IL-31-binding antibody variable domain

Abstract

The present invention relates to antibody variable domains that specifically bind to IL-31 and to multispecific antibodies, said multispecific antibodies comprising one or two of said antibody variable domains and at least one further binding domain that specifically binds to a target different to IL-31. The present invention further relates to a nucleic acid or two nucleic acids encoding said antibody variable domains or said multispecific antibodies, a vector comprising said nucleic acid or said nucleic acids, one or more host cells comprising said nucleic acid or nucleic acids or said vector or vectors, and to methods of producing said antibody variable domains or said multispecific antibodies. The present invention further relates to pharmaceutical compositions comprising said antibody variable domains or said multispecific antibodies and methods of use thereof.

Description

[Technical Field]

[0001] The present invention relates to antibody variable domains that specifically bind to IL-31 and to multispecific antibodies, wherein the multispecific antibodies comprise one or two of the antibody variable domains and at least one further binding domain that specifically binds to a target different from IL-31. The present invention further relates to one or two nucleic acids encoding the antibody variable domains or multispecific antibodies, a vector comprising the nucleic acid or nucleic acids, one or more host cells comprising the nucleic acid or nucleic acids or the vector(s), and methods for producing the antibody variable domains or multispecific antibodies. The present invention also relates to pharmaceutical compositions comprising the antibody variable domains or multispecific antibodies and methods for using them. [Background technology]

[0002] Interleukin-31 (IL-31) is a proinflammatory cytokine that helps induce cell-mediated immunity against pathogens. IL-31 is preferentially produced by TH2 cells. IL-31 signals through a heterodimeric receptor complex (IL-31R or IL31R) containing interleukin-31 receptor alpha (IL-31RA or IL31RA) and oncostatin M receptor beta (OSMRβ), which is expressed on immune and epithelial cells. Binding of IL-31 to this receptor complex results in activation of the JAK / STAT and P13K / AKT signaling pathways, and also activates different MAPK pathways (ERK, p38, and JNK).

[0003] IL-31 has been shown to be associated with various chronic inflammatory diseases. For example, overexpression of IL-31 in mice has been shown to cause dermatitis-like symptoms (see Dillon, et al., Nature Immunol. 5 (2004):752-760). Furthermore, in many chronic inflammatory diseases, such as atopic dermatitis (AD), IL-31 mediates the activation of nerve fibers in the patient's skin, resulting in an aggressive itch phenotype and exacerbating the symptoms of these diseases by causing scratching. See, for example, Oetjen et al., Cell 171 (2017) 217-228. Scratching can cause disruption of the skin barrier, allowing microbial pathogens to enter the skin and further promoting inflammation at the site.

[0004] Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by intense pruritus (i.e., severe itching) and scaly, dry, eczematous lesions. Severe disease can be extremely distressing and result in high socioeconomic costs due to significant psychological problems, significant sleep loss, and reduced quality of life. AD often begins in childhood, before the age of 5, and can persist into adulthood.

[0005] The pathophysiology of AD is influenced by a complex interplay between immunoglobulin E (IgE)-mediated sensitization, the immune system, and environmental factors. The primary skin defect may be an immune disorder that causes IgE-mediated sensitization, with epithelial barrier dysfunction being the result of both genetic mutations and local inflammation.

[0006] IL-31 has also been implicated in allergic asthma, allergic rhinitis, inflammatory bowel disease, malignant tumors, and osteoporosis (see Bagci et al., J Allergy Clin Immunol. 141 (2018):858-866).

[0007] Blockade of IL-31 / IL-31RA signaling with anti-IL-31RA antibodies, such as the anti-IL-31RA antibody nemolizumab, has been clinically proven effective in reducing itch in patients with AD (see Ruzicka et al., N Engl J Med. 376 (2017):2092-2093).

[0008] Additionally, the IL-31 neutralizing antibody BMS-981164 has been developed to provide an effective targeted therapy for the treatment of chronic pruritic skin conditions (see Lewis et al, J Eur Acad of Dermatol Venereol. 31 (2017) 142-150).

[0009] Although these anti-IL-31 therapies appear to be effective in treating the itch symptoms that occur in pruritic diseases, such as AD, they typically do not address the underlying causes of these diseases. Even in allergic, inflammatory, and autoimmune diseases associated with imbalances in IL-31 signaling, the efficacy of these anti-IL-31 therapies is often limited and / or the response rates are low to moderate, indicating that imbalances in IL-31 signaling are not the sole cause of these diseases. Therefore, to increase the response rate and / or efficacy of these therapies, other signaling pathways must also be addressed. Therefore, there is a critical need for additional IL-31-based treatment options for patients with such allergic, inflammatory, and autoimmune diseases.

[0010] More specifically, it would be desirable to have stable and potent anti-IL-31 antibody components at hand that can be easily incorporated into multispecific antibody formats that further interfere with other signaling pathways. The anti-IL-31 components should have high potency for inhibiting IL-31-mediated signaling. Furthermore, the components should have excellent biophysical properties, particularly high stability, to facilitate their efficient incorporation into multispecific antibodies suitable for pharmaceutical development.

[0011] In particular, the anti-IL-31 component, i.e., the antibody binding domain, must exhibit the following minimal characteristics: - It has a monovalent dissociation constant (K) of 5 nM or less as measured by surface plasmon resonance (SPR). D ) must bind to human IL-31, - It inhibits human IL-31-induced signaling with an IC of 30ng / ml or less as measured by the Path Hunter IL-31RA / SMRb dimerization assay. 50 must be hindered by - When prepared at a starting concentration of 10 mg / ml in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl, there should be no more than a 5% loss in monomer content after storage at 4°C or 40°C for at least 4 weeks.

[0012] Furthermore, the anti-IL-31 construct, when formulated in 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl, exhibits a melting temperature (T m ) is preferably 65°C or higher.

[0013] While methods for identifying anti-IL-31 antibody variable domains that meet one or two of the above criteria are known in the art, identifying such antibody variable domains that meet all of these criteria, let alone most or all of the criteria mentioned in section 7 below, can be challenging and unpredictable.

[0014] The object of the present invention is to provide novel antibody variable domains that are capable of specifically binding to IL-31 and efficiently reducing or eliminating IL-31-mediated activity, and at the same time are highly stable and can be easily incorporated into multispecific antibody formats.

[0015] The present inventors surprisingly discovered that an scFv based on the monoclonal rabbit antibody clone 50-09-D07 can potently block IL-31 signaling while exhibiting excellent stability. This antibody clone is one of a limited number of monoclonal rabbit antibody clones obtained from an extensive immunization campaign that have been confirmed to bind to IL-31 with high affinity. In particular, the scFv derived from clone 50-09-D07, which has a dissociation constant (KD) well below 1 nM, exhibits an IC of less than 30 nM. 50 These scFvs are able to neutralize IL-31-induced signaling at 10 mg / ml and are stored at 4°C and 40°C for 4 weeks without significant loss of protein and monomer content. This is particularly surprising considering the fact that none of the scFvs produced from other clones exhibited good pharmacological activity or high stability. Optimization of the pharmacological activity of these scFvs almost always resulted in a deterioration of their stability, and vice versa.

[0016] Therefore, scFvs based on clone 50-09-D07 are suitable building blocks that can be easily incorporated into multispecific antibody formats such as the Morrison format.

[0017] Thus, in a first aspect, the present invention relates to an antibody variable domain which specifically binds to IL-31, comprising: a) a VH chain having a sequence selected from SEQ ID NOs: 5, 6, and 7, and b) A VL chain having a sequence selected from SEQ ID NOs: 12 and 37.

[0018] In a second aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) one or two antibody variable domains as defined herein; b) at least one binding domain that specifically binds to a target different from IL-31.

[0019] In a third aspect, the present invention relates to one or two nucleic acids encoding an antibody variable domain or a multispecific antibody of the invention.

[0020] In a fourth aspect, the present invention relates to a vector or two vectors comprising said nucleic acid or said two nucleic acids of the invention.

[0021] In a fifth aspect, the present invention relates to a host cell or host cells comprising said vector or said two vectors of the invention.

[0022] In a sixth aspect, the present invention relates to a method for producing an antibody variable domain or a multispecific antibody of the invention, comprising the steps of: (i) providing a nucleic acid or two nucleic acids of the invention, or a vector or two vectors of the invention, expressing said nucleic acid or said two nucleic acids, or said vector or vectors, and collecting said antibody variable domain or said multispecific antibody from the expression system; or (ii) providing a host cell or multiple host cells of the invention, culturing said host cell or multiple host cells, and collecting said antibody variable domain or said multispecific antibody from the cell culture.

[0023] In a seventh aspect, the present invention relates to a pharmaceutical composition comprising an antibody variable domain or a multispecific antibody of the invention and a pharmaceutically acceptable carrier.

[0024] In an eighth aspect, the present invention relates to an antibody variable domain or a multispecific antibody according to the invention for use as a medicament.

[0025] In a ninth aspect, the present invention relates to an antibody variable domain or a multispecific antibody of the invention for use in the treatment of a disease, particularly a human disease, more particularly a human disease selected from allergic diseases, inflammatory diseases, and autoimmune diseases, particularly inflammatory diseases and autoimmune diseases.

[0026] In a tenth aspect, the present invention relates to a method for treating a disease, in particular a human disease, more particularly a human disease selected from allergic diseases, inflammatory diseases and autoimmune diseases, in particular inflammatory diseases and autoimmune diseases, comprising providing an antibody variable domain or a multispecific antibody of the invention to a patient in need thereof.

[0027] The aspects, advantageous features and preferred embodiments of the present invention summarized in the following items, each alone or in combination, further contribute to solving the object of the present invention. 1. An antibody variable domain that specifically binds to IL-31, comprising: a) variable heavy chain (VH), wherein the variable heavy chain comprises, from N-terminus to C-terminus, the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW represents a heavy chain framework region and each HCDR represents a heavy chain complementarity determining region, and wherein said HCDR1 having the sequence of SEQ ID NO: 1; said HCDR2 having a sequence selected from SEQ ID NO: 2 or 3, and said HCDR3 having the sequence of SEQ ID NO: 4; b) variable light chain (VL); wherein the variable light chain comprises, from N-terminus to C-terminus, the region LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, wherein each LFW represents a light chain framework region and each LCDR represents a light chain complementarity determining region, and wherein said LCDR1 having a sequence selected from SEQ ID NO: 9 or 36; said LCDR2 has the sequence of SEQ ID NO: 10, and The LCDR3 has the sequence of SEQ ID NO:11. 2. The antibody variable domain of item 1, wherein the variable heavy chain is a VH3 chain and / or the variable light chain is a Vκ1 light chain. 3. The antibody variable domain according to item 1, wherein - said variable heavy chain is a VH3 chain, - LFW1, LFW2, and LFW3 belong to the Vκ1 light chain subtype, and - LFW4 has a human Vλ sequence selected from SEQ ID NOs: 16 to 23. 4. The antibody variable domain according to any one of items 1 to 3, wherein the antibody variable domain is selected from Fab, Fv, scFv, DSFv, and scAB, preferably from Fab, Fv, scFv, and DSFv, in particular from Fab, scFv, and DSFv. 5. The antibody variable domain according to any one of items 1 to 4, wherein the antibody variable domain comprises: a) a VH chain having a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical to any one of the amino acid sequences selected from SEQ ID NOs: 5, 6, and 7; and b) A VL chain having a sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical to any one of the amino acid sequences selected from SEQ ID NOs: 12 and 37. 6. The antibody variable domain of any one of items 1 to 5, wherein the antibody variable domain blocks binding of IL31 to the interleukin-31 receptor alpha (IL-31RA) / oncostatin M receptor (OSMR) complex (IL-31RA / OSMR complex). 7. The antibody variable domain according to any one of items 1 to 5, wherein the antibody variable domain, when in scFv format, exhibits at least two of the following characteristics a. to d.: a. Human IL-31 has a monovalent dissociation constant (K) of 5 nM or less as measured by surface plasmon resonance (SPR). D ), especially 5 pM to 5 nM, especially 5 pM to 2 nM, especially 5 to 1000 pM monovalent K D and combine them with b. Cross-reactive with cynomolgus monkey (Macaca fascicularis) IL-31, specifically, exhibiting a monovalent K of 5 nM or less to cynomolgus monkey IL-31 as measured by SPR. D , especially 5 pM to 5 nM, especially 5 pM to 2 nM, especially 5 to 1000 pM monovalent K D and combine them with c. Human IL-31-induced signaling was inhibited with IC values ​​between 0.1 and 30 ng / ml as measured by the Path Hunter IL-31RA / SMRb dimerization assay. 50 , especially IC of 0.1 to 20 ng / ml 50 , especially IC of 0.1 to 10 ng / ml 50 and inhibits d. Binding of human IL-31 to human IL-31R was measured by competitive ELISA with an IC of 0.1-20 ng / ml. 50 , especially IC of 0.1 to 10 ng / ml 50 In particular, IC of 0.1 to 6 ng / ml 50 and shut it off. and wherein said antibody variable domain, when in scFv format, further exhibits at least two of the following characteristics e.-i.: e. a melting temperature (Tm) of at least 65°C, preferably at least 67°C, more preferably at least 69°C, as measured by differential scanning fluorimetry, particularly wherein said scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl; f. when the starting concentration of the scFv is 10 mg / ml, the loss of monomer content after 4 weeks of storage at 4°C is less than 5%, e.g., less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl; g. when the starting concentration of the scFv is 10 mg / ml, the loss of monomer content after 4 weeks of storage at 40°C is less than 5%, e.g. less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl; h. when the starting concentration of the scFv is 10 mg / ml, the loss of monomer content after three freeze-thaw cycles is less than 5%, e.g., less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl; i. when the starting concentration of the scFv is 10 mg / ml, there is less than 5% loss in protein content after 4 weeks of storage at 4°C or 40°C, such as less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly where the scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl. 8. The antibody variable domain of item 7, wherein the antibody variable domain, when in scFv format, exhibits at least characteristics a., c., f., and g. 9. The antibody variable domain according to item 7, wherein the antibody variable domain, when in scFv format, exhibits at least features a, c, d, e, f, and g, in particular at least features a to i. 10. The antibody variable domain according to any one of items 1 to 9, comprising: a) a VH chain having a sequence selected from SEQ ID NOs: 5, 6, and 7, and b) A VL chain having a sequence selected from SEQ ID NOs: 12 and 37. 11. The antibody variable domain according to any one of items 1 to 10, comprising: a) a VH chain having the sequence of SEQ ID NO: 5 and a VL chain having the sequence of SEQ ID NO: 12, or b) a VH chain having the sequence of SEQ ID NO: 6 and a VL chain having the sequence of SEQ ID NO: 12, or c) a VH chain having the sequence of SEQ ID NO: 7 and a VL chain having the sequence of SEQ ID NO: 12, or d) A VH chain having the sequence of SEQ ID NO: 5 and a VL chain having the sequence of SEQ ID NO: 37. 12. The antibody variable domain according to any one of items 1 to 11, which is an scFv antibody having a sequence selected from SEQ ID NOs: 27 to 29 and 31. 13. Multispecific antibodies, comprising: a) one or two antibody variable domains as defined in any one of items 1 to 12; b) at least one binding domain that specifically binds to a target different from IL-31. 14. The multispecific antibody according to item 13, which does not comprise an immunoglobulin Fc region. 15. The multispecific antibody according to item 14, wherein the multispecific antibody is in a format selected from the group consisting of tandem scDb (Tandab), linear dimeric scDb (LD-scDb), cyclic dimeric scDb (CD-scDb), tandem tri-scFv, tribody (Fab-(scFv)2), Fab-Fv2, triabody, scDb-scFv, tetrabody, di-diabody, tandem di-scFv, and MATCH. 16. The multispecific antibody according to item 14, which does not contain a CH1 and / or CL region. 17. The multispecific antibody according to item 16, wherein the multispecific antibody is in scDb-scFv, triabody, tetrabody, or MATCH format, in particular wherein the multispecific antibody is in MATCH or scDb-scFv format, more particularly wherein the multispecific antibody is in MATCH format, more particularly MATCH3 or MATCH4 format. 18. The multispecific antibody according to item 13, comprising an immunoglobulin Fc region. 19. The multispecific antibody according to item 18, wherein the immunoglobulin Fc region is selected from the IgG subclass, in particular the IgG subclasses IgG1 and IgG4, in particular IgG4. 20. The multispecific antibody according to item 19, wherein the format of the multispecific antibody is selected from a bivalent, bispecific IgG format, a trivalent, bispecific IgG format, and a tetravalent, bispecific IgG format; More specifically, the format of the multispecific antibody is selected from KiH-based IgG; DVD-Ig; CODV-IgG and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)), and even more specifically DVD-Ig and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)). 21. The multispecific antibody according to item 20, wherein the format of the multispecific antibody is selected from the Morrison-H format and the Morrison-L format. 22. The multispecific antibody according to any one of items 13 to 21, wherein the multispecific antibody comprises two antibody variable domains as defined in any one of items 1 to 12 and two binding domains that specifically bind to a second target different from IL-31. 23. One or two nucleic acids encoding any one of the antibody variable domains according to items 1 to 12 or the multispecific antibody of any one of items 13 to 22. 24. A vector or two vectors comprising the nucleic acid or the two nucleic acids according to item 23. 25. A host cell or a plurality of host cells comprising the vector or the two vectors according to item 24. 26. A method for producing an antibody variable domain according to any one of items 1 to 12 or a multispecific antibody according to items 13 to 22, comprising: (i) providing the nucleic acid or the two nucleic acids according to item 23, or the vector or the two vectors according to item 24, expressing the nucleic acid or the two nucleic acids, or the vector or the two vectors, and collecting the antibody variable domain or the multispecific antibody from the expression system; or (ii) providing a host cell or a plurality of host cells according to item 25, culturing the host cell or a plurality of host cells, and collecting the antibody variable domain or the multispecific antibody from the cell culture. 27. A pharmaceutical composition comprising the antibody variable domain according to any one of items 1 to 12 or the multispecific antibody according to any one of items 13 to 23, and a pharmaceutically acceptable salt carrier. 28. An antibody variable domain according to any one of items 1 to 12 or a multispecific antibody according to any one of items 13 to 22 for use as a medicament. 29. The antibody variable domain according to any one of items 1 to 12 or the multispecific antibody according to any one of items 13 to 22 for use in the treatment of a disease, in particular a human disease, more in particular a human disease selected from allergic diseases, inflammatory diseases and autoimmune diseases, in particular allergic diseases that cause pruritus, inflammatory diseases that cause pruritus and autoimmune diseases that cause pruritus. 30. An antibody variable domain or a multispecific antibody for use according to item 29, wherein the disease is selected from atopic dermatitis, acute allergic contact dermatitis, chronic spontaneous urticaria, bullous pemphigoid, alopecia areata, dermatomyositis, prurigo nodularis, psoriasis, and atopic asthma, in particular the disease is atopic dermatitis. 31. A method for treating a disease, particularly a human disease, more particularly a human disease selected from allergic diseases, inflammatory diseases, and autoimmune diseases, particularly inflammatory diseases and autoimmune diseases, comprising the step of administering to a patient in need thereof an antibody variable domain according to any one of items 1 to 12, or a multispecific antibody according to any one of items 13 to 22. 33. The method according to item 31, wherein the disease is selected from atopic dermatitis, acute allergic contact dermatitis, chronic spontaneous urticaria, bullous pemphigoid, alopecia areata, dermatomyositis, prurigo nodularis, psoriasis, and atopic asthma, in particular wherein the disease is atopic dermatitis. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 shows the potency of three scFVs to neutralize IL-31-induced signaling in an IL-31RA / OSMR dimerization assay. (A) PRO1641 (50-03-H07-sc03), (B) PRO1643 (50-09-D07-sc03), and (C) PRO1650 (50-35-B03-sc03) are potent inhibitors with IC50 values ​​comparable to BMS-981164 for neutralizing IL-31-induced signaling.

[0029] [Figure 2]Figure 2 shows the potency of scFvs to inhibit the interaction between IL-31 and IL-31RA in a competitive ELISA. Compared to dupilumab, the top-ranked scFvs (A) PRO1641 (50-03-H07-sc03), (B) PRO1643 (50-09-D07-sc03), and (C) PRO1650 (50-35-B03-sc03) are potent inhibitors of the IL-31 / IL-31RA interaction with IC50 values ​​comparable to BMS-981164.

[0030] [Figure 3] Figure 3 shows the potency of optimized scFvs based on PRO1643 (50-09-D07-sc03) in neutralizing IL-31-induced signaling in an IL-31RA / OSMR dimerization assay. (A) PRO1900 (50-09-D07-sc04) and PRO1901 (50-09-D07-sc05), and (C) PRO1903 (50-09-D07-sc07) are potent inhibitors with IC50 values ​​comparable to BMS-981164 in neutralizing IL-31-induced signaling. (B) PRO1902 (50-09-D07-sc06) lost inhibitory potency due to the optimization process. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description of the Invention Although anti-IL-31 therapies appear to be effective in treating the itchy symptoms that occur in pruritic diseases, these therapies typically do not address the underlying cause of the disease. Furthermore, even when these diseases are associated with an imbalance in IL-31 signaling, the effectiveness of these anti-IL-31 therapies is often limited in patients suffering from allergic, inflammatory, and autoimmune diseases, with low to moderate response rates. Therefore, there is a great need for additional IL-31-based treatment options for patients living with these allergic, inflammatory, and autoimmune diseases.

[0032] The present invention provides novel anti-IL-31 antibody variable domains comprising specific VL and VH chains. The variable domains are based on the monoclonal rabbit antibody clone 50-09-D07. This clone was selected from a limited number of rabbit monoclonal antibodies identified in an extensive immunization campaign and found to bind to IL-31 with high affinity. The scFv derived from the monoclonal rabbit antibody clone 50-09-D07 has a dissociation constant (K) much lower than 1 nM. D ) binds to IL-31 at an IC of less than 30 ng / ml 50 It can neutralize IL-31-induced signaling at 40°C and can be stored at 10 mg / ml for 4 weeks at 4°C and 40°C without significant loss of protein and monomer content.

[0033] To the best of the inventors' knowledge, there are no anti-IL-31 antibody variable domains in the prior art that have such advantageous properties.

[0034] The antibody variable domains of the present invention could also be successfully incorporated into a multispecific antibody format targeting IL-4R. These anti-IL-4R x IL-31 multispecific antibodies can bind to IL-31 with high affinity and potently inhibit IL-31-mediated signaling, while exhibiting highly advantageous biophysical properties, particularly excellent preparative and storage stability, at antibody concentrations well above 100 mg / ml. This also demonstrates that the antibody variable domains of the present invention, when incorporated into a multispecific antibody format, also provide advantageous biological and biophysical properties.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] The terms "comprising" and "including" are used herein in an open-ended and non-limiting sense, unless expressly stated otherwise. Thus, with respect to such latter embodiments, the term "comprising" encompasses the narrower term "consisting of."

[0037] In the context of the description of the invention (particularly in the context of the claims below), the terms "a," "an," "the," and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. For example, the term "cell" includes a plurality of cells, including mixtures thereof. When the plural is used for compounds, salts, etc., this is deemed to mean a single compound, salt, etc.

[0038] In one embodiment, the present invention provides a) a VH chain having a sequence selected from SEQ ID NOs: 5, 6, and 7, and b) An antibody variable domain that specifically binds to IL-31, comprising a VL chain having a sequence selected from SEQ ID NOs: 12 and 37.

[0039] As used herein, the term "antibody" and the like includes whole antibodies or single chains thereof; and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof; and molecules comprising antibody CDRs, VH regions, or VL regions (including but not limited to, and molecules comprising ABCDRs, VH regions, or VL regions (including but not limited to, multispecific antibodies). A naturally occurring "whole antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a The VH and VL regions are composed of a heavy chain and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) adjacent to more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0040] The term "antibody variable domain" as used herein refers to one or more portions of an intact antibody having the ability to specifically bind to a given antigen (e.g., IL-31). This can be an intact antibody or any single-chain antigen-binding fragment thereof (i.e., "antigen-binding portion"), as well as molecules comprising the CDRs, VH region, or VL region of an antibody. Specifically, in the case of the multispecific antibodies of the present invention, the term "antibody variable domain" as used herein refers to a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; an Fv fragment (Fv) consisting of the VL and VH domains of a single arm of an antibody; a disulfide-stabilized Fv fragment (DSFv); a single-chain Fv fragment (scFv); and an additional light chain constant domain (C) fused thereto. L ) refers to a single-chain Fv fragment (scAB) having a nucleotide sequence similar to that of the nucleotide sequence 1 ...

[0041] The term "complementarity-determining region" ("CDR") is used in accordance with the principles of Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)) ("IMGT" numbering scheme); and Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670. "A" refers to an amino acid sequence having boundaries determined using any of a number of well-known numbering schemes, including those described by the International Organization for Standardization (IOS) ("AHo" numbering). For example, in the classical format, Kabat numbers the CDR amino acid residues of the heavy chain variable domain (VH) as 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) as 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Chothia numbers the CDR amino acids of the VH as 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues of the VL as 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Combining the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of human VL.In IMGT, the CDR amino acid residues of the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues of the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering based on "Kabat"). In IMGT, the CDRs of an antibody can be determined using the program IMGT / DomainGapAlign.

[0042] In the context of the present invention, unless otherwise specified, the numbering system proposed by Honegger & Pluckthun ("AHo") is used (Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670). In particular, the following residues are defined as CDRs according to the AHo numbering scheme: LCDR1 (also referred to as CDR-L1): L24-L42; LCDR2 (also referred to as CDR-L2): L58-L72; LCDR3 (also referred to as CDR-L3): L107-L138; HCDR1 (also referred to as CDR-H1): H27-H42; HCDR2 (also referred to as CDR-H2): H57-H76; HCDR3 (also referred to as CDR-H3): H108-H138. For clarity, the Honegger & Plückthun numbering system takes into account the length diversity found in naturally occurring antibodies in both different VH and VL subfamilies, particularly in the CDRs, and provides for gaps in the sequences. Thus, in a particular antibody variable domain, not all positions 1 through 149 are typically occupied by an amino acid residue.

[0043] As used herein, the term "binding specificity" refers to the ability of an individual antibody to react with one antigenic determinant but not with a different antigenic determinant. As used herein, the terms "specifically bind to" or "specific for" refer to a measurable and reproducible interaction, such as the binding of an antibody to a target, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is one that binds to this target with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other targets. In its most general form (and when no defined reference is given), "specific binding" refers to the ability of an antibody to distinguish between the target of interest and unrelated molecules, as measured, for example, according to specificity assay methods known in the art. Such methods include, but are not limited to, Western blot, ELISA, RIA, ECL, IRMA, SPR (surface plasmon resonance) testing, and peptide scanning. For example, a standard ELISA assay can be performed. Scoring can be performed using standard colorimetric methods (e.g., secondary antibodies using horseradish peroxidase and tetramethylbenzidine using hydrogen peroxide). Reactions in specific wells are scored, for example, by optical density at 450 nm. A typical background (=negative reaction) is approximately 0.1 OD. A typical positive reaction is approximately 1 OD. This means that the ratio of positive to negative scores can be 10-fold or greater. As a further example, SPR assays can be performed in which a difference of at least 10-fold, and especially at least 100-fold, between background and signal indicates specific binding. Typically, binding specificity is determined using a set of approximately 3-5 unrelated molecules, such as milk powder or transferrin, rather than a single reference molecule.

[0044] In a further aspect, the present invention relates to a multispecific antibody comprising: a) one or two antibody variable domains as defined herein; b) at least one binding domain that specifically binds to a target different from IL-31, in particular wherein said at least one binding domain is hSA-BD and / or IL4R-BD.

[0045] In certain embodiments, the multispecific antibodies of the invention do not comprise an immunoglobulin Fc region.

[0046] In certain of these embodiments, the multispecific antibodies are preferably in a format selected from the group consisting of tandem scDb (Tandab), linear dimeric scDb (LD-scDb), cyclic dimeric scDb (CD-scDb), tandem tri-scFv, tribody (Fab-(scFv)2), Fab-Fv2, triabody, scDb-scFv, tetrabody, di-diabody, tandem di-scFv, and MATCH (described in WO 2016 / 0202457; Egan T., et al., MABS 9 (2017) 68-84). In particular, the multispecific antibodies of the present invention are in the MATCH format. More specifically, the multispecific antibodies of the present invention are in the MATCH3, MATCH4, or MATCH5 format.

[0047] As used herein, the term "immunoglobulin Fc region" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, i.e., the CH2 and CH3 domains of the heavy chain constant region. The term "Fc region" includes native sequence Fc regions and variant Fc regions, i.e., Fc regions that have been engineered to exhibit specific desired properties, such as altered Fc receptor binding function and / or reduced or inhibited Fab arm exchange. An example of such an engineered Fc region is knobs-into-holes (KiH) technology (see, e.g., Ridgway et al., Protein Eng. 9:617-21 (1996) and Spiess et al., J Biol Chem. 288(37):26583-93 (2013)). Native sequence Fc regions include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In particular, FcR is a native-sequence human FcR, which binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced ​​forms of these receptors), FcγRII receptors, including FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see M. Daeron, Annu. Rev. Immunol. 5:203-234 (1997)).FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capet et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. The terms "Fc receptor" or "FcR" also include the neonatal receptor FcRn, which is responsible for the transport of maternal IgG to the fetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994). Methods for measuring binding to FcRn are known (e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997); Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem. TJI (8): 6213-6 (2004); WO 2004 / 92219 (Hinton et al)). In vivo binding to FcRn and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice, or transfected human cell lines expressing human FcRn, or primates to which polypeptides with variant Fc regions are administered. WO 2004 / 42072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See, e.g., Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0048] In other specific embodiments, the multispecific antibodies of the invention comprise an immunoglobulin Fc region.

[0049] In a further particular embodiment, the multispecific antibody of the invention comprises an IgG region.

[0050] The term "IgG region" as used herein refers to the heavy and light chains of immunoglobulin G, i.e., the Fc region as defined above, and the Fab region, which consists of the VL, VH, CL, and CH1 domains. The term "IgG region" includes native sequence IgG regions, e.g., human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4, as well as engineered IgG regions that exhibit particular desired properties, such as those properties defined above for the Fc region.

[0051] In certain embodiments, the immunoglobulin Fc region comprised in the multispecific antibody of the invention is selected from the Fc regions of the IgG subclasses, in particular from the Fc regions of the IgG subclasses IgG1 and IgG4, and particularly from the Fc region of IgG4.

[0052] As used herein, the terms "binding domain," "antigen-binding fragment thereof," "antigen-binding portion," and the like of an antibody refer to one or more portions of an intact antibody that retain the ability to specifically bind to a particular antigen. The antigen-binding function of an antibody is performed by fragments of the intact antibody. Specifically, in the case of the multispecific antibodies of the present invention, the terms "binding domain," "antigen-binding fragment thereof," "antigen-binding portion," and the like as used herein refer to Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL, and CH1 domains; Fv fragments consisting of the VL and VH domains of a single antibody arm; disulfide-stabilized Fv fragments (DSFv); and single-chain Fv fragments (scFv). Preferably, the binding domains of the multispecific antibodies of the present invention are independently selected from Fab fragments, Fv fragments, scFv fragments, and single-chain Fv fragments (scFv). In certain embodiments, the binding domains of the antibodies of the present invention are independently selected from Fab fragments and single-chain Fv fragments (scFv). In other particular embodiments, the VL and VH domains of the scFv fragment are stabilized by an interdomain disulfide bond, and in particular, the VH domain comprises a single cysteine ​​residue at position 51 (AHo numbering) and the VL domain comprises a single cysteine ​​residue at position 141 (AHo numbering).

[0053] Suitably, antibody variable domains of the invention are isolated variable domains. Similarly, multispecific antibodies of the invention are isolated antibodies. As used herein, the term "isolated variable domain" or "isolated antibody" refers to a variable domain or antibody that is substantially free of other variable domains or other antibodies with different antigen specificities (e.g., an isolated antibody variable domain that specifically binds to IL-31 is substantially free of antibody variable domains that specifically bind to antigens other than IL-31). Furthermore, an isolated antibody variable domain or isolated antibody may be substantially free of other cellular material and / or chemicals.

[0054] Suitably, the antibody variable domains and multispecific antibodies of the invention are monoclonal antibody variable domains and antibodies. As used herein, the term "monoclonal antibody variable domain" or "monoclonal antibody" refers to variable domains or antibodies that have substantially identical amino acid sequences or are derived from the same genetic source. A monoclonal variable domain or antibody exhibits binding specificity and affinity for a particular epitope, or multiple binding specificities and multiple affinities for multiple specific epitopes.

[0055] The antibody variable domains and multispecific antibodies of the present invention include, but are not limited to, chimeric antibodies, human antibodies, and humanized antibody variable domains and antibodies.

[0056] As used herein, the term "chimeric antibody" or "chimeric antibody variable domain" refers to an antibody molecule or antibody variable domain in which (a) the constant region or a portion thereof has been altered, substituted, or exchanged so that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or (b) the variable region or a portion thereof has been altered, substituted, or exchanged with a variable region having a different or altered antigen specificity. For example, a murine antibody can be modified by replacing its constant region with that of a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity for antigen recognition while exhibiting reduced antigenicity in humans compared to the original murine antibody.

[0057] As used herein, the term "human antibody" or "human antibody variable domain" is intended to include antibodies or antibody variable domains having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody or antibody variable domain contains a constant region, the constant region also is derived from such a human sequence, e.g., a human germline sequence or a mutated version of a human germline sequence. Human antibodies and antibody variable domains of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by in vitro site-directed mutagenesis or in vivo somatic mutation). This definition of a human antibody or antibody variable domain specifically excludes humanized antibodies or antibody variable domains comprising non-human antigen-binding residues. Human antibodies and antibody variable domains can be produced using various techniques known in the art, such as phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1992); Marks et al., J. Mol. Biol, 222:581 (1991)). Human monoclonal antibodies and human monoclonal antibody variable domains can also be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol, 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodies and human antibody variable domains can be prepared by administering antigen to transgenic animals, such as immunized xenomouse, that have been engineered to produce such antibodies and antibody variable domains in response to antigen challenge but whose endogenous gene loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology).Also, for human antibodies produced via human B cell hybridoma technology, see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0058] As used herein, the term "humanized" antibody or "humanized" antibody variable domain refers to an antibody or antibody variable domain that retains the reactivity of a non-human antibody or antibody variable domain but is less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody or antibody variable domain with their human counterparts (i.e., the constant regions, as well as the framework portions of the variable regions). Further framework region modifications can be made within the human framework sequences, as well as within CDR sequences derived from the germline of another mammalian species. The humanized antibodies and antibody variable domains of the invention can include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo, or by conservative substitutions to facilitate stability or manufacturing). See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv. Immunol., 44:65-92, 1988; Verhoeyen et al., Science, 239:1534-1536, 1988; Padlan, Molec. Immun., 28:489-498, 1991; and Padlan, Molec. Immun., 31:169-217, 1994. Other examples of ergonomic technologies include, but are not limited to, the Xoma technology disclosed in U.S. Patent No. 5,766,886.

[0059] The term "recombinant humanized antibody" or "recombinant humanized antibody variable domain" as used herein includes all human antibodies and human antibody variable domains prepared, expressed, created, or isolated by recombinant means, e.g., antibodies and human antibody variable domains isolated from host cells that have been transformed to express the humanized antibody or human antibody variable domain, e.g., from a transfectoma, and antibodies and human antibody variable domains prepared, expressed, created, or isolated by other means, including splicing all or part of a human immunoglobulin gene sequence onto other DNA sequences.

[0060] Preferably, the antibody variable domains and multispecific antibodies of the invention are humanized, more preferably the antibody variable domains and multispecific antibodies of the invention are humanized and comprise CDRs of rabbit origin.

[0061] As used herein, the term "multispecific antibody" refers to an antibody that binds to two or more different epitopes on at least two or more different targets (e.g., IL-31 and IL-4R). Preferably, the multispecific antibodies of the present invention are bispecific. As used herein, the term "bispecific antibody" refers to an antibody that binds to at least two different epitopes on two different targets (e.g., IL-31 and IL-4R).

[0062] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. "Conformational" epitopes are distinguished from "linear" epitopes in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0063] As used herein, the term "conformational epitope" refers to amino acid residues of an antigen that are grouped together on the surface when the polypeptide chain folds to form the native protein.

[0064] The term "linear epitope" refers to an epitope in which all points of interaction between a protein and an interacting molecule (such as an antibody) occur linearly (continuously) along the primary amino acid sequence of the protein.

[0065] As used herein, the term "recognize" refers to an antibody or antigen-binding portion thereof that finds and interacts with (eg, binds to) that conformational epitope.

[0066] Suitably, the multispecific antibody of the invention comprises one or two antibody variable domains that specifically bind to IL-31, as defined herein, in particular the multispecific antibody of the invention comprises two antibody variable domains that specifically bind to IL-31, as defined herein.

[0067] The term "IL-31" or "IL31" specifically refers to human IL-31, which has UniProt ID number Q6EBC2. The antibody variable domains of the invention target human IL-31. In particular, the antibody variable domains of the invention target human and cynomolgus monkey (Macaca fascicularis) IL-31.

[0068] The antibody variable domains of the invention, when in scFv format, are characterized by the following parameters: a. Human IL-31 has a monovalent dissociation constant (K) of 5 nM or less as measured by surface plasmon resonance (SPR). D ), especially 5 pM to 5 nM, especially 5 pM to 2 nM, especially 5 to 1000 pM monovalent K D and combine them with b. Human IL-31-induced signaling was inhibited with IC50 values ​​between 0.1 and 30 ng / ml as measured by the Path Hunter IL-31RA / SMRb dimerization assay. 50 , especially IC of 0.1 to 20 ng / ml 50 , especially IC of 0.1 to 10 ng / ml 50 and inhibits c. When the starting concentration of the scFv is 10 mg / ml, the loss of monomer content after 4 weeks of storage at 4° C. is less than 5%, e.g., less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl; and d. When the starting concentration of the scFv is 10 mg / ml, there is less than 5% loss in monomer content after 4 weeks of storage at 40°C, such as less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl.

[0069] In specific embodiments, the antibody variable domains of the invention, when in scFv format, are characterized by the following parameters: a. Human IL-31 has a monovalent dissociation constant (K) of 5 nM or less as measured by surface plasmon resonance (SPR). D ), especially 5 pM to 5 nM, especially 5 pM to 2 nM, especially 5 to 1000 pM monovalent K D and combine them with b. Human IL-31-induced signaling was inhibited with IC50 values ​​between 0.1 and 30 ng / ml as measured by the Path Hunter IL-31RA / SMRb dimerization assay. 50 , especially IC of 0.3 to 20 ng / ml 50 , especially IC of 0.1 to 10 ng / ml 50 and inhibits c. a melting temperature (Tm) of at least 65°C, preferably at least 67°C, more preferably at least 69°C, as measured by differential scanning fluorimetry, particularly wherein said scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl; d. When the starting concentration of the scFv is 10 mg / ml, the loss of monomer content after 4 weeks of storage at 4° C. is less than 5%, e.g., less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl; and e. when the starting concentration of the scFv is 10 mg / ml, there is less than 5% loss in monomer content after 4 weeks of storage at 40°C, such as less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl.

[0070] In more specific embodiments, the antibody variable domains of the invention, when in scFv format, are characterized by the following parameters: a. Human IL-31 has a monovalent dissociation constant (K) of 5 nM or less as measured by surface plasmon resonance (SPR). D ), especially 5 pM to 5 nM, especially 5 pM to 2 nM, especially 5 to 1000 pM monovalent K D and combine them with b. Human IL-31-induced signaling was inhibited with IC50 values ​​between 0.1 and 30 ng / ml as measured by the Path Hunter IL-31RA / SMRb dimerization assay. 50 , especially IC of 0.1 to 20 ng / ml 50 , especially IC of 0.1 to 10 ng / ml 50 and inhibits c. Binding of human IL-31 to human IL-31R was measured by competitive ELISA with an IC of 0.1-20 ng / ml. 50 , especially IC of 0.1 to 10 ng / ml 50 , especially IC of 0.1 to 6 ng / ml 50 and shut it off. d. a melting temperature (Tm) of at least 65°C, preferably at least 67°C, more preferably at least 69°C, as measured by differential scanning fluorimetry, particularly wherein said scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl; e. when the starting concentration of the scFv is 10 mg / ml, the loss of monomer content after 4 weeks of storage at 4°C is less than 5%, e.g., less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl; and f. When the starting concentration of the scFv is 10 mg / ml, there is less than 5% loss in monomer content after 4 weeks of storage at 40°C, such as less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl.

[0071] In more specific embodiments, the antibody variable domains of the invention, when in scFv format, are characterized by the following parameters: a. Human IL-31 has a monovalent K of 5 nM or less as measured by surface plasmon resonance (SPR). D , especially 5 pM to 5 nM, especially 5 pM to 2 nM, especially 5 to 1000 pM monovalent K D and combine them with b. Cross-reactive with cynomolgus monkey (Macaca fascicularis) IL-31, specifically, exhibiting a monovalent K of 5 nM or less to cynomolgus monkey IL-31 as measured by SPR. D , especially 5 pM to 5 nM, especially 5 pM to 2 nM, especially 5 to 1000 pM monovalent K D and combine them with c. Human IL-31-induced signaling was inhibited with IC values ​​between 0.1 and 30 ng / ml as measured by the Path Hunter IL-31RA / SMRb dimerization assay. 50 , especially IC of 0.1 to 20 ng / ml 50 , especially IC of 0.1 to 10 ng / ml 50 and inhibits d. Binding of human IL-31 to human IL-31R was measured by competitive ELISA with an IC of 0.1-20 ng / ml. 50 , especially IC of 0.01 to 10 ng / ml 50 , especially IC of 0.1 to 6 ng / ml 50 and inhibits e. a melting temperature (Tm) of at least 65°C, preferably at least 67°C, more preferably at least 69°C, as measured by differential scanning fluorimetry, particularly wherein said scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl; f. when the starting concentration of the scFv is 10 mg / ml, the loss of monomer content after 4 weeks of storage at 4°C is less than 5%, e.g., less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl; g. when the starting concentration of the scFv is 10 mg / ml, the loss of monomer content after 4 weeks of storage at 40°C is less than 5%, e.g. less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl; h. when the starting concentration of the scFv is 10 mg / ml, the loss of monomer content after three freeze-thaw cycles is less than 5%, e.g., less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer at pH 6.4 containing 150 mM NaCl; i. when the starting concentration of the scFv is 10 mg / ml, there is less than 5% loss in protein content after 4 weeks of storage at 4°C or 40°C, such as less than 4%, less than 3%, less than 2%, preferably less than 1%, and particularly wherein the scFv is prepared in 50 mM phosphate citrate buffer, pH 6.4, containing 150 mM NaCl.

[0072] As used herein, the term "HEK-Blue cells" or "HEK-Blue" refers to commercially available human embryonic kidney cells that are transfected with and stably express an optimized secreted fetal alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by the NF-κB transcription factor. The level of SEAP protein released into the culture medium is typically used as a measure of NF-κB activation.

[0073] The term "affinity," as used herein, refers to the strength of interaction between an antibody or antibody variable domain and an antigen at a single antigenic site. Within each antigenic site, the variable region of an antibody or antibody variable domain "arm" interacts with the antigen at many sites through weak non-covalent forces. The more interactions, the stronger the affinity.

[0074] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antibody variable domain) and its binding partner (e.g., an antigen, or more specifically, an epitope on an antigen or an antigen). Unless otherwise specified, "binding affinity," "bind to," "binds to," or "binding to," as used herein, refer to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody variable domain and an antigen). The affinity of a molecule X for a partner Y is generally measured by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including the methods described herein. Low-affinity antibodies and antibody variable domains generally bind antigens slowly and tend to dissociate easily, whereas high-affinity antibodies generally bind antigens faster and tend to remain bound for longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific exemplary embodiments for measuring binding affinity, i.e., binding strength, are described below.

[0075] As used herein, "K assoc "," "K a " or "K on " is intended to refer to the association rate of a particular antibody-antigen interaction, while "K" as used herein is dis "," "K d " or "K off The term "K" is intended to refer to the off-rate of a particular antibody-antigen interaction. D The term "Kd Against K a The ratio of (i.e., K d / K a ) and expressed as a molar concentration (M). D " or "K D "value" or "KD" or "KD value" is, in one embodiment, measured using a surface plasmon resonance assay.

[0076] Affinity for recombinant human IL-31 and recombinant cynomolgus IL-31 was measured by surface plasmon resonance (SPR) measurements as described in paragraphs

[0189] (scFv) and

[0225] (multispecific molecules).

[0077] The antibody variable domains of the invention act as antagonists of IL-31. In other words, the antibody variable domains of the invention are inhibitors of IL-31-mediated signal transduction. As used herein, the terms "blocking agent" or "inhibitor" or "antagonist" refer to an antibody or antibody variable domain that inhibits or reduces the biological activity of the antigen to which it binds. The antibody variable domains of the invention bind to IL-31, thereby blocking the binding of IL-31 to IL-31R, thereby reducing IL-31R function.

[0078] DSF has been previously described (Egan, et al., MAbs, 9(1) (2017), 68-84; Niesen, et al., Nature Protocols, 2(9) (2007) 2212-2221). The midpoint of the thermal unfolding transition of the scFv construct is determined by differential scanning fluorimetry using the fluorescent dye SYPRO® Orange (see Wong & Raleigh, Protein Science 25 (2016) 1834-1840). Samples are prepared in phosphate-citrate buffer (pH 6.4) at a final protein concentration of 50 μg / ml, with a final concentration of 5× SYPRO® Orange in a total volume of 100 μl. 25 μl of the prepared sample is added in triplicate to a white-walled AB gene PCR plate. The assay is performed on a qPCR machine used as a thermal cycler, and fluorescence emission is detected using a custom dye calibration routine in the software. The PCR plate containing the test samples is heated from 25°C to 96°C in 1°C increments, with a 30-second pause after each temperature increase. The total assay time is approximately 2 hours. Tm is calculated using the GraphPad Prism software, using the mathematical second derivative method to calculate the inflection point of the curve. The reported Tm is the average of triplicate measurements.

[0079] The loss of monomer content is determined by calculating the area under the curve of the SE-HPLC chromatogram. SE-HPLC is a separation technique based on a solid stationary phase and a liquid mobile phase, as outlined in the United States Pharmacopeia (USP), Chapter 621. This method utilizes a hydrophobic stationary phase and an aqueous mobile phase to separate molecules based on their size and shape. The separation of molecules is determined by the void volume (V0) and total permeate volume (V) of a particular column. T) The SE-HPLC measurements were performed on a Chromaster HPLC system (Hitachi High-Technologies Corporation) equipped with automatic sample injection and a UV detector set to a detection wavelength of 280 nm. The instrument was controlled by the software EZChrom Elite (Agilent Technologies, version 3.3.2SP2), which also supported the analysis of the resulting chromatograms. Protein samples were clarified by centrifugation and kept at a temperature of 4–6°C in the autosampler before injection. For the analysis of scFv samples, a Shodex KW403-4F column (Showa Denko Inc., #F6989202) was used with a standardized buffered saline mobile phase (50 mM sodium phosphate pH 6.5, 300 mM sodium chloride) at a recommended flow rate of 0.35 ml / min. The amount of target sample per injection was 5 μg. Samples were detected by a UV detector at a wavelength of 280 nm, and data were recorded by the appropriate software suite. The resulting chromatograms were plotted as V0–V T range, thereby excluding matrix-related peaks with elution times greater than 10 minutes.

[0080] Suitably, the antibody variable domains of the invention are binding domains provided in this disclosure, including but not limited to the humanized antibody variable domains derived from rabbit antibody clone 50-09-D07, the sequences of which are listed in Table 1.

[0081] The term "multivalent antibody" refers to a single binding molecule having multiple valencies, where "valency" is described as the number of antigen-binding moieties that bind to an epitope on a target molecule. Thus, a single binding molecule can bind to multiple binding sites on a target molecule and / or multiple target molecules due to the presence of multiple copies of the corresponding antigen-binding moieties. Examples of multivalent antibodies include, but are not limited to, bivalent antibodies, trivalent antibodies, tetravalent antibodies, pentavalent antibodies, hexavalent antibodies, etc.

[0082] As used herein, the term "monovalent antibody" refers to an antibody that binds to a single target molecule, more specifically, a single epitope on a target molecule. Also, as used herein, the term "binding domain" or "monovalent binding domain" refers to a binding domain that binds to a single epitope on a target molecule.

[0083] In certain embodiments, a multispecific antibody of the invention comprises one antibody variable domain that specifically binds to IL-31, as defined herein, and one binding domain that binds to a target other than IL-31, i.e., the multispecific antibody of the invention is monovalent with respect to both IL-31 and the target other than IL-31.

[0084] In a further particular embodiment, the multispecific antibody of the invention comprises one antibody variable domain that specifically binds to IL-31, as defined herein, and two binding domains with the same binding specificity that bind to a target different from IL-31, i.e. the multispecific antibody of the invention is monovalent for its IL-31 specificity and bivalent for a target different from IL-31.

[0085] In a further particular embodiment, the multispecific antibody of the invention comprises two antibody variable domains that specifically bind to IL-31, as defined herein, and one binding domain that binds to a target other than IL-31, i.e. the multispecific antibody of the invention is bivalent for IL-31 specificity and monovalent for a target other than IL-31.

[0086] In a preferred embodiment, a multispecific antibody of the invention comprises two antibody variable domains that specifically bind to IL-31, as defined herein, and two binding domains that have the same binding specificity and that specifically bind to a target different from IL-31, i.e. the multispecific antibody of the invention is bivalent for IL-31 specificity and bivalent for a target different from IL-31.

[0087] When a multispecific antibody of the present invention comprises two binding domains that have the same binding specificity and specifically bind to targets different from IL-31, the two binding domains bind to the same epitope or different epitopes on the target molecule. Preferably, the two binding domains bind to the same epitope on the target molecule.

[0088] As used herein, the term "same epitope" refers to an individual protein determinant on a protein that can be specifically bound by multiple antibodies, where that individual protein determinant is identical, i.e., consists of the same chemically active surface groupings of molecules such as amino acids or sugar side chains that have the same three-dimensional structural characteristics as well as the same charge characteristics for each of the antibodies.

[0089] The term "distinct epitopes," as used herein with respect to a particular protein target, refers to individual protein determinants on the protein that can each specifically bind to a different antibody, where these individual protein determinants are not identical to the different antibodies, i.e., are composed of non-identical chemically active surface groupings of molecules such as amino acids or sugar side chains that have different three-dimensional structural characteristics as well as different charge characteristics. These different epitopes may or may not overlap.

[0090] In certain embodiments, the multispecific antibodies of the invention are bispecific and bivalent.

[0091] In a more particular embodiment, the multispecific antibodies of the invention are bispecific and trivalent.

[0092] Preferably, the multispecific antibodies of the invention are bispecific and tetravalent, ie bivalent for IL-31 and bivalent for a target different from IL-31.

[0093] In a particular embodiment, the present invention relates to a multispecific antibody comprising: a) two antibody variable domains as defined herein; b) two binding domains that have the same binding specificity and specifically bind to IL-31 and different targets, in particular, wherein said binding domains are hSA-BD or IL4R-BD; wherein the multispecific antibody comprises an IgG region.

[0094] Other variable domains for use in the present invention comprise amino acid sequences which have been mutated but which have in the CDR regions at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the CDR regions set forth in the sequences set forth in Table 1, provided that such other variable domains exhibit the functional characteristics of Paragraph 0068 and, optionally, additionally, Paragraphs 0069 to 0071. Other variable domains for use in the present invention comprise mutated amino acid sequences wherein not more than 1, 2, 3, 4, or 5 amino acids have been mutated in the CDR regions as compared to the CDR regions set forth in the sequences set forth in Table 1, provided that such other variable domains exhibit the functional characteristics of Paragraph 0068 and, optionally, additionally, Paragraphs 0069 to 0071.

[0095] Suitably, the VH domain of the binding domain of the present invention belongs to the VH3 or VH4 family. In one embodiment, the binding domain used in the present invention comprises a VH domain belonging to the VH3 family. In the context of the present invention, the term "belonging to the VHx family (or VLx family)" means that the framework sequences FR1 to FR3 show the highest homology to said VHx family (or VLx, respectively). Examples of VH and VL families are described in Knappik et al., J. Mol. Biol. 296 (2000) 57-86 or in WO 2019 / 057787. A specific example of a VH domain belonging to the VH3 family is shown in SEQ ID NO: 13, and a specific example of a VH domain belonging to the VH4 family is shown in SEQ ID NO: 14. In particular, the framework regions FR1 to FR3 from SEQ ID NO: 13 belong to the VH3 family (Table 2, regions marked in non-bold). Suitably, a VH belonging to the VH3 family as used herein is a VH comprising FR1 to FR3 that have at least 90%, more particularly at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to FR1 to FR3 of SEQ ID NO: 13. Alternative examples of VH3 and VH4 sequences, and examples of other VHx sequences, can be found in Knappik et al., J. Mol. Biol. 296 (2000) 57-86, or in WO 2019 / 057787.

[0096] Suitably, the VL domain of a binding domain used in the present invention comprises Vκ framework FR1, FR2, and FR3, particularly a Vκ1 or Vκ3 framework, particularly a Vκ1 framework FR1-FR3, and a framework FR4 selected from Vκ FR4. When the binding domain is in scFv format, the binding domain comprises Vκ framework FR1, FR2, and FR3, particularly a Vκ1 or Vκ3 framework, particularly a Vκ1 framework FR1-FR3, and a framework FR4 selected from Vκ FR4 and Vλ FR4, particularly Vλ FR4.

[0097] Suitable Vκ1 frameworks FR1-FR3 and an exemplary Vλ FR4 are set forth in SEQ ID NO: 15 (Table 2, FR regions are marked in non-bold). Alternative examples of Vκ1 sequences, and examples of Vκ2, Vκ3, or Vκ4 sequences, are found in Knappik et al., J. Mol. Biol. 296 (2000) 57-86. Suitable Vκ1 frameworks FR1-FR3 include amino acid sequences having at least 80, 90, or 95 percent identity to the amino acid sequences corresponding to FR1-FR3 and obtained from SEQ ID NO: 15 (Table 2, FR regions are marked in non-bold). Suitable Vλ FR4s are set forth in SEQ ID NOs: 16-22 and SEQ ID NO: 23 and include a single cysteine ​​residue, particularly if a second single cysteine ​​is present in the corresponding VH chain, particularly at position 51 (AHo numbering) of the VH, for interdomain disulfide bond formation. In one embodiment, the VL domain of the binding domain of the present invention, when in scFv format, comprises a Vλ FR4 comprising an amino acid sequence having at least 80, 90, or 95 percent identity to an amino acid sequence selected from any of SEQ ID NOs: 16 to 23, particularly SEQ ID NO: 16 or 23.

[0098] Antibody variable domains of the invention comprise a VH domain listed in Table 1. Suitably, an antibody variable domain of the invention comprises a VH amino acid sequence listed in Table 1, wherein no more than 5 amino acids, particularly no more than 4 amino acids, particularly no more than 3 amino acids, particularly no more than 2 amino acids, particularly no more than 1 amino acid in the framework sequences (i.e. sequences that are not CDR sequences) are mutated (wherein a mutation is an addition, substitution or deletion, as various non-limiting examples). Other binding domains for use in the present invention include mutated but VH regions that contain amino acids that have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to a VH region set forth in the corresponding sequence set forth in Table 1, and include VL domains that include at least positions 5 to 140 (AHo numbering), particularly at least positions 3 to 145, of one of the sequences set forth in Table 1, provided that such other variable domains exhibit the functional characteristics of paragraph 68 and, optionally, additionally, the functional characteristics of paragraphs 69 to 71.

[0099] In particular, antibody variable domains of the invention comprise a VL domain listed in one of Table 1. Suitably, antibody variable domains of the invention comprise a VL amino acid sequence listed in Table 1, wherein no more than 5 amino acids, particularly no more than 4 amino acids, particularly no more than 3 amino acids, particularly no more than 2 amino acids, particularly no more than 1 amino acid, are mutated (wherein a mutation is an addition, substitution, or mutation, as various non-limiting examples) in the framework sequences (i.e., sequences that are not CDR sequences). Other binding domains for use in the present invention include VL domains that are mutated but contain amino acids in the VL region that have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to a VL region set forth in a sequence set forth in Table 1, and that include at least positions 5 to 140 (AHo numbering), particularly at least positions 3 to 145, of one of the sequences set forth in Table 1, provided that such other variable domains exhibit the functional characteristics of paragraph 68 and, optionally, additionally, the functional characteristics of paragraphs 69 to 71.

[0100] Specific, but non-limiting, examples of antibody variable domains of the invention are scFv PRO1643, PRO1900, PRO1901, and PRO1903, the sequences of which are listed in Table 3.

[0101] Suitably, at least one binding domain of the multispecific antibody of the invention is selected from the group consisting of Fab, Fv, DSFv and scFv.

[0102] The antibody variable domains and binding domains comprised in the multispecific antibodies of the present invention can simultaneously bind to their respective antigens or receptors. The term "simultaneously" as used in this context refers to the simultaneous binding of at least one antibody variable domain that specifically binds to IL-31 with at least one binding domain that has specificity for a target different from IL-31.

[0103] Suitably, the antibody variable domains and binding domains comprised in the multispecific antibodies of the invention are operably linked.

[0104] As used herein, the term "operably linked" refers to two molecules (e.g., polypeptides, domains, binding domains) joined in a manner that maintains functional activity of each molecule. Two molecules can be "operably linked" whether they are directly or indirectly linked (e.g., via a linker, a moiety, or a linker to a moiety). The term "linker" refers to a peptide or other moiety optionally positioned between a binding domain or antibody variable domain used in the present invention. Many strategies can be used to covalently link molecules. These include, but are not limited to, polypeptide bonds between the N- and C-termini of proteins or protein domains, bonds via disulfide bonds, and bonds via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond generated by recombinant techniques or peptide synthesis. The selection of an appropriate linker for a particular case in which two polypeptide chains are connected depends on various parameters, including, but not limited to, the properties of the two polypeptide chains (e.g., whether they naturally oligomerize), the distance between the N- and C-termini to be connected, if known, and / or the stability of the linker against proteolysis and oxidation. Additionally, the linker may contain amino acid residues that provide flexibility.

[0105] In the context of the present invention, the term "polypeptide linker" refers to a linker consisting of a chain of amino acid residues linked by peptide bonds, connecting two domains, each attached to one end of the linker. The polypeptide linker must be of sufficient length to connect the two molecules in a manner that allows them to assume the correct conformation relative to one another and retain the desired activity. In certain embodiments, the polypeptide linker has a continuous chain of 2 to 30 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues). Furthermore, the amino acid residues selected for inclusion in the polypeptide linker must exhibit properties that do not significantly interfere with the activity of the polypeptide. Thus, the linker peptide as a whole must not exhibit charges incompatible with the activity of the polypeptide, interfere with internal folding, or form bonds or other interactions with the amino acid residues of one or more of the monomers that would seriously interfere with the binding of the receptor monomer domains. In certain embodiments, the polypeptide linker is an unstructured polypeptide. Useful linkers include glycine-serine, or GS linkers. A "Gly-Ser" or "GS" linker is a polymer of consecutive glycines and serine (e.g., (Gly-Ser) n , (GSGGS) n (GGGGS) n , and (GGGS) n(where n is an integer of at least 1), and refers to glycine-alanine polymers, alanine-serine polymers, and other flexible linkers understood by those of skill in the art (e.g., tethers for Shaker potassium channels, and a wide variety of other flexible linkers). Glycine-serine polymers are preferred because oligopeptides containing these amino acids are relatively unstructured and therefore may function as neutral tethers between components. Second, serine is hydrophilic, thereby allowing the solubilization of potentially globular glycine chains. Third, similar chains have been shown to be effective in linking subunits of recombinant proteins, such as single-chain antibodies.

[0106] In one group of embodiments, the multispecific antibodies of the invention comprise an immunoglobulin Fc region and are in a format selected from (scFv)2-Fc-(scFv)2 fusion (ADAPTIR), DVD-Ig, DART™, and TRIDENT™. The term "DART™" refers to an antibody format developed by MacroGenics that comprises an immunoglobulin Fc region polypeptide and one or two bispecific Fv binding domains fused to the N-terminus of one heavy chain of the Fc region or to the N-terminus of both Fc regions. The term "TRIDENT™" refers to an antibody format developed by MacroGenics that comprises an immunoglobulin Fc region polypeptide, one bispecific Fv binding domain, and one Fab fragment. Both the bispecific Fv binding domain and the Fab fragment are fused to the N-terminus of each of the two heavy chains of the Fc region polypeptide.

[0107] In another group of embodiments, the format of the multispecific antibodies of the invention is selected from a bivalent, trivalent, and tetravalent bispecific IgG format. In particular, the multispecific antibody format is a KiH-based IgG, such as DuoBodies (bispecific IgG prepared by Duobody technology) (MAbs. 2017 Feb / Mar;9(2):182-212. doi: 10.1080 / 19420862.2016.1268307); DVD-IgG; IgG-scFv fusions, such as CODV-IgG, Morrison (IgG CH3-scFv fusion (Morrison-H)), or IgG CL-scFv fusion (Morrison-L)), bsAb (scFv attached to the C-terminus of the light chain), Bs1Ab (scFv attached to the N-terminus of the light chain), Bs2Ab (scFv attached to the N-terminus of the heavy chain), Bs3Ab (scFv attached to the C-terminus of the heavy chain), Ts1Ab (scFv attached to the N-terminus of both the heavy and light chains), and Ts2Ab (dsscFv attached to the C-terminus of the heavy chain). More particularly, the format of the multispecific antibody is selected from KiH-based IgGs, such as DuoBodies; DVD-Ig; CODV-IgG, and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)), and even more particularly DVD-Ig and Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)).

[0108] In a specific embodiment of the present invention, the format of the multispecific antibody is selected from the Morrison format, i.e., Morrison-L format and Morrison-H format. The Morrison-L and Morrison-H formats used in the present invention are tetravalent bispecific molecule formats having an IgG Fc region, particularly an IgG4 Fc region. Two highly stable scFv binding domains (the light chain comprises Vκ FR1-FR3 in combination with Vλ FR4 (λcap), also referred to herein as λcap scFv) are fused to the C-terminus of the heavy chain (Morrison-H) or light chain (Morrison-L) via the linker L1.

[0109] The linker L1 is a peptide of 2 to 30 amino acids, more particularly 5 to 25 amino acids, and most particularly 10 to 20 amino acids. In certain embodiments, the linker L1 is a peptide of one or more units of four glycine amino acid residues and one serine amino acid residue (GGGGS). n where n=1, 2, 3, 4, or 5, in particular n=2.

[0110] In a particular embodiment of the invention, the multispecific antibody has the Morrison-L format as defined above. In another particular embodiment of the invention, the multispecific antibody has the Morrison-H format as defined above.

[0111] When the antibody variable domains comprised in the multispecific antibodies of the present invention are in the form of scFv fragments, these scFv fragments comprise a variable heavy chain domain (VH) and a variable light chain domain (VL) connected by a linker L2.

[0112] The linker L2 is a peptide of 10 to 40 amino acids, more particularly 15 to 30 amino acids, and most particularly 20 to 25 amino acids. In certain embodiments, the linker L2 is a peptide of four glycine amino acid residues and one serine amino acid residue (GGGGS). n (where n=1, 2, 3, 4, 5, 6, 7, or 8, in particular n=4).

[0113] Specific, but non-limiting, examples of multispecific antibodies of the invention in which the antibody variable domains comprised therein are Fab fragments are the Morrison-H antibodies PRO2198, PRO2199, the sequences of which are listed in Table 4.

[0114] The antibody variable domains and multispecific antibodies of the invention can be produced using any convenient antibody production method known in the art (for the production of bispecific constructs see e.g. Fischer, N. & Leger, O., Pathobiology 74 (2007) 3-14; for bispecific diabodies and tandem scFvs see Hornig, N. & Farber-Schwarz, A., Methods Mol. Biol. 907 (2012) 713-727 and WO 99 / 57150). Further specific examples of suitable methods for preparing bispecific constructs include, inter alia, the Genmab technology (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150) and the Merus technology (see de Kruif et al., Biotechnol. Bioeng. 106 (2010) 741-750). Methods for producing bispecific antibodies comprising a functional antibody Fc portion are also known in the art (see, for example, Zhu et al., Cancer Lett. 86 (1994) 127-134, and Suresh et al., Methods Enzymol. 121 (1986) 210-228).

[0115] These methods typically involve the generation of monoclonal antibodies or monoclonal antibody variable domains, for example by using hybridoma technology to fuse myeloma cells with spleen cells from mice immunized with the desired antigen (see, e.g., Yokoyama et al., Curr. Protoc. Immunol. Chapter 2, Unit 2.5, 2006), or by recombinant antibody engineering (repertoire cloning or phage display / yeast display) (see, e.g., Chames & Baty, FEMS Microbiol. Letters 189 (2000) 1-8), and combining antigen-binding domains or fragments or portions thereof of two or more different monoclonal antibodies to obtain bispecific or multispecific constructs using known molecular cloning techniques.

[0116] Multispecific antibodies of the invention can be prepared by conjugating the component binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-5-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., 1984 J. Exp. Med. 160: 1686; Liu, MA et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science 229:81-83, and Glennie et al., 1987 J. Immunol. 139: 2367-2375. Coupling agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill., USA).

[0117] Alternatively, two or more binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x Fab, mAb x scFv, mAb x DSFv, or mAb x Fv fusion protein. Methods for preparing multispecific antibodies and molecules are described, for example, in U.S. Patent Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

[0118] The binding of antibody variable domains and multispecific antibodies to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a protein-antibody complex of interest specifically by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0119] In a further aspect, the invention provides one or two nucleic acids encoding an antibody variable domain or a multispecific antibody of the invention, which nucleic acids may be optimized for expression in mammalian cells.

[0120] As used herein, the term "nucleic acid" is used interchangeably with the term "polynucleotide" and refers to one or more deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. This term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, natural, and non-natural, have similar binding properties compared to the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, a particular nucleic acid also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and nucleic acids with complementary sequences, as well as the sequence explicitly indicated. Specifically, as detailed below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0121] The present invention provides substantially purified nucleic acid molecules encoding polypeptides comprising the above-described antibody variable domains or multispecific antibody segments or domains. When expressed from an appropriate expression vector, the polypeptides encoded by these nucleic acid molecules are capable of exhibiting the antigen-binding ability of the multispecific antibody of the invention.

[0122] Polynucleotide sequences can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences encoding the multispecific antibodies of the invention or their variable or binding domains (e.g., the sequences described in the Examples below). Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method of U.S. Pat. No. 4,458,066. Methods for introducing mutations into polynucleotide sequences by PCR can be carried out, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0123] The present invention also provides expression vectors and host cells for producing the antibody variable domains or multispecific antibodies of the present invention.

[0124] The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments have been ligated. Another type of vector is a viral vector, in which additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0125] Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Because the plasmid is the most commonly used form of vector, "plasmid" and "vector" may be used interchangeably herein. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions. In this particular context, the term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Usually, it refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically adjacent or located in close proximity to the coding sequences whose transcription they enhance.

[0126] A variety of expression vectors can be used to express polynucleotides encoding antibody variable domains or multispecific antibody chains. Both viral-based and non-viral expression vectors can be used to produce antibodies or antibody variable domains in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors that typically carry expression cassettes for protein or RNA expression, and human artificial chromosomes (see, e.g., Harrington et al., Nat. Genet. 15:345, 1997). For example, non-viral vectors useful for expressing IL-31-binding polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHisA, B, and C, pcDNA3.1 / His, pEBVHisA, B, and C (Invitrogen, San Diego, CA, USA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, SV40-based vectors, papilloma viruses, HBP Epstein-Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV). See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68: 143, 1992.

[0127] The choice of expression vector depends on the host cell in which the vector is to be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the multispecific antibody chains or variable domains. In one embodiment, an inducible promoter is used to prevent expression of the inserted sequence except under inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population toward coding sequences whose expression products are better tolerated by the host cell. In addition to a promoter, other regulatory elements may be necessary or desirable for efficient expression of the multispecific antibody chains or variable domains. These elements usually include an ATG initiation codon and adjacent ribosome binding sites or other sequences. Furthermore, expression efficiency can be enhanced by including enhancers appropriate for the cell system used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0128] The vectors used typically encode antibody variable domains, including constant regions or portions thereof, or the light and heavy chains of a multispecific antibody, if present. Such vectors allow the variable regions to be expressed as fusion proteins with constant regions, thereby resulting in the production of an intact antibody and its antibody variable domains. Usually, such constant regions are human.

[0129] The term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0130] Host cells for harboring and expressing the antibody variable domains or multispecific antibodies of the invention can be either prokaryotic or eukaryotic. Escherichia coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the invention. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors, typically containing expression control sequences compatible with the host cell (e.g., an origin of replication), can also be made in these prokaryotic hosts. Additionally, any number of well-known promoters will be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or promoter systems derived from lambda phage. Promoters typically contain ribosome binding site sequences for initiating and completing transcription and translation, optionally with operator sequences, to control expression. Other microorganisms, such as yeast, can also be used to express the antibody variable domains or multispecific antibodies of the invention. Insect cells can also be used in conjunction with baculovirus vectors.

[0131] In one embodiment, mammalian host cells are used to express and produce the antibody variable domains or multispecific antibodies of the invention. For example, these can be either hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines harboring exogenous expression vectors. These include any normal mortal cell, or normal or abnormal immortal animal or human cells. For example, many suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell culture to express polypeptides is reviewed, for example, in Winnacker, FROM GENES TO CLONES, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or regulatable or controllable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRPpolIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (e.g., the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0132] Methods for introducing expression vectors containing polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cellular hosts (see generally Green, MR, and Sambrook, J., Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012)). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistics, virosomes, immunoliposomes, polycation-nucleic acid complexes, naked DNA, artificial virions, fusion to herpesvirus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced DNA uptake, and ex vivo transduction. Stable expression is often desired for long-term, high-yield production of recombinant proteins. For example, cell lines stably expressing the antibody variable domains or multispecific antibodies of the invention can be prepared using the expression vectors of the invention containing a viral origin of replication or endogenous expression elements and a selectable marker gene. After introduction of the vector, cells are grown in an enriched medium for 1-2 days and then switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection; its presence allows the growth of cells that successfully express the introduced sequences in the selective medium. Resistant, stably transfected cells can be grown using tissue culture techniques appropriate to the cell type. Thus, the invention provides methods for producing the variable domains or multispecific antibodies of the invention, comprising culturing host cells containing a nucleic acid or vector encoding the antibody variable domains or multispecific antibodies of the invention, thereby expressing the antibody variable domains or multispecific antibodies or fragments thereof of the present disclosure.

[0133] In one aspect, the invention relates to a method for producing antibody variable domains or multispecific antibodies of the invention, the method comprising the step of culturing host cells expressing nucleic acids encoding the antibody variable domains or multispecific antibodies of the invention. In particular, the invention relates to a method for producing antibody variable domains or multispecific antibodies of the invention, the method comprising the steps of (i) providing one or two nucleic acids encoding the antibody variable domains or multispecific antibodies of the invention, or one or two vectors encoding the antibody variable domains or multispecific antibodies of the invention, expressing the nucleic acid(s) or the vector(s), and collecting the antibody variable domains or multispecific antibodies from the expression system, or (ii) providing one or more host cells expressing one or two nucleic acids encoding the antibody variable domains or multispecific antibodies of the invention, culturing the host cell(s), and collecting the antibody variable domains or multispecific antibodies from the cell culture.

[0134] In a further aspect, the present invention relates to a pharmaceutical composition comprising a multispecific antibody of the present invention and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a medium or diluent that does not interfere with the structure of the antibody. A pharmaceutically acceptable carrier enhances or stabilizes the composition or facilitates preparation of the composition. Pharmaceutically acceptable carriers include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc.

[0135] Some such carriers allow the pharmaceutical composition to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral ingestion by a subject. Some such carriers allow the pharmaceutical composition to be formulated for injection, infusion, or topical administration. For example, a pharmaceutically acceptable carrier may be a sterile aqueous solution.

[0136] The pharmaceutical compositions of the present invention can be administered by various methods known in the art. The route and / or method of administration varies depending on the desired results. Administration may be intravenous, intramuscular, intraperitoneal, or subcutaneous, or may be administered in the vicinity of the target site. In a specific embodiment, administration is intramuscular or subcutaneous, particularly subcutaneous. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion), particularly intramuscular or subcutaneous administration. Depending on the route of administration, the active compound, i.e., the multispecific antibody of the present invention, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0137] The pharmaceutical compositions of the present invention can be prepared according to methods well known and routinely practiced in the art. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or effective dose of a multispecific antibody of the present invention is used in the pharmaceutical compositions of the present invention. The multispecific antibody of the present invention is formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated, each containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier.

[0138] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without causing toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, such as the activity of the particular composition of the present invention or its ester, salt, or amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and past medical history of the patient being treated, and similar factors.

[0139] Multispecific antibodies of the present invention are typically administered multiple times. The intervals between single doses can be weekly, monthly, or yearly. Irregular intervals can also be used, as indicated by measuring the blood levels of the multispecific antibodies of the present invention in the patient. Alternatively, the multispecific antibodies of the present invention can be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency of administration depend on the half-life of the antibody in the patient. Humanized antibodies generally exhibit a longer half-life than chimeric or non-human antibodies. The dosage and frequency of administration depend on whether the treatment is prophylactic or therapeutic. For prophylactic purposes, relatively low doses are administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for life. For therapeutic purposes, relatively high doses may be required at relatively short intervals until the progression of the disease is alleviated or halted, preferably until the patient shows partial or complete improvement in the symptoms of the disease. The patient can then undergo prophylactic therapy.

[0140] In one aspect, the invention relates to a multispecific antibody of the invention or a pharmaceutical composition of the invention for use as a medicament. In a suitable embodiment, the invention provides a multispecific antibody or a pharmaceutical composition for use in the treatment of an allergic disease, an inflammatory disease, and an autoimmune disease, in particular a disease selected from an inflammatory disease and an autoimmune disease.

[0141] In another aspect, the present invention provides a pharmaceutical composition for use in the manufacture of a medicament for the treatment of an allergic, inflammatory or autoimmune disease, particularly an inflammatory or autoimmune disease.

[0142] In another aspect, the invention relates to the use of a multispecific antibody or a pharmaceutical composition for treating an allergic, inflammatory, or autoimmune disease, in particular for treating an inflammatory or autoimmune disease in a subject in need thereof.

[0143] In another aspect, the present invention relates to a method for treating a subject, comprising administering to the subject a therapeutically effective amount of a multispecific antibody of the invention. In a suitable embodiment, the present invention relates to a method for treating an allergic disease, an inflammatory disease, or an autoimmune disease in a subject, in particular a method for treating an inflammatory disease or an autoimmune disease, comprising administering to the subject a therapeutically effective amount of a multispecific antibody of the invention.

[0144] The term "subject" includes human and non-human animals.

[0145] The term "animal" includes all vertebrates, e.g., non-human mammals, and non-human mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably herein.

[0146] As used herein, the terms "treatment," "treating," "treat," "treated," and the like refer to obtaining a desired pharmacological and / or physiological effect. This effect may be therapeutic in that it partially or completely cures a disease and / or side effects caused by the disease, or slows the progression of the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, e.g., a human, and includes: (a) inhibiting the disease, i.e., preventing its onset, and (b) relieving the disease, i.e., causing the disease to regress.

[0147] The terms "therapeutically effective amount" or "effective amount" refer to the amount of a drug that, when administered to a mammal or other subject for treating a disease, is sufficient to affect treatment for such disease. A "therapeutically effective amount" will vary depending on the drug, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0148] In one embodiment, the allergic disease, inflammatory disease, and autoimmune disease are selected from allergic diseases that cause itching, inflammatory diseases that cause itching, and autoimmune diseases that cause itching, particularly inflammatory diseases that cause itching and autoimmune diseases that cause itching.The terms "pruritus" and "itch" are used interchangeably herein and refer to the sensation that causes the desire or reflex to scratch.In the case of chronic skin diseases that cause itching, such as atopic dermatitis, dermatomycosis, psoriasis, and urticaria, scratching can be particularly problematic because persistent itching irritation can lead patients to constantly and / or excessively scratch the affected skin area, leading to skin damage and further deterioration of the skin surface.

[0149] As used herein, the term "allergic disease" or "allergy" refers to a number of conditions caused by hypersensitivity of the immune system to normally harmless substances in the environment.

[0150] As used herein, the term "inflammatory disease" often refers to a vast number of inflammatory disorders, i.e., inflammatory abnormalities characterized by prolonged inflammation, known as chronic inflammation. The term "inflammation" refers to the complex biological response of bodily tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. This is a defensive response involving immune cells, blood vessels, and molecular mediators. While a periodic inflammatory response is essential for the body to eliminate the initial cause of cellular injury, remove necrotic cells and tissue damaged by the initial injury or inflammatory process, and initiate tissue repair, inflammatory diseases are generally characterized by continued inflammation even in the absence of harmful stimuli.

[0151] As used herein, the term "autoimmune disease" refers to a condition resulting from an abnormal immune response against a functioning body part, which is the result of the presence of an autoimmune, i.e., autoreactive, immune response (e.g., autoantibodies, autoreactive T cells), with or without resulting damage or pathology, usually confined to a specific organ or involving specific tissues in various locations.

[0152] In certain embodiments, the inflammatory or autoimmune disease causing pruritus is selected from atopic dermatitis, acute allergic contact dermatitis, chronic spontaneous urticaria, bullous pemphigoid, alopecia areata, dermatomyositis, prurigo nodularis, psoriasis, and atopic asthma, particularly atopic dermatitis.

[0153] In another embodiment, the allergic, inflammatory, and autoimmune disease is selected from atopic dermatitis, acute allergic contact dermatitis, chronic spontaneous urticaria, bullous pemphigoid, alopecia areata, dermatomyositis, prurigo nodularis, psoriasis, and atopic asthma, particularly atopic dermatitis.

[0154] In another embodiment, the allergic disease, inflammatory disease, and autoimmune disease is selected from allergic asthma, allergic rhinitis, inflammatory airway disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, Crohn's disease, chronic nonhistamine-related urticaria, antihistamine-unresponsive mastocytosis, lichen simplex chronicus, seborrheic dermatitis, xerosis, dermatitis herpetiformis, lichen planus, and ulcerative colitis.

[0155] In another embodiment, the invention provides a multispecific antibody or a pharmaceutical composition as defined herein for use in the treatment of a disease which is a neuropathic pruritus selected from postherpetic neuralgia, postherpetic pruritus, dysesthesias, multiple sclerosis, and brachioradial pruritus.

[0156] In another embodiment, the present invention provides a multispecific antibody or pharmaceutical composition for use as an antipruritic agent for the treatment of systemic diseases accompanied by itch, such as cholestasis, chronic kidney disease, Hodgkin's disease, cutaneous T-cell lymphoma and other lymphomas or leukemias accompanied by chronic itch, polycythemia vera, hyperthyroidism, chronic post-arthropod itch (Id reaction), pregnancy-induced chronic itch (e.g., PUPPP), eosinophilic pustular folliculitis, drug hypersensitivity reactions, chronic pruritus of the elderly or dry skin itch (local, systemic), and post-burn scars. Sequence Listing (mutations designated according to the AHo numbering scheme; CDRs defined according to the Numab CDR definitions unless otherwise specified), selected from scar itch (post-burn itch), hereditary or nevus-induced chronic itch (e.g., Netherton syndrome, Darier's disease (morbus-Darier), Hailey-Hailey disease, inflammatory linear verrucous epidermal nevus (ILVEN), familial primary cutaneous amyloidosis, Olmsted syndrome), aquagenic pruritus, fiberglass dermatitis, mucous-induced chronic itch, chemotherapy-induced itch, and HIV. [Table 1] [Table 2] [Table 3] [Table 4]

[0157] Throughout the body of this application, in the event of a conflict between the body of the specification (e.g., Tables 1-4) and the sequence listing, the body of the specification shall control.

[0158] It is understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments according to the present invention are specifically embraced by the present invention and are disclosed herein as if every combination were individually and explicitly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each such subcombination were individually and explicitly disclosed herein.

[0159] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

[0160] To the extent possible under their respective patent laws, all patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference.

[0161] The following examples illustrate the invention described above, but are not intended to limit the scope of the invention in any way. Other test models known per se to those skilled in the art can also be used to determine the beneficial effects of the claimed invention. [Example]

[0162] Example 1: Generation and testing of anti-IL-31 molecules : Project Objective The goal of this project is to identify humanized monoclonal antibody variable domains that specifically bind to human IL-31 and neutralize its biological effects.

[0163] 1.1.Immunity To obtain optimal immune responses to human IL-31, two different immunization protocols were applied to a total of six rabbits. Rabbits were immunized with recombinantly produced and purified IL-31 (Sino Biological, catalog number 11557-H08H). Prior to immunization, the manufacturer analyzed the quality of the recombinant human IL-31 for 1) purity by SDS-page analysis and 2) bioactivity by measuring its ability to induce STAT3 activation in U87 cells. The first group (protocol 1) of three rabbits received four injections of 200 μg each over a 70-day period. The second group (protocol 2) of three rabbits received five injections of 200 μg each over a 112-day period. During the immunization process, the strength of the humoral immune response to the antigen was qualitatively assessed by determining the maximum dilution (titer) of each rabbit's serum that still resulted in detectable binding of polyclonal serum antibodies against the antigen. Serum antibody titers against the immobilized antigen (recombinant human IL-31) were assessed using an enzyme-linked immunosorbent assay (ELISA). All six rabbits immunized with purified human IL-31 had up to 3 × 10 7 EC 50 It showed a high titer.

[0164] 1.2.Sorting Prior to the hit identification procedure, flow cytometry-based sorting campaigns using protein G beads were performed in the presence of R-phycoerythrin (RPE)-labeled IL-31, which allows for the specific detection and isolation of high-affinity IL-31-binding B cells. These two sorting campaigns yielded a total of 4.58 × 10 cells from four rabbits. 7 and 4.53 x 10 7 Of these, a total of 3,520 and 3,696 B cells expressing IL-31-specific antibodies (IgG) were isolated and cultured individually as single clones for 3–4 weeks.

[0165] 1.3. Hit Identification Overview To identify hits, direct ELISA screening was performed to assess binding to recombinant human IL-31. 618 clones were found to bind to human IL-31. Binding to cynomolgus monkey IL-31 and mouse IL-31 was assessed by SPR alone. All supernatants were further analyzed for their ability to neutralize the biological activity of IL-31-induced signaling in a cell-based IL-31RA / OSMR dimerization assay and for blocking the interaction between human IL-31RA and human IL-31 in a competitive ELISA. Due to the lack of cross-reactivity with mouse IL-31, further analysis with mouse IL-31 was not performed.

[0166] Binding to human IL-31 by ELISA To identify hits, i.e., B cell clones producing antibodies that bind to IL-31, cell culture supernatants of 3,520 and 3,696 B cell clones from the two selections above were screened for the presence of antibodies that bind to human IL-31 by ELISA as described in section 1.3. Supernatants from 618 B cell clones produced signals above background.

[0167] Measurement of binding affinity to human IL-31 In the secondary hit identification procedure, information on the binding affinity of 618 monoclonal rabbit antibodies identified as positive during the primary screening to human IL-31 was determined by surface plasmon resonance (SPR).

[0168] For these affinity screenings by SPR, antibodies specific for the Fc region of rabbit IgG were immobilized on a sensor chip (SPR-2 affinity sensor, high-capacity amine, Sierra Sensors) using standard amine coupling procedures. Rabbit monoclonal antibodies in B cell supernatants were captured by immobilized anti-rabbit IgG antibodies. To allow sufficient capture, the IgG concentration in the B cell supernatants must be minimized. After capturing the monoclonal antibodies, human IL-31 was injected into the flow cell at a concentration of 90 nM for 3 minutes, and dissociation of the protein from the IgG captured on the sensor chip was allowed to proceed for 5 minutes. The apparent dissociation (k d ) rate constant and association (k a ) rate constant, and the apparent dissociation equilibrium constant (K D ) was calculated using a 1:1 Langmuir binding model with MASS-2 analysis software (Analyzer, Sierra Sensors).

[0169] The binding affinity of 584 anti-IL-31 antibodies to human IL-31 was measured. -12 Less than M ~ 2.11 x 10 -5 Dissociation constants (K D ) 2.7% of the antibodies had a K value of less than 0.5 nM. D showed.

[0170] Neutralization of human IL-31 in IL-31RA / OSMR dimerization assay and competitive ELISA To assess potency, a cell-based IL-31RA / OSMR dimerization assay (PathHunter assay) and a receptor-ligand competition ELISA were developed and adapted for use with B cell supernatants. The cell-based assay allows for assessment of blockade of IL-31-induced signaling, whereas the competition ELISA assesses only the interaction between IL-31RA and IL-31. Because these assays can be performed using B cell supernatant as a matrix and are sensitive and accurate, both assays were used for screening. The inhibitory activity of each B cell supernatant was tested using a single-well assay (a dose response was not performed). Therefore, the degree of inhibition observed depends not only on the IgG characteristics but also on the concentration of rabbit IgG in the B cell supernatant.

[0171] Blocking ELISA analysis yielded numerous neutralizing clones. Of 618 clones, 166 blocked the interaction of human IL-31 with human IL-31RA by >90%, and 143 clones inhibited by >80%. Based on a threshold of >30% inhibition, 103 clones inhibited IL-31-induced signaling in the PathHunter assay.

[0172] Species cross-reactivity (binding to cynomolgus monkey IL-31 by SPR) All 618 hits identified in the primary screen were analyzed for species cross-reactivity to cynomolgus IL-31 by SPR. Binding affinities were determined by surface plasmon resonance (SPR) using a MASS-2 SPR device (Sierra Sensors) as described above for human IL-31, except that 90 nM cynomolgus IL-31 was used instead of human IL-31.

[0173] 498 clones (80.5%) showed binding to cynomolgus IL-31. For affinity, the off-rate was below or close to the limit of the SPCR instrument, so accurate K values ​​for five antibodies were obtained. DThe value could not be determined. 120 clones showed no binding to cynomolgus IL-31. The binding antibody was 4.73 x 10 -12 M~3.39×10 -5 The equilibrium dissociation constant (K D ) 3.8% of all antibodies analyzed had a K below 500 pM. D 75.7% of rabbit monoclonal antibodies that bound to human IL-31 had affinity 10-fold lower than that for cynomolgus monkeys. The correlation between human and cynomolgus monkey IL-31 was good.

[0174] 1.4. Hit selection and hit confirmation Hit selection and RT-PCR As a prerequisite for hit confirmation, gene sequence analysis, and subsequent humanization of rabbit antibodies, it was necessary to retrieve the genetic information encoding the rabbit antibody variable domains. This was achieved by reverse transcription (RT) of the respective messenger RNAs into complementary DNA (cDNA), followed by amplification of the double-stranded DNA by polymerase chain reaction (PCR). Selection of B cell clones subjected to RT-PCR was primarily based on affinity for human IL-31 of less than 500 pM and neutralizing activity in IL-31 / IL-31RA competitive ELISA. Several clones with affinity greater than 500 pM but also good neutralizing properties were included in the selection.

[0175] Ninety-four sets of rabbit CDRs corresponding to 94 independent clones were selected and identified. Forty-three sets of rabbit CDRs corresponding to 43 independent clones were identified. The rabbit CDR sequences of all 43 sequenced clones were clustered into a phylogenetic tree. Forty sequences were non-overlapping based on the CDR regions, and three sequences contained a free cysteine. Three clones with identical sequences were excluded. As a result, a total of 40 clones were selected for expression as recombinant IgG.

[0176] Monoclonal antibody cloning and production Following the selection of clones for hit confirmation, rabbit antibodies were cloned, expressed, and purified for further characterization. Cloning of the corresponding light and heavy chain variable domains required in vitro ligation of the DNA fragments into appropriate mammalian expression vectors. The rabbit antibody heavy and light chain expression vectors were transfected into mammalian suspension cell lines for transient heterologous expression. The secreted rabbit IgG was then affinity purified, and the final product was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), UV absorbance at 280 nm, and size-exclusion high-performance liquid chromatography (SE-HPLC) to confirm identity, content, and purity. Thirty-seven of the 40 clones were successfully cloned into appropriate mammalian expression vectors and produced with good to high expression titers (3–19 μg protein / ml) and high monomer content (94.7–99.5%).

[0177] 1.5. Pharmacological characterization of monoclonal antibodies Affinity for human and cynomolgus monkey IL-31 The binding kinetics of 37 purified monoclonal rabbit antibodies to human and cynomolgus IL-31 were determined by SPR (MASS-2) analysis. Cynomolgus IL-31 was not commercially available and was produced on demand by Sino Biological. Each IgG was captured via anti-rabbit IgG bound to a carboxymethylated dextran surface, and the dose response of the analyte was measured. All but two IgGs were confirmed to bind to human IL-31. Twelve of the 35 target-binding antibodies had K values ​​below 500 pM. D Thirty-one of the 37 IgGs bound to cynomolgus IL-31 with high affinity, and 23 IgGs had K values ​​below 500 pM. D Nine of these IgGs further showed values ​​below 10 pM.

[0178] IL-31RA / OSMR dimerization assay (blockade of human IL-31-induced signaling) Furthermore, the effect of 37 rabbit monoclonal antibodies on IL-13-induced signaling through the IL-31RA / OSMR receptor was tested in the PathHunter dimerization assay from DiscoveryX. The potency (IC) to neutralize signaling induced by 10 ng / ml of IL-31 was 50 ) were analyzed for serial dilutions of all antibodies and compared to the potency of BMS-981164. Relative IC was used to compare IgGs from different assay plates. 50 These values ​​were used to calculate the IC of IgG relative to the reference molecule BMS-981164 included on each assay plate. 50 The relative IC values ​​were determined by calibrating the 50 :I C 50 , BMS-981164 / IC 50 , test antibody).

[0179] The IgGs inhibited IL-31-induced signaling with greater potency than BMS-981164 or up to 5-fold less potency. Two IgGs showed greater potency than BMS-981164, and two additional IgGs showed IC 50 The same was true for

[0180] Competitive ELISA (inhibition of hIL-31 binding to hIL-31RA) Inhibition of human IL-31 binding to human IL-31RA was assessed by competitive ELISA. For this purpose, IL-31RA was coated onto an ELISA plate. Biotinylated IL-31 was pre-incubated with a rabbit monoclonal antibody, and the mixture was added to the ELISA plate to allow binding to IL-31RA. Bound biotinylated IL-31 was then detected using streptavidin-HRP.

[0181] The competitive ELISA was sensitive enough to distinguish the rabbit antibodies with respect to potency, with a lower IC than BMS-981164 50Antibodies with IC values ​​were found. With the exception of two IgGs, all antibodies blocked the interaction between human IL-31 and IL-31RA, with some blocking only incompletely. Compared to BMS-981164, 20 rabbit antibodies more potently inhibited the interaction, and 12 antibodies showed similar potency or IC values ​​not more than 5-fold lower. 50 The values ​​were shown.

[0182] Selection of rabbit IgG for generation of humanized scFv Based on the pharmacodynamic properties of 37 characterized rabbit monoclonal antibodies, the best-performing clones were selected for humanization and lead candidate generation. The selection criteria for clones were: i) complete blockade of IL-31-induced signaling in an IL-31RA / OSMR dimerization assay (with one exception), ii) high affinity for human IL-31, iii) neutralization of the interaction between human IL-31 and human IL-31RA in a competitive ELISA, iv) cross-reactivity with cynomolgus monkey IL-31 by SPR, and v) sequence diversity. In addition to these criteria, the primary sequences of all 37 clones were screened for the presence of unpaired cysteines in the complementarity-determining regions (CDRs). Identification of unpaired cysteines excluded the commonly occurring cysteine ​​pair in CDR-H1 and CDR-H2, which is encoded in the rabbit germline repertoire and is thought to form disulfide bridges.

[0183] Because most neutralizing antibodies showed high affinity, the focus of rabbit antibody selection for humanization was the IL-31RA / OSMR dimerization assay. In the IL-31RA / OSMR dimerization assay, IC 50 Antibodies with a IgG antibody titer of 50 ng / ml or less were selected for humanization. A total of five promising clones were selected for reformatting and humanization.

[0184] 1.6. Humanization of scFv Five rabbit monoclonal antibody clones were selected for lead candidate generation. Humanization of these clones involved transferring rabbit CDRs onto one of Numab's proprietary human variable domain acceptor scaffolds. In this process, the amino acid sequences of six CDR regions were identified using Numab's CDR definitions (Table 5) and grafted onto Numab's proprietary, highly stable, fully human Vk1 / VH3 lambda cap acceptor framework, resulting in a construct called a "CDR graft." [Table 5]

[0185] The exclusive grafting of rabbit CDRs onto a human acceptor framework is the most basic grafting strategy. However, in some cases, a specific set of CDRs requires mutation of specific rabbit framework residues to maintain its full functionality. Therefore, in addition to "CDR grafting," additional grafting variants containing defined patterns of rabbit framework residues have been designed.

[0186] Humanized scFv constructs were designed and ordered as mammalian (CHO-S, pcDNA3.1) expression vectors at 1 mg scale from GeneUniversal (formerly General Biosystems). Plasmids were used for transient transfection of CHO-S cells as described below.

[0187] 1.7. Production of humanized scFv Expression of mammalian constructs was carried out in CHO-S cells using the CHOgro transient transfection kit (Mirus). After 5–7 days of expression at 37°C (when cell viability reached <70%), the culture was harvested by centrifugation followed by filtration. Protein was purified from the clarified culture supernatant by protein L affinity chromatography. As assessed by SE-HPLC analysis, all molecules exhibited at least one affinity chromatography fraction with a post-capture monomer content >95%, so no molecules required refinement by size-exclusion chromatography. Samples were re-buffered by dialysis into the final buffer (50 mM phosphate-citrate buffer, pH 6.4, containing 150 mM NaCl). Quality control of the produced material included SE-HPLC, UV spectroscopy, and HPLC. 280 Standard analytical methods such as SDS-PAGE were applied. The manufacturing characteristics of five sc03 constructs ("full grafts") that were deemed suitable for incorporation into a multispecific antibody format are summarized in Table 6. Table 7 summarizes the manufacturing characteristics of the different grafts of clone 50-35-B03. [Table 6] [Table 7]

[0188] 1.8. Pharmacodynamic characterization of suitable anti-IL-31 binding domains (scFv format) Humanized scFv antibodies that were assessed as suitable were analyzed for primary pharmacodynamic properties.

[0189] Affinity for human and cynomolgus monkey IL-31 The affinity of seven humanized scFvs for human and cynomolgus IL-31 was measured by SPR analysis on a T200 instrument (Biacore, GE Healthcare). Cynomolgus IL-31 was not commercially available and was therefore produced on demand by Sino Biological. Human and cynomolgus IL-31-His were captured via anti-His tag antibodies bound to a carboxylmethylated dextran surface, and the scFvs were injected as analytes. After each analyte injection cycle, the sensor chip was regenerated and new antigen was captured. The scFvs were measured using a dose-response multi-cycle kinetic assay in high-throughput mode using two concentrations (30 and 10 nM) diluted in running buffer. The resulting sensorgrams were fitted using a 1:1 binding model.

[0190] As shown in Table 8, binding to human IL-31 was confirmed for all seven humanized scFvs. [Table 8]

[0191] IL-31RA / OSMR dimerization assay (blockade of human IL-31-induced signaling) An IL-31RA / OSMR dimerization assay was used to test the ability of the humanized scFv to inhibit IL-31-induced signaling via the IL-31RA / OSMR heterodimer. 10,000 cells were seeded per well into a 96-well plate. The following day, serial dilutions of the scFv and control antibody BMS-981164 were added to the plate in the presence of 10 ng / ml IL-31. After 6 hours of incubation at 37°C and 5% CO2, detection solution was added, the plate was incubated for an additional hour, and luminescence was measured.

[0192] Seven scFvs were measured in a cellular assay, and the potency of the analyzed molecules was compared to BMS-981164, as described above.

[0193] Relative IC50 Values ​​were calculated in mass units (ng / ml) of BMS-981164 and scFv. Potency data are summarized in Table 9. Representative dose-response curves for PRO1641, PRO1643, and PRO1650 are shown in Table 1. [Table 9]

[0194] Competitive ELISA (inhibition of hIL-31 binding to hIL-31RA) The potency of the two humanized scFvs was further determined using a competitive ELISA. The potency of each scFv to inhibit the interaction of human IL-31 with human IL-31RA was assessed by ELISA using the same procedure as described above. As with the IL-31RA / OSMR dimerization assay, the individual IC 50 The values ​​are taken as the IC of the reference molecule BMS-981164. 50 The fully grafted scFvs inhibited the interaction of human IL-31 with human IL-31RA with potency similar to that of BMS-981164. Potency data are summarized in Table 10. Representative dose-response curves for PRO1641, PRO1643, and PRO1650 are shown in Table 2. [Table 10]

[0195] Overview of molecular selection for pharmacological characterization and detailed biophysical evaluation of scFvs Based on the above results of pharmacological characterization, PRO1645 exhibited the lowest binding affinity and was therefore excluded. The other six scFvs were selected for detailed biophysical evaluation to determine their developability and suitability for incorporation into a multispecific antibody format.

[0196] 1.9. Biophysical characterization of suitable anti-IL-31 binding domains (scFv format) Preparation of stability materials for stability measurements PRO1641, PRO1643, PRO1644, and PRO1650 were re-produced on a slightly larger scale (0.25 L expression volume) using the same manufacturing process described above to generate sufficient material for stability evaluation. For the other proteins selected for stability evaluation, the amount of material previously produced was sufficient to perform the stability evaluation. Samples were prepared in 50 mM phosphate-citrate buffer at pH 6.4 containing 150 mM NaCl (50 mM NaCiP, pH 6.4). After purification and dialysis, protein samples were concentrated to >10 mg / ml using 5 MWCO centrifugal concentration tubes.

[0197] As shown in Table 11, the monomer loss when concentrated to 10 mg / ml was 0.0 to 4.9%. [Table 11]

[0198] Storage stability test The humanized scFv was subjected to a 4-week stability study, in which the scFv was prepared at 10 mg / ml in aqueous buffer (final buffer, 50 mM NaCiP, 150 mM NaCl, pH 6.4) and stored at <-80°C, 4°C, and 40°C for 4 weeks. The proportion of monomers and oligomers in the preparations was assessed by integration of SE-HPLC peak areas at different time points of the study. Additionally, protein concentrations were assessed by UV spectroscopy at different time points. 280 Table 12 compares the endpoint measurements obtained on d14 and d28 of the study.

[0199] Three scFvs, namely PRO1641, preparation PRO1643, and PRO1644, showed less than 5% loss of monomer content at d28 of testing at 4°C. PRO1643 was the only scFv that did not show significant loss of monomer content at d14 and d28 of storage at 40°C. All other scFvs showed losses well above 5% even after 14 d of storage at 40°C. In summary, PRO1643, based on clone 50-09-D07, showed the greatest storage stability and did not show significant loss of monomer content or protein content at any temperature.

[0200] Freeze-thaw stability In addition to the storage stability studies described above, the suitability (colloidal stability) of six selected scFvs for freeze-thaw (F / T) cycles was evaluated.

[0201] For the F / T stability assessment, the same analytical methods (SE-HPLC, UV-Vis) and parameters (% monomer content and % monomer loss) as those used in the storage stability study were applied to track the quality of the molecule over three F / T cycles. Table 13 shows the progression of monomer content (%) and % monomer content loss over three F / T cycles. Since a dedicated freeze-thaw study was not performed, the freeze-thaw data obtained for the -80°C samples from the storage stability study taken over 28 days are shown in the table below. Since only three time points could be recorded per sample, freeze-thaw data are available for only three F / T cycles.

[0202] Thermal unfolding Thermal unfolding measurements of six scFvs were performed using differential scanning fluorescence (DSF). PRO1698 was excluded due to its poor storage stability. The resulting thermal unfolding midpoints (T m ) and the onset temperature of unfolding (T onset 10%) values ​​were determined by fitting the data to the Boltzmann equation. Table 14 summarizes the calculated melting temperatures measured by DSF. [Table 12] [Table 13] [Table 14]

[0203] Example 2: Selection and optimization of anti-IL-31 molecules for multispecific formats: 2.1. General Description of Anti-IL-31 Domain Selection The anti-IL-31 domain PRO1643 (50-09-D07-sc03) was initially selected for further development because it exhibited desirable pharmacodynamic properties and excellent stability. Nevertheless, the anti-IL-31 binding domain PRO1643 (50-09-D07-sc03) underwent further solubility improvements as shown in Section 2.2 below.

[0204] The anti-IL-31 binding domain applied in the final multispecific antibody does not cross-react with cynomolgus IL-31.

[0205] 2.2. Optimization of the anti-IL-31 domain The anti-IL-31 domain PRO1643 (50-09-D07-sc03) was further optimized for assembly into a multispecific format.

[0206] Although PRO1643 (50-09-D07-sc03) is a very stable anti-IL-31 scFv, attempts were made to further improve it in terms of solubility, long-term stability, and concentration behavior. Therefore, new variants of this molecule were designed. These variants are described below. Briefly, the CDRs or the hydrophobic patches at the former VH-CH interface were analyzed in detail with the aim of designing a molecule that would not impair binding affinity and would not introduce significant sequence defects (chemical and post-translational modifications), T cell epitopes, etc.

[0207] A total of four variants were designed and these are summarized in Table 15. [Table 15]

[0208] 2.3. Biophysical Characterization of Optimized Anti-IL-31 Binding Domains (scFv Format) Storage stability test PRO1900, PRO1901, PRO1902, and PRO1903 were subjected to the storage stability testing described above in Section 1.2.3, however, the last reading was obtained on day 14 and F / T and stability at -80°C were not evaluated.

[0209] As summarized in Table 16, scFv PRO1900, PRO1901, PRO1902, and PRO1903 exhibited excellent storage stability, with less than 0.2% loss in monomer content after 14 days of storage at 4° C. and less than 3% loss after 14 days of storage at 40° C. Additionally, none of these molecules showed significant loss in protein content at all temperatures and data points.

[0210] Thermal unfolding The thermal stability of PRO1900, PRO1901, PRO1902, and PRO1903 was analyzed by nanodynamic scanning fluorometry (nDSF) using a NanoTemper, and the onset temperature of unfolding (T onset ), the midpoint of unfolding (T m1 ), and scattering onset temperature could be determined. T m1 and T onset The results are summarized in Table 17. As can be inferred from Table 17, PRO1900, PRO1901, PRO1902, and PRO1903 exhibited high melting temperatures. [Table 16] [Table 17]

[0211] Concentrated solubility test PRO1643, PRO1900, PRO1901, PRO1902, and PRO1903 were concentrated to >10 mg / ml and >50 mg / ml using 5 MWCO centrifugal concentrator tubes as described herein.

[0212] As shown in Table 18, there was no substantial loss of monomer content upon concentration to 10 mg / ml and 50 mg / ml, respectively.

[0213] Storage stability test PRO1900, PRO1901, PRO1902, and PRO1903 were subjected to a two-week stability study, in which the scFv was prepared at 50 mg / ml in aqueous buffer (final buffer, 50 mM NaCiP, 150 mM NaCl, pH 6.4) and stored at 4°C and 40°C for two weeks. Monomeric and oligomeric fragments in the preparations were assessed by integration of SE-HPLC peak areas at different time points during the study. Additionally, protein concentrations were assessed by UV spectroscopy at different time points. 280 Table 19 shows the results of the d14 measurements of the test.

[0214] PRO1900, PRO1901, PRO1902, and PRO1903 exhibited excellent storage stability at 50 mg / ml with no substantial loss of monomer and protein content at 4°C, but also at 40°C.

[0215] 2.4. Pharmacodynamic Characterization of Optimized Anti-IL-31 Binding Domains (scFv Format) Affinity for human IL-31 As described above in section 1.8, the affinity of PRO1643 and the optimized anti-IL-31 scFv for human IL-31 was measured by SPR analysis on a T200 instrument (Biacore, GE Healthcare). The scFv was measured using a dose-response multi-cycle kinetic assay in high-throughput mode at two concentrations (30 and 10 nM) diluted in running buffer. The resulting sensorgrams were fitted using a 1:1 binding model.

[0216] As shown in Table 20, binding to human IL-31 and cynomolgus monkey IL-31 was confirmed for all optimized scFvs. [Table 18] [Table 19] [Table 20]

[0217] Path Hunter IL-31RA / 0SMR Dimerization Assay (Blockade of Human IL-31-Induced Signaling) PRO1643 and the optimized scFvs were tested for their ability to inhibit IL-31-induced signaling through the IL-31RA / 0SMR heterodimer using an IL-31RA / 0SMR dimerization assay. This assay was performed as described in Section 1.8. The potency of the analyzed molecules was compared to BMS-981164.

[0218] Relative IC 50 Values ​​were calculated in mass units (ng / ml) of BMS-981164 and scFv. Potency data are summarized in Table 21. As shown in Table 21, PRO1643 and optimized variants PRO1900, PRO1901, and PRO1903 were able to potently neutralize IL-31-induced signaling. Mutations introduced into variant PRO1902 to optimize solubility and stability clearly caused a disruption of its ability to inhibit IL-31-induced signaling. Dose-response curves for optimized scFvs PRO1900, PRO1901, PRO1902, and PRO1903 are shown in Figure 3. [Table 21]

[0219] Example 3: Morrison-H IgG4-based anti-IL-4R x IL-31 bispecific antibody Next, we further tested whether the anti-IL-31 antibody variable domains of the present invention also provide advantageous biological and biophysical properties when incorporated into a multispecific antibody format. Therefore, a multispecific antibody was designed based on the Morrison-H IgG4 format containing two anti-IL-31 antibody variable domains, as defined herein. Two IL-4R binding domains (IL4R-BDs) were selected as binding domains that specifically bind to targets different from IL-31.

[0220] 3.1. Morrison Format Design A series of anti-IL-4R x IL-31 bispecific antibodies with the Morrison-H format were designed, in which the Fc region is derived from the IgG subclass, IgG4.

[0221] Due to its cross-reactivity to cynomolgus IL-31, its excellent potency in blocking IL-31-mediated signaling, and its excellent biophysical properties, the anti-IL-31 scFv variable domain 50-09-D07-sc04 was selected as the scFv domain fused to the C-terminus of the heavy chain of the Morrison-H antibody. Similarly, due to their excellent biological and biophysical properties, the anti-IL-4R scFv variable domains 44-34-C10-sc08 and 44-34-C10-sc09 were selected as the Fab arm binding domains for the Morrison-H construct.

[0222] The identification, humanization, production, and final selection of the humanized anti-IL-4R binding domains 44-34-C10-sc08 and 44-34-C10-sc09 were performed using the same methods as described above for the anti-IL-31 antibody variable domains.

[0223] Two IgG4-(scFv)2 Morrison-H molecules, PRO2198 and PRO2199, shown in Table 22, were designed (Morrison-H). [Table 22]

[0224] Expression of the multispecific antibodies PRO2198 and PRO2199 was carried out in FreeStyle CHO-S cells using the transient CHOgro expression system (Mirus). The genes of interest were optimized for mammalian expression, synthesized, and cloned into a standard pcDNA3.1 vector. Expression cultures were grown in batches at 37°C for 6–7 days (cell viability <70%) using shake flasks. The culture supernatant was separated from the cells by centrifugation followed by 0.22 μm sterile filtration. The target protein was captured from the clarified culture supernatant by protein L or A affinity chromatography, followed by a polishing step of size exclusion chromatography (if fractions containing the appropriate monomer content, as assessed by SE-HPLC, were not already available after capture). Quality control of the produced material included SE-HPLC, SDS-PAGE, and UV spectroscopy. 280 Standard analytical methods were used.

[0225] 3.2. Affinity for human and cynomolgus monkey IL-31 The binding kinetics (including affinity) of the bispecific Morrison-H antibodies PRO2198 and PRO2199 to recombinant human IL-31 protein (Peprotech) was measured by SPR analysis on a T200 instrument (Biacore, Cytiva). In this SPR experiment, the Morrison antibodies were injected over a carboxylmethylated dextran surface (CM5 sensor chip; Biacore, Cytiva) onto which human recombinant IL-4R protein (ECD with an Fc tag, R&D Systems) was immobilized, and a titration series of human IL-31 was injected as the analyte. The affinity for human IL-31 was measured using a single-cycle kinetic assay with injections of five consecutive analyte concentrations ranging from 0.05 to 30 nM diluted in running buffer (Hepes-buffered saline, 0.05% Tween-20, pH 7.5) without regeneration of the sensor chip after analyte injection. The kinetics and apparent dissociation equilibrium constant (K) were measured. DCalculation of R and measurement of curve fitting quality were performed as described above for IL-4R. Binding levels were calculated as the maximum stable binding achieved normalized to the theoretical R. Cross-reactivity to cynomolgus IL-31 was measured using the same setup as for the analysis of human IL-31 affinity, except that recombinant cynomolgus IL-31 (His-tagged ECD, Acro Biosystems) protein was used as the analyte instead of human IL-31.

[0226] The prerequisite for the SPR setup used to measure binding kinetics to recombinant IL-31 is the capture of Morrison antibodies via immobilized human IL-4R. All Morrison antibodies tested so far have shown stable binding to immobilized human IL-4R, thus proving the validity of this setup. The rationale for using this SPR setup is to ensure uniform orientation of the captured Morrison molecules while minimizing steric hindrance of the IL-31 binding site.

[0227] Having demonstrated stable capture of Morrison antibody via recombinant human IL-4R protein, IL-31 was injected as an analyte and the binding kinetics of IL-31 to the captured Morrison molecules were calculated.

[0228] As shown in Table 23, high affinity binding to human IL-31 and cynomolgus IL-31 was demonstrated for the Morrison-H antibodies PRO2198 and PRO2199. 3.3. Evaluating Potency in Inhibiting Human IL-31-Induced Receptor Dimerization (PathHunter™ eXpress IL31RA / OSMRb Assay)

[0229] PathHunter IL-31RA / OSMRb Dimerization Assay The ability of Morrison-H antibodies PRO2198 and PRO2199 to inhibit IL-31-induced signaling via IL-31RA / OSMR was assessed in an IL-31RA / OSMR dimerization assay. 10,000 cells per well were seeded into a 96-well plate. The following day, 3-fold serial dilutions of the molecules and the control antibody BMS-981164, ranging in concentration from 1,000 to 0.2 ng / ml, were added to the plate in the presence of 10 ng / ml IL-31. After 6 hours of incubation at 37°C and 5% CO, detection solution was added, the plates were incubated for an additional hour, and luminescence was measured. All antibodies were also tested for inhibition of IL-31-induced signaling by excess IL-4R (at 50 nM) in the assay medium. [Table 23] [Table 24] Inhibition of human IL-31 A summary of all efficacy data is provided in Table 24. Mean relative IC from replicate analyses 50 Values ​​(pM) are also shown. Both Morrison-H antibodies inhibited IL-31-induced signaling with potency comparable to that of BMS-981164. Efficient blockade of the interaction of IL-31 with IL-31R was maintained when the anti-IL-4R domain was bound to IL-4R.

[0230] 3.4. Biophysical Characterization of PRO2198 and PRO2199: 3.4.1.Thermal stability PRO2198 and PRO2199 were analyzed for thermal stability between pH 5 and pH 8.5. Both thermal unfolding and the onset of thermal aggregation were measured. During thermal unfolding, all molecules exhibited multiple transitions due to the multi-domain structure of the molecules. For simplicity, only the first melting midpoint is shown. The results are summarized in Table 25. [Table 25]

[0231] The start of unfolding (T onset ) and first melting point (T m1 ) was highest at pH 7 for all molecules. Changing to pH 8.5 only slightly decreased the thermal stability. As the pH became more acidic, the thermal stability decreased. However, at pH 5, the onset of unfolding was all above 55°C.

[0232] Thermal aggregation was observed only at pH 7 and pH 8.5. At pH 5.0, both Morrison antibodies did not form larger aggregates, even in the unfolded state. At neutral and basic pH, the onset of aggregation was above the initial melting temperature, suggesting a non-native aggregation mechanism.

[0233] 3.4.2. High-concentration stability test For high concentration stability studies, PRO2198 was formulated in two formulation buffers. Prepare buffer F1: 20 mM acetate, pH 5.5; Prepare buffer F2: 20 mM citrate, 50 mM NaCl, pH 5.5.

[0234] Solubility - Protein Concentration PRO2198 could be concentrated to greater than 100 mg / ml without signs of precipitation or reaching its solubility limit. Furthermore, PRO2198 showed no detectable decrease in protein concentration, i.e., PRO2198 was sufficiently soluble to reach and maintain target concentrations above 100 mg / ml for at least 4 weeks at 4°C and 25°C.

[0235] Monomer stability at various temperatures PRO2198 was prepared in F1 and F2 and concentrated to above 100 mg / ml. The concentrated samples were stored at 4°C, 25°C, and 40°C for up to 4 weeks. At different time points, the monomer content was analyzed by SE-HPLC. The results are summarized in Table 26. [Table 26]

[0236] 3.5. General Methods Used for Biophysical Characterization of PRO2198 and PRO2199 buffer exchange Buffer exchange was performed by dialysis: the antibody was dialyzed in a Spectra Pro3 dialysis membrane (Spectrum Laboratories) using at least a 200-fold excess of dialysis buffer.

[0237] Antibody concentration Antibodies were concentrated using centrifugal concentrators with a molecular weight cutoff (MWCO) of 10 kDa or 30 kDa. Samples were centrifuged in 5-minute steps at 22°C until the target concentration was reached. Samples were resuspended between steps.

[0238] Protein concentration determination The concentrations of protein samples were determined using a Tecan plate reader and NanoQuant plate. Buffer was used as a blank, which was subtracted from the absorbance measured at 280 nm. Each measurement was corrected for scattering caused by visible particles, which was measured at 310 nm. Corrected values ​​were normalized to a 1 cm path length, and the theoretical extinction coefficient of the corresponding protein was used to calculate the protein concentration. For protein samples with concentrations greater than 10 mg / ml, the samples were diluted at least 10-fold with the corresponding buffer, or to a nominal concentration of 1 mg / ml.

[0239] keep To assess protein stability at different temperatures, samples were incubated at 4°C, 25°C, and 40°C. 4°C storage was performed in a refrigerator with a nominal temperature of 4°C. For storage at 25°C and 40°C, samples were placed in humidity-controlled cabinets at 65% rH and 75% rH, respectively.

[0240] Dynamic Light Scattering Dynamic light scattering is used to determine the diffusion coefficients of molecules and particles in solution, which allows the calculation of the hydrodynamic radius (Rh) of molecules in solution as well as providing a sensitive method to detect the formation of higher order oligomers.

[0241] In high-concentration stability studies, Rh and polydispersity were determined at target concentrations, providing information on self-association, oligomerization, and potential increases in viscosity. Measurements were not corrected for the viscosity of the buffer or sample; instead, the viscosity of water was used to calculate the Rh of the sample.

[0242] Thermal unfolding by nanoDSF Thermal unfolding using TSA was determined by the change in Sypro Orange fluorescence intensity. The dye's fluorescence is sensitive to hydrophobic interactions. As the protein unfolds, hydrophobic amino acids become exposed to the solvent, resulting in an increase in Sypro Orange fluorescence. The resulting thermal unfolding midpoint (T m ) and the onset temperature (T onset is the temperature at minimum signal + 0.1 * (maximum signal minus minimum signal) was determined by fitting the data to the Boltzmann equation.

[0243] Monomer content by SE-HPLC Monomer content was determined by analytical size exclusion chromatography using a Shodex KW403-4F column, run in 50 mM sodium phosphate, 300 mM NaCl, pH 6.5. For analysis, 5 μg of sample was injected and absorbance recorded at 280 nm. Sample quality is reported as the relative percent of monomer, HMWS, and LMWS.

Claims

1. An antibody variable domain that specifically binds to IL-31, comprising: below: a) a VH chain having the sequence of SEQ ID NO: 6 and a VL chain having the sequence of SEQ ID NO: 12, or b) a VH chain having the sequence SEQ ID NO: 5 and a VL chain having the sequence SEQ ID NO: 37; An antibody variable domain comprising:

2. The antibody variable domain of claim 1 , selected from FAB, Fv, scFv, and DSFv.

3. 3. The antibody variable domain of claim 2, selected from the scFv antibodies of SEQ ID NOs: 28 and 31.

4. below: a) one or two antibody variable domains as defined in any one of claims 1 to 3; and b) at least one binding domain that specifically binds to a target different from IL-31; A multispecific antibody comprising:

5. The multispecific antibody of claim 4, which does not contain an immunoglobulin Fc region.

6. Tandem scDb (Tandab), linear dimer scDb (LD-scDb), cyclic dimer scDb (CD-scDb), tandem tri-scFv, tribody (Fab-(scFv) ２ ), Fab-Fv ２ 6. The multispecific antibody of claim 5, which is in a format selected from the group consisting of triabody, scDb-scFv, tetrabody, di-diabody, tandem-di-scFv, and MATCH.

7. 5. The multispecific antibody of claim 4, comprising an immunoglobulin Fc region selected from the IgG subclasses IgG1 and IgG4, in particular IgG4.

8. The multispecific antibody formats include KiH-based IgG, DVD-IgG, CODV-IgG, and Morrison (IgG CH ３ 8. The multispecific antibody according to claim 7, wherein the antibody is selected from IgG CL-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L), in particular from Morrison-H and Morrison-L.

9. 9. The multispecific antibody of any one of claims 4 to 8, comprising two antibody variable domains as defined in any one of claims 1 to 3 and two binding domains that specifically bind to a second target different from IL-31.

10. One or two nucleic acids encoding an antibody variable domain according to any one of claims 1 to 3 or a multispecific antibody according to any one of claims 4 to 9,

11. A vector or two vectors comprising the nucleic acid or the two nucleic acids according to claim 10.

12. A host cell or a plurality of host cells comprising the vector or the two vectors of claim 11.

13. A pharmaceutical composition comprising the antibody variable domain of any one of claims 1 to 3 or the multispecific antibody of any one of claims 4 to 9, and a pharmaceutically acceptable carrier.

14. An antibody variable domain according to any one of claims 1 to 3 or a multispecific antibody according to any one of claims 4 to 9 for use as a medicament.

15. 10. An antibody variable domain according to any one of claims 1 to 3 or a multispecific antibody according to any one of claims 4 to 9 for use in the treatment of a disease, particularly a human disease, more particularly a human disease selected from allergic diseases, inflammatory diseases and autoimmune diseases, particularly pruritus-induced allergic diseases, pruritus-induced inflammatory diseases and pruritus-induced autoimmune diseases.

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