (using monoclonal 21-4). Binding measurements were performed using a Biacore T100 with a CM5 T120416 sensor chip (running buffer HBS-EP+, 25°C). Since the surface of directly immobilized mAb21-4-3 was found to be inefficiently unregenerative, 21-4 was captured by immobilized rabbit anti-mouse IgG. The anti-mouse IgG used was a polyclonal rabbit anti-mouse IgG antibody (GE Healthcare; Cat#BR-1008-38; Lot#10056316) that reacts with all IgG subclasses, IgA and IgM. Immobilization of anti-mouse IgG was performed using manual amine coupling (Biacore, amine coupling kit) with 7 minutes of EDC / NHS injection for activation and 7 minutes of 1M ethanolamine HCl (pH 8.5) injection for deactivation. Binding conditions are listed in Table XI. Based on the protein immobilization level and molecular weight (MW), the theoretical R of mAb21-4-3 binding to immobilized anti-mouse IgG was calculated. max The RU was approximately 13,000 RU (when one mAb21-4-3 molecule is bound to one anti-mouse IgG molecule).

[0232] [Table 13]

[0233] The conditions used for the binding experiment (Biacore T100) using the immobilized 21-4 in this manner are shown in Table XII. The surface of anti-mouse IgG was successfully regenerated after capture of mAb21-4-3 and injection of all samples (baseline levels increased slightly after each regeneration).

[0234]

Table 14

[0235] Using the above protocol, the binding data of 21-4 shown in Table X were created. (After adjusting the measured RU value for the molecular weight of the ISV, protein or polypeptide according to the formula ([measured RU] / [molecular weight (MW) of the protein]) x 10 6 and then) considering the absolute value of RU, it was found that the nanobodies or nanobody constructs described in Table X (which have added alanine residues and showed >90% reduction in binding to both 21-4 and the interfering factor) generally showed RU values of 30 RU to 400 RU (the corresponding reference nanobodies or polypeptides - listed in Figure 9 - have RU values greater than 1000, usually greater than 1500 and often greater than 2000).

[0236] Based on this, an (adjusted) RU value of less than 500 in this assay was considered to clearly indicate an ISV (or a protein or polypeptide containing at least one IS described herein) that is not (essentially) bound by the interfering factor in a form that can interfere with the ADA assay.

[0237] The entire contents of all references (including reference documents, issued patents, published patent applications and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference herein, particularly for the teachings specifically referred to herein.

Claims

1. An immunoglobulin single variable domain (ISV) that is a nanobody, or contains a VH sequence (i.e., is not a nanobody), or is derived from a VH sequence, and has the sequence VTVSS(X) ｎ A monovariate immunoglobulin domain (ISV) having a C-terminus; Or a protein or polypeptide containing such an ISV (preferably such a nanobody) at its C-terminus. Here, in the formula: - n = 1, 2, or 3 (preferably 1 or 2), each X = Ala or Gly; or - n = 1, 2 or 3 (preferably 1 or 2), each X = Ala; or - n = 1, 2 or 3 (preferably 1 or 2), each X = Gly; or - n=2 or 3, at least one X=Ala or Gly (the remaining amino acid residue X is independently selected from any naturally occurring amino acid, preferably independently selected from Val, Leu and / or Ile; or - n = 2 or 3, all X except one = Ala or Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acids, preferably independently selected from Val, Leu and / or Ile).

2. - n = 1, 2, or 3 (preferably 1 or 2), each X = Ala or Gly; or - n = 1, 2 or 3 (preferably 1 or 2), each X = Ala; or - n = 1, 2, or 3 (preferably 1 or 2), each X = Gly The immunoglobulin single variable domain (ISV), protein, or polypeptide according to claim 1.

3. The immunoglobulin single variable domain (ISV), protein, or polypeptide according to claim 1, wherein X is not cysteine.

4. An immunoglobulin monovariate domain (ISV) that is a nanobody or contains or derived from a VH sequence and has a C-terminus of the sequence VTVSS(X)n; or a protein or polypeptide having such an ISV (preferably such a nanobody) at its C-terminus. Here, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4 or 5 (preferably 1 or 2, for example 1), and each X is an amino acid residue (preferably naturally occurring) independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), except that X is not cysteine.

5. The immunoglobulin monovariate domain (ISV), protein, or polypeptide according to any one of claims 1 to 4, wherein the (C-terminal) ISV is a nanobody.

6. The binding by 21-4 has an RU value of less than 500, and this RU value is measured using Biacore according to the protocol shown in Example 9, and the measured RU value is expressed as ISV, and the formula for the molecular weight of the protein or polypeptide is ([measured RU] / [molecular weight of protein (MW)] x 10 ６ An immunoglobulin single variable domain (ISV), protein, or polypeptide according to any one of claims 1 to 5, which is adjusted and determined according to ).

7. A method for predicting whether an ISV or a protein or polypeptide containing at least one ISV causes protein interference in an immunoassay such as an ADA assay, (i) A step of contacting the ISV or protein / polypeptide with an antibody obtained from a human subject, which has been selected / isolated based on its ability to recognize the C-terminus of the ISV and / or its ability to bind to the C-terminus of the ISV; and (ii) A step in which the ISV, protein, or polypeptide in the immunoassay is determined to be bound to the antibody. A method comprising performing an immunoassay that includes at least the following.

8. The method according to claim 7, wherein the ISV is a nanobody, or is a VH domain, or contains a VH domain (i.e., other than a nanobody).

9. The method according to claim 7 or 8, wherein the ISV is a nanobody.

10. The method according to claim 7 or 9, wherein the protein or polypeptide has the ISV at its C-terminus.

11. The method according to any one of claims 7 to 10, wherein in step (ii), the fact that the ISV, protein, or polypeptide binds to the antibody means that the ISV, protein, or polypeptide can cause such protein interference (or has a high or increased risk of causing such protein interference).

12. The method according to claim 7 or 11, wherein the antibody is a polyclonal antibody.

13. The method according to any one of claims 7 to 12, wherein the antibody is a polyclonal antibody obtained from a biological sample which is suitable as a starting material for obtaining a polyclonal antibody obtained from a human subject, and the polyclonal antibody is obtained by a method comprising at least one step of (immuno) affinity chromatography in which an affinity matrix holding an ISV or a protein or polypeptide containing at least one ISV is used and / or an ISV or a protein or polypeptide containing at least one ISV is used as an affinity moiety or antigen, and optionally comprising one or more further steps (to be performed before and / or after the affinity step) for isolating and / or purifying the polyclonal antibody from the sample.

14. The method according to claim 13, wherein an ISV or protein or polypeptide containing at least one ISV, which is held on an affinity matrix and / or used as an affinity moiety or antigen, is an ISV or protein or polypeptide containing at least one ISV whose C-terminus is terminated with the amino acid sequence VTVSS (SEQ ID NO: 33), or an ISV or protein or polypeptide containing at least one ISV, which is held on an affinity matrix and / or used as an affinity moiety or antigen, has an ISV or nanobody terminated with the amino acid sequence VTVSS (SEQ ID NO: 33) at its C-terminus.

15. The method according to claim 13 or 14, wherein the ISV or protein or polypeptide containing at least one ISV, which is held on an affinity matrix and / or used as an affinity moiety or antigen, has an ISV or protein or polypeptide containing at least one ISV that terminates at its C-terminus with the amino acid sequence VTVSS (SEQ ID NO: 33) and has a proline residue at position 14; or the method according to claim 13 or 14, wherein the ISV or protein or polypeptide containing at least one ISV, which is held on an affinity matrix and / or used as an affinity moiety or antigen, has an ISV or protein or polypeptide containing at least one ISV that terminates at its C-terminus with the amino acid sequence VTVSS (SEQ ID NO: 33) and has a proline residue at position 14.

16. The method according to any one of claims 13 to 15, wherein the ISV or nanobody held on an affinity matrix and / or used as an affinity moiety or antigen is a sequence-optimized and / or humanized nanobody (e.g., sequence-optimized and / or humanized VHH or camelized VH, e.g., camelized human VH); or the ISV-based drug or nanobody-based drug held on an affinity matrix and / or used as an affinity moiety or antigen has an ISV or nanobody at its C-terminus that is a sequence-optimized and / or humanized nanobody (e.g., sequence-optimized and / or humanized VHH or camelized VH, e.g., camelized human VH).

17. The method according to claim 15 or 16, wherein the ISV or nanobody held on an affinity matrix and / or used as an affinity moiety or antigen is a sequence-optimized and / or humanized nanobody whose C-terminus is terminated with the amino acid sequence VTVSS (SEQ ID NO: 33) and has a proline residue at position 14 introduced as part of the humanization and / or sequence optimization of the corresponding naturally occurring VHH; or the method according to claim 15 or 16, wherein the ISV drug or nanobody drug held on an affinity matrix and / or used as an affinity moiety or antigen is a sequence-optimized and / or humanized nanobody whose C-terminus is terminated with the amino acid sequence VTVSS (SEQ ID NO: 33) and has a proline residue at position 14 introduced as part of the humanization and / or sequence optimization of the corresponding naturally occurring VHH.

18. The method according to any one of claims 7 to 11, wherein the antibody is a monoclonal antibody.

19. The method according to any one of claims 7 to 11 or 18, wherein the antibody is a polyclonal antibody obtained from a human subject and starting from a biological sample suitable as a starting material for obtaining a monoclonal, and the antibody is obtained by a method comprising at least one screening or selection step used to screen and select a monoclonal antibody that binds to the ISV, nanobody, ISV-based drug or nanobody-based drug (particularly to the C-terminus thereof), and optionally one or more further steps (performed before and / or after the screening and / or selection step) for isolating and / or purifying the monoclonal antibody from the sample.

20. The method according to claim 19, wherein the ISV or nanobody used in the screening or selection step terminates at its C-terminus with the amino acid sequence VTVSS (SEQ ID NO: 33), or the ISV-based drug or nanobody-based drug used in the screening or selection step has an ISV or nanobody at its C-terminus that terminates at the amino acid sequence VTVSS (SEQ ID NO: 33).

21. The method according to claim 19 or 20, wherein the ISV or nanobody used in the screening or selection step terminates at its C-terminus with the amino acid sequence VTVSS (SEQ ID NO: 33) and has a proline residue at position 14, or the ISV-based drug or nanobody-based drug used in the screening or selection step has an ISV or nanobody at its C-terminus that terminates at its C-terminus with the amino acid sequence VTVSS (SEQ ID NO: 33) and has a proline residue at position 14.

22. The method according to claim 19, 20, or 21, wherein the ISV or nanobody used in the screening or selection step is a sequence-optimized and / or humanized nanobody (e.g., sequence-optimized and / or humanized VHH or camelized VH, e.g., camelized human VH); or the ISV-based drug or nanobody-based drug used in the screening or selection step has an ISV or nanobody at its C-terminus that is a sequence-optimized and / or humanized nanobody (e.g., sequence-optimized and / or humanized VHH or camelized VH, e.g., camelized human VH).

23. The method according to any one of claims 21 to 22, wherein the ISV or nanobody used in the screening or selection step is a sequence-optimized and / or humanized nanobody whose C-terminus terminates with the amino acid sequence VTVSS (SEQ ID NO: 33) and has a proline residue at position 14 that has been introduced as part of the humanization and / or sequence optimization of the corresponding naturally occurring VHH; or the method according to any one of claims 21 to 22, wherein the ISV drug or nanobody drug used in the screening or selection step is a sequence-optimized and / or humanized nanobody whose C-terminus terminates with the amino acid sequence VTVSS (SEQ ID NO: 33) and has a proline residue at position 14 that has been introduced as part of the humanization and / or sequence optimization of the corresponding naturally occurring VHH.

24. A method that can be used to predict whether an ISV or a protein or polypeptide containing at least one ISV will cause protein interference in an immunoassay (or will have a high or increased tendency to cause protein interference) (and / or whether interfering factors present in human blood or serum will bind to the ISV or the protein or polypeptide containing at least one ISV), (i) A step of contacting the monoclonal antibody 21-4 (i.e., used as an "analytical antibody") with the ISV or nanobody (or an ISV-based drug or a nanobody-based drug); and (ii) A step in determining whether the monoclonal antibody 21-4 binds to the ISV or nanobody (or ISV-based drug or nanobody-based drug) in the immunoassay. A method comprising performing an immunoassay that includes at least the following.

25. The method according to claim 24, wherein the ISV is a nanobody, or is a VH domain, or contains a VH domain (other than a nanobody).

26. The method according to claim 24 or 25, wherein the ISV is a nanobody.

27. The method according to any one of claims 24 to 26, wherein the protein or polypeptide has the ISV at its C-terminus.

28. The method according to any one of claims 24 to 27, as performed according to the protocol shown in Example 9.

29. A pharmaceutical composition comprising an ISV, protein, or polypeptide according to any one of claims 1 to 6, and at least one suitable carrier, diluent, or excipient.

30. - The composition, ISV, protein, or polypeptide is intended to treat a chronic disease in humans, and / or - The ISV, protein, polypeptide is intended to be present in the circulatory system of the subject to which it is administered (i.e., at a therapeutically active dose) for a period of at least one week, preferably at least two weeks, for example, at least one month; and / or - The ISV, protein, polypeptide has a half-life in a human subject of at least 3 days, for example, at least 1 week, up to 10 days or more (preferably expressed as t1 / 2-β); and / or - The pharmaceutical composition according to claim 29, wherein the ISV, protein, polypeptide, or pharmaceutical composition is intended to be administered to a human being over a period of at least three days, for example, at least one week, for example, at least two weeks, or at least one month, or a longer period (i.e., at least three months, at least six months, or at least one year), or further administered in two or more doses chronically.

31. An ISV, protein, or polypeptide according to any one of claims 1 to 6, for use in the treatment of a disease in humans.

32. - The ISV, protein, or polypeptide is intended to treat a chronic disease in humans, and / or - The ISV, protein, polypeptide is intended to be present in the circulatory system of the subject to which it is administered (i.e., at a therapeutically active dose) for a period of at least one week, preferably at least two weeks, for example, at least one month; and / or - The ISV, protein, polypeptide has a half-life in a human subject of at least 3 days, for example, at least 1 week, up to 10 days or more (preferably expressed as t1 / 2-β); and / or - The ISV, protein, or polypeptide according to any one of claims 1 to 6 and / or 31, which is intended to be administered to a human being over a period of at least three days, for example, at least one week, for example, at least two weeks, or at least one month, or a longer period (i.e., at least three months, at least six months, or at least one year), or further administered in two or more chronic doses.

33. The use of an ISV or protein or polypeptide according to any one of claims 1 to 6, 31 or 32 in the preparation of a pharmaceutical composition, particularly in the preparation of the pharmaceutical composition according to claim 29 or 30.

34. - The ISV, protein, or polypeptide is intended to treat a chronic disease in humans, and / or - The ISV, protein, polypeptide is intended to be present in the circulatory system of the subject to which it is administered (i.e., at a therapeutically active dose) for a period of at least one week, preferably at least two weeks, for example, at least one month; and / or - The ISV, protein, polypeptide has a half-life in a human subject of at least 3 days, for example, at least 1 week, up to 10 days or more (preferably expressed as t1 / 2-β); and / or - The use according to claim 33, wherein the ISV, protein, or polypeptide is intended to be administered to a human being over a period of at least three days, for example, at least one week, for example, at least two weeks, or at least one month, or a longer period (i.e., at least three months, at least six months, or at least one year), or further administered in two or more chronic doses.

35. A treatment method comprising administering an ISV or protein or polypeptide according to any of claims 1 to 6, 31 or 32, or a pharmaceutical composition according to claim 29 or 30, to a human subject (for example, a patient requiring such treatment).

36. - The pharmaceutical composition, ISV, protein, or polypeptide is intended to treat a chronic disease in humans, and / or - The ISV, protein, polypeptide is intended to be present in the circulatory system of the subject to which it is administered (i.e., at a therapeutically active dose) for a period of at least one week, preferably at least two weeks, for example, at least one month; and / or - The ISV, protein, polypeptide has a half-life in a human subject of at least 3 days, for example, at least 1 week, up to 10 days or more (preferably expressed as t1 / 2-β); and / or - The treatment method according to claim 35, wherein the pharmaceutical composition, ISV, protein, or polypeptide is intended to be administered to a human being over a period of at least three days, for example, at least one week, for example, at least two weeks, or at least one month, or a longer period (i.e., at least three months, at least six months, or at least one year), or further administered in two or more chronic doses.

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