Formulations comprising lunsekimig

Abstract

The present invention relates to pharmaceutical compositions of polypeptides, e.g., immunoglobulin single variable domains. The present invention also relates to methods of preparation, methods for storage and uses of the compositions. The invention further relates to dosage unit forms and medical uses of the compositions. In particular, the present invention relates to pharmaceutical compositions comprising lunsekimig and an amino acid, such as arginine.

Description

[0001] 271 136 ul2 / u52

[0002] Formulations for ISVDs With High Dipol Moments

[0003] 1 Field of the invention

[0004] The present invention relates to pharmaceutical compositions of polypeptides, e.g., immunoglobulin single variable domains, such as Nanobody® molecules.

[0005] The present invention also relates to methods of preparation, methods for storage and uses of the compositions. The invention further relates to dosage unit forms and medical uses of the compositions.

[0006] 2 Background of the invention

[0007] Immunoglobulin single variable domains (ISVDs), such as camelid VHH domains, camelized VH domains or humanized VHH domains, represent a rapidly growing class of antibody therapeutics.

[0008] Polypeptides such as ISVDs are typically formulated in liquid compositions to be administered to a patient. However, it has been observed that formulations comprising this type of drugs may be viscous as the concentration of the drug increases and furthermore show a high opalescence, which is above the highest pharmacopoeial opalescence reference standard available (STD IV).

[0009] Opalescence can have different causes and raises several questions and concerns regarding drug efficacy and safety. In addition to reducing the aesthetic appeal of a formulation, high opalescence makes it difficult to detect possible microbial contamination. Moreover, the presence of aggregates in pharmaceuticals can reduce the product quality and efficacy and seriously impact the patient safety.

[0010] Opalescence can be defined as a type of dichroism (reflection of iridescent light) in highly dispersed systems due to the Rayleigh scattering, the product results as a visual turbid solution exhibiting a blue sheen under high light exposure and against a black background.

[0011] Furthermore, opalescence has also been described as being due to molecular crowding and excluded volume effects when increasing the concentration of protein molecules. This molecular crowding is driven by self-attractions via nonspecific or weak interactions. As a result, the protein's diffusion is slowed down which tends to increase solutions viscosities. The diffusion of proteins in highly concentrated solutions is not directed solely by the Brownian motion, but also to long- and short-range protein-protein interactions (electrostatic, ionic, Van der Waals, etc.). These nonspecific attractive interactions can 271 136 ul2 / u52 result in reversible self-association and may lead to aggregation, high viscosity, opalescence and / or phase separation.

[0012] It has also been described that kinetically driven liquid / liquid phase separation (LLPS) is associated with opalescence. In that case the opalescence itself could be caused by the loss of homogeneity due to the formation of droplets of condensed phases (Wang et al. 2011, PNAS 108(40), 16606-16611). Even if not visible by the eye, these microdroplets of the protein-rich-phase are scattering visible light strongly which results in an opalescent shine. These droplets, also referred as density / concentration fluctuations, increase the light scattering. This is similar to what aggregation or particulates in suspension will do in solution and that is described herein as turbidity. In the case of opalescence arising without aggregation, the Rayleigh scattering property is inherent to the product itself (microdroplets or molecular crowding effects).

[0013] Opalescence is dependent on the nature of the protein and mostly determined by its capacity to self-interact via weak attractions. High opalescence is often observed for molecules with high dipole moments due to the resulting electrostatic interactions. The formulation plays a crucial role in mitigating such high opalescence levels and the associated issues. Numerous formulation factors can have an impact on protein-protein interactions: ionic strength, pH, protein concentration, nature of the excipients can alter these interactions and therefore impact the way proteins behave in solution.

[0014] Arginine has been reported to decrease the opalescence of compositions comprising immunoglobulin G in Oki et al., International Journal of Biological Macromolecules 2018, 118, 1708-1712. However, polypeptides such as ISVDs behave differently in a formulation due to their difference in size and structure resulting in different electrochemical properties. Therefore it is not possible to predict their interaction with charged amino acids such as arginine.

[0015] There remains a need for providing formulations for polypeptides such as ISVDs, and in particular ISVDs with high dipole moments, which allow for achieving high concentrations of the polypeptide in the formulations, while maintaining the opalescence, the viscosity and the LLPS of the composition within the regulatory limits for administration as well as a good stability during storage.

[0016] 3 Summary of the invention

[0017] The present invention provides a pharmaceutical composition comprising a polypeptide comprising at least two portions whose isoelectric points differ by at least two pH units, and an amino acid having an isoelectric point of 7 or higher or a salt thereof. 271 136 ul2 / u52

[0018] The present inventors have found that when using polypeptides displaying a dipole moment within the molecule, good reduction of opalescence can be achieved by addition of an amino acid with an isoelectric point of 7 or higher even when the polypeptide is present at high concentration and in the presence of various other pharmaceutical excipients.

[0019] It has been found that for these kinds of polypeptides, electrostatic self-attraction may be modulated by formulation changes and in particular by introducing an amino acid having an isoelectric point of 7 or higher, without compromising the secondary structure of the polypeptide. By adding such an amino acid, opalescence of the product may be decreased to a more desirable level according to pharmacopoeial standards. The viscosity and the LLPS may be also reduced and an acceptable formulation for pharmaceutical use may be obtained with good stability over time, acceptable osmolality and high polypeptide concentration.

[0020] As a result, the compositions of the invention ensure a high concentration of polypeptides, such as 150 mg / mL, while maintaining properties acceptable for pharmaceutical administration such as opalescence, viscosity, LLPS and stability upon storage. They are suitable for pharmaceutical preparations and compatible with pharmaceutically acceptable diluents, such as saline, Ringer's solution or glucose / dextrose solution.

[0021] In particular, the present invention provides the following embodiments: i) A pharmaceutical composition comprising: a polypeptide that comprises at least two portions whose isoelectric points differ by at least two pH units, and an amino acid having an isoelectric point of 7 or higher, or a salt thereof. ii) The pharmaceutical composition according to i), wherein the amino acid is L- arginine or a salt thereof, optionally wherein the amino acid is L-arginine hydrochloride. iii) The pharmaceutical composition according to i) or ii), wherein the pH of the pharmaceutical composition is 5 or more and 11 or less. iv) The pharmaceutical composition according to any one of i) to iii), wherein the polypeptide comprises one or more portions having an isoelectric point of 6 or lower and one or more portions having an isoelectric point of 8 or higher. v) The pharmaceutical composition according to any one of i) to iv), wherein each of the two portions includes at least 50 contiguous amino acid residues. 271 136 ul2 / u52 vi) The pharmaceutical composition according to any one of i) to v), wherein at least one portion of the polypeptide is an immunoglobulin single variable domain (ISVD). vii) The pharmaceutical composition according to any one of i) to vi), wherein at least one portion of the polypeptide is an ISVD having an isoelectric point of 6 or lower and at least one portion of the polypeptide is an ISVD having an isoelectric point of 8 or higher. viii) The pharmaceutical composition according to any one of i) to vii), further comprising a buffer and one or more nonionic surfactants. ix) The pharmaceutical composition according to viii), wherein the buffer is selected from sodium phosphate buffer, HEPES buffer, sodium acetate buffer and histidine hydrochloride buffer, and the nonionic surfactant is selected from polysorbates and block copolymers of polyethylene and polypropylene glycol, optionally wherein the nonionic surfactant is polysorbate 20 and / or Poloxamer 188. x) The pharmaceutical composition according to any one of i) to ix), wherein the amino acid is present at a concentration of at least 50 mM. xi) The pharmaceutical composition according to x), wherein the amino acid is present at a concentration of at least 200 mM. xii) The pharmaceutical composition according to any one of i) to xi), wherein the polypeptide is present at a concentration of at least 10 mg / mL. xiii) The pharmaceutical composition according to any one of i) to xii), wherein the polypeptide is present at a concentration of at least 50 mg / mL. xiv) The pharmaceutical composition according to any one of i) to xiii), wherein the polypeptide is present at a concentration of at least 100 mg / mL. xv) The pharmaceutical composition according to any one of i) to xiv), comprising: the polypeptide at a concentration of 100 mg / mL to 300 mg / mL; the amino acid at a concentration of 200 mM to 500 mM, wherein the amino acid is L-arginine or a salt thereof; sodium phosphate buffer at a concentration of 5 mM to 20 mM;

[0022] Poloxamer 188 at a concentration of 0.01 % (w / v) to 0.1 % (w / v) and wherein the composition has a pH of 6 to 8. 271 136 ul2 / u52 xvi) The pharmaceutical composition according to any one of i) to xv), wherein the polypeptide comprises or consists of at least four ISVDs, wherein two ISVDs specifically bind IL-13 and two ISVDs specifically bind TSLP, wherein each of said at least four ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least four ISVDs are optionally linked via one or more peptidic linkers, and wherein: a first ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 7, a CDR2 that is the amino acid sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid sequence of SEQ ID NO: 17, a second ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid sequence of SEQ ID NO: 18, a third ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of SEQ ID NO: 19, and a fourth ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 11, a CDR2 that is the amino acid sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid sequence of SEQ ID NO: 21. xvii) The pharmaceutical composition according to xvi), wherein the polypeptide further comprises a fifth ISVD that binds to human serum albumin, wherein the fifth ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 10, a CDR2 that is the amino acid sequence of SEQ ID NO: 15; and a CDR3 that is the amino acid sequence of SEQ ID NO: 20; and the fifth ISVD is positioned between the third and the fourth ISVD. xviii) The pharmaceutical composition according to any one of i) to xvii), wherein the polypeptide comprises or consists of the amino acid sequence SEQ ID NO:1. 271 136 ul2 / u52 xix) The pharmaceutical composition according to any one of i) to xviii), wherein the polypeptide is lunsekimig. xx) The pharmaceutical composition according to any one of i) to xix), comprising: the polypeptide at a concentration of 150 mg / mL, wherein the polypeptide is lunsekimig, the amino acid at a concentration of 230 mM, wherein the amino acid is L-arginine hydrochloride, sodium phosphate buffer at a concentration of 10 mM,

[0023] Poloxamer 188 at a concentration of 0.02 % (w / v); and wherein the composition has a pH of 7.0. xxi) The pharmaceutical composition according to any one of i) to xv), wherein at least one portion of the polypeptide is an ISVD having an isoelectric point of 8 or higher, and at least one portion of the polypeptide is an antibody Fc region having an isoelectric point of 6 or lower. xxii) The pharmaceutical composition according to xxi), wherein the antibody Fc region is an lgG4 Fc region. xxiii). The pharmaceutical composition according to xxi) or xxii), wherein the polypeptide comprises at least one portion having an isoelectric point of 8 or higher which is an ISVD, at least one portion having an isoelectric point of 8.5 or higher which is an ISVD, at least one portion having an isoelectric point of 6 or lower which is an antibody Fc region, and at least one portion having an isoelectric point of 6.5 or lower which is an antibody Fc region xxiv) The pharmaceutical composition according to any one of i) to xxiii) for use in the treatment of asthma, chronic obstructive pulmonary disease, atopic dermatitis or chronic rhinosinusitis with nasal polyps in a subject.

[0024] 4 Brief description of the figures

[0025] Figure 1 Schematic illustration of the immunoglobulin single variable domains (ISVDs) of lunsekimig (SAR443765) and their isoelectric points.

[0026] Figure 2 Graphical representation of KD analysis of Comparative Example la and Examples la-e.

[0027] Figure 3 Graphical representation of KD analysis of Comparative Examples la-e.

[0028] Figure 4 Schematic illustration of the ISVD-Fc constructs tested in Example 1 iii). 271 136 ul2 / u52

[0029] Figure 5 Graphical representation of KD analysis of Examples lk, m, and o.

[0030] Figure 6 Visual appearance of formulations containing Example II and histidine 10 mM in combination with increasing concentrations of arginine.

[0031] Figure 7 Visual appearance of Example 4b including reference standards and buffer against black and white background.

[0032] Figure 8 Graphical representation of OD500 results of Example 5.

[0033] Figure 9 Graphical representation of OD500 results of Comparative Example 5.

[0034] Figure 10 Visual appearance of Example 5.

[0035] Figure 11 Visual appearance of Example 7a and Example 7b after 4 and 24 weeks storage against a black background.

[0036] Figure 12 Graphical representation of opalescence (NTU) measured during the stability study of Example 7 at 5°C and 25°C.

[0037] Figure 13 (a) / (b) Graphical representation of opalescence (NTU) of Example 8.

[0038] 5 Detailed description of the invention

[0039] 5.1 Definitions

[0040] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person.

[0041] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition that comprises a list of components is not necessarily limited to only those components but may include other components that are not expressly listed or inherent to such a composition. The terms "comprises", "comprising", "includes", "including", "has", "having" or any other variation thereof also cover the disclosed embodiment having no further additional components (i.e., consisting of those components).

[0042] Also, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to be non-restrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0043] Further, as used herein, the term weight percent (% w / v) is based on the total volume of the composition unless specified otherwise. The term mg / mL refers on the amount of component per volume in mL of the total composition. 271 136 ul2 / u52

[0044] 5.2 Pharmaceutical composition

[0045] The composition of the invention is a pharmaceutical composition. The term "formulation" is also used herein to refer to the pharmaceutical composition. The term "pharmaceutical" means that the composition is suitable for pharmaceutical use.

[0046] In particular, the pharmaceutical composition may be a liquid composition. The term "liquid" is used throughout this disclosure to mean that the composition or ingredient in question takes the form of a liquid at NIST STP, that is, at 25°C and 1 atm (101.325 kPa).

[0047] That is, the pharmaceutical composition may contain a liquid carrier. The liquid carrier may be an aqueous liquid, for example water.

[0048] Water that may be comprised in the pharmaceutical composition is for example water for injection, in an amount sufficient to achieve the specified concentrations of the respective ingredients.

[0049] The pharmaceutical composition may be sterile. For example, the components of the composition can be combined to form a solution and then the solution can be sterile filtered to provide the sterile composition.

[0050] 5.3 Polypeptides

[0051] The pharmaceutical composition comprises a polypeptide comprising at least two portions whose isoelectric points differ by at least two pH units. The pharmaceutical composition may comprise one or more polypeptide(s) satisfying this requirement.

[0052] The term "polypeptide" as used in this disclosure refers to an organic compound comprising a chain of amino acids bond together with peptide bonds (also referred as "amino acid residues" herein). The polypeptide may comprise at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, or at least 400 amino acids.

[0053] The weight average molecular weight of the polypeptide may be 20 kDa or more, 30 kDa or more, 35 kDa or more, 40 kDa or more, or 50 kDa or more. Further, the weight average molecular weight of the polypeptide may be 200 kDa or less, 150 kDa or less, 100 kDa or less, or 90 kDa or less.

[0054] Any of the disclosed lower limits for the weight average molecular weight of polypeptide may be combined with any of the disclosed upper limits to define further suitable weight average molecular weight ranges for the purposes of this invention. For instance, 271 136 ul2 / u52 exemplary ranges for the weight average molecular weight of polypeptide include 20 kDa to 150 kDa, 20 kDa to 100 kDa, 40 kDa to 150 kDa, and 40 kDa to 100 kDa.

[0055] In one embodiment of the present invention, the weight average molecular weight of polypeptide is 50 kDa to 90 kDa.

[0056] If more than one polypeptide satisfying the isoelectric point characteristics of the present invention is present, each polypeptide has a weight average molecular weight within the above ranges.

[0057] 5.3.1 Isoelectric point of the portions of the polypeptide

[0058] The polypeptide used in the pharmaceutical composition comprises at least two portions whose isoelectric points differ by at least two pH units.

[0059] The term "isoelectric point" as used in this disclosure refers to the pH at which a particular molecule or a portion of a molecule carries no net electrical charge. The isoelectric point (pl) of the polypeptide may be determined by any means known to those skilled in the art. For example, the pl may be determined by denatured isoelectric focusing.

[0060] The term "portion" or "fragment" as used interchangeably in this disclosure refers to a part of the polypeptide which comprises less than the full-length amino acid sequence. Portions may include an amino acid sequence of at least 50 contiguous amino acid residues, at least 75 contiguous amino acid residues, or at least 100 contiguous amino acid residues. Further, the portion may include 500 contiguous amino acid residues or less, 300 contiguous amino acid residues or less, 250 contiguous amino acid residues or less, 200 contiguous amino acid residues or less, or 150 contiguous amino acid residues or less.

[0061] In one embodiment, the portion includes 100 to 150 contiguous amino acid residues.

[0062] In particular, the "portion" or "fragment" can be a protein domain, such as an ISVD, a Fc fragment, a Fab fragment, a single chain Fv, an immunoglobulin domain, and a coiled-coil domain. In particular, the "portion" or "fragment" can be an ISVD or a Fc domain.

[0063] For example, the polypeptide may comprise one or more portions having an isoelectric point of 6 or lower and one or more portions having an isoelectric point of 8 or higher. I n one embodiment, the polypeptide comprises two portions having an isoelectric point of 6 or lower and two portions having an isoelectric point of 8 or higher. Further, in one embodiment, the polypeptide further comprises at least one portion having an isoelectric point above 6 and below 8, for example between 6.5 and 7.5. 271 136 ul2 / u52

[0064] For example, at least one portion of the polypeptide is an immunoglobulin single variable domain (ISVD) as defined below. For example, at least one portion having an isoelectric point of 6 or lower is an ISVD and at least one portion having an isoelectric point of 8 or higher is an ISVD. Further, in one embodiment the polypeptide comprises at least two portions having an isoelectric point of 6 or lower which are ISVDs and at least two portions having an isoelectric point of 8 or higher which are ISVDs. In one embodiment, the polypeptide further comprises at least one portion having an isoelectric point above 6 and below 8 which is an ISVD.

[0065] In one embodiment, the polypeptide comprises 4 to 6 portions each being an ISVD, wherein at least two portions have an isoelectric point of 6 or lower and at least two portions have an isoelectric point of 8 or higher. Further, in one embodiment, the polypeptide further comprises at least one portion having an isoelectric point above 6 and below 8, for example between 6.5 and 7.5, which is an ISVD.

[0066] In another embodiment, at least one portion of the polypeptide is an immunoglobulin single variable domain (ISVD) as defined below and another portion of the polypeptide is an antibody Fc region. For example, at least one portion having an isoelectric point of 6 or lower is an antibody Fc region, such as an lgG4 Fc region, and at least one portion having an isoelectric point of 8 or higher is an ISVD. In one embodiment, at least one portion having an isoelectric point of 6.5 or lower is an antibody Fc region, such as an lgG4 Fc region, and at least one portion having an isoelectric point of 8.5 or higher is an ISVD. In one embodiment, at least one portion having an isoelectric point of 7 or lower is an antibody Fc region, such as an lgG4 Fc region, and at least one portion having an isoelectric point of 9 or higher is an ISVD.

[0067] In one embodiment, the polypeptide comprises at least two portions which are ISVDs and at least two portions which are antibody Fc regions. In one embodiment, the polypeptide comprises at least one portion having an isoelectric point of 8 or higher which is an ISVD, at least one portion having an isoelectric point of 8.5 or higher which is an ISVD, at least one portion having an isoelectric point of 6 or lower which is an antibody Fc region, and at least one portion having an isoelectric point of 6.5 or lower which is an antibody Fc region. The polypeptide may further comprise a portion having an isoelectric point of 7 or higher which is an ISVD.

[0068] Further, in one embodiment the polypeptide comprises at least one portion having an isoelectric point of preferably 6 or lower, or 6.5 or lower, or 7 or lower, which is an antibody Fc region, such as an lgG4 Fc region, and at least two portions independently having an isoelectric point of preferably 8 or higher, or 8.5 or higher, or 9 or higher, which 271 136 ul2 / u52 are ISVDs. In one embodiment, the polypeptide further comprises at least one portion having an isoelectric point above 6 and below 8 which is an ISVD.

[0069] In some embodiments, one portion has an isoelectric point of 3 or lower, and another portion has an isoelectric point of 5 or higher. In some embodiments, one portion has an isoelectric point of 4 or lower, and another portion has an isoelectric point of 6 or higher. In some embodiments, one portion has an isoelectric point of 5 or lower, and another portion has an isoelectric point of 7 or higher. In some embodiments, one portion has an isoelectric point of 6 or lower, and another portion has an isoelectric point of 8 or higher. In some embodiments, one portion has an isoelectric point of 7 or lower, and another portion has an isoelectric point of 9 or higher. In some embodiments, one portion has an isoelectric point of 8 or lower, and another portion has an isoelectric point of 10 or higher. In some embodiments, one portion has an isoelectric point of 9 or lower, and another portion has an isoelectric point of 11 or higher. In some embodiments, one or more portion(s) having an isoelectric point as defined above is an ISVD. In some embodiments, one or more portion(s) having an isoelectric point as defined above is an antibody Fc region, for example, an lgG4 Fc region.

[0070] 5.3.2 Immunoglobulin single variable domains (ISVDs)

[0071] 5.3.2.1 General definitions

[0072] The polypeptide may comprise immunoglobulin single variable domains (ISVDs).

[0073] The term "immunoglobulin single variable domain" (ISVD), interchangeably used with "single variable domain", defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets ISVDs apart from "conventional" immunoglobulins (e.g., monoclonal antibodies) or their fragments (such as Fab, Fab', Ffab' , scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation.

[0074] In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulfide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain 271 136 ul2 / u52 antibody, would normally not be regarded as an ISVD, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.

[0075] In contrast, ISVDs are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain. The binding site of an ISVD is formed by a single VH, a single VHH or single VL domain. Hence, the antigen binding site of an ISVD is formed by no more than three CDRs.

[0076] As such, the single variable domain may be a light chain variable domain sequence (e.g., a V sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).

[0077] An ISVD can for example be a heavy chain ISVD, such as a VH, VHH, including a camelized VH or humanized VHH. In one embodiment, it is a VHH, including a camelized VH or humanized VHH. Heavy chain ISVDs can be derived from a conventional four-chain antibody or from a heavy chain antibody.

[0078] For example, the ISVD may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody® ISVD (as defined herein and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof.

[0079] In particular, the ISVD may be a Nanobody® ISVD (such as a VHH, including a humanized VHH or camelized VH) or a suitable fragment thereof. Nanobody® and Nanobodies® are registered trademarks of Ablynx N.V.

[0080] "VHH domains", also known as VHHS, VHH antibody fragments and VHH immunoglobulins, have originally been described as the antigen binding immunoglobulin variable domain of "heavy chain antibodies" (i.e., of "antibodies devoid of light chains"; Hamers-Casterman et al. 1993 (Nature 363: 446-448). The term "VHH domain" has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains") 271 136 ul2 / u52 and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHH'S, reference is made to the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74: 277-302).

[0081] For the term "dAb's" and "domain antibody", reference is for example made to Ward et al. 1989 (Nature 341: 544), to Holt et al. 2003 (Trends Biotechnol. 21: 484); as well as to for example WO 2004 / 068820, WO 2006 / 030220, WO 2006 / 003388 and other published patent applications of Domantis Ltd. It should also be noted that single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 2005 / 18629).

[0082] Typically, the generation of immunoglobulins involves the immunization of experimental animals, fusion of immunoglobulin producing cells to create hybridomas and screening for the desired specificities. Alternatively, immunoglobulins can be generated by screening of naive, immune or synthetic libraries e.g., by phage display.

[0083] The generation of immunoglobulin sequences, such as VHHs, has been described extensively in various publications, among which WO 1994 / 04678, Hamers-Casterman et al. 1993 (Nature 363: 446-448) and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74: 277-302, 2001). In these methods, camelids are immunized with the target antigen in order to induce an immune response against said target antigen. The repertoire of VHHs obtained from said immunization is further screened for VHHs that bind the target antigen.

[0084] In these instances, the generation of antibodies requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources, or in the course of recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0085] Immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences can be used herein. Also, fully human, humanized or chimeric sequences can be used in the method described herein. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g., camelized dAb as described by Ward et al. 1989 (Nature 341: 544), WO 1994 / 04678, and Davis and Riechmann (1994, Febs Lett., 339:285-290; and 1996, Prot. Eng., 9:531-537) can be used herein. Moreover, the ISVDs are fused forming a multivalent and / or multispecific construct (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, 271 136 ul2 / u52 reference is also made to Conrath et al. 2001 (J. Biol. Chem., Vol. 276, 10. 7346-7350) as well as to for example WO 1996 / 34103 and WO 1999 / 23221).

[0086] A "humanized VHH" comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g., indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the prior art (e.g., WO 2008 / 020079). Again, it should be noted that such humanized VHHS can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material.

[0087] A "camelized VH" comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a (camelid) heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the description in the prior art (e.g., Davies and Riechman 1994, FEBS 339: 285; 1995, Biotechnol. 13: 475; 1996, Prot. Eng. 9: 531; and Riechman 1999, J. Immunol. Methods 231: 25). Such "camelizing" substitutions are inserted at amino acid positions that form and / or are present at the VH- VL interface, and / or at the so-called Camelidae hallmark residues, as defined herein (see for example WO 1994 / 04678 and Davies and Riechmann 1994 and 1996, supra). In one embodiment, the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is a VH sequence from a mammal, such as the VH sequence of a human being, such as a VH3 sequence. However, it should be noted that such camelized VH can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.

[0088] The structure of an immunoglobulin single variable domain sequence can be considered to be comprised of four framework regions ("FRs"), which are referred to in the art and herein as "Framework region 1" ("FR1"); as "Framework region 2" ("FR2"); as "Framework region 3" ("FR3"); and as "Framework region 4" ("FR4"), respectively; which framework 271 136 ul2 / u52 regions are interrupted by three complementary determining regions ("CDRs"), which are referred to in the art and herein as "Complementarity Determining Region 1" ("CDR1"); as "Complementarity Determining Region 2" ("CDR2"); and as "Complementarity Determining Region 3" ("CDR3"), respectively.

[0089] In such an immunoglobulin sequence, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.

[0090] The framework sequences are (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences may be framework sequences derived from a light chain variable domain (e.g., a V sequence) and / or from a heavy chain variable domain (e.g., a VH- sequence or VHH sequence). In one particular aspect, the framework sequences are either framework sequences that have been derived from a VHH-sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences that have been camelized (as defined herein).

[0091] In particular, the framework sequences present in the ISVD sequence described herein may contain one or more of hallmark residues (as defined herein), such that the ISVD sequence is a Nanobody® ISVD, such as e.g., a VHH, including a humanized VHH or camelized VH. Non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

[0092] The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0093] However, it should be noted that the ISVDs described herein is not limited as to the origin of the ISVD sequence (or of the nucleotide sequence used to express it), nor as to the way that the ISVD sequence or nucleotide sequence is (or has been) generated or obtained. Thus, the ISVD sequences may be naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences. In a specific but non-limiting aspect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or 271 136 ul2 / u52 rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized VH sequences), as well as ISVDs that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.

[0094] Similarly, nucleotide sequences may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.

[0095] Generally, Nanobody® ISVDs (in particular VHH sequences, including (partially) humanized VHH sequences and camelized VH sequences) can be characterized by the presence of one or more "Hallmark residues" (as described herein) in one or more of the framework sequences (again as further described herein). Thus, generally, a Nanobody® ISVD can be defined as an immunoglobulin sequence with the (general) structure:

[0096] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the Hallmark residues are as further defined herein.

[0097] In particular, a Nanobody® ISVD can be an immunoglobulin sequence with the (general) structure:

[0098] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the framework sequences are as further defined herein. 271 136 ul2 / u52

[0099] More in particular, a Nanobody® ISVD can be an immunoglobulin sequence with the (general) structure:

[0100] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A below.

[0101] Table A: Hallmark Residues in Nanobody® ISVDs 271 136 ul2 / u52

[0102] In one embodiment, the immunoglobulin single variable domain has certain amino acid substitutions in the framework regions effective in preventing or reducing binding of so- called "pre-existing antibodies" to the polypeptides. ISVDs in which (i) the amino acid residue at position 112 is one of K or Q; and / or (ii) the amino acid residue at position 89 is T; and / or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is for example V have been described in WO2015 / 173325.

[0103] 5.3.2.2 ISVDs in the polypeptides

[0104] In the present invention, the ISVDs may form part of a protein or polypeptide, which may comprise or essentially consist of one or more (at least one) ISVDs and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). The term "immunoglobulin single variable domain" may also encompass such polypeptides. The one or more ISVDs may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acids that can serve as a binding unit, so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al. 2001 (J. Biol. Chem. 276: 7346), as well as to for example WO 1996 / 34103, WO 1999 / 23221 and WO 2010 / 115998). 271 136 ul2 / u52

[0105] The polypeptides may comprise or essentially consist of one ISVD, as outlined above. Such polypeptides are also referred to herein as monovalent polypeptides.

[0106] The term "multivalent" indicates the presence of multiple ISVDs in a polypeptide. In one embodiment, the polypeptide is "bivalent", i.e., comprises or consists of two ISVDs. In one embodiment, the polypeptide is "trivalent", i.e., comprises or consists of three ISVDs. In another embodiment, the polypeptide is "tetravalent", i.e. comprises or consists of four ISVDs. The polypeptide can thus be "bivalent", "trivalent", "tetravalent", "pentavalent", "hexavalent", "heptavalent", "octavalent", "nonavalent", etc., i.e., the polypeptide comprises or consists of two, three, four, five, six, seven, eight, nine, etc., ISVDs, respectively. In one embodiment the multivalent ISVD polypeptide is trivalent. In another embodiment the multivalent ISVD polypeptide is tetravalent. In still another embodiment, the multivalent ISVD polypeptide is pentavalent.

[0107] In one embodiment, the multivalent ISVD polypeptide can also be multispecific. The term "multispecific" refers to binding to multiple different target molecules (also referred to as antigens). The multivalent ISVD polypeptide can thus be "bispecific", "trispecific", "tetraspecific", etc., i.e., can bind to two, three, four, etc., different target molecules, respectively.

[0108] For example, the polypeptide may be bispecific-trivalent, such as a polypeptide comprising or consisting of three ISVDs, wherein two ISVDs bind to a first target and one ISVD binds to a second target different from the first target. In another example, the polypeptide may be trispecific-tetravalent, such as a polypeptide comprising or consisting of four ISVDs, wherein one ISVD binds to a first target, two ISVDs bind to a second target different from the first target and one ISVD binds to a third target different from the first and the second target. In still another example, the polypeptide may be trispecific- pentavalent, such as a polypeptide comprising or consisting of five ISVDs, wherein two ISVDs bind to a first target, two ISVDs bind to a second target different from the first target and one ISVD binds to a third target different from the first and the second target.

[0109] In one embodiment, the multivalent ISVD polypeptide can also be multiparatopic. The term "multiparatopic" refers to binding to multiple different epitopes on the same target molecules (also referred to as antigens). The multivalent ISVD polypeptide can thus be "biparatopic", "triparatopic", etc., i.e., can bind to two, three, etc., different epitopes on the same target molecules, respectively.

[0110] In another aspect, the polypeptide of the invention that comprises or essentially consists of one or more ISVDs (or suitable fragments thereof), may further comprise one or more 271 136 ul2 / u52 other groups, residues, moieties or binding units. Such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the immunoglobulin single variable domain (and / or to the polypeptide in which it is present) and may or may not modify the properties of the immunoglobulin single variable domain.

[0111] For example, such further groups, residues, moieties or binding units may be one or more additional amino acids, such that the compound, construct or polypeptide is a (fusion) protein or (fusion) polypeptide. In another but non-limiting aspect, said one or more other groups, residues, moieties or binding units are immunoglobulins. In a further aspect, said one or more other groups, residues, moieties or binding units are ISVDs, such as ISVDs chosen from the group consisting of VHH, including humanized VHHs, such as VHs, including human VHs, camelized VHs and camelized human VHs, such as domain antibodies, single domain antibodies, and / or "dAb"s.

[0112] Alternatively, such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and / or pharmacologically active. For example, and without limitation, such groups may be linked to the one or more immunoglobulin single variable domain so as to provide a "derivative" of the immunoglobulin single variable domain.

[0113] In another embodiment, said further residues may be effective in preventing or reducing binding of so-called "pre-existing antibodies" to the polypeptides. For this purpose, the polypeptides and constructs may contain a C-terminal extension (X)n (in which n is 1 to 10, or 1 to 5, such as 1, 2, 3, 4 or 5 (and in particular 1 or 2, such as 1); and each X is an (for example naturally occurring) amino acid residue that is independently chosen, and for example independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I), for which reference is made to WO 2012 / 175741. Accordingly, the polypeptide may further comprise a C-terminal extension (X)n, in which n is 1 to 5, such as 1, 2, 3, 4 or 5, and in which X is a naturally occurring amino acid, or no cysteine.

[0114] In the polypeptides described above, the one or more immunoglobulin single variable domains and the one or more groups, residues, moieties or binding units may be linked directly to each other and / or via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are amino acids, the linkers may also be an amino acid, so that the resulting polypeptide is a fusion protein or fusion polypeptide. 271 136 ul2 / u52

[0115] As used herein, the term "linker" denotes a peptide that fuses together two or more ISVDs into a single molecule. The use of linkers to connect two or more (poly)peptides is well known in the art. Further exemplary peptidic linkers are shown in Table B. One often used class of peptidic linker are known as the "Gly-Ser" or "GS" linkers. These are linkers that essentially consist of glycine (G) and serine (S) residues, and usually comprise one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO: 23) motif (for example, having the formula (Gly-Gly-Gly-Gly-Ser)n in which n may be 1, 2, 3, 4, 5, 6, 7 or more). Some often-used examples of such GS linkers are 9GS linkers (GGGGSGGGS, SEQ ID NO: 26), 15GS linkers (n=3) and 35GS linkers (n=7). Reference is for example made to Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10): 1357-1369) and Klein et al. 2014 (Protein Eng. Des.

[0116] Sei. 27 (10): 325-330).

[0117] Table B: Linker sequences ("ID" refers to the SEQ ID NO as used herein) 271 136 ul2 / u52

[0118] 5.3.3 Binding affinity to IL-13 and TSLP

[0119] In one embodiment, the polypeptide binds interleukin-13 (IL-13) and thymic stromal lymphopoietin (TSLP). More specifically, the polypeptide may contain at least one ISVD which specifically binds TSLP and one ISVD which specifically binds IL-13.

[0120] In relation to the compounds of the present invention, binding to IL-13 and TSLP means specific binding to IL-13 and TSLP. The binding of a compound to its target can be determined based on affinity. The affinity denotes the strength or stability of a molecular interaction. The affinity is commonly given as by the dissociation constant, which has units of mol / liter (or M). The affinity can also be expressed as an association constant which equals to the reciprocal of the dissociation constant and has units of (mol / liter)1(or M1).

[0121] The affinity is a measure for the binding strength between a moiety and a binding site on the target molecule: the lower the value of the dissociation constant, the stronger the binding strength between a target molecule and a targeting moiety.

[0122] Typically, binding units used in the present invention will bind to their targets with a dissociation constant of IO-5to 1012moles / liter or less, or 10-7to 1012moles / liter or less, or IO-8to 1012moles / liter (i.e. with an association constant of 105to 1012liter / moles or more, or 107to 1012liter / moles or more, or 108to 1012liter / moles).

[0123] Any dissociation constant value greater than IO-4mol / liter (or any association constant value lower than 104liters / mol) is generally considered to indicate non-specific interaction.

[0124] The dissociation constant for biological interactions, such as the binding of immunoglobulin sequences to an antigen, which are considered specific are typically in the range of 10-5moles / liter (10000 nM or lOpM) to 1012moles / liter (0.001 nM or 1 pM) or less.

[0125] Accordingly, specific / selective binding may mean that - using the same measurement method, e.g., SPR - a compound binds to IL13 and / or TSLP with a dissociation constant value of 10-5to 1012moles / liter or less and binds to related cytokines with a dissociation 271 136 ul2 / u52 constant value greater than IO-4moles / liter. Examples of I L13 related targets are human IL4. Examples of related cytokines for TSLP are human IL7. Thus, in some embodiments, the compound used in the present invention binds to IL13 with a dissociation constant value of 10-5to 1012moles / liter or less and binds to IL4 of the same species with a dissociation constant value greater than IO-4moles / liter, and binds to TSLP with a dissociation constant value of 10-5to 1012moles / liter or less and binds to human IL7 of the same species with a dissociation constant value greater than IO-4moles / liter.

[0126] In some embodiments, the polypeptides used in the present invention have at least half the binding affinity, or at least the same binding affinity, to human IL13 and to human TSLP as compared to a polypeptide consisting of the amino acid of SEQ ID NO: 1, wherein the binding affinity is measured using the same method, such as SPR.

[0127] Specific binding to a certain target from a certain species does not exclude that the binding unit can also specifically bind to the analogous target from a different species. For example, specific binding to human IL13 does not exclude that the binding unit (or a polypeptide comprising the same) can also specifically bind to IL13 from cynomolgus monkeys. Likewise, for example, specific binding to human TSLP does not exclude that the binding unit (or a polypeptide comprising the same) can also specifically bind to TSLP from cynomolgus monkeys ("cyno").

[0128] Specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.

[0129] The dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned below. In this respect, it will also be clear that it may not be possible to measure dissociation constants of more than IO-4moles / liter or 10-3moles / liter (e.g., of 10-2moles / liter). Optionally, as will also be clear to the skilled person, the (actual or apparent) dissociation constant may be calculated on the basis of the (actual or apparent) association constant, by means of the relationship [(dissociation constant) = l / (association constant)].

[0130] The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see for example Ober et al. 2001, Intern. Immunology 13: 271 136 ul2 / u52

[0131] 1551-1559). The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding kon, kOff measurements and hence dissociation constant (or association constant) values. This can for example be performed using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. (1991 Biotechniques 11: 620-627), Johnsson et al. (1995, J. Mol. Recognit. 8: 125-131), and Johnnson et al. (1991, Anal. Biochem. 198: 268- 277).

[0132] Another well-known biosensor technique to determine affinities of biomolecular interactions is bio-layer interferometry (BLI) (see for example Abdiche et al. 2008, Anal. Biochem. 377: 209-217). The term "bio-layer Interferometry" or "BLI", as used herein, refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam). A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since the interactions can be measured in real-time, association and dissociation rates and affinities can be determined. BLI can for example be performed using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0133] Alternatively, affinities can be measured in Kinetic Exclusion Assay (KinExA) (see for example Drake et al. 2004, Anal. Biochem., 328: 35-43), using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). The term "KinExA", as used herein, refers to a solution-based method to measure true equilibrium binding affinity and kinetics of unmodified molecules. Equilibrated solutions of an antibody / antigen complex are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).

[0134] The GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74). 271 136 ul2 / u52

[0135] In one embodiment, the polypeptide comprises or consists of at least four ISVDs, wherein two ISVDs specifically bind IL-13 and two ISVDs specifically bind TSLP, wherein each of said at least four ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least four ISVDs are optionally linked via one or more peptide linkers, and wherein: a first ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 7, a CDR2 that is the amino acid sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid sequence of SEQ ID NO: 17, a second ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid sequence of SEQ ID NO: 18, a third ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of SEQ ID NO: 19, and a fourth ISVD comprises: a CDR1 that is the amino acid sequence of SEQ I D NO: 11, a CDR2 that is the amino acid sequence of SEQ I D NO: 16 and a CDR3 that is the amino acid sequence of SEQ ID NO: 21.

[0136] SEQ ID NO: 7 is GRTFSSYRMG; SEQ ID NO: 12 is ALSGDGYSTY; SEQ ID NO: 17 is KLQYVSGWSYDYPY.

[0137] SEQ ID NO: 8 is GFTFNNYAMK; SEQ ID NO: 13 is SITTGGGSTD; SEQ ID NO: 18 is VPFGYYSEHFSGLSFDY.

[0138] SEQ ID NO: 9 is GSGFGVNILY; SEQ ID NO: 14 is SITSGGITN; SEQ ID NO: 19 is RNIFDGTTE.

[0139] SEQ ID NO: 11 is GFTFADYDYDIG; SEQ ID NO: 16 is CISNRDGSTY; SEQ ID NO: 21 is EIHCDDYGVENFDFDP.

[0140] In some embodiments, the compound comprises: a first ISVD comprising the amino acid sequence of SEQ ID NO: 2, a second ISVD comprising the amino acid sequence of SEQ ID NO: 3, 271 136 ul2 / u52 a third ISVD comprising the amino acid sequence of SEQ ID NO: 4, and a fourth ISVD comprising the amino acid sequence of SEQ ID NO: 6.

[0141] In some embodiments, the order of the ISVDs above indicates their relative position to each other considered from the N-terminus to the C-terminus of said polypeptide.

[0142] In a further embodiment, the polypeptide further comprises a fifth ISVD that binds to human serum albumin, wherein the fifth ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 10, a CDR2 that is the amino acid sequence of SEQ ID NO: 15; and a CDR3 that is the amino acid sequence of SEQ ID NO: 20; and the fifth ISVD is positioned between the third and the fourth ISVD.

[0143] In some embodiments, the compound comprises: a first ISVD comprising the amino acid sequence of SEQ ID NO: 2, a second ISVD comprising the amino acid sequence of SEQ ID NO: 3, a third ISVD comprising the amino acid sequence of SEQ ID NO: 4, a fourth ISVD comprising the amino acid sequence of SEQ ID NO: 6, and a further ISVD that binds to human serum albumin and comprises the amino acid sequence of SEQ ID NO: 5.

[0144] An exemplary polypeptide that binds TSLP and IL-13 is the polypeptide consisting of SEQ ID NO: 1, also called lunsekimig or SAR443765. A schematic illustration of the ISVDs of lunsekimig and their isoelectric points is displayed in Figure 1.

[0145] In some embodiments, the polypeptide comprised in the pharmaceutical composition is lunsekimig (SEQ ID NO: 1).

[0146] SEQ ID NO: 1 is:

[0147] DVQLVESGGGVVQPGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAALSGDGYSTYTANSVKG RFTISRDNSKNTVYLQMNSLRPEDTALYYCAAKLQYVSGWSYDYPYWGQGTLVTVSSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFNNYAMKWV RQAPGKGLEWVSSITTGGGSTDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCANVPFGYYS EHFSGLSFDYRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSGFGVNILYWY RQAAGI ERELIASITSGGITNYVDSVKGRFTISRDNSENTMYLQIVINSLRAEDTGLYYCASRNIFDGTTE WGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGIVISWVRQAPGKG PEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVS 271 136 ul2 / u52

[0148] SGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFADYDYDIGWFRQAPGKEREGVSCISNR DGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAVEIHCDDYGVENFDFDPWGQGTLV TVSSA.

[0149] 5.3.4 Amount of polypeptide

[0150] The pharmaceutical composition of the present invention may contain the polypeptide in a concentration of at least 50 mg / mL.

[0151] The concentration of polypeptide may be at least 10 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, or 120 mg / mL. Further, the concentration of polypeptide may be 300 mg / mL or less, 250 mg / mL or less or 200 mg / mL or less.

[0152] Any of the disclosed lower limits for the concentration of polypeptide may be combined with any of the disclosed upper limits to define further suitable concentration ranges for the purposes of this invention. As an example, further exemplary ranges for the concentration of polypeptide include 10 mg / mL to 300 mg / mL, 10 mg / mL to 250 mg / mL, 10 mg / mL to 200 mg / mL, 50 mg / mL to 300 mg / mL, 50 mg / mL to 250 mg / mL, 70 mg / mL to 300 mg / mL, 70 mg / mL to 250 mg / mL, 80 mg / mL to 250 mg / mL, 80 mg / mL to 200 mg / mL, 90 mg / mL to 250 mg / mL, 90 mg / mL to 200 mg / mL, 100 mg / mL to 250 mg / mL and 100 mg / mL to 200 mg / mL.

[0153] In one embodiment of the present invention, the concentration of polypeptide in the pharmaceutical composition is 100 mg / mL to 200 mg / mL.

[0154] In another embodiment of the present invention, the concentration of polypeptide in the pharmaceutical composition is 150 mg / mL.

[0155] The pharmaceutical composition of the present invention may contain one or more of the above-described polypeptides. The concentration of polypeptide described herein refers to the total amount of polypeptide or polypeptides with respect to the volume of pharmaceutical composition.

[0156] The concentration of polypeptide may be determined, for example, by measuring the absorbance of a sample containing the polypeptide at 280 nm.

[0157] 5.4 Amino acid

[0158] The pharmaceutical composition comprises an amino acid having an isoelectric point of 7 or higher, or a salt thereof.

[0159] It has to be noted that the amino acid is not part of the polypeptide described above, but is an isolated amino acid, i.e. not linked to other amino acids by a peptide bond. 271 136 ul2 / u52

[0160] The term "isoelectric point" is as defined above for the polypeptide and may be determined as defined above. For example, the pharmaceutical composition may comprise an amino acid having an isoelectric point of 8 or more, 8.5 or more, 9 or more, 9.5 or more, or 10 or more.

[0161] Examples of amino acids for use in the pharmaceutical composition include natural and synthetic amino acids. The natural amino acids include L-arginine, D-arginine, L-histidine, D-histidine, L-lysine, D-lysine, as well as salts thereof and mixtures thereof. For example, the amino acid is selected from L-arginine, L-arginine hydrochloride, L-histidine, L- histidine hydrochloride, L-lysine, L-lysine hydrochloride, and mixtures thereof. In particular, the amino acid may be L-arginine, L-arginine hydrochloride, or a mixture thereof. In one embodiment, the amino acid is L-arginine hydrochloride.

[0162] The pharmaceutical composition of the invention may contain the amino acid at a concentration of at least 50 mM. As used herein, the concentration of the amino acid refers to the concentration of the amino acid free base.

[0163] The concentration of the amino acid may be at least 100 mM, 120 mM, 130 mM, 150 mM, 200 mM, or 230 mM. Further, the concentration of the amino acid may be 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, or 300 mM or less.

[0164] Any of the disclosed lower limits for the concentration of amino acid may be combined with any of the disclosed upper limits to define further suitable concentration ranges for the purposes of this invention. As an example, further exemplary ranges for the concentration of amino acid in the composition are 100 mM to 500 mM, 100 mM to 450 mM, 100 mM to 400 mM, 100 mM to 350 mM, 120 mM to 500 mM, 120 mM to 450 mM, 120 mM to 400 mM, 120 mM to 350 mM, 130 mM to 500 mM, 130 mM to 450 mM, 130 mM to 400 mM, 130 mM to 350 mM, 150 mM to 500 mM, 150 mM to 450 mM, 150 mM to 400 mM, 150 mM to 350 mM, 200 mM to 500 mM, 200 mM to 450 mM, 200 mM to 400 mM, 200 mM to 350 mM, 200 mM to 300 mM, 200 mM to 250 mM.

[0165] In some embodiments, the concentration of the amino acid in the pharmaceutical composition is at least 200 mM. In some embodiments, the concentration of the amino acid in the pharmaceutical composition is higher than 200 mM. In one embodiment of the present invention, the concentration of the amino acid in the pharmaceutical composition is 200 mM to 500 mM.

[0166] In another embodiment, the concentration of the amino acid in the pharmaceutical composition is 230 mM to 500 mM. 271 136 ul2 / u52

[0167] In particular, the concentration of the amino acid in the pharmaceutical composition may be 230 mM.

[0168] The pharmaceutical composition of the present invention may contain one or more of the above-described amino acids. The concentration of amino acid described herein refers to the total amount of amino acid or amino acids with respect to the volume of pharmaceutical composition.

[0169] 5.5 pH of the pharmaceutical composition

[0170] The pharmaceutical composition may have a pH of 5 or more, 6 or more, or 6.5 or more. Further, the pharmaceutical composition may have a pH of 11 or less, 10 or less, 8 or less, or 7 or less. In one embodiment of the present invention the pH of the pharmaceutical composition is 5 or more and 11 or less including 5 to 11. In another embodiment, the pharmaceutical composition has a pH of 7.

[0171] The pH of the composition may be measured by any means known to those of skill in the art. A means for measuring pH is using a pH meter with a microelectrode. The pH of the formulation may be adjusted using any means known in the art. Exemplary chemicals for altering the pH of the formulations are hydrochloric acid (HCI) and sodium hydroxide (NaOH).

[0172] 5.6 Buffer

[0173] The pharmaceutical composition may contain a buffer (also referred here as "buffering agent"), in particular a pharmaceutically acceptable buffer.

[0174] A "buffer" or "buffering agent" as referred to herein refers to an agent that allows a solution in which it is contained to resist changes in pH when a small amount of acid or base is added.

[0175] Examples of buffers for use in the pharmaceutical composition are phosphate buffer, citrate buffer, (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) (HEPES) buffer, sodium acetate buffer and histidine hydrochloride buffer.

[0176] For example, the pharmaceutical composition may comprise a phosphate buffer, for example a sodium phosphate buffer. The phosphate buffer may be made in situ by mixing phosphoric acid and sodium hydroxide, or it may be composed of a dihydrogen phosphate salt and a monohydrogen phosphate salt, e.g., sodium dihydrogen phosphate and disodium hydrogen phosphate or, more specifically, sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate. 271 136 ul2 / u52

[0177] The term "sodium phosphate buffer" as used herein refers to a mixture of disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4). Disodium hydrogen phosphate and sodium dihydrogen phosphate are combined in the respective amounts required to form a suitable buffer system, which allows for adjusting the desired pH of the pharmaceutical composition of the present invention.

[0178] In one embodiment, the buffer in the pharmaceutical composition of the present invention is sodium phosphate buffer or HEPES buffer.

[0179] The buffer may be present in a concentration of 1 mM or more, 3 mM or more, 5 mM or more, 8 mM or more, 10 mM or more, or 15 mM or more. Further, the concentration of the buffer may be 100 mM or less, 75 mM or less, 50 mM or less, 40 mM or less, 35 mM or less, or 25 mM or less.

[0180] In one embodiment of the present invention, the composition comprises sodium phosphate buffer at a concentration of 5 mM to 20 mM.

[0181] In another embodiment of the present invention, the composition comprises sodium phosphate buffer at a concentration of 10 mM.

[0182] In one embodiment of the present invention, the composition comprises HEPES buffer at a concentration of 5 mM to 20 mM.

[0183] In another embodiment of the present invention, the composition comprises HEPES buffer at a concentration of 10 mM.

[0184] The pharmaceutical composition may comprise one or more buffer(s). For example, the pharmaceutical composition may comprise two buffers, e.g., sodium phosphate buffer and HEPES buffer. If present, the buffers may be contained in different molar ratios, for example ranging from 5:1 to 1:5, or from 2:1 to 1:2 for two buffers, depending on the desired pH value of the composition. The concentration of buffer described herein refers to the total amount of buffer or buffers with respect to the volume of pharmaceutical composition.

[0185] 5.7 Nonionic surfactant

[0186] The pharmaceutical composition may contain a nonionic surfactant. Nonionic surfactants are defined as surfactants that have polar head groups that are not electrically charged when dissolved in water.

[0187] Examples of nonionic surfactants that may be used in the pharmaceutical composition are polysorbates and block copolymers such as copolymers of polyethylene and polypropylene glycol such as poloxamers. Examples of polysorbates include polysorbate 271 136 ul2 / u52

[0188] 20 (Tween 20), polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80. Examples of block copolymers are Poloxamer 188 (Polyethylene glycol)-block- poly(propylene glycol)-block-poly(ethylene glycol)), Poloxamer 407 (Poly(ethylene glycol)- block-poly(propylene glycol)-block-poly(ethylene glycol)), Synperonic® F-108 (Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), and Pluronic® P-123 (Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)).

[0189] The composition may comprise one or more non-ionic surfactants. For example, the nonionic surfactant may be polysorbate 20 and / or Poloxamer 188. In one embodiment of the present invention, the non-ionic surfactant is Poloxamer 188.

[0190] The non-ionic surfactant may be present in the pharmaceutical composition in an amount of at least 0.001 % (w / v), at least 0.005 % (w / v), at least 0.01 % (w / v), or at least 0.015 % (w / v). Further, the non-ionic surfactant may be present in the formulations in an amount of 0.2 % (w / v) or less, 0.15 % (w / v) or less, 0.1 % (w / v) or less, or 0.05 % (w / v) or less.

[0191] Any of the disclosed lower limits for the amount of non-ionic surfactant may be combined with any of the disclosed upper limits to define further suitable amount ranges for the purposes of this invention. As an example, further exemplary ranges for the amount of non-ionic surfactant in the composition are 0.005 % (w / v) to 0.2 % (w / v), 0.005 % (w / v) to 0.15 % (w / v), 0.005 % (w / v) to 0.1 % (w / v), 0.01 % (w / v) to 0.15 % (w / v), and 0.01 (w / v) to 0.1 % (w / v).

[0192] In one embodiment of the present invention, the amount of non-ionic surfactant in the pharmaceutical composition is 0.01 (w / v) to 0.2 % (w / v).

[0193] In another embodiment of the present invention, the amount of non-ionic surfactant in the pharmaceutical composition is 0.01 (w / v) to 0.1 % (w / v). When added in an amount within the range of 0.01 (w / v) to 0.1 % (w / v), poloxamers and in particular Poloxamer P188 can have a positive influence on opalescence compared to other, e.g. higher, poloxamer concentrations.

[0194] In one embodiment of the present invention, the amount of non-ionic surfactant in the pharmaceutical composition is 0.02 % (w / v).

[0195] In one embodiment, the amount of non-ionic surfactant in the pharmaceutical composition of the present invention is 0.02 % (w / v) and the non-ionic surfactant is Poloxamer 188 or polysorbate 20. In another embodiment, the amount of non-ionic surfactant in the pharmaceutical composition is 0.02 % (w / v) and the non-ionic surfactant is Poloxamer 188. 271 136 ul2 / u52

[0196] The pharmaceutical composition of the present invention may contain one or more of the above-described non-ionic surfactants. The concentration of non-ionic surfactant described herein refers to the total amount of non-ionic surfactant or non-ionic surfactants with respect to the weight of pharmaceutical composition.

[0197] 5.8 Other components

[0198] The pharmaceutical composition may further comprise other pharmaceutically acceptable excipients.

[0199] The pharmaceutical composition may comprise saccharides, sugars, glycerol, sorbitol, mannitol, sodium chloride, potassium chloride, magnesium chloride, other inorganic salts, sodium L-glutamate, sodium L-aspartate, and mixtures thereof.

[0200] For example, the pharmaceutical composition may comprise sucrose, sorbitol, sodium chloride, sodium L-glutamate, sodium L-aspartate and mixtures thereof.

[0201] If sucrose is present, its concentration in the pharmaceutical composition may be 0.5 % (w / v) or more, 1 % (w / v) or more, 3 % (w / v) or more, or 4 % (w / v) or more. Further, the concentration of sucrose in the pharmaceutical composition may be 20 % (w / v) or less, 15 % (w / v) or less, or 10 % (w / v) or less.

[0202] If sorbitol is present, its concentration in the pharmaceutical composition may be 0.5 % (w / v) or more, 1 % (w / v) or more, 3 % (w / v) or more, or 4 % (w / v) or more. Further, the concentration of sucrose in the pharmaceutical composition may be 20 % (w / v) or less, 15 % (w / v) or less, or 10 % (w / v) or less.

[0203] If sodium chloride is present, its concentration in the pharmaceutical composition may be 20 mM or more, 30 mM or more, 50 mM or more, 75 mM or more, or 100 mM or more. Further, the concentration of sodium chloride in the pharmaceutical composition may be 500 mM or less, 450 mM or less, 400 mM or less, or 300 mM or less.

[0204] If sodium L-glutamate is present, its concentration in the pharmaceutical composition may be 5 mM or more, 10 mM or more, 15 mM or more, or 20 mM or more. Further, the concentration of sodium L-glutamate in the pharmaceutical composition may be 300 mM or less, 250 mM or less, 200 mM or less, or 150 mM or less.

[0205] If sodium L-aspartate is present, its concentration in the pharmaceutical composition may be 5 mM or more, 10 mM or more, 15 mM or more, or 20 mM or more. Further, the concentration of sodium L-aspartate in the pharmaceutical composition may be 300 mM or less, 250 mM or less, 200 mM or less, or 150 mM or less. 271 136 ul2 / u52

[0206] Furthermore, other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980) may be included in the formulation.

[0207] In particular, the formulations of the invention may comprise other excipients including, but not limited to, water for injection, diluents, solubilizing agents, soothing agents, additional buffers, inorganic or organic salts, antioxidants, or the like.

[0208] In some embodiments, however, the formulations of the invention comprise no other excipients, except those described above.

[0209] In a particular embodiment, the formulation is substantially free of preservatives, although, in alternative embodiments, preservatives may be added as necessary.

[0210] 5.9 Exemplary formulations

[0211] In one embodiment, the invention provides a pharmaceutical composition comprising: the polypeptide at a concentration of 150 mg / mL or more, wherein the polypeptide comprises at least two portions having an isoelectric point of 6 or lower which are ISVDs and at least two portions having an isoelectric point of 8 or higher which are ISVDs; the amino acid at a concentration of 150 mM or more, wherein the amino acid is L-arginine or a salt thereof.

[0212] In one embodiment, the invention provides a pharmaceutical composition comprising: the polypeptide at a concentration of 100 mg / mL to 300 mg / mL; the amino acid at a concentration of 100 mM to 500 mM, wherein the amino acid is L-arginine or a salt thereof; sodium phosphate buffer at a concentration of 5 mM to 20 mM;

[0213] Poloxamer 188 at a concentration of 0.01 % (w / v) to 0.1 % (w / v) and wherein the composition has a pH of 6 to 8.

[0214] In one embodiment, the invention provides a pharmaceutical composition comprising: the polypeptide at a concentration of 100 mg / mL to 300 mg / mL; the amino acid at a concentration of 200 mM to 500 mM, wherein the amino acid is L-arginine or a salt thereof; sodium phosphate buffer at a concentration of 5 mM to 20 mM; 271 136 ul2 / u52

[0215] Poloxamer 188 at a concentration of 0.01 % (w / v) to 0.1 % (w / v); and wherein the composition has a pH of 6 to 8.

[0216] In one embodiment, the invention provides a pharmaceutical composition comprising: the polypeptide at a concentration of 100 mg / mL to 300 mg / mL; the amino acid at a concentration of 230 mM to 500 mM, wherein the amino acid is L-arginine or a salt thereof; sodium phosphate buffer at a concentration of 5 mM to 20 mM;

[0217] Poloxamer 188 at a concentration of 0.01 % (w / v) to 0.1 % (w / v); and wherein the composition has a pH of 6 to 8.

[0218] In one embodiment, the invention provides a pharmaceutical composition comprising: the polypeptide at a concentration of 150 mg / mL, wherein the polypeptide comprises or consists of the amino acid sequence SEQ ID NO:1, the amino acid at a concentration of 230 mM, wherein the amino acid is L-arginine hydrochloride, sodium phosphate buffer at a concentration of 10 mM,

[0219] Poloxamer 188 at a concentration of 0.02 % (w / v); and wherein the composition has a pH of 7.0.

[0220] In one embodiment, the pharmaceutical composition consists of: the polypeptide at a concentration of 150 mg / mL, wherein the polypeptide is lunsekimig, the amino acid at a concentration of 230 mM, wherein the amino acid is L-arginine hydrochloride, sodium phosphate buffer at a concentration of 10 mM,

[0221] Poloxamer 188 at a concentration of 0.02 % (w / v), and water, wherein the composition has a pH of 7.0.

[0222] In one embodiment, the invention provides a pharmaceutical composition comprising: the polypeptide at a concentration of 100 mg / mL to 300 mg / mL; 271 136 ul2 / u52 the amino acid at a concentration of 200 mM to 500 mM, wherein the amino acid is L-arginine or a salt thereof;

[0223] HEPES buffer at a concentration of 5 mM to 20 mM;

[0224] Poloxamer 188 at a concentration of 0.01 % (w / v) to 0.1 % (w / v) and wherein the composition has a pH of 6 to 8.

[0225] In one embodiment, the invention provides a pharmaceutical composition comprising: the polypeptide at a concentration of 100 mg / mL to 300 mg / mL; the amino acid at a concentration of 230 mM to 500 mM, wherein the amino acid is L-arginine or a salt thereof;

[0226] HEPES buffer at a concentration of 5 mM to 20 mM;

[0227] Poloxamer 188 at a concentration of 0.01 % (w / v) to 0.1 % (w / v); and wherein the composition has a pH of 6 to 8.

[0228] In one embodiment, the invention provides a pharmaceutical composition comprising: the polypeptide at a concentration of 150 mg / mL, wherein the polypeptide comprises or consists of the amino acid sequence SEQ ID NO:1, the amino acid at a concentration of 230 mM, wherein the amino acid is L-arginine hydrochloride,

[0229] HEPES buffer at a concentration of 10 mM,

[0230] Poloxamer 188 at a concentration of 0.02 % (w / v); and wherein the composition has a pH of 7.0.

[0231] In one embodiment, the invention provides a pharmaceutical composition comprising: the polypeptide at a concentration of 150 mg / mL, wherein the polypeptide is lunsekimig, the amino acid at a concentration of 230 mM, wherein the amino acid is L-arginine hydrochloride,

[0232] HEPES buffer at a concentration of 10 mM,

[0233] Poloxamer 188 at a concentration of 0.02 % (w / v); and wherein the composition has a pH of 7.0.

[0234] In one embodiment, the invention provides a pharmaceutical composition comprising: 271 136 ul2 / u52 the polypeptide at a concentration of 50 mg / mL to 100 mg / mL, wherein the polypeptide comprises or consists of the amino acid sequence SEQ ID NO:1, the amino acid at a concentration of 230 mM, wherein the amino acid is L-arginine hydrochloride, sodium phosphate buffer at a concentration of 10 mM,

[0235] Poloxamer 188 at a concentration of 0.02 % (w / v); and wherein the composition has a pH of 7.0.

[0236] In one embodiment, the invention provides a pharmaceutical composition comprising: the polypeptide at a concentration of 50 mg / mL to 100 mg / mL, wherein the polypeptide comprises or consists of the amino acid sequence SEQ ID NO:1, the amino acid at a concentration of 230 mM, wherein the amino acid is L-arginine hydrochloride,

[0237] HEPES buffer at a concentration of 10 mM,

[0238] Poloxamer 188 at a concentration of 0.02 % (w / v); and wherein the composition has a pH of 7.0.

[0239] 5.10 Applications

[0240] The pharmaceutical compositions of the present invention are suitable for use in the treatment of asthma, chronic obstructive pulmonary disease, atopic dermatitis or chronic rhinosinusitis with nasal polyps in a subject.

[0241] The subject of the treatment of the present invention can be any animal, and more specifically a mammal. Among mammals, a distinction can be made between humans and non-human mammals. Non-human animals may be for example companion animals (e.g., dogs, cats), livestock (e.g., bovine, equine, ovine, caprine, or porcine animals), or animals used generally for research purposes and / or for producing antibodies (e.g., mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates, such as cynomolgus monkeys, or camelids, such as llama or alpaca). In one embodiment, the subject is a human subject.

[0242] Compositions according to the present invention may be administered to a subject by any suitable route of administration, for example by enteral (such as oral or rectal) or parenteral (such as epicutaneous, sublingual, buccal, nasal, intra-articular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous (SC), transdermal, or 271 136 ul2 / u52 transmucosal) administration. In one embodiment, substances are administered by parenteral administration, such as intramuscular, subcutaneous or intradermal administration. In one embodiment, subcutaneous administration is used.

[0243] Accordingly, in one embodiment, the pharmaceutical composition of the present invention is suitable for administration parenterally, intravenously, intramuscularly, intradermally, subcutaneously, or a combination thereof.

[0244] One or more doses can be administered. If more than one dose is administered, the doses can be administered in suitable intervals in order to maximize the effect of the polypeptide.

[0245] 5.11 Examples

[0246] The following examples and comparative examples investigate the effect of certain excipients in different amounts on the quality of formulations comprising Lunsekimig (SAR443765), further ISVD constructs, and ISVD-Fc constructs.

[0247] 5.11.1 Example 1 (KD / colloidal stability) i) Lunsekimig

[0248] The properties of Lunsekimig (also referred to as SAR443765, see also Figure 1) used in the examples are listed in Table 1. Lunsekimig comprises ISVDs whose isoelectric points differ by several pH units.

[0249] [Table 1]

[0250] Formulations containing 100 mg / mL lunsekimig, 0.01% w / v Tween 20, 20 mM sodium phosphate buffer and the excipients listed in Table 2 were prepared. Each formulation had a pH of 7.0.

[0251] For each formulation, the diffusion interaction parameter (KD) was determined by dynamic light scattering (DLS). Results are reported in Table 2 as well as in Figure 2 and Figure 3 for the samples containing L-arginine hydrochloride and sucrose, respectively. 271 136 ul2 / u52

[0252] [Table 2]

[0253] Higher KD values indicate less self-attraction between molecules in solution and are thus more desirable. Lower (more negative) values for this parameter are indicative of more self-attraction between the molecules. As shown in the table above, the highest KDs were obtained with formulations containing L-arginine hydrochloride, wherein the positive effect on KD increased with increasing concentrations of L-arginine hydrochloride. The addition of sucrose, even in high amount, did not result in such improvements.

[0254] Accordingly, it was concluded that L-arginine hydrochloride decreases self-attractions between the nanobody molecules even when added in minor amounts. ii) ISVD constructs

[0255] Five formulations, each containing a different ISVD construct at 10 mg / mL, were prepared in 15 mM histidine at pH 6.5 and 4 % sucrose (formulation A), then in 15 mM histidine at pH 6.5 and 230 mM L-arginine HCI through buffer exchange (formulation B). The diffusion interaction parameter (KD) was determined by dynamic light scattering (DLS) on serial dilutions of the sample (10 mg / mL, 7.5 mg / mL, 5 mg / mL, 2.5 mg / mL, 1 mg / mL). The pl of each building block (BB) of the respective construct and of each construct as a whole, as well as the KD results for each formulation are reported in Table 3. 271 136 ul2 / u52

[0256] [Table 3] iii) ISVD-Fc constructs

[0257] Five different ISVD-Fc constructs were obtained. The building blocks and the experimentally determined pl of the constructs are reported in Table 4. Their structure is schematically depicted in Figure 4.

[0258] [Table 4] 271 136 ul2 / u52

[0259] The isoelectric point of the ISVD-Fc constructs was further determined based on the net charge of each building block at increasing pH values. Table 5 exemplarily reports the determination for construct Example II. [Table 5] 271 136 ul2 / u52

[0260] The net charge of the totality of the ISVD portions and the net charge of the totality of the Fc portions of Example II at each pH value is reported in Table 6, alongside the overall net charge of the construct.

[0261] [Table 6] 271 136 ul2 / u52

[0262] The values reported in Table 5 and Table 6 were determined by different computational calculations based on the protein sequence of the respective building blocks.

[0263] As shown above, Example II contains five building blocks, three of which are ISVDs and two of which are Fc portions. The three ISVDs portions have a pl of about 8.5, between 8.5 and 9, and between 8 and 8.5, respectively. The Fc portions have a pl of between 5.5 and 6, and between 6 and 6.5, respectively.

[0264] The Fc portions and the building block BB are common to all ISVD-Fc constructs shown in Table 4. Accordingly, each of the ISVD-Fc constructs has at least one portion having a pl of between 5.5 and 6 (the first Fc portion), one portion having a pl of between 6 and 6.5 (the second Fc portion) and one portion having a pl of between 8 and 8.5 (the building block BB).

[0265] Formulations containing three exemplary constructs from Table 4, Example lk, Example lm and Example lo, were prepared in 10 mM histidine and spiked with (i) 8% sucrose, (ii) 150 mM arginine, or (iii) 150 mM NaCI. The observed KD values for each formulation are reported in Figure 5. From this, it was confirmed that the addition of arginine achieved the highest increase in KD for all constructs.

[0266] The construct Example II was selected for a further testing with increasing concentrations of arginine. The construct was formulated at 10 mg / mL with 10 mM histidine at pH 6.0, and arginine (150 mM, 250 mM or 500 mM).

[0267] The visual appearance of the samples is reported in Figure 6. From left to right in Figure 6, the samples contain: histidine 10 mM; histidine 10 mM and arginine 150 mM, histidine 10 mM and arginine 250 mM; histidine 10 mM and arginine 500 mM; histidine 10 mM with the same volume as in the sample including arginine 500 mM.

[0268] The visual inspection of the samples revealed that all tested arginine concentrations reduced opalescence and white coloring, and the resulting samples were clear. The opalescence was reduced more quickly with higher concentrations of arginine during the preparation of the sample.

[0269] 5.11.2 Example 2 (Viscosity)

[0270] Formulations of lunsekimig having the composition reported in Table 7 were prepared.

[0271] The viscosity was measured by DLS at 25 °C and the results are reported in Table 7. 271 136 ul2 / u52

[0272] [Table 7]

[0273] All formulations containing L-arginine hydrochloride or L-histidine showed a beneficial viscosity allowing for subcutaneous administration. The lowest viscosity was obtained for Example 2a, which contained 125 mM L-arginine hydrochloride.

[0274] 5.11.3 Example 3 (Viscosity)

[0275] Formulations of lunsekimig having the composition reported in Table 8 were prepared.

[0276] The viscosity was determined by DLS at 25 °C and the results are reported in Table 8.

[0277] [Table 8]

[0278] In the presence of L-arginine hydrochloride, the viscosity of the tested formulations containing the nanobody at 100 mg / mL or at 150 mg / mL was no more than 10.3 cP, which is acceptable for subcutaneous administration. 271 136 ul2 / u52

[0279] L-arginine hydrochloride thus enabled adjusting the viscosity of the respective formulations even when the nanobody was present at a high concentration.

[0280] 5.11.4 Example 4 (Opalescence, osmolality, viscosity)

[0281] (i) Opalescence

[0282] The following formulation was prepared: 100 mg / mL lunsekimig, 20 mM sodium phosphate buffer, 125 mM L-arginine hydrochloride, 0.01% (w / v) Tween 20, pH 7.0 (Example 4a).

[0283] The opalescence of this formulation was above that of the Ph. Eur. opalescence standard IV (STD IV).

[0284] L-arginine hydrochloride was further added to the formulation to obtain a final composition containing 100 mg / mL lunsekimig, 20 mM sodium phosphate buffer, 230 mM L-arginine hydrochloride, 0.01% (w / v) Tween 20, pH 6.8 (Example 4b). The opalescence was determined by visual inspection and comparison with Ph. Eur. Standards and the result are shown in Figure 7. In Figure 7, "FB" refers to the composition of Example 4b without lunsekimig and "CER 2 SP-mimic TO" corresponds to Example 4b. "REF STD III" and "REF STD IV" are the Ph. Eur. standards III and IV. Visual inspection showed that the degree of clarity of Example 4b was between STD III and IV. This was confirmed when measured with a nephelometer. The opalescence (NTU; Nephelometric Turbidity Unit) of different batches with the same composition as Example 4b was between the opalescence (NTU) of STD III and STD IV were measured at 8 and 12 NTU, respectively.

[0285] Increasing the L-arginine hydrochloride concentration thus was advantageous to reduce the opalescence levels of lunsekimig-comprising formulations.

[0286] (ii) Osmolality and viscosity

[0287] Osmolality of the composition of Example 4b was measured by using an osmometer. Viscosity was measured using a Brookfield rheometer. The osmolality was 477 mOsm / kg, i.e., within the hypertonic range of up to 600 mOsm / kg being acceptable for subcutaneous injection. The viscosity was 3.6 cP and thus suitable for subcutaneous injection as well.

[0288] 5.11.5 Example 5 and Comparative Example 5 (Turbidity)

[0289] Formulations containing lunsekimig at 100 mg / mL, 0.01% (w / v) Tween 20, 20 mM sodium phosphate buffer and the excipients listed in Table 9 were prepared. The pH of each 271 136 ul2 / u52 formulation was 7.0. Turbidity was determined by measuring the optical density (OD) at 500 nm. The results are reported in Table 9 as well as Figure 8 and Figure 9 for the samples containing arginine and sucrose, respectively. The visual appearance of the samples containing arginine are also shown in Figure 10 (from left to right: arginine 0 mM, 10 mM, 50 mM, 125 mM, 230 mM, 500 mM).

[0290] [Table 9] 271 136 ul2 / u52

[0291] 5.11.6 Example 6 (Stability, Opalescence)

[0292] To evaluate stability, formulations containing lunsekimig at 100 mg / mL were prepared with the excipient composition reported in Table 10.

[0293] [Table 10]

[0294] The opalescence level of the different batches was measured according to Ph. Eur. at TO and during storage by visual inspection and with a nephelometer. Results are shown in Table 11.

[0295] [Table 11] 271 136 ul2 / u52

[0296] The opalescence of the formulation of Example 6a was lower than that of the formulation of Example 6b. For both examples, original opalescence (NTU) was maintained during storage under different conditions.

[0297] 5.11.7 Example 7 (Osmolality, viscosity, opalescence)

[0298] Formulations containing lunsekimig at 150 mg / mL were prepared with the excipient compositions reported in Table 12.

[0299] [Table 12]

[0300] Osmolality was measured using an osmometer. The viscosity of each formulation was determined using a rheometer at 25 °C. As reported in Table 13, osmolality was below the limit of 600 mOsm / kg for Examples 7a and 7b and viscosity was within a suitable range of from 9.7 to 11.5 cP for all tested formulations.

[0301] [Table 13]

[0302] The opalescence was determined for Examples 7a and 7b by visual inspection and measured with a nephelometer at different temperatures and storage times. The appearance of the samples is shown in Figure 11 and the variation in the observed NTU is reported in Figure 12. The opalescence at all instances remained stable between the opalescence (NTU) of STD III and STD IV. 271 136 ul2 / u52

[0303] For Examples 7c, the opalescence was measured with a nephelometer. It was well below the opalescence of STD IV with a NTU ratio of sample / STD IV of 0.73. Poloxamer P188 added in an amount within the range of 0.01 (w / v) to 0.1 % (w / v) can thus have a positive influence on opalescence compared to other poloxamer concentrations.

[0304] The results show that both sodium phosphate buffer and HEPES are suitable buffers and that both Polysorbate 20 and Poloxamer 188 are suitable nonionic surfactants for use in formulations comprising lunsekimig in combination with L-arginine as excipient.

[0305] 5.11.8 Example 8 (Opalescence)

[0306] Formulations containing increasing concentrations of lunsekimig and with the excipient composition reported in Table 14 were prepared.

[0307] [Table 14]

[0308] The opalescence was measured with a nephelometer. The results are shown in Figure 13a and Figure 13b.

[0309] From Figure 13a, it can be seen that with increasing concentrations of L-arginine hydrochloride, the opalescence decreases and may be maintained at the desired standard even with increasing concentrations of lunsekimig.

[0310] From Figure 13b, it can be seen that both buffers, i.e., sodium phosphate buffer and HEPES buffer work equivalently well regardless of the concentration of lunsekimig. 271 136 ul2 / u52

[0311] 5.11.9 List of tables

[0312] Table 1. Properties of lunsekimig (SAR443765)

[0313] Table 2. Formulations and KD characterization of Examples la-e and Comparative Examples la-e

[0314] Table 3. pl of the building blocks of the constructs of Example 1 ii) and KD of the tested formulations

[0315] Table 4. Properties of the ISVD-Fc constructs of Example 1 iii)

[0316] Table 5. pl of ISVD-Fc construct of Example II (I)

[0317] Table 6. pl of ISVD-Fc construct of Example II (II)

[0318] Table 7. Formulations of Example 2 and viscosity measurement

[0319] Table 8. Formulations of Example 3 and viscosity measurement

[0320] Table 9. Formulations and opalescence measurement (OD 500) of Example 5 and Comparative Example 5

[0321] Table 10. Compositions of Example 6

[0322] Table 11. Opalescence measurement result of Example 6

[0323] Table 12. Compositions of Example 7

[0324] Table 13. Characterization of compositions of Example 7

[0325] Table 14. Compositions of Example 8

Claims

1. 271 136 ul2 / u52CLAIMS1. A pharmaceutical composition comprising: a polypeptide that comprises at least two portions whose isoelectric points differ by at least two pH units, and an amino acid having an isoelectric point of 7 or higher, or a salt thereof.

2. The pharmaceutical composition according to claim 1, wherein the amino acid is L- arginine or a salt thereof, optionally wherein the amino acid is L-arginine hydrochloride.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pH of the pharmaceutical composition is 5 or more and 11 or less.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the polypeptide comprises one or more portions having an isoelectric point of 6 or lower and one or more portions having an isoelectric point of 8 or higher.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein each of the two portions includes at least 50 contiguous amino acid residues.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein at least one portion of the polypeptide is an immunoglobulin single variable domain (ISVD).

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein at least one portion of the polypeptide is an ISVD having an isoelectric point of 6 or lower and at least one portion of the polypeptide is an ISVD having an isoelectric point of 8 or higher.

8. The pharmaceutical composition according to any one of claims 1 to 7, further comprising a buffer and one or more nonionic surfactants.271 136 ul2 / u529. The pharmaceutical composition according to claim 8, wherein the buffer is selected from sodium phosphate buffer, HEPES buffer, sodium acetate buffer and histidine hydrochloride buffer, and the nonionic surfactant is selected from polysorbates and block copolymers of polyethylene and polypropylene glycol, optionally wherein the nonionic surfactant is polysorbate 20 and / or Poloxamer 188.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the amino acid is present at a concentration of at least 50 mM.

11. The pharmaceutical composition according to claim 10, wherein the amino acid is present at a concentration of at least 200 mM.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the polypeptide is present at a concentration of at least 10 mg / mL.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the polypeptide is present at a concentration of at least 50 mg / mL.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the polypeptide is present at a concentration of at least 100 mg / mL.

15. The pharmaceutical composition according to any one of claims 1 to 14, comprising: the polypeptide at a concentration of 100 mg / mL to 300 mg / mL; the amino acid at a concentration of 200 mM to 500 mM, wherein the amino acid is L-arginine or a salt thereof; sodium phosphate buffer at a concentration of 5 mM to 20 mM;Poloxamer 188 at a concentration of 0.01 % (w / v) to 0.1 % (w / v) and wherein the composition has a pH of 6 to 8.271 136 ul2 / u5216. The pharmaceutical composition according to any one of claims 1 to 15, wherein the polypeptide comprises or consists of at least four ISVDs, wherein two ISVDs specifically bind IL-13 and two ISVDs specifically bind TSLP, wherein each of said at least four ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least four ISVDs are optionally linked via one or more peptidic linkers, and wherein: a first ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 7, a CDR2 that is the amino acid sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid sequence of SEQ ID NO: 17, a second ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid sequence of SEQ ID NO: 18, a third ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of SEQ ID NO: 19, and a fourth ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 11, a CDR2 that is the amino acid sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid sequence of SEQ ID NO: 21.

17. The pharmaceutical composition according to claim 16, wherein the polypeptide further comprises a fifth ISVD that binds to human serum albumin, wherein the fifth ISVD comprises: a CDR1 that is the amino acid sequence of SEQ ID NO: 10, a CDR2 that is the amino acid sequence of SEQ ID NO: 15; and a CDR3 that is the amino acid sequence of SEQ ID NO: 20; and the fifth ISVD is positioned between the third and the fourth ISVD.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the polypeptide comprises or consists of the amino acid sequence SEQ ID NO:1.271 136 ul2 / u5219. The pharmaceutical composition according to any one of claims 1 to 18, wherein the polypeptide is lunsekimig.

20. The pharmaceutical composition according to any one of claims 1 to 19, comprising: the polypeptide at a concentration of 150 mg / mL, wherein the polypeptide is lunsekimig, the amino acid at a concentration of 230 mM, wherein the amino acid is L-arginine hydrochloride, sodium phosphate buffer at a concentration of 10 mM,Poloxamer 188 at a concentration of 0.02 % (w / v); and wherein the composition has a pH of 7.0.

21. The pharmaceutical composition according to any one of claims 1 to 15, wherein at least one portion of the polypeptide is an ISVD having an isoelectric point of 8 or higher, and at least one portion of the polypeptide is an antibody Fc region having an isoelectric point of 6 or lower.

22. The pharmaceutical composition according to claim 21, wherein the antibody Fc region is an lgG4 Fc region.

23. The pharmaceutical composition according to claim 21 or 22, wherein the polypeptide comprises at least one portion having an isoelectric point of 8 or higher which is an ISVD, at least one portion having an isoelectric point of 8.5 or higher which is an ISVD, at least one portion having an isoelectric point of 6 or lower which is an antibody Fc region, and at least one portion having an isoelectric point of 6.5 or lower which is an antibody Fc region.

24. The pharmaceutical composition according to any one of claims 1 to 23 for use in the treatment of asthma, chronic obstructive pulmonary disease, atopic dermatitis or chronic rhinosinusitis with nasal polyps in a subject.

Images