WO2011161263A1 - Pharmaceutical compositions for cutaneous administration - Google Patents

Abstract

The field of invention relates to semi-solid pharmaceutical composition for administration onto the skin comprising immunoglobulin single variable domains or constructs thereof.

Description

PHARMACEUTICAL COMPOSITIONS FOR CUTANEOUS ADMINISTRATION

FIELD OF THE INVENTION

The field of invention relates to semi-solid pharmaceutical compositions for administration onto the skin comprising immunoglobulin single variable domains or constructs thereof.

BACKGROUND OF THE INVENTION

The administration of pharmaceutical compositions such as semi-solid pharmaceutical compositions comprising immunoglobuli single variable domains or constructs thereof to the skin of an animal such as a human (also herein referred to as "cutaneous administration"), can be used i) to avoid systemic side effects when high doses are required at a localized area, e.g. psoriasis treatment, or ii) as an alternative systemic administration route, in order to e.g. avoid hepatic processing. Furthermore, the use of a pharmaceutical compositions with a high amount of the active principle such as an immunoglobulin single variable domain or construct thereof directed against a therapeutic target, is desirable in order to minimize the frequency of administration and the amount of the pharmaceutical composition that needs to be applied.

However, semi-solid pharmaceutical compositions with a high amount of immunoglobulin single variable domains or constructs thereof pose several problems. One problem is the instability due to degradation or variant formation of the immunoglobulin. With the addition of sucrose and appropriate buffers (e.g. WO 2011/026948) or sequence optimization of the immunoglobulin single variable domain (e.g. WO 2009/095235), this problem has been addressed to a certain, extent for liquids. However the long term stability of immunoglobulin single variable domains in semi-solid pharmaceutical composition has not been explored and it is unclear whether such an approach that had some success for liquids will hold true also for semi-solid pharm ceutical compositions. Another problem is the instability due to the formation of protein aggregates in particular at high concentration of the protein. Emulsifying agents such as surfactants have been reported to help to minimize this issue in liquid solutions. US7147854 discloses some optimized topical pharmaceutical compositions containing surfactants and conventional antibodies but also indicate that their stability is not sufficient for long term storage or long term applications (see Table 17: Data from the accelerated stability test). SUMMARY OF THE INVENTION

While the prior art indicates numerous example of excipients that can be suitably employed to create pharmaceutical compositions of immunoglobulins for use in injectabies, few immunoglobulins have been formulated for cutaneous administration.

Applicants have discovered that immunoglobulin single variable domains (and constructs thereof) in combinations with non ionic surfactants are particularly suited for concentrated semi-solid pharmaceutical compositions for cutaneous use. Optionally, said pharmaceutical composition may further be complemented with sugar, i.e. in particular with high amounts of sugar (in order to stabilize the polypeptides).

The invention provides semi-solid pharmaceutical compositions for the administration of therapeutic molecules comprising immunoglobulin variable domain(s). The semi-solid pharmaceutical compositions allow for the local and/or systemic delivery of the therapeutic molecules comprising immunoglobulin variable domain(s). The semi -solid pharmaceutical compositions can be deli ered locally to the skin, for instance in order to treat psoriasis or other skin diseases, by administering an effective dose of the semi-solid pharmaceutical composition in an effective interval directly to the diseased skin and the semi-solid pharmaceutical, composition comprises the one or more therapeutic molecules that include one or more immunoglobulin variable domains. In some embodiments the semi-soiid pharmaceutical compositions allow for the systemic delivery of the therapeutic molecules by non-invasive means. In some embodiments the semi-soiid pharmaceutical composition comprises one or more excipients. Excipients provide the physical environment for the delivery of the therapeutic molecules as a semi-solid pharmaceutical composition. It was surprisingly found, as described herein, that immunoglobulin single variable domain-based therapeutic molecules could be formulated at a high amount of the total mass of the pharmaceutical composition, while retaining stability, solubility, and potency (such as molecule integrity and functional such as inhibiting properties) of the unformulated therapeutic molecule. Accordingly, in a preferred aspect, the immunoglobulin variable domain is an immunoglobulin single variable domain.

BRIEF DESCRIPTION OF THE DRAWINGS The figures are illustrative only and are not required for enablement of the invention disclosed herein. Fig. 1 shows a SEC chromatogram of a PBS extract of a cream containing the P231L0075 Nanobody construct, after 8 weeks storage at 4°C. An overlay with the reference sample, the liquid pharmaceutical composition stored at -20°C, is shown. After storage a small increase of the percentage oligomers was detected.

Fig. 2 shows an RPC chromatogram of a PBS extract of a cream containing the P23IL0075 Nanobody construct, after 8 weeks storage at 4°C. An overlay with the reference sample, the liquid pharmaceutical composition stored at -20°C, is shown. After storage a very small increase of some impurities was detected.

Fig. 3 shows a SEC chromatogram of a PBS extract of a cream containing the P231L0087 Nanobody construct, after 4 weeks storage at 25°C. An overlay with the reference sample, the liquid pharmaceutical composition stored at -20°C, is shown. After storage a small increase of the percentage oligomers was detected.

Fig. 4 shows a RPC chromatogram of a PBS extract of a cream containing P231L0087 Nanobody construct, after 4 weeks storage at 25°C. An overlay with the reference sample, the liquid pharmaceutical composition stored at -20°C, is shown. After storage an increase of the relative amount of impurities was detected.

Fig. 5 shows the inhibition of IL-23 binding to 1L-23R by the P23IL0075 Nanobody construct before (= P231L0075 Reference) and after lyophilisation (= P23IL0075

Lyophiiized). Also included is P23IL0075 after being formulated in a cream as lyophilized material and subsequent extraction into PBS (= P23IL0075 Cream extract (r=0)).

Fig. 6 shows the binding of P23IL0075 Nanobody construct to HSA. before (= P231L0075 Reference) and after lyophilisation (= P23IL0075 Lyophilized). Also included is P231L0075 after being formulated in a cream as lyophilized material and subsequent extraction into PBS (= P23IL0075 Cream extract (t=0)).

Fig. 7 shows the binding of the P23IL0087 Nanobody construct to HSA, before (=

P231L0087 Reference) and after being formulated in a cream as lyophilized material and subsequent extraction into PBS (= P231L0087 Cream extract (t=0)). Fig. 8 shows the inhibition of IL-23 binding to IL-23R by the P23IL0075 Nanobody construct 2 weeks after being formuiated in cream and storage at 4°C (= P23IL0075 Cream extract (t=2w; 4°C) as compared to the reference batch (=P23IL0075 Reference).

Fig. 9 shows the inhibition of IL-23 binding to IL-23R by the P23IL0075 Nanobody construct after being formulated in cream. For P23IL0075, analysis is performed in PBS extracts from a cream pharmaceutical composition which was stored at 4°C for 8 weeks (= P23IL0075 Cream Extract (t=8w; 4°C)) as compared to the reference batch (=P23IL0075 Reference). In addition, a PBS extract from P23IL0075 in a cream pharmaceutical composition prepared without intermediate lyophilisation of P23IL0O75 was included (= P23IL0O75 Cream Extract (no lyoph; t=0)).

Fig. 10 shows the binding of the P23IL0075 Nanobody construct to HSA (= P23IL0075 Cream, extract (t=2w; 4°C) or 8 weeks (= P23IL0075 Cream Extract (t=8w; 4°C)) after being formuiated in cream and storage at 4°C as compared to the reference batch (HP23I.L0075 Reference). In addition, a PBS extract from P23IL0075 in a cream pharmaceutical composition prepared without intermediate lyophilisation of P23IL0075 was included (= P23IL0075 Cream Extract (no lyoph; t=0)).

Fig. 1 1 shows the inhibition of IL-23 binding to IL-23R by the P23IL0087 Nanobody construct after being formulated in cream. For P23IL0087, analysis is performed in PBS extracts from a 4 week cream pharmaceutical composition which was stored at 4°C (= P23IL0087 Cream extract (t=4w; 4°C) or 25°C (=P23IL0087 Cream extract (t =4w; 25°C), as compared to the reference batch (-Reference P23IL0087).

Fig. 12 shows the binding of the P23IL0087 Nanobody construct to HSA after being formulated in cream. Analysis is perforated in PBS extracts from a 2 (= P23IL0087 Cream extract (t=2w; 4°C) or 4 week (= P23IL0087 Cream extract (t= w; 4°C) cream

pharmaceutical composition which was stored at 4°C, or from a 4 week cream

pharmaceutical composition stored at 25°C (=P23IL0087 Cream extract (t = w; 25°C)), as compared to the reference batch (=Reference P23IL0087). DETAILED DESCRIPTION OF THE INVENTION

In one aspect the invention provides semi-solid pharmaceutical compositions for cutaneous administration of therapeutic molecules comprising immunoglobulin variable domains, and therapeutic molecules based on other protein scaffolds, without significantly compromising stability or biological activity, in certain aspects, the invention provides semisolid pharmaceutical compositions and methods for the administration of therapeutic- molecules that include one or more immunoglobulin single variable domains, or other protein scaffolds, and constructs thereof.

Pharmaceutical compos it ions

In one aspect, the semi -solid pharmaceutical compositions including one or more therapeutic molecules and one or more excipients are provided. The one or more therapeutic molecules may include any immunoglobulin, variable domains, and preferably one or more immunoglobulin single variable domains.

The semi-solid pharmaceutical compositions include at least 10 mg of the one or more therapeutic molecules per g semi-solid pharmaceutical compositions, and the one or more therapeutic molecules include one or more immunoglobulin variable domains, such as one or more immunoglobulin single variable domains. In some embodiments, the semi-solid pharmaceutical composition includes at least 15 mg, more preferably 20 mg, more preferably 25 mg, more preferably 30 mg, more preferably 40 mg or even more preferably 50 mg of the one or more therapeutic molecules per gram of said semi-solid pharmaceutical composition.

In one aspect the invention provides semi-solid pharmaceutical compositions for the administration of therapeutic molecules comprising immunoglobulin variable domain(s). such as immunoglobulin single variable domains. The semi-solid pharmaceutical compositions allow for the local delivery of the therapeutic molecules comprising immunoglobulin variable domain(s). such as immunoglobulin single variable domains. The semi-solid pharmaceutical, compositions can be delivered locally to the skin, for instance in order to treat psoriasis or other skin diseases, by administering an effective dose of the semisolid pharmaceutical composition in an effective interval directly to the diseased skin and the semi-solid pharmaceutical composition comprises the one or more therapeutic molecules that include one or more immunoglobulin variable domains, such as immunoglobulin single variable domains. In some embodiments the semi-solid phaimaceutical compositions allow for the delivery of the therapeutic molecules by non-invasive means. In some embodiments the semi-solid pharmaceutical composition comprises one or more excipients. Excipients provide the physical environment for the delivery of the therapeutic molecules as a semi-solid pharmaceutical composition. It was surprisingly found, as described herein, that

immunoglobulin single variable domain-based therapeutic molecules could be formulated at a high amount of the total mass of the pharmaceutical composition, while retaining stability, solubility, and potency (such as molecule integrity and functional such as inhibiting properties) of the unformulated therapeutic molecule.

In one aspect the invention provides semi-solid pharmaceutical compositions for the administration of therapeutic molecules comprising immunoglobulin variable domain(s), such as immunoglobulin single variable domains. The semi-solid pharmaceutical compositions allow for the transdermal delivery of the therapeutic molecules comprising immunoglobulin variable domain(s), such as immunoglobulin single variable domains. The semi-solid pharmaceutical compositions can be transdermal^ delivered by administering an. effective dose of the semi-solid pharmaceutical composition in an effective interval to the skin and the semi-solid pharmaceutical composition comprises the one or more therapeutic molecules that include one or more immunoglobulin variable domains, such as

immunoglobulin single variable domains. In some embodiments the semi-solid

pharmaceutical compositions allow for the systemic delivery of the therapeutic molecules. In some embodiments the semi-solid pharmaceutical composition comprises one or more excipients. Excipients provide the physical environment for the delivery of the therapeutic molecules as a semi-solid pharmaceutical composition. It was surprisingly found, as described herein, that immunoglobulin single variable domain-based therapeutic molecules could be formulated at a high percentage of the total mass of the pharmaceutical composition, while retaining stability, solubility, and potency (such as pharmacodynamic and

pharmacokinetic properties) of the unformulated therapeutic molecule.

Thus the invention provides for semi-solid pharmaceutical compositions with higher percentages of therapeutic molecules comprising immunoglobulin variable domains such as e.g. immunoglobulin single variable domains, and therapeutic molecules based on other protein scaffolds than previously described. The invention also accordingly provides for semi-solid pharmaceutical compositions that are stable. Thus, the methods of the invention allow for the prolonged delivery of more therapeutic molecules in a semi-solid

pharmaceutical composition. The semi-solid pharmaceutical compositions comprise one or multiple excipients. The excipients used in the pharmaceutical composition can have a variety of functions, including, but not limited to, carriers, lyoprotectants, emulsifying agents, oil phase ingredients, viscosity adjusters and preservatives. Suitable excipients are, in particular, pharmaceutically acceptable carriers which include, but are not limited to. a neutral sterile cream, a base cream., a gel, a jelly, an ointment, or a combination thereof. The preferred pharmaceutically acceptable carrier is a base cream, which contains an emulsifying agent, an oil-phase ingredient, and a water-phase ingredient. The preferred emulsifying agent includes, but is not limited to, a non- ionic surfactant, e.g. polyethylene glycol derivatives (e.g. cetomacrogol 1000) and polyoxyethylene derivatives such as polysorbate 20 or 80 alone or in combination with fatty alcohols such as cetyl alcohol, stearyl alcohol, and cerostearylalcohol. The preferred oil-phase ingredient includes, but is not limited to white vaselin, liquid paraffin, and dimethicone. The preferred water-phase ingredient includes, but is not limited to water, glycerol and propyleneglycol. The preferred preservative includes, but is not limited to, sorbic acid, potassium sorbate, benzoic acid, parabens and sodium benzoate.

Optionally, the pharmaceutical composition may also contain suitable stabilizers or agents which increase the solubility of the compounds of the invention to allow for the preparation of highly concentrated solutions. The preferred stabiliser includes, but is not limited to, carbohydrates such as sucrose, lactose and trehalose, sugar alcohols such as raannitol and sorbitol, amino acids such as histidine, glycine, phenylalanine and arginine. Furthermore, it is also an aspect of the present invention that a suitable buffer such as a histidine buffer may further stabilize and/or solubilize the compounds of the invention within the semi-solid formulation. The preferred pharmaceutically acceptable cream formulation may help protect and maintain the activity of the immunoglobulin single variable domains and/or constructs thereof (also herein referred to as "compounds of the invention") for at least two years at appropriate temperature e.g. 4°C or room temperature.

Optionally, the compounds of the invention may have undergone a sequence optimization procedure wherein (in particular) the framework residues will have been amended to minimize possible chemical instabilities such as e.g. the risk of pyroglutamate formation, oxidation or isomerization of the therapeutic molecules (reference is also made to methods described in WO 2009/095235, the content of which is hereby incoiporated in its entirety). The semi-solid pharmaceutical compositions described herein can have a variety of concentrations. For instance, the semi-solid pharmaceutical compositions used according to methods of the invention can be at least about 15 mg, more preferably at least about 20 mg, more preferably at least about 25 mg, more preferably at least about 30 mg, more preferably at least about 40 mg or even more preferably at least about 50 mg of the one or more therapeutic molecules per g said semi-solid pharmaceutical composition. Because the pharmaceutical compositions described herein allow for a high drug load of the therapeutic molecules, the semi-solid pharmaceutical compositions provide, for example, for the delivery of at least about 0.1 mg therapeutic molecule per cm² or more such as e.g. between 0.01 to 0.5 mg therapeutic molecule or more per cm².

Integri , Stability and Deliver}/

It was surprisingly found, as described herein, that the therapeutic molecules in the described pharmaceutical compositions have unexpectedly retained most of their integrity. In some embodiments, after formulation the integrity of the one or more therapeutic molecules at a concentration of at least about 20 mg of therapeutic molecules per gram semi-solid composition was greater than 95% when measured by either % main peak on SEC (see also experimental part example 3.3 for more details on measuring integrity, e.g. Table 5). In some embodiments, after formulation the integrity of the one or more therapeutic molecules at a concentration of at least about 20 mg per gram semi-solid composition was greater than 80% when measured by either % extraction recovery (see also experimental part 3.3 for more details on measuring integrity, e.g. Table 5). In some embodiments_., after formulation the integrity of the one or more therapeutic molecules at a concentration of at least about 20 mg per gram semi- solid composition was greater than 75% when measured by either % main peak on RPC (see also experimental part 3.3 for more details on measuring integrity, e.g. Table 5).

Furthermore, it was surprisingly found, as described herein, that the tiierapeutic molecules in the described pharmaceutical compositions were unexpectedly stable and retained potency. In some embodiments, the potency of the one or more therapeutic molecul es was greater than 95% after formulation at a concentration of at least about 20 mg of therapeutic molecules per g semi-solid composition and storage at 25°C over 4 weeks (see also experimental part 4 and 5 for more details on measuring potency, e.g. Tables 6-13). In. some embodiments, the potency of the one or more therapeutic molecules was greater than 99% after formulation at a concentration of at least about 20 nig of therapeutic molecules per g semi-solid composition and storage at 25°C over 4 weeks (see also experimental part example 5 for more details on measuring potency, e.g. Tables 9-13).

In some embodiments, the therapeutic molecules are lyophilized prior to use in the pharmaceutical compositions described herein. Prior to the instant invention it was not recognized, or expected, that therapeutic molecules comprising immunoglobulin variable domains couid be lyophilized and reconstituted without loss of potency.

In one aspect the invention provides pharmaceutical compositions for transdermal and/or local delivery of therapeutic molecules comprising immunoglobulin variable domains, such as immunoglobulin single variable domains. In one aspect the invention therefore provides methods for the systemic and/or local delivery of immunoglobulin variable domains using semi-solid pharmaceutical compositions. It was surprisingly found, as described herein, that the invention provides pharmaceutical compositions in which, after formulation, the potency of the one or more therapeutic molecules at a concentration, of at least about 20 mg per gram semi-solid composition is not affected, such as upon delivery to a subject. For instance, a semi-solid pharmaceutical composition of 1 g comprising at least about 20 mg of therapeutic molecule, constructed and arranged as described herein, is capable of delivering to a subject at least about 0.1 mg therapeutic molecule per cm² or more such as e.g. between 0.01 to 0.5 mg therapeutic molecule or more per cm².

Potency of the therapeutic molecule can be measured before preparation of the pharmaceutical composition, after preparation of the pharmaceutical composition, before administration and/or after administration of the pharmaceutical composition. For example, the binding properties of the therapeutic molecule can be measured using the methods and assays described in the examples below (see e.g. examples 4 and 5). In some embodiments, the therapeutic molecule has at least about 90% of the potency after preparation of the pharmaceutical composition as compared to prior to the preparation of the pharmaceutical composition. In other embodiments, the therapeutic molecule has at least about 90% of the potency after administration of the pharmaceutical composition as compared to prior to administration of the pharmaceutical composition. In still other embodiments, the therapeutic molecule has at least about 90% of the potency after administration of the pharmaceutical composition as compared to prior to preparation of the pharmaceutical composition. Potency of the therapeutic molecule in the pharmaceutical compositions described herein can be retained to levels (compared as described herein) of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or even to substantially 100% potency. Therapeutic amount

An effective amount of therapeutic is a dosage of the therapeutic molecules comprising the immunoglobulin variable domain (or other protein scaffold) sufficient to provide a medically desirable result. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and other factors within the knowledge and expertise of the health practitioner. For example, an effective amount for treating a disease or condition would be an amount sufficient to lessen the progression of or inhibit the disease or condition, or its symptoms.

Immunoglobulin variable domains

In one aspect the invention provides methods, pharmaceutical compositions and devices for the administration, such as cutaneous administration, of therapeutic molecules comprising immunoglobulin variable domains. In some embodiments the immunoglobulin variable domain (also referred to as ' mmunoglobulin, variable domain of the invention") is a VH, VL, VHH, camelized VH, camelized VL, or VHH that is optimized for stability, potency, manufacturability and similarity to human framework regions. In some embodiments the immunoglobulin variable domain is a VHH that is optimized for stability, potency, manufacturability and similarity to human framework regions. In some embodiments the therapeutic molecule comprises a multi valent and/or multispecific construct.

Unless indicated otherwise, the term "immunoglobulin variable domain" is used as a genera] term to include heavy chain, variable domain sequences. More specifically, the immunoglobulin variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody from e.g., human or heavy chain variable domain sequences that are derived from a heavy chain antibody from e.g., camels, dromedaries, alpacas or llamas, ccording to the invention, the immunoglobulin variable domains can be domain antibodies, or immunoglobulin sequences that are suitable for use as domain antibodies, single domain antibodies, or immunoglobulin sequences that are suitable for use as single domain antibodies or immunoglobulin sequences that are suitable for use as compounds termed "dAbs", or "Nanobodies" (NANOBODY® and NANOBODIES© are registered trademarks of Ablynx N.V.) in the field and preferably are NANOBODIES®. Immunoglobulin variable domains include camelid derived immunoglobulin variable domains and functional, optimized variants thereof. Functional and optimized variants include variants that bind the same epitope and variants that are optimized for better potency in binding, optimized for human framework regions, chemical stability and

manufacturability. The immunoglobulin variable domains provided by the invention are preferably in essentially isolated form (as defined herein), or form pari of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more immunoglobulin variable domains of the in vention and which may optionally further comprise one or more further immunoglobulin variable domains (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more immunoglobulin variable domains of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further immunoglobulin variable domains that can. serve as a binding unit (i.e., against one or more other antigens such as e.g. soluble antigens, membrane antigens, serum proteins such as e.g. serum albumin and/or antigens on the same target in order to result in therapeutic proteins or polypeptides that are multispecific), so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, all as described herein. Such a protein or polypeptide (or herei also referred to as "Agent of the Invention") may also be in essentially isolated form (as defined herein).

The invention includes immunoglobulin variable domains of different origin, comprising mouse, rat, rabbit, donkey, shark, human and camelid immunoglobulin variable domains. The invention also includes fully human, humanized or chimeric immunoglobulin variable domains. For example, the invention comprises camelid immunoglobulin variable domains and humanized, camelid immunoglobulin variable domains, or camelized domain antibodies, e.g., camelized VH sequences such as described by Hamers et al. (see for example WO 94/04678; Davies and Riechmann (FEBS Lett. 339: 285-290, 1994 and Prat. Eng. 9(6): 531-537, 1996); and Muyldermans et al. TIBS 26(4): 230-235, 2001). Moreover, the invention comprises fused immunoglobulin variable domains, e.g., forming a multivalent and/ or multispecific construct (e.g., for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al, J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001 , as well as to for example WO 96/34103 and WO 99/232 1), and immunoglobulin, variable domains comprising tags or other functional moieties, e.g., toxins, labels, radiochemicals, etc., which are derivable from the immunoglobulin variable domains of the present invention.

The immunoglobulin variable domain and structure of a immunoglobulin variable domain can - without however being limited thereto - be comprised of four framework regions or "PR's", which are referred to in the art and herein as "Framework region 1 " or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region l"or "CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively. Furthermore, the total number of amino acid residues in an immunoglobulin variable domain can be in the region of 1 10- 120, is preferably 1 12- 1 15, and is most preferably 1 13. it should however be noted that parts, fragments, analogs or derivatives (as further described herein) of a immunoglobulin variable domain are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein.

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

In contrast, the binding site of an immunoglobulin single variable domain is formed by a single VH or VL dom in. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. The term "immunoglobulin single variable domain" and "single variable domain" hence does not comprise conventional immunoglobulins or their fragments which require interaction of at least two variable domains for the formation of an antigen binding site. However, these terms do comprise fragments of conventional immunoglobulins wherein the antigen binding site is formed by a single variable domain.

As such, the single variable domain may for example comprise a light chain variable domain sequence (e.g. a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e. a functional antigen binding unit thai essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit, as is for example the case for the variable domains that are present in for example conventional antibodies and scFv fragments that need to interact with another variable domain - e.g. through a VH/VL interaction - to form a functional antigen binding domain).

In one embodiment of the invention, the immunoglobulin single variable domains are light chain variable domain sequences (e.g. a VL-sequence), or heavy chain, variable domain sequences (e.g. a VH-sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody.

For example, the single variable domain or immunoglobulin single variable domain (or an amino acid sequence that is suitable for use as an immunoglobulin single variable domain) may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a VHH sequence); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited herein, as well as to EP 0 368 684. For the term "dAb's", reference is for example made to Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), to Holt et al. (Trends BiotechnoL, 2003, 21(l l):484-490); as well as to for example WO 04/068820, WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. It should also be noted that, al though less preferred in the context of the present invention because they are not of mammalian origin, single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05/18629).

In particular, the immunoglobulin single variable domain may be a Nanobody® (as defined herein) or a suitable fragment thereof. [Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N. V.] For a general description of

Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in WO 08/020079 (page 16).

The total number of amino acid residues in a Nanobody can be in the region of 1 10- 120, is preferably i 12-1 5, and is most preferably 1 13. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) of a anobody are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein.

For a further description of VHH's and Nanobodies, reference is made to the review article by Muyldermans in Reviews in Molecular Biotechnology 74(2001), 277-302; as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1 134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/2181.7, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Aigonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1 433 793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551 , WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. Reference is also made to the further prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41 -43 of the International application WO 06/040153, which list and references are incorporated herein by reference. As described in these references, Nanobodies (in particular VHH sequences and partially humanized Nanobodies) can in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences. A further description of the

Nanobodies, including humanization and/or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multi valent constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of the Nanobodies and their preparations can be found e.g. in WO 08/101985 and WO 08/142164.

Thus, in the meaning of the present invention, the term "immunoglobulin single variable domain" or "single variable domain" comprises polypeptides which are derived from a non-human source, preferably a cameiid, preferably a camel heavy chain antibody. They may be humanized, as previously described. Moreover, the term comprises polypeptides derived from non-cameiid sources, e.g. mouse or human, which have been "camelized", as previously described.

The term "immunoglobulin single variable domain" encompasses immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and cameiid immunoglobulin sequences. It also includes fully human, humanized or chimeric immunoglobulin sequences. For example, it comprises cameiid immunoglobulin sequences and humanized cameiid immunoglobulin sequences, or camelized immunoglobulin single variable domains, e.g. camelized dAb as described by Ward et al (see for example WO 94/04678 and Davics and Riechmann (1994 and 1996)).

As used herein, the terms "immunoglobulin variable domain(s)", "immunoglobulin single variable domain(s)" and "Agent(s) of the Invention" refer to both the nucleic acid sequences coding for the polypeptide and the polypeptide per se. Any more limiting meaning will be apparent from the particular context.

Protein Scaffolds

It should be appreciated that the pharmaceutical compositions, devices and methods for the therapeutic molecules disclosed herein also include polypeptides comprising one or more protein scaffolds. Protein scaffolds, as used herein, includes both antibody-based scaffolds and non-antibody-based scaffolds, Protein scaffolds comprise antigen-binding polypeptides that, in turn, comprise at least one stretch of amino acid residues that correspond to an antibody CDR sequence (i.e., as part of its antigen binding site). Suitable protein scaffolds for presenting antigen-binding sequences will be clear to the skilled person. Non- limiting examples of protein scaffold embraced by the invention are immunoglobulin-based scaffolds, protein scaffolds derived from protein A domains (such as Affibodies™), tendamistat. fibronectiii, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binz et al, Nat. Biotech 2005, Vol 23 : 1257), Anticalins,

DARPins (designed ankyrin repeat proteins), Adnectins, Chemokine binding proteins identified in ticks, Affilins, CH2 domains, Centyrins (protein fold with significant structural homology to lg domains with loops analogous to CDRs) and Fynomers (single domain protein scaffolds). See also, Hosse et al. (Protein Science, 2006, 15: 14-27).

Therapeutic molecule, multivalent and multispecific constructs

A therapeutic molecule, as used herein, is any molecule, such as a polypeptide, that comprises one or more immunoglobulin variable domains, such as an immunoglobulin single variable domain, including multivalent or multispecific constructs comprising

immunoglobulin (single) variable domains. In addition, a therapeutic molecule, as used herein, also includes polypeptides that comprise one or more of the antibody based-scaffolds and non-antibody based scaffolds described herein. It should be appreciated that therapeutic molecules, as used herein, also include diagnostic molecules.

In order to further improve the avidity (i. e., for a desired antigen) of the

immunoglobulin (single) variable domains, and/or to provide constructs that can bind to two or more different antigens, two or more immunoglobulin (single) variable domains can be combined i a single polypeptide construct, resulting in a multivalent and/or multispecific polypeptide construct. The immunoglobulin (single) variable domains can be coupled to each other directly (e.g., as a fusion protein.) or using polypeptide or non-polypeptide linkers. In addition to increasing the binding to the target antigen, multiple binding to a target can also increase the therapeutic activity of the multivalent polypeptide relative to the individual immunoglobulin (single) variable domains. Multivalency does not have to be limited to two immunoglobulin (single) variable domains, and as such, multivalent polypeptide constructs can be trimers and tetramers etc. of the same or different immunoglobulin (single) variable domains. In some embodiments immunoglobulin (single) variable domains that bind to different targets are coupled to each other resulting in multispecific polypeptide constructs. In some embodiments one of the immunoglobulin (single) variable domains of the multispecific polypepti de constructs binds a serum protein, thereby increasing the half-life of the polypeptide construct. In some embodiments the serum protein is serum, albumin, serum immunoglobulin, thyroxine-binding protein, transferrin or fibrinogen. In some embodiments the multispecific polypeptide constructs also can comprise two or more immunoglobulin (single) variable domains that bind to the same target, thereby increasing the affinity for binding to a single antigen.

Immunoglobulin (single) variable domains (or multivalent and/or multispecific polypeptide constructs) can also be coupled to polypeptides other than antibodies. Coupling of the immunoglobulin (single) variable domains to non-antibody polypeptides can provide the immunoglobuhn (single) variable domains with an extra functionality and/or can increase their half-life. Individual immunoglobulin (single) variable domains can be small and can sometimes be disposed of in the body through the kidneys. Disposal through the kidneys may diminish the therapeutic effectiveness of the immunoglobulin (single) variable domains. Filtration by the kidneys can be prevented or reduced by increasing the size of individual immunoglobulin (single) variable domains through multimerization as described above or through coupling to a larger protein, preferably a stable protein found in the bloodstream, like albumin. Coupling immunoglobulin (single) variable domains to larger serum proteins will increase the half-life of the immunoglobulin (single) variable domains. In some embodiments the serum protein is serum albumin, serum immunoglobulin, thyroxine-binding protein, transferrin or fibrinogen. In another embodiment immunoglobulin (single) variable domains are coupled to a polypeptide that would give them additional functionalities. Examples include, but are not limited to, signaling peptides, binding peptides, peptide receptor ligands and functional, enzymes.

immunoglobulin (single) variable domains (or multivalent and/or rnultispecific polypeptide constructs) can also be coupled to a non -polypeptide group. In one embodiment, the non-polypeptide group is a toxic agent, in another embodiment, the non-polypeptide group is a tracer. The non-polypeptide groups can be coupled to the immunoglobulin (single) variable domain through a linker as is described below.

Coupling the immunoglobulin (single) variable domains to a toxic agent will allow for delivery of the toxin to the antigen. This methodology can be used to introduce a toxic agent to a site where it is most effective (e.g., inside a tumor cell, or on the membrane of a tumor cell). The methodology can also be used to rid the bloodstream of unwanted products. The immunoglobulin (single) variable domains can bind to an unwanted antigen, which can be inactivated by the toxic agent that is attached to the immunoglobulin (single) variable domains. In contrast, if the toxic agent were not attached to an immunoglobulin (single) variable domains, it would not get in close proximity to its target product, or would not stay in sufficiently close proximity long enough, to inactivate the target. Immunoglobulin (single) variable domains can also be coupled to tracers. This will allow for the monitoring of a specific target in the body. For instance, an immunoglobulin (single) variable domains that binds to a tumor antigen can be coupled to a radioactive tracer. The amount of radioactivity retained in the body and the localization of the tracer will help diagnose the amount of tumor cells in the body and can help determine the progress of a specific treatment regimen (See also below).

Amino acid linkers for use in multivalent and multispecific polypeptides will be clear to the skilled person, and for example include Gly-Ser linkers, for example of the type (Gly_xSei-y)_z, such as for example (Gly₄Ser)₃ or (Gly₃Ser₂)3, as described in WO 99/42077, hinge-like regions such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences. Linkers can also provide some functionality for the multivalent or multispecific polypeptides. For example, linkers containing one or more charged amino acid residues can provide improved hydrophilic properties, whereas linkers that form or contain small epitopes or tags can be used for the purposes of detection, identification and/or purification.

Immunoglobulin (single) variable domains can be connected to each other, to other polypeptides or to non-polypeptides groups through a number of non-peptide linkers. In one embodiment, the linker comprises an amido linker moiety, an amino linker moiety, a carbonyl linker moiety, a carbamate linker moiety, a urea linker moiety, an ether linker moiety, a disulphi.de linker moiety, a succinamidyl linker moiety, a succinyl linker moiety, and combinations thereof. In other embodiments, the linker moiety is an ester including: carbonate (-OC(O)O— ), succinoyl, phosphate esters (— 0— (O)POH-- 0— ), sulfonate esters, and combinations thereof. Diagnostics

In one embodiment, semi-solid pharmaceutical compositions are used to administer immunoglobulin variable domains, such as immunoglobulin single variable domains used for diagnostics. Coupling a tracer to an immunoglobulin (single) variable domain will allow for the determination of an amount and/or location of a specific antigen in vivo or in vitro. The tracer can be, but is not limited to an agent of fluorescent or radioactive origin. The diagnostic administered with the semi-solid pharmaceutical, compositions can be used to determine the amount and/or location of a variety of antigens, for instance a solid tumor cell marker or peptide marker in the bloodstream. In most diagnostic assays, the diagnostics needs to be administered a set time prior to the assay. While the readout of the diagnostic assay will likely need to be performed by a health care official, being able to self administer the diagnostic dose will allow for one less trip to the clinic and savings in time and cost.

Immunoglobulin (single) variable domains are preferred because of their stability and small size. They could for instance enter a tumor cell, allowing for a more complete diagnostic picture.

Diseases and disorders

A variety of diseases and disorders can be treated using immunoglobulin variable domains, such as immunoglobulin single variable domains as delivered according to the methods, pharmaceutical compositions and devices of the invention. Exemplary diseases include inflammatory disorders, aggregation-mediated disorders, cancers, autoimmune diseases, neurodegenerative disorders, genetic disorders,

"Inflammatory disorders" include diseases such as psoriasis, rheumatoid arthritis, Crohn's disease, mastocytosis, asthmas, multiple sclerosis, inflammatory bowel syndrome and allergic rhinitis (see, e.g., US published application 2006/0058340). For example, in one embodiment, the semi-solid pharmaceutical composition is used to administer (polypeptides comprising one or more) immunoglobulin (single) variable domains against IL-23, against TNF-alpha, against IL-6, against RANK-L, against 1L-6R, or against any other protein or target involved in the 1L~6 pathway, as for example described in WO 2009/068627, WO 2004/041862, US 2005/0054001 , US 2006/0034845, US 60/682332, WO 03/050531, WO 03/054016, US 60/782243, US 60/782246, WO 06/122786, WO 04/003019 and WO 03/002609.

For the prevention and treatment of inflammatory disorders such as psoriasis, for example, immunoglobulm (single) variable domains against 1L23 (as for example described in WO 2009/068627) may be formulated as and administered using the semi-solid pharmaceutical composition disclosed herein.

A variety of cancers can be treated according to the methods, pharmaceutical compositions and device of the invention. The cancer may be a carcinoma or a sarcoma but it is not so limited. For example, the cancer may be basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma,

CMS cancer, colon and rectum cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, cancer of the head and neck, gastric cancer, intra-epithelial neoplasm, kidney cancer, larynx cancer, leukemia, acute lymphoid leukemia, acute myeloid leukemia, chronic lymphoid leukemia, chronic myeloid leukemia, cutaneous T-cell leukemia, hairy cell leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, follicular lymphoma, Fiodgkin's lymphoma, Non-

Hodgkin's lymphoma, melanoma, myeloma, multiple myeloma, neuroblastoma, oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, cancer of the respiratory system, retinoblastoma, rhabdomyosarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, cancer of the urinary system and uterine cancer (see, e.g., US published application 2006/0019923).

An "immune disorder" includes adult respiratory distress syndrome, arteriosclerosis, asthma, atherosclerosis, cholecystitis, cirrhosis, Crohn's disease, diabetes mellitus, emphysema, hypereosinophilia, inflammation, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, psoriasis, rheumatoid arthritis, scleroderma, and ulcerative colitis (see, e.g., US published application 2003/0175754).

"Genetic disorders" include Aarskog-Scott syndrome, Aase syndrome,

achondroplasia, acrodysostosis, addiction, adreno-leukodystrophy, albinism, ablepharon- macrostomia syndrome, alagille syndrome, alkaptonuria, alpha- 1 antitrypsin, deficiency, Alport's syndrome, Alzheimer disease, asthma, autoimmune polyglandular syndrome, androgen insensitivity syndrome, Angelman syndrome, ataxia, ataxia telangiectasia, atherosclerosis, attention deficit hyperactivity disorder (ADHD), autism, baldness, Batten disease, Beckwith-Wiedemann syndrome, Best disease, bipolar disorder, brachydactyly, breast cancer, Burkitt lymphoma, chronic myeloid leukemia, Charcot-Marie-Tooth disease, Crohn's disease, cleft lip, Cockayne syndrome, Coffin Lowry syndrome, colon cancer, congenital adrenal hyperplasia, Cornelia de Lange syndrome, Costello syndrome, Cowden syndrome, craniofrontonasal dysplasia, Crigler-Najjar syndrome, Creutzfeldt-Jakob disease, cystic fibrosis, deafness, depression, diabetes, diastropbic dysplasia, DiGeorge syndrome, Down's syndrome, dyslexia, Duchenne muscular dystrophy, Dubowitz syndrome, ectodermal dysplasia Ellis-van Creveld syndrome, Ehlers-Danlos, epidermolysis bullosa, epilepsy, essential tremor, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Friedreich's ataxia, Gaucher disease, glaucoma, glucose galactose malabsorption, glutaricaciduri , gyrate atrophy, Goldberg Shprintzen syndrome (velocardiofacial syndrome), Gorlin syndrome, Hailey-Hailey disease, hemihypertrophy, hemochromatosis, hemophilia, hereditary motor and sensory neuropathy (HMSN), hereditary non polyposis colorectal cancer (HNPCC), Huntington's disease, immunodeficiency with hyper-IgM, juvenile onset diabetes, Klinefelter's syndrome, Kabuki syndrome, Leigh's disease, long QT syndrome, lung cancer, malignant melanoma, manic depression, Marfan syndrome, Menkes syndrome, miscarriage, mucopolysaccharide disease, multiple endocrine neoplasia, multiple sclerosis, muscular dystrophy, myotrophic lateral scierosis, myotonic dystrophy, neurofibromatosis, Niemann-Pick disease, Noonan syndrome, obesity, ovarian cancer, pancreatic cancer, Parkinson disease, paroxysmal nocturnal hemoglobinuria, Pendred syndrome, peroneal muscular atrophy, phenylketonuria (PKU), polycystic kidney disease, Prader-Willi syndrome, primary biliary cirrhosis, prostate cancer. REAR syndrome, Refsum disease, retinitis pigmentosa, retinoblastoma, Rett syndrome, Sanfilippo syndrome, schizophrenia, severe combined immunodeficiency, sickle cell anemia, spina bifida, spinal muscular atrophy, spinocerebellar atrophy, SRY: sex determination, sudden adult death syndrome, Tangier disease, Tay-Sachs disease, thrombocytopenia absent radius syndrome, Townes-Brocks syndrome, tuberous sclerosis, Turner syndrome, Usher syndrome, von Hippel-Lindau syndrome, Waardenburg syndrome, Weaver syndrome, Werner syndrome, Williams syndrome, Wilson's disease, xeroderma pigmentosum or Zellweger syndrome, (see, e.g., 2005/0281781).

In addition, molecules that can be targeted ("targets") by the immunoglobulin variable domains, such as immunoglobulin single variable domains delivered according to the methods of the invention include, for example, targets of foreign origin, host derived cellular targets, and host derived non-cellular targets. Exemplary targets are listed below.

Foreign target agents include drugs, especially drugs subject to abuse such as heroin and other opiates, PCP, barbiturates, cocaine and derivatives thereof, benzodiazepines, etc., poisons, toxins such as heavy metals like mercury and lead, chemotherapeutic agents, paracetamol, digoxin, free radicals, arsenic, bacterial toxins such as LPS and other gram negative toxins, Staphylococcus Toxins, Toxin A, Tetanus toxins, Diphtheria toxin and Pertussis toxins, plant and marine toxins, virulence factors, such as aerobactins, radioactive compounds or pathogenic microbes or fragments thereof, including infectious viruses, such as hepatitis B, A, C, E and delta. CMV, HSV (type 3, 2 & 6), EBV, varicella zoster virus (VZV), HIV-1, -2 and other retroviruses, adenovirus, rotavirus, influenzae, rhinovirus.

parvovirus, rubella, measles, polio, reovirus, orthomyxovirus, paramyxovirus, papovavirus, poxvirus and picornavirus, prions, protists such as plasmodia tissue factor, toxoplasma, filaria, kala-azar, bilharziose, entamoeba histolitica and giardia, and bacteria, particularly gram-negative bacteria responsible for sepsis and nosocomial infections such as E. coli,

Acynetobacter, Pseudomonas, Proteus and Klebsiella, but also gram positive bacteria such as staphylococcus, streptococcus, etc. Meningococcus il Mycobacteria, Chlamydiae, Legionnella and Anaerobes, fungi such as Candida, Pneumocystis carini, and Aspergillus, and Mycoplasma such as Hominis and Ureaplasma urealyticum (see, e.g., US patent 5,843,440).

Host derived cellular and non-cellular targets against to which the immunoglobulin (single) variable domains and constructs that are administered may be directed will be clear to the skilled person and for example include, but are not limited to, all targets for which NANOBODIES®, dAb's or otiier immunoglobulin variable domains (or polypeptides comprising the same) have been proposed in the art (such as IL-23, TNF-alpha, Von Wiiiebrand factor, interleukins such as 1L-6, amyloid-beta, etc., as well as the other targets mentioned in. the prior art referred to herein), as well as more generally cellular and non- cellular targets for which antibodies or antibody fragments (including but not-limited, to ScFv constructs) have been proposed in the art.

Matins of the semi-solid pharmaceutical compositions

The present invention can be further understood with reference to steps executed to manufacture a semi-solid pharmaceutical composition in accordance with a preferred embodiment of the present invention. The manufacturing flow may start by providing a water mixture that preferably contains at least water and possibly a buffer such, as e.g. a histidine or phosphate buffer. In a preferred embodiment, the water mixture occupies at least about 70% by mass of the pharmaceutical composition. When included, various other non-oil elements are preferably mixed with, the water mixture. For example, the composition may optionally include preservatives such as e.g. potassium sorbate in small amounts. Such ingredients, when included, are preferably dissolved in the water mixture.

in addition to providing the water mixture and any optional non-oily elements, the manufacturing flow preferably includes dispersing an oil based phase into the water based phase. Oils and fatty components such as selected e.g. from the ones disclosed and described herein, may be used. When included, certain non-oily elements are preferably mixed with the oil phase prior to heating. For example, the cream, may optionally include cetomacrogol and cetostearylalcohol or other emusifying agents. Such, an ingredient, when included, is preferably mixed together with the aforementioned oils. After the oils and optional non-oiiy elements have been mixed, the oil phase is heated, preferably on a low heat in a double boiler, to a temperature below the boiling point of the mixture. Once the oil mixture is hot, the oil mixture is then blended or otherwise mixed with, the water mixture (at approximately the same temperature) to produce the cream, thereby ending the manufacturing flow for purposes of the present invention. One skilled in the art will readily recognize that, while the logic flow diagram as described herein depicts the mixing and heating of the various phases, the phases may be alternatively mixed and heated, and the oil mixture created, before or contemporaneous with creation of the water mixture. Therefore, the order of heating or/and mixing should not be interpreted to limit the appended claims in any way.

The therapeutic molecule can be incorporated into the semi-solid pharmaceutical composition or cream in the fonn of a liquid or in lyophilized state. In one embodiment, the therapeutic molecule is added to the cream as a liquid. In this embodiment, the therapeutic molecule is prepared in an appropriate buffer at the appropriate concentration and the liquid buffer is mixed with the cream in the appropriate amount and concentration (e.g. 0.5 mL of buffer (20 mg/mL of therapeutic molecule) is mixed with 1 gram of cream). In another preferred embodiment, therapeutic molecule is added to the cream after lyophi zation of the therapeutic molecule. This method allows for the preparation of semi-solid formulation with higher concentrations of therapeutic molecule. In this embodiment, the lyophilized therapeutic molecule is mixed with the cream in the appropriate amounts. Definitions

A "stable" semi-solid pharmaceutical composition is one in which the protein such as the immunoglobulin single variable domain or a construct thereof retains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art. Stability can be measured at a selected temperature for a selected time period. For rapid screening, the pharmaceutical composition may be kept at 37~40°C for 2 weeks to 1 month, at which time protein stability is measured. Where the phaiTnaceutical composition is to be stored at 2 to 8 °C, generally depending on the intended shelve life the phaiTnaceutical composition should e.g. be stable at 30°C or 37-40°C for at least one month and/or stable for at least 2 years at 2 to 8 °C. For example, the extent of aggregation, may be one wherein less than about 10% and preferably less than about 5% of the protein is present as an aggregate in the pharmaceutical composition.

Aggregation as used in the present invention means the development of high molecular weight aggregates, i.e. aggregates with an apparent molecular weight of more/higher than the apparent molecular weight observed in SE-HPLC analysis for the therapeutic molecule of the invention in comparison with molecular weight markers.

Aggregation can be assessed by various methods known in the art. Without being limiting, examples include high performance size exclusion chromatography (SEC), subvisible particle counting, analytical uitracentrifugation (AUC), dynamic light scattering (DLS), static light scattering (SLS), elastic light scattering, OD320/OD280 measurement, Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence and/or differential scanning calorimetry techniques.

In other embodiments, any increase in aggregate formation following lyophilization and storage of the lyophilized pharmaceutical composition can be determined. For example, a "stable" lyophilized pharmaceutical composition may be one wherein the increase in aggregate in the lyophilized composition is less than about 10% and preferably less than about 5%, when the lyophilized composition is stored at 2 to 8 °C for at least 8 weeks, preferably at least one year.

For rapid screening, the pharmaceutical composition may be kept at 37-40°C for 2 weeks to 1 month, at which time protein stability, is measured. For example, a "stable" lyophilized pharmaceutical composition may be one wherein the increase in aggregate in the lyophilized composition is less than about 10% and preferably less than about 5%, when the lyophilized composition is stored at 37-40 °C for 2 weeks to one month.

In other embodiments, stability of the pharmaceutical composition may be measured using a biological assay. The potency and/or biological activity of a biological or therapeutic molecule describes the specific ability or capacity of said biological or therapeutic molecule to achieve a defined biological effect. The potency and biological activities of the therapeutic molecules of the invention can be assessed by various assays including any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved. Suitable in vitro assays will be clear to the skilled person, and for example include ELISA; FACS binding assay; Biacore; competition binding assay (AlphaScreen®, Perkin Elmer, Massachusetts, USA; FMAT); SEQ ID NO's: 1 to 3 interact with IL-23 and block the interaction, of this ligand with its receptor. The potency of SEQ ID NO's: 1 to 3 for blocking the respective ligand/receptor interaction can be determined, e.g. by ELISA, Biacore, AlphaScreen®.

For example, in one embodiment, Biacore kinetic analysis uses Surface Plasmon Resonance (SPR) technology to monitor macromolecular interactions in real time and is used to determine the binding on and off rates of polypeptides of the formulation of the invention to their target. Biacore kinetic analysis comprises analyzing the binding and dissociation of the target from chips with immobilized polypeptides of the invention on their surface. A typical Biacore kinetic study invoives the injection of 250 μΐ, of polypeptide reagent at varying concentration in HBS buffer containing 0.005% Tween 20 over a sensor chip surface, onto which has been immobilized the antigen. In the BlAcore 3000 system, the ligand is immobilized on carboxymethylated dextran over a gold surface, while the second partner (analyte) is captured as it flows over the immobilized ligand surface. The immobilized ligands are remarkably resilient and maintain their biological activity. The bound analytes can be stripped from the immobilized ligand without affecting its activity to allow many cycles of binding and regeneration on the same immobilized surface. Interaction is detected in real time via SPR and at high sensitivity. Because the same affinity may reflect different on-rates and off -rates, this instrument excels over most other affinity measuring methods in that it measures on-rates (ka) and off-rates (kd). Concentration determination experiments arc also feasible.

The semi-solid formulations of the present invention exhibits almost no loss in biological activities and/or potency of the therapeutic molecules during the prolonged storage under the conditions described above, as assessed by various immunological assays including, for example, enzyme-linked immunosorbent assay (ELISA). The therapeutic molecules in the semi-solid formulations of the present invention retain, even under the above defined stress conditions (such as storage under certain temperature stress for defined periods) more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, or more than 99.5% of their initial biological activities and/or potencies (e.g., the ability to bind IL-23 and/or HSA) of the therapeutic molecule prior to the storage. In some embodiments, the polypeptides in the formulation of the invention retain under the above defined stress conditions at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% of the biological activity and/or potency (e.g., the ability to bind to IL-23 and/or HSA) compared to the therapeutic molecules present in a reference formulation prior to the storage.

A "stable" pharmaceutical composition can be one wherein the loss in potency in a biological assay is less than 10%, preferably less than about 5% when the composition is stored at 2 to 8 °C for at least 8 weeks, preferably at least one year.

For rapid screening, the pharmaceutical composition may be kept at 37-40°C for 2 weeks to 1 month, at which time protein stability is measured. A "stable" pharmaceutical composition can be one wherein the loss in potency is less than 30%, less than 25%, preferably less than 20%, less than 1 %, most preferably less than 10%, less than 5% when the composition is stored at 37-40 °C for 2 weeks to one month. "Semisolid" pharmaceutical systems comprise a body of products, which when applied to the skin or accessible mucous membranes tend to alleviate or treat a pathological condition or offer protection against a harmful environment. They have the property to cling to the skin or mucous membrane for a protracted period of time to e ert their therapeutic effect through protection and occlusion. The adhesion is due to their plastic rheologic behavior which allows semisolid to retain their shape and cling as film until acted upon by an outside force. "Semisolid" dosage forms usually are intended for localized, drug delivery. In the past few years, however, these forms also have been explored for the systemic delivery of various drugs. They can. be applied topically to the skin, cornea, rectal tissue, nasal mucosa, vagina, buccal tissue, urethral membrane, and external ear lining (ref: Encyclopedia of Pharmaceutical Technology, third edition, 2006, ISBN: 978-0-8493-9399-0 (hardback) 978- 0-8493-9398-3 (electronic)).

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching thai is referenced hereinabove.

EXAMPLES

Example 1: Semi-solid pharmaceutical compositions comprising anti-IL23 Nanobody constructs

Example la: Lyophilisation of Nanobody constructs P23IL0Q75, P23IL0087 and P23IL0095

The process was identical for the three anti-IL23 Nanobody constructs P23IL0075

(SEQ ID NO: 1), P23IL0087 (SEQ ID NO: 2) and P23IL0095 (SEQ ID NO: 3) (Table A).

After purification, the protein was concentrated and buffer-excha ged by cross flow to a concentration of approximately 20 mg/niL in a 10 mM histidine pH6 buffer with 10 % sucrose. Aliquots of 2.5 mL were Iyophilized in 6R vials using an EPSILON 2-4 LSC

(Christ®) freeze dryer. The lyophilization program is shown in Table I , When the iyophilisation process was completed, the pressure inside the Iyophilizer was increased to 200 mbar before closing the vials and opening the iyophilizer to remove the vials. The Iyophilized powder was stored in the closed vials at 5°C until further use.

To check the quality of the Nanobody constructs after the lyophilization process, one vial of each lyophilised product was resuspended in. 2.5ml Milii-Q (3V5Q) water and the purity analyzed by size exclusion chromatography (SEC) and by reversed phase chromatography (RPC). The obtained chromatographic profiles looked exactly the same as for the start sample, so no aggregation or chemical modifications induced by lyophilisation were detected. In Tabie 2 the percent main peak for both analyses is shown and no significant differences are seen. Example 1 b: Preparation of the base cream

Water was heated to 80°C and potassium sorbate was dissolved in the amounts as listed in Table 3 to result in the aqueous phase. In a separate vessel, cetostearyialcohol, cetomacrogol 1000, white vaselin and liquid paraffin were heated to 80°C and stirred until homogenous to result in the lipid phase. Then, the aqueous phase was added to the liquid phase at a temperature of 65°C while stirring. Finally, the preparation was stirred and cooled down to room temperature to form the semi-solid base cream (also referred to as

"cetomacrogol cream").

Example lc: Preparation of the Nanobody containing cream

Incorporation of lyophilized Nanobody construct in a cream:

The incorporation of the Nanobody in the cream was performed as follows: 120 to

133mg of lyophilized powder containing approximately 20 mg of the Nanobody constructs was weighed and cetomacrogol cream of Example lb was added to give 1 gram final pharmaceutical composition in total. The powder readily dissolved in the cream, the mixture was mixed manually with a spatula. The cream was afterwards brought into a 1-mL syringe and stored at 4°C or at 25°C. Details of the preparation are shown in Table 4.

Incorporation of Nanobody as a liquid in a cream:

From the liquid formulation of the Nanobody at approximately 20 mg mL in a 10 mM histidine pH6 buffer with 10 % sucrose, 0.5 mL was take and cetomacrogol cream (non- buffered) was added ad 1 gram. Details of the preparation are shown in Tabie 4. Example 2: Extraction and analysis of the Nanobody construct from the cream formulation

For the analysis, between 80 and 130 mg of the cream was weighed in a 1.5-mL eppendorf to which 1 mL of PBS buffer was added. The material was extracted by gentle rotation of the cream during 1 hour at room temperature. Afterwards the lipid and water phase were allowed to separate, and the water phase was transferred to another vial. The extract was analyzed as such by reversed phase chromatography (RPC), by size exclusion chromatography (SEC) and potency analysis.

Example 3 : Analysis of the Nanobody cream formulations

Example 3.1 Purity assay of the Nanobodies by Size Exclusion High Performance Liquid Chromatography (SE-HPLC)

The SE-HPLC assay consisted of a pre-packed silica gel TSKgel G2000SW_XL column equipped with a guard column pre-column filter, a mobile phase consisting of 10 mM Na Phosphate, 0.3M arginine and 0.005%NaN₃ at pH6, and UV detection at 280 nm. The relative amount of protein purity was expressed as area %, and was calculated by dividing the peak area by the total integrated area.

Example 3.2 Purity assay and quantification of the Nanobodv constructs bv Reversed Phase High Performance Liquid Chromatography (RP-HPLC)

In the RP-HPLC assay a Zorbax 300SB-C8 column (Agilent Technologies, Palo Alto, US) was used. The amount of the protein was determined by measuring the light absorbance of the components eluting from the RP-HPLC column and comparison with a reference sample. The identity of the Nanobody constructs was confirmed by comparing the relative elution time from the RP-HPLC column. The relative amount of protein purity was expressed as area %, and was calculated by dividing the peak area by the total (main peak + impurities) integrated area. Example 3.3 Analysis of the integrity of the anti-IL23 Nanobodies after yophilization. incorporation and storage in a cream

After each preparation step an aliquot of the sample was analyzed in order to check the integrity of the molecule. Lyophilized material was analyzed after reconstitution with milli-Q water, and the creams were extracted with PBS buffer. The data are presented in Table 2 and Table 5.

The recovery of the Nanobody construct was calculated as if all of the Nanobody construct in the cream would be extracted by the 1 mL PBS volume. In case the creams were made from lyophilized powder, the recovery was between 76 and 83%. One exception was the cream analyzed after 4 weeks storage at 25°C, which was concentrated due to evaporation during storage. In case the cream was made with the liquid formulation, the extraction recovery was close to 100 %. The extraction recoveries were calculated based on the RPC peak area's for quantification of the Nanobody concentration in the extract. These concentrations were also used for the potency analysis.

The integrity of the material was analyzed by size exclusion chromatography (SEC) and by reversed phase chromatography (RPC). In Figure 1 to Figure 4 examples of the samples stored for 8 weeks at 4°C and for 4 weeks at 25°C are shown. These were the only samples where some degradation could be observed compared to the reference sample.

In Table 2 and Table 5 the percent main peak integrated from all analyzed samples is shown. It was concluded that for all samples analyzed directly after lyophilizaiion or after cream preparation, and after up to 2 weeks storage in the cream at 4°C, the purity in both chromatography methods was the same as for the reference sample. A difference up to 2% in the RPC integrated area can easily be due to integration variability, the reproducibility of the integration is hampered by the bad resolution with the pre-peaks. Only for the samples stored for 8 weeks at 4°C or for 4 weeks at 25°C, small differences in the chromatographic profiles were seen (Figure 1 to Figure 4). In these samples the purity as determined on SEC decreased by maximum 1.5% (25 °C sample), the purity as determined on RPC decreased with 4% (see also Table 5).

Example 4: Potency analysis of anti-IL-23 Nanobody following lyophilization and formulation into cream

The potency of the anti-hIL-23 Nanobody construct P23ILO075 was analyzed after lyophilisation and after formulation as lyophilized material into cetomacrogol cream (20 mg g). Therefore, analysis was performed in a neutralization ELISA, measuring the potency of the IL-23 binding moieties of P23IL0075 Nanobody construct for their capacity to inhibit the interaction of human IL-23 to a hIL-23R-Fc chimera. Briefly, 96-well microliter plates were coated overnight at 4°C with anti-Fc Nanobody and blocked for 30 min with

Superblock T20 (PBS) blocking buffer. Different concentrations of compound were pre- incubated with 4 ng/mL recombinant human IL-23 (rhIL-23,eBioscience, 34-8239) for 30 min in Assay Diluent 3 (AD3, Immunohistochemistry Technologies, LLC) prior to subsequent 45 min incubation with 300 ng/mL human IL-23R/Fc chimera (R&D Systems, 1400-IR) in AD3, after which the mixtures were transferred to the coated, wells. After further incubation for 30 minutes at room temperature, residual bound. rhIL-23 was detected for 45 min at RT with a 1/4000 dilution of biotin.yi.ated anti-human IL-12 p40/p70 monoclonal antibody (eBiosciences, 13-7129) in PBS containing 1 % Superblock T20, followed by a 30 min incubation at RT with 0.067 μg/mL Strep-HRP (Thermo Scientific, 21126) in PBS containing 10% Superblock T20. Visualization was obtained with (undiluted) esTMB (SDT reagents Gemiany) and the coloring reaction was stopped by adding IN HCL after which the optical density was measured at 450nm.

The potency of P23IL0075 after lyophilisation followed by subsequent reconstitution in. PBS was compared with the potency of the P23IL0075 reference batch formulated as liquid solution in l OmM pH6 Histidine + 10% sucrose buffer. In addition, the potency of P23IL0075 was verified after formulation of lyophilized material into cream (20 mg/g) followed by subsequent extraction in PBS.

The results demonstrate that after lyophilisation and formulation into cream, P23IL0075 still concentration-dependently and completely blocks the interaction of 1L-23 with its receptor (Figure 5). Based on the evaluation of the 1C50 values and its lower and upper limits of the 95% confidence interval (LLC1 and ULCI), no drop in potency was observed after lyophilisation or formulation into cream (Table 6). A 4 parameter logistic regression was used as fitting model based on non-linear analysis using Graphpad Prism software.

in addition, the samples were analyzed in a human serum albumin (=HSA) binding ELISA, measuring the potency of the albumin-binding moiety (=ALB) of P23IL0075 Nanobody construct for its capacity to bind HSA. in addition samples from a similar anti-IL- 23 Nanobody construct, P231L0087, were included in the analysis.

Briefly, 100 g/mL HSA (Sigma, A7223) in PBS was coated overnight at 4°C onto a

96-well Maxisorp ELISA plate (Nunc, 430341 ) by passive adsorption. After blocking excess binding sites on the plates with Superblock T20 (PBS) (Thermo Scientific, 37516), a dilution series of the Nanobody construct was applied. Triplicates were measured and each replicate was assessed on a different plate. Bound Nanobody construct was subsequently detected using a bivalent anti-Nanobody construct recognizing Nanobody constructs directly conjugated to HRP. Visualisation was obtained with 1 /3 diluted esTMB (SDT reagents Germany) and the colouring reaction was stopped by adding IN HCL after which the optical density was measured at 450nm.

The potency of P23IL0075 after lyophilisation followed by subsequent reconstitution in PBS was compared with the potency of the P23IL0075 reference batch formulated as liquid solution in lOmM pH6 Histidine + 10% sucrose buffer, respectively. Also after lyophilisation and formulation into cream, P23IL0075 still concentration-dependently and saturably interacted with immobilized HSA (Figure 6). Based on the evaluation of the EC50 values and its lower and upper limits of the 95% confidence interval (LLC1 and ULC1), no drop in potency to bind HSA was observed after lyophilisation or formulation into cream (Table). Similar results were obtained for P23IL0087 (Figure 7, Table).

Example 5: Stability analysis of anti lilL-23 Nanobody formulated in cetomacrogoi creams by means of potency determination

In a next step, the stability of the anti-hIL~23 Nanobodies P23IL0075 and P23IL0087 as formulated in cream (20mg/g) was analyzed in the inhibition, potency EL1SA and HSA binding ELISA, as described in Example 4. Therefore, the potency of P23IL0075 was determined following extraction, after 2 and 8 weeks storage at 4°C, from the cream formulation. In addition, the potency of P23IL0075 was verified after processing the Nanobody reference batch directly into the cream without a lyophilisation step.

The results in the inhibition potency EILSA demonstrate that 2 and 8 weeks after formulation in cream and storage at 4°C, P231L0075 still concentration-dependently and completely blocked the interaction of IL-23 with its receptor (Figure 8 and Figure 9). Also, formulation into cream without intermediate lyophilisation resulted in. concentration- dependent and complete neutralization of IL-23. Based on the evaluation of the IC50 values and its lower and upper limits of the 95% confi dence interval (LLC1 and ULCI) of the described P231L0075 samples, no drop in potency was observed as compared to the reference batch (Table 9 and Table 10).

Analysis of the same samples in the HSA binding ELISA also showed no drop in potency of the HSA binding moiety of P23IL0075 after storage for 2 or 8 weeks P231L0075 still concentration-dependently and saturably interacted with immobilized HSA (Table 1 1, Figure 10). Only when P23IL0075 was fonnulated in a cream without intermediate lyophilisation, a small drop in potency was observed for HSA binding capacity.

Also for another anti-hIL-23 Nanobody construct, P23IL0087, the potency and stability was analyzed after formulation in a cream. For this Nanobody construct, the potency was analyzed 4 weeks after cream formulation and storage at 4°C and 25°C.

In line with the observations for P23IL0075, the results in the inhibition potency ELISA indicated that in cream fonnulated P23IL0087 still concentration-dependently and completely blocked the interaction of IL-23 with its receptor, even if the cream was stored for 4 weeks at 25°C (Table 12, Figure 11). Based on the evaluation of the IC50 values and its lower and upper limits of the 95% confidence interval (LLCI and ULCI) of the P23IL0087 samples, no drop in potency was observed as compared to the reference batch (Table 12).

Analysis of these cream samples in the HSA binding EL1SA also showed no drop in potency of the HSA binding moiety of P23IL0087 after storage for 2 or 4 weeks at 4 or 25°C. P23IL0087 still concentration-dependently and saturably interacted with immobilized HSA (Figure 12. Table).

In conclusion, the P23IL0075 and P23IL0087 Nanobody constructs did not show any loss in potency after being formulated in cream and stored for 8 and 4 weeks at 4 or 25 °C, respectively.

Equivalents

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in. addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference in their entirety, particularly for the use or subject matter referenced herein.

TABLES

Table A: Sequence Listing

P23IL0075; SEQ ID N0:1

EVQLLESGGGLVQPGGSLRLSCAASGRIPSLPASGNIFNLLTIAWYRQAPGKGRELVATINSGSRTYYADSVKG RFTISRDNSKKTLYLQMNSLRPEDTAVYyCQTSGSGSPNFWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQP GHSLRLSCAASGPTFSSFGMSWVRQAPGKGLE VSSISGSGSDTLYADSVKGRFTISRDHA TTLYLQMNSLRP EDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTLSSYAMG FR QAPGKGREFVARISQGGTAIYYADSVKGRFTISRDNSIiNTLYLQMNSLRPEDTAVYYCA DPSPYYRGSAYLLS

GSYDSWGQGTLVTVSS

P23IL0075; SEQ ID NO :

GAGGTGCAATTGTTAGAGTCCGGTGGTGGTTTAGTTCAACCTGGTGG TCCTTGAGATTGTCTTGTGCTGCCTC CGGTAGAATTTTCTCTCTGCCCGCCTCCGG AACATTTTCAACCTGCTGACTATCGCTTGGTATAGACAGGCTC CAGGAAAGGGTAGAGAGTTGGTTGCTACCATCAACTCCGGTTCCAGAACTTACTACGCCGACTCCGTTAAAGGA AGATTCACGATCTCCAGAGACAACTCCAAGAAAACCCTGTACCTGCAGATGAACTCTCTTAGACCAGAGGACAC CGCTGTTTACTACTGTCAAACCTCTGGTTCTGGTTCTCCAAACTTCTGGGGTCAAGGTACTCTGGTTACTGTTT CCTCTGGTGGTGGTGGTTCTGGTGGTGGATCTGAGGTTCAGTTGGTTGAATCCGGTGGAGGATTGGTTCAACCC GGTAACTCTTTGAGACTTTCCTGTGCCGCTTCTGGTTTTACTTTCTCCTCCTTCGGAATGTCTTGGGTTAGACA AGCGCCAGGTAAAGGATTGGAGTGGGTTTCCTCTATTTCTGGTTCCGGTTCCGATACTTTGTACGCTGATAGTG TCAAGGGAAGATTCACTATCAGTAGAGACAACGCTAAGAGCACTCTGTACTTGCAAATGAATTCATTAAGACCC GAGGACACTGCCGTCTATTATTGTACTATCGGTGGTTCTTTGTCCAGATCTTCCCAGGGAACTTTGGTTACAG? TAGTTCCGGTGGTGGAGGTAGTGGTGGAGGTTCCGAAGTTCAGTTGTTGGAAAGTGGTGGTGGACTTGTTCAAC CAGGTGGAAGTTTGAGATTGAGTTGCGCCGCTTCCGGTAGAACTTTGTCCTCTTACGCCATGGGTTGGTTTAGA CAGGCACCCGGTAAAGGTAGAGAATTCGTGgCCAGAATCTCCCAAGGTGGTACTGCTATCTACTACGCTGATTC AGTGAAAGGAAGATTCACAATTTCTAGAGATAACAGTAAGAACACACTTTACCTTCAAATGAACAGTCTTAGAC CCGAAGATACAGCCGTGTACTACTGTGCTAAGGACCCATCTCCATACTACAGAGGTTCCGCCTACTTGTTGTCT

GGTTC TACGACTCTTGGGGACAGGGAACTCTGGTCACCGTCTCCTCA

P23IL0087; SEQ ID NO:2

EVQLLESGGGLVQPGGSLRLSCAASGRIFSLPASGNIFNLLTIAWYRQAPGKGRELVATINSGSRTYYADSVKG RFTISRDNS KTVYLQMNSLRPEDTAVYYCQTSGSGSPNFWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQP GWSLRLSCAASGFTESSFGMSWVRQAPG GLEWVSSISGSGSDTLYADSV GRFTISRDNAKTTLYLQMNSLRP EDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTLSSYAMGWFR QAPGKG EFVARISQGGTAIYYADSV GRF ISRDNSK TLYLQMNSLRPEDTAVYYCAKDPSPYYRGSAYLLS

GSYDSWGQGTLVTVSS

P23ILO0B7; SEQ ID NO : 5 :

GAGGTGCaattgctggagtctgggGGAGGATTGGTACAACCAGGAGGTTCTTTGAGACTGTCTTGTGCAGCTTC TGGTCGTATATTCAGTTTGCCTGCTAGTGGGAATATTTTTAACCTGCTCACTATCGCTTGGTATCGTCAAGCTG CAGGTAAAGGTAGAGAGCTTGTTGCCACGATTAACTCTGGTTCCAGGACATATTACGCTGATAGCGTTAAGGGC AGATTCACAATCTCGCGAGATAATTCGAAAAAAACTGTTTACCTGCAAATGAACAGCCTTAGACCAGAAGATAC TGCCGTCTATTACTGTCAAACTTCAGGGTCTGGTAGTCCCAACTTTTGGGGTCAAgggaccCtggtcaeggtet cctcaggaggtggcggatccggcggaggtagtgaggtgcagctggtggagtctgggGGTGGATTGGTTCAACCT GGAAATAGCCTGAGACTATCTTGTGCAGCTTCAGGTTTCACCTTTTCCTCATTCGGTATGAGTTGGGTTAGACA AGCTCCAGGAAAGGGTTTGGAATGGGTCTCGTCAATTTCTGGTAGCGGATCTGATACGTTGTACGCTGACTCAG TAAAGGGAAGGTTCACTATTTCGCGTGACAATGCCAAAACCACGTTGTACTTGCAGATGAATTCACTCAGACCT GAAGATACCGCTGTGTACTATTGCACGATAGGTGGATCACTTAGTCGGTCTAGTCAAgggaccctggtcacggt ctcctcaggaggtggcgggtccggaggaggtagtgaggtgcagctgctggagtctgggGGAGGTTTGGTACAAC CAGGTGGATCTCTGAGATTGTCTTGTGCTGCTTCTGGAAGAACACTTTCCTCGTATGCTATGGGATGGTTTAGA CAGGCACCAGGTAAAGGCAGGGAATTTGTAGCACGAATCAGTCAAGGAGGaACCGCAATTTATTACGCAGACTC TGTAAAGGGTCGTTTTACAATATCCCGAGATAACAGTAAGAATACCCTCTACCTCCAAATGAACTCCCTAAGAC CAGAAGACACAGCCGTTTATTATTGCGCTAAAGATCCATCACCCTACTATCGAGGATCTGCGTATCTTTTGTCT

GGgTCCTACGATTCTTGGGGACAAgggaccctggtcacCGTCTCCTCA

P23IL0095; SEQ ID NO : 3

EVQLLESGGGLVQPGGSLRLSCAASGRIFSLPASGNIFNLLTIAWYRQAPGKGRELVATINSGSRTYYADSVKG RFTISRDNSKKTVYLQMNSLRPEDTAWYCQTSGSGSPNFWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTLSSYAMGWFRQAPGKGREFVARISQGGTAIYYAD

SVKGRFTISRDNSKNTLYLQMNSLRPBDTAVYYCAKDPSPYYRGSAYLLSGSYDSWGQGTLVTVSS

P23IL0O95 is SEQ ID NO: 6 GAGGTGCaattgctggagtCtgggGGAGGATTGGTACAACCAGGAGGTTCTTTGAGACTGTCTTGTGCAGCTTC TGGTCGTATATTCAGTTTGCCTGCTAGTGGGAATATTTTTAACCTGCTCACTATCGCTTGGTATCGTCAAGCTC CAGGTAAAGGTAGAGAGCTTGTTGCCACGATTAACTCTGGTTCCAGGACATATTACGCTGATAGCGTTAAGGGC AGATTCACAATCTCGCGAGATAATTCGAAAAAAACTGTTTACCTGCAAATGAACAGCCTTAGACCAGAAGATAC TGCCGTCTATTACTGTCAAACTTCAGGGTCTGGTAGTCCCAACTTTTGGGGTCAAgggaccctggtcacggtCt cctcaggaggtggcggatccggcggaggaggtagtGGTGGTGGAGGTTCTGGTGGTGGAGGATCAGGTGGAGGT GGAAGTGGAGGTGGTGGTTCAGGAGGTGGTGGTTCAgaggtgcagctgctggagtctgggGGAGGTTTGGTACA ACCAGGTGGATCTCTGAGATTGTCTTGTGCTGCTTCTGGAAGAACACTTTCCTCGTATGCTATGGGATGGTTTA GACAGGCACCAGGTAAAGGCAGGGAATTTGTAGCACGAATCAGTCAAGGAGGaACCGCAATTTATTACGCAGAC TCTGTAAAGGGTCGTTTTACAATATCCCGAGATAACAGTAAGAATACCC CTACCTCCAAATGAACTCCCTAAG ACCAGAAGACACAGCCGTTTATTATTGCGCTAAAGATCCATCACCCTACTATCGAGGATCTGCGTATCTTTTGT CTGGgTCCTACGATTCTTGGGGACAAgggaccctqgtcacCGTCTCGTCA

Table 1 : Lyophilization process program

Table 2: Overview of the QC data on the Nanobody constructs during the different steps of formulation. The iyophilized samples were reconstituted to their original volume with MQ water, and the creams were extracted with PBS prior to analysis

n.a.: Not Analyzed

Table 3 : Composition of base cream (Cetomacrogol cream)

Tabic 4: Preparation of the Creams

Tabic 5: Overview of the stability data obtained after storage of the creams up to 8 weeks at 4°C and up to 4 weeks at 25°C

"Over-estimated because the cream was stored in open conditions and evaporation of water led to concentration of the cream,

n.a.: Not Analyzed Tabic 3. Overview of the IC50 values (nM) of the P23IL0075 Nanobody construct in the IL-23/IL-23R inhibition potency ELISA before and after lyophilisation, and after formulation in cream. Lower limits and upper limits of the 95% confidence interval

(LLCI. and ULCI) are indicated.

Table 7. Overview of the ECSO values (nM) of the P231L0075 Nanobody construct in the HSA potency binding ELISA before and after lyophilisation and after formulation in cream. Lower limits and upper limits of the 95% confidence interval (LLCI and ULCI) are indicated.

Table 8. Overview of the ECSO values (nM) of the P23IL0087 Nanobody construct in the USA potency binding ELISA before and after formulation in cream. Lower limits and upper limits of the 95% confidence interval (LLCI and ULCI) are indicated.

T able 9. Overview of the IC50 values (nM) of th P23IL0075 Nanobody construct in the IL-23/IL-23R inhibition potency ELISA before and after a 2 week formulation in cream. Lower limits and upper limits of the 95% confidence interval (LLCl and ULCI) are indicated.

Table 10. Overview of the 1C50 values (nM) of P231L0075 the Nanobody construct in the IL-23/IL-23R inhibition potency ELISA before and after formulation in cream after storage for 8 weeks. Lower limits and upper limits of the 95% confidence interval (LLCl and ULCI) are indicated.

Table I I. Overview of the EC50 values (nM) of the P23IL0075 Nanobody construct in the HSA binding potency ELISA before and after formulation in cream after storage for 2 or 8 weeks at 4°C. Lower limits and upper limits of the 95% confidence interval (LLCl and ULCI) are indicated.

Table 4. Overview of the IC50 values (nM) of the P23IL0087 anobody construct in the IL-23/IL-23R inhibition potency ELISA before and after formulation in cream after storage for 4 weeks at 4 or 25°C. Lower limits and upper limits of the 95% confidence interval (LLCI and ULCI) are indicated.

Table 13. Overvie of the EC50 values (nM) of the P231L0087 Nanobody construct in the HSA binding potency ELISA before and after formulation in cream after storage for 2 or 4 weeks at 4 or 25°C. Lower limits and upper limits of the 95% confidence interval (LLC 1 and ULCI) are indicated.

We claim:

Claims

A semi-solid pharmaceutical composition, comprising one or more therapeutic molecules and one or more excipients, wherein the semi-solid pharmaceutical composition comprises at least 15 mg of the one or more therapeutic molecules per gram of said composition, and wherein the one or more therapeutic molecules comprise one or more immunoglobulin single variable domains, and wherein said semi-solid pharmaceutical composition comprises an aqueous and a lipid phase.

The semi -solid pharmaceutical composition of claim 1, wherein the semi-solid pharmaceutical composition comprises at least 20 mg of the one or more therapeutic molecules per gram of said composition.

The semi-solid pharmaceutical composition of claim 1 or 2, wherein the one or more therapeutic molecules have been lyophilized prior to formulation.

The semi-solid pharmaceutical composition of any one of claims 1 to 3, wherein the semi-solid pharmaceutical composition is administered to the subject's skin, cornea, rectal tissue, nasal mucosa, vagina, buccal tissue, urethral membrane, and external lining, more preferably to the subject's skin.

The semi-solid pharmaceutical, composition of any one of claims 1 to 4, wherein the semi-solid pharmaceutical composition additionally comprises a non-ionic surfactant.

The semi-solid pharmaceutical composition, of any one of claims 1 to 5, wherein the integrity of the one or more therapeutic molecules is greater than 95% after formulation at a concentration of at least about 20 mg of therapeutic molecule per gram of said composition, the integrity as measured as % of the main peak on SEC.

The semi-solid pharmaceutical composition of any one of claims 1 to 6, wherein the integrity of the one or more therapeutic molecules is greater than 99% after formulation at a concentration of at least about 20 mg of therapeutic molecule per gram, of said composition, the integrity as measured as % of the main peak on SEC.

The semi-solid pharmaceutical composition of any one of claims 1 to 7, wherein the potency of the one or more therapeutic molecules is greater than 95% after formulation at a concentration of at least about 20 mg of therapeutic molecule per gram of said composition, wherein the potency is measured as the capacity to inhibit the interaction of the target to its ligand or receptor.

9. The semi-solid pharmaceutical composition, of any one of claims 1 to 8, wherei the potency of the one or more therapeutic molecules is greater than 99% after formulation at a concentration of at least about 20 mg of therapeutic molecule per gram of said composition, wherein the potency is measured as the capacity to inhibit the interaction, of the target to its ligand or receptor.

10. The semi-solid pharmaceutical composition of any one of claims 1 to 9, wherein the composition is stable over a period of 8 weeks at 4°C.

1 1. The semi-solid pharmaceutical composition of claim 10, wherein aggregate formation is less tha 10%, preferably less than 5% of the therapeutic molecule.

12. The semi-solid pharmaceutical composition of claim 10, wherein the loss of potency of the therapeutic molecule is less than 5%.

13. The semi-solid pharmaceutical composition of any one of claims 1 to 12, wherein the composition is stable over a period of 2 weeks at 40°C.

14. The semi-solid pharmaceutical composition of claim 13. wherein aggregate formation is less than 10%, preferably less than 5% of the therapeutic molecule.

15. The semi-solid pharmaceutical composition of claim. 13, wherein the loss of potency of the therapeutic molecule is less than 20%.

16. The semi-solid pharmaceutical composition of any one of claims 1 to 15, wherein the composition comprises a carrier that is selected from the group consisting of neutral sterile cream, base cream, gel, jelly, ointment; and/or a lypoprotectant that is selected from the group consi ting of sugars and sugar alcohols.

17. The semi-solid pharmaceutical composition of claim 1 , wherein the carrier is a base cream and/or the lypoprotectant is sucrose.

18. The semi-solid pharmaceutical composition of any one of claims 1 to 17. wherein administration of the semi-solid pharmaceutical composition to a subject results, per administration, event, in the local delivery to the subject of at least 0.1 mg of the one or more therapeutic molecules or more per cm^" skin or other accessible mucous membrane.

19. The semi-solid pharmaceutical composition of any one of claims 1 to 17, wherein the semi-solid pharmaceutical composition is constructed and arranged to, when administered to a subject, locally deliver to the subject 0, 1 mg or more of the one or more therapeutic molecules per cnr skin or other accessible mucous membrane.

20. The semi-solid pharmaceutical composition of any one of claims 1 to 19, wherein the immunoglobulin single variable domain, is a VH, VL, VHH, camelized VH, camelized VL, or VHH that is optimized for stability, potency, manufacturability and similarity to human framework regions.

21. The semi-solid pharmaceutical composition of any one of claims 1 to 20, wherein the immunoglobulin single variable domain is a VHH that is optimized for stability, potency, manufacturability and similarity to human framework regions.

22. The semi-solid pharmaceutical composition of any one of claims 1 to 21, wherein the therapeutic molecule consists of or comprises a multivalent and/or multispecific construct.

23. Method for the preparation of a semi-solid pharmaceutical composition of any of claims 1 to 22 comprising the steps of incorporating a therapeutic molecule comprising or consisting of an immunoglobulin single variable domain into a semi-solid

pharmaceutical composition or cream.

24. Method of claim 23, wherein the therapeutic molecule is incorporated into the semisolid pharmaceutical composition in the form of a liquid.

25. Method of claim 23, wherein the therapeutic molecule is incorporated into the semisolid pharmaceutical composition, in lyophi!ized state.

26. Method of any of claims 23 to 25 comprising the steps of.

Preparation of a water mixture containing at least water, preferably a buffer; and possibly additional non-oil elements; - Preparation of an oil based phase (e.g. with vaselin and/or paraffin); and possibly including certain non-oily elements such as cetomacrogol, cetostearylalcohoi and/or other emusifying agents;

- Dispersing the oil based phase into the water based phase;

- Incorporating the therapeutic molecule into the cream obtained.

27. A method for administering to a subject the semi-solid pharmaceutical composition of any one of claims 1 -22, comprising administering to the subject's skin or other accessible mucous membrane the semi-solid phannaceuticai. composition of any one of claims 1-22.

28. The method of claim 23, wherein the one or more therapeutic molecules retain at least about 90% of the potency after formulation in the semi-solid phannaceuticai composition compared to prior to formulation.

29. The method of claim 23, wherein the one or more therapeutic molecules retain at least about 95% of the potency after formulation in the semi-solid pharmaceutical composition compared to prior to formulation.

30. The method of claim 238, wherein the one or more therapeutic molecules retain at least about 99% of the potency after formulation in the semi -solid phannaceuticai composition compared to prior to formulation.