WO2015086830A1

WO2015086830A1 - Skin penetrating polypeptides

Info

Publication number: WO2015086830A1
Application number: PCT/EP2014/077602
Authority: WO
Inventors: Lorenz Vogt; Thomas Jung; Titus Kretzschmar; Stefanie GRABULOVSKI
Original assignee: Delenex Therapeutics Ag
Priority date: 2013-12-13
Filing date: 2014-12-12
Publication date: 2015-06-18

Abstract

The invention relates to polypeptides, in particular antigen-binding polypeptides, capable of penetrating skin. Also provided are compositions comprising such polypeptides as well as uses of said polypeptides.

Description

Skin penetrating polypeptides Description

The invention relates to polypeptides capable of penetrating skin,

compositions comprising said polypeptides, and uses of said polypeptides.

Background of the invention

Human skin has a multilayered structure which serves as a protective barrier. The epidermis is the outermost layer, providing the most important and effective barrier to the external environment. The epidermis itself comprises different layers, among them the stratum corneum (SC), a layer of dead cells in a highly lipophilic environment that is considered as major barrier for the topical applications of drugs. It is currently believed that molecules with a mass of 500 Da or more cannot penetrate skin. Therefore, the number of drugs effectively treating skin diseases via the topical route of administration are limited and strategies to improve penetration are needed (Brown MB et al., Methods Mol Biol. 2008;437:1 19-39).

Chronic skin diseases impact the quality of life of the affected persons, both physically as well as psychologically. Psoriasis, for example, is a frequent disease with a prevalence of 2-3% in the Western population. While milder forms can be treated topically with glucocorticoids and/or vitamin D3- analogues, more severe forms require systemic treatment including cyclosporine, methotrexate or eventually biologies (Prieto-Perez R. et al, Pharmacogenomics 2013 Oct; 14(13):1 623-1 634). The introduction of biological therapies for the systemic treatment of severe psoriasis involving the use of monoclonal antibodies targeting e.g. CD2 (alefacept), TNF alpha (adalimumab, etanercept, and infliximab), IL-12 and IL-23 (ustekinumab; see Papoutsaki M and Costanzo A., BioDrugs 2013 Jan; 27 Suppl 1 :3-12) has substantially addressed the medical need in this group of patients. While highly effective, these drugs are associated with a number of potentially severe and serious adverse events. Therefore, the benefit/risk profile of these drugs are not applicable to the majority of psoriasis patients presenting with milder forms of psoriasis. These patients require effective topical therapies, which can be used long-term to address the chronicity of the disease and have an improved benefit/risk profile. The available drugs do not address this unmet medical need and novel, effective and innovative drugs have not emerged over the last decades (Parkins and Burden, Br J Dermatol 2013; 168:925).

Thus, biological therapies for topical use would fill the existing gap in the treatment of psoriasis and other chronic inflammatory skin diseases.

Summary of the invention

It has been found that polypeptides being much larger than 500 Da are capable of penetrating skin after topical application in buffer solution and reach a therapeutically relevant level. Therefore, medical or cosmetic treatment with biologies via topical administration routes becomes a viable option.

The polypeptides described herein have preferably a molecular weight of at least about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa or 30 kDa, most preferably of about 21 to about 29 kDa. Even more preferably, such polypeptides have a globular shape.

Such polypeptides are preferably antigen-binding polypeptides such as antibody fragments or alternative antibody scaffolds. Preferably, said antigen-binding polypeptides bind to a target selected from the group consisting of TNF alpha, VEGF, IL-1 alpha and beta, IL-4, IL-6, IL-8, IL-12, IL-13, IL-17A, IL-18, IL-23, p40, IL-31 or IL-36. These targets have been validated as clinically relevant in chronic skin diseases such as psoriasis, acne and atopic dermatitis. More preferably, such antigen-binding polypeptides are highly potent in neutralizing the target molecule and/or in binding to its target molecule.

Preferably, the polypeptides are particularly stable, thus having a low tendency to aggregate. The polypeptides have preferably a low tendency to induce an immune response against the polypeptide upon administration to a subject in need thereof. The polypeptides distribute effectively within the intercellular space, in contrast to full-length antibodies, thus reaching the source of inflammation even via the systemic route of administration.

Such polypeptides are useful for the topical treatment of skin diseases, thereby making biological therapy available to a larger range of patients. Thus, the invention provides pharmaceutical compositions comprising the polypeptides disclosed herein, containers comprising such compositions as uses of said

polypeptides in the treatment of skin diseases. Brief description of drawings

Figure 1 shows the permeation capacity of scFv versus IgG in an RHE model. The level of scFv and IgG in ng/mL was determined in the receptor fluid of the RHE chamber (see Figure 8) at time point t = 48 hours. Data were cleaned from outliers (40 data points were eliminated by ROUT software). Lane 1 : scFvl . Lane 2:

Infliximab. Lane 3: scFv2. Lane 4: Canakinumab. Lane 5: scFv3. Lane 6:

Bevacizumab. Lane 7: scFv4. Lane 8: Ustekinumab.

Figure 2 shows a 2-photon microscopy (PM) image of the basal epidermal cell layer of RHE tissue exposed with labeled scFvl . The basal epidermal layer consists of normal human keratinocytes. Figures 2A and B show an identical section of the basal epidermal cell layer. Figure 2A: Chanel detecting the AlexaFluor 594 labeled scFvl compound. Figure 2B: Chanel detecting the autofluorescence of keratin.

Labeled scFvl (Figure 2A) is located in the intercellular space of the tissue. Figure 3 shows cross-sections of minipig skin exposed in vivo to labeled scFvl . After 30 hours of exposure skin biopsies were taken and analyzed by confocal laser scanning microscopy. Figures 3A to 3D show an identical section of the epidermis and dermis. Labeled scFvl is penetrating in the upper layer of skin and evenly distributed in the extracellular part of the epidermis. Figure 3A: Chanel detecting the AlexaFluor 594 labeled scFvl compound. Figure 3B: Chanel detecting autofluorescence of keratin. Figure 3C: Phase contrast of the cross section. Figure 3D: Chanel detecting the DAPI labeled cell nuclei. Figure 4 shows the penetration via the hair follicle (marked by arrow) in cross- sections of minipig skin exposed for 30 hours with labeled scFvl in vivo. Skin biopsies were taken and analyzed by confocal laser scanning microscopy. Labeled scFvl is located around the hair follicle (arrow) in the lower layers of the skin

(dermis) and the epidermis. Figure 4A: Chanel detecting the AlexaFluor 594 labeled scFvl compound. Figure 4B: Chanel detecting autofluorescence of keratin. Figure 4C: Phase contrast image of the cross section. Figure 4D: Chanel detecting the DAPI labeled cell nuclei.

Figure 5 shows a SDS-PAGE gel stained with Coomassie blue of native scFvl after incubation with kallikrein 5 (KLK5). Lane 1 : 0.2 mg/ml of native scFvl incubated for 1 hour at 37°C with 0.001 mg/ml of KLK5. Lane 2: 0.2 mg/ml of native scFvl incubated for 3 hours at 37°C with 0.001 mg/ml of KLK5. Lane 3: 0.2 mg/ml of native scFvl incubated for 19 hours at 37°C with 0.001 mg/ml of KLK5. Lane 4: 0.2 mg/ml of native scFvl incubated for 3 hours at 37°C with 0.005 mg/ml of KLK5. Lane 5: 0.2 mg/ml of untreated native scFvl . Lane 6: Molecular weight marker.

Figure 6 shows a SDS-PAGE gel stained with Coomassie blue of native scFvl after incubation with kallikrein 7 (KLK7). Lane 1 : molecular weight marker. Lane 2: 0.2 mg/ml of untreated native scFvl incubated overnight at 37°C. Lane 3: 0.2 mg/ml of native scFvl incubated for 3 hours at 37°C with 0.0025 mg/ml of KLK7. Lane 4: 0.2 mg/ml of native scFvl incubated overnight at 37°C with 0.0025 mg/ml of KLK7. Lane 5: 0.2 mg/ml of native scFvl incubated for 3 hours at 37°C with 0.01 mg/ml of KLK7. Lane 6: 0.2 mg/ml of native scFvl incubated overnight at 37°C with

thermolysin.

Figure 7 shows a SDS-PAGE gel stained with Coomassie blue of native scFvl after incubation with trypsin. Lane 1 : molecular weight marker. Lane 2: 0.1 mg/ml of native scFvl incubated for 1 hour at 37°C with 0.02 mg/mL of trypsin. Lane 3: 0.1 mg/ml of native scFvl incubated for 2 hours at 37°C with 0.02 mg/ml of trypsin. Lane 4: 0.1 mg/ml of native scFvl incubated for 4 hours at 37°C with 0.02 mg/ml of trypsin. Lane 5: 0.1 mg/ml of native scFvl incubated overnight at 37°C with 0.02 mg/ml of trypsin. Lane 6: 0.1 mg/ml of native scFvl incubated overnight at 37°C. Figure 8 shows a schematic view of an RHE set-up 1 wherein a culture insert 2 comprising reconstituted human epidermis tissue 3 on a polycarbonate filter is hanging in a reservoir 4 comprising a chemically defined medium 5. Figure 9 shows the expression of the pro-inflammatory molecules TNF alpha,

IL-17A, IL-12, IL-23, IL-22, IL-27, I FN gamma, IL-1 beta, IL-6, IL-8 and CCL7 in skin biopsies taken at Day 14 after repeated topical administration of scFvl and placebo onto psoriatic plaques, see example 5. Pre-treatment with tape-stripping was performed weekly on one of these plaques. Data are expressed as fold-change over placebo. Black bars: samples from tape-stripped plaques; grey bars: samples from non-stripped plaques.

Detailed description

So that the invention may be more readily understood, certain terms are first defined. Unless otherwise defined within the specification, all technical and scientific terms used herein have their art-recognized meaning. Although similar or equivalent methods and materials to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present

specification, including definitions, will prevail. The materials, methods, and examples are illustrative only and not intended to be limiting.

Definitions

The term "polypeptide" as used herein refers to a compound made up of amino acids residues or amino acid analogs, linked by peptide bonds comprising at least 30 amino acids or analogs. The term "polypeptide" also includes molecules which contain more than one polypeptide that are joined together, covalently or noncovalently. Thus, the term protein is included within the definition of polypeptide and both terms are used interchangeably. Polypeptides can, e.g., be obtained (i) by isolation and purification from cells where they are produced naturally, (ii) by enzymatic (e.g., proteolytic) cleavage or fusion and/or (iii) by recombinant expression of nucleic acids encoding such polypeptides. Polypeptides can also be obtained by chemical synthesis or other known protocols for producing polypeptides. Within the scope of the present invention, an "antigen-binding polypeptide" refers to antibody fragments, non-antibody scaffolds, and/or other binding

compounds. Such antigen-binding polypeptide can be monovalent or multivalent, i.e. having one or more antigen binding sites. Non-limiting examples of monovalent antigen-binding polypeptide include scFv, Fab fragments, dAb, VHH, DARPins, affilins and nanobodies. A multivalent antigen-binding polypeptide can have two, three, four or more antigen binding sites whereby one or more different antigens can be recognized. F(ab')2 fragments, bis-scFv and diabodies are non-limiting examples of multivalent antigen-binding polypeptide; in said exemplary multivalent antigen- binding polypeptide, two binding sites are present, i.e. the antigen-binding

polypeptide is bivalent.

In one embodiment, the multivalent antigen-binding polypeptide is bispecific, i.e. the binding member is directed against two different targets or two different target sites on one target molecule. Bispecific antibodies are, e.g., reviewed in Muller, D. and Kontermann, R.E. Bispecific antibodies (Edited by Dubel, S. Weinheim: Wiley- VCH, 2007. ISBN 3527314539. p. 345-378). In another embodiment, the multivalent antigen-binding polypeptide comprises more than two, e.g., three or four different binding sites for three or four, respectively, different antigens. Such antigen-binding polypeptide is multivalent and multispecific, in particular tri- or tetra-specific, respectively.

"Antibody fragments" comprise portions of a full-length immunoglobulin retaining the targeting specificity of said immunoglobulin. Many but not all antibody fragments lack at least partially the constant region (Fc region) of the full-length immunoglobulin. In some embodiments, antibody fragments are produced by digestion of the full-length immunoglobulin. An antibody fragment may also be a synthetic or recombinant construct comprising parts of the immunoglobulin or immunoglobulin chains (see e.g. Holliger, P. and Hudson, Nature Biotechnology 2005; 23(9): 1 126-1 136). Examples of antibody fragments, without being limited to, include scFv, Fab, Fv, Fab', F(ab')2 fragments, dAb, VHH, nanobodies, V(NAR) or minimal recognition units. "Single chain variable fragments" or "single chain antibodies" or "scFv" are one type of antibody fragments. scFv are fusion proteins comprising the VH and VL of immunoglobulins connected by a linker. They thus lack the constant Fc region present in full-length immunoglobulins. The scFv disclosed herein have the general orientation VL-linker-VH.

"Non-antibody scaffolds" are antigen-binding polypeptides which are e.g.

described in Fielder, M. and Skerra, A. Non-antibody scaffolds. Edited by DLIBEL, S. Weinheim: Wiley-VCH, 2007. ISBN 3527314539. p. 467-500; or Gilbreth, R.N. and Koide, S. Current Opinion in Structural Biology 2012; 22: 413-420. Non-limiting examples include affibodies, affilin molecules, AdNectin, Anticalin, DARPins, Knottin, Kunitz-type domain, Avimer, Tetranectin and trans-body.

The "IC50" or "half-maximum inhibitory concentration" is a measure of antagonist drug potency and describes quantitatively the effectiveness of a

compound to inhibit a biological or biochemical function. This measure indicates how much of the compound is needed to inhibit by 50% a certain biological or biochemical process. Although no direct indicator of affinity, both values are correlated and can be determined via the Cheng-Prusoff equation (Cheng Y. and Prusoff W.H.,

Biochemical Pharmacology 1973; 22: 3099-3108; Rammes, G., et al., PLOS one 2009; 4:1 -14; Zhen J., et al., J Neuroscience Methods 2010; 188: 32-38).

The term "KD" refers to the dissociation equilibrium constant of the interaction between antigen and antigen-binding polypeptide. Said constant may e.g. be determined using surface plasmon resonance (SPR) technology in a BIACORE instrument or in an ATTANA instrument.

"Humanized" antibody fragments refer to antibody fragments comprising one or more, typically all six CDR regions of a non-human parent antibody or variants thereof, and of which the framework is, e.g., (i) a human framework, potentially comprising one or more framework residues of the non-human parent antibody, or (i a framework from a non-human antibody modified to increase similarity to naturally produced human frameworks. Methods of humanizing antibodies are known in the art, see e.g. Leger, O. and Saldanha, J. Antibody Drug Discovery. Edited by WOOD, C. London: Imperial College Press, 201 1 . ISBN 18481 66281 . p. 1 -23.

"Framework" (FR) refers to the scaffold of the variable immunoglobulin domain, either the variable light chain (VL) or variable heavy chain (VH), embedding the respective CDRs. A VL and/or VH framework typically comprises four framework sections, FR1 , FR2, FR3 and FR4, flanking the CDR regions. Thus, as known in the art, a VL has the general structure: (FR-L1 ) - (CDR-L1 ) - (FR-L2) - (CDR-L2) - (FR- L3) - (CDR-L3) - (FR-L4), whereas a VH has the general structure: (FR-H1 ) - (CDR- H1 ) - (FR-H2) - (CDR-H2) - (FR-H3) - (CDR-H3) - (FR-H4).

"CDR" refers to the hypervariable region(s) of an immunoglobulin or fragment thereof which mainly contribute to antigen binding. Typically, an antigen binding site comprises six CDRs, embedded into a framework scaffold. Herein, the CDRs of the VL are referred to as CDR-L1 , CDR-L2 and CDR-L3 whereas the CDRs of the VH are referred to as CDR-H1 , CDR-H2 and CDR-H3. These can be identified as described in KABAT, E.A., et al. (Sequences of Proteins of Immunological Interest. 5th edition. Edited by U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. NIH Publications, 1991 . p. 91 -3242). CDR-H1 as used herein, however, differs from the Kabat definition in that it starts with position 27 and ends prior to position 36.

An "isolated" antigen-binding polypeptide or nucleic acid is one being identified and separated and/or recovered from at least one component of its natural environment.

The term "identity" as used herein refers to the sequence match between two proteins or nucleic acids. The protein or nucleic acid sequences to be compared are aligned to give maximum identity, for example using bioinformatics tools such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). When the same position in the sequences to be compared is occupied by the same nucleobase or amino acid residue, then the respective molecules are identical at that very position. Accordingly, the "percent identity" is a function of the number of matching positions divided by the number of positions compared and multiplied by 100%. For instance, if 6 out of 10 sequence positions are identical, then the identity is 60%. The percent identity between two protein sequences can, e.g., be determined using the Needleman and Wunsch algorithm (Needleman, S.B. and Wunsch, CD., J Mol Biol. 1970; 48: 443-453) which has been incorporated into EMBOSS Needle, using a BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extend penalty" of 0.5, a false "end gap penalty", an "end gap open penalty" of 10 and an "end gap extend penalty" of 0.5. Two molecules having the same primary amino acid or nucleic acid sequence are identical irrespective of any chemical and/or biological modification. For example, two antibodies having the same primary amino acid sequence but different glycosylation patterns are identical by this definition. In case of nucleic acids, for example, two molecules having the same sequence but different linkage components such as thiophosphate instead of phosphate are identical by this definition.

A "variant" refers to an amino acid or nucleic acid sequence which differs from the parental sequence by virtue of addition (including insertions), deletion and/or substitution of one or more amino acid residues or nucleobases while retaining at least one desired activity of the parent sequence disclosed herein. In the case of antigen-binding polypeptides such desired activity may include specific antigen binding. Similarly, a variant nucleic acid sequence may be modified when compared to the parent sequence by virtue of addition, deletion and/or substitution of one or more nucleobases, but the encoded antibody retains the desired activity as described above. Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed.

As used herein, the term "effective amount" refers to an amount of the polypeptide or biologically effective compound that is capable of treating the symptoms of the described skin condition. The specific dose of a compound administered according to this invention is typically determined by the particular circumstances surrounding the case including, e.g., the polypeptide or biologically effective compound administered, the route of administration, the state of being of the subject, and the severity of the condition being treated.

Various aspects of the invention are described in further detail in the following subsections. It is understood that the various embodiments, preferences and ranges may be combined at will. Further, depending of the specific embodiment, selected definitions, embodiments or ranges may not apply.

Polypeptides

In one aspect, the present invention provides polypeptides capable of penetrating into the epidermis at pharmacological relevant levels after topical application. Said polypeptides are capable of skin penetration without the presence of penetration enhancers or penetration enhancing delivery systems (e.g., liposomes), although such systems or enhancers might be used in order to even improve skin penetration.

Compounds applied on the skin surface can potentially penetrate via three pathways: (i) through sweat ducts, (ii) via hair follicles and associated sebaceous glands or (iii) across the stratum corneum. These different routes are not mutually exclusive. In most cases the penetration of the compound is a combination of pathways and the relative amounts are depended on the physicochemical properties of the penetrant. The penetration via the stratum corneum is divided in two routes: transcellular and intercellular. It has been found that the polypeptides disclosed herein penetrate preferably via the intercellular route (see Figure 2 for according imaging data). Polypeptides penetrating via the stratum corneum are mainly targeting the upper layers of the epidermis as can be derived from Figure 3. As shown in Figure 4 polypeptides penetrating via sweat ducts or hair follicles are also reaching deeper skin layers (dermis). Penetration via the hair follicle is e.g. beneficial for the local treatment of acne vulgaris by polypeptides.

Skin penetration, and in particular permeation through the stratum corneum, may e.g. be assessed in Reconstructed Human Epidermis (RHE) models. RHE tissues are widely used and established to study skin permeation of compounds in vitro. These tissues are readily available and characterized and therefore circumvent the limitations of using fresh full skin samples from human subjects. RHE tissues are in vitro skin models composed of epidermis and stratum corneum. Some structures and layers, as for example dermis, sweat pores, hair structures and nerve fibers, present in normal human skin are missing in RHE skin models. Nevertheless, RHE skin tissues and human skin are highly similar in their histological composition. As demonstrated by Lotte et al. (Skin Pharmacol Appl Physiol. 2002, 15 Suppl 1 : p.18-30) a correlation for penetration and/or permeation of different compounds exists when comparing human skin and RHE tissues in respective assays.

In RHE models, percutaneous permeation of different compounds applied in aqueous solution can be assessed and these models are particularly useful for ranking the skin penetration potential of different compounds.

RHE can e.g. be obtained from StratiCELL (Les Isnes, Belgium) or Episkin (Lyon, France). StratiCELL RHE model consists of living epidermal tissue, produced in culture inserts on polycarbonate filters with a size of 0.63 cm². Tissues are cultivated at the air-liquid interface in serum-free and chemically defined medium. StratiCELL RHE are fully stratified and differentiated as demonstrated by the expression profile of keratin 14, keratin 10 and involcurin. StratiCELL tissues also synthesize IL-1 a and IL-8 following the exposure to irritant or sensibilizing agents, respectively. The Episkin kit comprises several RHE units having a size of 1 .07 cm² or 0.38 cm² onto which products can be directly applied. Each reconstructed skin unit consists of type I collagen matrix, representing the dermis, surfaced with a film of type IV collagen, onto which is laid a stratified and differentiated epidermis derived from human keratinocytes.

Preferably, the penetration efficacy of the polypeptide is characterized by a flux of about 0.70 ng/(h cm²) (coefficient of variation (CV) of 4%) when said polypeptide is applied at a concentration of 1 .0% onto the RHE. Additionally or alternatively, the penetration efficacy of the polypeptide is characterized by a flux of at least about 0.07, preferably about 0.36 ng/(h cm²) (CV of 97%) when said polypeptide is applied at a concentration of 0.5% onto the RHE and/or a flux of about 0.05 ng/(h cm²) (CV of 38%) when said polypeptide is applied at a concentration of 0.1 % onto the RHE.

Additionally or alternatively, the penetration efficacy of the polypeptide reaches a dose in the receptor fluid of about 0.13%o (CV of 58%) when said polypeptide is applied at a concentration of 1 .0% onto the RHE. Additionally or alternatively, the penetration efficacy of the polypeptide reaches a dose determined in the receptor fluid of at least about 0.04 _o, preferably about 0.05 _o, 0.06 _o, 0.07 _o, 0.08 _o, and most preferably about 0.09 _o (CV 87%) when said polypeptide is applied at a concentration of 0.5% onto the RHE. Additionally or alternatively, the penetration efficacy of the polypeptide reaches a dose in the receptor fluid of about 0.06 _o (CV 31 %) when said polypeptide is applied at a concentration of 0.1 % onto the RHE.

The polypeptides of the instant invention have also been shown to penetrate skin of minipigs as well as of human psoriasis patients after topical application in vivo. Thus, the polypeptides described herein can be used for the topical treatment of skin diseases, in particular of psoriasis.

Psoriasis is an inflammatory skin disease characterized by keratinocyte hyperplasia, epidermal thickness, and infiltration of dermal T cells and leukocytes (Wang, C.Q.F. et al (2013), J Inv Derm 133, 2741 -2752). Five main types of psoriasis are known, being plaque, guttate, inverse, pustular, and erythrodermic psoriasis, where plaque psoriasis is the most common form of psoriasis. Histopathologically, psoriatic skin differs from normal skin in the following: dermal inflammatory infiltrates; parakeratosis; absence of a granular layer; rete ridges (epidermal thickenings) being regularly elongated with thickening of the lower portions; and long edematous and often club shaped papillae and the presence of microabscesses (Sehgal, V.N (2008), Textbook of Clinical Dermatology, Jaypee Brothers Medical Publishers on page 133).

Preferably, the polypeptides described herein have a molecular weight of at least about 5 kDa, preferably at least about 10 kDa, 15 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa or 27 kDa. Additionally or alternatively, the polypeptides have a molecular weight below about 150 kDa, more preferably below about 100 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa, 30 kDa, 29 kDa, 28 kDa, 27 kDa or 26 kDa. Even more preferably, the polypeptides have a molecular weight between about 21 kDa and about 27 kDa. Most preferably, the molecular weight is between about 25.5 and 26.5 kDa.

The polypeptides may have different shapes, i.e. being globular or fibrous. In a preferred embodiment, the polypeptide is globular. Globular polypeptides are generally compact, soluble, and about spherical in shape. Preferably, the polypeptides distribute effectively into the intercellular space, thus reaching the source of inflammation even via the systemic route of

administration. Given a normal blood volume of at least 5.8 liters in a normal 75 kg human subject and a corresponding volume of extracellular fluid of 15 L, "effective distribution" means a volume of distribution at steady state of the polypeptide of higher than 5.8 L, more preferably higher than about 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 or 18 liters as measured in human beings. In particular, the polypeptides are capable of penetrating skin in the absence of a penetration enhancer or any vehicles for enhanced drug delivery to skin (e.g., liposomes).

Preferably, the polypeptide is stable. As used herein the term "stable" refers to the biophysical property of the polypeptide to remain monomeric in solution after prolonged incubation and/or incubation at elevated temperature. Unstable

polypeptides tend to dimerize or oligomerize and even precipitate, thereby

decreasing shelf-life and becoming less suitable for pharmaceutical applications. Preferably, the polypeptide remains stable at higher concentrations, for example, they remain monomeric at least to 50%, preferably at least to 55%, 60%, 65%, 70% and most preferably to at least 75% after being incubated for 2 weeks at room temperature and/or 4°C at a concentration of about 10 mg/ml in saline buffered solution, preferably in PBS at pH 7.2. In one embodiment, the in-use stability of the polypeptide is characterized by a monomer content of at least 75%, more preferably at least 80%, 85% or 90%, at a concentration of 5 mg/ml and a temperature of 2-8°C and/or room temperature for period of at least 21 days in a composition as described herein, preferably in a hydrogel.

In its native form, the polypeptides are preferably resistant to proteases, such as skin-derived serine proteases and/or trypsin. Non limiting examples of serine proteases include kallikreins, such as kallikrein 5 and/or kallekrein 7. Kallikreins are highly expressed in the epidermis of psoriasis patients. Such protease resistance correlates with a low tendency to induce an immune response upon administration to a subject in need thereof. In one embodiment, the size of the polypeptide in its native form remains intact to at least 80%, preferably to at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, more preferably to at least 100%, after overnight incubation at 37°C with a given protease. Additionally or alternatively, the function of the polypeptide in its native form remains intact to at least 80%, preferably to at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, more preferably to at least 100%, after overnight incubation at 37°C with a given protease.

The polypeptides disclosed herein are preferably antigen-binding

polypeptides. Exemplary targets of such antigen-binding polypeptides include antigens involved in psoriasis (Prieto-Perez R. et al, Autoimmune Dis.

2013;2013:613086), acne vulgaris (Kistowska et al., J Invest Dermatol 2014,

134:677), atopic dermatitis (Beck et al., NEJM 2014, 371 :130), portwine stain

(Laquer et al., Lasers Surg Med 2013, 45:67), alopecia areata, basal cell carcinoma, melanoma, Bowen's disease, congenital erythropoietic porphyria, contact dermatitis, Darier's disease, epidermolysis bullosa simplex, erythropoietic protoporphyria, hailey- hailey disease, herpes simplex, hidradenitis suppurativa (HASLUND, P. et al (2009), Acta Derm Venereol. 2009 Nov;89(6):595-600), hirsutism, hyperhidrosis, ichthyosis, impetigo, keloids (Gira et al., J Am Acad Dermatol 2004; 50:850), keratosis pilaris, lichen planus (LeCleach and Chosidow, NEJM 2012; 366:723), lichen sclerosus (Limpers et al., Exp Rev Clin Immunol 2014, 10:231 ), melasma, pemphigus vulgaris, verrucas, pityriasis lichenoides, polymorphic light eruprion, pyroderma gangrenosum (Goodarzi H. et al (2012), Adv Wound Care (New Rochelle), Oct;1 (5):194-199), rosacea (Steinhoff et al., J Invest Dermatol Symp Proc 2007; 15:2), scabies, shingles, squamous cell carcinoma and/or vitiligo. Examples of such antigens include, without being limited to, TNF alpha, VEGF, IL-1 alpha and beta, IL-4, IL-6, IL-8, IL-12, IL-13, IL-17A, IL-18, IL-23, p40, IL-31 or IL-36.

Thus, in one embodiment, the antigen that is bound by the antigen-binding polypeptide is TNF alpha (tumor necrosis factor alpha). Also known as cachetectin, said naturally occurring mammalian cytokine is a major mediator of inflammatory, immunological and pathopysiological reactions. A large number of disorders are associated with up-regulated TNF alpha levels, many of them at significant medical importance. Skin diseases involving elevated TNF alpha levels include, without being limited to, psoriasis, hidradermitits supperativa and pyoderma gangrenosum (Karampetsou et al. 2010; QJM 103:917). The antigen-binding polypeptides of the instant invention are particularly suitable for the treatment of such diseases, as they can be applied locally or topically to the skin. Examples of suitable antigen-binding polypeptides which bind specifically to TNF alpha are e.g. described in

PCT/CH2006/000300, filed June 6, 2006; or in PCT/CH2009/000219, filed June 25, 2009 and include, e.g., scFvl and variants thereof.

The scFvl , an anti-TNF alpha scFv comprising SEQ ID No. : 1 , is

characterized by a molecular weight of about 26.3 kDa and a pi of 7.80. It showed a similar TNF alpha inhibitory potency to infliximab (Remicade®; see Urech D.M et al., Ann Rheum Dis. 2010 Feb; 69: 443-449). In one embodiment, the antigen-binding polypeptide comprises the VL of SEQ ID No.: 1 or a variant thereof. Additionally or alternatively, the antigen-binding polypeptide comprises the VH of SEQ ID No.: 1 , or a variant thereof.

In another embodiment, the antigen that is bound by the antigen-binding polypeptide is VEGF (vascular endothelial growth factor). VEGF is a known regulator of angiogenesis and neovascularization. Angiogenesis is an important aspect of tumor growth as the developing tumor requires an adequate supply of oxygen and nutrients which are provided by the growth of new blood vessels. However, VEGF is not only involved in tumor growth; skin diseases involving elevated VEGF levels include, without being limited to, portwine stain, rosacea, Kaposi sarcoma or keloid (Patel et al., Semin Cutan Med 2012; 31 :98). The antigen-binding polypeptides of the instant invention are particularly suitable for the treatment of such diseases, as it can be applied locally or topically to the skin. Examples of suitable antigen-binding polypeptides which bind specifically to VEGF are described in PCT/CH2009/000220, filed June 25, 2009 and include, e.g., scFv3 and variants thereof.

The scFv3, anti-VEGF-scFv comprising SEQ ID No. : 3, is characterized by a molecular weight of about 26.3 kDa and a pi of 4.93. In vitro, it binds to all isoforms of VEGFA, including VEGF1 65, with an affinity KD of 28.4 pM. It inhibits VEGF induced HUVEC proliferation with an ICso of 0.19 nM. In one embodiment, the antigen-binding polypeptide comprises the VL of SEQ ID No.: 3 or a variant thereof. Additionally or alternatively, the antigen-binding polypeptide comprises the VH of SEQ ID No.: 3, or a variant thereof.

In still another embodiment, the antigen that is bound by the antigen-binding polypeptide is IL-1 beta (interleukin 1 beta). IL-1 beta is a pro-inflammatory cytokine which is produced as a precursor by activated macrophages. Upon proteolytic cleavage, signal transduction is initiated by binding of the active form to the IL-1 receptor type I (IL-1 R1 ) which in turn associates with the transmembrane IL-1 receptor accessory protein (IL-1 RAP). The formed complex is competent of signal transduction. Being a key mediator in the inflammatory response, the cytokine affects a number of cellular activities such as cell proliferation, differentiation, and apoptosis. Therefore, IL-1 beta has been considered an important target for a variety of pharmaceuticals. Examples of skin diseases involving elevated IL-1 beta levels include, without being limited to, acne vulgaris (Qin et al., J Invest Dermatol epub July 2013). Suitable antigen-binding polypeptides which specifically bind IL-1 beta are described in US 14/072,1 65 and WO2014/068132, both filed November 5, 2013 and include scFv2 and variants thereof and/or scFv5 and variants thereof.

The scFv2, an anti-IL-1 beta-scFv comprising SEQ ID No. : 2, is characterized by a molecular weight of about 25.6 kDa and a pi of 8.32. Its potency ICso for inhibiting the biological effect of human IL-1 beta is in the range of 3 pM ± 1 .05, as determined by inhibiting the IL-1 beta stimulated release of IL-6 from human fibroblasts. In one embodiment, the antigen-binding polypeptide comprises the VL of SEQ ID No. : 2 or a variant thereof. Additionally or alternatively, the antigen-binding polypeptide comprises the VH of SEQ ID No. : 2, or a variant thereof. A variant of scFv2 is scFv5, also targeting IL-1 beta, however, with a much improved potency: the ICso was determined to be < 0.6 ± 0.4 pM. Working at the detection limit of the assay, the ICso value is believed to be even lower. scFv5 comprises SEQ ID No. : 4 and has a pi of 8.9.

In still another embodiment, the antigen that is bound by the antigen-binding polypeptide is p40. p40 is a subunit of both, human interleukin 12 (IL-12) and interleukin 23 (IL-23), and also known as natural killer cell stimulatory factor 2, or cytotoxic lymphocyte maturation factor 2. IL-12 is a cytokine expressed by activated macrophages that serve as an essential inducer of Th1 cells development. The cytokine acts on T and natural killer cells, and has a broad array of biological activities. Neutralization of p40 is an approach for the treatment of e.g. psoriasis. Suitable antigen-binding polypeptides which specifically bind p40 include scFv4 or variants thereof. In one embodiment, the antigen-binding polypeptide comprises the VL of scFv4 or a variant thereof. Additionally or alternatively, the antigen-binding polypeptide comprises the VH of scFv4, or a variant thereof.

Additionally or alternatively, the antigen-binding polypeptides may be directed against targets involved in the aging process of skin. For example, transcription factor N F-KB (nuclear factor-κΒ) has been identified as a crucial regulator of gene expression associated with ageing. Blocking N F-κΒ activity in the epidermis of aged mice reverted tissue characteristics and the global gene expression program to those of young mice (Adler AS et al., Genes Dev 2007; 21 : 3244-3257). Polypeptides binding to N F-κΒ may therefore be used to decelerate the degenerative effects of ageing processes.

The antigen-binding polypeptide is preferably not a full-length immunoglobulin. Full-length immunoglobulins typically have a molecular weight of about 150 kDa. In one embodiment, the antigen-binding polypeptide is antibody fragment or alternative antibody scaffold. Antibody fragments include, without being limited to, scFv, Fab, Fv, Fab', F(ab')2 fragments, dAb, VHH, nanobodies, V(NAR) or minimal recognition units. In a much preferred embodiment, said antibody fragment is a scFv.

Alternatively, said antigen-binding polypeptide is an alternative antibody scaffold. Examples thereof include, without being limited to, affibodies, affilin molecules, AdNectin, Anticalin, DARPins, fynomers, Knottins, Kunitz-type domains, Avimers, Tetranectins or a trans-bodies. In one embodiment, the antigen-binding polypeptide is a bispecific molecule, such as a tandem scFv, a diabody, a scFv diabody or a Dart. Such bispecific molecule may e.g. bind to the same target or to different target molecules. Antigen-binding polypeptides having more than two binding-sites are also contemplated herein.

Much preferred antigen-polypeptides are scFv. Due to their small size and their stability parameters, protease-resistance and thermal stability, the scFv described herein are capable of efficiently penetrating tissues. Further, they display a decreased retention in the systemic circulation as they are unable to bind to Fc receptors such as FcRn, eventually leading to high renal clearance rates. These characteristics of good tissue penetration with subsequent even distribution in the tissue and the rapid elimination from the systemic circulation are particularly advantageous for chronic topical diseases. This practical utility has however been severely limited in the past by low stability and low biological potency of recombinant, humanized scFv which is overcome by the scFv described herein which are particularly stable and highly potent.

In one embodiment, the polypeptide is a scFv selected for enhanced stability and solubility in a reducing environment. A reducing environment sets stringent conditions for polypeptide stability and solubility and therefore, polypeptides stable in an intracellular environment are also under oxidizing conditions. For example, stability and solubility in a reducing environment can be assessed using a yeast Quality Control (QC) - System (see e.g., PCT Publication WO 2001 /48017; U.S. Application Nos. 2001 /0024831 and US 2003/0096306; US Patent Nos 7,258,985 and 7,258,986). In one exemplary embodiment of said QC system, a scFv library is fused to the activation domain (AD) of the Gal4 yeast transcription factor, which is in turn fused to a portion of the so-called Gall 1 p protein (1 1 p). The scFv-AD-Gal1 1 p fusion construct is then transformed into host cells which contain the Gal4 DNA- binding domain (DBD; Gal4(1 -100)). Gall 1 p is known to directly bind to Gal4(1 -100) (see Barberis et al., Cell, 81 : 359 (1995)). The transformed host cells are cultivated under conditions suitable for expressing the scFv fusion protein and that allow for cell survival only in the case that the scFv fusion protein is stable and soluble enough to interact with Gal4(1 -100) and thereby form a functional transcription factor containing an AD linked to a DBD. Thereby, scFvs expressed in the surviving cells have frameworks that are stable and soluble in a reducing environment. In one embodiment, the antigen-binding polypeptide, in particular the scFv, does not comprise SEQ ID No. 1 and/or 3.

The antigen-binding polypeptide has preferably an affinity KD of less than 10 nM, preferably less than 1 nM, less than 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 10 pM, 5 pM and most preferably below 1 pM. Such parameter may be determined by well-known methods in the art, such as surface plasmon resonance (BIACORE) or via an Attana instrument.

The antigen-binding polypeptide has preferably a potency of less than 10 nM, preferably less than 1 nM, less than 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 10 pM, 5 pM and most preferably below 1 pM in neutralizing its target. Such parameter may be determined by well-known methods in the art, such as cell-based assays.

Most preferred are antigen-binding polypeptides having a high potency. Such antigen-binding polypeptide have the advantage that small amounts of polypeptides achieve a therapeutic effect. Thus, preferably, the antigen-binding polypeptide inhibits the biological effect of its target with an ICso of lower than about 1 nM, preferably lower than about 500 pM, 400 pM, 300 pM, 100 pM, 50 pM, 25 pM, 10 pM, most preferably lower than about 1 pM.

The antigen-binding polypeptides comprise preferably a human or humanized framework sequence. For example, in case the antigen-binding polypeptide is a scFv, it preferably comprises the VL framework sequences as set forth in SEQ ID Nos. 5 to 8 or variants thereof and/or the VH framework sequences as set forth in SEQ ID Nos. 9 to 12, or variants thereof. For example, such variant may comprise any one of SEQ ID Nos: 13 to 20. Alternatively, the scFv may comprise the VL framework sequences as set forth in SEQ ID Nos. 21 to 24 and/or the VH framework sequences as set forth in SEQ ID Nos. 25 to 26, or variants thereof. Such variants typically have at least 90% sequence identity, more preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the overall VL or VH framework sequence. Recombinant scFv may e.g. be produced by grafting the CDRs of an antibody stemming from rabbit or any other lagomorph onto a framework comprising at least one of the sequences as set forth in SEQ ID Nos. : 5 to 20; preferably all four light chain and all four heavy chain framework sequences FR1 -4 being selected from the group consisting of SEQ ID Nos. : 5 to 20. Rabbit antibodies mostly show significant higher affinities when compared to rodent antibodies and are therefore a preferred starting point for engineering humanized scFv therapeutics. In particular VH SEQ ID Nos: 9-12 and 13-1 6 in combination with VL SEQ ID Nos: 5-8 have been described as universal acceptor framework for the grafting of rabbit CDRs (see, e.g.,

PCT/CH2009/000222, filed June 25, 2009 or BORRAS, L. et al, JBC 2010; 285(12): p. 9054-9066). scFv comprising in particular at least one, preferably at least 2,3, 4, 5, 6, 7, more preferably all 8 framework sequences of the group consisting of SEQ ID Nos. 5-28 remain monomeric even at high concentrations and/or for prolonged periods of time, such as 2, 3, 4, 5, 6, 7, 8 or more weeks in saline buffered solution, preferably PBS at 4°C or room temperature.

Variant sequences of the polypeptides described herein typically have at least 90% sequence identity, more preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the overall parent sequence and bind to the same target, preferably the same epitope. More preferably, the potency and/or affinity of such variant is substantially the same as the parent polypeptide, even more preferably at least about 50%, 60%, 70%, 80% or at least 90% of the potency and/or affinity value of the parent polypeptide. Additionally or alternatively, the variant is at least as resistant to proteases as the parent polypeptide. For example, the size of the variant in its native form remains intact to at least 80%, preferably to at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, more preferably to at least 100%, after overnight incubation at 37°C with a given protease. Additionally or alternatively, the function of the variant in its native form remains intact to at least 80%, preferably to at least 85%, 90%, 95% 96%, 97%, 98%, 99%, more preferably to at least 100%, after overnight incubation at 37°C with a given protease. Additionally or alternatively, the variant shows the same monomeric behavior as the parent polypeptide.

Variants of the polypeptides provided herein may be prepared by protein and/or chemical engineering, introducing appropriate modifications into the nucleic acid sequence encoding the polypeptide, or by protein/peptide synthesis. Any combination(s) of deletions, substitutions, additions and insertions can be made to the framework or to the CDRs, provided that the generated antibody possesses the desired characteristics for which it can be screened using appropriate methods. Of particular interest are substitutions, preferably conservative substitutions. As used herein, the term "conservative modifications" refers to modifications that are physically, biologically, chemically or functionally similar to the corresponding reference, e.g., has a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like. Such

conservative modifications include, but are not limited to, one or more nucleobases and amino acid substitutions, additions and deletions. For example, conservative amino acid substitutions include those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, in antigen- binding polypeptides the amino acid residues being non-essential with regard to binding to an antigen can be replaced with another amino acid residue from the same side chain family, e.g. serine may be substituted for threonine. Amino acid residues are usually divided into families based on common, similar side-chain properties, such as:

1 . nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, methionine),

2. uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, proline, cysteine, tryptophan),

3. basic side chains (e.g., lysine, arginine, histidine, proline),

4. acidic side chains (e.g., aspartic acid, glutamic acid),

5. beta-branched side chains (e.g. , threonine, valine, isoleucine) and

6. aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

A conservative substitution may also involve the use of a non-natural amino acid. Preferred conservative substitutions include:

1 . Substituting alanine (A) by valine (V);

2. Substituting arginine (R) by lysine (K);

3. Substituting asparagine (N) by glutamine (Q);

4. Substituting aspartic acid (D) by glutamic acid (E);

5. Substituting cysteine (C) by serine (S);

6. Substituting glutamic acid (E) by aspartic acid (D);

7. Substituting glycine (G) by alanine (A);

8. Substituting histidine (H) by arginine (R) or lysine (K)

9. Substituting isoleucine (I) by leucine (L);

10. Substituting methionine (M) by leucine (L); 1 1 . Substituting phenylalanine (F) by tyrosine (Y);

12. Substituting proline (P) by alanine (A);

13. Substituting serine (S) by threonine (T);

14. Substituting tryptophan (W) by tyrosine (Y);

15. Substituting phenylalanine (F) by tryptophan (W);

and/or

1 6. Substituting valine (V) by leucine (L)

and vice versa. The polypeptide described herein may comprise one or more, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of such

conservative substitutions.

Non-conservative substitutions may lead to more substantial changes, e.g., with respect to the charge, dipole moment, size, hydrophilicity, hydrophobicity or conformation of the polypeptide. In one embodiment the polypeptide comprises one or more, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of such non-conservative substitutions.

Conservative and non-conservative modifications can be introduced into parental polypeptide sequences by a variety of standard techniques known in the art, such as combinatorial chemistry, site-directed DNA mutagenesis, PCR-mediated and/or cassette mutagenesis, peptide/protein chemical synthesis, chemical reaction specifically modifying reactive groups in the parental binding member. The variants can be tested by routine methods for their chemical, biological, biophysical and/or biochemical properties.

Modifications may be present in the CDRs or in the framework sequences. For example, the CDRs provided herein may comprise one, two, three, four, five or even more modifications. For example, the CDR-L1 , CDR-L2 and CDR-L3 sequences taken as a whole are at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more preferably 99% identical to the CDRs provided herein. Additionally or alternatively, the CDR-H1 , CDR-H2 and CDR-H3 sequences taken as a whole are at least 80%, preferably at least 81 %, 82%, 83%, 84%, 95%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more preferably 99% identical to the CDRs provided herein.

Nucleic Acids, vectors, host cells and method of production

The polypeptides described herein are encoded by a single nucleic acid or by two or more nucleic acids, for example each encoding at least one variable region. Knowing the sequence of the antibody or of its parts, cDNAs encoding the

polypeptide sequence can be generated by methods well known in the art, e.g. by gene synthesis. These cDNAs can be cloned by standard cloning and mutagenesis techniques into a suitable vector such as an expression vector or a cloning vector. Optionally, for scFv and the like, the variable light chain is encoded by a separate nucleic acid than the variable heavy chain of the polypeptides. Further, additional sequences such as tags (e.g., a His-tag), linkers, the coding sequence of a second binding specificity or another functional polypeptide such as an enzyme to generate a fusion construct or a bispecific molecule may be included into the genetic construct.

Based on the cloning strategy chosen, genetic constructs may generate a polypeptide having one or more additional residues at the N-terminal or C-terminal end. For example, an N-terminal methionine derived from the start codon or an additional alanine may be present in an expressed polypeptide, unless it has been clipped off post-translationally. It is therefore to be understood that the polypeptides disclosed herein comprise the disclosed sequences rather than consist of them.

Basic protocols of standard cloning, mutagenesis and molecular biology techniques are described in, e.g., Molecular Cloning, A Laboratory Manual (Green,

M. and Sambrook, J. Molecular Cloning: a Laboratory Manual. 4th edition. Cold

Spring Harbor Laboratory, 2012. ISBN 19361 13422.).

Appropriate host cells for the expression of the genetic constructs can be prokaryotic or eukaryotic. Suitable prokaryotic host cells are gram-negative or gram- positive and include species of the Escherichia, Erwinina, Enterobacter, Klebsiella,

Pseudomonas or Bacillus families. Much preferred is Escherichia coli, in particular E. coli strains BL21 (DE3) (Life Technologies™, cat. no. C6000-03) and Origami™

2(DE3) (Novagen, cat. no 71345). If post-translational modifications such as glycosylation or phosphorylation are desired, eukaryotic host cells are preferable. For example, eukaryotic microbes such as commonly used Saccharomyces cerevisiae or Pichia pastoris strains may serve as host cells. Host cells can also include plant or animal cells, in particular insect or mammalian cells. Suitable mammalian cells include, without being limited to, Chinese Hamster Ovary Cells (CHO), Human Embryonic Kidney Cells (HEK), Human

Umbilical Vein Endothelial Cells (HUVEC) or NSO myeloma cells.

The polypeptides can be produced by expression in a suitable host cell. For example, the expression vectors described above are introduced into a host cell by standard techniques such as electroporation or chemical transformation. The transformed cells are then cultivated under conditions adequate for recombinant protein expression, typically in appropriate nutritional media, optionally modified for inducing promoters, selecting transformants, or amplifying encoding sequences of interest. The antibody is recovered from the culture and optionally purified using standard techniques in the art. The yield of recombinant protein may be improved by optimizing media and culture conditions such as temperature or oxygen supply. In prokaryotes the antibody can be produced in the periplasm, intracellular^ as inclusion bodies or be secreted into the medium. Upon harvest, the protein can be purified using methods well known in that art such as gel filtration, ion exchange chromatography, reversed phase chromatography, hydrophobic interaction, mixed mode chromatography and/or affinity chromatography.

In one embodiment the polypeptide is produced in a cell-free system. This typically involves in vitro transcription followed by in vitro translation of nucleic acid product templates encoding the proteins described herein, e.g., plasmid DNA or PCR product templates. For example, crude lysates from growing cells are used, providing the necessary enzymes as well as the cellular protein synthesis machinery. The necessary building blocks such as amino acids or nucleobases as well as energy delivering molecules and others can be exogenously supplied. Cell-free expression systems can, for example, be based on lysed rabbit reticulocytes (e.g., Rabbit Reticulocyte Lysate System, Promega, cat. no. L4540), HeLa cells (e.g., 1 -Step Human In Vitro Translation Kit, Thermo Scientific, cat. no. 88881 ), insect cells (e.g., EasyXpress Insect Kit II, Qiagen, cat. no. 32561 ), wheat germs (e.g., Wheat Germ Extract, Promega, cat. no. L4380), or E.coli cells (e.g., PURExpress® In Vitro Protein Synthesis Kit, NEB, cat. no. E6800S). Also, optimized cell-free antibody expression systems for improved disulfide bond generation can be used for production.

Commercially available kits include insect cell lysates (e.g., EasyXpress Disulfide Insect Kit, Qiagen, cat. no. 32582) or E.coli cell lysates (e.g., EasyXpress Disulfide E. coli Kit, Qiagen, cat. no. 32572). Cell-free protein synthesis has, e.g., the advantage of being fast, achieving high product yields, allowing for easy modification of reaction conditions, forming a low degree of or even no byproducts. Cell-free protein synthesis may involve biological and/or chemical steps which cannot be conducted in purely biological or chemical production systems. For example, non-natural or chemically-modified amino acids can be incorporated into the protein at desired positions. ScFv- toxin fusion proteins have been successfully produced in cell-free systems (Nicholls, P. J. et al., J Biol Chem 1993; 268: 5302-5308). Thus, in one embodiment a method of producing the polypeptide described herein is provided comprising the steps of (a) providing a cell-free system, (b) providing a nucleic acid product template encoding the polypeptide described herein, (c) allowing for transcription and translation of said nucleic acid product template; (d) recovering; and optionally (e) purifying said polypeptide. Additionally or alternatively, a method of producing the polypeptide described herein comprises at least one step of chemical synthesis. For example, the method may be entirely chemical. In another embodiment, the cell-based or the cell-free production systems described above comprise such at least one step of chemical synthesis.

In a preferred embodiment the polypeptides described herein are produced in a cell-based system using an expression vector for intracellular expression in E. coli. Upon expression the polypeptide is generated as inclusion bodies within the cells which are separated from further cell particles followed by solubilisation in a denaturing agent such as guanidine hydrochloride (GndHCI) and refolded by renaturation procedures well known to the skilled person.

Chemical and/or biological modifications In one aspect the polypeptide of the instant invention is chemically and/or biologically modified. Such modification may comprise, but is not limited to, glycosylation, PEGylation, HESylation, Albumin fusion technology, PASylation, labelling with dyes and/or radioisotopes, conjugation with enzymes and/or toxins, phosphorylation, hydroxylation and/or sulfation. Likewise, the nucleic acid sequence, the vector and/or the host cell described above can be modified accordingly.

Chemical and/or biological modifications may be conducted to optimize pharmacodynamics or water solubility of the polypeptide or to lower its side effects. For example, PEGylation, PASylation and/or HESylation may be applied to slow down renal clearance and thereby increase plasma half-life time of the polypeptide. Additionally or alternatively, a modification may add a different functionality to the polypeptide, e.g. a toxin to more efficiently combat cancer cells, or a detection molecule for diagnostic purposes.

Glycosylation refers to a process that attaches carbohydrates to proteins. In biological systems, this process is performed enzymatically within the cell as a form of co-translational and/or post-translational modification. A polypeptide can also be chemically glycosylated. Typically, but not limited to, glycosylation is (i) N-linked to a nitrogen of asparagine or arginine side-chains; (ii) O-linked to the hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains; (iii) involves the attachment of xylose, fucose, mannose, and N-acetylglucosamine to a phospho- serine; or (iv) in form of C-mannosylation wherein a mannose sugar is added to a tryptophan residue found in a specific recognition sequence. Glycosylation patterns can, e.g., be controlled by choosing appropriate cell lines, culturing media, protein engineering manufacturing modes and process strategies (Hossler, P., Glycobiology 2009; 19(9): 936-949).

Protein engineering to control or alter the glycosylation pattern may involve the deletion and/or the addition of one or more glycosylation sites. The creation of glycosylation sites can conveniently be accomplished by introducing the

corresponding enzymatic recognition sequence into the amino acid sequence of the antibody or by adding or substituting one or more of the above enumerated amino acid residues. It may be desirable to PEGylate the polypeptide. PEGylation may alter the pharmacodynamic and pharmacokinetic properties of a protein. Polyethylene-glycol (PEG) of an appropriate molecular weight is covalently attached to the protein backbone (see, e.g., Pasut, G. and Veronese, F., J Controlled Release 2012; 1 61 (2): 461 -472). PEGylation may additionally reduce the immunogenicity by shielding the PEGylated protein from the immune system and/or alter its pharmacokinetics by, e.g. increasing the in vivo stability of the polypeptide, protecting it from proteolytic degradation, extending its half-life time and by altering its biodistribution.

Similar effects may be achieved by PEG Mimetics, e.g., HESylating or

PASylating the antibody. HESylation utilises hydroxyethyl starch ("HES") derivatives, whereas during PASylation the polypeptide becomes linked to conformationally disordered polypeptide sequences composed of the amino acids proline, alanine and serine. Said PEG Mimetics and related compounds are, e.g., described in BINDER, U. and Skerra, A. Half-Life Extension of Therapeutic Proteins via Genetic Fusion to Recombinant PEG Mimetics (in: Therapeutic Proteins: Strategies to Modulate Their Plasma Half-Lives. Edited by Kontermann, R., Weinheim, Germany: Wiley-VCH, 2012. ISBN: 9783527328499. p. 63-81 ).

The polypeptide may include an epitope and in particular a salvage receptor binding epitope. Such salvage receptor binding epitope typically refers to an epitope of the Fc region of an IgG molecule (e.g., lgG1 , lgG2, lgG3, or lgG4) and has the effect of increasing the in vivo half-life of the molecule.

Additionally or alternatively, the polypeptide is labelled with or conjugated to a second moiety which ascribes ancillary functions following target binding. Said second moiety may, e.g., have an additional immunological effector function, be effective in drug targeting or useful for detection. The second moiety can, e.g., be chemically linked or fused genetically to the antibody using known methods in the art. Such second moiety may also have the function to further improve skin penetration. For example, a cell-penetrating peptide such as the TAT sequence may be added or conjugated to the polypeptide. For the purposes of clarity, it is however to be understood that the presence of such second moiety such as a cell-penetrating peptide or even a compound typically used in the arts as penetration enhancer is optional as the polypeptides described herein can penetrate skin without the presence of a penetration enhancer. Molecules which may serve as second moiety include, without being limited to, radionuclides, also called radioisotopes (e.g., 35S 32P, 14C, 18F, 1 1 1 1, 1251, 223Ra, 227Th); apoenzymes; enzymes (such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase or angiogenin); co-factors; peptides (e.g., HIS-tags); proteins (incl. lectins); carbohydrates (incl. mannose-6-phosphate tag); fluorophores (including fluorescein isothiocyanate (FITC); phycoerythrin; green/blue/red and other fluorescent proteins; allophycocyanin (APC)); chromophores; vitamins (including biotin); chelators; antimetabolites (e.g., methotrexate), liposomes; toxins including cytotoxic drugs such as taxol, gramicidin D or colchicine; or a radiotoxin. A labelled polypeptide is particularly useful for in vitro and in vivo detection or diagnostic purposes. For example, a polypeptide labelled with a suitable

radioisotope, enzyme, fluorophore or chromophore can be detected by

radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or flow cytometry-based single cell analysis (e.g., FACS analysis), respectively. Similarly, the nucleic acids and/or vectors disclosed herein can be used for detection or diagnostic purposes, e.g. using labelled fragments thereof as probes in hybridization assays. Labelling protocols may, e.g., be found in Johnson, I. and Spence, M. T.Z., Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies. Life Technologies, 2010. ISBN: 098292791 6.

Compositions

The invention also extends to compositions for use in the medical or cosmetic treatment of a skin disorder. Such compositions comprises the polypeptides disclosed herein, preferably the antigen-binding polypeptides that are immuno- reactive with a target antigen associated with a skin disease.

Hence, in one embodiment, a pharmaceutical composition for topical application to the skin is provided, said formulation comprising an antigen-binding polypeptide that is not a full-length immunoglobulin, such as a recombinant humanized scFv. The antigen-binding polypeptide is preferably non-glycosylated. More preferably, the antigen-binding polypeptide has a molecular weight of about 21 to about 29 kDa. Most preferably, the antigen-binding polypeptide specifically binds to TNF alpha, VEGF, IL-1 alpha and beta, IL-4, IL-6, IL-8, IL-12, IL-13, IL-17A, IL-18, IL-23, p40, IL-31 and/or IL-36and is preferably a recombinant humanized scFv antigen-binding polypeptide thereof.

The compositions of the present invention typically comprise at least one further compound, such as a dermatologically acceptable carrier. Although the polypeptides may be used in a simple buffer solution, topical administration may require a more viscous formulation so that the application to skin results more easily. Thus, in one embodiment, the compositions has a viscosity of at least 250 mPa.s, more preferably at least 500, 1 ,000, 2,000, 2500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000 or 9,000 mPa.s as determined by the rotation viscometer method with a Brookfield Spindel SC4-34 at 12 rpm, 25°C and a torsion of 49.9% (see Ph. Eur. 2013, method 2.2.10). A dermatologically acceptable carrier can be selected from variety of well-known carriers, including, without being limited to, polyols, synthetic or vegetable oils, starches, starch derivatives, sugars, or emulsifiers. In embodiments, the composition is not suitable for injection (e.g., the formulation need not be formulated to permit passage through a 20 gauge or smaller gauge needle). In another embodiment, the composition is not suitable for oral administration (e.g., is not in a tablet or capsule formulated for oral administration, formulated to comprise flavorants or gastrointestinal protectants). Thus, in one aspect, a composition is provided comprising a dermatologically acceptable carrier and/or a substantially stable and protease resistant polypeptide, preferably an antigen-binding polypeptide, wherein said polypeptide is at a

concentration of about 0.1 mg/ml or higher or less. Preferably, the composition is substantially free of aggregates. In one embodiment, said polypeptide is at a concentration of about 0.01 mg/ml or less, or 1 mg/ml, 2.5 mg/ml, 5 mg/ml, 10 mg/ml or greater.

In another embodiment, said polypeptide does not aggregate or dimerize by 1 % or more (e.g., 1 %, 2%, 5%, 10%, 20% or more) when stored in phosphate buffer solution at about 4°C at a concentration of 1 mg/ml or more ( e.g., 1 mg/ml, 2 mg/ml), 5 mg/ml, 7 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 50 mg/ml or more) when compared to the starting point during a period of one week or less. The monomeric state is an important feature of the polypeptides disclosed herein. The degree of aggregation or dimerization may e.g. be assessed by size exclusion chromatography.

The composition may comprise at least one further therapeutically active compound, such as a biologic and/or chemical entity. For example, the composition may comprise a variety of antigen-binding molecules directed against different targets involved in the disease to be treated. In one embodiment, the polypeptides disclosed herein are combined with corticosteroids and vitamin D3- analogues.

Illustrative examples of corticosteroids include, without being limited to, calcipotriene, clobetasol, betamethasone, dexamethasone, tazarotene, hydrocortisone,

fludrocortisone, methylprednisolone, prednisolone and prednisone. Suitable vitamin D3-analogues include, without being limited to calcipotriol and calcitriol.

Further ingredients that may optionally be present in such composition include, without being limited to, antioxidants, detergents, emollients, emulsifiers, humectants, minerals, moisturizers and hydration agents, penetration agents, preservatives, natural or synthetic oils, solvents, surfactants, fragrances, thickeners, vitamins, waxes, powders. Said ingredients may or may not have a pharmacological or a cosmetic effect. If compositions intended for multiple dosing are prepared, the composition typically comprises a preservative.

Preferably, the compositions disclosed herein maintain (or do not appreciably alter) the beneficial physicochemical properties of the polypeptides disclosed herein as they exist in the non-formulated state. For example, the formulations of the invention maintain the beneficial physicochemical property of the polypeptide following administration of the formulated polypeptide to a subject. Thus, the compositions of the invention maximize the therapeutic potential of the polypeptide. In some embodiments, the polypeptide maintains about the same potency, solubility and monomeric behaviors as observed in its native form in a simple buffer system for at least about 5 days or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more days) following the administration of the composition comprising said polypeptide to a subject.

In one embodiment, the pharmacologically efficacious level corresponds to an approximately 2:1 molar ratio of target antigen :antigen-binding polypeptide. In another embodiment, the pharmacologically efficacious level corresponds to an approximately 1 :1 molar ratio of target antigen :antigen-binding polypeptide. In still another embodiment, the pharmacologically efficacious level corresponds to an approximately 1 :2 molar ratio of target antigen :antigen-binding polypeptide or smaller, e.g., 1 :3, 1 :4, 1 :5, 1 :10, 1 :20 molar ratio of target antigen:antigen-binding polypeptide.

In one embodiment, the polypeptide reaches a pharmacodynamically efficacious level in the skin, preferably in the epidermis, more preferably in the dermis, of a subject for at least about 2 days or more following administration of the composition to the subject. In one embodiment, said efficacious level corresponds to an approximately 2:1 molar ratio of target antigen:antigen-binding polypeptide. In another embodiment, said efficacious level corresponds to an approximately 1 :1 molar ratio of target antigen :antigen-binding polypeptide. In still another embodiment, said efficacious level corresponds to an approximately 1 :2 or smaller molar ratio (e.g., 1 :3, 1 :4 etc.) of target antigen:antigen-binding polypeptide.

Due to the described penetration and/or permeation properties of the polypeptide, said pharmaceutical composition is preferably formulated for direct topical application on skin. Dosage forms include aerosols, creams, dispersions, emollients, oil/water emulsions (including microemulsions), foams, gels, jellys, lotions, ointments, patches including, e.g., transdermal patches, powders, unguents, water-based solutions, sera, suspensions or other typical solid or liquid compositions or combinations thereof used for application to skin. In one embodiment, the pharmaceutical composition if formulated as a hydrogel. Semi-solid preparations such as hydrogels typically comprise a gelling agent. Suitable gelling agents include, without being limited to, poloxamers, starch, cellulose derivatives, carbomers and magnesium-aluminium silicates.

In one embodiment, the composition has a pH of 6 or lower, such as about pH 5.5, 5 or 4.5. An acidic pH is advantageous for topical application to skin. Normal skin pH ranges from 4.5 to 6.5, and is thus slightly acidic. The acidity of the skin is termed the "acid mantle" and is maintained by sebaceous glands, sweat glands, normal skin flora, among others. The acidic mantle serves many protective functions, such as protecting skin from growth of microorganisms. Thus, a composition should not disrupt the acidic mantle and has therefore a mildly acidic pH. Moreover, applying a mildly acidic compositions soothes the skin, helping to retain moisture which is important for penetration of biologically active compounds.

Such compositions may be prepared by any of the methods known in pharmacy and typically involve the step of admixing the polypeptide described herein with a liquid or solid carrier (or both).

Further provided is a container comprising the compositions disclosed herein wherein the container is labeled for use. The container may e.g. be labelled for therapeutic or for cosmetic use.

Also provided is a kit comprising the container above and written instructions for use. Uses

The polypeptides described herein can be used in the treatment of skin diseases. Thus, in another aspect, the invention provides a method of treating a skin disease, comprising the step of administering to a subject a pharmaceutically effective amount of the polypeptide described herein. Preferably, the polypeptide are used in a pharmaceutical composition formulated for topical application to skin, comprising a pharmaceutically effective amount of such polypeptide. Suitable compositions are outlined above. Such compositions can easily be administered in a variety of administration forms suitable for direct topical application on skin. Upon formulation, compositions will be administered in a manner compatible with the formulated dosage and in such amount and frequency as is therapeutically effective. Thus, in one aspect, it is provided a composition (preferably a pharmaceutical composition) comprising one or more polypeptides described herein for use in a method of treating a subject having a skin disease comprising topically administering to the skin an effective amount of said composition. Although the polypeptides of the instant invention show good skin penetration properties, passive drug delivery methods might be limiting and active methods of drug delivery might be preferable, e.g. to reach deeper dermis. Such active methods include, without being limited to, electroporation, iontophoresis, sonophoresis, phonophoresis, laser radiation, photomechanical waves, radio-frequency,

magnetophoresis, thermophoresis, microneedle based devices, skin puncture and perforation, needleless injection and suction ablation (see Brown et al., Methods Mol Biol. 2008;437:1 19-39 for a detailed discussion of such methods). Also contemplated herein are combinations of passive and active drug delivery methods. The skin disease referred to above may be a chronic skin disease, e.g. an inflammatory skin disease, or an acute injury such as burned and photodamaged skin. Examples of skin diseases include, without being limited to, psoriasis, acne, portwine stain, alopecia areata, basal cell carcinoma, melanoma, Bowen's disease, congenital erythropoietic porphyria, contact dermatitis, Darier's disease, atopic eczema, epidermolysis bullosa simplex, erythropoietic protoporphyria, hailey-hailey disease, herpes simplex, hidradenitis suppurativa, hirsutism, hyperhidrosis, ichthyosis, impetigo, keloids, keratosis pilaris, lichen planus, lichen sclerosus, melasma, pemphigus vulgaris, verrucas, pityriasis lichenoides, polymorphic light eruprion, wats, Kaposi's sarcoma, pyoderma gangrenosum, rosacea, scabies, shingles, squamous cell carcinoma and vitiligo.

The term "subject" is known in the art and as used herein, refers to a warmblooded animal, more preferably a mammal, e.g., non-human animals such as horse, monkey, dog, cat, pig, mouse, rat, cattle, sheep, in addition to human beings. In a preferred embodiment, said subject is human. The subject is preferably a subject in need of such treatment.

In some embodiments, it might be desirable to include one or more

pretreatment and/or concomitant treatment steps before or while administering the polypeptide. Such pretreatment step may involve the removal of stratum corneum (SC) layers. The thickness of the SC varies throughout the body and also from person to person. To enhance penetration of the polypeptide into the skin, it might be desirable to remove at least partially the SC barrier. SC can e.g. be removed by tape stripping, skin abrasion such as microdermabrasion, skin stretching, by using emollients alpha-hydroxy acids and/or beta-hydroxy acids. Effective removal of the SC may e.g. be quantified by the measurement of the transepidermal water loss (TEWL). The TEWL reflects the skin water-holding capacity quantity of water that passes from the body towards the outside and is thus a measure of the effectiveness of the SC's barrier function. The TEWL can be determined using e.g., a Tewameter® TM300, (Courage + Khazaka Electronics GmbH) instrument.

Also provided is a method of inhibiting an inflammatory cytokine in the skin by administering topically a pharmaceutically effective amount of a cytokine-binding polypeptide in a pharmaceutical composition to a subject in need thereof. It is understood that the definitions of the subject given above, the polypeptides given above and the pharmaceutical composition given above also apply to this method. The polypeptides of the instant invention, in particular the scFv of SEQ ID No. 1 , have been shown to penetrate psoriatic skin and inhibit mRNA production of inflammatory cytokines such as TNF alpha, IL-1 beta, IL-6, IL-8, IL-12, IL-17A, IL-22, IL-23, IL-27, IFN gamma and CCL7. Thus, cytokine-binding polypeptides described herein can be used in the treatment of psoriasis or other skin diseases caused by inflammatory cytokines. The polypeptides are typically used in the production of a medicament that can be topically applied to the skin and comprises an effective amount of said polypeptide.

As outlined above, inflammatory cytokines involved in skin diseases include, without being limited to, TNF alpha, VEGF, IL-1 alpha and beta, IL-4, IL-6, IL-8, IL- 12, IL-13, IL-17A, IL-18, IL-23, p40, IL-31 or IL-36. The mentioned targets have been validated as clinically relevant in chronic diseases such as psoriasis, acne and atopic dermatitis. In psoriatic skin, for example, a multitude of inflammatory mediators are overexpressed that contribute to psoriatic skin inflammation, including cytokines such as tumor necrosis factor (TNF), IL-17, IL-1 a, IL-6, IL22 and transforming growth factor-b1 (Wang, C.Q.F. (2013), J Inv Dermatol 133, 2741 -2752). Further

inflammatory skin diseases in which the mentioned cytokines mediate inflammation include, without being limited to, rosacea, lichenoid disorders such as lichen planus, erythroderma and Stevens-Johnson syndrome and toxic epidermal necrolysis. In one embodiment, the cytokine is a cytokine of the Th17 pathway.

Accordingly, the cytokine-binding polypeptides are polypeptides targeting such cytokines, e.g., TNF alpha-binding polypeptides, VEGF-binding polypeptides etc. In one embodiment, the polypeptide targets TNF alpha; preferably, the polypeptide comprises the VL and VH regions of SEQ ID No. 1 or variants thereof. In another embodiment, the polypeptide targets IL-1 beta; preferably, the polypeptide comprises the VL and VH regions of SEQ ID No. 2 or 4 or variants thereof. In another

embodiment, the polypeptide targets VEGF; preferably, the polypeptide comprises the VL and VH regions of SEQ ID No. 3 or variants thereof. In another embodiment, a combination of two or more polypeptides is administered.

Additionally or alternatively to a pharmaceutical effect, the polypeptide of the present invention may have a cosmetic effect. Hence, the polypeptides described herein may also be applied in cosmetic uses, e.g. to improve acne, to improve overall skin appearance, to alleviate sun burns, to alleviate the effects of the aging process of skin and the like.

Depending on the particular subject, composition and/or mode of

administration, the actual dosage levels of the polypeptides in the pharmaceutical composition may be varied so as to obtain an amount of the polypeptide which is effective to achieve the desired therapeutic response without being toxic to the subject and/or without provoking any allergic response or skin irritation. The selected dosage level will depend on a number of factors well known in the medical arts, including, without being limited to, the age, sex, weight, condition, prior medical history and general health of the subject being treated, the duration of the treatment and the activity of the particular composition being used. Also, the characteristics of the polypeptide, such as potency and penetration efficacy, influence the dosage level. The skilled person is well able to balance these parameters and determine the appropriate dosage level by routine methods in the art.

Sequence listing

The sequences disclosed herein are: SEQ ID No. 1 - scFvl

DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYT GVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGG GSGGGGSGGGGSSGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNW VRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTA VYYCARE RG DAM DYWGQGTLVTVSS

SEQ ID No. 2 - scFv2

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASG

VPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGG

SGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWV

RQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVY

YCARERAIFSGDFVLWGQGTLVTVSS

SEQ ID No. 3 - scFv3

EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGV

PSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGG

GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMT

WVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCAGGDHNSGWGLDIWGQGTLVTVSS

SEQ ID No. 4 - scFv5

EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKLLIYRASNLASG VPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFGQGTKLTVLGGGGG SGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSNSAMAWV RQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATY YCARERAIFSGDFALWGQGTLVTVSS

SEQ ID No.: 5- FR-L1 of FW1.4

EIVMTQSPSTLSASVGDRVIITC

SEQ ID No.: 6- FR-L2 of FW1.4

WYQQKPGKAPKLLIY

SEQ ID No.: 7- FR-L3 of FW1.4

GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC

SEQ ID No.:8-FR-L4 of FW1.4

FGQGTKLTVLG

SEQ ID No.:9-FR-H1 of rFW1.4

EVQLVESGGGLVQPGGSLRLSCTASG

SEQ ID No.: 10- FR-H2 of rFW1.4

WVRQAPGKGLEWVG

SEQ ID No.: 11 - FR-H3 of rFW1.4

RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR

SEQ ID No.: 12-FR-H4 of rFW1.4

WGQGTLVTVSS

SEQ ID No.: 13- FR-H1 of rFW1.4(V2)

EVQLVESGGGLVQPGGSLRLSCTVSG

SEQ ID No.: 14 - FR-H2 of rFW1.4(V2)

WVRQAPGKGLEWVG SEQ ID No.: 15- FR-H3 of rFW1.4(V2) RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR

SEQ ID No.: 16-FR-H4 of rFW1.4(V2) WGQGTLVTVSS

SEQ ID No.: 17- FR-H1 of rFW1.4-SST EVQLVESGGGSVQPGGSLRLSCTASG

SEQ ID No.: 18 - FR-H2 of rFW1.4-SST WVRQAPGKGLEWVG

SEQ ID No.: 19 - FR-H3 of rFW1.4-SST RFTISRDTSKNTVYLQMNSLRAEDTASYYCAR

SEQ ID No.: 20 - FR-H4 of rFW1.4-SST WGQGTTVTVSS

SEQ ID No.21 - VL-FR1 of scFvl

DIVMTQSPSSLSASVGDRVTLTC

SEQ ID No.22 - VL-FR2 of scFvl

WYQQRPGKAPKLLIY

SEQ ID No.23 - VL-FR3 of scFvl

GVPSRFSGRGYGTDFTLTISSLQPEDVAVYYC

SEQ ID No.24 - VL-FR4 of scFvl

FGQGTKLEVKR

SEQ ID No.25 - VH-FR1 of scFvl

QVQLVQSGAEVKKPGASVKVSCTASG SEQ ID No. 26 - VH-FR2 of scFvl

WVRQAPGKGLEWMG

SEQ ID No. 27 - VH-FR3 of scFvl

R FT FS L ETS AST V YM E LTS LTS D DTAVYYCA R

SEQ ID No. 28 - VH-FR4 of scFvl

WGQGTLVTVSS Examples

Example 1 - RHE model

Four different scFvs, namely scFvl (anti-TNF alpha inhibitor, see SEQ ID No. 1 ), scFv2 (anti-IL-1 beta inhibitor, see SEQ ID No. 2), scFv3 (anti-VEGF inhibitor, see SEQ ID No. 3) and scFv4 (an anti-p40-inhibitor comprising variants of the framework regions SEQ ID Nos 5 - 12)) were subcloned into bacterial vectors, expressed as inclusion bodies in BL21 E. coli bacteria, refolded and purified by chromatographic techniques.

To measure differences in their permeation behavior, the scFvs and

corresponding monoclonal IgG antibodies (the anti-TNF alpha inhibitor infliximab

(Remicade®, Janssen Biotech Inc.), the anti-IL-1 beta inhibitor canakinumab (Maris®, Novartis), the anti-VEGF-inhibitor bevacizumab (Avastin®, Roche) and the anti-p40- inhibitor ustekinumab (Stelara®, Janssen-Cilag)) were applied in aqueous buffer onto reconstructed human epidermis tissues at a concentration of 5 mg/ml each.

Samples of 200 to 300 μί. were taken from the receptor fluid at time points T = 0 hours, 4 hours, 8 hours, 24 hours, 28 hours, 32 hours and 48 hours. Removed receptor fluid was substituted by fresh cell culture medium. Figure 8 depicts the experimental set-up and shows a scheme of a hanging insert with reconstructed human epidermis tissue.

Cell viability and integrity was assessed in the following manner: To monitor viability and integrity of reconstructed human epidermis samples, trans-epidermal electrical resistance was measured. Skin samples were aseptically transferred into 6- well plates pre-f illed with 5 ml_ of pre-warmed PBS at 37°C for measurement of resistance values. Into each insert, pre-warmed PBS (37°C) was pipetted and resistance values were measured via an epithelial voltometer (EVOM², World

Precision Instruments). Usually, TEER was determined at time point t=0 (blank only) and at the very end of the experiment.

Further, viability of RHE cells was controlled using the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega, G3580) in the end of the experiment.

The determination of the caffeine flux served as control for permeation. For said purpose, 25 μΙ_ (StratiCELL), respectively 43 μί. (Episkin) of 1 % caffeine solution in citrate buffer (pH 6) was applied onto the cell layer after having collected time point T = 48 hours. At time points T = Oh, 0.5h, 1 h, 2h or T = 0, 1 , 2, 3h, medium samples were collected. Removed medium was replaced by fresh medium. Caffeine content in the medium was determined by measuring absorbance at 270nm.

As a negative control, 0.5% Dextran-FITC solution was applied onto the stratum corneum of the RHE (25 μΙ (StratiCELL) or 43 μί. (Episkin)). Samples are taken at time points t = Oh, 4h, 8h, 24h, 28h, 32h, 48h.

Results

All tested scFvs permeated the artificial skin model whereas full-length immunoglobulins did not as evidenced by the statistical significant difference between scFv and IgG amount detected 48 hours after application, whereas no statistical significant differences among diverse scFvs could be detected by an ANOVA one way test, see Figure 1 and Table 1 . Table 1 summarizes the applied compounds to RHE tissues, the total number of data points submitted to an outlier identification program and to the 1 way ANOVA test (PRISM software).

Table 1

Protein ID Experiments # Data points # Outliers # 0.5% scFvl 24 1 1 1 1 1

0.5% Infliximab 5 22 5

0.5% scFv2 7 32 4

0.5% Canakinumab 5 27 4

0.5% scFv3 8 36 6

0.5% Bevacizumab 5 26 4

0.5% scFv4 5 27 0

0.5% Ustekinumab 4 24 6

Total 305 40

Permeation efficacy

Table 2 summarizes the data on flux and dose in receptor fluid.

Table 2

Standard

deviation

1 % scFvl Average flux [ng/h cm²] 0.70 0.03

Average dose in 0.13 0.07

receptor fluid [%₀]

0.5% scFvl Average flux [ng/h cm²] 0.30 0.20

Average dose in 0.09 0.08

receptor fluid [%₀]

0.1 % scFvl Average flux [ng/h cm²] 0.05 0.02

Average dose in 0.06 0.02

receptor fluid [%₀]

0.5% scFv2 Average flux [ng/h cm²] 0.1 6 0.05

Average dose in 0.05 0.03

receptor fluid [%₀]

0.5% scFv4 Average flux [ng/h cm²] 0.36 0.35 Average dose in 0.08 0.01

receptor fluid [%₀]

0.5% scFv3 Average flux [ng/h cm²] 0.07 0.05

Average dose in 0.04 0.02

receptor fluid [%₀]

The polypeptides showing penetration in this model have a pi in the range of 4.93 to 8.32. Therefore, the studies demonstrated that the penetration is independent of the pi of the molecule.

This study shows that engineered recombinant antibody fragments can successfully penetrate skin after topical administration in buffer solution. The polypeptides reach a therapeutically relevant level and moreover, retain full antigen binding activity, thereby offering the possibility of new approaches towards

therapeutic intervention on skin. The study further demonstrates that full-length antibodies do not penetrate skin (see Figure 1 ).

Example 2 - Imaging

RHE tissues were purchased from StratiCell Belgium and fluorescently-labeled scFvl (FluorAlexa 594, Molecular Probes) was applied on the stratum corneum layers (top). The labeled scFvl was removed after 8 hours and the surface washed with PBS. After 48 hours the RHE tissues were subjected to two-photon microscopy (2-PM) analysis. The penetration of scFvl is confirmed as seen in Figure 2. scFvl is located in the intercellular space of the basal layer (Figure 2A). The three- dimensional reconstruction of all 2-PM layers is depicting the distribution of scFvl along the vertical axis. As expected a very strong scFvl signal is observed in the upper layers (stratum corneum). Nevertheless, scFvl is located in the well- differentiated epidermis consisting of basal layer several spinous and granular layers. The images confirm the permeation of scFvl across the RHE tissues, permeation was quantified by ELISA.

In vivo skin penetration studies were performed with Gottingen minipigs supplied by Ellegaard, 4261 Dalmose, Denmark. Fluorescently-labeled scFvl (FluorAlexa 594, Molecular Probes) was topically applied three times as a gel-like solution on the dorsal part of the minipig skin. Half of the application sites were pretreated by tape stripping using D-Squame® (Skin Sampling Discs -D100, diameter 22 mm, CuDerM). After 30 hours of treatment, the residual scFvl compound on the skin surface was removed. After washing and disinfection, biopsies were taken on the center of the treatment area using 6 mm punch-out needle between 7 and 10 mm depth. The skin biopsies were stored frozen below minus 60°C. Cyrosections of 50 μηπ thickness from the skin biopsies were analyzed by confocal laser scanning microscopy. The penetration of the polypeptide in the upper layers of the skin (epidermis) is confirmed as seen in Figure 3. The penetration of the polypeptide via the hair follicle in the lower layers of the skin (dermis) is confirmed as depicted in Figure 4.

According to Figure 2, the intercellular route is preferred by the polypeptides. Polypeptides penetrating via the stratum corneum are mainly targeting the upper layers of the epidermis as can be derived from Figure 3. As shown in Figure 4 polypeptides penetrating via hair follicles or sweat ducts are also reaching deeper skin layers (dermis). Example 3 - Pharmacokinetics of scFvl

scFvl was administered intravenously to healthy human subjects as a single dose and pharmacokinetic parameters were determined.

Table 3 summarizes some pharmacokinetic parameters of scFvl 1 mg/mk versus adalimumab and canakinumab following intravenous administration in healthy volunteer subjects. Of particular interest is the fact that scFvl distributes into the entire extracellular space since the volume of distribution is in the range of 1 6-17 L, while both adalimumab and canakinumab distribute only into the vascular space (volume of distribution in the range of 5-7 L). This indicates that scFvl better than IgG antibodies has the intrinsic ability to penetrate efficiently into all human tissues and organs and therefore to reach the site of inflammation rapidly and with high concentrations.

Table 3 scFvl Adalimumab Canakinumab

1 mg/kg 1 mg/kg^* (population)+

Half-life [h] 17 ± 3 357 ± 218 748 ± 81

Cmax [ug/ml] 1 6.5 ± 1 .3 34.6 ± 8.8 n.a.

CL [L/d] 75 ± 10 0.288 0.182 ± 0.05

Vdss [L] 1 6.5 ± 2.1 4.9 ± 1 .5 7.08 ± 2.1

CL = Clearance; Vdss = Volume of distribution at steady state

All data shown as mean ± SD; n.a. = not applicable

scFvl (n=6) and Adalimumab (n=15) were administered i.v. to healthy subjects Data for Canakinumab are derived from healthy and diseased subjects

* BLA Adalimumab, Clinical Pharmacology, Study DE024

+ Clin Pharmacokinetics 2012; 51 :e1

Example 4 - Protease resistance of scFvl

Native and heat-denatured scFvl was treated with recombinant human kallikrein 5 (R&D systems, cat. nr. 1 108-SE-010) (50-250 nM), recombinant human kallikrein 7 (R&D systems, cat. nr. 2624-SE) (100 to 400 nM) or recombinant trypsin (Sigma Aldrich, cat. nr. T1426) for various periods of time at 37°C. Heat denaturation of scFvl was performed at 95°C for 5 minutes. Recombinant human kallikrein 7 was pre-activated by thermolysin according to the manufacturer's protocol before incubating with scFvl . After incubation of scFvl with either kallikrein 5, kallikrein 7 or trypsin, samples were subjected to SDS-PAGE analysis and gels were stained with Coomassie Blue. No degradation of native scFvl by kallikrein 5 was observed, even at high concentrations and after overnight exposure. Furthermore, no detectable degradation products were seen when heat-denatured scFvl was exposed to kallikrein 5 for 19 hours. Only very little degradation of scFvl by kallikrein 7 was observed after overnight exposure. The heat-denatured form of scFvl was partially cleaved by kallikrein 7 after overnight incubation at 37°C. Little degradation of scFvl by trypsin was observed. These data suggest that native scFvl in its normal globular conformation is stable and almost completely resistant to degradation by skin-related proteolytic enzymes. Example 5 - scFvl penetrates in vivo into psoriatic skin

In a clinical trial, a total of 59 patients with chronic, mild-moderate psoriasis were randomized to receive in a double-blind way either 0.5% of scFvl or placebo in a hydrogel formulation. Two plaques per patient were selected for treatment where in one plaque the stratum corneum was partially removed with tape stripping on a weekly basis. This procedure was selected to increase skin penetration of study drug. The second plaque was kept na^'ive. Study drug was administered topically twice a day over 4 weeks on both plaques. Gene expression data in skin biopsies taken at Day 14 demonstrated a scFvl -mediated highly-significant reduction of TNF alpha as compared to placebo (see Figure 9). In addition, TNF alpha-dependent cytokine genes such as IL-1 beta, IL-6, IL-8, IL-12, IL-17A, IL-22, IL-23, IL-27, I FN gamma and CCL7 were also decreased in comparison to placebo. Thus, the data indicate that locally-administered compound sufficiently inhibits skin-derived TNF alpha in a way that biological down-stream effects are detectable, i.e. on the Th17 pathway. Although clinically, signs and symptoms of psoriasis were not different between scFvl and placebo treatment, the clinical trial demonstrates in vivo in humans that scFvl permeates into inflamed skin and mediates a biological response. While there are shown and described presently preferred embodiments of the invention, it is to be understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims. Since numerous modifications and alternative embodiments of the present invention will be readily apparent to those skilled in the art, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the best mode for carrying out the present invention. Accordingly, all suitable modifications and equivalents may be considered to fall within the scope of the following claims.

Claims

I . A polypeptide capable of penetrating skin in the absence of a penetration enhancer.

2. The polypeptide of claim 1 , wherein the skin is psoriatic skin.

3. The polypeptide of claim 1 , that permeates through reconstituted human epidermis.

4. The polypeptide of any one of claims 1 to 3, being an antigen-binding polypeptide.

5. The antigen-binding polypeptide of claim 4, wherein said polypeptide is not a full- length immunoglobulin.

6. The antigen-binding polypeptide of any one of claims 4 or 5, being an antibody fragment or an non-antibody scaffold.

7. The antibody fragment of claim 6, being a scFv, Fab fragments, dAb, VHH or nanobody.

8. The non-antibody scaffold of claim 6, being affibodies, an affilin molecule, an AdNectin, an Anticalin, a DARPin, a fynomer, an Knottin, a Kunitz-type domain, an Avimer, a Tetranectin or a trans-body.

9. The antigen-binding polypeptide of any one of the preceding claims, binding to TNF alpha, VEGF, IL-1 alpha and beta, IL-4, IL-6, IL-8, IL-12, IL-13, IL-17A, IL-18, IL- 23, p40, IL-31 and/or IL-36.

10. The polypeptide of any one of the preceding claims, having a globular shape.

I I . The polypeptide of any one of the preceding claims, having a molecular weight of at least 5 kDa.

12. The polypeptide of claim 1 1 , having a molecular weight of about 21 kDa to 29 kDa.

13. The polypeptide of any one of the preceding claims, remaining monomeric at least to 50% after being incubated for 2 weeks at room temperature at a

concentration of about 10 mg/ml in PBS at pH 7.2.

14. The polypeptide of any one of the preceding claims, being resistant to proteases.

15. The polypeptide of any one of the preceding claims, being capable of distributing effectively into tissue.

16. The antigen-binding polypeptide of any one of claims 4 to 15, comprising a framework sequence as defined in SEQ ID Nos. : 5 to 8, or a variant thereof, or as defined in SEQ ID Nos. : 9 to 12, or a variant thereof.

17. The antigen-binding polypeptide of any one of claims 4 to 16, comprising SEQ ID No. : 1 , SEQ ID No. : 2, SEQ ID No. : 3 or SEQ ID No. : 4, or a variant thereof, respectively.

18. The polypeptide of any one of the preceding claims, being formulated in a buffer, a gel, a hydrogel, an emulsion or in aqueous solution.

19. The polypeptide of any one of the preceding claims for use in the treatment or diagnostics of a skin disease or for use in cosmetics.

20. The polypeptide of claim 19, wherein said skin disease is selected from the group consisting of psoriasis, acne, portwine stain, alopecia areata, basal cell carcinoma, melanoma, Bowen's disease, congenital erythropoietic porphyria, contact dermatitis, Darier's disease, atopic eczema, epidermolysis bullosa simplex, erythropoietic protoporphyria, hailey-hailey disease, herpes simplex, hidradenitis suppurativa, hirsutism, hyperhidrosis, ichthyosis, impetigo, keloids, keratosis pilaris, lichen planus, lichen sclerosus, melasma, pemphigus vulgaris, verrucas, pityriasis lichenoides, polymorphic light eruprion, wats, Kaposi's sarcoma, pyoderma gangrenosum, rosacea, scabies, shingles, squamous cell carcinoma, vitiligo.

21 . A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 18.

22. The composition of claim 21 , wherein said polypeptide is at a concentration of about 1 mg/ml or higher and substantially free of aggregates.

23. A dermatologically acceptable composition of claim 21 or 22, comprising the polypeptide of any one of claims 1 to 18 and a dermatologically acceptable excipient.

24. The composition of claim 23, wherein the composition is a hydrogel or cream.

25. The composition of any one of claims 21 to 24, wherein the dynamic viscosity of the composition is equal to or higher than 250 mPa.s.

26. The composition of any one of claims 21 to 25, wherein the composition is not suitable for injectable, optical or oral administration.

27. The composition of any one of claims 21 or 26, comprising at least one further therapeutically active compound.

28. A container comprising the pharmaceutical composition of any one of claims 21 to 27.

29. A kit comprising the container of claim 28 and instructions for use.

30. A method of treating a skin disease, comprising the step of applying topically to a subject in need thereof a pharmaceutically effective amount of the antigen-binding polypeptide of any one of claims 1 to 18 or a composition of any one of claims 21 -27.

31 . The method of claim 30, wherein said disease is psoriasis, acne, portwine stain, alopecia areata, basal cell carcinoma, melanoma, Bowen's disease, congenital erythropoietic porphyria, contact dermatitis, Darier's disease, atopic eczema, epidermolysis bullosa simplex, erythropoietic protoporphyria, hailey-hailey disease, herpes simplex, hidradenitis suppurativa, hirsutism, hyperhidrosis, ichthyosis, impetigo, keloids, keratosis pilaris, lichen planus, lichen sclerosus, melasma, pemphigus vulgaris, verrucas, pityriasis lichenoides, polymorphic light eruprion, wats, Kaposi's sarcoma, pyoderma gangrenosum, rosacea, scabies, shingles, squamous cell carcinoma, vitiligo.

32. The method of any one of claims 30 to 31 , wherein the polypeptide is directed against a cytokine, in particular against TNF alpha.

33. The method of claim 32, wherein said polypeptide comprises SEQ ID No. 1 .

34. The method of claim 33, wherein said disease is psoriasis.

35. A method of inhibiting an inflammatory cytokine in the skin by administering topically a pharmaceutically effective amount of the composition of any one of claims 21 to 27.

36. The method of claim 35, wherein said composition comprises a therapeutically effective amount of a polypeptide directed against TNF alpha.

37. The method of claim 36, wherein said polypeptide comprises SEQ ID No. 1 .

38. The method of any one of claims 30 to 37, wherein said polypeptide is combined with at least one additional therapeutically active compound.

39. Use of the polypeptide of any one of claims 1 to 18 for the production of a medicament for the treatment of skin diseases.

40. The use of claim 39, wherein said skin disease is psoriasis, acne, portwine stain, alopecia areata, basal cell carcinoma, melanoma, Bowen's disease, congenital erythropoietic porphyria, contact dermatitis, Darier's disease, atopic eczema, epidermolysis bullosa simplex, erythropoietic protoporphyria, hailey-hailey disease, herpes simplex, hidradenitis suppurativa, hirsutism, hyperhidrosis, ichthyosis, impetigo, keloids, keratosis pilaris, lichen planus, lichen sclerosus, melasma, pemphigus vulgaris, verrucas, pityriasis lichenoides, polymorphic light eruprion, wats, Kaposi's sarcoma, pyoderma gangrenosum, rosacea, scabies, shingles, squamous cell carcinoma, vitiligo.

41 . The use of any one of claims 39 or 40, wherein said medicament is applied topically onto skin.

42. The use of any one of claims 39 to 41 , wherein the medicament comprises at least one additional therapeutically active compound.