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

Abstract

The present invention provides, and in certain specific but non-limiting aspects relates to: methods for modifying and / or improving ISV's so as to eliminate or reduce their propensity to generate said protein interference or said signal; - modifications that can be introduced into ISV's that eliminate or reduce their propensity to generate said protein interference or said signal; - ISV's that are specifically selected (e.g., using the assays described herein) so as to not have or have a lower / reduced propensity to generate said protein interference or said signal; - ISV's that are modified and / or improved so as to not have or have a lower / reduced propensity to generate said protein interference or said signal.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201280030902.9, filed on June 25, 2012, entitled "Technology for predicting, detecting and reducing nonspecific protein interference in assays involving immunoglobulin single variable domains". Technical Field

[0002] This invention relates to the field of immunoglobulin single variable domain. Background Technology

[0003] Immunoglobulin single variable domain, or "ISV," is generally defined in this paper as an amino acid sequence whose...

[0004] - Contains immunoglobulin folds or is capable of forming immunoglobulin folds (i.e., through folding) under suitable conditions (such as physiological conditions), thereby forming immunoglobulin variable domains (e.g., VH, VL, or VHH domains).

[0005] and

[0006] - Formation (or the ability to form under such suitable conditions) of immunoglobulin variable domains containing functional antigen-binding sites (in the sense that they do not need to interact with another immunoglobulin variable domain (such as VH-VL interaction) to form a functional antigen-binding site).

[0007] Some examples of immunoglobulin single variable domains known in the art are VHHs and / or (other) nanobodies, dAbs, and (single) domain antibodies. Among these, various nanobodies are in Phase I and Phase II clinical trials as of the date of this application. This makes it important to obtain reliable assays for analyzing biological samples from individuals treated with ISVs (such as clinical trial subjects, and patients treated with ISVs post-marketing).

[0008] This is important not only for management purposes, but also for treating patients with biological drugs, as clinicians who prescribe such treatments also want reliable assays to monitor all aspects of the treatment.

[0009] For example, in the clinical development of biopharmaceutical molecules, it is important to assess their immunogenic potential, and specifically the extent to which they can elicit so-called “anti-drug antibodies” or “ADAs”. This can be measured using so-called “anti-drug antibodies” or “ADAs” (immunoassays) (see, for example, 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). The ADA assays and the methods used to perform them are standard common sense in the pharmaceutical field and are routinely used in the clinical development of biopharmaceutical products (and are also required by various regulatory agencies worldwide).

[0010] For example, as described on pages 3 and 4 of Mire-Sluis's article, and as illustrated, for example, in the figures of Peng's article, many different forms of ADA assays are known, such as "ELISA-bridging form," "ELISA-direct form," "indirect form," radioimmunoprecipitation assay (RIP), "surface plasmon resonance," and "electrochemiluminescence-bridging form." Other forms of ADA immunoassays are readily apparent to those skilled in the art.

[0011] Technicians are also familiar with many different commercially available technology platforms that have proven suitable for establishing and performing ADA assays. These include, but are not limited to, the MSD platform (Mesoscale), Gyrolab (Gyros), and the Octet platform (Fortebio).

[0012] Some non-limiting examples of ADA measurement methods are also included. Figure 1A-1C It is shown schematically in the middle.

[0013] Generally, it should be noted that in these ADA assays used to detect or measure ADAs against ISVs, the ISV is used as an "analytical reagent" (i.e., a compound used to detect the presence of any ADAs in the test sample), and the ADAs are "antigens" (i.e., compounds to be detected in the test sample). Therefore, in these assays, the ISV is usually / frequently bound to a carrier (such as an ELISA plate), while the ADAs (if present) are present in the sample used for the assay.

[0014] To better understand the invention described herein, it should also be noted—by contrast—that in the methods used herein for predicting 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, as further described herein) is used as an “analytical reagent” (i.e., as a tool to detect whether a given ISV binds; and thereby to detect whether it has a higher or increased risk of causing protein interference). Therefore, in this method according to the invention, the antibody used as the analytical reagent (also referred to herein as an “analytical antibody”) is typically bound to a carrier (i.e., bound to an ELISA plate), and the ISV is (present) in the sample to be tested. However, it should generally be noted that the invention is not limited to assays in which the “analytical antibody” is bound to a carrier. For example, in alternative methods of performing the assay according to the invention (as shown, for example, in Figure 1 and described in the examples), the analytical antibody is alternatively used as a bridging agent, and thus in solution rather than bound to the plate (although it is indirectly bound to the plate via an ISV coated on the plate). However, in the specific bridging assay described in the embodiments (which is a competitive assay), the analytical antibody is still used as the analytical reagent (i.e., to determine whether the target ISV binds; and thus to determine whether it has a higher or increased risk of producing protein interference). It is also envisioned that, based on the further disclosure herein, those skilled in the art can design other assays in which the analytical antibody can be used as the analytical reagent to determine whether a given ISV can bind; and thus to determine whether it has a higher or increased risk of producing protein interference.

[0015] As a result of studies on single-chain Fv's or "ScFv's" (which are constructs containing a single variable domain of immunoglobulin and, similar to ISV's, do not associate with a constant domain), it has been described in the art that the C-terminus of the variable domain of immunoglobulin forms a hydrophobic patch, which is embedded in the interface between the variable and constant domains in the antibody, but becomes solvent-exposed when the variable domain does not associate with the constant domain (Nieba et al., Protein Engineering, 10, 435-444 (1997)). It has also been described that the exposed C-terminus can form a B-cell epitope that can generate anti-drug antibodies and / or interact with (novel and / or pre-existing) anti-drug antibodies (WO 11 / 07586), the presence of which can then be determined using the aforementioned ADA assay. For this reason, it has been proposed to mutate some amino acid residues of the portion forming the C-terminus of the variable domain to reduce the hydrophobicity and / or remove the epitope. For example, Nieba et al. proposed mutating positions 11, 14, 41, 84, 87 and / or 89 of the VH region (according to Kabat numbering), while in WO11 / 07586, positions 99, 101 and / or 148 of the VL domain (AHo numbering) or positions 12, 97, 98, 99, 103 and / or 144 of the VH domain (also AHo numbering - these positions correspond to positions 11, 83, 84, 85, 89 and 103 according to Kabat).

[0016] However, none of these references are aware that certain proteins present in the blood or serum of the subjects can interfere with the ADA assay involving ISVs, and therefore, these references do not address (or provide a protocol for) the question of how to avoid non-specific protein interference in the ADA assay so that the ADA assay can be used to determine the true presence / degree of anti-drug antibodies (new or existing) in the test sample. Summary of the Invention

[0017] In contrast, the present invention provides methods and assays that readily enable those skilled in the art to predict whether an immunoglobulin single variable domain is prone to nonspecific protein interference in ADA assays. The methods and assays described herein also allow those skilled in the art to readily test (proposed) modifications to the variable domain, thereby predicting whether any such (proposed) modification reduces or substantially eliminates such protein interference, should a variable domain be found to be prone to or at risk of interference in ADA assays.

[0018] This invention also describes numerous modifications that can be made to the variable domain to reduce or substantially avoid such protein interference. According to a non-limiting aspect, such modifications include adding a limited amount (as further described herein) of an amino acid residue (as further described herein) to the C-terminus of the variable domain. Surprisingly, it has been found that for many different variable domains or constructs based thereon, even adding a single amino acid residue to the C-terminus (such as a single alanine residue) can adequately or even substantially completely eliminate the protein interference problem in ADA assays, even if the addition of one such amino acid is insufficient on its own to “cover” or “embed” the hydrophobic sheet present at the C-terminus of ISVs according to Nieba et al. Similarly, but not intended to limit the invention in any way or to any mechanism or interpretation, it is also considered that the addition of one such amino acid is insufficient to “cover” or “embed” any B-cell epitopes that may be present at the C-terminus of the variable domain according to WO 11 / 07586. It should also be noted that, although according to this specific aspect of the invention, the addition of a limited amount of amino acids or even a single amino acid (i.e., without any substitution within the C-terminal region itself, as proposed by Nieba et al. and WO 11 / 07586) to the C-terminus of the variable domain can—and in many cases—significantly reduce or even substantially eliminate the problem of nonspecific protein interference, such additions at the C-terminus in combination with C-terminal mutations are also within the scope of this aspect of the invention. However, in this aspect, it should also be noted that the invention is not specifically limited to the underlying principles behind these mutations. For example, it is well known in the art to mutate amino acid residues within the C-terminus (including those positions clearly mentioned by Nieba et al. and in WO 11 / 07586) thereby affecting the variable domain (including, but not limited to, V... HH Humanize the structural domain or thereby modify V H The domain is "camel-sourced" (for example, refer to WO 08 / 020079 and some other applications of Ablynx NV mentioned in this paper).

[0019] The methods, assays, and modifications taught herein are intended to be applicable to any variable domain that is not linked to a constant domain (or otherwise associated with a constant domain (or other groups or peptide segments that function to “mask,” cover, or “embed” the C-terminal region of the variable domain) and, more generally, to any variable domain having a solvent-exposed C-terminal region. However, according to a preferred, but non-limiting, aspect of the invention, the methods, assays, and modifications can be specifically applied to heavy-chain variable domains (V... H (structural domain), and according to a specific aspect of the invention, applied to V HHStructural domain.

[0020] It is also believed that the methods, assays, and modifications described herein can be suitably applied to protein constructs comprising one or more variable domains, and specifically to constructs in which the variable domains form the C-terminal portion of the construct, or, in the case of the methods and assays described herein, specifically to constructs in which the C-terminal region of the variable domain is solvent-exposed. Furthermore, according to a preferred but non-limiting aspect of the invention, the methods, assays, and modifications are applied to constructs in which V H Structural domain (and specifically, V) HH The structural domains that form the C-terminal portion of the construct, or in the case of the methods and assays of the present invention, are solvent-exposed.

[0021] Some non-limiting examples of the constructs are multivalent, multispecific (e.g., bispecific), or multi-complementary (e.g., bicomplementary) constructs that contain two or more directly connected ISVs or connected via one or more suitable connectors (also referred to as V, depending on a particular aspect). H or V HH The ISVs (interconnected by structural domains) form the C-end portion of the construct. For example, but not limited to, the construct may entirely comprise V-type structures that are also directly connected or connected via one or more suitable connectors. H Structural domains, specifically nanobodies (i.e., V... HH Structural domain, humanized V HH V with domain or camel-derived form H (Domains). For some non-limiting examples of the general teachings on these constructs and how to construct them (specifically based on nanobodies), see, for example, the review articles Conrath et al., JBC 276, 10(9), 7346 (2001) and Muyldermans. Reviews in Mol. Biotechnol., 74:27 (2001).

[0022] However, it is also thought that the invention can be applied, for example, to other constructs having solvent-exposed variable domains and specifically to constructs having variable domains at their C-terminus, such as, for example, single-chain Fv's, specifically ScFv's having their heavy-chain variable domains at their C-terminus.

[0023] In the specification and claims of this invention, terms such as “ISV,” “analytical reagent,” and “protein interference” have the meanings further defined herein.

[0024] Specifically, the ISV described herein may be a nanobody, or (other) ISV (i.e., different from a nanobody) that has or contains a VH domain; and preferably a nanobody.

[0025] Furthermore, any protein or polypeptide containing an ISV (such as an ISV-based drug) preferably has such an ISV (or at least one) at its C-terminus. Moreover, the ISV may specifically be a nanobody, or (other) ISV (i.e., different from a nanobody) that is or contains a VH domain; and preferably a nanobody.

[0026] The invention described herein is specifically intended and suitable for application to ISVs comprising, based on, and / or derived from heavy chain variable domains, such as VH domains (including human VH domains) and nanobodies such as VHH domains (including humanized and sequence-optimized VHH domains) or camel-derived VH domains. These can be synthetic (e.g., obtained from synthetic libraries and / or based on fixed framework regions), semi-synthetic (e.g., humanized, camel-derived, or sequence-optimized, or obtained from natural VH or VHH domains via affinity maturation or CDR transplantation), or entirely naturally occurring VH or VHH domains. Therefore, the invention relates to ISVs that are VH or VHH domains, based on VH or VHH domains, and / or derived from VH or VHH domains, as further described herein.

[0027] In developing this invention, it has been found that protein interference may be present in some assays (e.g., in ADA immunoassays) used to analyze biological samples (such as blood samples, including whole blood, serum and plasma, tears, bronchoalveolar fluid / BALF, cerebrospinal fluid, or other samples of biological fluids), and such protein interference may produce nonspecific signals in some such assays and / or in some such samples. It has been found that such protein interference is present not only in samples from subjects who have received ISVs (and specifically, with nanobodies; or with proteins, peptides, or other biological agents containing at least one of said ISVs or nanobodies) and / or subjects who have been administered them (e.g., patients or clinical trial subjects), but also in subjects who have never received ISVs (illustrating that the interference may be due to nonspecific protein-protein interactions with pre-existing proteins rather than with any newly emerging ADAs).

[0028] Although such protein interference and / or the signal has been found in these assays not to be associated with any change or reduction in the pharmacological properties of ISVs (such as pharmacokinetic / PK or pharmacodynamic / PD properties), there is a need for techniques that can predict, detect, reduce, and / or (if possible) avoid such nonspecific protein interference. This is the general objective of the present invention.

[0029] Specifically, the present invention provides, and relates to in certain specific but non-limiting aspects:

[0030] - An assay that can be used to predict whether a given ISV will suffer from such protein interference and / or produce such (non-specific) signals in such an assay (e.g., in the ADA immunoassay). Such a predictive assay can, for example, be used to test whether a given ISV has a tendency to produce such protein interference and / or such signals; to select ISVs that are less likely to produce or produce such protein interference or such signals; as such an assay or test that can be used to test whether certain modifications to an ISV (completely or partially) reduce its tendency to produce such interference or such signals; and / or as such an assay or test that can be used to guide the modification or improvement of an ISV to reduce its tendency to produce such protein interference or signals.

[0031] - Used to modify and / or improve ISVs to eliminate or reduce their tendency to produce such protein interference or such signals;

[0032] - Modifications that can be introduced into ISVs to eliminate or reduce their tendency to produce such protein interference or such signals;

[0033] - ISVs can be specifically selected (e.g., using the assays described herein) so as to have no or a lower / reduced tendency to produce such protein interference or such signals;

[0034] - Modifications and / or improvements to ISVs that do not have or have a lower / reduced tendency to produce such protein interference or such signals.

[0035] For example, in a first non-limiting aspect, the present invention relates to a method for predicting whether a given ISV or nanobody (or ISV-based or nanobody-based drug) will produce (or have a higher or increased risk of producing) protein interference in an immunoassay (i.e., after its administration to a subject, a sample of the biofluid is obtained from the subject, and the sample is subjected to an immunoassay as further described herein), the method comprising performing an immunoassay that includes at least the following steps:

[0036] (i) contacting the ISV or nanobody (or ISV-based or nanobody-based drug) with an antibody obtained from a human subject and selected based on its ability to recognize and / or bind to the C-terminus of the ISV or nanobody, generating and / or separating (“analytical antibody”); and (ii) determining whether the ISV or nanobody (or ISV-based or nanobody-based drug) binds to the antibody in the immunoassay.

[0037] In this approach, when the ISV, nanobody, ISV-based drug, or nanobody-based drug binds to the analytical antibody, it is expected that the ISV, nanobody, ISV-based drug, or nanobody-based drug will produce (or have a higher or increased risk of producing) such protein interference (as further described herein). Based on this, for example, the ISV, nanobody, ISV-based drug, or nanobody-based drug can be modified or improved to reduce or eliminate its tendency to produce such protein interference (which can also be determined using the assay described above), and some strategies for modifying the ISV, nanobody, ISV-based drug, or nanobody-based drug as described herein (e.g., including linking a small number of amino acid residues to the C-terminus and / or introducing one or more specific amino acid substitutions) can be used.

[0038] Therefore, in general, this invention 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 produce protein interference and / or as tools to improve ISVs, thereby reducing or avoiding their tendency to produce protein interference. In doing so, this invention also provides those skilled in the art with means of selecting ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs based on their lower or reduced ability (or lack thereof) to produce protein interference. Therefore, this invention provides those skilled in the art with important assays and tools that can be used in optimizing and developing ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs.

[0039] Furthermore, as further described herein, the present invention teaches those skilled in the art numerous methods in which ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs can be modified or improved to reduce or avoid their tendency to produce protein interference. Therefore, the present invention generally also yields modified and / or improved ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs with reduced, lower, or no tendency to produce protein interference.

[0040] As further described herein, the present invention can be specifically used to predict whether a given ISV or nanobody (or ISV-based or nanobody-based drug) will cause protein interference in an immunoassay, and more specifically in an ADA assay (as further described herein). The ADA assay can generally be, for example, an ADA assay for detecting or measuring ADAs against ISVs, and can specifically be an ADA assay for detecting or measuring ADAs against the ISVs used in steps (i) and (ii) above.

[0041] Furthermore, as mentioned herein, the ISV described herein can specifically be a nanobody or (other) ISV (i.e., different from a nanobody) that has or contains a VH domain; and preferably is a nanobody.

[0042] Furthermore, any protein or polypeptide containing an ISV (such as an ISV-based drug) preferably has such an ISV (or at least one) at its C-terminus. Moreover, the ISV may specifically be a nanobody or (other) ISV (i.e., different from a nanobody) that is or contains a VH domain; preferably a nanobody.

[0043] The samples tested in the immunoassay or ADA assay are also referred to herein as “test samples” or “assay samples”. To avoid confusion, terms such as “test samples” or “assay samples” should not be confused with biological samples used herein as raw materials for obtaining the (polyclonal or monoclonal) “analytical antibodies” used in this invention.

[0044] In a particularly preferred but non-limiting aspect, the invention can be used to predict whether a given ISV or nanobody (or ISV-based or nanobody-based drug) will produce protein interference (as further described herein) in an immunoassay involving the use of said ISV (and specifically, in an ADA assay). Furthermore, said ADA assay can generally be, for example, an ADA assay for detecting or measuring ADA's against ISVs, and can specifically be an ADA assay for detecting or measuring ADA's against the ISVs used in steps (i) and (ii) above.

[0045] In even more specific but non-limiting aspects, the invention can be used to predict whether a given ISV or nanobody (or ISV-based or nanobody-based drug) will produce protein interference in an immunoassay (and specifically, in an ADA assay) (as further described herein), said immunoassay being intended to determine or measure whether said sample contains any ADAs against the ISV. Furthermore, for example, said immunoassay can be a known type of ADA assay (with reference, for example, to prior art concerning ADA assays cited herein) (performed to determine or measure whether any ADAs against said ISV are present in a “test sample”), said test sample being a sample of a biological fluid obtained from a subject who has been administered said ISV (as further described herein) (as described herein).

[0046] As further described herein, in all these aspects (and other aspects of the invention described herein), the invention can also be used to select ISVs that are less likely to produce or produce such protein interference in the immunoassay or ADA assay; as such an assay or test, it can be used to test whether certain modifications of an ISV (completely or partially) reduce its tendency to produce such interference in the immunoassay or ADA assay; and / or as such an assay or test, it can be used to guide the modification or improvement of ISVs to reduce their tendency to produce such protein interference in the immunoassay or ADA assay.

[0047] Other aspects, embodiments, advantages, and applications of the invention will become clear from the further description herein.

[0048] In the specification of this invention, whenever the term "ISV" is used, it should be understood as:

[0049] - The ISV is preferably a nanobody, wherein the term "nanobody" is generally as defined in WO08 / 020079 or WO09 / 138519, and therefore in a specific context generally refers to a VHH, a humanized VHH, or a camel-derived VH (such as a camel-derived human VH), or generally refers to a 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 are therefore also referred to as... and / or );

[0050] The term “ISV” in its broadest sense also includes “ISV-based biopharmaceuticals,” and when the ISV is a nanobody, it includes “nanobody-based biopharmaceuticals.” “ISV-based biopharmaceuticals” are defined herein as proteins, peptides, or other biopharmaceuticals comprising at least one (e.g., one, two, or three) ISVs or substantially composed of them. Similarly, “nanobody-based biopharmaceuticals” are defined as proteins, peptides, or other biopharmaceuticals comprising at least one (e.g., one, two, or three) nanobodies or substantially composed of them. Regarding the term “ISV,” whenever the term “ISV-based biopharmaceuticals” is used, it should be understood that the ISV-based biopharmaceutical is preferably a nanobody-based biopharmaceutical. In the context of this invention, "ISV-based biologics" and "nanobody-based biologics" can be, for example, monovalent, bivalent (or multivalent), bispecific (or multispecific), and bicomplementary (or multicomplementary) ISV constructs or nanobody constructs, respectively. Furthermore, in addition to one or more (e.g., one, two, or three) ISVs or nanobodies, any ISV-based biologic or nanobody-based biologic optionally includes one or more (e.g., one or two) other therapeutic structural portions and / or one or more (e.g., one or two) other structural portions that affect the pharmacokinetic or pharmacodynamic properties (e.g., their half-life) of the ISV-based biologic or nanobody-based biologic. Suitable examples of these other therapeutic agents or other structural portions will be apparent to those skilled in the art, and generally may include, for example, any therapeutically active protein, peptide, or other binding domain or binding unit, and, for example, modifications such as those in WO Those described on pages 149-152 of 09 / 138159. ISV-based or nanobody-based bioproducts are preferably therapeutic agents or intended for use as therapeutic agents (including prevention and diagnostics), and for this purpose preferably contain at least one ISV targeting a therapeutically relevant target (e.g., RANK-L, vWF, IgE, RSV, CXCR4, IL-23, or other interleukins, etc.). Some specific, but not limiting, examples of such ISV-based or nanobody-based bioproducts are, for example, reference to various applications of Ablynx NV (e.g., but not limited to WO 2004 / 062551, WO 2006 / 122825, WO 2008 / 020079, and WO 2009 / 068627), and, for example (but not limited to) applications such as WO 06 / 038027, WO 06 / 059108, WO 07 / 063308, WO 07 / 063311, WO 07 / 066016 and WO 07 / 085814.Furthermore, in this application specification, unless otherwise clearly stated, all terms mentioned herein have the meanings given in WO 09 / 138519 (or in the prior art referenced in WO 09 / 138519) or WO 08 / 020079 (or in the prior art referenced in WO 08 / 020079). Additionally, if a method or technique is not specifically described herein, it may be performed as described in WO 09 / 138519 (or in the prior art referenced in WO 09 / 138519) or WO 08 / 020079 (or in the prior art referenced in WO 08 / 020079).

[0051] Specifically, the following terms have the same meanings as those given on pages 62-75 of WO 09 / 138519, and / or may be determined in the manner described on that page, if applicable: “agonist”, “antagonist”, “reverse agonist”, “nonpolar, uncharged amino acid residue”, “polar, uncharged amino acid residue”, “polar, charged amino acid residue”, “sequence identity”, “exactly identical” and “amino acid difference” (when referring to a comparison of two amino acid sequences), “(in) substantially separate (form)”, “domain”, “binding domain”, “antigenic determinant”, “epitope”, “target” or “target” (antigen)”, “specificity” and “half-life”. Furthermore, the terms “regulation” and “to be regulated”, “interacting site”, “specific to”, “cross-block”, “cross-blocked” and “cross-blocking”, and “substantially pH-independent” are as defined on pages 74-79 of the applicant’s WO 10 / 130832 (and / or may be as described on that page). Additionally, when referring to the constructs, compounds, proteins, or peptides of the present invention, terms such as “monovalent”, “divalent” (or “multivalent”), “bispecific” (or “multispecific”), and “dual complementary” (or “multiple complementary”) may have the meanings described in WO 09 / 138.519, WO 10 / 130832, or WO 08 / 020079.

[0052] As used herein, the term "half-life" in relation to ISV, nanobody, ISV-based biologic, nanobody-based biologic, or any other amino acid sequence, compound, or peptide may generally be defined as described in paragraph o) on page 57 of WO 08 / 020079, and as mentioned herein, refers to the time required for a 50% reduction in serum concentration of the amino acid sequence, compound, or peptide in vivo, for example, due to degradation of the sequence or compound by natural mechanisms and / or clearance or chelation of the sequence or compound. The in vivo half-life of the amino acid sequence, compound, or peptide of the present invention may be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and may generally be described, for example, as described in paragraph o) on page 57 of WO 08 / 020079. Also as mentioned in paragraph o) on page 57 of WO 08 / 020079, half-life may be expressed using parameters such as t1 / 2-α, t1 / 2-β, and area under the curve (AUC). In this regard, it should be noted that the term “half-life” as used herein specifically refers to t1 / 2-β or terminal half-life (where t1 / 2-α and / or AUC may be disregarded). See, for example, the experimental section below and standard manuals such as Kenneth, A et al.: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Also see “Pharmacokinetics”, M. Gibaldi & D. Perron, Marcel Dekker, 2nd ed. (1982). Similarly, the term “increased half-life” or “increased half-life” is also defined as in paragraph o) on page 57 of WO 08 / 020079 and specifically refers to an increase in t1 / 2-β, with or without an increase in t1 / 2-α and / or AUC, or both.

[0053] When a term is not specifically defined herein, it shall have the common meaning known to those skilled in the art. For example, reference standard manuals include Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd edition), Volumes 1-3, Cold Spring Harbor Laboratory (1989); F. Ausubel et al., eds., "Current protocols in molecular biology", Green Publishing & Wiley Interscience, New York (1987); Lewin, "Genes II", John Wiley & Sons, New York, NY (1985); Old et al., "Principles of Gene Manipulation: An Introduction to Genetic Engineering", 2nd edition, University of California Press, Berkeley, CA (1981); Roitt et al., "Immunology" (6th edition), Mosby / Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential Immunology (Roitt's Basic Immunology), 10th edition. Blackwell Publishing, UK (2001); and Janeway et al., "Immunobiology" (6th edition), Garland Science Publishing / Churchill Livingstone, New York (2005), and refer to the general background techniques cited herein.

[0054] Furthermore, in this paper, the amino acid residues of nanobodies are numbered according to the general numbering method for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from camels in Riechmann and Muyldermans, J. Immunol. Methods, June 23, 2000; 240(1-2):185-195; or as mentioned in this paper. According to this numbering scheme, FR1 of the nanobody contains amino acid residues at positions 1-30, CDR1 of the nanobody contains amino acid residues at positions 31-35, FR2 of the nanobody contains amino acid residues at positions 36-49, CDR2 of the nanobody contains amino acid residues at positions 50-65, FR3 of the nanobody contains amino acid residues at positions 66-94, CDR3 of the nanobody contains amino acid residues at positions 95-102, and FR4 of the nanobody contains amino acid residues at positions 103-113. [In this respect, it should be noted—as is well known in the art with respect to VH domains and VHH domains—that the total number of amino acid residues in each CDR's can vary and may not correspond to the total number of amino acid residues specified by the Kabat numbering scheme (i.e., one or more positions according to the Kabat numbering scheme may not be filled by the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering scheme). This generally means that the Kabat numbering scheme may or may not correspond to the actual number of amino acid residues in the actual sequence.] However, it is generally believed that, according to the Kabat numbering system and without considering the number of amino acid residues in CDRs, position 1 according to the Kabat numbering system corresponds to the beginning of FR1 and vice versa, position 36 according to the Kabat numbering system corresponds to the beginning of FR2 and vice versa, position 66 according to the Kabat numbering system corresponds to the beginning of FR3 and vice versa, and position 103 according to the Kabat numbering system corresponds to the beginning of FR4 and vice versa.

[0055] An alternative method for numbering the amino acid residues of the VH domain (which can also be applied in a manner similar to that used for VHH domains from camels and nanobodies) is the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in the specification, aspects and figures of this invention, the numbering method according to Kabat applied to the VHH domain by Riechmann and Muyldermans will be followed unless otherwise indicated.

[0056] It should also be noted that the accompanying drawings, any sequence listings and experimental sections / examples are for further illustrative purposes only and should not be construed or interpreted in any way as limiting the scope of the invention and / or the appended claims, unless otherwise clearly stated herein.

[0057] It should also be noted that the present invention is not specifically limited to any cause, explanation, hypothesis, or mechanism of protein interference (and / or signals generated in immunoassays) observed in the present invention or that can be reduced according to the present invention. However, it is believed that the blood or serum (or other biological fluids, such as those mentioned herein) of certain individuals or groups of individuals may contain certain (pre-existing) proteins that, in certain circumstances, may (non-specifically) bind to ISVs, resulting in interfering signals in certain assays (used to analyze blood or serum samples obtained from said individuals). This is particularly based on observations made in the development of the present invention, namely that the non-specific protein interference addressed by the present invention is present not only in samples obtained from subjects who have been pre-administered with ISVs, but also in samples obtained from subjects who have not been pre-administered with ISVs.

[0058] Specifically, based on observations at the time of the invention's development, and although the invention is not limited thereto, it is considered that the (pre-existing) protein can specifically (be able to) bind to the C-terminus of the ISV (in full-length conventional 4-chain monoclonal antibodies and in "heavy chain only" antibodies found in camels, it is linked to the remainder of the antibody - i.e., linked to the CH1 region in conventional monoclonal antibodies - and linked to the hinge region in camel heavy chain antibodies - and thus can be protected from such protein interference in the full-length antibody).

[0059] This is confirmed by the discoveries made by the inventors of this invention during the formation of the invention (as further described herein), namely that certain (simple) modifications to the C-terminus of ISVs can substantially reduce or substantially prevent such protein interference. Therefore, the methods for modifying ISVs in this manner, and the ISVs already modified in this manner, form other aspects of the invention, as further described herein.

[0060] Specifically, this invention can be used to reduce or avoid protein interference and / or signaling due to nonspecific binding in immunoassays performed on biological samples (such as blood or serum samples) obtained from a subject who has been administered a (biologic) drug (again, the sample is referred to herein as a "test sample" or "assay sample"). Some examples are immunoassays used to characterize drug disposition and antibody formation after administration of a biological drug to a subject, such as those mentioned in the "Guideline on the Clinical Investigation of the Pharmacokinetics of Therapeutic Proteins" (document CHMP / EWP / 89249 / 2004, dated January 27, 2007) issued by the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMEA). As noted on pages 4 and 5 of that document:

[0061] "Several potential defects have been identified that could lead to erroneous characterization of drug handling and antibody formation. The following issues should be considered [...]:"

[0062] Immunoassay

[0063] Drug assay:

[0064] [...]

[0065] (iii) Interference from endogenous substances.

[0066] (iv) Interference from plasma components or anti-drug antibodies binding to the analyte and inhibiting complementary binding to the capture antibody.

[0067] This invention can be specifically used to predict, reduce, or avoid this type of interference in immunoassays used to analyze test / assay samples of biological fluids taken from subjects who have been administered ISVs (and specifically nanobodies; or ISV-based or nanobodies-based bioproducts, as further described herein).

[0068] This invention can be specifically used to predict, reduce, or avoid this type of (nonspecific) protein interference in immunoassays used to characterize drug treatment and / or to determine the formation of any ADA's (anti-drug) antibodies. In this regard, it should be noted that generally, in the specification and appended claims of this invention, when phrases such as "predict, reduce, or avoid protein interference" are used, this includes not only predicting, reducing, or avoiding such protein interference itself, but generally also predicting, reducing, or avoiding the occurrence of nonspecific signals in immunoassays (such as those in which (nonspecific) signals associated with protein interference may be present, such as in ADA assays), and specifically in said immunoassays, predicting, reducing, or avoiding the occurrence of nonspecific signals that, when observed in said nonspecific signals, are generally attributed to, associated with, and / or considered as indications of (nonspecific) protein interference. In this regard, it should generally be noted that, as mentioned herein, the invention is not particularly limited to any cause, explanation, hypothesis, or mechanism.

[0069] In one specific, but non-limiting aspect, the invention can be used to predict, avoid, or reduce protein interference in “anti-drug antibody” or “ADA” assays performed on a biological fluid sample (i.e., a “test sample”) taken from a subject who has been administered ISVs (and specifically nanobodies; or ISV-based or nanobody-based bioproducts, as further defined herein).

[0070] In another specific but non-limiting aspect, the invention can be used to predict, avoid, or reduce protein interference (and / or typically associated nonspecific signals) 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 (and specifically nanobodies; or ISV-based or nanobody-based bioproducts, as further defined herein). Specifically, the invention can be used to predict, avoid, or reduce protein interference in said “anti-drug antibody” or “ADA” assays performed on a sample of biological fluid (i.e., a “test sample”), more specifically on a sample of biological fluid already obtained from a subject who has been administered one or more ISVs or nanobodies (or ISV-based or nanobody-based bioproducts, as further defined herein). For example, the present invention can be used to predict, avoid, or reduce such protein interference in said “anti-drug antibody” or “ADA” assays used to detect, measure, and / or characterize the presence of (any) anti-drug antibody against an ISV or nanobody (or an ISV-based biologic or nanobody-based biologic, as further defined herein), which has been administered to a subject (from whom a sample is obtained) (in the context of a clinical trial and / or in the context of treatment).

[0071] Therefore, in one specific, but non-limiting aspect, the invention can be used to predict, avoid, or reduce such protein interference (and / or typically associated nonspecific signals) in biological samples (i.e., “test samples”) obtained from subjects who have been administered one or more of the ISVs or nanobodies (or ISV-based or nanobodies-based bioproducts, as further defined herein), wherein the samples are suitable for and / or intended for use in immunological assays, such as the ADA assay. As mentioned, the biological sample may be blood (including whole blood, serum, or plasma), tears, bronchoalveolar fluid / BALF, cerebrospinal fluid, or any other suitable biological fluid or sample for use in immunoassays, and specifically ADA assays.

[0072] In one specific, but non-limiting aspect, the test sample may be obtained from a subject who has received multiple administrations (e.g., at least 1-3 individual administrations over a period of at least 10 days, such as at least one month or longer) of an ISV, nanobody, ISV-based biologic (as further defined herein), or nanobody-based biologic (as further defined herein) and / or long-term treatment therewith (i.e., treatment over a period of at least 10 days, such as at least one month). The ISV, nanobody, ISV-based biologic, or nanobody-based biologic may be administered to the subject, for example, in the context of treatment or in the context of a clinical trial.

[0073] In one specific, but non-limiting aspect, the test sample may be obtained from a subject who has been administered (and / or provided) an ISV, nanobody, ISV-based biologic, or nanobody-based biologic that has an increased half-life (as defined herein and compared to a monovalent ISV) in the subject who has been administered it, for example, at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as a half-life of at least 10 days.

[0074] For example, but not limited to, an increased half-life can be provided to the ISV, nanobody, ISV-based biologic, or nanobody-based biologic by functionalization and / or by including structural portions or binding units that increase the half-life of the construct. Those skilled in the art will know examples of such functionalizations, structural portions, or binding units, which may, for example, be as described herein, and may include, for example, PEGylation, fusion with serum albumin, or fusion with a peptide or binding unit that can bind to a serum protein such as serum albumin. The serum-albumin-binding peptide or binding domain may be any suitable serum-albumin-binding peptide or binding domain capable of increasing the half-life of the construct (compared to the same construct without the serum-albumin-binding peptide or binding domain), and may specifically be a serum-albumin-binding peptide as described in the applicant's WO2008 / 068280 (and specifically in the applicant's WO 2009 / 127691 and unpublished U.S. application 61 / 301,819), or a serum-albumin-binding ISV (such as a serum-albumin-binding nanobody; for example, Alb-1 or a humanized form of Alb-1 such as Alb-8, for which see, for example, WO 06 / 122787).

[0075] Therefore, in one specific but non-limiting aspect, the biological sample may be obtained from a subject who has been administered such an ISV, nanobody, ISV-based biologic, or nanobody-based biologic, which comprises (human) serum albumin-binding peptide or binding domain.

[0076] As mentioned above, in a non-limiting aspect, the present invention generally relates to a method for predicting whether a given ISV or nanobody (or ISV-based or nanobody-based drug) will produce (or have a high or increased tendency to produce) protein interference (as further described herein) in an immunoassay (i.e., after the ISV is administered to a subject, a biofluid sample is obtained from the subject, and the biofluid is subjected to an immunoassay as further described herein), the method comprising performing an immunoassay including at least the following steps:

[0077] (i) Contacting the ISV or nanobody (or ISV-based or nanobody-based drug) with an antibody (“analytical antibody”) obtained from a human subject and selected, generated, and / or isolated based on its ability to recognize and / or bind to the C-terminus of the ISV or nanobody; and

[0078] (ii) Determine whether the ISV or nanobody (or ISV-based or nanobody-based drug) binds to the antibody in the immunoassay.

[0079] Furthermore, as mentioned herein, the ISV described herein can specifically be a nanobody or (other) ISV (i.e., different from a nanobody) that has or contains a VH domain; preferably a nanobody.

[0080] Furthermore, any protein or polypeptide containing an ISV (such as an ISV-based drug) preferably has said (or at least one) such ISV at its C-terminus. Moreover, the ISV may specifically be a nanobody or (other) ISV (i.e., different from a nanobody) that is or contains a VH domain; preferably a nanobody.

[0081] In an alternative implementation, as further described herein, instead of the aforementioned antibody derived from human subjects, a monoclonal antibody referred to herein as "21-4-3" (or simply "21-4", see SEQ ID NO's 35 and 36 for the VH and VL sequences) may be used. Using hybridoma technology, 21-4 was generated starting with mice immunized with the nanobody construct of SEQ ID NO:98 in WO 2006 / 122825, as further described in Example 7, and a hybridoma cell line expressing 21-4 (designated "ABH0015") was deposited on June 4, 2012 at BCCM, Ghent, Belgium, accession number LMBP 9680CB. It has been shown that monoclonal 21-4 recognizes the C-terminus of the nanobody construct of SEQ ID NO:98 in WO 2006 / 122825, the C-terminus of which is a nanobody (humanized V) generated targeting von Willebrand factor (vWF). HH The composition is as follows. 21-4 was initially developed as an analytical reagent for detecting protein nanobodies (specifically, the nanobodies construct of SEQ ID NO:98 in WO 2006 / 122825) in (serum) samples; surprisingly, it has now been found that 21-4 can also be used to predict whether ISVs are prone to nonspecific protein interference (especially for the nanobodies construct of SEQ ID NO:98 in WO 2006 / 122825 or for some other comparable (mouse) monoclonal antibodies produced against other nanobodies).

[0082] Specifically, it has been found that when measured according to the protocol given in Example 9, if the measurement of the binding of 21-4 to ISV (or to a protein or peptide containing ISV at its C-terminus, or a similar protein or peptide as mentioned herein) produces an RU value of less than 500 (in accordance with the formula [measured RU] / [protein MW] x 10), 6 After adjusting for the molecular weight of the protein (the RU value), then the ISV or protein may not be prone to protein interference (within the confidence level provided by the data presented in the examples below). For the purposes of the above formula, MW can be calculated as the sum of all MW's of all amino acid residues present in the ISV.

[0083] Therefore, the binding of any ISV, protein, or polypeptide described herein with 21-4 preferably has an RU value of less than 500 (determined according to the scheme presented in Example 9, and after adjusting the measured RU value according to the above formula with respect to the molecular weight of the ISV or protein used).

[0084] Therefore, this aspect of the invention generally relates to a method for predicting whether a given ISV or nanobody (or ISV-based or nanobody-based drug) will produce (or has a high or increased tendency to produce) protein interference (as further described herein) in an immunoassay (i.e., after the ISV is administered to a subject, a biofluid sample is obtained from the subject, and the biofluid is subjected to an immunoassay as further described herein), the method comprising performing an immunoassay including at least the following steps:

[0085] (i) Contact the ISV or nanobody (or ISV-based or nanobody-based drug) with monoclonal antibody 21-4 (i.e., used as an "analytical antibody"); and

[0086] (ii) Determine whether the ISV or nanobody (or ISV-based or nanobody-based drug) binds to monoclonal antibody 21-4 in the immunoassay.

[0087] The method can be specifically performed using BiaCore or similar technologies, and more specifically using the scheme presented in Example 9. As mentioned herein, when the binding of an ISV or ISV-based drug in this scheme shows an RU value of less than 500 (in accordance with the formula [measured RU] / [protein MW] x 10), 6 After adjusting the RU value for the molecular weight of the protein, the ISV or ISV-based protein is unlikely to bind to any interfering factors present in human blood or serum and / or is unlikely to have a tendency to be subject to nonspecific protein interference in the ADA assay (i.e., within the confidence level presented in the Experimental section below).

[0088] Furthermore, as mentioned herein, the ISV described herein can specifically be a nanobody or (other) ISV (i.e., different from a nanobody) that has or contains a VH domain; preferably a nanobody.

[0089] Furthermore, any protein or polypeptide containing an ISV (such as an ISV-based drug) preferably has said (or at least one) such ISV at its C-terminus. Moreover, the ISV may specifically be a nanobody or (other) ISV (i.e., different from a nanobody) that is or contains a VH domain; preferably a nanobody.

[0090] As mentioned in this article, the method described above using 21-4 can also be used to determine whether an ISV or a protein or peptide containing an ISV binds to (or has a tendency to bind to) interfering factors present in human blood or serum.

[0091] Furthermore, as mentioned herein, the method described in 21-4 is also considered to be useful for predicting whether any protein or polypeptide (such as an antibody fragment or ScFv) having a VH domain at its C-terminus binds to (or has a tendency to bind to) interfering factors present in human blood or serum and / or has a tendency to be subject to protein interference in ADA assays.

[0092] In addition to 21-4, antibodies or antibody fragments (such as suitable Fab fragments) containing the heavy and light chain variable domains of 21-4 (see SEQ ID NO's: 35 and 36, respectively) or even just the CDR sequence of 21-4 (appropriately transplanted into other suitable VH and VK frameworks) may also be used in the methods described herein.

[0093] As further described herein, the present invention can be specifically used to predict whether a given ISV or nanobody (or ISV-based or nanobody-based drug) will produce protein interference in an immunoassay (which is an ADA assay) (as further described herein). The ADA assay can generally be, for example, an ADA assay for detecting or measuring ADAs against ISVs, and can specifically be an ADA assay for detecting or measuring ADAs against ISVs used in steps (i) and (ii) above.

[0094] In a particularly preferred but non-limiting aspect, the invention can be used to predict whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) will produce protein interference (as further described herein) in an immunoassay involving the use of said ISV (and specifically, in an ADA assay). Furthermore, said ADA assay can generally be, for example, an ADA assay for detecting or measuring ADAs against ISVs, and can specifically be an ADA assay for detecting or measuring ADAs against ISVs used in steps (i) and (ii) above.

[0095] In even more specific but non-limiting aspects, the present invention can be used to predict whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) will produce protein interference (as further described herein) in an immunoassay including the use of said ISV (and specifically, in an ADA assay). For example, said immunoassay may be an ADA assay (i.e., including ISV) that performs an assay or measures whether any ADAs against said ISV are present in a test sample, wherein said sample is a biological fluid sample (as described herein) obtained from a subject who has been administered said ISV (as further described herein). For example, as further mentioned herein, said sample (i.e., “test sample”) may be a blood sample (including whole blood, serum or plasma), tears, bronchoalveolar fluid / BALF, cerebrospinal fluid or any other suitable biological fluid, and may specifically be a biological sample suitable and / or intended for use in an immunological assay, such as an ADA assay.

[0096] As further described herein, in all these aspects (and other aspects of the invention as described herein), the invention can also be used to select ISVs that are less likely to produce or produce such protein interference in the immunoassay or ADA assay; as such an assay or test, it can be used to test whether certain modifications to an ISV (completely or partially) reduce its tendency to produce such interference in the immunoassay or ADA assay; and / or as such an assay or test, it can be used to guide the modification or improvement of ISVs to reduce their tendency to produce such protein interference in the immunoassay or ADA assay.

[0097] As mentioned, step (i) of the method of the present invention includes contacting the ISV or nanobody (or an ISV-based or nanobody-based drug) with an antibody obtained from a human subject and selected / separated based on its ability to recognize and / or bind to the C-terminus of the ISV or nanobody (as further described herein). In step (i) of the method described herein, the "ISV or nanobody (or ISV-based or nanobody-based drug)" is used as an antigen (i.e., as the substance to be detected) in an immunoassay. Furthermore, in step (i), the "antibody obtained from a human subject and selected / separated based on its ability to recognize and / or bind to the C-terminus of the ISV or nanobody" is used as an analytical reagent (i.e., used in the same manner as other antibodies in an immunoassay to detect the presence of the antigen they target).

[0098] As already mentioned, and for a better understanding of the invention described herein, it should be noted that in step (i), the ISV is typically used as an “antigen” (i.e., as the compound to be detected), and the “analytical antibody” is used as an analytical reagent (i.e., as a means of detecting whether a given ISV binds; and therefore as a means of detecting whether a given ISV has a higher or increased risk of causing protein interference). For example, when step (i) is performed in the form of an ELISA, the “antibody / analytical reagent” is typically bound to a carrier (i.e., bound to an ELISA plate), and the ISV is present in the sample to be tested.

[0099] In contrast, it should be noted that in these ADA assays used to detect or measure ADAs against ISVs, the ISV is used as an "analytical reagent" (i.e., a compound used to detect the presence of any ADAs), and the ADAs are "antigens" (i.e., compounds to be detected). Therefore, in these assays, the ISV is typically / frequently bound to a carrier (such as an ELISA plate), while the ADAs (if present) are present in the sample used for the assay.

[0100] However, as already mentioned, it should generally be noted that the invention is not limited to assays in which the "analytical antibody" binds to the carrier. For example, in alternative methods of performing the assay according to the invention (as shown in Example 5), the analytical antibody is alternatively used as a bridging agent and is therefore in solution rather than bound to the plate (although it is indirectly bound to the plate via an ISV coated on it). However, also in the specific bridging assay described in Example 5 (which is a competitive assay), the analytical antibody is still used as the analytical reagent (i.e., to determine whether the target ISV binds; and thus to determine whether it has a higher or increased risk of producing protein interference). It is also contemplated that, based on the further disclosure herein, those skilled in the art can design other assays in which the analytical antibody can be used as the analytical reagent to determine whether a given ISV can bind; and thereby to determine whether it has a higher or increased risk of producing protein interference.

[0101] The “analytical antibody” used in step (i) can be a polyclonal antibody or a monoclonal antibody.

[0102] When the analytical antibody is a polyclonal antibody, it can be, for example, a polyclonal antibody (preparation) obtained / purified / separated from a biological sample (such as blood, plasma, B cells, or other suitable biological sample or fluid from which polyclonal antibodies can be appropriately separated) obtained from a human subject. This can be, for example, a suitable biological sample obtained from a human subject who has been administered at least one ISV (such as the ISV used in step (i), but this is neither desired nor necessary), and can also be (and preferably) a suitable biological sample from a human subject who has never received or been treated with an ISV. More importantly, the polyclonal antibody is obtained from the biological sample by a method comprising at least one affinity step (and one or more other steps known per se for obtaining / purifying / separating polyclonal antibodies) using an affinity matrix or affinity column carrying an ISV as an affinity structural motif. For example, polyclonal antibodies can be obtained from the biological sample by affinity chromatography using an affinity column carrying an ISV, as described, for example, in Example 2. This can be performed, for example, using an affinity matrix carrying ISV as an antigen, using well-known techniques for isolating antibodies from biological samples using immunoaffinity chromatography. These techniques are well-known in the art, and suitable examples will be apparent to those skilled in the art based on the disclosure herein.

[0103] The polyclonal antibody (formulation) may specifically be IgG (or IgG fraction).

[0104] For example, it could be a polyclonal antibody that uses an ISV (and specifically nanobodies, such as VHHs, humanized and / or sequence-optimized VHHs, or camel-derived VHs, such as camel-derived human VHs) without a C-terminal marker (i.e., where the C-terminus is terminated with the amino acid sequence VTVSS (SEQ ID NO:33)) as an antigen bound to an affinity matrix, obtained by a method including (immuno)affinity chromatography performed on a sample of a biofluid obtained from a human subject. Specifically, the ISV used as the antigen bound to the affinity matrix could be a humanized or sequence-optimized VHH (or alternatively, a correspondingly camel-derived human VH), wherein the C-terminus is terminated with the amino acid sequence VTVSS (SEQ ID NO:33). In one specific, but non-limiting aspect, the ISV used as an antigen bound to the affinity matrix can be a humanized or sequence-optimized VHH, which, as a result of such humanization or sequence optimization, includes a proline (P) residue at position 14, wherein the corresponding “naive” VHH includes alanine (A) at position 14 (in other words, the ISV used as an antigen is a humanized form of VHH that naturally includes alanine at position 14, said alanine residue, as a result of humanization and / or sequence optimization, has been substituted with a proline (P) residue). As a result of such humanization or sequence optimization, the ISV used as an antigen may also include one or more other amino acid substitutions, such as those commonly described in WO 08 / 020079 or WO 09 / 138519.

[0105] Some specific examples of ISVs that can be used as antigens to generate / isolate the “analytical antibodies” used in this invention are given in SEQ ID NOs:1 and 2.

[0106] In addition, methods for obtaining polyclonal antibodies may include, in addition to the (immuno)affinity step, one or more other steps (performed before or after the affinity step) for isolating / purifying polyclonal antibodies from biological samples. Furthermore, those skilled in the art are aware of these steps and techniques used to perform them.

[0107] Therefore, in one aspect, the present invention includes the method as further described herein, comprising steps (i) and (ii) as described herein, wherein an "analytical antibody" (i.e., an antibody obtained from a human subject and selected / isolated based on its ability to recognize and / or bind to the C-terminus of an ISV or nanobody) is obtained from a biological sample obtained from a human subject ( wherein the biological sample is a sample suitable for a method of generating / isolating antibodies from said sample), wherein an ISV (and preferably a nanobody) is used as an antigen, and preferably, an ISV is used as an antigen containing the amino acid sequence VTVSS (SEQ ID NO: 33) as a C-terminal sequence, and more preferably, humanized and / or sequence-optimized nanobodies are used as antigens containing the amino acid sequence VTVSS (SEQ ID NO: 33) as a C-terminal sequence, and even more preferably, humanized and / or sequence-optimized nanobodies are used as such antigens containing the amino acid sequence VTVSS (SEQ ID NO: 33) as a C-terminal sequence. Nanobodies containing the amino acid sequence VTVSS (SEQ ID NO:33) as the C-terminal sequence and a proline residue at position 14, such as nanobodies containing the amino acid sequence VTVSS (SEQ ID NO:33) as the C-terminal sequence and a proline residue at position 14, the proline residue being introduced into the nanobodies as a result of the humanization and / or sequence optimization (e.g., to replace the naturally occurring alanine residue at said position in a humanized and / or sequence-optimized VHH).

[0108] The aforementioned ISVs can also be used in methods for isolating monoclonal antibodies (which also begin with suitable biological samples obtained from humans), and these monoclonal antibodies are suitable for use as "analytical antibodies" in this invention.

[0109] For example, the monoclonal antibody may be obtained from blood, B cells or other suitable samples and materials for antibody isolation, and may be selected based on its ability to recognize and / or bind to ISVs or nanobodies (C-terminals) (wherein, again, the ISV(s) used as antigens in the screening and / or selection process are preferably as described in the preceding paragraphs, including the preferred selection indicated with respect to the ISV / antigen). Such screening and selection can be carried out in any suitable manner, for example by using B-cell selection and / or expansion techniques (which are substantially the same as or suitably similar to the B-cell selection techniques described in EP 0488 470, WO 92 / 02551, EP 1 633 787, WO 01 / 55216, WO 02 / 26829, WO 04 / 051268, WO 04 / 102198 or WO 04 / 106377), or by using techniques similar to those described in WO 06 / 079372 (but using human B-cells instead of camel B-cells).

[0110] Once one or more B cells expressing suitable antibodies have been identified / isolated, the antibodies can be isolated, expressed, and / or produced in any suitable manner. For example, the B cells can be proliferated indefinitely as hybridomas to produce one or more desired antibodies (using techniques well-known per se for generating hybridomas from selected B cells), and the one or more antibodies can then be isolated from the hybridoma (culture supernatant) again using suitable techniques well-established in the art and described in various books and manuals, and in addition, in the patent publications mentioned in the foregoing paragraphs.

[0111] Alternatively, the B cells can be amplified using B cell amplification techniques known per se, and the antibody / multiple antibodies can be separated from the amplified B cells (culture supernatant). Furthermore, this can be done using suitable techniques well-established in the art and described in various books and manuals, and in addition, in the patent publications mentioned in the foregoing paragraphs.

[0112] In another alternative embodiment, DNA encoding one or more antibodies for the target can be obtained directly (e.g., using suitable single-cell PCR cloning techniques) or after appropriate amplification of the desired B cells from the B cells or other suitable cells. The DNA can then be appropriately expressed in suitable host cells or host organisms to provide the desired one or more antibodies. Furthermore, this can be done using suitable techniques well-established in the art and described in various books and manuals, and in addition, in the patent publications mentioned in the foregoing paragraphs.

[0113] Monoclonal antibodies suitable for use as "analytical antibodies" can also be generated by a method comprising all repertoire clones (starting from a suitable sample obtained from a human subject) and all repertoire clones of antibody screening clones relating to an ISV used as an antigen (wherein, again, the ISV(s) used as antigens in the screening and / or selection process preferably include preferred selections specified with respect to the ISV / antigen, as described in the preceding paragraphs). Methods for all repertoire clones and various techniques for displaying all repertoire clones relating to the selected and screened clones (such as phage display, ribosome display, and yeast display) are well known to those skilled in the art and are described, for example, in EP 0589877, EP 0 774 511, WO 90 / 14430, and EP 0368 684, as well as in various books concerning subjects.

[0114] Typically, 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 raw material for obtaining polyclonal or monoclonal antibodies, respectively). In one specific but non-limiting aspect, the sample can, for example, be obtained from a woman, and specifically from a postmenopausal woman. Thus, in one specific but non-limiting aspect, the analytical antibodies used in steps (i) and (ii) above are obtained from a biological sample obtained / from a postmenopausal woman (or derived from antibodies already obtained / from a postmenopausal woman).

[0115] Furthermore, the biological sample used as the starting point for obtaining (polyclonal or monoclonal) analytical antibodies can be obtained from subjects who have been pre-administered ISV (e.g., as part of a clinical trial or treatment), but preferably from subjects who have not been pre-administered ISV.

[0116] However, it should be noted that the present invention is not particularly limited to the source of one or more analytical antibodies used, and it has been demonstrated that in some cases, other suitable analytical antibodies can be obtained (generated, isolated) from other sources using the techniques described herein, including commercially available human blood or plasma (and even blood, plasma, or B cells from other mammalian or primate species such as baboons or cynomolgus monkeys).

[0117] As mentioned above, the (polyclonal or monoclonal) analytical antibodies used in steps (i) and (ii) should be such that they can recognize or bind to the C-terminus of ISVs or nanobodies, and are most preferably selected and / or separated based on their ability to bind to the C-terminus of ISVs or nanobodies.

[0118] If possible Figure 2 As observed, 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), and therefore 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. Further, as can be seen from... Figure 2 As observed, at least some amino acid residues in the VTVSS sequence (SEQ ID NO:33) are part of the putative epitope on the ISV, and among other residues, it includes amino acid residues at position 14 (and adjacent / near amino acid residues in the amino acid sequence, such as positions 11, 13, and 15), and may also include amino acid residues at position 83 (and adjacent / near amino acid residues in the amino acid sequence, such as positions 82, 82a, 82b, and 84) and / or amino acid residues at position 108 (and adjacent / near amino acid residues in the amino acid sequence, such as position 107). Position 109 is the first V of the C-terminal VTVSS (SEQ ID NO:33) sequence, and it has been shown, for example, that position 110 may also have an effect on protein interference. In this article, this is also referred to as the “C-terminal region”. It is understood that the C-terminal region contains at least the C-terminal sequence VTVSS (SEQ ID NO:33) and an amino acid residue at position 14, and may also contain amino acid residues at positions 83 and 108, and may also contain amino acid residues at positions 13, 15, 82b, 83, 84 and 107.

[0119] As also mentioned, and not limited to any hypothesis or interpretation, in full-length 4-chain monoclonal antibodies, or in those full-length heavy chain antibodies such as those present in camels, the C-terminal of the VH or VHH domain is attached to the remainder of the antibody—that is, to the CH1 region in conventional monoclonal antibodies or to the hinge region in camel heavy chain antibodies, respectively—and is therefore immune to such protein interference and / or covered by VH / VL interactions (in conventional 4-chain antibodies), thus making the “C-terminal region” generally not solvent-exposed and / or accessible as an interaction site for proteins present in the blood, serum, or body of a person who has been administered the ISV. However, if an ISV or nanobody is used on its own (i.e., not linked to any other part of the antibody), or if an ISV-based drug or nanobody-based drug is used, the drug carries an ISV or nanobody at its C-terminus, the C-terminal epitope of which is available for (non-specific) interaction with other proteins, and is not limited to any hypothesis or interpretation that such a C-terminal region may currently be susceptible to (non-specific) protein interaction with one or more proteins (e.g., one or more IgGs) pre-existing in the “test sample” to be tested, and this may result in protein interference and / or non-specific signaling in immunoassays (and specifically in ADA assays).

[0120] As mentioned, the methods described herein can be used to predict, reduce or avoid such protein interactions, and can also serve as a tool to guide the modification of ISVs, nanobodies, ISV-based drugs or nanobodies to provide them with (partial or preferably substantially all) a reduced tendency to produce such protein interference.

[0121] As is clear from the foregoing paragraphs, and not limited to any hypothesis or interpretation, it is particularly expected (and is part of the teachings of this invention) that modifications to the “C-terminal region” will alter (and preferably reduce) the tendency of ISVs to suffer from such non-specific protein interactions, and this has been observed experimentally (see, for example, the experimental results described in Examples 1C and 3 below).

[0122] Based on this, and not limited to any hypothesis or interpretation, the present invention also teaches certain modifications that can be introduced into the C-terminal region of ISVs, nanobodies, ISV-based drugs, or nanobodies for this purpose (their (potential) effectiveness can be tested using the methods described herein). Furthermore, based on the teachings herein, it is believed that those skilled in the art can select, design, or propose other (candidate) modifications to the C-terminal region that can be introduced for this purpose (and their (potential) effectiveness can also be tested using the methods described herein).

[0123] Returning to the analytical antibody used in this invention, it is preferably a (polyclonal or monoclonal) antibody that recognizes the C-terminal region of an ISV (as defined above), and specifically, but not limited to, the C-terminal region of a nanobody.

[0124] For example, in one specific but non-limiting aspect, the “analytical antibody” may be a polyclonal or monoclonal antibody that recognizes (and / or is able to bind to, and specifically specifically binds to) the C-terminal region of an ISV or nanobody (where the C-terminus of the sequence terminates 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 (which may be a different ISV, but preferably the same ISV), wherein one or more other amino acid residues (such as 1-5 amino acid residues, or alternatively small peptide sequences or even other polypeptides or proteins) are linked to the C-terminal VTVSS (SEQ ID NO:33).

[0125] In another, more specific, but still non-limiting aspect, the “analytical antibody” can be a polyclonal or monoclonal antibody that recognizes (and / or is able to bind to, and specifically specifically binds to) the C-terminal region of an ISV or nanobody (wherein the C-terminus of the sequence terminates with a VTVSS (SEQ ID NO: 33), and where position 14 is an amino acid not naturally present at position 14 and / or is a modified amino acid compared to the amino acid naturally present at position 14 (e.g., as a result of humanization, camelification, and / or sequence optimization)), but does not recognize (and / or cannot specifically bind to) the C-terminal region of such an ISV or nanobody (which may be a different ISV but preferably the same ISV), wherein one or more of the C-terminal VTVSS (SEQ ID NO: 33) are present. NO:33) is linked to other amino acid residues (such as 1-5 amino acid residues, or alternatively small peptide sequences or even other polypeptides or proteins; and / or wherein position 14 is an amino acid naturally present at position 14 (e.g., alanine, or proline when ISV naturally contains proline at position 14).

[0126] For example, an "analytical antibody" can also be a polyclonal or monoclonal antibody that recognizes (and / or is able to bind to, and specifically specifically binds to) the C-terminal region of an ISV or nanobody (where the C-terminus of the sequence is terminated by VTVSS (SEQ ID NO:33), and where position 14 is proline (and specifically, when position 14 is modified to proline, for example, as a result of humanization, camelification, and / or sequence optimization)), but does not recognize the C-terminal region of an ISV or nanobody (which may be a different ISV but preferably the same ISV) containing one or more other amino acid residues linked to the C-terminal VTVSS (SEQ ID NO:33) (such as 1-5 amino acid residues, or alternatively small peptide sequences or even other polypeptides or proteins; and / or where position 14 is alanine).

[0127] "Analytical antibody" can also be a polyclonal or monoclonal antibody that recognizes (and / or is able to bind to, and specifically specifically binds to) the C-terminal region of an ISV or nanobody (where the C-terminus of the sequence is terminated by VTVSS (SEQ ID NO:33), and where position 14 is proline (and specifically, where the proline residue is naturally present in the ISV at that position), but does not recognize the C-terminal region of such ISV or nanobody (which may be a different ISV but preferably the same ISV) containing one or more other amino acid residues linked to the C-terminal VTVSS (SEQ ID NO:33) (such as 1-5 amino acid residues, or alternatively small peptide sequences or even other polypeptides or proteins, where position 14 is still (naturally present or unmodified) proline).

[0128] The “analytical antibody” may also be, for example, a polyclonal or monoclonal antibody that recognizes the sequence (C-terminal region) of the ISV referred to herein as “Nb 3.4” (SEQ ID NO:5), but does not recognize the sequence (C-terminal region) of the ISV referred to herein as “Nb 3.1” (SEQ ID NO:3) and / or (and preferably) does not recognize the sequence of the ISV referred to herein as “Nb 3.2” (SEQ ID NO:4).

[0129] For the purposes described above, any suitable binding assay (such as Biacore) can be used to determine whether the "analytical antibody" recognizes ISV or nanobody (and / or can or cannot (specifically) bind to ISV or nanobody), but the BIACORE assay described in Example 3 or the ADA assay described in Example 5, such as the ADA bridging / competitive assay, can also be used to determine this (see also: Figures 1A to 1C and specifically Figure 1B ).

[0130] Based on the disclosure herein, those skilled in the art will understand suitable forms / techniques for performing the aforementioned determinations, which include, for example, but are not limited to:

[0131] - Colorimetric assays, such as ELISA using analytical antibodies directly or indirectly coated on the plate and detecting bound ISVs with monoclonal or polyclonal anti-ISV antibodies. Other useful alternative techniques for this setup include, but are not limited to, electrochemiluminescence (MSD platform), fluorescence (DELFIA, GYROS), and other methods that rely on secondary detection of bound ISVs.

[0132] - Surface plasmon resonance (e.g., BIACORE) or other real-time biosensor methods (i.e., methods other than those using SPR) that use directly or indirectly immobilized analytical antibodies and monitor the binding of subsequently injected / applied ISVs. These methods do not require further detection of bound ISVs. A representative method for performing this type of assay is described in Example 3.

[0133] - The competitive behavior of ISVs is analyzed in a bridging assay (ADA assay) by replacing the ADA containing biological fluid with an analytical antibody. Different techniques such as ELISA and the MSD platform can be used for the bridging assay. A representative method for this type of assay is illustrated in... Figures 1A to 1C As shown in the figure, and also described in Example 5, a specific example of this determination method is presented.

[0134] - Any chromatography method in which the analytical antibody is immobilized on the chromatography matrix and the ISV is specifically captured / separated from the solution.

[0135] Once a suitable analytical antibody is obtained using one of the methods described herein or in a specific example (or a method substantially equivalent thereof), the analytical antibody can be used to determine, by performing steps (i) and (ii) above, whether a given ISV or nanobody (or ISV-based drug or nanobody-based drug) will produce (or has a high or increased tendency to produce) protein interference (as defined herein). Also as described herein, this typically includes contacting the ISV, nanobody, ISV-based drug or nanobody-based drug with the analytical antibody and determining whether the ISV, nanobody, ISV-based drug or nanobody-based drug is recognized by (and / or binds to, and specifically specifically binds to) the analytical antibody (and specifically determines whether the C-terminal region of the ISV or nanobody, or any ISV or nanobody forming the C-terminus of the ISV-based drug or nanobody-based drug, is recognized by the analytical antibody).

[0136] The method can be performed using any suitable technique for determining whether an antigen (in the context of this invention, an ISV, nanobody, ISV-based drug, or nanobody-based drug) binds to an antibody, and the suitable (immunoassay) technique is known to those skilled in the art. Some non-limiting examples are suitable ELISA techniques (including, for example, sandwich ELISAs), wherein, depending on the form of ELISA used (as known to those skilled in the art), the analytical antibody or ISV can be coated on a plate and the analytical antibody or ISV can be detectably labeled. Other techniques may include, for example, the use of a BIAcore instrument (where the analytical antibody or ISV can also be coated on a chip, see, for example, Example 3). Another alternative method may be a competitive bridging assay (as illustrated, for example, in Example 5), in which the ability of an ISV to compete (or vice versa) with other known ISVs, nanobodies, ISV-based drugs, or nanobody-based drugs that bind to the analytical antibody is tested. Those skilled in the art will understand, based on the disclosure herein, 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.

[0137] Based on the disclosure herein, it is also clear that the present invention (and specifically the analytical antibodies used herein) can be used to determine whether a given ISV, nanobody, ISV-based drug, or nanobody-based drug contains such an interaction site (e.g., an interaction site present at or within the C-terminal region, and / or an interaction site forming part of the C-terminal region) capable of interacting 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 performing an immunoassay such as an ADA assay (specifically, an ADA assay for determining the presence of any ADAs 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 in this invention, the ISV, nanobody, ISV-based drug, or nanobody-based drug is very likely to contain such (accessible or exposed) interaction sites, and thus has a tendency to produce such protein interference (as defined herein) when used in such immunoassays or ADA assays for testing test samples. As will be apparent to those skilled in the art, this is a problem that should preferably be avoided, and if possible, this tendency to produce such protein interference can be substantially reduced or substantially eliminated by selecting / using another ISV, nanobody, ISV-based drug, or nanobody-based drug, or by modifying the ISV, nanobody, ISV-based drug, or nanobody-based drug (again, this can be avoided using the methods and analytical antibody assays disclosed herein).

[0138] As will be apparent to those skilled in the art based on the disclosure herein, such modifications may include, for example, one or more modifications (such as amino acid insertion, addition, deletion, or substitution) to the interaction sites on the ISV, nanobody, ISV-based drug, or nanobody-based substance, thereby reducing or eliminating its ability to interact with (non-specific) proteins that may be present in the test sample. Furthermore, this can be done through a limited trial-and-error process by introducing one or more modifications and then testing whether this ability is reduced using the methods disclosed herein and the analytical antibody. For example, multiple such modifications may be introduced, and then the ability of the modified ISV to bind to the analytical antibody may be compared with the ability of the original / unmodified ISV to bind to the analytical antibody. Alternatively, using a competitive bridging approach (as illustrated, for example, in Example 5) or using BIAcore (see, for example, Example 3), the ability of the modified ISV to compete with the original ISV for binding to the analytical antibody may be determined.

[0139] Once again, and although the invention is not limited to any hypothesis or interpretation, based on experimental evidence presented in the examples below, the inventors have found that such interaction sites may be located on / near the C-terminal region (as defined herein) or in a portion of the C-terminal region (or in a portion of the C-terminal region forming such an interaction site). This is, for example, at least in part, based on the observation that if an ISV has a tendency to produce such protein interference and has VTVSS (SEQ ID NO: 33) as an amino acid residue at its C-terminus, then attaching a limited number of amino acid residues (e.g., 1-10, e.g., 1-5, e.g., 1, 2, 3, 4, or 5), or alternatively, a labeled peptide or other peptide, protein, or other structural portion to such a C-terminus will generally substantially reduce or substantially eliminate the tendency. In some cases, it has been found that even adding 1, 2, or 3 amino acid residues to the C-terminal VTVSS (SEQ ID NO: 33) (which can be any suitable amino acid or combination of amino acids, each independently selected from any naturally occurring amino acid, such as those listed in Table A-2 on page 64 of WO 09 / 138519, for example, but not limited to, alanine, glycine, valine, leucine, or isoleucine) can substantially reduce or eliminate the aforementioned tendency. This is also based in part on the observation that in some cases, if VHH naturally contains an alanine residue at position 14 (which, as mentioned, forms the C-terminal region; see...) Figure 2 Naturally occurring VHHs typically do not have (or have a low) tendency to produce such protein interference; however, corresponding VHHs (in which the alanine at position 14 has been replaced by a proline residue) (e.g. for humanization or sequence optimization purposes) may result in an increased tendency to produce such protein interference (i.e., compared to VHHs with alanine at position 14).

[0140] In one aspect, the present invention relates to such VHHs, nanobodies (as defined herein, and specifically humanized VHHs or camel-derived VHs, such as camel-derived human VHs) or other ISVs (or ISV-based or nanobody-based pharmaceuticals having a VHH, nanobody, or other ISV at their C-terminus) that are modified (e.g., by introducing one or more amino acid substitutions, additions, or deletions), and specifically modified in the C-terminal region (e.g., by substituting or adding one or more amino acids in the C-terminal region) such that (i) they have a substantially reduced tendency to produce protein interference (as defined herein) (e.g., a reduction tendency that is at least statistically relevant); and / or (ii) they have a substantially reduced ability to bind to analytical antibodies (as described herein and used in Examples 2 and 3 and 5) in the methods of the invention described herein (e.g., the specific assays described in Examples 3 or 5), preferably compared to the same but unmodified VHHs, nanobodies, or ISVs in both cases.

[0141] Therefore, in one aspect, the present invention relates to such VHHs, nanobodies (as defined herein, and specifically humanized VHHs or camel-derived VHs, such as camel-derived human VHs) or other ISVs (or ISV-based or nanobody-based pharmaceuticals having a VHH, nanobody, or other ISV at their C-terminus) that are VHHs or VH domains (i.e., such ISVs are VH domains or derived from VH domains) and / or based on or derived from the amino acid sequence of a VHH or VH domain, wherein the VHH, nanobody, or ISV comprises the amino acid sequence VTVSS(X) at its C-terminus. n (SEQ ID NO:34), where n is 1-10, preferably 1-5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1), and where each X is (preferably naturally occurring) an amino acid residue, independently selected (and preferably independently selected independently from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I); however, other (preferably naturally occurring) amino acid residues or combinations of the aforementioned preferred amino acid residues with other amino acid residues (such as serine, proline, threonine and / or lysine) may also be used, as can be observed from the data below). Preferably, it has the amino acid sequence VTVSS(X) at its C-terminus. nThe VHH, nanobody, or ISV of (SEQ ID NO:34) is such that (i) it has a substantially reduced (e.g., at least statistically significantly reduced) tendency to produce protein interference (as defined herein); and / or such that (ii) it has a substantially reduced ability to bind to analytical antibodies (e.g., polyclonal antibodies as described herein) in the methods of the invention described herein (e.g., the specific assays described in Examples 3 or 5), preferably compared to the same VHH, nanobody, or ISV but having the amino acid sequence VTVSS (SEQ ID NO:33) at its C-terminus. Refer, for example, to the assays and data listed in Example 3.

[0142] The aforementioned VHHs, nanobodies, or (other) ISVs are preferably such that they have an RU value of less than 500 for binding to 21-4 (determined according to the scheme presented in Example 9 and after measuring the RU value with respect to molecular weight adjustment). It should also be noted that any C-terminal sequence VTVSS(X) is excluded whenever mentioned in the specification or claims herein. n (Including any one of the above aspects (a)-(p) according to a specific aspect of the invention), none of the amino acids X are cysteine ​​residues.

[0143] For example, in some preferred aspects, the C-terminus of the ISV or the construct containing the ISV (when the C-terminus is a VH-derived ISV, VHH, or nanobody) can be:

[0144] (a)VTVSS(X) n , where n = 1 and X = Ala;

[0145] (b)VTVSS(X) n Where n = 2 and each X = Ala;

[0146] (c)VTVSS(X) n Where n = 3 and each X = Ala;

[0147] (d)VTVSS(X) n , where n = 2 and at least one X = Ala (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0148] (e)VTVSS(X) n , wherein n = 3 and at least one X = Ala (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0149] (f)VTVSS(X) n , where n = 3 and at least two X = Ala (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0150] (g)VTVSS(X) n where n = 1 and X = Gly;

[0151] (h)VTVSS(X) n Where n = 2 and each X = Gly;

[0152] (i)VTVSS(X) n Where n = 3 and each X = Gly;

[0153] (j)VTVSS(X) n , wherein n = 2 and at least one X = Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0154] (k)VTVSS(X) n , wherein n = 3 and at least one X = Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0155] (l)VTVSS(X) n , where n = 3 and at least two X = Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0156] (m)VTVSS(X) n Where n = 2 and each X = Ala or Gly;

[0157] (n)VTVSS(X) n Where n = 3 and each X = Ala or Gly;

[0158] (o)VTVSS(X) n Where n = 3 and at least one X = Ala or Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile); or

[0159] (p)VTVSS(X) n, wherein n = 3 and at least two X = Ala or Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0160] Among them, aspects (a), (b), (c), (g), (h), (i), (m) and (n) are particularly preferred, wherein aspects n = 1 or 2 are preferred and aspects n = 1 are particularly preferred.

[0161] It should be noted that any C-terminal sequence VTVSS(X) is not mentioned in the specification or claims herein. n (Including any one of the above aspects (a)-(p) according to a specific aspect of the invention), none of the amino acids X are cysteine ​​residues.

[0162] Therefore, in a preferred aspect, the present invention relates to such immunoglobulin single variable domain (ISV) as nanobodies or other ISVs (preferably nanobodies) containing or derived from a VH sequence, having the sequence VTVSS(X). n The C-terminus of the nanobody, wherein n = 1 and X = Ala (or containing the ISV (and preferably the nanobody) protein or polypeptide at its C-terminus).

[0163] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the X, where n = 2 and each X = Ala (or a protein or polypeptide containing the ISV (and preferably the nanobody) at its C-terminus).

[0164] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the nanobody, wherein n = 2 and at least one X = Ala (the remaining amino acid residues X are independently selected from any naturally occurring amino acid but preferably independently selected from Val, Leu and / or Ile) (or a protein or polypeptide containing the ISV (preferably the nanobody) at its C-terminus).

[0165] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the nanobody, wherein n = 3 and at least one X = Ala (the remaining amino acid residues X are independently selected from any naturally occurring amino acid but preferably independently selected from Val, Leu and / or Ile) (or a protein or polypeptide containing the ISV (preferably the nanobody) at its C-terminus).

[0166] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the protein or polypeptide containing the ISV (preferably the nanobody) at its C-terminus.

[0167] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the nanobody, wherein n = 3 and X = Ala (or a protein or polypeptide containing the ISV (preferably the nanobody) at its C-terminus).

[0168] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of which n = 1 and X = Gly (or a protein or polypeptide containing the ISV (preferably the nanobody) at its C-terminus).

[0169] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the nanobody, wherein n = 2 and each X = Gly (or contains a protein or polypeptide of the ISV (preferably the nanobody) at its C-terminus).

[0170] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the nanobody, wherein n = 3 and each X = Gly (or contains a protein or polypeptide of the ISV (preferably the nanobody) at its C-terminus).

[0171] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the nanobody, wherein n=2 and at least one X=Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid but preferably independently selected from Val, Leu and / or Ile) (or a protein or polypeptide containing the ISV (preferably the nanobody) at its C-terminus).

[0172] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the nanobody, wherein n = 3 and at least one X = Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid but preferably independently selected from Val, Leu and / or Ile) (or a protein or polypeptide containing the ISV (preferably the nanobody) at its C-terminus).

[0173] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the protein or polypeptide containing the ISV (preferably the nanobody) at its C-terminus.

[0174] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). nThe C-terminus of the nanobody, wherein n = 2 and each X = Ala or Gly (or contains a protein or polypeptide of the ISV (preferably the nanobody) at its C-terminus).

[0175] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the nanobody, wherein n = 3 and each X = Ala or Gly (or contains a protein or polypeptide of the ISV (preferably the nanobody) at its C-terminus).

[0176] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the protein or polypeptide containing the ISV (preferably the nanobody) at its C-terminus. This is where n = 3 and at least one X = Ala or Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu, and / or Ile) or the C-terminus of the protein or polypeptide containing the ISV (preferably the nanobody).

[0177] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus of the protein or polypeptide containing the ISV (preferably the nanobody) at its C-terminus.

[0178] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably the nanobody), having the sequence VTVSS(X). n The C-terminals are n = 1, 2 or 3, where each X = Ala or Gly.

[0179] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably the nanobody), the VH sequence having the sequence VTVSS(X). n The C-terminus, of which

[0180] -n = 1, 2, or 3, where each X = Ala or Gly; or

[0181] -n = 2 or 3, where all but one of X = Ala or Gly (the remaining amino acids)

[0182] Residue X is independently selected from any naturally occurring amino acid, but preferably independently selected from Val.

[0183] Leu and / or Ile)

[0184] Or it may contain a protein or polypeptide of the ISV (preferably the nanobody) at its C-terminus.

[0185] In another preferred aspect, the invention relates to immunoglobulin single variable domain (ISV) such that it is a nanobody or an (other) ISV containing or derived from a VH sequence (preferably a nanobody), having the sequence VTVSS(X). n The C-terminus, where:

[0186] -n = 1, 2, or 3, where each X = Ala or Gly; or

[0187] -n = 2 or 3, wherein at least one X = Ala or Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0188] -n = 2 or 3, where all but one of X = Ala or Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0189] Alternatively, it may contain a protein or polypeptide of the ISV (preferably the nanobody) at its C-terminus.

[0190] In the foregoing, the (other) ISVs referring to “ISVs containing or derived from the VH sequence” are meant to be any such ISV containing or derived from the VH sequence that is not a nanobody (i.e., not a VHH, a humanized VHH, or a camel-derived VH). For example, such (other) ISVs may be, for instance, VH-based (single)-domain antibodies, VH-based dAb™, or VH-based microbodies (see WO 00 / 29004).

[0191] Furthermore, it should be noted that whenever an ISV mentioned herein has a C-terminal sequence VTVSS(X) according to a specific aspect of the invention, n (Including but not limited to the ISVs mentioned in the foregoing), in sequence VTVSS(X) n There is not a single amino acid X that is a cysteine ​​residue.

[0192] As further described herein, any of the proteins or polypeptides may be, for example, constructs comprising two or more ISVs (such as two or more nanobodies) optionally linked by one or more suitable linkers. Thus, the constructs may be, for example, bivalent, trivalent, tetravalent, or pentavalent constructs (such as bivalent, trivalent, tetravalent, or pentavalent nanobody constructs), and may be, for example, bivalent, trivalent, tetravalent, or pentavalent constructs (such as bivalent, trivalent, tetravalent, or pentavalent nanobody constructs) that are bispecific, trispecific, or bicomplementary constructs (including, for example, monospecific, bispecific, or bicomplementary constructs that may also bind to serum albumin (preferably) or other serum proteins to prolong half-life).

[0193] Furthermore, according to each of the above aspects of nanobodies, ISVs and proteins / peptides are preferably such that they have a RU value of less than 500 for binding to 21-4 (based on the scheme proposed in Example 9, and determined after adjusting the measured RU value with respect to the molecular weight of the ISV or protein used according to the formula proposed above).

[0194] As mentioned herein, the present invention is also considered applicable to other proteins or peptides having a VH domain at their C-terminus (and specifically antibody fragments such as Fab fragments or other antibody-based proteins or peptides, such as ScFv's). Therefore, in another aspect, the present invention relates to proteins or peptides (such as ScFv) having a VH domain at their C-terminus, the VH domain having the amino acid sequence VTVSS(X) at its C-terminus. n(SEQ ID NO:34), where n is 1-10, preferably 1-5, such as 1, 2, 3, 4 or 5, and where each X is (preferably naturally occurring) an amino acid residue, said amino acid residue being independently selected (and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I). Furthermore, according to some specific aspects, the C-terminus may be described according to any one of (a)-(p) above, and preferably according to one of (a), (b), (c), (g), (h), (i), (m) or (n), where n is 1, 2 or 3 and preferably 1 or 2.

[0195] Furthermore, the proteins or polypeptides are preferably such that they have an RU value of less than 500 for binding to 21-4 (determined according to the scheme presented in Example 9, and after adjusting the measured RU value according to the formula presented above with respect to the molecular weight of the ISV or protein used). Moreover, again, according to a specific aspect of this aspect of the invention, in the C-terminal sequence VTVSS(X)... n None of the amino acids X in the formula are cysteine ​​residues.

[0196] The present invention also relates to pharmaceutical compositions comprising an ISV (and preferably a therapeutic ISV) or a protein or polypeptide containing at least one ISV (and preferably at least one therapeutic ISV) and at least one suitable carrier, diluent, or excipient (i.e., suitable for pharmaceutical use), and optionally one or more other active substances, wherein the ISV, protein, or polypeptide is as further described herein (i.e., an ISV, protein, or polypeptide according to one or more aspects described herein, and specifically an ISV, protein, or polypeptide according to one or more aspects described on the preceding pages; and more specifically an ISV, protein, or polypeptide having a C-terminal / sequence according to one or more aspects described herein). The composition, carrier, diluent, or excipient may be, for example, as described in WO 08 / 020079 with respect to pharmaceutical compositions comprising nanobodies or containing at least one nanobodies (and also, as already mentioned, according to the present invention, the ISV is preferably a nanobodies).

[0197] The present invention also relates to an ISV or a protein or polypeptide comprising at least one ISV for treating a disease in a person (e.g., a patient requiring said treatment), wherein said ISV, protein or polypeptide is as further described herein (i.e., an ISV, protein or polypeptide according to one or more aspects described herein, and specifically an ISV, protein or polypeptide according to one or more aspects described on the preceding pages; and more specifically an ISV, protein or polypeptide having a C-terminal / sequence according to one or more aspects described herein).

[0198] The present invention also relates to the use of an ISV or a protein or polypeptide comprising at least one ISV for the preparation of a pharmaceutical composition, wherein the ISV, protein or polypeptide is as further described herein (i.e., an ISV, protein or polypeptide according to one or more aspects described herein, and specifically an ISV, protein or polypeptide according to one or more aspects described on the preceding page; and more specifically an ISV, protein or polypeptide having a C-terminal / sequence according to one or more aspects described herein).

[0199] The present invention also relates to a treatment method comprising administering to a human subject (e.g., a patient requiring the treatment) an ISV or a protein or polypeptide comprising at least one ISV in a formulation of a pharmaceutical composition, wherein the ISV, protein, or polypeptide is as further described herein (i.e., an ISV, protein, or polypeptide according to one or more aspects described herein, and specifically according to one or more aspects described on the preceding pages; and more specifically, an ISV, protein, or polypeptide having a C-terminal / sequence according to one or more aspects described herein; or a pharmaceutical composition comprising at least one of said ISV, protein, or polypeptide (as described above).

[0200] Regarding the foregoing, it is clear that the therapeutic applications of the ISVs, proteins, and peptides described herein are a very important aspect of the present invention, because such therapeutic applications (or the clinical development of the ISVs, proteins, and peptides for such therapeutic applications) may include using an ADA assay to determine whether the ISV, protein, or peptide is immunogenic (i.e., capable of producing ADAs when administered to human subjects). In this regard, it is clear that the question of potential immunogenicity needs to be addressed particularly when the therapeutic agent is used for a longer period (over weeks, months, or years) and / or has a half-life in human subjects of at least 3 days, such as at least one week, and up to 10 days or more (preferably expressed as t1 / 2-β).

[0201] Therefore, according to a specific aspect of the invention, an ISV, protein, peptide, or pharmaceutical composition as described herein is intended for use in treating a chronic disease in a human, and / or such an ISV, protein, or peptide is intended to be present in the cycle of a subject (i.e., at a pharmacologically active level) for at least one week, preferably at least two weeks, such as at least one month; and / or such an ISV, protein, or peptide is such that it has a half-life (preferably expressed as t1 / 2-β) in a human subject for at least 3 days, such as at least one week, and up to 10 days or more; and / or such an ISV, protein, peptide, or pharmaceutical composition is intended to be administered to a human as two or more doses for at least 3 days, such as at least one week, for example at least two weeks or at least one month, or even longer (i.e. at least 3 months, at least 6 months or at least one year), or even for a long period of time.

[0202] The present invention also relates to a method for (substantially) reducing or substantially eliminating the tendency of ISVs, nanobodies, or ISV-based drugs or nanobodies-based drugs to produce protein interference, said method comprising at least the following steps:

[0203] - Using a method that includes at least steps (i) and (ii) as mentioned herein, optionally determine the tendency of an ISV, nanobody, ISV-based drug or nanobody-based drug to produce protein interference;

[0204] - Modify the ISV, nanobody, ISV-based drug or nanobody-based drug by introducing one or more amino acid substitutions, additions or deletions into the ISV or nanobody, or by introducing one or more amino acid substitutions, additions or deletions into the C-terminal ISV or nanobody (if present) of the ISV-based drug or nanobody-based drug; and specifically by introducing one or more amino acid substitutions or additions into the C-terminal region of the ISV or nanobody, or by introducing one or more amino acid substitutions or additions into the C-terminal region of the C-terminal ISV or nanobody (if present) of the ISV-based drug or nanobody-based drug, for example by adding 1-10, such as 1-5, such as 1, 2, 3, 4 or 5 amino acid residues to the C-terminus of the sequence, each of which is independently selected from any naturally occurring amino acid (such as those listed in Table A-2 on page 64 of WO 09 / 138519, such as but not limited to alanine, glycine, valine, leucine or isoleucine);

[0205] - Determine the tendency of such modified ISV, nanobody, ISV-based drug, or nanobody-based drug to produce protein interference using a method that includes at least steps (i) and (ii) as mentioned herein; optionally, in such a manner that the tendency of such modified ISV, nanobody, ISV-based drug, or nanobody-based drug to produce protein interference is compared with the tendency of the initiating ISV, nanobody, ISV-based drug, or nanobody-based drug to produce protein interference (including, but not limited to, comparing them in competitive assays relating to binding to analytical antibodies as described herein). Alternatively, the methods described herein involving the use of 21-4 may be used. Attached Figure Description

[0206] The present invention will now be further described by way of the following non-limiting preferred aspects, embodiments and drawings, wherein:

[0207] - Figure 1A-1C The illustrations show some non-limiting examples of ADA assay methods. Some representative but non-limiting methods for performing these assays are mentioned in Example 4.

[0208] - Figure 2 The schematic diagram shows a representative 3D structure of ISVs, such as nanobodies.

[0209] - Figure 3 The binding curves show the binding of NB's 3.4-3.9 (SEQ ID NO's.5-10) to the immobilized polyclonal antibody obtained in Example 2 (obtained using the BIACORE assay described in Example 3).

[0210] - Figure 4 The binding curves (obtained using the BIACORE assay described in Example 3) show the binding of NB's 3.4, 3.11, 3.12 and 3.13 (SEQ ID NO's: 5, 12, 13 and 14) to the immobilized polyclonal antibody obtained in Example 2.

[0211] - Figure 5 The binding curves (obtained using the BIACORE assay described in Example 3) show the binding of NB's 3.4, 3.14 and 3.15 (SEQ ID NO's: 5, 15 and 16) to the immobilized polyclonal antibody obtained in Example 2.

[0212] - Figure 6 The binding curves show the binding of NB's 3.1, 3.2 and 3.4 (SEQ ID NO's: 3, 4 and 5) to the immobilized polyclonal antibody obtained in Example 2 (obtained using the BIACORE assay described in Example 3).

[0213] - Figure 7 The binding curves show the binding of NB's 4.1 and 4.2 (SEQ ID NO's: 17 and 18) to the immobilized polyclonal antibody obtained in Example 2 (obtained using the BIACORE assay described in Example 3).

[0214] - Figure 8 The binding curves (obtained using the BIACORE assay described in Example 3) show the binding of NB's 6.1, 6.2, 6.4 and 6.5 (SEQ ID NO's 19-22) to the immobilized polyclonal antibody obtained in Example 2.

[0215] - Figure 9 A table is provided that displays the sequences (SEQ ID NO's: 37-89) used in Example 8 and shows the corresponding reference sequences.

[0216] The sequences mentioned in this specification and claims are listed in Table A below (SEQ ID NO's: 1-37) and in Figure 9 Listed in (SEQ ID NO's:38-89).

[0217] Table A

[0218]

[0219] Table A (continued)

[0220]

[0221] Table A (continued)

[0222]

[0223] Table A (continued)

[0224]

[0225] Table A (continued)

[0226]

[0227] Table A (continued)

[0228] Detailed Implementation

[0229] Experimental section:

[0230] Example 1: Generation of polyclonal analytical antibodies.

[0231] Polyclonal antibodies (IgG fraction) that can be used as "analytical antibodies" are produced as follows:

[0232] A. Identification of suitable plasma samples for the isolation of polyclonal antibodies

[0233] The presence of antibodies against ISV that can be used as analytical antibodies in this invention was assessed using twenty plasma samples from healthy individuals who had never been treated with ISV.

[0234] In this embodiment, the ISV used at the outset is SEQ ID NO:1. Subsequently, to confirm that the interaction is not specific to this particular ISV, but rather a non-specific protein-protein interaction that may occur with many ISVs, the following assay was repeated with other ISVs (see paragraph C below). As an alternative to SEQ ID NO:1, SEQ ID NO:2 may also be used, for example.

[0235] The assay used was a bridging assay based on ECL (electrochemiluminescence), which used a biotinylated ISV (a biotinylated variant of SEQ ID NO:1) for capture and a sulfo-labeled ISV for detection of anti-drug antibodies. A similar approach was also used for the ADA assay. ISV biotinylation and sulfo-labeling were performed using standard coupling chemistry on primary amines, following the manufacturer's instructions, with sulfo-NHS-LC-Biotin (Pierce) and sulfo-tag NHS-Ester (MSD), respectively. Plasma samples were diluted 1 / 5 in PBS / 0.1% casein and incubated in 96-well polypropylene plates at 37°C, 600 RPM for 30 minutes. Next, the sample (50 μL) was diluted 1 / 3 in 100 μL of a 1:1 mixture of 2 μg / ml biotinylated and 2 μg / ml sulfonated ISV (SEQ ID NO:1) and incubated at room temperature, 600 RPM for 1 hour. The MSD was then... 96-well standard streptomycin protein plate, 150 μL / well Block with T20 for 1 hour, followed by washing 3 times with PBS / 0.05% Tween 20 (=wash buffer). Transfer the sample / 1:1 mixture (biotinylated and sulfonated ISV (SEQ ID NO:1) (50.0 μL)) from the polypropylene plate to an MSD plate and incubate at room temperature, 600 rpm for 1 hour. Wash the plate 3 times, then add 2x read buffer (MSD) (150 μL / well) and read ECL units (ECLU) on an MSD instrument (Sector Imager 2400 reader). Use the screening cutoff point determined during method validation to screen samples as positive or negative. Use appropriate statistical analysis as recommended in the guidance on the development of ADA assays (Shankar, 2008), based on background values ​​from 118 single plasma samples from healthy individuals who have never been treated with ISV. Use nonparametric evaluation and after excluding outliers, based on 95 th Cutoff value for percentage calculation.

[0236] Six plasma samples were clearly assessed 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).

[0237] These samples were further analyzed in a drug replacement setting (confirmatory assay) to confirm the specificity of the positive screening results (Table II). Therefore, the sample was diluted 1 / 5 in PBS / 0.1% casein containing 12.5 μg / mL ISV (SEQ ID NO:1) and incubated in a 96-well polypropylene plate at 37°C, 600 RPM for 30 min. Next, the sample (50 μL) was diluted 1 / 3 in a 1:1 mixture (100 μL) of 2 μg / mL biotinylated and 2 μg / mL sulfonated ISV (SEQ ID NO:1) and incubated at room temperature, 600 RPM for 1 h. Subsequently, the sample 1:1 / mixture (biotinylated and sulfonated ISV) (50.0 μL) was transferred from the polypropylene plate to a blocked MSD as described above for the screening assay. 96-well standard streptoacidin plates were incubated at room temperature, 600 rpm for 1 hour. The plates were washed three times, and then 2x read buffer (MSD) (150 μL / well) was added. ECL units (ECLU) were measured on an MSD instrument (Sector Imager 2400 reader). Samples were confirmed as true positives using the confirmatory cutoff determined during method validation, and the confirmatory cutoff was calculated based on the ECL response of 118 single plasma samples from healthy individuals who had never been treated with ISV (enhanced with 50 μg / ml ISV) using appropriate statistical analysis as recommended in the guidance on the development of ADA assays (Shankar, 2008). The 50% minimum signal reduction was calculated based on a 99% confidence interval.

[0238] Samples that were positive in both the ECL-based bridging assay and the drug displacement set-up assay were selected as the source for generating polyclonal antibodies using affinity chromatography.

[0239] Table I: Screening results of 20 plasma samples in the ADA ISV assay

[0240] IHuP#002-001-ABL-01 13081 IHuP#002-001-ABL-02 56 IHuP#002-001-ABL-03 272 IHuP#002-001-ABL-04 125 IHuP#002-001-ABL-05 70 IHuP#002-001-ABL-06 99 IHuP#002-001-ABL-07 170 IHuP#002-001-ABL-08 659358 IHuP#002-001-ABL-09 798 IHuP#002-001-ABL-10 1101 IHuP#002-001-ABL-11 83 IHuP#002-001-ABL-12 72 IHuP#002-001-ABL-13 403 IHuP#002-001-ABL-14 62 IHuP#002-001-ABL-15 1141 IHuP#002-001-ABL-16 159 IHuP#002-001-ABL-17 72 IHuP#002-001-ABL-18 170 IHuP#002-001-ABL-19 4503 IHuP#002-001-ABL-20 8243

[0241] Table II: Confirmatory screening plasma samples in confirmatory assays. 50% of the confirmatory cutoff points were used for outcome evaluation. One sample was not confirmed as a true positive sample.

[0242]

[0243] The above-described ECL-based bridging assay was also used to evaluate three additional serum samples from individuals who were not treated with ISV, and this was confirmed using a drug replacement setting assay.

[0244] Two serum samples were clearly assessed as positive in the ECL-based bridging assay: IHUS#B09032311A3 and IHUS#B09032311A20 (Table III). Further analysis of the two positively screened samples was performed in a drug replacement setting to confirm the specificity of the positive screening results.

[0245] Table III: Screening and confirmatory results of three serum samples and corresponding IgG purification fractions

[0246]

[0247] B. Purified polyclonal IgG fractions were produced.

[0248] Polyclonal IgG was purified from samples IHUS#B09032311A3 and IHUS#B09032311A20 (see above) using Protein G HP Spin Trap Columns (GE Healthcare) according to the manufacturer's instructions. Briefly, after removing the stock solution from the column by centrifugation (100x g for 30 seconds), the column was equilibrated by adding binding buffer (20 mM sodium phosphate, pH 7.0). After centrifugation, a solution containing the desired polyclonal antibody (up to 1 mg in 600 μl) was added, and the column was incubated for 4 minutes while gently mixing. The column was then centrifuged, and the cells were washed twice with binding buffer (600 μl) consecutively, followed by centrifugation. The antibody was eluted by centrifugation in 30 μl of neutralization buffer (1 M Tris-HCl, pH 9.0) after adding 400 μl of elution buffer (0.1 M glycine-HCl, pH 2.7) and mixing by inversion.

[0249] To confirm that the obtained IgG fraction participates in nonspecific binding to ISV(s), the purified IgG antibody was analyzed in the above-described ECL-based bridging assay and confirmed using the drug replacement setting assay as described in A). In both samples (IHUS#B09032311A3 and IHUS#B09032311A20), the purified IgG antibody was confirmed to participate in nonspecific binding, resulting in a positive signal in the assay (Table III). This confirms that the purified polyclonal IgG can be used as an "analytical antibody" and used unchanged in the assays of Examples 3 and 5.

[0250] C. Nonspecific binding with other ISVs

[0251] To determine whether the observed protein interference is specific to a single ISV, and / or specific to a particular region, epitope, or antigenic determinant on an ISV, and / or specific to certain mutations in wild-type ISVs (such as one or more humanized mutations), plasma samples IHUS#B09032311A3, IHUS#B09032311A20, and IHUS#B09032311A1 were used to repeat ECL-based bridging assays and drug replacement setting assays (all described under Section A, with SEQ ID NO:1 used as the sulfonated ISV). Because these plasma samples contain the polyclonal "analytical" antibodies isolated under Section B) above, this also provides information about the specificity, selectivity, and epitope recognition of the said polyclonal analytical antibodies.

[0252] Eight ISVs were tested (SEQ ID NOs 23-30, see Table A above), one of which was the wild-type VHH (SEQ ID NO: 23), and the other seven ISVs were humanized forms of the wild-type sequence with different humanized substitutions. Two ISVs (SEQ ID NOs: 29 and 30) also contained additional amino acid residues at their C-termini (1 and 3 additional alanine residues, respectively).

[0253] The data are shown in Table IV. Without limitation to any interpretation or hypothesis, it can be observed that changes in the C-terminal region (as defined herein) can significantly and strongly affect the degree to which the plasma sample used can produce protein interference. For example, it can be observed that adding one or three amino acid residues to the C-terminus can strongly reduce the tendency for protein interference to occur (e.g., compared to a 90% reduction with respect to SEQ ID NO:28, only 18% and 13% reductions were observed in ECLU assays using sample IHUS#B09032311A3 with respect to SEQ ID NO's:29 and 30, respectively, where SEQ ID NO:28 is the corresponding humanized variant without any amino acid residues added to the C-terminus). Similarly, the introduction of a proline residue at position 14 of the wild-type sequence can also significantly and strongly affect the degree to which the plasma sample used can produce protein interference (e.g., compared to a 91% reduction with respect to SEQ ID NO:24, sample IHUS#B09032311A3 using the wild-type sequence of SEQ ID NO's:23, a wild-type sequence with an A14P substitution, showed only a 20% reduction in ECLU assay). K83R and Q108L, which are also substitutions close to the C-terminus, also lead to some degree of increased tendency to produce protein interference, but not as much as A14P substitution, and the overall combined effect of A14P+K83R+Q108L substitution is offset by the addition of one or more amino acid residues at the C-terminus (again, comparing data for SEQ ID NO's:29 and 30 with data for other humanized variants).

[0254] Based on this data, it can also be concluded that, clearly, polyclonal assay antibodies typically recognize the C-terminal region of ISVs (as defined in this paper). (See also...) Figure 2 It was observed that position 14 (and to a lesser extent, positions 83 and 108) also form part of the C-terminal region of the ISV (when considering the three-dimensional ternary structure of the ISV).

[0255] Table IV: Evaluation of different nanobody variants as competitors in the ISV ADA assay using analytical antibodies

[0256]

[0257] Table IV (continued)

[0258]

[0259] Example 2: Analysis of antibody affinity purification

[0260] This embodiment describes two methods for isolating analytical antibodies capable of recognizing and / or binding to the C-terminus of ISVs from biofluids derived from human subjects. The antibodies were isolated from four different serum samples characterized in that they induced a positive signal in the ADA assay tested according to Example 1.

[0261] Starting with serum samples, each of these methods provides a purified formulation of the interfering factor, which can be used as an analytical antibody in the methods described herein. These methods can also be used more generally for other purposes to purify the interfering factor (e.g., the interfering factor purified using the protocol below was also experimentally used in Example 8 to demonstrate that the binding of the ISV or ISV-construct to monoclonal 21-4 predicts the binding of the same ISV or ISV-construct to the interfering factor, and thus predicts the tendency of the ISV or ISV-construct to suffer nonspecific protein interference in the ADA assay).

[0262] Example 2A: Purification using protein A and affinity chromatography

[0263] In the first step, IgG antibody fractions were enriched from serum samples using protein A affinity chromatography. Typical columns used for this enrichment include HiTrap MabselectSure and MabSelectXtra (GE Healthcare); PorosMabCapture A (Applied Biosystems). In all experiments, IgG antibodies were purified from serum samples in an automated and similar manner. Chromatographic protocols were performed on an AKTA purification system (GE Healthcare), and data were recorded in real-time using UNICORN protein purification software (GE Healthcare). Briefly, serum samples were diluted 1:1 with D-PBS (Dulbecco's phosphate-buffered saline) and filtered through a 0.22 μm filter before being loaded onto the column at a fixed flow rate of 0.5 mL / min. The column was washed with D-PBS at a flow rate of 0.5 mL / min to remove non-specifically bound components within 5 column volumes. IgG fractions were eluted by acidic elution using 100 mM glycine pH 2.6 buffer at a flow rate of 0.5 mL / min. After elution, the fraction was neutralized using 1.5 M Tris buffer at pH 8.8. SDS-PAGE was then performed to confirm the separation of IgG antibodies in the eluent.

[0264] In the second step, interfering IgGs were further enriched by applying protein A-purified IgG fractions from four different sera to an ISV-conjugated affinity column. More specifically, interfering IgGs were further enriched by binding to a column containing an ISV having the sequence of SEQ ID NO:1. For this purpose, the ISV was covalently linked to a Sepharose 4 fast flow (GE Healthcare) using a CNBr (cyanogen bromide) coupling method according to the manufacturer's protocol. Affinity purification was performed automatically and in a similar manner in all experiments. Chromatographic protocols were performed on an AKTA purification system and recorded in UNICORN. Briefly, IgG-enriched samples (up to 10 mL loading volumes) were loaded onto the column at a fixed flow rate of 0.5 mL / min. The column was washed with D-PBS at a flow rate of 0.5 mL / min to remove non-specifically bound components within 5 column volumes. ISV-bound components were eluted by acidic elution at a flow rate of 0.5 mL / min using 100 mM glycine pH 2.6 buffer. After elution, the fraction was neutralized with 1.5M Tris buffer at pH 8.8. SDS-PAGE analysis of the fraction confirmed the separation of IgG antibodies in the eluent (data not shown).

[0265] These sub-sub ...

[0266] Example 2B: Using CaptureSelect TM Purification by chromatography

[0267] Alternatively, interfering factors can be recovered from plasma and the commercially available IgA binding affinity resin CaptureSelecthIgA can be used. TM (BAC BV) (based on a camel-derived heavy-chain variable domain (VHH)) was purified. Subsequently, an 'IgA fraction' containing IgA and interfering IgG was loaded onto a Protein A column to remove the IgA fraction. The Protein A column was treated according to standard IgG purification conditions (run buffer: PBS; elution buffer: 100 mM glycine, pH 2.7; neutralization after elution with 1 M Tris). The interfering factor was recovered from the Prot A elution buffer in >95% yield.

[0268] In a variant of this method, another CaptureSelect affinity resin is used (CaptureSelect Alpha-1 antitrypsin resin, a commercially available affinity resin based on VHH that does not target any antibody-associated proteins). This resin provides high interfering factor binding potency and allows for selective two-step elution: antitrypsin elution using a neutral pH elution buffer of 2.0 M MgCl2, followed by interfering factor elution via an acidic step (0.1 M glycine, pH 3.0, similar to protein A / G elution conditions; neutralization using 1.5 M Tris). This one-step purification produces up to 15 μg of interfering IgG1 per mL of highly interfering plasma, which is approximately 0.3% of the total IgG present. Optionally, the neutralized interfering fraction can be desalted and further purified using a size exclusion column equilibrated in D-PBS.

[0269] Example 3: The effect of different ISV substitutions on the tendency of ISVs to cause protein interference

[0270] As mentioned in the foregoing description, the present invention provides certain assays and techniques for assessing whether a given ISV has a tendency to produce protein interference. These assays and techniques include the ECL-based bridging assay and drug replacement setting assay used in Example 1, the BIACORE assay described in Example 3, and the bridging / competitive ADA assay described in other examples below.

[0271] As also mentioned in the foregoing description, these assays can also be used to determine whether specific alterations (such as amino acid deletions, substitutions, or additions) affect (and preferably reduce) the tendency of a given ISV to produce protein interference. Some of these alterations, based on the disclosure herein and the experimental data listed in Example 1 and the experimental evidence listed in Example 3, are clear or become clear to those skilled in the art.

[0272] As indicated by the data generated in Example 1, certain mutations appear to occur in or near the C-terminal region of the ISV (as defined herein) that can (strongly) affect its tendency to produce protein interference. For example, adding a few amino acid residues to the C-terminus (such as 1 or 3 alanine residues) appears to strongly reduce the tendency of the ISV to produce protein interference and appears to even counteract the presence of other substitutions (e.g., in or near the C-terminus) that appear to increase the tendency to produce protein interference (e.g., A14P substitution).

[0273] In this Example 3, the analytical polyclonal antibody produced in Example 2 was used to investigate the effects of other substitutions and the addition of an additional amino acid to the C-terminus by comparing relevant ISVs with different substitutions. This was achieved using Biacore from GE Healthcare. TM The T100 biosensor analyzes the kinetics of interactions between each ISV studied and the analyzed polyclonal antibody via surface plasmon resonance (SPR). The ISVs tested in Example 3 are those listed in SEQ ID NOs 3-22 (see Table A above and Table V below).

[0274] In a typical experiment, a 10 μg / ml polyclonal antibody solution was prepared in 10 mM NaOAc pH 5.0. Next, following the manufacturer's protocol, the polyclonal antibody was immobilized on a CM5 sensor chip using amine coupling via the EDC / NHS method (EDC = N-ethyl-N'-[3-diethylamino-propyl]-carbodiimide; NHS = N-hydroxysuccinimide). The immobilized amount provided approximately 2700 response units (RU). Then, a 500 nM immobilized concentration of ISV was injected onto the surface at a flow rate of 45 μl / min for 120 seconds. Because an effective regeneration buffer could not 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 the flow cell bound to the polyclonal antibody. The blank flow cell was activated / inactivated in a similar manner to the flow cell for the polyclonal antibody, but without the addition of protein. In addition, blank injections (HBS-EP + running buffer (HBS = Hepes buffered saline: GE Healthcare) were subtracted to correct for possible baseline drift.

[0275] To examine the effect of adding amino acid residues to the C-terminus, the effects of adding 1 or 2 alanine residues and 1, 2 or 3 glycine residues were investigated by comparing the binding of ISVs with different additions using analytical polyclonal antibodies produced as described in Example 2. The ISVs produced and tested for this purpose were NBs 3.4-3.9 (SEQ ID NOs: 5-10).

[0276] As a representative example of the types of data obtained, Figure 3 The binding of NB's 3.4–3.9 to the immobilized polyclonal antibody is shown. Table V summarizes the results obtained.

[0277] Table V

[0278]

[0279] **: Binding signal obtained at the end of injection (= maximum RU signal)

[0280] (1) In this numbering scheme, position 113 is the last “S” of the C-terminal VTVSS base sequence, and positions 114, 115 and 116 are the (downstream) positions immediately following position 113.

[0281] To examine the effects of (other) substitutions in the C-terminal region, the same analytical polyclonal antibody as described above was used to investigate the effects of different substitutions by comparing relevant ISVs containing these substitutions. The analysis was performed as described above.

[0282] The ISVs containing the substitutions tested were NB's 3.1, 3.2 and 3.4 (SEQ ID NO's 3, 4 and 5); NB's 3.10-3.15 (SEQ ID NO's 11-16), which were compared with NB 3.4; NB's 4.1 and 4.2 (SEQ ID NO's 17 and 18) and NB's 6.1, 6.2, 6.4 and 6.5 (SEQ ID NO's 19-22).

[0283] As a representative example of the types of data obtained:

[0284] - Figure 4 The binding of NB's 3.4, 3.11, 3.12 and 3.13 to immobilized polyclonal antibodies is shown;

[0285] - Figure 5 The binding of NB's 3.4, 3.14, and 3.15 to immobilized polyclonal antibodies is shown.

[0286] - Figure 6 The binding of NB's 3.1, 3.2, and 3.4 to immobilized polyclonal antibodies is shown.

[0287] - Figure 7 The binding of NB's 4.1 and 4.2 to immobilized polyclonal antibodies is shown;

[0288] - Figure 8 The binding of NB's 6.1, 6.2, 6.4 and 6.5 to immobilized polyclonal antibodies is shown.

[0289] Tables VI, VII, and VIII summarize the results obtained.

[0290] Table VI

[0291]

[0292] **: Binding signal obtained at the end of injection (= maximum RU signal)

[0293] (1) According to Kabat's numbering system

[0294] Table VII

[0295]

[0296] **: Binding signal obtained at the end of injection (= maximum RU signal)

[0297] (1) According to Kabat's numbering system

[0298] Table VIII

[0299]

[0300] *: If it is "+", this ISV contains other amino acids at the C-terminus of VTVSS.

[0301] **: Binding signal obtained at the end of injection (= maximum RU signal)

[0302] (1): According to the numbering method of Kabat (corresponding to the amino acid at position 87 of SEQ ID NO's 3-5).

[0303] (2): According to the numbering method of Kabat (the amino acids corresponding to positions 123 of SEQ ID NO's 3-5).

[0304] (3): According to the numbering method of Kabat (corresponding to the amino acid at position 86 of SEQ ID NO's 17 and 18).

[0305] (4): According to the numbering method of Kabat (the amino acid at position 116 corresponding to SEQ ID NO's 17 and 18).

[0306] (5): According to the numbering method of Kabat (corresponding to the amino acid at position 86 of SEQ ID NO's 19-22).

[0307] (6): According to the numbering method of Kabat (the amino acid at position 112 corresponding to SEQ ID NO's 19-22).

[0308] Furthermore, without limitation any specific hypothesis or explanation, the above data show that various substitutions of the C-terminal region of ISV (as defined herein) can alter / improve its tendency to produce protein interference.

[0309] Example 4: A representative method for performing the ADA determination shown in Figure 1

[0310] This embodiment provides some representative but non-limiting conditions that can be used for the competitive / bridging ADA determination method schematically shown in Figure 1:

[0311] -in solution Figure 1A ADA assay: 100% matrix sample, 30°C, 37°C, acid treatment with acetic acid in 10% matrix, 5°C, room temperature, pre-incubation / acid neutralization.

[0312] Sample: ISV-sulfonyl(:Tris) 1:1:1 (1:0, 9:0, 9:0, 1), 1 h, room temperature; on plate, 1 h, room temperature; wash 3x, read buffer 4 times.

[0313] -in solution Figure 1B ADA assay: Sample 20% matrix, 30°C, 37°C, preheating.

[0314] Sample: ISV--sulfonyl 1:1:1, 1 h, room temperature, on plate for 1 h, room temperature, wash 3 times, read buffer twice.

[0315] - Figure 1C Continuous ADA assay: ISV-Bio capture, 1 h, room temperature, wash 3 times, sample 20% matrix, 15°C, room temperature, on plate: 2 h, room temperature, wash 3X, detect ALX-0141-sulfonyl, 1 h, room temperature, wash 3 times, read buffer 4X.

[0316] Example 5: Predicting ISV sensitivity to nonspecific protein interference using analytical antibodies

[0317] This embodiment describes a bridging / competitive ADA assay using analytical antibodies, which can be used to predict the sensitivity of ISVs to nonspecific protein interference.

[0318] The ISV to be tested was diluted to a concentration of 10 μg / ml and incubated with 400 ng / ml of analytical antibody, purified according to Example 2, and incubated in a 96-well polypropylene plate at 37°C and 600 rpm. Next, the sample (50 μL) was diluted 1 / 3 in 100 μL of a 1:1 mixture of 2 μg / ml biotinylated and 2 μg / ml sulfonated ISV and incubated at 600 RPM for 1 hour at room temperature. The MSD was then... 96-well standard streptomycin protein plate, 150 μL / well T20 was blocked at room temperature for 1 hour, followed by washing three times with PBS / 0.05% Tween20 (wash buffer). A 1:1 mixture of sample and sulfonated ISV (50.0 μL) was transferred from the polypropylene plate to an MSD plate and incubated at 600 rpm for 1 hour at room temperature. The plate was washed three times, then 2x read buffer (MSD) (150 μL / well) was added, and ECL units (ECLUs) were read on an MSD instrument (Sector Imager 2400 reader).

[0319] Using this assay, ISVs of SEQ ID NOs 23-30 were tested and compared. 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 produce protein interference, but also confirm the effect of substitution in the C-terminal region observed in the previous embodiments. As can be observed, adding three (or at least one) alanine residues to the C-terminus of fully humanized ISVs eliminates their ability to compete with the analytical antibody for binding. Mutating position 14 from alanine to proline in wild-type ISV variants significantly increases their ability to compete as a competitor in the assay (i.e., making the ISV variant more susceptible to nonspecific protein interference), while mutations at positions 83 and 108 did not significantly affect the sensitivity of ISVs to nonspecific protein interference.

[0320] Table IX

[0321]

[0322] Example 6: Effect of adding amino acids to the C-terminus of anti-OX40L nanobodies on their OX40L blocking potential

[0323] This embodiment demonstrates that C-terminal extension has no effect on the activity or blocking potential of nanobodies.

[0324] The in vitro potential of the trivalent bispecific sequence-optimized anti-OX40L nanobody Nb 3.16 (SEQ ID NO:31) was compared with that of the corresponding nanobody Nb 3.17 (SEQ ID NO:32), which contains an additional Ala at its C-terminus.

[0325] The first assay, the T-cell activation assay, was performed as follows. PBMCs were isolated from buffy coats (Red Cross, Ghent, Belgium) from healthy donors using Ficoll Paque Plus reagent (GE Healthcare) and washed with RPMI 1640 complete medium (RPMI 1640 + GlutaMAX + 25 mM HEPES + 10% fetal bovine serum + 1% penicillin / streptomycin; Invitrogen). PBMCs (1 x 10⁻⁶ cells / ml) were stimulated with phytohemagglutinin (PHA-L; final concentration 0.6 μg / ml). 5 (cells / well), then add 1x10 4 CHO cells expressing hOX40L (irradiated with a γ-scintillation counter at 3000 RAD; UZ Gent, Belgium) and anti-OX40L nanobody were incubated in RPMI 1640 complete medium at 37°C for 22 h in a CO2 incubator. IL2 production by PBMCs was measured by ELISA. Wells of Maxisorp plates were coated overnight at 4°C with anti-human IL2 monoclonal antibody (BD Biosciences). After washing and blocking the coated wells, a 1 / 2 dilution of cell supernatant was added. As standards, 1 / 2 serial dilutions of recombinant human IL2 (BD Biosciences) starting at 2000 pg / ml were used for detection using biotinylated anti-human IL2 monoclonal antibody (BD Biosciences), HRP-conjugated streptavidin (Thermo Scientific), and esTMB (SDT Reagents). The reaction was terminated with 1N HCl, and OD was read at 450 nm. As expected, the potential of the trivalent bispecific sequence-optimized nanobody Nb 3.17 (IC50 = 0.13 nM, 95% CI = 0.098–0.17 nM) is comparable to that of Nb 3.16 (IC50 = 0.10 nM, 95% CI = 0.071–0.15 nM).

[0326] In the second ELISA-based competitive assay, a series of diluted nanobody nanobodies (1.5 μM–0.083 pM) were pre-incubated overnight at room temperature with 100 ng / ml human OX40 / Fc (R&D Systems) and 10 ng / ml biotinylated human OX40L (R&D Systems; in-house biotinylated as described in Example 1) in PBS + 0.1% BSA + 0.01% Tween-20. Next, the samples were incubated on Maxisorp plates coated with 10 μg / ml anti-human Fc nanobody (in-house generated) and blocked with PBS + 1% BSA + 0.1% Tween-20. The binding of human OX40 / Fc was detected using HRP-conjugated streptavidin (ThermoScientific) and sTMB (SDT Reagents). The reaction was terminated with 1N HCl and the OD was read at 450 nm. According to cell-based assays, the potential of the trivalent bispecific sequence-optimized nanobody Nb 3.17 (IC50 = 0.178 nM, 95% CI = 0.152–0.200 nM) is comparable to that of Nb 3.16 (IC50 = 0.179 nM, 95% CI = 0.149–0.215 nM).

[0327] Example 7: Production of monoclonal antibody 21-4-3.

[0328] During a 39-day period, two different mouse strains (BALB / c and NMRI - 3 mice per group) were intraperitoneally immunized with the nanobody construct of SEQ ID NO:98 in WO 2006 / 122825, which was in the form of an oil-in-water emulsion of equal volume of antigen and Fluhr's complete or incomplete adjuvant, with booster treatments until a suitable antiserum titer was obtained.

[0329] After asphyxiation of stimulated mice in CO2, the spleen was removed under aseptic conditions, and a single-cell suspension of the conjoined spleen was prepared. Splenic cells and myeloma cells were washed several times with DMEM and fused in the presence of 1 ml of 50% (w / v) PEG 3350 (splenic cell to SP2 / 0 ratio 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 thus generated were resuspended in CGM containing 20% ​​FCS and aminopterin (HAT medium) and plated (140 μl / well) into eight 96-well Corning-Costar tissue culture plates containing 140 μl / well CGM (20% FCS) and peritoneal excudate cells as feeder cells. The plates were incubated for 10 days in complete growth medium (CGM) containing DMEM and additives 2-mercaptoethanol, L-glutamine, stabilized glutamine, HT, and non-essential amino acids (at supplier-recommended concentrations) and different concentrations of FCS (10%, 15%, or 20%). During this period, the cells were fed twice with HAT medium. Cell culture supernatants from hybridoma cells typically contain 1-20 μg / ml of antibody, which is tested in a binding ELISA to confirm binding to the nanobody construct of SEQ ID NO:98 in WO 2006 / 122825.

[0330] Cells from the positive IgG-generating wells were transferred to the wells of a 48-well plate and cultured for 2–4 days (depending on cell growth characteristics). Binding ELISAs for ALX081 and human / cynomolgus monkey IgG were performed to exclude nonspecific conjugates. Hybridoma cells expressing conjugates of the nanobody construct specific to SEQ ID NO:98 in WO 2006 / 122825 were cloned twice using limiting dilution. After fusion and re-screening, 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 cloned again (twice).

[0331] Clone 21-4 (one of the clones that stably produces antibodies against ALX-081 after the second cloning) was named “ABH0015” and deposited on June 4, 2012, at the Belgian Coordinated Collections of Microorganisms (BCCM) in Ghent, Belgium, accession number LMBP9680CB. A mouse monoclonal strain derived from ABH0015 was designated 21-4-3. Isotype determination of 21-4-3 showed the IgG1 heavy chain and κ light chain, which were sequenced (see SEQ ID NO's:35 and 36, respectively). 21-4-3 showed binding to the C-terminal region of the nanobody construct of SEQ ID NO:98 in WO 2006 / 122825 (data not shown).

[0332] Example 8: 21-4 Binding with ISV predicts the tendency of ISV to suffer from non-specific protein interference.

[0333] This example, together with Example 9 below, demonstrates that the binding of monoclonal 21-4 to ISV can be used to predict (within the degree of certainty specified in this example) whether a given ISV is prone to nonspecific protein interference (e.g., in the ADA assay).

[0334] This Example 8 specifically shows that 21-4 can be used to predict whether certain suggested modifications to a given ISV (such as adding one or more amino acid residues to the C-terminus of the ISV and / or replacing one or more amino acid substitutions in the C-terminal region of the ISV) will result in a reduction in the ISV's tendency to suffer from nonspecific protein interference.

[0335] In short, a group of 53 different nanobodies and nanobody constructs were tested (see...). Figure 9 The binding of the same nanobody and nanobody construct to monoclonal 21-4-3 was also tested. The binding of the same nanobody and nanobody construct to purified preparations of interfering factors derived from three different human donors (referred to herein as “donor 8”, “donor 19”, and “donor” 30) was also tested to observe whether there was any association between binding to 21-4 and binding to the purified interfering factor.

[0336] It was confirmed that the combination of ISV with 21-4 can indeed be used to predict the combination of the same ISV with the interference factor (within the total confidence range provided by the data presented in this paper).

[0337] To verify this, as detailed in the experimental data presented below, 53 nanobodies or nanobodies constructs (as described below) were measured using a Biacore T100 (according to the protocol presented below). Figure 9The combination of SEQ ID NO's (38-89) with 21-4, and the combination of this with a reference nanobody or construct (also listed in SEQ ID NO's:38-89) with 21-4, and with .... Figure 9 The results are listed below for comparison, such as those measured using the same Biacore instrument and the same protocol.

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347] For each of the 53 nanobodies or nanobodies constructs tested, a reference was selected such that, when compared with the reference, the tested nanobodies or nanobodies constructs had one or more additional amino acid residues at the C-terminus (these amino acid residues were added to test the effect of the addition on protein interference, and specifically to reduce the interference) and / or one or more mutations in the C-terminal region (e.g., humanization results compared with the reference).

[0348] The results are expressed as the percentage reduction in the binding of a given nanobody (also measured in RU units) compared to the binding of a reference (measured in RU units) – for example, if the measured binding level (RU) of the reference nanobody is 276 and the binding level of the given nanobody (also expressed in RU units) is 9, then the reduction in binding level is at the level of [9 RU / 276 RU] x 100% = 3%, which means a 97% reduction compared to the reference (100%).

[0349] Similarly, the binding of purified interfering factors from each of the three donors to each of the 53 nanobodies or nanobodies constructs was measured using the same Biacore instrument, and compared to the binding of purified interfering factors to the same reference nanobodies or constructs. The results are similarly expressed as a reduction in the percentage of binding of the interfering factor to a given nanobodies or nanobodies construct compared to a reference.

[0350] It was found that for virtually all nanobodies or nanobody constructs (where one or more amino acid residues were added to the C-terminus compared to the reference), the binding of interfering factors was drastically reduced. This again confirms that adding one or more amino acid residues to the C-terminus of an ISV (VTVSS) can reduce non-specific protein interference in ADA assays. It was also found that in most cases, substitution only within the C-terminal region (i.e., not adding one or more amino acid residues to the C-terminus) generally did not have a similarly drastic effect on the binding of interfering factors compared to the reference.

[0351] Further analysis of the data determined that, compared to the reference, the reduction in binding to 21-4 was correlated in all cases with a reduction in binding to each of the three different preparations of the purified interfering factor compared to the reference. Such correlations were found.

[0352] For example, of the 54 nanobodies or nanobody constructs tested, 36 showed a reduction of more than 70% in binding to 21-4 compared to their respective reference sequences (for most of these 36 nanobodies or nanobody constructs having one or more additional amino acid residues at the C-terminus, in some cases with substitution combinations within the C-terminal region). Of these 36, 32 also showed a reduction of more than 50% in binding to the interfering factor compared to the reference (and in many cases, specifically for nanobodies or nanobody constructs with one or more amino acid residues added to the C-terminus, the reduction was much greater than 50%, such as more than 70% or even more than 90%, see data provided in Table X). This confirms that in 32 out of 36 cases (i.e., 89%), a reduction of more than 70% in binding to 21-4 (compared to reference = 100%) predicted a reduction of more than 50% in binding to the interfering factor (compared to the same reference). For clarity, in each case, the reduction will be calculated as 100% - [the percentage of reduction level achieved by the tested nanobody given in the table below].

[0353] Similarly, of the 53 nanobodies or nanobodies constructed tested, 33 showed a reduction of over 90% in binding to 21-4 compared to their respective reference sequences (again, this applies to most of these 33 nanobodies or nanobodies constructed with one or more additional amino acid residues at the C-terminus, in some cases with substitution combinations within the C-terminal region). Of these 33, 32 also showed a reduction of over 50% in binding to the interfering factor compared to their respective reference sequences. This confirms that in 32 out of 33 cases (i.e., 97%), a reduction of over 90% in binding to 21-4 (compared to the reference) predicted a reduction of over 50% in binding to the interfering factor (compared to the same reference).

[0354] It should also be noted that the reduction of more than 50% in the binding of interfering factors (as confirmed by a reduction of more than 70% in the binding with 21-4) means that the interfering factors essentially no longer interfere with the ADA assay for the target ISV: experimental evidence using the ADA assay shows that when the binding to interfering factors is reduced by more than 45%, no significant effect of the presence of the interfering factors on the ADA assay can be observed. In this respect, it will also be clear to those skilled in the art that this is even more true when the binding to interfering factors is reduced to well greater than 50% (e.g., more than 70% or even more than 90%), as observed in some cases (see also the data listed herein).

[0355] In fact, a reduction of more than 45% in the binding of 21-4 has been found to indicate a reduction of more than 45% in the binding of interfering factors, which, as mentioned, means that the interfering factors no longer interfere with the ADA assay.

[0356] Furthermore, the data presented herein regarding the correlation between the binding (reduction) to 21-4 and the binding (reduction) to the interference factor also allows the inventors to set an absolute value for the binding to 21-4 below which it can be expected (within the confidence range provided by the data presented in Example 8) that the ISV or ISV-based construct will not readily bind to the interference factor in a manner that could interfere with the ADA assay. This value is 500 RU (as determined and calculated as presented in Example 9 below).

[0357] The following is the purification of monoclonal antibody 21-4 from the hybridoma culture medium obtained in Example 7 above: Hybridoma cells secreting monoclonal antibody 21-4-3 were cultured in a rotary flask in 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 clarified supernatant was filtered and mouse IgG1 was captured on a Protein A column (HiTrap MabSelectSuRe, 5 mL, GE Healthcare) at a reduced flow rate of 2 mL / min. The bound antibody was eluted in 0.1 M citrate buffer pH 3.0, and the elution fraction was directly neutralized with 1 mL of 1 M TRIS pH 9 (5 mL). The purity of the antibody was confirmed by reducing and non-reducing SDS-PAGE.

[0358] The purified preparations of the interfering factors from donors 8 and 19 were obtained from serum samples from said donors via affinity purification, essentially as described in Example 2A. The interfering factor from donor 30 was obtained from a serum sample from donor 30, essentially as described in Example 2B.

[0359] To determine the binding of 21-4 with each of the nanobodies or nanobodies constructs, the scheme described in Example 9 was used.

[0360] Using interference factors from each of donors 8, 19, and 30 directly immobilized on the CM5 sensor chip, the binding of the interference factors from the three donors to each of the nanobodies or nanobodies constructs was determined using a Biacore T100, essentially as described in Example 3.

[0361] Example 9: A scheme for predicting whether an ISV is subject to nonspecific protein interference (using Monoclonal 21-4)

[0362] Binding measurements were performed using a Biacore T100 sensor chip with a CM5 T120416 sensor chip and running buffer HBS-EP+ at 25°C. 21-4 was captured by immobilized rabbit anti-mouse IgG, as directly immobilized mAb21-4-3 surfaces were found to be inefficiently regenerated. 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 artificial amine conjugation, with 7-minute injections of EDC / NHS for activation and 7-minute injections of 1M ethanolamine HCl pH 8.5 for inactivation (Biacore, Amine Conjugation Kit). Binding conditions are listed in Table XI. The theoretical Rw for the binding of mAb21-4-3 to immobilized anti-mouse IgG is given by the protein immobilization level and MW. max It is ~13000RU (when one mAb21-4-3 molecule binds to one anti-mouse IgG molecule).

[0363] Table XI

[0364]

[0365] Conditions for the binding assay (Biacore T100) using 21-4 immobilized in the manner described are provided in Table XII. Anti-mouse IgG surface could be successfully regenerated after capturing mAb 21-4-3 and injecting all samples (with a limited increase in baseline levels after each regeneration).

[0366] Table XII

[0367]

[0368]

[0369] The above scheme was used to generate the binding data presented in Table X, 21-4. When considering the absolute value of RU (according to the formula ([measured RU] / [protein MW] x 10)... 6 (Regarding the RU values ​​measured for molecular weight adjustments of ISVs, proteins, or peptides), it was found that the nanobodies and nanobody constructs mentioned in Table X (with added alanine residues and showing >90% reduction in binding to 21-4 and interfering factors) typically provide RU values ​​between 30 RU and 400 RU (regarding the corresponding reference nanobodies or peptides - such as in...). Figure 9 The list includes RU values ​​that are more than 1,000, often more than 1,500, and often more than 2,000.

[0370] Based on this, it is believed that an RU value of less than 500 (adjusted) in this assay will clearly indicate that the ISV (or a protein or peptide containing at least one IS, as described herein) will not be bound by interfering factors in a manner that interferes with the ADA assay.

[0371] All references cited throughout this application (including bibliographic references, granted patents, published patent applications, and co-pending patent applications) are hereby clearly incorporated herein by reference, especially in relation to the teachings mentioned herein.

Claims

1. A bioproduct based on an immunoglobulin single variable domain (ISV), comprising one or more ISVs and additionally comprising one or more other structural portions that increase the half-life of the ISV-based bioproduct, said other structural portions being selected from polyethylene glycol, serum albumin, and peptides or binding units capable of binding to serum proteins, wherein said ISV-based bioproduct has an ISV at its C-terminus, the ISV having the sequence VTVSS(X)n at its C-terminus, wherein (X)n is A, AA, AAA, G, GG, GGG, AS, AST, ASTK, ASP, AP, APT, W, or L, and wherein each ISV is VHH or camel-derived VH.

2. The ISV-based bioproduct according to claim 1, wherein each ISV is a sequence-optimized VHH, a humanized VHH, or a camel-derived human VH.

3. The ISV-based bioproduct of claim 1, wherein one or more other structural portions comprise a peptide or binding unit that can bind to a serum protein.

4. The ISV-based bioproduct according to claim 3, wherein the serum protein is serum albumin.

5. The ISV-based bioproduct of claim 1, wherein one or more other structural portions comprise an ISV that binds to serum albumin.

6. The ISV-based biological product according to claim 1, having a half-life of at least 3 days in human subjects, expressed as t1 / 2-β.

7. The ISV-based biological product according to claim 1, used in a treatment method.

8. The ISV-based biological product according to claim 1, used in a treatment method, wherein the treatment includes treating chronic diseases in humans.

9. A pharmaceutical composition comprising: an ISV-based biological product according to any one of claims 1 to 8, and at least one suitable carrier, diluent or excipient.

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