Antibodies of KLK5

Abstract

The present invention relates to antibodies that bind to and inhibit KLK5 and methods of using them to treat diseases caused by an imbalance of KLK5. In particular, the present invention relates to inhibitory anti-KLK5 antibodies and their use in the treatment of Netherton's disease, atopic dermatitis and cancer.

Description

[Technical Field]

[0001] The present invention relates to an antibody that binds to and inhibits KLK5, and to a method of using the antibody to treat diseases caused by KLK5 dysregulation. In particular, the present invention relates to an anti-KLK5 antibody, and its use in the treatment of ichthyosis such as Netherton's disease and congenital ichthyosis, atopic dermatitis, and cancer. [Background technology]

[0002] Kallikrein-related peptidases (known as KLKs) constitute a single family of 15 highly conserved trypsin or chymotrypsin-like serine proteases, encoded by the largest contiguous cluster of protease-coding genes (chromosome 19q13.4) in the human genome (Sotiropoulou G. et al., 2009; JBC 284:48, 32989-94).

[0003] KLK proteins are synthesized as inactive pre-proforms that are proteolytically processed to secrete an inactive proform. Such proforms are then activated into mature peptidases by specific proteolytic removal of the N-terminal propeptide, either by other KLK or endopeptidases, or by autocatalytic cleavage such as KLK5. KLK5 possesses trypsin-like activity. Its pre-proform contains a 29-amino acid signal peptide followed by a 37-amino acid activation peptide. The active mature form consists of 237 amino acids, including a mature enzyme containing a serine-protease domain responsible for protease activity (Michael IP et al., 2005; JBC 280:15, 14628-35).

[0004] KLK5 is found in many tissues, but appears to be most abundantly expressed in the skin. Along with KLK7, KLK5 is expressed at the upper spine and granular levels of the skin, where keratinocytes undergo terminal differentiation and are converted into corneocytes that make up the stratum corneum. The stratum corneum functions as a barrier against the external environment and is maintained by the constant replacement of corneocytes shed through the desquamation process. KLK5 can activate pro-KLK7 and other kallikreins, so its role in desquamation is essential.

[0005] After activation, mature KLK5 is inactivated by the endogenous inhibitor lymphoepithelial Cazal-type inhibitor (LEKTI) encoded by the SPINK5 gene (Chavans P et al., 2005; Nat Genet 37, 56-65). LEKTI contains 15 serine protease inhibitory domains that form a close complex with KLK5. This close interaction with acidic pH, which releases active KLK5 from the complex, is influenced by pH fluctuations (Deraison C et al. 2007; Mol Biol Cell 18 3607-19).

[0006] Loss-of-function mutations in the SPINK5 gene cause Netherton syndrome, a rare autosomal recessive skin disorder characterized by ichthyosis-like symptoms with severe inflammation, skin desquamation, elevated IgE levels, and certain allergic symptoms (Hovnanian A. 2013; Cell Tissue Res 351 289-300). Following increased epidermal protease activity, LEKTI deficiency leads to stratum corneum exfoliation caused by KLK5 activity against desmogleins and desmosomes, which results in high permeability to various allergens that cause atopic dermatitis-like lesions. KLK5 activity against KLK7 also contributes to an imperfect skin barrier, resulting in allergen and microbial permeability and IL-1β production.

[0007] SPINK5 - / -The mice reproduce a phenotype that closely resembles Netherton syndrome, replicating the appearance of diseased skin and inflammation (Yant T et al.; 2004, Genes Dev 18 2354-58). SPINK5 in Netherton syndrome patients. - / - The epidermis exhibits uncompetitive KLK5 and KLK7 protease activity, which is thought to sustain activation of pro-inflammatory and pro-signaling pathways, including the KLK5-PAR2-TSLP (thymic interstitial lymphopoietin) axis. SPINK5 - / - and KLK5 - / - In both mice, KLK5 knockout was sufficient to correct these skin symptoms associated with LEKTI knockout, demonstrating the important role of KLK5 in skin homeostasis.

[0008] In recent years, several studies have reported a genetic link between atopic dermatitis (AD) and LEKTI polymorphisms, indicating the expression of abnormal variants of LEKTI (Hovnanian A. 2013; Cell Tissue Res 351 289-300).

[0009] To date, KLK7 inhibitors are in clinical development, but no KLK5-specific therapies exist. Other approaches aimed at replacing LEKTI are being pursued, including gene addition using SPINK5 lentiviral or adenoviral vectors, and autografts of genetically corrected patient keratinocytes (Di WL. et al.; 2011, Mol Ther 19 408-16). [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] Therefore, anti-KLK5 therapies, such as passive immunotherapy aimed at inhibiting KLK5, which can exert therapeutic effects in diseases related to or caused by KLK5 dysregulation, remain necessary.

Means for Solving the Problem

[0011] The present invention addresses the above need by providing an inhibitory anti-KLK5 antibody according to the following embodiments.

[0012] Embodiment 1: A monoclonal antibody that binds to kallikrein 5 (KLK5), comprising a variable light chain and a variable heavy chain, and a. The variable light chain comprises CDR-L1 containing SEQ ID NO: 1 or 7 or 8 or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and b. The variable heavy chain comprises CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs: 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27 or 29, the above monoclonal antibody.

[0013] Embodiment 2: a. The variable light chain comprises CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, b. The variable heavy chain comprises CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 10 or 14 or 23, the antibody according to Embodiment 1.

[0014] Embodiment 3: a. The variable light chain comprises CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, b. The variable heavy chain comprises CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 23, the antibody according to Embodiment 1 or 2.

[0015] Embodiment 4: The antibody according to any one of Embodiments 1 to 3, wherein the antibody is a chimeric antibody or a humanized antibody.

[0016] Embodiment 5: The antibody according to any one of Embodiments 1 to 4, wherein the antibody is a full-length antibody.

[0017] Embodiment 6: The antibody according to embodiment 5, wherein the full-length antibody is selected from IgG1, IgG4 or IgG4P.

[0018] Embodiment 7: The antibody according to any one of embodiments 1 to 4, wherein the antibody is selected from Fab, Fab’, F(ab’)2, scFv, dAb or V HH The antibody according to any one of embodiments 1 to 4, wherein the antibody is selected from Fab, Fab’, F(ab’)2, scFv, dAb or V

[0019] Embodiment 8: The antibody is a. a variable light chain comprising SEQ ID NO: 30 or SEQ ID NO: 34 or SEQ ID NO: 38 or SEQ ID NO: 42 or SEQ ID NO: 46, and / or b. a variable heavy chain comprising SEQ ID NO: 32 or SEQ ID NO: 50 or SEQ ID NO: 54 or SEQ ID NO: 58 or SEQ ID NO: 62 or SEQ ID NO: 66 or SEQ ID NO: 70 or SEQ ID NO: 74 or SEQ ID NO: 78 or SEQ ID NO: 82 or SEQ ID NO: 86 or SEQ ID NO: 90 or SEQ ID NO: 94 or SEQ ID NO: 98 or SEQ ID NO: 102 or SEQ ID NO: 106 or SEQ ID NO: 110 or SEQ ID NO: 114 or SEQ ID NO: 118 or SEQ ID NO: 122 or SEQ ID NO: 126 or SEQ ID NO: 130 or SEQ ID NO: 134, preferably SEQ ID NO: 32 or SEQ ID NO: 50 or SEQ ID NO: 54 or SEQ ID NO: 58 or SEQ ID NO: 62 or SEQ ID NO: 66 or SEQ ID NO: 70 or SEQ ID NO: 74 or SEQ ID NO: 78 or SEQ ID NO: 82 or SEQ ID NO: 86 or SEQ ID NO: 90 or SEQ ID NO: 94 or SEQ ID NO: 98 or SEQ ID NO: 106 or SEQ ID NO: 110 or SEQ ID NO: 114 or SEQ ID NO: 118 or SEQ ID NO: 126 or SEQ ID NO: 134, The antibody according to any one of embodiments 1 to 7.

[0020] <00OO124>Embodiment 9: The antibody is a. a variable light chain comprising SEQ ID NO: 38, and / or b. a variable heavy chain comprising SEQ ID NO: 110, The antibody according to any one of embodiments 1 to 8.

[0021] Embodiment 10: The antibody is a. a light chain comprising SEQ ID NO: 36 or SEQ ID NO: 40 or SEQ ID NO: 44 or SEQ ID NO: 48, and b. a heavy chain comprising SEQ ID NO: 52 or SEQ ID NO: 56 or SEQ ID NO: 60 or SEQ ID NO: 64 or SEQ ID NO: 68 or SEQ ID NO: 72 or SEQ ID NO: 76 or SEQ ID NO: 80 or SEQ ID NO: 84 or SEQ ID NO: 88 or SEQ ID NO: 92 or SEQ ID NO: 96 or SEQ ID NO: 100 or SEQ ID NO: 104 or SEQ ID NO: 108 or SEQ ID NO: 112 or SEQ ID NO: 116 or SEQ ID NO: 120 or SEQ ID NO: 124 or SEQ ID NO: 128 or SEQ ID NO: 132 or SEQ ID NO: 136, preferably SEQ ID NO: 52 or SEQ ID NO: 56 or SEQ ID NO: 60 or SEQ ID NO: 64 or SEQ ID NO: 68 or SEQ ID NO: 72 or SEQ ID NO: 76 or SEQ ID NO: 80 or SEQ ID NO: 84 or SEQ ID NO: 88 or SEQ ID NO: 92 or SEQ ID NO: 96 or SEQ ID NO: 100 or SEQ ID NO: 108 or SEQ ID NO: 112 or SEQ ID NO: 116 or SEQ ID NO: 120 or SEQ ID NO: 128 or SEQ ID NO: 136, The antibody according to any one of embodiments 1 to 6 or 8 or 9.

[0022] Embodiment 11: The antibody is a. Light chain containing Sequence ID No. 40, and b. The antibody according to any one of Embodiments 1 to 8, comprising a heavy chain containing SEQ ID NO: 112.

[0023] Embodiment 12: The antibody according to any one of Embodiments 1 to 11, wherein KLK5 is human KLK5 containing SEQ ID NO: 142, 143, or 144, or cynomolgus monkey KLK5 containing SEQ ID NO: 151.

[0024] Embodiment 13: The antibody according to any one of Embodiments 1 to 12, wherein the antibody binds to kallikrein 5 (KLK5) and binds to a human KLK5 epitope comprising at least one, preferably at least two amino acid residues from the group consisting of Leu212, Ser213, Gln214, Lys215, Arg216, Glu218, Asp219, Ala220, Pro222, Gly233, Pro269, Asn270, and Pro272, with reference to Sequence ID No. 142.

[0025] Embodiment 14: The antibody according to Embodiment 13, wherein the epitope is characterized by X-ray crystallography.

[0026] Embodiment 15: The antibody is a. Inhibit or reduce the protease activity of KLK5, and / or b. When KLK5 binds to LEKTI or a fragment of LEKTI, it binds to KLK5 and / or c. KLK5 binding does not compete with LEKTI or LEKTI fragments, and / or d. The antibody according to any one of Embodiments 1 to 14, which forms a complex with LEKTI or KLK5 conjugated to a fragment of LEKTI.

[0027] Embodiment 16: The antibody according to Embodiment 15, wherein the LEKTI fragment is a human LEKTI domain 5 containing amino acids 1-64 of SEQ ID NO: 145 or a LEKTI domain 8 containing amino acids 1-71 of SEQ ID NO: 152.

[0028] Embodiment 17: The antibody according to any one of Embodiments 1 to 16, wherein the antibody binds to human KLK5, preferably human KLK5 comprising SEQ ID NO: 144, and cynomolgus monkey (cyno) KLK5, preferably cynomolgus monkey KLK5 comprising SEQ ID NO: 151.

[0029] Embodiment 18: The antibody according to any one of Embodiments 1 to 17, wherein the antibody does not bind to human or cynomolgus kallikrein 2 (KLK2), or human or cynomolgus kallikrein 4 (KLK4), or human or cynomolgus kallikrein 7 (KLK7).

[0030] Embodiment 19: Competing for binding of the antibody described in any one of Embodiments 1 to 18 to KLK5, and a. Regarding binding to KLK5, the antibody described in any one of Embodiments 1 to 18 is cross-blocked, or the antibody described in any one of Embodiments 1 to 18 is cross-blocked, b. An antibody that binds to KLK5 to the same epitope as the antibody described in any one of Embodiments 1 to 18, The antibody comprises a heavy chain variable region having at least 90% identity or similarity to the sequence of SEQ ID NO: 38, and / or a light chain variable region having at least 90% identity or similarity to the sequence of SEQ ID NO: 110.

[0031] Embodiment 20: An isolated polynucleotide encoding the antibody described in any one of Embodiments 1 to 18.

[0032] Embodiment 21: Polynucleotides are a. A light chain variable region in which polynucleotides are i. At least 90% identical to sequence number 31, 35, 39, 43, or 47, or ii. Including SEQ ID NOs. 31, 35, 39, 43, or 47, or iii. A light chain variable region essentially consisting of sequence numbers 31, 35, 39, 43, or 47, or b. A heavy chain variable region in which polynucleotides are i. At least 90% identical to international airports 33, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, or 135, or ii. Including international airports 33 or 51 or 55 or 59 or 63 or 67 or 71 or 75 or 79 or 83 or 87 or 91 or 95 or 99 or 103 or 107 or 111 or 115 or 119 or 123 or 127 or 131 or 135, or iii. A heavy chain variable region essentially consisting of international airports 33 or 51 or 55 or 59 or 63 or 67 or 71 or 75 or 79 or 83 or 87 or 91 or 95 or 99 or 103 or 107 or 111 or 115 or 119 or 123 or 127 or 131 or 135, or c. A light chain, which consists of polynucleotides, i. At least 90% identical to sequence number 37, 41, 45, or 49, or ii. Including sequence numbers 37, 41, 45, or 49, or iii. Light chains essentially consisting of sequence numbers 37, 41, 45, or 49, or d. A heavy chain, and the polynucleotides are i. At least 90% identical to international airports 53 or 57 or 61 or 65 or 69 or 73 or 77 or 81 or 85 or 89 or 93 or 97 or 101 or 105 or 109 or 113 or 117 or 121 or 125 or 129 or 133 or 137, or ii. International Airports including 53 or 57 or 61 or 65 or 69 or 73 or 77 or 81 or 85 or 89 or 93 or 97 or 101 or 105 or 109 or 113 or 117 or 121 or 125 or 129 or 133 or 137, or iii. Isolated polynucleotides according to Embodiment 20, which essentially comprise the number 53 or 57 or 61 or 65 or 69 or 73 or 77 or 81 or 85 or 89 or 93 or 97 or 101 or 105 or 109 or 113 or 117 or 121 or 125 or 129 or 133 or 137, and which encode a heavy chain.

[0033] Embodiment 22: A cloning vector or expression vector comprising one or more polynucleotides as described in any one of Embodiments 20 or 21.

[0034] Embodiment 23: A host cell, a. One or more polynucleotides as described in either Embodiment 20 or 21, or b. A host cell comprising one or more expression vectors as described in Embodiment 22.

[0035] Embodiment 24: A method for producing an antibody according to any one of Embodiments 1 to 18, comprising culturing a host cell according to Embodiment 23 under conditions suitable for antibody production, and isolating the antibody produced by the host cell.

[0036] Embodiment 25: A pharmaceutical composition comprising the antibody described in any one of Embodiments 1 to 18 and one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0037] Embodiment 26: An antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 18, or a pharmaceutical composition according to Embodiment 25, for use in therapeutic purposes.

[0038] Embodiment 27: An antibody according to any one of Embodiments 1 to 18 or a pharmaceutical composition according to Embodiment 25 for use in the treatment of a disease characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition.

[0039] Embodiment 28: The antibody for use according to Embodiment 27, wherein the disease is selected from Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or a combination thereof.

[0040] Embodiment 29: The antibody for use according to Embodiment 28, wherein the disease is Netherton syndrome.

[0041] Embodiment 30: The antibody for use according to Embodiment 28, wherein the disease is atopic dermatitis.

[0042] Embodiment 31: A method for treating a disease characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition in a patient, comprising the step of administering to the patient a therapeutically effective amount of an antibody according to any one of Embodiments 1 to 18 or a pharmaceutical composition according to Embodiment 25.

[0043] Embodiment 32: The method according to Embodiment 31, wherein the disease is selected from Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer.

[0044] Embodiment 33: The antibody for use according to Embodiment 32, wherein the disease is Netherton syndrome.

[0045] Embodiment 34: The antibody for use according to Embodiment 32, wherein the disease is atopic dermatitis. [Brief explanation of the drawing]

[0046] [Figure 1]This figure illustrates the inhibition of IP-1 release from KLK5-stimulated keratinocytes by antibody 10273. The amount of IP-1 released into the culture medium after keratinocyte stimulation with KLK5 was determined in the presence or absence of rabbit antibody 10273. Antibody 10273 inhibited IP-1 release, while the isotype control antibody did not show any inhibitory effect. [Figure 2] This figure plots Kobs values ​​against substrate concentrations of antibody 10273 and LEKTI-D5 Fc protein. The data shown is for 2nM antibody 10273 and 2nM LEKTI D5 Fc. The slope indicates that antibody 10273 is a non-competitive inhibitor, while the LEKTI protein is a competitive inhibitor. [Figure 3A] Panels A and B of the size exclusion chromatography show the elution profiles for human KLK5 alone (solid trace), rabbit Fab antibody 10273 alone (dotted trace), human KLK5 + LEKTI-D5 Fc (long dashed trace, panel A) or human KLK5 + LEKTI D8 Fc (long dashed trace, panel B), and human KLK5 + LEKTI D5 Fc + rabbit Fab antibody 10273 (short dashed trace, panel A) or human KLK5 + LEKTI D8 Fc + rabbit Fab antibody 10273 (short dashed trace, panel B). [Figure 3B] Panels A and B of the size exclusion chromatography show the elution profiles for human KLK5 alone (solid trace), rabbit Fab antibody 10273 alone (dotted trace), human KLK5 + LEKTI-D5 Fc (long dashed trace, panel A) or human KLK5 + LEKTI D8 Fc (long dashed trace, panel B), and human KLK5 + LEKTI D5 Fc + rabbit Fab antibody 10273 (short dashed trace, panel A) or human KLK5 + LEKTI D8 Fc + rabbit Fab antibody 10273 (short dashed trace, panel B). [Figure 4A]This figure shows the SDS-PAGE of the peak fraction from SEC shown in Figure 3A. Lane 1 is the MW marker, lanes 2 and 3 are the fractions of the two-component KLK5+LEKTI D5 complex, and lanes 4 and 5 are the peak fractions of the three-component KLK5+LEKTI D5+rabbit Fab antibody 10273 complex. [Figure 4B] Figure 3B shows the SDS-PAGE of the peak fractions from SEC. Lane 1 is the MW marker, lanes 2 and 3 are the peak fractions of the two-component KLK5+LEKTI D8 complex, and lanes 4 and 5 are the peak fractions of the three-component KLK5+LEKTI D8+rabbit Fab antibody 10273 complex. [Figure 5] This figure shows the SDS-PAGE of KLK5 produced for X-ray crystallography studies. Lane M is the MW marker. Lane 1 is human KLK5 purified from a culture grown in the presence of kifunensin (kif). Lane 2 is human KLK5 purified from the kifunensin culture and treated with endoglycosidase H (endoH). [Figure 6] This is a schematic diagram of the human KLK5 epitope in a complex with rabbit Fab antibody 10273. The Fab heavy and light chains are shown as dark and light surfaces, respectively, and as a transparent surface in the schematic diagram. The residues that form the epitope on human KLK5 bound to antibody 10273—Leu212(163), Ser213(164), Gln214(165), Lys215(166), Arg216(167), Glu218(169), Asp219(170), Ala220(171), Pro222(173), Gly233(184), Pro269(223), Asn270(224), and Pro272(226)—are shown as black bars. [Figure 7] This figure shows two orientations of the crystalline structure of human KLK5 in complex with rabbit Fab antibodies 10236 and 10273. Human KLK5 is shown as a ribbon shape, and rabbit Fab antibodies 10236 and 10273 are shown as three-dimensional surfaces. [Figure 8]This figure shows the humanization of the rabbit variable light chain sequence of antibody 10273. Grafts 10273gL2, 10273gL2 Q1R, 10273gL2 Q1K, and 10273gL2 Q1H are humanized grafts of the rabbit variable light chain of antibody 10273 using the IGKV1D-13 human germline as the acceptor framework. CDRs are shown in bold / underlined. Mutations in CDRL1 resulting in an overall increase in pI are shown in bold / underlined, and Q1R, Q1K, or Q1H are highlighted. [Figure 9] This figure shows the humanization of the rabbit variable heavy chain sequence of antibody 10273. Grafts 10273gH1, gH4, gH5, gH8, gH10, and gH11 are humanized rabbit variable heavy chain grafts of antibody 10273 using the variable heavy chain with the IGHV3-66 human germline as the acceptor framework. CDRs are shown in bold / underlined. Donor residues are shown in bold / italicized, with V24, I48, G49, K71, S73, and V78 shaded in gray. Mutations in CDR-H3 to remove a potential DP hydrolysis site (D116E) or to increase pI (D116N) are shown in bold / underlined and highlighted. [Figure 10] This figure shows the inhibition of human KLK5 by humanized grafts of antibody 10273. Humanized graft variants of antibody 10273 were assayed to determine the extent to which they inhibited KLK5 activity. Percentage inhibition values ​​were calculated, and the data were plotted as shown in Example 8. No Inh = No inhibitor, No Sub = No substrate, No Enz = No enzyme. [Figure 11] This figure shows the effect of vortex on the aggregation stability of humanized antibody 10273 in two buffers of different compositions and pH levels. [Modes for carrying out the invention]

[0047] Herein, the disclosure will be described with reference to certain non-limiting aspects and embodiments thereof, as well as to certain figures and examples.

[0048] Technical terms are used by common sense unless otherwise specified. When a particular meaning is conveyed by a particular term, the definition of that term is given in the context in which it is used.

[0049] When the term “comprising” is used herein and in the claims, it does not preclude other elements. For the purposes of this disclosure, the term “consisting of” is considered a preferred concretization of the term “comprising of.”

[0050] When an indefinite or definite article, such as "a," "an," or "the," is used to refer to a singular noun, unless otherwise specified, it includes the plural form of that noun.

[0051] As used herein, terms such as “treatment” and “to treat” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely cures a disease and / or adverse effects resulting from the disease. Thus, treatment encompasses any treatment of a disease in mammals, in particular humans, and includes (a) preventing the onset of the disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease, (b) inhibiting the disease, i.e., stopping the onset of the disease, and (c) reducing the disease, i.e., causing a regression of the disease.

[0052] The "therapeutic dose" refers to the amount of anti-alpha-synuclein antibody or its antigen-binding fragment that, when administered to a mammal or other subject to treat a disease, is sufficient to produce such treatment for the disease. The therapeutic dose varies depending on the anti-alpha-synuclein antibody or its antigen-binding fragment, the disease and its severity, and the age and weight of the subject being treated.

[0053] The term "isolated" throughout this specification means, in some cases, that an antibody, antigen-binding fragment, or polynucleotide exists in a physical environment different from that which may occur in nature.

[0054] In a first aspect of the present invention, an antibody that binds to kallikrein 5 (KLK5) is provided, the monoclonal antibody comprising a variable light chain and a variable heavy chain, and a. The variable light chain includes CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and b. The variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or one of SEQ ID NOs from 10 to 29, preferably one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29.

[0055] Kallikrein 5 (KLK5, KLK-L2, SCTE, or any other known synonym) possesses trypsin-like activity. It is expressed in a preproform and contains a 29-amino acid signal peptide followed by a 37-amino acid propeptide sequence, as measured by the bioinformatics tool SignalP 5.0 (http: / / www.cbs.dtu.dk / services / SignalP / index.php). Cleavage of the propeptide produces an active mature enzyme consisting of 237 amino acids, which has an active site with a catalytic triplicate of residues typical of serine proteases (Michael IP et al., 2005; JBC 280:15, 14628-35).

[0056] Unless otherwise specified, the term KLK5 refers to any natural preform and proform (i.e., untreated KLK5 containing the signal sequence and activating peptide), alternative splicing or natural variants, mutants and KLK5 from other species (e.g., mouse, cynomolgus monkey), and active KLK5 (resulting from self-cleavage or otherwise). Where human KLK5 is specified, human KLK5 includes the sequence shown in SEQ ID NO: 144 (active human KLK5). Other KLK5 sequences referred to herein include SEQ ID NO: 143 (human KLK5 proform lacking the signal sequence) or SEQ ID NO: 142 (full-length human KLK5 with the signal sequence and propeptide sequence), the sequence corresponding to Uniprot Q9Y337, or natural variants containing mutations at positions 55 and 153 (see SEQ ID NO: 142). Examples of these mutations include human KLK5 containing residues 23-293 as shown in Sequence ID No. 142, which has a Gly-to-Arg mutation at residue 55 (G55R) and / or an Asp-to-Asn mutation at residue 153 (D153N).

[0057] The antibody according to the present invention is a monoclonal antibody. Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler & Milstein, 1975, Nature, 256:495-497), trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4:72), and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

[0058] Antibodies for use in the present invention can also be produced using a monolymphocyte antibody method by cloning and expressing immunoglobulin variable region cDNA produced from a monolymphocyte selected to produce a specific antibody, for example, by the method described in Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-7848l, International Publication No. 92 / 02551, International Publication No. 2004 / 051268, and International Publication No. 2004 / 106377.

[0059] In one embodiment, the antibody that binds to kallikrein 5 (KLK5) includes a variable light chain comprising CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 10, 14, or 23.

[0060] In one preferred embodiment, the antibody that binds to kallikrein 5 (KLK5) includes a variable light chain comprising CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, as well as a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 23.

[0061] The antibody according to the present invention contains complementarity-determining regions (CDRs) that are three derived from the heavy chain and three derived from the light chain. Generally, the CDRs are located within a framework and together form a variable region. By convention, the CDRs in the heavy chain variable region of an antibody or its antigen-binding fragment are called CDR-H1, CDR-H2, and CDR-H3, and the CDRs in the light chain variable region are called CDR-L1, CDR-L2, and CDR-L3. They are numbered sequentially from the N-terminus to the C-terminus of each chain.

[0062] CDRs are numbered according to the system conventionally devised by Kabat et al. This system is described in Kabat et al., 1991, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al."). This numbering system is used herein unless otherwise indicated.

[0063] The naming of Kabat residues does not necessarily directly correspond to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering corresponding to the shortening or insertion of structural components, regardless of the framework of the basic variable domain structure or the complementarity-determining region (CDR). The correct Kabat numbering of residues can be determined for a given antibody by aligning homologous residues in the antibody sequence with the "standard" Kabat numbering sequence.

[0064] The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia, C. and Lesk, AMJ Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Therefore, unless otherwise specified, "CDR-H1" as used herein is intended to refer to residues 26-35 as described by the combination of the Kabat numbering system and Chothia's topological loop definition.

[0065] The CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3), according to the Kabat numbering system.

[0066] In addition to the CDR loop, a fourth loop exists between CDR-2 (CDR-L2 or CDR-H2) and CDR-3 (CDR-L3 or CDR-H3), which is formed by Framework 3 (FR3). The Kabat numbering system defines Framework 3 as the heavy chain positions 66-94 and the light chain positions 57-88.

[0067] As used in the context of this disclosure, the term “antibody” includes whole antibodies and their functionally active fragments, also called antigen-binding fragments, which are molecules containing an antigen-binding domain that specifically binds to an antigen. The characteristics described herein with respect to antibodies also apply to antigen-binding fragments unless otherwise indicated in the context. Antibodies may be monoclonal, polyvalent, multispecific, bispecific, fully human, humanized, or chimeric (or derived therefrom).

[0068] Whole antibodies, also known as "immunoglobulins (Ig)," generally refer to intact or full-length antibodies, i.e., antibodies comprising two heavy chains and two light chains interconnected by disulfide bonds that assemble to define a characteristic Y-shaped three-dimensional structure. Classical natural whole antibodies are monospecific in that they bind to one antigen type and bivalent in that they have two independent antigen-binding domains. The terms "intact antibody," "full-length antibody," and "whole antibody" are used interchangeably to refer to monospecific bivalent antibodies having a structure similar to that of a natural antibody, including the Fc region as defined herein.

[0069] Each light chain consists of a light chain variable region (hereinafter abbreviated as VL) and a light chain constant region (CL). Each heavy chain, depending on the Ig class, consists of a heavy chain variable region (hereinafter abbreviated as VH) and a heavy chain constant region (CH) consisting of three constant domains CH1, CH2, and CH3, or four constant domains CH1, CH2, CH3, and CH4. The “class” of Ig or antibody refers to the type of constant region, including IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, and IgG4. The constant region of an antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors including the first component (Clq) of the classical complement system.

[0070] As used herein, the terms “constant region” or “constant domain” are used interchangeably to refer to the domain of an antibody located outside the variable region. The constant domain is identical in all antibodies of the same isotype, but differs from isotype to isotype. Typically, the constant region of a heavy chain is formed from three or four constant domains, CH1-hinge-CH2-CH3-, and possibly CH4, from the N-terminus to the C-terminus.

[0071] The constant region domain of the antibody molecule of the present invention, if present, can be selected considering the function of the proposed antibody, particularly any effector function that may be required. For example, the constant region domain may be a human IgA, IgD, IgE, IgG, or IgM domain. In particular, if the antibody is intended for therapeutic use and antibody effector function is required, the human IgG constant region domain, especially those of the IgG1 and IgG3 isotypes, can be used. Alternatively, if the antibody is intended for therapeutic purposes and antibody effector function is not required, the IgG2 and IgG4 isotypes can be used. It will be understood that sequence variants of these constant region domains can also be used. For example, IgG4 in which the serine (numbered according to the Kabat numbering system) proline at position 241 has been changed is described by Angal et al. (Angal et al., 1993). A single amino acid substitution eliminates the heterogeneity of chimeric mouse / human (IgG4) antibodies observed during SDS-PAGE analysis (Mol Immunol 30, 105-108), and this can be used. This is referred to herein as IgG4P. This single amino acid substitution disrupts the natural tendency of the IgG4 molecule's heavy chain to exchange and produce a chimeric molecule.

[0072] The terms “Fc region,” “Fc fragment,” or simply “Fc” are used interchangeably to refer to the C-terminal region of an antibody, including the constant region of the antibody excluding the first constant-region immunoglobulin domain. Therefore, Fc refers to the last two constant domains, CH2 and CH3, of IgA, IgD, and IgG, or the last three constant domains of IgE and IgM, as well as the N-terminus of the flexible hinge to these domains. The human IgG1 heavy chain Fc region is defined herein as including residue C226 to its carboxyl terminus, with numbering following the EU index as defined by Kabat. In the context of human IgG1, according to the EU index as defined by Kabat, the lower hinge refers to positions 226–236, the CH2 domain to positions 237–340, and the CH3 domain to positions 341–447. The corresponding Fc regions of other immunoglobulins can be identified by sequence alignment.

[0073] In the context of this disclosure, the constant region or Fc region, if present, may be natural as defined above, or may be modified in various ways, insofar as it includes a functional FcR-binding domain, preferably a functional FcRn-binding domain. Preferably, the modified constant region or Fc region improves functionality and / or pharmacokinetics. The modification may include deletion of a specific portion of the Fc fragment. The modification may further include various amino acid substitutions that can affect the biological properties of the antibody. Mutations to increase FcRn binding, and therefore in vivo half-life, may also be present. The modification may further include modification of the antibody's glycosylation profile. In the natural Fc fragment, the N-glycan bound to the asparagine residue at position 297 (Asn297) is glycosylated in the CH2 domain present in each of the two heavy chains. In the context of this disclosure, the antibody may be genetically engineered and glycosylated to have a specific glycosylation profile that results in, for example, improved properties, such as improved effector function, or improved serum half-life.

[0074] Antibody antigen-binding fragments include single-chain antibodies (e.g., scFv and dsscFv), Fab, Fab', F(ab')2, Fv, single-domain antibodies, or nanobodies (e.g., VH or VL, or VHH or VNAR). Other antibody fragments for use in the present invention include Fab and Fab' fragments described in International Publication Nos. 2011 / 117648, 2005 / 003169, 2005 / 003170, and 2005 / 003171 (all incorporated herein by reference).

[0075] Methods for producing and manufacturing these antibody fragments are known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0076] A typical “Fab' fragment” or “Fab'” as used herein comprises a heavy-chain and a light-chain pair, the heavy chain comprising a variable region VH, a constant domain CH1, and a native or modified hinge region, and the light chain comprising a variable region VL and a constant domain CL. Dimers of Fab' according to this disclosure produce F(ab')2, for example, by which dimerization may be hinge-mediated.

[0077] As used herein, the term "single-domain antibody" refers to an antibody fragment consisting of a single monomeric variable antibody domain. Examples of single-domain antibodies include VH, VL, VHH, or V-NAR.

[0078] "Fv" refers to two variable domains, such as congeneral pairs or affinity maturation variable domains, i.e., cooperative variable domains such as VH and VL pairs.

[0079] As used herein, "single-stranded variable fragment" or "scFv" refers to a single-stranded variable fragment stabilized by a peptide linker between the VH variable domain and the VL variable domain.

[0080] As used herein, “disulfide-stabilized single-stranded variable fragment” or “dsscFv” refers to a single-stranded variable fragment stabilized by a peptide linker between the VH and VL variable domains, and also containing an interdomain disulfide bond between VH and VL (see, for example, Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012, International Publication No. 2007109254).

[0081] The disulfide bond between the variable domains VH and VL is located between two of the residues listed below (unless otherwise indicated in context, Kabat numbering is used in the following list) (Protein Science 6,781-788 Zhu et al(1997), Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012, J Biochem. 118,825-831 Luo et al(1995), FEBS Letters 377 135-139 Young et al(1995), Proc.Natl.Acad.Sci.USA Vol.90 pp.7538-7542 Brinkmann et al(1993), Proteins 19,35-47 Jung et al(1994) Biochemistry 29 1362-1367, Glockshuber et al (1990). When Kabat numbering is referenced, the relevant reference is Kabat et al., 1991 (5 th edition, Bethesda, Md.), in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.

[0082] VH37+VL95C, ·VH44+VL100, VH44 + VL105, VH45+VL87, • VH55 + VL101, ·VH100+VL50, · VH100b+VL4 VH98+VL46, VH101+VL46, VH105+VL43, VH106+VL57, and includes one or more corresponding positions in a pair of variable regions located within the molecule.

[0083] As used herein, the term “antibody” also includes monovalent antibodies, i.e., antibodies containing only one antigen-binding domain (for example, a one-arm antibody containing interconnected full-length heavy and full-length light chains, also called a “half-antibody”).

[0084] The term "antibody" also encompasses polyvalent antibodies, including those with multiple specificities, such as bispecific, tripspecific, or multispecific antibodies.

[0085] As used herein, "multispecific" or "multi-specific antibody" refers to an antibody described herein having at least two binding domains, i.e., two or more binding domains, for example, two or three binding domains, where at least two binding domains independently bind to two different antigens or two different epitopes on the same antigen (also known as multiparatopic). Multispecific antibodies are generally monovalent for each specificity (antigen). The multispecific antibodies described herein include monovalent and, for example, bivalent, trivalent, and tetravalent multispecific antibodies.

[0086] As used herein, “antigen-binding domain” refers to a portion of an antibody comprising one or more variable domains that specifically interact with a target antigen, for example, a pair of variable domains VH and VL, in whole or in part. The binding domain may include a single-domain antibody. In one embodiment, each binding domain is monovalent. Preferably, each binding domain comprises one or fewer VH and one VL.

[0087] Various multispecific antibody formats are known in the art. Although various classifications have been proposed, multispecific IgG antibody formats generally include, for example, bispecific IgG, IgG adducts, multispecific (e.g., bispecific) antibody fragments, multispecific (e.g., bispecific) fusion proteins, and multispecific (e.g., bispecific) antibody conjugates, as described, for example, Spiess et al., Mol Immunol. 67(2015):95-106.

[0088] Techniques for producing bispecific antibodies include, but are not limited to, CrossMab technology (Klein et al., Methods 154(2019)21-31), Knobs-in-holes engineering (e.g., International Publication No. 1996027011, International Publication No. 1998050431), DuoBody technology (e.g., International Publication No. 2011131746), and Azymetric technology (e.g., International Publication No. 2012058768). Further techniques for producing bispecific antibodies are described, for example, in Godar et al., 2018, Expert Opinion on Therapeutic Patents, 28:3, 251-276. Examples of bispecific antibodies include CrossMab antibodies, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, Knobs-in-holes common LC, Knobs-in-holes assembly, charge pair, Fab arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body, and orthogonal Fab.

[0089] Additive IgG classically comprises full-length IgG modified by adding additional antigen-binding domains or antigen-binding fragments to the N-terminus and / or C-terminus of the heavy and / or light chains of IgG. Examples of such additional antigen-binding fragments include sdAb antibodies (e.g., VH or VL), Fv, scFv, dsscFv, Fab, and scFav. Additional IgG antibody formats include, for example, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-Fv, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-IgG, Zybody, and DVI-IgG (four-in-one), as described in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106.

[0090] For example, as described in Spiess et al., for bispecific antibodies. Mol Immunol. 67(2015):95-106, examples of multispecific antibody fragments include nanobodies, nanobodies-HAS, BiTE, bispecific antibodies, DART, TandAb, sc bispecific antibodies, sc-bispecific antibodies-CH3, bispecific antibodies-CH3, triple bodies, mini antibodies, mini bodies, Tri Bi mini bodies, scFv-CH3KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc bispecific antibodies-Fc, bispecific antibodies-Fc, tandem scFv-Fc, and intracellular antibodies.

[0091] Examples of multispecific fusion proteins include Dock and Lock, ImmTAC, HSAbody, sc bispecific antibody-HAS, and tandem scFv-Toxin. Examples of multispecific antibody conjugates include IgG-IgG, Cov-X-Body, and scFv1-PEG-scFv2.

[0092] Additional multispecific antibody formats, such as tandem scFv, triplebody, Fab-VHH, taFv-Fc, scFv4-Ig, scFv2-Fcab, and scFv4-IgG, are described, for example, in Brinkmann and Kontermann, mAbs, 9:2, 182-212 (2017), particularly in Figure 2. Vibodies, tripodies, and methods for producing them are disclosed, for example, in International Publication No. 99 / 37791.

[0093] The added IgG and added Fab each comprise a whole IgG or Fab fragment that has been modified by adding at least one additional antigen-binding domain (e.g., two, three, or four additional antigen-binding domains), such as a single-domain antibody (e.g., VH or VL, or VHH), scFv, dsscFv, or dsFv to the N-terminus and / or C-terminus of the heavy and / or light chain of the IgG or Fab, as described, for example, in International Publication Nos. 2009 / 040562, 2010035012, 2011 / 030107, 2011 / 061492, 2011 / 061246, and 2011 / 086091, to the N-terminus and / or C-terminus of the heavy and / or light chain of the IgG or Fab. In particular, the Fab-Fv format was first disclosed in International Publication No. 2009 / 040562, and its disulfide-stabilized version, Fab-dsFv, was first disclosed in International Publication No. 2010 / 035012. Single-linker Fab-dsFv, in which dsFv is linked to Fab via a single linker between the VL or VH domain of Fv and the C-terminus of the LC or HC of Fab, was first disclosed in International Publication No. 2014 / 096390, which is incorporated herein by reference. Additive IgG, including full-length IgG1 manipulated by adding dsFv to the C-terminus of the heavy or light chain of IgG, was first disclosed in International Publication No. 2015 / 197789, which is incorporated herein by reference.

[0094] Alternatively, another multispecific format includes a Fab linked to two scFv or dsscFv, each scFv or dsscFv bound to the same or different target (e.g., one scFv or dsscFv that binds to a therapeutic target and another scFv or dsscFv that extends the half-life by binding to albumin, for example). Such antibody fragments are described in International Publication 2015 / 197772, which is incorporated herein by reference in its entirety. Another format includes a Fab linked to just one scFv or dsscFv, as described in International Publication 2013 / 068571 and Dave et al., Mabs, 8(7)1319-1335(2016), which are incorporated herein by reference.

[0095] Other known formats of multispecific antibodies include: As used herein, a bispecific antibody refers to two Fv pairs having two Fv-linkers such that the VH of the first Fv is linked to the VL of the second Fv, and the VL of the first Fv is linked to the VH of the second Fv, a first VH / VL pair, and a further VH / VL pair.

[0096] As used herein, a triabody refers to a format similar to that of a bispecific antibody containing three Fvs and three inter-Fv linkers.

[0097] As used herein, a tetrabody refers to a format similar to that of a bispecific antibody containing four Fvs and four inter-Fv linkers.

[0098] As used herein, a tandem scFv refers to at least two scFvs linked via a single linker such that a single inter-Fv linker exists.

[0099] As used herein, tandem scFv-Fc refers to at least two tandem scFvs, each attached, for example, via a hinge, to the N-terminus of the CH2 domain of a constant region fragment-CH2CH3.

[0100] As used herein, Fab-Fv refers to an Fv fragment having variable regions attached to the C-terminuses of CH1 in the heavy chain and CL in the light chain, respectively. The format can be provided as a PEGylated version.

[0101] Fab'-Fv, as used herein, is equivalent to FabFv, where the Fab portion is replaced by Fab'. The format can be provided as its PEGylated version.

[0102] As used herein, Fab-dsFv refers to a FabFv in which an intracellular disulfide bond stabilizes the added C-terminal variable region. The format can be provided as a PEGylated version thereof.

[0103] As used herein, Fab-scFv is a Fab molecule in which scFv is attached to the C-terminus of either the light chain or the heavy chain.

[0104] As used herein, Fab'-scFv is a Fab' molecule in which scFv is attached to the C-terminus of either the light chain or the heavy chain.

[0105] As used herein, DiFab refers to two Fab molecules linked via the C-terminus of a heavy chain.

[0106] As used herein, DiFab' refers to two Fab' molecules linked via one or more disulfide bonds within their hinge region.

[0107] As used herein, an sc bispecific antibody is a bispecific antibody containing an intra-Fv linker such that the molecule contains three linkers, the VH and VL ends of which are each linked to one of the variable regions of a further Fv pair, forming a typical scFv.

[0108] When used herein, Sc bispecific antibody-Fc is two sc bispecific antibodies, each of which is attached, for example, via a hinge, to the N-terminus of the CH2 domain of the constant region fragment-CH2CH3.

[0109] As used herein, ScFv-Fc-scFv refers to four scFvs, each of which is attached to both the N-terminus and C-terminus of both the heavy and light chains of the -CH2CH3 fragment.

[0110] As used herein, Sc bispecific antibody-CH3 refers to two sc bispecific antibody molecules, each linked to a CH3 domain, for example, via a hinge.

[0111] When used herein, IgG-scFv is a full-length antibody having an scFv at the C-terminus of each heavy chain or each light chain.

[0112] When used herein, scFv-IgG is a full-length antibody having scFv at the N-terminus of each heavy chain or each light chain.

[0113] When used herein, V-IgG is a full-length antibody having a variable domain at the N-terminus of each heavy chain or each light chain.

[0114] As used herein, IgG-V is a full-length antibody having a variable domain at the C-terminus of each heavy chain or each light chain.

[0115] DVD-Ig (also known as dual V-domain IgG) is a full-length antibody that has four additional variable domains, one at the N-terminus of each heavy chain and one at the N-terminus of each light chain.

[0116] The monoclonal antibody according to the present invention is preferably a full-length antibody. More preferably, the full-length antibody is selected from IgG1, IgG4, or IgG4P.

[0117] In another embodiment, the monoclonal antibody is selected from Fab, Fab', F(ab')2, scFv, dAb, or VHH.

[0118] In one embodiment, the antibody according to the present invention may include a framework region of the animal in which the antibody was produced. For example, if the antibody is produced in a rabbit, the antibody includes the CDR defined above and the framework region of the rabbit antibody, for example, a light chain variable region according to SEQ ID NO: 30 (its nucleotide sequence is shown in SEQ ID NO: 31) and a heavy chain variable region according to SEQ ID NO: 32 (its nucleotide sequence is shown in SEQ ID NO: 33).

[0119] In one embodiment, the antibody may be a chimeric or humanized antibody. Alternatively, the antibody may be human.

[0120] Chimeric antibodies are typically produced using recombinant DNA methods. DNA can be modified by replacing the coding sequences of the human L and H chains with the corresponding non-human (e.g., mouse or rabbit) H and L constant regions (Morrison; PNAS 81, 6851 (1984)).

[0121] Human antibodies include heavy or light chain variable regions or full-length heavy or light chains that are “products” of or “derived” from a particular germline sequence, if the variable region or full-length chain of the antibody is obtained from a system using human germline immunoglobulin genes. Such systems include immunizing transgenic mice possessing human immunoglobulin genes with the antigen of interest, or screening human immunoglobulin gene libraries presented on phages with the antigen of interest. Human antibodies or fragments thereof that are “products” of or “derived” from a human germline immunoglobulin sequence can be identified by comparing the amino acid sequence of the human antibody with the amino acid sequence of the human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is most closely sequenced to the human antibody sequence (i.e., maximum % identity). Human antibodies that are “products” of or “derived” from a particular human germline immunoglobulin sequence may have amino acid differences compared to the germline sequence, for example, due to the intentional introduction of naturally occurring somatic mutations or site-directed mutations. However, selected human antibodies typically contain amino acid residues that identify them as human, such that their amino acid sequence is at least 90% identical to the amino acid sequence encoded by the human germline immunoglobulin gene when compared to the germline immunoglobulin amino acid sequence of another species (e.g., mouse germline sequence). In specific cases, human antibodies may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, human antibodies derived from a particular human germline sequence show a difference of 10 or fewer amino acids from the amino acid sequence encoded by the human germline immunoglobulin gene. In specific cases, human antibodies may show a difference of 5 or fewer amino acids from the amino acid sequence encoded by the germline immunoglobulin gene, or even 4, 3, 2, or 1 or fewer amino acids.

[0122] Human antibodies can be produced by many methods known to those skilled in the art. Human antibodies can be produced by hybridoma using human myeloma or mouse-human heteromyeloma cell lines (Kozbor, J Immunol; (1984) 133:3001; Brodeur, Monoclonal Isolated Antibody Production Techniques and Applications, pp51-63, Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both of which utilize the human variable region repertoire (Winter G; (1994) Annu Rev Immunol 12:433-455, Green LL, (1999) J Immunol Methods 231:1 1-23).

[0123] In one preferred embodiment of the present invention, the antibody according to the present invention is humanized.

[0124] In one preferred embodiment, the monoclonal antibody bound to kallikrein 5 (KLK5) comprises a variable light chain and a variable heavy chain, and a. The variable light chain includes CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and b. The variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs: 10-29, preferably any one of 10, 11, 13-16, 18, 20, 22-25, 27, or 29. The antibody is humanized, and more preferably the variable light chain comprises CDR-L1 containing SEQ ID NO: 7, and the variable heavy chain comprises CDR-H3 containing SEQ ID NO: 23.

[0125] In a more preferred embodiment, the monoclonal antibody according to the present invention is preferably a full-length antibody conjugated to kallikrein 5 (KLK5) and containing a variable light chain and a variable heavy chain, and a. The variable light chain includes CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and b. The variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or one of SEQ ID NOs from 10 to 29, preferably one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29. The antibody is humanized, more preferably the variable light chain comprises CDR-L1 containing SEQ ID NO: 7, and the variable heavy chain comprises CDR-H3 containing SEQ ID NO: 23. Even more preferably the full-length antibody is selected from IgG1, IgG4, or IgG4P.

[0126] As used herein, the term “humanized” antibody refers to an antibody whose heavy chain and / or light chain comprises one or more CDRs (optionally including one or more modified CDRs) derived from a donor antibody (e.g., a non-human antibody such as a mouse or rabbit monoclonal antibody) transplanted into the heavy chain and / or light chain variable region framework of an acceptor antibody (e.g., a human antibody). For a review, see Vaughan et al., Nature Biotechnology, 16, 535-539, 1998. In one embodiment, rather than the entire CDR being transferred, only one or more specificity-determining residues from any one of the CDRs described above are transferred into the human antibody framework (e.g., see Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only specificity-determining residues from the above one or more CDRs are transferred into the human antibody framework. In another embodiment, only specificity-determining residues from each of the above CDRs are transferred into the human antibody framework.

[0127] When a CDR is implanted, any suitable acceptor variable region framework sequence can be used, taking into account the class / type of the donor antibody from which the CDR originates, and including mouse, primate, and human framework regions.

[0128] Preferably, the humanized monoclonal antibody according to the present invention has a variable domain comprising a human acceptor framework region and one or more CDRs specifically provided herein. Thus, in one embodiment, a humanized monoclonal antibody that binds to KLK5 is provided, wherein the variable domain comprises a human acceptor framework region and a non-human donor CDR.

[0129] Examples of human frameworks that can be used in the present invention include KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al., previously mentioned). For example, KOL and NEWM can be used in the heavy chain, REI in the light chain, and EU, LAY, and POM can be used in both the heavy and light chains. Alternatively, human germline sequences can be used, which are available at http: / / www.imgt.org / .

[0130] In the humanized antibody according to the present invention, the acceptor heavy chain and light chain do not necessarily have to originate from the same antibody, and if desired, a composite chain having framework regions derived from different chains may be included.

[0131] The framework region suitable for the light chain of the humanized monoclonal antibody according to the present invention includes SEQ ID NO: 138 and is derived from the human germline IGKV1D-13 JK4, whose nucleotide sequence is shown in SEQ ID NO: 139.

[0132] The framework region suitable for the heavy chain of the humanized monoclonal antibody according to the present invention includes the sequence shown in SEQ ID NO: 140, and its nucleotide sequence is derived from the human germline IGHV3-66 JH6 shown in SEQ ID NO: 141.

[0133] Therefore, in one embodiment, a humanized monoclonal antibody that binds to KLK5 is provided, the antibody comprising a variable light chain and a variable heavy chain, and a. The variable light chain includes CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and b. The variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs: 10-29, preferably any one of 10, 11, 13-16, 18, 20, 22-25, 27, or 29. More preferably, the variable light chain comprises CDR-L1 containing SEQ ID NO: 7, the variable heavy chain comprises CDR-H3 containing SEQ ID NO: 23, the light chain framework region is derived from human germline IGKV1D-13 JK4 containing SEQ ID NO: 138, and the heavy chain framework region is derived from human germline IGHV3-66 JH6 containing SEQ ID NO: 140.

[0134] In the humanized monoclonal antibody according to the present invention, the framework region does not have to have the exact same sequence as that of the acceptor antibody. For example, abnormal residues may be replaced with residues that occur more frequently for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework region may be modified so that they correspond to residues found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such modifications should be kept to the minimum necessary to restore the affinity of the donor antibody. A protocol for selecting residues in the acceptor framework region that may need to be modified is described in International Publication No. 91 / 09967 (which is incorporated herein by reference).

[0135] Therefore, in one embodiment, one, two, three, four, five, six, seven, or eight residues in the framework are replaced with alternative amino acid residues.

[0136] Therefore, in one embodiment, a humanized monoclonal antibody according to the present invention is provided, wherein at least the residues at positions 24, 48, 49, 71, 73, and 78 of the variable heavy chain (see SEQ ID NO: 140) are donor residues.

[0137] Preferably, the residue at position 24 of the variable heavy chain is a valine residue (instead of alanine), the residue at position 48 is isoleucine (instead of valine), the residue at position 49 is glycine (instead of serine), the residue at position 71 is lysine (instead of arginine), the residue at position 73 is serine (instead of asparagine), and the residue at position 78 is valine (instead of leucine).

[0138] Therefore, a humanized monoclonal antibody that binds to KLK5 is provided, and the antibody is, a. The variable light chain comprises CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and b. The variable heavy chain comprises CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs: 10-29, preferably any one of 10, 11, 13-16, 18, 20, 22-25, 27, or 29. More preferably, the variable light chain comprises CDR-L1 containing SEQ ID NO: 7, the variable heavy chain comprises CDR-H3 containing SEQ ID NO: 23, the light chain framework region is derived from human germline IGKV1D-13 JK4 containing SEQ ID NO: 138, the heavy chain framework region is derived from human germline IGHV3-66 JH6 containing SEQ ID NO: 140, and the amino acid residues at positions 24, 48, 49, 71, 73 and 78 of the variable heavy chain (see SEQ ID NO: 140) are donor residues.

[0139] Therefore, in one embodiment, the humanized monoclonal antibody that binds to KLK5 is a. Variable light chains including sequence numbers 34, 38, 42, or 46, and b. Includes a variable heavy chain containing the following attendance codes: 50 or 54 or 58 or 62 or 66 or 70 or 74 or 78 or 82 or 86 or 90 or 94 or 98 or 102 or 106 or 110 or 114 or 118 or 122 or 126 or 130 or 134, preferably 32 or 50 or 54 or 58 or 62 or 66 or 70 or 74 or 78 or 82 or 86 or 90 or 94 or 98 or 106 or 110 or 114 or 118 or 126 or 134.

[0140] Preferably, the humanized monoclonal antibody that binds to KLK5 is a. Variable light chains including Sequence ID No. 38, and b. Contains variable heavy chains including sequence number 110.

[0141] In one embodiment, the present invention provides an antibody comprising a sequence that is 80% similar or identical to a sequence disclosed herein, for example, a related sequence, for example, a variable domain sequence, a CDR sequence, or a sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar or identical to a part or all of a variable domain sequence excluding a CDR. In one embodiment, the related sequence is SEQ ID NO: 38. In one embodiment, the related sequence is SEQ ID NO: 110.

[0142] In one embodiment, a monoclonal antibody that binds to KLK5 comprises a light chain and a heavy chain, wherein the variable light chain comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence contained in SEQ ID NO: 38, and / or the variable heavy chain comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence contained in SEQ ID NO: 110.

[0143] As used herein, “identity,” “same,” or its grammatical variations thereof indicate that at any particular position in the aligned sequences, amino acid residues are identical between sequences. As used herein, “similarity,” “similar,” or its grammatical variations thereof indicate that at any particular position in the aligned sequences, amino acid residues are of similar type between sequences. For example, leucine may be used instead of isoleucine or valine. Other amino acids that may be substituted for each other include, but are not limited to, - Phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains), - Lysine, arginine, and histidine (amino acids with basic side chains), - Aspartic acid and glutamic acid (amino acids with acidic side chains), -Asparagine and glutamine (amino acids with amide side chains), and - Examples include cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0144] The degree of identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; BLAST™ software available from NCBI (Altschul, SFet al.,1990, J.Mol.Biol.215:403-410, Gish,W.&States,DJ1993,Nature Genet.3:266-272.Madden,TLet al.,1996,Meth.Enzymol.266:131-141, Altschul,SFet al.,1997,Nucleic Acids Res.25:3389-3402, Zhang, J. & Madden, TL1997, Genome Res.7:649-656).

[0145] In one embodiment, the antibody is preferably a full-length antibody selected from IgG1 and IgG4 or IgG4P.

[0146] Therefore, the present invention binds to kallikrein 5 (KLK5), and a. The variable light chain comprises CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and b. The variable heavy chain comprises CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs: 10-29, preferably any one of 10, 11, 13-16, 18, 20, 22-25, 27, or 29. More preferably, a full-length humanized monoclonal antibody is provided, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO: 7, and the variable heavy chain comprises CDR-H3 containing SEQ ID NO: 21, and the antibody is an IgG4P isoform.

[0147] The present invention also combines with KLK5, a. Light chains containing sequence numbers 36, 40, 44, or 48, and b. Provides a full-length humanized monoclonal antibody comprising a heavy chain containing the following frequencies: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134.

[0148] Preferably, the full-length humanized monoclonal antibody that binds to KLK5 is a. Light chain containing Sequence ID No. 40, and b. Contains a heavy chain including sequence number 112.

[0149] In one embodiment, the monoclonal antibody that binds to KLK5 is a light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar to or identical to the sequence shown in SEQ ID NO: 40, wherein the antibody has a sequence containing SEQ ID NO: 7 (or SEQ ID NO: 1, 8, or 9) for CDR-L1, a sequence containing SEQ ID NO: 2 for CDR-L2, and a sequence containing SEQ ID NO: 3 for CDR-L3, and SEQ ID NO: 112 A heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar to or identical to the sequence shown in, wherein the antibody has a sequence that includes SEQ ID NO: 4 for CDR-H1, SEQ ID NO: 5 for CDR-H2, and SEQ ID NO: 6 or one of SEQ ID NOs from 10 to 29 for CDR-H3, preferably one of SEQ ID NOs from 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, comprising the above heavy chain.

[0150] In yet another embodiment, the monoclonal antibody that binds to KLK5 is a Fab' fragment comprising a light chain variable region containing SEQ ID NO: 38 and a heavy chain variable region containing SEQ ID NO: 110.

[0151] In another embodiment, the monoclonal antibody that binds to KLK5 is a full-length IgG4 antibody comprising a light chain containing SEQ ID NO: 40 and a heavy chain containing SEQ ID NO: 112.

[0152] It will be understood by those skilled in the art that antibodies can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the antibody. Such modifications can include variations of glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A frequent modification is the loss of a carboxyl-terminal basic residue (such as lysine or arginine) by the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Therefore, the C-terminal lysine of the antibody heavy chain may not be present.

[0153] In one embodiment, the C-terminal amino acid derived from the antibody is cleaved during post-translational modification.

[0154] In one embodiment, the N-terminal amino acid derived from the antibody is cleaved during post-translational modification.

[0155] In another embodiment, the monoclonal antibody according to the present invention preferably binds to human KLK5 including SEQ ID NO: 144, 143, or 142, and also binds to cynomolgus monkey KLK5, preferably cynomolgus monkey KLK5 including SEQ ID NO: 151.

[0156] In one embodiment, the monoclonal antibody is conjugated to human and / or cynomolgus kallikrein 5 (KLK5), the antibody comprises a variable light chain and a variable heavy chain, and a. The variable light chain includes CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3; the variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29; b. The variable light chain includes color codes 30 or 34 or 38 or 42 or 46, and the variable heavy chain includes color codes 32 or 50 or 54 or 58 or 62 or 66 or 70 or 74 or 78 or 82 or 86 or 90 or 94 or 98 or 102 or 106 or 110 or 114 or 118 or 122 or 126 or 130 or 134, preferably 32 or 50 or 54 or 58 or 62 or 66 or 70 or 74 or 78 or 82 or 86 or 90 or 94 or 98 or 106 or 110 or 114 or 118 or 126 or 134, or c. The monoclonal antibody is a full-length antibody comprising a light chain and a heavy chain, wherein the light chain comprises SEQ ID NOs. 36, 40, 44, or 48, and the heavy chain comprises SEQ ID NOs. 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136.

[0157] In a preferred embodiment, the monoclonal antibody is conjugated to human and / or cynomolgus kallikrein 5 (KLK5), and the antibody comprises a variable light chain and a variable heavy chain. a. The variable light chain includes CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and the variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 23, or b. The variable light chain includes SEQ ID NO: 38, and the variable heavy chain includes SEQ ID NO: 110, or c. The monoclonal antibody is a full-length antibody containing a light chain and a heavy chain, the light chain containing SEQ ID NO: 40 and the heavy chain containing SEQ ID NO: 112.

[0158] In another embodiment, the monoclonal antibody according to the present invention does not bind to human or cynomolgus kallikrein 2 (KLK2), human or cynomolgus kallikrein 4 (KLK4), or human or cynomolgus kallikrein 7 (KLK7). In other words, the antibody is specific to KLK5 and not specific to other kallikreins.

[0159] As used herein, "specific" refers to an antibody that recognizes only a specific antigen, or an antibody that has a significantly higher binding affinity to a specific antigen (e.g., KLK5) compared to binding to a nonspecific antigen (gamma-synuclein and beta-synuclein), for example, at least 5, 6, 7, 8, 9, or 10 times higher binding affinity.

[0160] In one embodiment, the monoclonal antibody binds to human and / or cynomolgus monkey KLK5 but not to human or cynomolgus monkey KLK2, or human or cynomolgus monkey KLK4, or human or cynomolgus monkey KLK7, and the antibody includes a variable light chain and a variable heavy chain, and a. The variable light chain includes CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and the variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or b. The variable light chain includes color codes 30, 34, 38, 42, or 46, and the variable heavy chain includes color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or c. The monoclonal antibody is a full-length antibody comprising a light chain and a heavy chain, wherein the light chain comprises SEQ ID NOs. 36, 40, 44, or 48, and the heavy chain comprises SEQ ID NOs. 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136.

[0161] In a preferred embodiment, the monoclonal antibody binds to human and / or cynomolgus monkey KLK5 but not to human or cynomolgus monkey KLK2, or human or cynomolgus monkey KLK4, or human or cynomolgus monkey KLK7, and the antibody comprises a variable light chain and a variable heavy chain. a. The variable light chain includes CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and the variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 23, or b. The variable light chain includes SEQ ID NO: 38, and the variable heavy chain includes SEQ ID NO: 110, or c. The monoclonal antibody is a full-length antibody containing a light chain and a heavy chain, the light chain containing SEQ ID NO: 40 and the heavy chain containing SEQ ID NO: 112.

[0162] In one embodiment, according to the present invention, the binding of the antibody to KLK5 is such that the dissociation constant (K) is approximately 7 nM or less, preferably 500 pM or less, and preferably approximately 400 pM or less. D ) is a characteristic feature.

[0163] The term "K" used in this specification D The term "K" is d vs K a (That is, K d / K a This refers to the dissociation constant, which is obtained from the ratio of ) and expressed as molar concentration (M). d and K a These refer to the dissociation rate and association rate of a specific antigen-antibody (or its antigen-binding fragment) interaction, respectively. D The value can be determined using methods well established in the art. Antibody K DThe method for determining affinity is by using surface plasmon resonance, for example, the Biacore® system described in the examples herein, and recombinant KLK5 or a suitable fusion protein / polypeptide. In one example, affinity is measured using recombinant KLK5 described in the examples herein. In surface plasmon resonance, the target molecule is immobilized on a solid phase and exposed to a ligand in a mobile phase moving along a flow cell. When the ligand binds to the immobilized target, the local refractive index changes, changing the SPR angle, which can be monitored in real time by detecting the change in the intensity of the reflected light. By analyzing the rate of change of the SPR signal, the apparent rate constants of the association and dissociation phases of the binding reaction can be obtained. The ratio of these values ​​gives the apparent equilibrium constant (affinity) (see, e.g., Wolff et al, Cancer Res. 53:2560-65 (1993)).

[0164] In one embodiment, the antibody according to the present invention has a higher binding affinity to human KLK5 than cynomolgus monkey or mouse KLK5 (i.e., a lower K). D ) has. The term "affinity" refers to the strength of the interaction between the antibody and KLK5.

[0165] In one embodiment, the monoclonal antibody according to the present invention blocks KLK5 protease activity at an IC of less than 800 pM. 50 Preferably, the monoclonal antibody according to the present invention has an IC50 of less than 18 pM to block KLK5 protease activity in the in vitro assay described herein. 50 It has.

[0166] ICs used in this specification 50 In this invention, the term IC refers to the maximum half-molecule inhibitory concentration, which is a measure of the effectiveness of a substance such as an antibody in inhibiting a specific biological or biochemical function, which is the protease activity of KLK5. 50 This is a quantitative measure that indicates the extent to which a particular substance is needed to inhibit a given biological process, function, or activity by half.

[0167] In another embodiment of the present invention, a monoclonal antibody that binds to KLK5, wherein the antibody comprises a variable light chain and a variable heavy chain, and a. The variable light chain includes CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3. b. The variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs: 10-29, preferably any one of 10, 11, 13-16, 18, 20, 22-25, 27, or 29. Antibodies inhibit or reduce the protease activity of KLK5.

[0168] Within this invention, the term “inhibit” (and its grammatical variation) refers to the effect that the antibody according to the present invention has on the biological activity of KLK5. Preferably, the biological activity of KLK5 is protease activity, preferably serine protease activity. This effect results in complete or partial inhibition of the serine protease activity of KLK5.

[0169] While we do not wish to be bound by theory, the monoclonal antibody according to the present invention binds to KLK5 and inhibits (e.g., completely or partially) or reduces the protease activity (preferably serine protease activity) of KLK5, and / or i) When KLK5 binds to LEKTI or a fragment of LEKTI, it binds to KLK5 and / or ii) KLK5 binding does not compete with LEKTI or LEKTI fragments, and / or iii) It is thought that KLK5 will form a complex with LEKTI or a fragment of LEKTI (i.e., it will form a complex containing the antibody of the present invention, KLK5 and LEKTI, or a fragment of LEKTI).

[0170] The term "form a complex" (and any grammatical variation thereof) means that if KLK5 is already bound to another protein such as LEKTI, or a fragment of LEKTI, or another antibody or an antibody fragment such as Fab, then the antibody according to the present invention can bind to KLK5.

[0171] Preferably, the antibody claimed herein inhibits or reduces the protease activity of KLK5 and / or, when KLK5 binds to LEKTI or a fragment of LEKTI, binds to KLK5 and / or does not compete with LEKTI or a fragment of LEKTI for binding to KLK5 and / or forms a complex with KLK5 bound to LEKTI or a fragment of LEKTI.

[0172] In this invention, the term "LEKTI" refers to a Cazal-type 5 lymphoepithelial inhibitor consisting of 15 domains, which is cleaved into smaller functional fragments by proprotein-converting enzymes such as furin, a protease, yielding LEKTI fragments consisting of one or more domains. These fragments are secreted into the extracellular space, where they can form inhibitory complexes with proteases such as KLK5. LEKTI is also known as the serine protease inhibitor Cazal-type 5 (SPINK5), and is a protein encoded by the SPINK5 gene in humans. In humans, three splice variants of LEKTI mRNA are produced, resulting in full-length, long, and short isoforms of the protein, differing only in their COOH-terminal regions.

[0173] SPINK5 is a member of a gene family cluster located on chromosome 5q32 that encodes a serine protease inhibitor. This includes other epidermal proteins SPINK6 and LEKTI-2 (SPINK9), which are also included in the present invention under the term "LEKTI".

[0174] An advantage of antibodies that can bind to KLK5 and inhibit its biological (i.e., protease) activity, but do not compete with LEKTI or LEKTI fragments for KLK5 binding, is that they can enable inhibition of KLK5 activity under conditions where LEKTI dissociates from the KLK5:LEKTI complex, such as in an increasingly acidic environment from the basal layer to the stratum corneum of the epidermis.

[0175] In these embodiments, the LEKTI fragment is preferably a human LEKTI domain 5 containing amino acids 1-64 of SEQ ID NO: 145 or a LEKTI domain 8 containing amino acids 1-71 of SEQ ID NO: 152.

[0176] In one embodiment, the monoclonal antibody according to the present invention binds to a human KLK5 epitope comprising at least one, preferably at least two or more amino acid residues from the group consisting of Leu212, Ser213, Gln214, Lys215, Arg216, Glu218, Asp219, Ala220, Pro222, Gly233, Pro269, Asn270, and Pro272, with reference to SEQ ID NO: 142. Preferably, the epitope is characterized by X-ray crystallography. The numbers in parentheses correspond to protease nomenclature.

[0177] Within this invention, the term "epitope" is used interchangeably for both structural epitopes and linear epitopes. Structural epitopes consist of discontinuous portions of the primary amino acid sequence of an antigen, while linear epitopes are formed by sequences of continuous amino acids.

[0178] The epitopes can be identified in combination with any of the antibodies provided by the present invention by any suitable epitope mapping method known in the art. Examples of such methods include screening peptides of various lengths derived from full-length KLK5 for binding to the antibodies of the present invention or fragments thereof, and identifying the smallest fragments that can specifically bind to the antibody, containing the sequence of the epitope recognized by the antibody. KLK5 peptides can be produced synthetically or by proteolytic digestion of KLK5. Peptides that bind to the antibody can be identified, for example, by mass spectrometry. The antibody-bound epitope can be identified using methods such as NMR spectroscopy or X-ray crystallography. Typically, when epitope determination is performed by X-ray crystallography, amino acid residues of the antigen within 4 Å of the CDR are considered to be the amino acid residue portion of the epitope. Once identified, the epitope can be useful in preparing fragments that bind to the antibodies of the present invention and, if necessary, can be used as immunogens to obtain further antibodies that bind to the same epitope.

[0179] The epitopes described in the embodiments and models illustrating the present invention are preferably epitopes characterized by X-ray crystallography.

[0180] Furthermore, the present invention also provides, with reference to Sequence ID No. 142, an antibody that competes for binding to KLK5, preferably human KLK5, by cross-blocking with or being cross-blocked by an antibody that binds to an epitope of human KLK5 comprising the amino acid residues Leu212, Ser213, Gln214, Lys215, Arg216, Glu218, Asp219, Ala220, Pro222, Gly233, Pro269, Asn270, and Pro272, wherein the antibody is 1. A variable light chain and a variable heavy chain, wherein the variable light chain includes CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and the variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 23, or 2. Includes a variable light chain containing Sequence ID No. 38 and a variable heavy chain containing Sequence ID No. 110.

[0181] In one embodiment, such a competing antibody has a heavy chain variable region having at least 80% identity or similarity to the sequence containing SEQ ID NO: 110, and / or a light chain variable region having at least 80% identity or similarity to the sequence containing SEQ ID NO: 38.

[0182] In the present invention, an antibody that "competes," "crossblocks," "is crossblocked," or "bounds to the same epitope on human KLK5" (and any grammatical variation thereof) refers to an antibody that cannot form a complex with KLK5 bound to the antibody of the present invention.

[0183] Competing antibodies can be identified using any suitable method in the art, for example, by using a competitive ELISA or BIAcore assay, such that the binding of KLK5 by the competing antibody prevents the binding of the antibody of the present invention, or vice versa, by cross-blocking or being cross-blocked. Such a competitive assay can use isolated natural or recombinant KLK5 or a suitable fusion protein / polypeptide thereof. In one example, recombinant human active KLK5 (e.g., including SEQ ID NO: 144) is used to measure competition.

[0184] The present invention also provides, as described and claimed herein, an antibody that forms a complex with KLK5, wherein KLK, preferably human KLK5, is conjugated to (or may be conjugated to) another antibody, and the other antibody is 1. Variable light chains including CDR-L1 containing SEQ ID NO: 175, CDR-L2 containing SEQ ID NO: 176, and CDR-L3 containing SEQ ID NO: 177, and variable heavy chains including CDR-H1 containing SEQ ID NO: 178, CDR-H2 containing SEQ ID NO: 179, and CDR-H3 containing SEQ ID NO: 160, and / or 2. A variable light chain containing SEQ ID NO: 161 and a variable heavy chain containing SEQ ID NO: 163, and / or 3. Includes a variable light chain encoded by a nucleotide containing SEQ ID NO: 162 and a variable heavy chain encoded by a nucleotide containing SEQ ID NO: 164.

[0185] In one embodiment, the antibody according to the present invention is bound to KLK5, 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO: 1 or 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and the variable heavy chain comprises CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NOs: 10 to 29, preferably 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or 2. A variable light chain comprising a color code 30 or 34 or 38 or 42 or 46, and a variable heavy chain comprising a color code 32 or 50 or 54 or 58 or 62 or 66 or 70 or 74 or 78 or 82 or 86 or 90 or 94 or 98 or 102 or 106 or 110 or 114 or 118 or 122 or 126 or 130 or 134, preferably 32 or 50 or 54 or 58 or 62 or 66 or 70 or 74 or 78 or 82 or 86 or 90 or 94 or 98 or 106 or 110 or 114 or 118 or 126 or 134. The antibody forms a complex with KLK5, preferably human KLK5, which is bound to another antibody, 1. A variable light chain including CDR-L1 containing SEQ ID NO: 175, CDR-L2 containing SEQ ID NO: 176, and CDR-L3 containing SEQ ID NO: 177, and a variable heavy chain including CDR-H1 containing SEQ ID NO: 178, CDR-H2 containing SEQ ID NO: 179, and CDR-H3 containing SEQ ID NO: 160, and / or 2. A variable light chain containing SEQ ID NO: 161 and a variable heavy chain containing SEQ ID NO: 163, and / or 3. Includes a variable light chain encoded by a nucleotide containing SEQ ID NO: 162 and a variable heavy chain encoded by a nucleotide containing SEQ ID NO: 164.

[0186] Furthermore, the present invention also, a. KLK5, preferably human KLK5, and b. Binds to KLK5, 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO: 1 or 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and the variable heavy chain comprises CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NOs: 10 to 29, preferably 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or 2. An antibody comprising a variable light chain containing the color code 30 or 34 or 38 or 42 or 46, and a variable heavy chain containing the color codes 32 or 50 or 54 or 58 or 62 or 66 or 70 or 74 or 78 or 82 or 86 or 90 or 94 or 98 or 102 or 106 or 110 or 114 or 118 or 122 or 126 or 130 or 134, preferably 32 or 50 or 54 or 58 or 62 or 66 or 70 or 74 or 78 or 82 or 86 or 90 or 94 or 98 or 106 or 110 or 114 or 118 or 126 or 134, and It binds to c.KLK5, 1. Variable light chains comprising CDR-L1 containing SEQ ID NO: 175, CDR-L2 containing SEQ ID NO: 176, and CDR-L3 containing SEQ ID NO: 177, and variable heavy chains comprising CDR-H1 containing SEQ ID NO: 178, CDR-H2 containing SEQ ID NO: 179, and CDR-H3 containing SEQ ID NO: 160, wherein the antibody may be humanized, and / or 2. A variable light chain containing SEQ ID NO: 161 and a variable heavy chain containing SEQ ID NO: 163, and / or 3. A KLK5 antibody complex comprising another antibody comprising a variable light chain encoded by a nucleotide containing SEQ ID NO: 162 and a variable heavy chain encoded by a nucleotide containing SEQ ID NO: 164.

[0187] It should be understood that so-called "other antibodies" bind to KLK5, preferably human KLK5, at non-overlapping epitopes, unlike those described herein, as shown in the following examples. In this respect, such antibodies do not compete with each other.

[0188] The antibodies according to the present invention can be obtained using any suitable method known in the art. Antibodies that specifically recognize KLK5 can be produced using KLK5 containing the fusion protein, or cells expressing KLK5 (recombinantly or naturally). Various forms of KLK5 described herein can be used.

[0189] In one embodiment, the antigen used is active KLK5, which is preferably produced as described in the following example.

[0190] KLK5 or fragments thereof for use in immunizing a host can be prepared from genetically modified host cells, including expression systems, by methods known in the art, or recovered from natural biological sources. In some examples, KLK5 or fragments thereof may be part of a larger protein, such as a fusion protein fused to an affinity tag, for example.

[0191] Antibodies produced against KLK5 according to the present invention can be obtained by administering KLK5 to animals, preferably non-human animals, using known conventional protocols when animal immunization is required (see, for example, Handbook of Experimental Immunology, DM Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep, cattle, camels, or pigs, can be immunized. However, mice, rabbits, pigs, and rats are generally the most suitable.

[0192] Antibody screening can be performed using assays that measure binding to KLK5 and / or assays that measure inhibition of KLK5 biological activity, preferably KLK5 protease activity.

[0193] Antibodies, including monoclonal antibodies, contain acidic and / or basic functional groups, thereby conferring a net positive or negative charge to the molecule. The overall amount of "observable" charge depends on the absolute amino acid sequence of the entity, the local environment of the charged groups in the 3D structure, and the environmental conditions of the molecule. The isoelectric point (pI) is the pH at which a particular molecule or its surface that can come into contact with its solvent has no net charge. In one example, the KLK5-conjugated monoclonal antibody according to the present invention can be manipulated to have a suitable isoelectric point. This allows for the acquisition of an antibody with more robust properties, particularly a suitable solubility and / or stability profile and / or improved purification properties.

[0194] Therefore, in one embodiment, the monoclonal antibody that binds to KLK5 is a. A variable light chain including color codes 30, 34, 38, 42, or 46, and a variable heavy chain including color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or b. A light chain comprising a frequency code 36 or 40 or 44 or 48, and a heavy chain comprising a frequency code 52 or 56 or 60 or 64 or 68 or 72 or 76 or 80 or 84 or 88 or 92 or 96 or 100 or 104 or 108 or 112 or 116 or 120 or 124 or 128 or 132 or 136, preferably 52 or 56 or 60 or 64 or 68 or 72 or 76 or 80 or 84 or 88 or 92 or 96 or 100 or 108 or 112 or 116 or 120 or 128 or 136, Monoclonal antibodies are engineered to have an isoelectric point different from that of the initially identified antibody.

[0195] Antibodies can be manipulated by substituting amino acid residues, for example, by substituting one or more basic amino acid residues for acidic amino acid residues. Alternatively, basic amino acid residues may be introduced, or acidic amino acid residues may be removed. Or, if the molecule has an unacceptably high pI value, acidic residues may be introduced as needed to lower the pI. When manipulating the pI, it is important that care must be taken to maintain the desired activity of the antibody or fragment. Thus, in one embodiment, the manipulated antibody has the same or substantially the same activity as the "unmodified" antibody or fragment.

[0196] **The isoelectric point of an antibody can be predicted using programs such as ExPASY (http: / / www.expasy.ch / tools / pi_tool.html) and http: / / www.iut-arles.up.univ-mrs.fr / w3bb / d_abim / compo-p.html.

[0197] It will be understood that the affinity of the antibody provided by the present invention can be modified using any suitable method known in the art. Accordingly, the present invention also relates to variants of antibodies having improved affinity for KLK5, particularly human KLK5. Such variants can be identified through CDR mutations (Yang et al., J.Mol.Biol., 254, 392-403, 1995), strand shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), use of mutant strains of Escherichia coli (Low et al., J.Mol.Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr.Opin.Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J.Mol.Biol., 256, 77-88, 1996), and sexual PCR (Crameri et al.) This can be obtained by several affinity maturation protocols, including one described in al., Nature, 391, 288-291, 1998).

[0198] If desired, the antibody according to the present invention may be conjugated to one or more effector molecules. It will be understood that the effector molecule may include a single effector molecule or two or more such molecules linked to form a single portion that can bind to the antibody of the present invention. If it is desirable to obtain an antibody fragment linked to an effector molecule, this can be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment is linked to the effector molecule directly or via a coupling agent. Techniques for conjugating such effector molecules to antibodies are known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58; and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemical procedures include, for example, those described in International Publications 93 / 06231, 92 / 22583, 89 / 00195, 89 / 01476, and 03 / 031581. Alternatively, if the effector molecule is a protein or polypeptide, it can be linked using recombinant DNA procedures, for example, as described in International Publication 86 / 01533 and European Patent 0392745.

[0199] As used herein, the term effector molecule includes reporter groups such as anti-cancer drugs, drugs, toxins, biologically active proteins such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and their fragments such as DNA, RNA and its fragments, radionuclides, particularly radioactive iodides, radioisotopes, chelated metals, nanoparticles, and fluorescent compounds or compounds detectable by NMR or ESR spectroscopy.

[0200] Examples of effector molecules include cytotoxic or cytotoxic agents, including any drug that is harmful to cells (e.g., causes cell death). Examples include combrestatin, drastatin, epothilone, staurosporine, mytansinoids, spongestatin, rhizoxin, halichondrin, loridine, hemiasterin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologs.

[0201] Effector molecules also include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, anthramycin (AMC), calicheamicin or duocalmycin), and antimitotic agents (e.g., vincristine and vinblastine).

[0202] Other effector molecules include chelated radionuclides such as 111In and 90Y, Lu177, bismuth-213, californium-252, iridium-192, and tungsten-188 / rhenium-188, or drugs such as alkylphosphocholines, topoisomerase I inhibitors, taxoids, and suramin, but are not limited to these.

[0203] Other effector molecules include proteins, peptides, and enzymes. Target enzymes include, but are not limited to, proteolytic enzymes, hydrolytic enzymes, lyases, isomerases, and transferases. Target proteins, polypeptides, and peptides include, but are not limited to, immunoglobulins, toxins, e.g., abrin, lysine A, Pseudomonas exotoxin, or diphtheria toxin; proteins, e.g., insulin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, or tissue plasminogen activator; thrombotic agents or anti-angiogenic agents, e.g., angiostatin or endostatin; or biological response modifiers, e.g., lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), nerve growth factor (NGF), or other growth factors and immunoglobulins.

[0204] Other effector molecules include, for example, detectable substances useful for diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radionuclides, positron-emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents, see U.S. Patent No. 4,741,900 in general. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; suitable luminescent substances include luminol; suitable bioluminescent substances include luciferase, luciferin, and aequorin; and suitable radionuclides include 125I, 131I, 111In, and 99Tc.

[0205] In another example, effector molecules can increase the half-life of an antibody in vivo and / or decrease its immunogenicity and / or enhance the delivery of the antibody across the epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumins, albumin-binding proteins, or albumin-binding compounds, such as those described in International Publication No. 05 / 117984.

[0206] If the effector molecule is a polymer, it may generally be a synthetic or naturally occurring polymer, such as a substituted linear or branched polyalkylene, polyalkene, or polyoxyalkylene polymer, or a branched or unbranched polysaccharide, such as a homo or heteropolysaccharide.

[0207] Specific optional substituents that may be present on the above synthetic polymer include one or more hydroxy, methyl, or methoxy groups.

[0208] Specific examples of synthetic polymers include substituted or branched linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), or derivatives thereof, particularly substituted or branched poly(ethylene glycol), such as methoxypoly(ethylene glycol) or derivatives thereof.

[0209] Specific examples of naturally occurring polymers include lactose, amylose, dextran, glycogen, or their derivatives.

[0210] In one embodiment, the polymer is albumin or a fragment thereof, for example, human serum albumin or a fragment thereof.

[0211] As used herein, “derivatives” are intended to include reactive derivatives, such as thiol-selective reactive groups, including maleimide. The reactive group may be linked directly to the polymer or via a linker segment. It will be understood that residues of such a group may, in some cases, form part of the product as linking groups between the antibody fragment and the polymer.

[0212] The polymer size can be varied as desired, but generally, the average molecular weight range is 500 Da to 50,000 Da, e.g., 5,000 to 40,000 Da, e.g., 20,000 to 40,000 Da. The polymer size can be selected particularly based on the intended use of the product, e.g., its ability to localize to specific tissues such as tumors or its ability to extend the circulating half-life (see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545 for a review). Therefore, for example, if the product is intended to leave the circulation and penetrate tissues for use in the treatment of tumors, it may be advantageous to use a low molecular weight polymer, for example, with a molecular weight of about 5,000 Da. For applications where the product remains in circulation, it may be advantageous to use a high molecular weight polymer, for example, with a molecular weight in the range of 20,000 Da to 40,000 Da.

[0213] Suitable polymers include polyalkylene polymers, such as poly(ethylene glycol), or in particular methoxypoly(ethylene glycol) or its derivatives, especially those having a molecular weight in the range of about 15,000 Da to about 40,000 Da.

[0214] In one example, the antibody according to the present invention is bound to a poly(ethylene glycol) (PEG) moiety. In a particular embodiment, the antibody and PEG molecule according to the present invention can be bound via any available amino acid side chain or terminal amino acid functional group located in the antibody fragment, such as any free amino, imino, thiol, hydroxyl, or carboxyl group. Such amino acids may be naturally present in the antibody fragment or can be engineered into the antibody using recombinant DNA methods (see, for example, U.S. Patent No. 5,219,996, U.S. Patent No. 5,667,425, International Publication No. 98 / 25971, and International Publication No. 2008 / 038024). In one example, the antibody according to the present invention is a modified Fab fragment, where modification is the addition of one or more amino acids to the C-terminus of its heavy chain to allow attachment of an effector molecule. Preferably, the additional amino acids form a modified hinge region containing one or more cysteine ​​residues to which the effector molecule can be bound. Multiple sites can be used to bind two or more PEG molecules.

[0215] Suitablely, the PEG molecule is covalently bonded via a thiol group on at least one cysteine ​​residue located in the antibody fragment. Each polymer molecule bound to the modified antibody fragment can be covalently bonded to the sulfur atom of a cysteine ​​residue located in the fragment. The covalent bond is generally a disulfide bond, or more specifically, a sulfur-carbon bond. When a thiol group is used as a bonding site, a appropriately activated effector molecule, such as a thiol-selective derivative like maleimide or a cysteine ​​derivative, can be used. The activated polymer can be used as a starting material in the preparation of the polymer-modified antibody fragment described above. The activated polymer may be any polymer containing a thiol-reactive group, such as an α-halocarboxylic acid or ester, e.g., iodoacetamide, imide, e.g., maleimide, vinyl sulfone, or disulfide. Such starting materials can be commercially available (e.g., Nektar, formerly Shearwater Polymers Inc., Huntsville, Alabama, USA) or prepared from commercially available starting materials using conventional chemical procedures. Specific PEG molecules include 20K methoxy-PEG-amine (which can be obtained from Nektar, formerly Shearwater, Rapp Polymere, and SunBio) and M-PEG-SPA (which can be obtained from Nektar, formerly Shearwater).

[0216] In one embodiment, the antibody is PEGylated, i.e., a modified Fab fragment, Fab' fragment, or diFab, covalently bonded with PEG (poly(ethyleneglycol)), according to a method disclosed, for example, in European Patent No. 0948544 or European Patent No. 1090037 ("Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York; "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC; and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York; see also Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545). In one example, PEG is bound to a cysteine ​​within the hinge region. In another example, the PEG-modified Fab fragment has a maleimide group covalently bonded to a single thiol group within the modified hinge region. Lysine residues may also be covalently bonded to the maleimide group, and each amine group on the lysine residue may be bonded to a methoxypoly(ethylene glycol) polymer having a molecular weight of approximately 20,000 Da. Therefore, the total molecular weight of PEG bonded to the Fab fragment may be approximately 40,000 Da.

[0217] A specific PEG molecule is PEG2MAL40K (Nektar, formerly obtainable from Shearwater), which is 2-[3-(N-maleimide)propionamide]ethylamide of N,N'-bis(methoxypoly(ethylene glycol) molecular weight 20,000) modified lysine.

[0218] An alternative source of PEG linker is an NOF supplying GL2-400MA3 (where m is 5 in the following structure) and GL2-400MA (where m is 2), with n being approximately 450. [ka]

[0219] Therefore, in one embodiment, the PEG is 2,3-bis(methylpolyoxyethylene-oxy)-1-{[3-(6-maleimide-1-oxohexyl)amino]propyloxy}hexane(2-arm branched PEG, -CH2)3NHCO(CH2)5-MAL, known as SUNBRIGHT GL2-400MA3, with a molecular weight of 40,000.

[0220] Further alternative PEG effector molecules of the following types are: [ka] It can be obtained from Dr. Reddy, NOF, and Jenkem.

[0221] In one embodiment, the Fab or Fab' according to the present invention is conjugated to a PEG molecule.

[0222] In one embodiment, the disclosure provides a Fab'PEG molecule comprising one or more PEG polymers, such as one or more polymers, including 40 kDa polymers.

[0223] Fab’-PEG molecules according to the present disclosure can be particularly advantageous in that they have a half-life independent of the Fc fragment. In one embodiment, a Fab’ conjugated to a polymer such as a PEG molecule, a starch molecule, or an albumin molecule is provided. In one embodiment, a scFv conjugated to a polymer such as a PEG molecule, a starch molecule, or an albumin molecule is provided. In one embodiment, a Fab or Fab’ according to the present disclosure is conjugated to human serum albumin. In one embodiment, an antibody or fragment is conjugated to a starch molecule, for example, to increase its half-life. A method of conjugating starch to a protein described in U.S. Patent No. 8,017,739, which is incorporated herein by reference.

[0224] The present invention also provides an isolated polynucleotide encoding an antibody according to the present invention. The isolated polynucleotide according to the present invention can include, for example, synthetic DNA, cDNA, genomic DNA, or any combination thereof generated by chemical treatment.

[0225] Using standard techniques in molecular biology, a DNA sequence encoding an antibody of the present invention can be prepared. The desired DNA sequence can be synthesized completely or partially using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques can be used as needed.

[0226] In one embodiment, the isolated polynucleotide according to the present invention is a. a light chain variable region, wherein the polynucleotide is i. at least 90% identical to SEQ ID NO: 31 or 35 or 39 or 43 or 47, or ii. includes SEQ ID NO: 31 or 35 or 39 or 43 or 47, or iii. consists essentially of SEQ ID NO: 31 or 35 or 39 or 43 or 47, a light chain variable region, or b. a heavy chain variable region, wherein the polynucleotide is i. At least 90% identical to SEQ ID NO: 33 or 51 or 55 or 59 or 63 or 67 or 71 or 75 or 79 or 83 or 87 or 91 or 95 or 99 or 103 or 107 or 111 or 115 or 119 or 123 or 127 or 131 or 135, or ii. Comprising SEQ ID NO: 33 or 51 or 55 or 59 or 63 or 67 or 71 or 75 or 79 or 83 or 87 or 91 or 95 or 99 or 103 or 107 or 111 or 115 or 119 or 123 or 127 or 131 or 135, or iii. A heavy chain variable region consisting essentially of SEQ ID NO: 33 or 51 or 55 or 59 or 63 or 67 or 71 or 75 or 79 or 83 or 87 or 91 or 95 or 99 or 103 or 107 or 111 or 115 or 119 or 123 or 127 or 131 or 135, c. A light chain, wherein the polynucleotide is i. At least 90% identical to SEQ ID NO: 37 or 41 or 45 or 49, or ii. Comprising SEQ ID NO: 37 or 41 or 45 or 49, or iii. A light chain consisting essentially of SEQ ID NO: 37 or 41 or 45 or 49, d. A heavy chain, wherein the polynucleotide is i. At least 90% identical to SEQ ID NO: 53 or 57 or 61 or 65 or 69 or 73 or 77 or​​​​​​ In one embodiment, the present invention provides isolated polynucleotides encoding the variable heavy chain of the antibody Fab' fragment or IgG1 or IgG4 antibody of the present invention, comprising the sequence shown in 33 or 51 or 55 or 59 or 63 or 67 or 71 or 75 or 79 or 83 or 87 or 91 or 95 or 99 or 103 or 107 or 111 or 115 or 119 or 123 or 127 or 131 or 135. Also provided are isolated polynucleotides encoding the variable light chain of the antibody Fab' fragment or IgG1 or IgG4 antibody of the present invention, comprising the sequence shown in SEQ ID NO: 31 or 35 or 39 or 43 or 47.

[0228] In another embodiment, the present invention provides isolated polynucleotides encoding the heavy and light chains of the IgG4(P) antibody of the present invention, wherein the polynucleotide encoding the heavy chain comprises a sequence shown in SEQ ID NO: 53 or 57 or 61 or 65 or 69 or 73 or 77 or 81 or 85 or 89 or 93 or 97 or 101 or 105 or 109 or 113 or 117 or 121 or 125 or 129 or 133 or 137, and the polynucleotide encoding the light chain comprises a sequence shown in SEQ ID NO: 37 or 41 or 45 or 49.

[0229] The present invention also provides cloning or expression vectors comprising one or more polynucleotides described herein. For example, a cloning or expression vector according to the present invention comprises one or more isolated polynucleotides comprising a sequence selected from SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, or 137.

[0230] General methods for constructing vectors, transfection methods, and culture methods are known to those skilled in the art. For further information, see the Maniatis Manual in “Current Protocols in Molecular Biology”, 1999, FMAusubel (ed), Wiley Interscience, New York and Cold Spring Harbor Publishing.

[0231] Also provided are host cells comprising one or more isolated polynucleotide sequences according to the present invention, or one or more cloning vectors or expression vectors comprising one or more isolated polynucleotide sequences encoding the antibodies of the present invention. Any suitable host cell / vector system can be used for the expression of the polynucleotide sequences encoding the antibodies of the present invention. Bacteria, e.g., Escherichia coli (E. coli), and other microbial systems can be used, or eukaryotes, e.g., mammals, host cell expression systems can be used. Suitable mammalian host cells include CHO, myeloma, or hybridoma cells.

[0232] Suitable types of Chinese hamster ovary (CHO cells) for use in the present invention include dhfr-CHO cells such as CHO-DG44 cells and CHO-DXB11 cells, which can be used with a DHFR-selective marker, or CHO and CHO-K1 cells including CHOK1-SV, which can be used with a glutamine synthase-selective marker. Other cell types used for antibody expression include lymphocyte cell lines, such as NSO myeloma cells and SP2 cells, and COS cells. Host cells can be stably transformed or transfected with the isolated polynucleotide sequence or expression vector according to the present invention.

[0233] In one embodiment, the host cells according to the present invention are CHO-DG44 cells stably transfected with an expression vector containing an isolated polynucleotide sequence comprising the isolated polynucleotide sequence of the present invention, preferably comprising SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, or 137.

[0234] The present invention also provides a method for producing a KLK5-binding antibody according to the present invention, comprising culturing host cells according to the present invention under conditions suitable for the production of a monoclonal antibody, and isolating the monoclonal antibody thus produced.

[0235] Antibodies may contain only heavy chains or only light chains, in which case only the heavy chain or light chain polynucleotide sequence must be used to transfect host cells. For the production of antibodies containing both heavy and light chains, cell lines can be transfected with two vectors: a first vector encoding the light chain and a second vector encoding the heavy chain. Alternatively, a single vector containing polynucleotide sequences encoding both light and heavy chains can be used.

[0236] Therefore, a method is provided for culturing host cells, expressing antibodies, isolating antibodies, and optionally purifying them to obtain isolated antibodies. Accordingly, in one embodiment, an isolated monoclonal antibody that binds to KLK5, preferably human KLK5, for example, a humanized monoclonal antibody, in particular the antibody according to the present invention, is provided, which is substantially purified, in particular, does not contain or substantially contains endotoxin and / or host cell proteins or DNA. Monoclonal antibodies bind to KLK, preferably human KLK5, and a. A variable light chain comprising CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or b. A variable light chain including color codes 30, 34, 38, 42, or 46, and a variable heavy chain including color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or c. A light chain and a heavy chain, wherein the light chain contains color codes 36, 40, 44, or 48, and the heavy chain contains color codes 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136. Includes.

[0237] "Substantially endotoxin-free" generally means that the endotoxin content is 1 EU or less per mg of antibody product, for example, 0.5 or 0.1 EU per mg of product.

[0238] "Substantially free of host cell proteins or DNA" generally means, where necessary, that the host cell protein and / or DNA content is 400 μg or less per 1 mg of antibody product, for example, 100 μg or less per 1 mg, and especially 20 μg per 1 mg.

[0239] Since the antibodies of the present invention are useful in the treatment, diagnosis, and / or prevention of pathological conditions, the present invention also provides pharmaceutical compositions or diagnostic compositions comprising the antibodies according to the present invention in combination with one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0240] Preferably, the pharmaceutical composition or diagnostic composition contains an antibody that binds to KLK5, preferably human KLK5, and the antibody is a. A variable light chain comprising CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or b. A variable light chain including color codes 30, 34, 38, 42, or 46, and a variable heavy chain including color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or c. A light chain and a heavy chain, wherein the light chain contains color codes 36, 40, 44, or 48, and the heavy chain contains color codes 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136. Includes.

[0241] In one embodiment, the antibody according to the present invention is the sole active ingredient. In another embodiment, the antibody according to the present invention is combined with one or more additional active ingredients. Alternatively, the pharmaceutical composition contains the antibody according to the present invention as the sole active ingredient and can be administered individually to a patient in combination with other therapeutic agents, diagnostic agents or palliative agents (e.g., simultaneously, sequentially, or separately).

[0242] In another embodiment, the pharmaceutical composition is an antibody that binds to KLK5, preferably human KLK5, and a. a variable light chain comprising CDR-L1 containing SEQ ID NO: 1 or 7 or 8 or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing any one of SEQ ID NO: 6 or SEQ ID NOs: 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27 or 29, or b. a variable light chain containing SEQ ID NO: 30 or 34 or 38 or 42 or 46, and a variable heavy chain containing SEQ ID NO: 32 or 50 or 54 or 58 or 62 or 66 or 70 or 74 or 78 or 82 or 86 or 90 or 94 or 98 or 102 or 106 or 110 or 114 or 118 or 122 or 126 or 130 or 134, preferably 32 or 50 or 54 or 58 or 62 or 66 or 70 or 74 or 78 or 82 or 86 or 90 or 94 or 98 or 106 or 110 or 114 or 118 or 126 or 134, or c. a monoclonal antibody which is a full-length antibody containing a light chain and a heavy chain, the light chain containing SEQ ID NO: 36 or 40 or 44 or 48, and the heavy chain containing SEQ ID NO: 52 or 56 or 60 or 64 or 68 or 72 or 76 or 80 or 84 or 88 or 92 or 96 or 100 or 104 or 108 or 112 or 116 or 120 or 124 or 128 or 132 or 136, preferably 52 or 56 or 60 or 64 or 68 or 72 or 76 or 80 or 84 or 88 or 92 or 96 or 100 or 108 or 112 or 116 or 120 or 128 or 136, said monoclonal antibody The above antibodies, The same includes one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0243] Preferably, the pharmaceutical composition includes an antibody that binds to KLK5, preferably human KLK5, and comprises an antibody that includes the light chain variable region of SEQ ID NO: 38 and the heavy chain variable region of SEQ ID NO: 110.

[0244] The pharmaceutical composition according to the present invention can be appropriately administered to a patient to determine the required therapeutically effective dose.

[0245] As used herein, the term “therapeutic dose” refers to the amount of therapeutic agent required to treat, improve, or prevent a target disease or condition, or to demonstrate a detectable therapeutic or prophylactic effect. For any antibody, the therapeutic dose can be initially estimated using a cell culture assay or an animal model, typically rodents, rabbits, dogs, pigs, or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes of administration in humans.

[0246] The precise therapeutically effective dose for human subjects depends on the severity of the disease state, the subject's general health status, age, weight and sex, diet, timing and frequency of administration, drug combination, response sensitivity, and tolerance / response to treatment. Generally, the therapeutically effective dose is between 0.01 mg / kg and 500 mg / kg, for example, 0.1 mg / kg to 200 mg / kg, such as 100 mg / kg. The pharmaceutical composition can be conveniently provided in unit dose form containing a predetermined amount of the active agent of the present invention per dose.

[0247] A pharmaceutically acceptable carrier in a therapeutic composition may further include liquids such as water, saline solution, glycerol, and ethanol. Furthermore, auxiliary substances such as wetting agents, emulsifiers, or pH buffers may be present in such a composition. Such carriers can be used to formulate the pharmaceutical composition as tablets, pills, coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions for patient ingestion.

[0248] Suitable forms for administration include, for example, intravenous, inhalable, or subcutaneous forms suitable for parenteral administration by injection or infusion, such as by bolus injection or continuous infusion. When the product is for injection or infusion, it may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain formulation agents such as suspending agents, preservatives, stabilizers, and / or dispersants. Alternatively, the antibody according to the present invention may be in a dry form for reconstitution with a suitable sterile liquid before use. A solid form suitable for dissolution in a liquid vehicle or suspension in a liquid vehicle before injection can also be prepared.

[0249] When formulated, the composition of the present invention can be directly administered to a target. Therefore, the use of antibodies according to the present invention for the manufacture of pharmaceuticals is provided herein.

[0250] Preferably, the pharmaceutical composition according to the present invention is suitable for administration to human subjects.

[0251] Therefore, in another embodiment, the present invention binds to KLK5, preferably human KLK5, a. A variable light chain comprising CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or b. A variable light chain including color codes 30, 34, 38, 42, or 46, and a variable heavy chain including color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or c. A monoclonal antibody is provided comprising a light chain and a heavy chain, wherein the light chain contains SEQ ID NO: 36, 40, 44, or 48, and the heavy chain contains SEQ ID NO: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136.

[0252] In preferred embodiments, the monoclonal antibody that binds to KLK5, preferably human KLK5, is 1. A variable light chain and a variable heavy chain, wherein the variable light chain includes CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and the variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 23, or 2. A variable light chain containing SEQ ID NO: 38, and a variable heavy chain containing SEQ ID NO: 110, or 3. Includes a light chain containing SEQ ID NO: 40 and a heavy chain containing SEQ ID NO: 112.

[0253] In particular, therapeutic use includes use in the treatment of one or more diseases characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition.

[0254] In yet another aspect, the present invention provides a method for treating one or more diseases characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition in a patient, the method comprising administering to the patient a therapeutically effective amount of a monoclonal antibody bound to KLK5, the antibody bound to KLK5, preferably human KLK5, and a. A variable light chain comprising CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or b. A variable light chain including color codes 30, 34, 38, 42, or 46, and a variable heavy chain including color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or c. A light chain and a heavy chain, wherein the light chain contains color codes 36, 40, 44, or 48, and the heavy chain contains color codes 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136. Includes.

[0255] In yet another embodiment, a monoclonal antibody conjugated to KLK5, preferably human KLK5, or a pharmaceutical composition comprising a monoclonal antibody, wherein the monoclonal antibody or composition is for use in the treatment of one or more diseases characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition, and the antibody is a. A variable light chain comprising CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or b. A variable light chain including color codes 30, 34, 38, 42, or 46, and a variable heavy chain including color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or c. A light chain and a heavy chain, wherein the light chain contains color codes 36, 40, 44, or 48, and the heavy chain contains color codes 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136. Includes.

[0256] In one preferred embodiment, the present invention provides a method for treating one or more diseases characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition in a patient, the method comprising a substance bound to KLK5, preferably human KLK5, 1. A variable light chain and a variable heavy chain, wherein the variable light chain includes CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and the variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 23, or 2. A variable light chain containing SEQ ID NO: 38, and a variable heavy chain containing SEQ ID NO: 110, or 3. This includes administering to the patient a therapeutically effective dose of an antibody comprising a light chain containing SEQ ID NO: 40 and a heavy chain containing SEQ ID NO: 112.

[0257] In another preferred embodiment, a monoclonal antibody conjugating to KLK5, preferably human KLK5, or a pharmaceutical composition comprising a monoclonal antibody, wherein the antibody or pharmaceutical composition is for use in the treatment of one or more diseases characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition, and the antibody is 1. A variable light chain and a variable heavy chain, wherein the variable light chain includes CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and the variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 23, or 2. A variable light chain containing SEQ ID NO: 38, and a variable heavy chain containing SEQ ID NO: 110, or 3. Includes a light chain containing SEQ ID NO: 40 and a heavy chain containing SEQ ID NO: 112.

[0258] Preferably, one or more diseases characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition are selected from Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or a combination thereof.

[0259] Therefore, the present invention provides a method for treating Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or a combination thereof, in a patient, the method comprising administering to the patient a therapeutically effective amount of a monoclonal antibody or a pharmaceutical composition comprising a monoclonal antibody that binds to KLK5, preferably human KLK5, the antibody or pharmaceutical composition is a. A variable light chain comprising CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or b. A variable light chain including color codes 30, 34, 38, 42, or 46, and a variable heavy chain including color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or c. A light chain and a heavy chain, wherein the light chain contains color codes 36, 40, 44, or 48, and the heavy chain contains color codes 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136. Includes.

[0260] More preferably, the method is for the treatment of Netherton syndrome and / or atopic dermatitis.

[0261] In another embodiment, a monoclonal antibody conjugating to KLK5, preferably human KLK5, or a pharmaceutical composition comprising a monoclonal antibody is provided, the antibody or pharmaceutical composition being used for the treatment of Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer or a combination thereof, the monoclonal antibody or pharmaceutical composition is provided, a. A variable light chain comprising CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or b. A variable light chain including color codes 30, 34, 38, 42, or 46, and a variable heavy chain including color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or c. A light chain and a heavy chain, wherein the light chain contains color codes 36, 40, 44, or 48, and the heavy chain contains color codes 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136. Includes.

[0262] More preferably, the antibodies are intended for use in the treatment of Netherton syndrome and / or atopic dermatitis.

[0263] In another preferred embodiment, the present invention provides a method for treating Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or a combination thereof, in a patient, the method comprising administering to the patient a therapeutically effective amount of an antibody or monoclonal antibody conjugated to KLK5, preferably human KLK5, wherein the antibody or pharmaceutical composition is 1. A variable light chain and a variable heavy chain, wherein the variable light chain includes CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and the variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 23, or the above variable light chain and variable heavy chain, or 2. A variable light chain containing SEQ ID NO: 38, and a variable heavy chain containing SEQ ID NO: 110, or 3. Light chain containing SEQ ID NO: 40 and heavy chain containing SEQ ID NO: 112 Includes.

[0264] More preferably, the method is for the treatment of Netherton syndrome and / or atopic dermatitis.

[0265] In another preferred embodiment, a pharmaceutical composition comprising an antibody or monoclonal antibody that binds to KLK5, preferably human KLK5, wherein the antibody or pharmaceutical composition is for use in the treatment of Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer or a combination thereof, and the antibody or pharmaceutical composition is 1. A variable light chain and a variable heavy chain, wherein the variable light chain includes CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and the variable heavy chain includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 23, or 2. A variable light chain containing SEQ ID NO: 38, and a variable heavy chain containing SEQ ID NO: 110, or 3. Includes a light chain containing SEQ ID NO: 40 and a heavy chain containing SEQ ID NO: 112.

[0266] More preferably, the antibodies are intended for use in the treatment of Netherton syndrome and / or atopic dermatitis.

[0267] Alternatively, the present invention also provides the use of a pharmaceutical composition comprising an antibody or monoclonal antibody that binds to KLK5, preferably human KLK5, the antibody or pharmaceutical composition for the manufacture of a drug for treating one or more diseases characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition, such dysregulation being preferably Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, e.g., ovarian cancer or bladder cancer or a combination thereof, more preferably Netherton syndrome and / or atopic dermatitis, and the antibody is a. A variable light chain comprising CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or b. A variable light chain including color codes 30, 34, 38, 42, or 46, and a variable heavy chain including color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or c. A light chain and a heavy chain, wherein the light chain contains color codes 36, 40, 44, or 48, and the heavy chain contains color codes 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136. Includes.

[0268] Furthermore, the present invention provides for the use of an antibody that binds to KLK5 as a diagnostic active agent or in a diagnostic assay for diagnosing, for example, Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, wherein the antibody binds to KLK5, preferably human KLK5.

[0269] More preferably, monoclonal antibodies a. A variable light chain comprising CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or b. A variable light chain including color codes 30, 34, 38, 42, or 46, and a variable heavy chain including color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or c. A light chain and a heavy chain, wherein the light chain contains color codes 36, 40, 44, or 48, and the heavy chain contains color codes 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136. Includes.

[0270] Diagnosis can preferably be performed on a biological sample. “Biological sample” encompasses various sample types obtained from an individual and can be used in a diagnostic assay or monitoring assay. The definition includes blood, such as plasma and serum, as well as other liquid samples of biological origin, such as urine and saliva, cerebrospinal fluid, solid tissue samples, such as biopsy specimens, such as skin biopsies or tissue cultures, or cells and their offspring derived therefrom. This definition also includes samples that have been manipulated in any way after their procurement, for example, by treatment with reagents, solubilization, or concentration of specific components such as polynucleotides.

[0271] Diagnostic tests can preferably be performed on biological samples that have not come into contact with the human or animal body. Such diagnostic tests are also called in vitro tests. In vitro diagnostic tests may rely on an in vitro method for detecting KLK5 in a biological sample obtained from an individual, comprising the steps of i) contacting the biological sample with the antibody described herein, and ii) detecting the binding of the antibody to KLK5. By comparing the detected KLK5 level or the presence of a specific post-translational modification form of KLK5 (including any proform) with a suitable control, one or more diseases characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition can be identified. Thus, using such detection methods, it can be determined whether a subject (including an embryo or fetus) has or is at risk of developing a disease characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition.

[0272] Therefore, the present invention provides an antibody that binds to KLK5, preferably human KLK5, and is intended for use in the diagnosis of one or more diseases characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition, preferably Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, and the antibody is a. A variable light chain comprising CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and a variable heavy chain comprising CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29, preferably any one of 10, 11, 13 to 16, 18, 20, 22 to 25, 27, or 29, or b. A variable light chain including color codes 30, 34, 38, 42, or 46, and a variable heavy chain including color codes 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134, preferably 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134, or c. A light chain and a heavy chain, wherein the light chain contains the color code 36, 40, 44, or 48, and the heavy chain contains the color codes 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136, preferably 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136. Includes.

[0273] Table 1 shows the sequences included in the present invention. Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 [Table 1-29] [Table 1-30] [Table 1-31] [Table 1-32] [Table 1-33] [Table 1-34] [Table 1-35]

[0274] The present invention will now be further described by reference to embodiments shown in the attached drawings. [Examples]

[0275] Example 1: Cloning, expression, and purification of kallikrein protein and LEKTI domain The optimized nucleotide sequence encoding the protein specified by Sequence ID No. 142 was cloned into an autologous mammalian expression vector using the HindIII / EcoRI site to create a vector encoding the untagged human KLK5 protein.

[0276] By similarly cloning the mouse and cynomolgus monkey (cyno) KLK5 sequences, we were able to produce the active forms of these proteins, including sequence numbers 150 and 151, respectively.

[0277] Domains 5 (D5) and 8 (D8) of human LEKTI (Uniprot Q9NQ38), containing residues 292-353 and 490-558 respectively (according to the numbering in Uniprot), were cloned and expressed for use as reference proteins in in vitro assays.

[0278] Both human LEKTI domain 5 and domain 8 nucleotide sequences, optimized for expression in mammalian cells, were separately cloned into autologous mammalian expression vectors encoding rabbit Fc tags using the HindIII / XhoI site. Vectors encoding either the LEKTI domain 5 sequence with a C-terminal rabbit Fc tag (SEQ ID NO: 145) or the LEKTI domain 8 sequence with a C-terminal rabbit Fc tag (SEQ ID NO: 152) were then constructed. The encoded proteins are called LEKTI D5 rabbit Fc and LEKTI D8 rabbit Fc, respectively.

[0279] KLK5, LEKTI domain 5, and LEKTI domain 8 rabbit Fc fusion protein were expressed by transient transfection using the Expi 293® expression system (Life Technologies®) according to the manufacturer's protocol. During expression, KLK5 self-activates, resulting in active KLK5 (containing residues I67-S293 of SEQ ID NO: 144 or SEQ ID NO: 142) in the supernatant. Cells were harvested 5 days after transfection, and the supernatant was immediately used for purification. The supernatant containing human (or mouse or cynomolgus monkey) active KLK5 was diluted 4-fold with buffer A (50 mM Tris pH 7.0, 50 mM NaCl) and loaded onto a HiTrap SP HP cation exchange column. The bound protein was eluted using buffer A (50 mM Tris pH 7.0, 50 mM NaCl) and buffer B (50 mM Tris pH 7.0, 1 M NaCl) over a salt gradient generated over a total of 10 column volumes. The fraction containing purified human (or mouse or cynomolgus monkey) active KLK5 was pooled, concentrated, and further purified by size exclusion chromatography on an S200 26 / 60 column equilibrated with 20 mM Tris, 150 mM NaCl, and 5% glycerol at pH 7.2. SDS-PAGE analysis indicated that the protein underwent glycosylation during expression. Mass spectrometry analysis yielded the expected molecular weight.

[0280] The supernatant containing human LEKTI D5 rabbit Fc fusion protein (according to SEQ ID NO: 145) was first subjected to Protein A affinity chromatography. The supernatant was loaded onto a 5 ml Hitrap® Protein A column. The bound protein was eluted with 1 M citrate buffer, pH 2.0, and the fraction was neutralized with 2 M Tris-HCl, pH 8.5. The fraction containing LEKTI D5 rabbit Fc fusion protein was pooled, concentrated, and further purified by size exclusion chromatography using an S200 26 / 60 column equilibrated with PBS. Subsequently, the fraction containing human LEKTI D5 rabbit Fc fusion protein was pooled and concentrated. LEKTI D8 rabbit Fc fusion protein (according to SEQ ID NO: 152) was similarly purified from the supernatant of transfected cell cultures.

[0281] LEKTI D5 Fab fusion molecules (as defined by SEQ ID NOs. 169 and 170) were expressed and purified by cation exchange chromatography. The LEKTI domain 5-nucleotide sequence flanked at the 5' and 3' ends by a sequence encoding the Gly4Ser linker was incorporated into the albumin-specific Fab heavy chain sequence framework 3 (described in International Publication No. 2020011868, incorporated herein by reference), and a tag encoding the 10×His sequence was also placed at the 3' end of the Fab H chain. The LEKTI D5 Fab fusion heavy chain was optimized for expression in mammalian cells, cloned into an autologous expression vector, and co-transfected in CHOSXE cells with an appropriate light chain optimized for mammalian expression. Transfected cells were cultured in a ventilated flask at 32°C for 13 days. The supernatant was collected, concentrated, and buffered with 20 mM Tris, 50 mM NaCl, pH 7.0 before being loaded onto an SP Sepharose HP column. The bound protein was eluted using a salt gradient generated over a total of 10 column volumes with buffer A (20 mM Tris pH 7.0, 50 mM NaCl) and buffer B (20 mM Tris pH 7.0, 1 M NaCl). The fraction containing the LEKTI D5 Fab fusion was pooled and further purified by size exclusion chromatography using an S200 column equilibrated with PBS pH 7.4. The related fractions were pooled.

[0282] Human and cynomolgus monkey nucleotide sequences encoding full-length KLK7 were expressed in a manner similar to that of human KLK5 to generate pro-KLK7 (including SEQ ID NOs. 146 and 148, respectively). Unlike KLK5, KLK7 does not self-activate during expression; therefore, the active forms of human, mouse, and cynomolgus monkey KLK7 (including SEQ ID NOs. 147, 186, and 149, respectively) were prepared by cleaving the propeptide sequence from purified protein using thermolysin. Human, mouse, or cynomolgus monkey pro-KLK7 (including SEQ ID NOs. 146, 185, and 148, respectively) were diluted to 1 mg / ml in activation buffer (50 mM Tris pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij35). Thermolysin (25 mg) manufactured by Sigma® was resuspended in 25 ml of digestion buffer (50 M Tris pH 8.0, 0.5 mM CaCl2), and added to each KLK7 protein in a 1:10 ratio at 37°C for 45 minutes. The mixture was then mixed with anion exchange DEAE resin (GE Life Sciences®) to bind to and remove the thermolysin. The flow-through was recovered as active (human, mouse, or cynomolgus monkey) KLK7.

[0283] The active forms of human, mouse, and cynomolgus monkey KLK7 were buffered with 50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, and 1 mM EDTA, and concentrated to approximately 3.2 mg / ml.

[0284] Human KLK2 was supplied as an active protein from R&D Systems (trademark) (catalog number 4104-SE-010).

[0285] Human KLK4 was supplied as a proform from R&D Systems (trademark) (catalog number 1719-SE) and activated as follows: Human pro-KLK4 was diluted to 200 μg / mL in 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5, and bacterial thermolysin supplied by R&D Systems (trademark) (catalog number 3097-ZN) was diluted to 2 μg / mL in the same buffer. Equal volumes of pro-human KLK4 and thermolysin were combined and incubated at room temperature for 10 minutes to activate. The reaction was stopped with EDTA to a final concentration of 10 mM.

[0286] Example 2: Antibody production through immunization with KLK5 Female New Zealand white rabbits (over 2 kg) were subcutaneously immunized with 100 μg of 0.4 mg / mL human active KLK5 and human active KLK7 (expressed according to Example 1) mixed with an equal volume of complete Freund's adjuvant (Sigma®). The animals were given boost injections containing 100 μg of the same immunogen mixed with an equal volume of incomplete Freund's adjuvant (Sigma®) at 21-day intervals. Fourteen days after the final boost, single-cell suspensions of spleen, bone marrow, and peripheral blood mononuclear cells (PBMCs) were prepared and the study was terminated when they were frozen at -80°C in 10% dimethyl sulfoxide (DMSO) in fetal bovine serum (FCS).

[0287] B cell cultures were prepared using the same method as described in Tickle et al., 2015 J Biomol Screen:20(4), 492-497. In short, lymph node cells, spleen cells, or peripheral blood mononuclear cells (PBMCs) derived from immunized animals were cultured at a density of 2000 cells per well in barcoded 96-well tissue culture plates containing 200 μl / well RPMI 1640 medium (Gibco®) supplemented with 10% FCS (Sigma Aldrich®), 2% HEPES solution (Sigma Aldrich®), 2% L-glutamine solution (Gibco®), 1% penicillin / streptomycin solution (Gibco®), 0.2% Normocin (Invivogen®), and 0.1% β-mercaptoethanol (Gibco®), using supporting cells expressing CD40L and IL-2, with or without B cell-stimulated supernatant (BSS). B cells (B cells in which PBMCs) were cultured for 6 days in the presence of the mitotic stimulants Phorbol-12-myristart-13-acetate (PMA) and phytohemagglutinin-L (PHA-L) to produce B cells (BSS), and the supernatant was collected. The plates were incubated at 37°C and 5% CO2 for 6 days. Cultures were set up using B cells derived from all immunized animals, totaling approximately 1 × 10⁶ cells. 9 We screened individual B cells.

[0288] Six days later, the supernatant was screened for binding to human KLK5 (generated as in Example 1) by a multi-uniform, homogeneous fluorescence-based binding assay using Sol-R2 streptavidin beads (TTP Labtech®) coated with biotinylated human KLK5 as the target antigen source and Sol-R4 streptavidin beads (TTP Labtech®) coated with relevant KLK7 for counterscreening. Biotinylation of the protein was performed using Lightning-Link Rapid Biotin Type B (Expedeon®) with a 5-fold molar excess of protein compared to the provider protocol, avoiding complete modification of all lysine residues. A total of 10 μL of supernatant from barcoded 96-well tissue culture plates was transferred to barcoded 384-well black-wall assay plates containing the biotinylated KLK-coated Sol-R beads and FITC conjugate goat anti-rabbit Fc fragment specific (Jackson ImmunoResearch®) using an Agilent Bravo liquid handler. After one hour of incubation, the plate was read using a mirror ball device (TTP-Labtech®).

[0289] Following primary screening, supernatants positive for KLK5 binding were immobilized onto 96-well barcoded master plates using a Beckman Coulter BiomekNXP® hit-picking robot, and B cells from cell culture plates were frozen at -80°C. The immobilized supernatants were first re-screened by fluorescence microvolume assay (FMAT) to confirm binding to human KLK5. Briefly, 10 μL of supernatant was transferred to barcoded black Greiner plates. 50 μL / plate of 10 μm super avidin (Bangs Beads®) was coated with human biotinylated KLK5 and mixed with Alexa-647® goat anti-rabbit IgG Fc fragment specific (Jackson ImmunoResearch®). The supernatant was then added, and the plates were read using an Applied Biosystems® Cellular Detection System 8200.

[0290] Multiple antibodies were identified as binding to KLK5, and their ability to specifically inhibit KLK5 and their specificity for KLK5 compared to other kallikreins were subsequently investigated.

[0291] Example 3: Identification of KLK5 inhibitory antibodies A screening assay was developed to identify antibodies that can specifically inhibit KLK5 activity among proteases and protease inhibitors present in the complex B cell supernatant. Nunc Maxisorp black 384 (Sigma Aldrich®) was coated with 10 μg / mL of F(ab')2 fragment goat anti-rabbit IgG Fc fragment specific (Jackson ImmunoResearch®) in carbonate buffer and left overnight at 4°C. The plates were washed three times with a Biotek® plate washer containing PBS / 0.1% Tween-20 and blocked in 20 μL / well PBS / 1% BSA at room temperature for 1 hour. Next, B cell supernatant was added to the plate, and 25 μL of 1 nM LEKTI D5 rabbit Fc fusion protein was added to the control well as a positive control for inhibition, and assay buffer A (50 mM Tris, 150 mM NaCl, 0.05% (v / v) Tween-20, pH 7.6) was added to a separate set of control wells as a negative control for inhibition. The plate was incubated overnight at room temperature and then washed three times with a Biotek® plate washer containing PBS / 0.1% Tween-20. 10 μL of 250 pM human KLK5 in assay buffer A was added to each well, and the plate was incubated overnight at room temperature to allow complete association. Boc-VPR-AMC substrate (Cambridge Research Biochemicals®) in assay buffer A was added to the wells to a final concentration of 600 μM, and fluorescence (λ) was measured after 4 hours using a PHERAStar FSX (BMG Labtech®) plate reader. ex 380nm λ em The wavelength was determined to be 430nm.

[0292] The data was analyzed, and the inhibition rate of KLK5 activity was determined using the following formula.

number

[0293] If the test is based on the fluorescence value of the test antibody, a positive result is the average fluorescence value of the positive control for the inhibitory well, and a negative result is the average fluorescence value of the negative control for the inhibitory well.

[0294] Supernatants showing more than 40% inhibition were considered hits. This represented approximately 4% of all screened supernatants. These antibodies were selected for variable region recovery.

[0295] A deconvolution step had to be performed to identify specific antibody-secreting cells and enable the recovery of antibody variable region genes from a heterogeneous population of activated B cells. Fluorescence focusing (Clargo et al., 2014) was used. In short, antibody-secreting cells were statically incubated at 37°C for 1 hour in the presence of streptavidin beads coated with biotinylated human KLK5 (New England Biolabs®) and goat anti-rabbit Fc fragment-specific FITC conjugates (Jackson ImmunoResearch®). Antigen-specific antibody-secreting cells were then identified from the surrounding fluorescence halo. Multiple individual B cell clones identified using an Olympus microscope were then collected with an Eppendorf® micromanipulator and placed in PCR tubes. cDNA was obtained from single cells by standard RT-PCR, and subsequent PCR of the variable immunoglobulin sequences of the heavy and light chains was performed using immunoglobulin gene-specific primers. Subsequently, nested PCR incorporating the duplicated vector site was performed to directly clone the variable region into rabbit IgG (VH) or rabbit κ (VL) mammalian expression vectors. The heavy and light chain constructs were co-transfected into ExpiHEK-293 cells using ExpiFectamine® (Life Technologies®) and recombinant antibody expressed in 30 ml of a 125 ml Erlenmeyer® flask. After 5-7 days of culture, the supernatant was collected, and the antibody was purified by protein A affinity capture using an AKTA pure chromatography system. A 1 ml protein A HiTrap MabSelect SuRe column (GE Healthcare) was mounted on the system, and after equilibrating the column with PBS pH 7.4, the cell culture supernatant was applied to the column at a flow rate of 0.25 ml / min. Next, the column was washed with PBS pH 7.4, the bound material was eluted with sodium citrate pH 3.4, and neutralized with an appropriate volume of 2 M Tris-HCl pH 8.5. The eluted fraction was buffered with PBS (Sigma) pH 7.4 and passed through a 0.22 μm filter.The final purified material was assayed for endotoxins using A280 scanning, SE-UPLC (BEH200 method), and the PTS Endosafe system.

[0296] From this analysis, rabbit antibodies 10236 and 10273 showed potent inhibition and were selected for further characterization.

[0297] Example 4: Identification of KLK5-specific inhibitory antibodies Next, purified rabbit antibodies 10236 and 10273 were screened to confirm their inhibitory activity against KLK5, and their specificity for KLK5 was determined by using a panel of human sequenced kallikrein family members, including KLK2, KLK4, and KLK7, along with mouse and cynomolgus monkey KLK5 and KLK7. A 10-point semi-logarithmic dilution series ranging from 600 nM to 20 pM was prepared for each antibody, and 5 μL was transferred to a black 384-well assay plate (Corning®, catalog no. 3575) using a Beckman Coulter FX® and Multidrop System. 15 μL of activated recombinant kalkrein protein was added to the corresponding wells to achieve the following final assay concentrations in assay buffer A (50 mM Tris, 150 mM NaCl, 200 μM EDTA, 0.05% (v / v) Tween-20, pH 7.6): 60 pM human KLK5, 250 pM human KLK7, 500 pM human KLK2, 30 pM human KLK4, 30 pM cynomolgus monkey KLK5, 500 pM cynomolgus monkey KLK7, 30 pM mouse KLK5, or 10 nM mouse KLK7. As a control, 20 μL of assay buffer A was added to the wells for 0% activity, and LEKTI D5 rabbit Fc was used as a positive control for inhibition within the same antibody concentration range. 15 μL of human KLK5 in 5 μL of assay buffer A was used for 100% activity. After incubating the plates overnight at room temperature, the following peptide substrates were added using a multidrop device: Boc-VPR-AMC (Cambridge Research Biochemicals®) for human KLK5 (300 μM), human KLK2 (30 μM), mouse KLK5 (300 μM), and cynomolgus monkey KLK5 (450 μM); KHLF-AMC (Cambridge Research Biochemicals®) for human and cynomolgus monkey KLK7 (90 μM and 150 μM, respectively); PFR-AMC (R&D Systems®) for human KLK4 (200 μM); and Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (R&D Systems®) for mouse KLK7 (150 μM).The samples were incubated for 4 hours, and λ was measured for Boc-VPR-AMC, PFR-AMC, and KHLF-AMC. ex 380nm and λ em At 430nm, for Mca-RPKPVE-Nval-WRK(Dnp)-NH2, λ ex 320nm and λ em The data was read at 400 nm using a Pherastar FSX plate reader (BMG Labtech®). The data was analyzed to determine the inhibition rate described in Example 3. The data was plotted against the concentration of the test antibody, and the IC50 (Genedata Screener®) was determined by fitting a 4-parameter sigmoid.

[0298] In addition to rabbit antibody 10236, for this measurement, the polynucleotide sequences of the rabbit variable regions of antibodies 10236 and 10273 were cloned into a modified mouse C kappa vector containing the S171C mutation to reproduce additional disulfide bonds found in the rabbit VK light chain but not present in the mouse constant region (sequence numbers 155 and 156 for rabbit antibody 10273 mIgG, and sequence numbers 159 and 160 for rabbit antibody 10236 mIgG). As a result, antibodies including sequence numbers 157 and 158 for rabbit antibody 10236 mIgG and sequence numbers 153 and 154 for rabbit antibody 10273 mIgG were obtained.

[0299] Rabbit antibodies 10236 and 10273 mIgG showed potent inhibition of human KLK5 and no activity (i.e., <40% threshold according to the selection criteria of Example 2) against other human family members tested (human KLK2, 4, and 7). Potent inhibition of cynomolgus monkey KLK5 was also demonstrated, but not against cynomolgus monkey KLK7. No inhibitory activity was evident against either mouse KLK5 or KLK7. IC for rabbit antibodies 10236, 10273, and LEKTI D5 rabbit Fc. 50 The results are shown in Table 2. [Table 2]

[0300] Example 5: Determination of affinity for KLK5-specific antibodies The dynamics of mouse IgG molecules binding to human KLK5 were evaluated by surface plasmon resonance (Biacore T200) at 25°C.

[0301] A goat anti-mouse IgG Fc-specific antibody (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip to a level of approximately 7000 RU via amine coupling chemistry. Each analytical cycle consisted of capturing anti-KLK5 IgG molecules onto the anti-Fc surface, injecting KLK5 analyte (prepared by the inventors) at 30 μl / min for 300 seconds, followed by dissociation for 600 seconds. At the end of each cycle, the surface was regenerated at a flow rate of 10 μl / min using 60 seconds of injection of 50 mM HCl, followed by 30 seconds of injection of 5 mM NaOH and a final 60 seconds of injection of 50 mM HCl. Human KLK5 was titrated to a final concentration of 300 mM in HBS-EP+ running buffer (GE Healthcare) supplemented with NaCl, ranging from 20 nM to 0.25 nM (4 × 3-fold serial dilutions). Buffer blank injections were included to account for instrument noise and drift.

[0302] Kinetic parameters were determined using a 1:1 coupling model with Biacore T200 evaluation software.

[0303] Table 3 shows the affinities of rabbit antibodies 10236 and 10273. [Table 3]

[0304] Example 6: Characterization of antibody 10273 KLK5-PAR2 cell assay of rabbit antibody 10273 KLK5 has been shown to activate protease-activated receptor-2 (PAR2) receptors on the surface of keratinocytes (K. Oikonomopoulou et al. Kallikrein-mediated cell signalling: targeting proteinase-activated receptors (PAR). Biol Chem, 387 (2006), pp. 817-824). This leads to an inflammatory cascade driven by NFKB and the release of related cytokines such as TSLP.

[0305] Since PAR2 is a Gq-coupled G protein-coupled receptor (GPCR), its activation leads to phospholipase signaling and inositol monophosphate (IP-1) production. Activation of endogenous PAR2 expressed on HaCat keratinocytes upon exposure to recombinant KLK5 was monitored by detecting IP1 using a Cisbio assay kit.

[0306] Confluent HaCat cells were harvested, seeded at 10,000 cells / well in 384 Fluoblock plates (Corning®), and cultured overnight in DMEM medium + 10% FBS + 2 mM L-glutamine + penicillin / streptomycin (Life Technologies®) at 37°C and 5% CO2, followed by treatment according to the IP-One Gq assay protocol (Cisbio®). The antibody to be tested was serially diluted with 1× stimulating buffer (IP-One Gq assay kit, Cisbio®) from the highest concentration of 2 μM and incubated in the presence of 200 nM human recombinant KLK5 at 37°C for 1 hour. The antibody / KLK5 mixture was added to HaCat cells, and inositol monophosphate (IP1) was detected according to the IP-One Gq assay protocol, with fluorescence read at 665 nM and 620 nM using a Synergy Neo plate reader.

[0307] Antibody 10273 was able to almost completely inhibit IP1 release from KLK5-treated HaCat cells (Figure 1), showing similar maximum inhibition of IP1 release, but with greater potency compared to the LEKTI protein.

[0308] Mechanism of action of antibody 10273 Non-competitive enzyme inhibitors reduce enzyme activity but can bind equally well to the enzyme in the presence or absence of the substrate. Both the inhibitor and substrate can bind to the enzyme simultaneously, but they cannot form cleaved products; the enzyme-substrate-inhibitor complex can only be broken down into either the enzyme-substrate or the enzyme-inhibitor complex. With non-competitive inhibitors, the inhibition rate is not affected by increasing substrate concentration.

[0309] Antibody 10273 or LEKTI-D5 Fc protein was prepared in assay buffer (150 mM NaCl, 50 mM Tris, 200 μM EDTA, 0.05% (v / v) Tween-20, pH 7.6) at an IC50 of 300, 30, or 3 times that of KLK5 (see above). 10 μL of antibody was added to a Corning low-binding black low-flange 384-well assay plate (Corning®). 10 μL of five sequential dilutions of 30 mM to 300 μM Boc-VPR-AMC (Cambridge Research Biochemicals®) were added to the plate. Using a Pherastar FSX plate reader (BMG Labtech®), the reaction was simultaneously initiated by injecting either 10 μL of 1.8 nM KLK5 (Boc-VPR-AMC <1 mM) or 180 pM KLK5 (Boc-VPR-AMC >1 mM), and fluorescence (λex 380 nm λem 430 nm) was monitored every 30 seconds. The final reaction conditions included antibody 10273 or LEKTI-D5 Fc protein at 100, 10, or 1-fold IC50 relative to the determined KLK5 (as described above), serial dilutions of Boc-VPR-AMC ranging from 10 mM to 100 μM, and 60 or 600 pM KLK5. Negative controls were prepared by replacing the antibody or KLK5 with assay buffer.

[0310] The data was analyzed by subtracting the background fluorescence at each time point and plotting the fluorescence against time. The data was then applied to the following formula (GraphPad Prism®, GraphPad software).

number

[0311] This resulted in the observed time-dependent inhibition rate k obs We can decide, here v i v is the initial reaction rate, s This is the final rate. To determine the inhibition mechanism, k is used relative to the substrate concentration. obs The values ​​were plotted (Figure 2).

[0312] The inhibition rate of KLK5 by antibody 10273 did not change with increasing substrate concentration, demonstrating that antibody 10273 is a non-competitive inhibitor of KLK5 (Figure 2A). LEKTI-D5 Fc protein showed a decrease in inhibition rate with increasing substrate concentration, demonstrating a competitive mechanism of action (Figure 2B).

[0313] LEKTI binding to KLK5 in the presence of antibody 10273 Surface plasmon resonance (SPR) experiments were performed to determine whether antibody 10273 competes with the LEKTI D5 protein for binding to human KLK5. These assays allowed us to compare the affinity of the LEKTI D5 Fab fusion to the KLK5 protein alone with the affinity of human KLK5 complexed with antibody 10273.

[0314] Kinetic measurements of the binding of LEKTI D5 Fab fusion protein to human KLK5 were obtained using Biacore T200 (GE Life Sciences®). To prepare the surface, a CM5 chip (GE Life Sciences®) was first injected with an EDC / NHS (GE Life Sciences®) mixture for 5 minutes (30 μL).-1 ), followed by 100 μg mL of acetate buffer at pH 5.0. -1 The chip surface was activated by injection of a LEKTI D5 Fab fusion (UCB) (GE Life Sciences®), and an 80RU immobilized LEKTI D5 Fab fusion was obtained on the chip surface. Finally, the surface was inactivated by injection of 1M ethanolamine hydrochloride-NaOH, pH 8.5. Next, the concentration of human KLK5 in HBS-EP buffer (GE Life Sciences®) was increased from 0.32 nM to 32 nM and injected in a single-cycle kinetic mode. The kinetics were determined by subtracting the values ​​obtained from the buffer-only injection from the values ​​obtained for KLK5 injection and applying them to the 1:1 binding model in BIAcore evaluation software (GE Life Sciences®).

[0315] To determine whether human LEKTI can bind to human KLK5 when it is conjugated by rabbit antibody 10273, an antibody capture surface was prepared using goat anti-rabbit Fc polyclonal as described in Example 4, and then Ab10273 was captured. Next, 20 nM of human KLK5 was injected until the surface reached saturation. Then, LEKTI D5 Fab fusion protein (produced as described in Example 1) was injected at concentrations of 30 pM to 100 nM. The values ​​obtained with buffer only were first subtracted from the values ​​obtained with the analyte, and then fitted to a 1:1 binding kinetic model in Biacore® evaluation software (GE Life Sciences®).

[0316] For reference, the LEKTI D5 Fab fusion protein was immobilized on the chip surface before monitoring its interaction with human KLK5. The human LEKTI D5 Fab fusion protein was able to bind to human KLK5 with an affinity of 19.7 nM when human KLK5 was already complexed with rabbit antibody 10273 (Table 4). The affinity of human LEKTI to human KLK5 was higher (40 pM) in the absence of rabbit antibody 10273, but this analysis demonstrates that rabbit antibody 10273 can provide further inhibitory activity to human KLK5 by binding to human KLK5 in or out of the presence of human LEKTI. [Table 4]

[0317] LEKTI-KLK5-antibody 10273 complex formation KLK5 was produced in HEK293 cells as a secreted protein with an N-terminal TEV-cleavable 8xHis tag. The protein was first extracted from conditioned medium using Ni. 2+ Purified by affinity chromatography. Ni containing KLK5. 2+ The fraction from the column was pooled, digested with TEV protease to remove the His tag, then a second Ni affinity step was performed to remove the TEV protease, and the cleaved KLK5 was passed through the column. 2+ The flow-through fraction from the column was concentrated and run on a size exclusion column in 50 mM Tris pH 7, 50 mM NaCl, 1 mM EDTA, and 5% glycerol. The KLK5 fraction from SEC was pooled, concentrated to approximately 10 mg / ml, and stored at -80°C.

[0318] LEKTI domain 5 (LEKTI D5 Fc TEV) according to SEQ ID NO: 173 and LEKTI domain 8 (LEKTI D8 Fc TEV) according to SEQ ID NO: 174 were produced in HEK293 cells as secreted proteins with a C-terminal TEV-cleavable Fc tag. These proteins were purified by passing them through conditioned medium on Protein A beads. The bound proteins were eluted with 0.1 M citrate, pH 2.0, and the fraction was neutralized by adding 2 M Tris-HCl, pH 8.5. The fractions from the Protein A column containing either LEKTI domain 5 or domain 8 were pooled, and the Fc tag was removed with TEV protease to obtain LEKTI D5 or LEKTI D8. The cleaved proteins were concentrated to approximately 15 mg / ml for size exclusion chromatography. SEC was performed in PBS, pH 7.2. The LEKTI-containing fractions were pooled, concentrated to approximately 10 mg / ml, and stored frozen at -80°C.

[0319] Rabbit Fab antibody 10273 was expressed as a secreted protein in HEK293 cells. Expression constructs containing SEQ ID NOs. 166 and 168 were co-transfected in a 1:1 molar ratio. Secreted Fab (including SEQ ID NOs. 165 and 167) was purified by passing conditioned medium over protein G beads and eluted with 0.1 M glycine, pH 2.7. The fraction was neutralized by adding 2 M Tris-HCl, pH 8.5. The protein was dialyzed to PBS, pH 7.2, then concentrated to approximately 10 mg / ml and stored frozen at -80°C.

[0320] First, 25 μM KLK5 was incubated with 25 μM LEKTI D5 or LEKTI D8 on ice for 60 minutes, then 25 μM rabbit Fab antibody 10273 was added and incubated on ice for another 60 minutes to form a complex of KLK5, LEKTI D5 or LEKTI D8, and rabbit Fab antibody 10273. The mixture was injected into a Superdex 200 size exclusion column equilibrated in PBS, pH 7.2, and connected to an HPLC system. Peak fractions were collected for analysis by SDS-PAGE. Figures 3A and 3B show SEC chromatograms of human KLK5 alone (solid trace, far right), rabbit Fab antibody 10273 alone (dotted trace), a two-component complex of human KLK + LEKTI D5 or D8 (Figure 3A or Figure 3B, respectively, long dashed line), and a three-component complex of KLK5 + LEKTI D5 or D8 + rabbit Fab antibody 10273 (Figure 3A or Figure 3B, respectively, short dashed line, far left).

[0321] As shown in Figures 4A and 4B, the molecular weight (MW) of each peak component was determined by SDS-PAGE.

[0322] Complexes between KLK5, LEKTI D5, or LEKTI D8 and rabbit Fab antibody 10273 were readily formed by mixing human KLK5 with each LEKTI fragment individually, and then incubating the two-component complexes with rabbit Fab antibody 10273 in a 1:1:1 ratio. Two-component and three-component complexes were observed on SEC and by SDS-PAGE of the peak fraction, demonstrating their stability and suitability for isolation / purification from other species.

[0323] Example 7: Crystallization of the KLK5 / Fab antibody 10273 / Fab antibody 10236 complex For crystallographic studies, human KLK5 was expressed by transient transfection using the Expi293® expression system (Life Technologies®) according to the manufacturer's protocol, with kifunensin (Sigma®) added at a final concentration of 5 mM. Kifunensin is a potent inhibitor of the mannosidase I enzyme and is primarily used in cell culture to produce high-mannose glycoproteins.

[0324] During KLK5 expression, the protein self-activates, yielding active KLK5 protein (residues I67-S293 of SEQ ID NO: 142 or SEQ ID NO: 144 (UniProt Q9Y337 numbering)) in the supernatant. Cells were harvested 5 days after transfection, and the supernatant was immediately used for purification. The supernatant containing human KLK5 was diluted 4-fold with buffer A (50 mM Tris pH 7.0, 50 mM NaCl) and loaded onto a HiTrap SP HP cation exchange column. The bound protein was eluted over 10 column volumes using a buffer B (50 mM Tris pH 7.0, 1 M NaCl) gradient. The fraction containing purified human KLK5 was pooled, concentrated, and further purified by size exclusion chromatography on an S200 26 / 60 equilibrated with 20 mM Tris pH 7.2 and 150 mM NaCl. KLK5 was characterized by SDS-PAGE and migrated to a location on the gel that matched the expected molecular weight (MW) of approximately 35–38 kDa for a highly mannose glycosylated protein.

[0325] Next, human KLK5 protein was treated with endoglycosidase H (Endo H) protein in a 1:100 ratio and incubated overnight at 4°C to form a homogeneous deglycosylated KLK5 protein for structural studies. Endo Glycosidase H (Endo H) is a recombinant glycosidase cloned from Streptomyces plicatus and overexpressed in Escherichia coli (E. coli). Endo H cleaves a limited number of hybrid oligosaccharides derived from the high-mannose chitobiose core and N-linked glycoproteins. Endo H does not cleave complex glycans. Enzymatic cleavage occurs between two N-acetylglucosamine residues in the core of the oligosaccharide diacetylchitobiose, leaving one N-acetylglucosamine residue on asparagine. This step was performed to ensure that homogeneous human KLK5 was available for crystallographic studies. KLK5 was characterized by SDS-PAGE (Figure 5) and migrated to a location on the gel that was consistent with the predicted molecular weight (MW) of the deglycosylated protein, approximately 25 kDa.

[0326] Rabbit Fab antibody 10273 was expressed as described in Example 6. Rabbit Fab antibody 10236 (including SEQ ID NOs. 181 and 183) was expressed as described for rabbit Fab antibody 10273. In short, expression constructs including SEQ ID NOs. 182 and 184 were co-transfected in a 1:1 molar ratio. Each secreted Fab protein was purified by passing conditioned medium over protein G beads and eluted with 0.1 M glycine, pH 2.7. The fractions were neutralized by adding 2 M Tris-HCl, pH 8.5. Each individual protein was dialyzed to PBS, pH 7.2, concentrated to approximately 10 mg / ml, and stored frozen at -80°C.

[0327] A 1:1.5:1.5 human KLK5 / rabbit Fab antibody 10273 / rabbit Fab antibody 10236 conjugate was prepared, incubated overnight at 4°C, and purified by size exclusion chromatography (20 mM Tris, 150 mM NaCl, pH 7.2 elution buffer). A single peak containing the conjugate was concentrated to approximately 10.8 mg / ml before crystallization.

[0328] The crystallization conditions for the human KLK5 / rabbit Fab antibody 10273 / rabbit Fab antibody 10236 conjugate were identified using several commercially available crystallization screenings. These were performed in a sitting drop format using a Swissci 96-well 2-droplet MRC crystallization plate (supplied from Molecular Dimensions, catalog no. MD11-00-100). First, 75 μL of each crystallization solution from the screen was packed into the reservoir using a Microlab STAR liquid processing system (Hamilton). Then, 300 nL of the human KLK5 / rabbit Fab antibody 10273 / rabbit Fab antibody 10236 conjugate and 300 nL of the reservoir solution were dispensed into the wells of the crystallization plate using a Mosquito liquid handler (TTP LabTech). Single crystals were obtained under condition 16 (well B4) of the MIDAS+HT-96 screen (Molecular Dimensions, catalog no. MD1-107). This condition contains 45% v / v pentaerythritol propoxilate (5 / 4), 0.2 M NaCl, and 0.1 M MES monohydrate at pH 6.0. The crystals were rapidly frozen in liquid nitrogen, and diffraction data were collected at beamline I03 (Diamond Light Source, UK). The data were indexed and integrated using XDS (Kabsch, W. XDS. Acta Cryst. D66, 125-132 (2010)), and then scaled using AIMLESS (2. Evans PR, Murshudov GN. How good are my data and what is the resolution? Acta Crystallogr D Biol Crystallogr. 2013; 69 (Pt7): 1204-1214).The structure of the human KLK5 / rabbit Fab antibody 10273 / rabbit Fab antibody 10236 complex was elucidated by molecular substitution using the Phaser (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Stornini, LC, &Read, RJPhaser crystallographic software. J.Appl.Cryst. (2007). 40, 658-674) of the Phenix software suite (Adams PD, Afonine PV, Bunkoczi G, et al. The Phenix software for automated determination of macromolecular structures. Methods. 2011;55(1):94-106). In this procedure, the KLK5 structure 2PSX (Debela M, Goettig P, Magdolen V, Huber R, Schechter NM, Bode W. Structural basis of the zinc inhibition of human tissue kallikrein 5. J Mol Biol. 2007 Nov 2;373(4):1017-31) and a proprietary Fab model were used as molecular substitution templates.Until acceptable Rwork, Rfree, and Ramachandran statistics (analyzed using Molprobity (Williams et al. (2018) MolProbity: More and better reference data for improved all-atom structure validation. Protein Science 27:293-315)) are obtained, Coot (P. Emsley; B. Lohkamp; WG Scott; Cowtan (2010). “Features and Development of Coot”. Acta Crystallographica. D66:486-501) and phenix.refine (Towards automated crystallographic structure refinement with phenix.refine. PVAfonine, RWG Rosse-Kunstleve, N. Echols, JJ Headadd, NWMoriarty, M. Mustaakimov, TCT Terwilliger, A. Urzhumtsev, PH Zwart, and PDA Dams Acta Crystallogr D Biol Crystallogr) Models 68,352-67 (2012)) were used in the following cycle of manual model completion and improvement.

[0329] The human KLK5 / rabbit Fab antibody 10273 / rabbit Fab antibody 10236 complex was observed as a crystalline asymmetric unit. Epitopes on KLK5 recognized by the Fab10273 and Fab10236 molecules were determined using NCONT within the CCP4 software suite. KLK5 amino acid numbering is based on UnitProtKB entry Q9 Y337, with the standard protease numbering based on chymotrypsinogen in parentheses. Table 5 shows refinement statistics at the time the invention was first described. [Table 5]

[0330] The human KLK5 epitope, conjugated by rabbit Fab antibody 10273 at a contact distance of 4 Å, is SEQ ID NO: 142 Refer to the following and it is composed of residues Leu212(163), Ser213(164), Gln214(165), Lys215(142), Arg216(167), Glu218(169), Asp219(170), Ala220(171), Pro222(173), Gly233(184), Pro269(223), Asn270(224), and Pro272(226), with the numbers in parentheses corresponding to the protease nomenclature. The binding site is shown in detail in Figure 6.

[0331] Other amino acid residues located near the epitope but within a 5 Å contact distance between human KLK5 and rabbit Fab antibody 10273 include Ala181(132), Val211(162), Tyr221(172), Asp234(185), and Arg271(225), with the numbers in parentheses corresponding to protease nomenclature, as shown in SEQ ID NO: 142. As shown in Figure 7, antibodies 10236 and 10273 have very distinct and non-overlapping binding sites and bind to different epitopes on human KLK5.

[0332] Example 8: Humanization and characterization of antibody 10273 Humanization of Ab10273 Rabbit antibody 10273 was humanized by transplanting a CDR from the rabbit V region into a human germline antibody V region framework. Several framework residues from the rabbit V region were also retained in the humanized sequence to restore antibody activity. These residues were selected using the protocol outlined by Adair et al. (1991) (Humanized antibodies. International Publication No. 91 / 09967). The alignment of the rabbit antibody (donor) V region sequence and the human germline (acceptor) V region sequence, along with the designed humanized sequence, is shown in Figures 8 and 9. The CDRs transplanted from the donor to the acceptor sequence are as defined by Kabat (Kabat et al., 1987), except for CDR-H1 (see Adair et al., 1991 Humanized antibodies. International Publication No. 91 / 09967), where the Chothia / Kabat combined definition is used.

[0333] For antibody 10273, the human V region IGKV1D-13+JK4J region (IMGT, http: / / www.imgt.org / ) was selected as the acceptor for the light chain CDR. All framework residues in the humanized light chain graft gL2 are derived from human germline genes (Figure 8).

[0334] The human V region IGHV3-66+JH6J region (IMGT, http: / / www.imgt.org / ) was selected as the acceptor for the heavy chain CDR of antibody 10273. As with many rabbit antibodies, the VH gene of antibody 10273 is shorter than that of the selected human acceptor. When aligned with the human acceptor sequence, framework 1 of the VH region of antibody 10273 lacks the N-terminal residue retained in the humanized antibody (Figure 9). Framework 3 of the 10273 rabbit VH region also lacks two residues (75 and 76) in the loop between the β-sheet chains D and E, and in the humanized graft, the gap is filled with the corresponding residues from the selected human acceptor sequence (lysine 75, K75; asparagine 76, N76) (Figure 9). All framework residues in the 10273 heavy chain humanized graft are derived from human germline genes, with the exception of one or more residues from the group containing residues 24, 48, 49, 71, 73, and 78, which retain donor residues valine (V24), isoleucine (I48), glycine (G49), lysine (K71), serine (S73), or valine (V78), respectively. Retention of residue G49 was essential for the full potency of the humanized antibody. Potential hydrolysis sites in CDRH3 were modified in graft gH3 by substituting the aspartic acid residue at position 116 with glutamic acid (D116E).

[0335] The pI of the humanized 10273 antibody is approximately 6.2. To facilitate the removal of impurities by ion-exchange chromatography during downstream processing, the pI was increased by mutating residue 1 in CDRL1 from glutamine (Q) to either arginine (R), lysine (K), or histidine (H), referring to SEQ ID NO: 1 of graft gL2. Furthermore, the pI was further increased by mutating residue 19 in CDRH3 (refer to SEQ ID NO: 6) from aspartic acid (D) to asparagine (N), and unexpectedly, the mutation in CDRH3(D19N) also resulted in an increased affinity for KLK5.

[0336] In the graft antibody 10273 gL2-Q24RgH1-D116N, CDR-H3 contains six tyrosine residues. Mutants of this graft were prepared, and each tyrosine residue was replaced with a phenylalanine residue to generate grafts 10273 gL2-Q1RgH1-D19N-Y4F, 10273 gL2-Q1RgH1-D19N-Y6F, 10273 gL2-Q1RgH1-D19N-Y9F, 10273 gL2-Q1RgH1-D19N-Y12F, 10273 gL2-Q1RgH1-D19N-Y15F, and 10273 gL2-Q1RgH1-D19N-Y16F. Further variants were prepared by mutating the threonine preceding the double tyrosine at positions 15 and 16, as shown in Sequence ID No. 6, resulting in grafts 10273 gL2-Q1RgH1-D19N-T14V and 10273 gL2-Q1RgH1-D19N-T14S.

[0337] The dynamics of these grafts regarding their binding to human KLK5 were evaluated by surface plasmon resonance (Biacore T200) at 25°C.

[0338] A goat anti-human IgG Fc-specific antibody (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip to a level of approximately 7000 RU via amine coupling chemistry. Each analytical cycle consisted of capturing anti-KLK5 IgG molecules onto the anti-Fc surface, injecting KLK5 analyte (prepared by the inventors) at 30 μl / min for 180 seconds, followed by dissociation for 600 seconds. At the end of each cycle, the surface was regenerated at a flow rate of 10 μl / min using 60 seconds of injection of 50 mM HCl, followed by 30 seconds of injection of 5 mM NaOH and a final 60 seconds of injection of 50 mM HCl. Human KLK5 was titrated in NaCl-supplemented HBS-EP+ running buffer (GE Healthcare) from 20 nM to 0.74 nM (3 × 3 serial dilutions) up to a final concentration of 300 mM. Buffer blank injections were included to account for instrument noise and drift.

[0339] Kinetic parameters were determined using a 1:1 coupling model with Biacore T200 evaluation software.

[0340] Table 6 shows that not all mutations result in antibodies that can still bind to KLK5 with the same or near-same affinity as the parental or graft antibodies from which they are derived. [Table 6]

[0341] Profiling of humanized antibodies KLK5 selectivity A series of studies were conducted to confirm that humanization of rabbit antibody 10273 did not alter its KLK5 selectivity or decrease its affinity or inhibitory activity compared to other kallikreins.

[0342] Next, the purified antibodies were screened to confirm their inhibitory activity against KLK5 according to the method described in Example 4. The antibodies were tested in a 10-point semi-logarithmic dilution series ranging from 600 nM to 20 pM. Using a Beckman Coulter FX® and Multidrop System, 5 μL of each antibody was transferred to a black 384-well assay plate (Corning®, catalog no. 3575) and assay buffer A (150 mM NaCl, 50 mM Tris, 200 μM By adding 15 μL of selected activated recombinant kallikrein enzyme in EDTA, 0.05% (v / v) Tween-20, pH 7.6, the following final assay concentrations were achieved: human KLK5 (UCB) at 60 pM, KLK7 (UCB) at 250 pM, KLK2 (R&D) at 500 pM, KLK4 (UCB) at 30 pM, and cynomolgus monkey recombinant KLK5 (UCB) at 30 pM, cynomolgus monkey KLK7 at 500 pM, mouse KLK5 (UCB) at 30 pM, and mouse KLK7 (UCB) at 5 nM. The enzymes prepared in UCB are shown in parentheses and prepared as described above in Example 1. The sources of commercially available enzymes are shown in parentheses.

[0343] Only 20 μL of assay buffer A was added to the wells for 0% activity. LEKTI D5 rabbit Fc (UCB prepared as described above) was used as the baseline for inhibitory activity; therefore, 5 μL of LEKTI D5 rabbit Fc in the same concentration range used for the 10236 Abs antibody was added to 15 μL of kallikrein enzyme. 15 μL of human KLK5 added to 5 μL of assay buffer A was used as the 100% activity baseline.

[0344] Antibodies and kallikrein were incubated overnight at room temperature. Using multidrops, the following peptide substrates were added: Boc-VPR-AMC (Cambridge Research Biochemicals®) for human KLK5 (300 μM), human KLK2 (30 μM), mouse KLK5 (300 μM), and cynomolgus monkey KLK5 (450 μM); KHLF-AMC (Cambridge Research Biochemicals®) for human and cynomolgus monkey KLK7 (90 μM and 150 μM, respectively); PFR-AMC (R&D Systems®) for human KLK4 (200 μM); and Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (R&D Systems®) for mouse KLK7 (150 μM). The samples were incubated for 4 hours, and for Boc-VPR-AMC, PFR-AMC, and KHLF-AMC, λ ex 380nm and λ em At 430nm, for Mca-RPKPVE-Nval-WRK(Dnp)-NH2, λ ex 320nm and λ em The data was read at 400 nm using a Pherastar FSX plate reader (BMG Labtech®). The data was analyzed to determine the inhibition rate described in Example 3. The data was plotted against the concentration of the test antibody, and the IC50 (Genedata Screener®) was determined by fitting a 4-parameter sigmoid.

[0345] Humanized grafts of Ab 10273 retained specificity for KLK5 and showed little to no inhibition of other KLK family members tested (data not shown).

[0346] Inhibition level of KLK5 by humanized grafts A capture assay was developed to confirm the level of KLK5 activity inhibition mediated by the humanized Ab 273 graft variant.

[0347] Nunc Maxisorp black 384-well plates (Sigma Aldrich®) were coated with 10 μg / mL of F(ab')2 fragment goat anti-human IgG Fcγ fragment-specific (Jackson ImmunoResearch®) in carbonate coating buffer and left overnight at 4°C. The plates were washed three times with Biotek® plate washer containing PBS and 0.005% Tween-20, and the plates were blocked at room temperature for 1 hour in 20 μL / well PBS containing 1% BSA. After washing the plates as described above, 10 μl of 5 nM antibody (diluted from stock using assay buffer 150 mM NaCl, 50 mM Tris, 200 μM EDTA, 0.005% (v / v) Tween-20, pH 7.6) was added to the appropriate wells. The plates were sealed and incubated overnight at room temperature, then washed. Next, 10 μl of 250 pM KLK5 (diluted from stock in assay buffer) was added to the relevant wells, incubated at room temperature for 4 hours, and then 10 μl of 600 mM BVPR-AMC substrate was added. The antibody was removed, and the "maximum activity" control well was replaced with assay buffer, while the "minimum activity" control well was left without substrate or enzyme (replaced with assay buffer). Fluorescence (λ) ex 380nm λ em The 430nm wavelength was read every hour over a period of 4 hours using a PheraStar FSX plate reader.

[0348] The data was analyzed to obtain the inhibition rate of KLK5 values. A normalized baseline-corrected dataset was obtained by subtracting the 0-value at each time point from its equivalent 4-hour value. The normalized fluorescence values ​​were converted to inhibition percentages using the following formula.

number

[0349] The data was plotted to generate the bar graph shown in Figure 10. The inhibition percentage values ​​ranged from 95% to 97%, demonstrating that the antibody 10273 graft was able to almost completely inhibit KLK5 enzyme activity.

[0350] Example 9: Biophysical characterization of humanized antibodies Characterization of antibodies by mass spectrometry Identification of each antibody was confirmed by intact mass measurement of heavy and light chains using LC-MS with a Waters ACQUITY UPLC system equipped with a Xevo G2 Q-ToF mass spectrometer. The sample (approximately 5 μg) was reduced with 5 mM tris(2-carboxyethyl)phosphine (TCEP) in 150 mM ammonium acetate for 40 minutes at 37°C. The LC column was a Waters BioResolveT RP mAb polyphenyl, 450 Å, 2.7 μm, held at 80°C, equilibrated with 95% solvent A (water / 0.02% trifluoroacetic acid (TFA) / 0.08% formic acid) and 5% solvent B (95% acetonitrile / 5% water / 0.02% TFA / 0.08% formic acid) at a flow rate of 0.6 mL / min. The protein was eluted over 8.8 minutes with a gradient of 5% to 50% solvent B, followed by washing with 95% solvent B and re-equilibriumization. UV data was acquired at 280 nm. The MS conditions were as follows: ion mode: ESI positive ion, resolution mode, mass range: 400-5000 m / z, and external calibration with NaI. The data was analyzed using Waters MassLynx and MaxEnt software.

[0351] The intact mass (reduced chain) showed that the observed mass of the light chain was consistent with the predicted mass. However, for all 10273 molecules except 10273 gL2-Q1RgH1-D19N-Y9F, a difference of +80 Daltons was observed between the observed heavy chain mass and the predicted heavy chain mass (Table 7).

[0352] This was identified as a post-translational modification of Tyr9 in the heavy chain and confirmed to be sulfation by Western blotting with an anti-sulfotyrosine antibody. Furthermore, incubation of 10273 gL2-Q1RgH1-D19N with abalone sulfatase at 37°C for 1 hour resulted in a reduction of approximately 17% in the proportion of +80Da modification, as determined by intact mass spectrometry. [Table 7]

[0353] Thermal stability (Tm) measurement The melting temperature (Tm) or the midpoint temperature of unfolding was determined using a thermofluor assay.

[0354] In the Thermofluor assay, the protein unfolding process was monitored by using the fluorescent dye SYPRO® orange, which binds to hydrophobic regions exposed as the temperature increases. The reaction mixture contained 5 μL of 30×SYPRO® Orange Protein Gel Stain (Thermofisher Scientific, S6651), diluted from a 5000-fold concentrate in test buffer. 45 μL of 10273 Ab sample at 0.2 mg / mL in PBS pH 7.4 was added to the dye and mixed. 10 μL of this solution was dispensed in a quadruple-strand into a 384 PCR optical well plate and run on a QuantStudio7 real-time PCR system (Thermofisher®). The PCR system heating device was set to 20°C and increased to 99°C at a rate of 1.1°C / min. The charge-coupled device monitored the fluorescence changes in the wells. The increase in fluorescence intensity was plotted, and the inflection point of the slope was used to generate the apparent midpoint temperature (Tm).

[0355] Two unfolding transitions were observed for all antibodies. The first domain may be attributable to the CH2 domain, and the second domain may be attributable to the average Tm of the Fab unfolding domain and the CH3 domain.

[0356] There was no difference in thermal stability among the 10273 IgG4P antibodies (Table 8). As expected, increased thermal stability was observed for the IgG1 format of antibody 10273 gL2gH1 compared to the corresponding IgG4P format. (Heads et al “Relative stabilities of IgG1 and IgG4 Fab domains: influence of the light-heavy interchain disulfide bond architecture”. Protein Sci 2012;21:1315-22). [Table 8]

[0357] Further biophysical characterization was performed on three humanized IgG4P antibodies, antibody 10273 gL2H1, gL2Q1R-gH1D19N, and gL2Q1R-gH4D19N, as well as the IgG1-type antibody 10273 gL2gH1.

[0358] Experimental isoelectric point (pI) measurement pI was experimentally determined using the iCE3® Total Capillary Imaging Capillary Isoelectric Focusing (cIEF) system (ProteinSimple). Sample 10273 Ab was prepared by mixing 30 μL of sample (from 1 mg / mL stock in HPLC-grade water), 35 μL of 1% methylcellulose solution (ProteinSimple, 101876), 4 μL of pH 3-10 Pharmalite (ProteinSimple, 042-848), 0.5 μL of 4.65 synthetic pI marker, 0.5 μL of 9.77 synthetic pI marker (ProteinSimple, 102223 and 102219), and 12.5 μL of 8M urea solution (Sigma Aldrich®). The final volume was 100 μL using HPLC-grade water. The sample was focused at 1.5kV for 1 minute, followed by 3kV for 5 minutes, and a 280nm image of the capillary was acquired using Protein Simple software. The resulting electrophoresis maps were analyzed using iCE3 software, and pI values ​​were assigned (linear relationship between pI markers).

[0359] Higher pI was observed as a result of mutations in the light chain Q1R and heavy chain D19N. Differences in isotype, i.e., IgG1 instead of IgG4P, also resulted in increased experimental pI. Both mutations and isotype changes can be utilized during production for the removal of host cell proteins in the first ion exchange step (AEX). The increased pI also allows for formulation in more common buffers (around pH 5-6). [Table 9]

[0360] Hydrophobic interaction chromatography (HIC) Hydrophobic interaction chromatography (HIC) separates molecules in order of increasing hydrophobicity. Molecules bind to a hydrophobic stationary phase in the presence of a high concentration of polar salt and desorb to the mobile phase as the salt concentration decreases. Longer retention times correspond to greater apparent hydrophobicity.

[0361] A 2 mg / mL 10273 Ab sample was diluted 1:2 with 1.6 M ammonium sulfate and PBS (pH 7.4). 10 μg (10 μL) of the sample was injected into a Dionex ProPac™ HIC-10 column (100 mm × 4.6 mm) connected in series to an Agilent 1200 two-component HPLC equipped with a fluorescence detector. Separation was monitored by intrinsic fluorescence (excitation and emission wavelengths, 280 nm and 340 nm, respectively). The samples were analyzed using buffer A (0.8 M ammonium sulfate, 100 mM phosphate, pH 7.4) and buffer B (100 mM phosphate, pH 7.4) with the following gradient elution: (i) retention at 0% B for 2 minutes, (ii) linear gradient from 0 to 100% B over 30 minutes (0.8 mL / min), and (iii) washing the column at 100% B for 2 minutes and re-equilibrium at 0% B for 10 minutes before the next sample injection. The column temperature was maintained at 20°C. The retention time (in minutes) is shown in Table 10. [Table 10]

[0362] Only slight differences in retention time, i.e., apparent hydrophobicity, were observed among the 10273 different antibodies, all of which are thought to have shown above-average measurements and therefore potentially exhibit a tendency to aggregate (Jain et al “Biophysical properties of the clinical-stage antibody landscape” Proc Natl Acad Sci US A.2017 Jan 31;114(5):944-949.doi:10.1073 / pnas.1616408114.Epub 2017 Jan 17). Therefore, all molecules showed a greater tendency to bind to the HIC matrix. The HIC matrix has been used as an alternative step to ion exchange in manufacturing.

[0363] Solubility measurement using polyethylene glycol (PEG) Understanding colloidal stability (solubility) can be derived by investigating the effect of polyethylene glycol (PEG) precipitation. Using PEG, protein solubility was reduced in a quantitatively definable manner by increasing the PEG concentration (w / v) and measuring the amount of protein remaining in the solution. This assay helps mimic the effect of high-concentration solubility without using conventional concentration methods.

[0364] A 40% (w / v) stock solution of PEG3350 (Merck, 202444) was prepared in PBS pH 7.4, 50 mM sodium acetate, 125 mM sodium chloride pH 5.0 (common storage buffer), and 50 mM histidine, 250 mM proline pH 5.5 (common pre-formulation buffer). Continuous titration was performed using an assist plus liquid processing robot (Integra, 4505) to obtain PEG3350 concentrations ranging from 40% to 15.4%. To minimize non-equilibrium precipitation, sample preparation consisted of mixing the 10273 Ab sample and the PEG solution in a 1:1 volume ratio. 35 μL of the PEG3350 stock solution was added to 96-well v-bottom PCR plates (A1-H1) using a liquid processing robot. 35 μL of 2 mg / mL 10273 Ab solution was added to the PEG stock solution to obtain a test concentration of 1 mg / mL. This solution was mixed by automated, slow, repeated pipetting and incubated at 37°C for 0.5 hours to redissolve non-equilibrium aggregates. The sample was then incubated at 20°C for 24 hours. Subsequently, the sample plate was centrifuged at 20°C and 4000×g for 1 hour. 50 μL of the supernatant was dispensed into a UV-Star®, half-area, 96-well, μClear®, microplate (Greiner, 675801). Protein concentrations were determined by UV spectrophotometric analysis at 280 nm using a FLUOstar® Omega multi-detection microplate reader (BMG LABTECH). The obtained values ​​were plotted using GraphPad Prism version 7.04, and the PEG midpoint (PEG) score was derived from the midpoint of the sigmoid dose-response (variable gradient) fit.

[0365] The data is shown in Table 11. A higher PEG midpoint (%) indicates a higher likelihood of stability / solubility at higher concentrations.

[0366] At pH 7.4, 10273 gL2gH1(IgG4P) showed the highest PEG midpoint (maximum predicted solubility), but the lowest solubility at acetate pH 5. This trend differed from that of the corresponding IgG1 molecule, where there was no difference in predicted solubility between PBS pH 7.4 and acetate pH 5. Both 10273 gL2-Q1RgH1-D19N and 10273 gL2-Q1RgH4-D19N showed similar PEG midpoints in PBS pH 7.4 and histidine pH 5.5 buffer, but decreased at acetate pH 5. [Table 11]

[0367] Effect of stress at the gas-liquid interface (aggregation assay) Proteins tend to unfold when exposed to an air-liquid interface where their hydrophobic surface is presented to a hydrophobic environment (air) and their hydrophilic surface is presented to a hydrophilic environment (water). Agitating the protein solution creates a large gas-liquid interface that can promote aggregation. This assay helps mimic the stress molecules experience during production (e.g., ultrafiltration) and provides stringent conditions for attempting to distinguish between different antibody molecules.

[0368] Samples were stressed in PBS pH 7.4 or 50 mM sodium acetate and 125 mM sodium chloride pH 5 by vortexing using an Eppendorf Thermomixer Comfort®. Before vortexing, the concentration was adjusted to 1 mg / mL using an appropriate extinction coefficient (1.46 Abs 280 nm, 1 mg / mL, path length 1 cm), and absorbance at 280 nm, 340 nm, and 595 nm was obtained using a Varian Cary 50-Bio spectrophotometer® to establish the reading at time 0. Each sample was subdivided into 1.5 mL conical Eppendorf® capped tubes (3 × 250 μL) and vortexed at 1400 rpm, 25 °C for 4 hours. Aggregation (turbidity) was monitored by measuring the sample at 595 nm using a Varian Cary® 50-Bio spectrophotometer.

[0369] Table 12 and Figure 11 show the aggregation tendencies of different antibodies in two buffer solutions over 4 hours. [Table 12]

[0370] IgG1 10273gL2gH1 showed maximum agglutination stability in both buffers, and generally, all 10273 antibodies were more agglutination-stable at PBS pH 7.4.

[0371] Chemical denaturation (unfolding) of guanidine hydrochloride using circular dichroism (CD) As a test of robustness, a chemical denaturing agent (guanidine hydrochloride) was used to unfold the antibody.

[0372] Using DragonFly® (TTP labtech, Cambridge, UK), guanidine HCl was titrated at 0–4 M across 48 wells of a 96-well plate. The sample was then added at 7.5 μM (final) in a total volume of 80 μL, and equilibrated overnight at room temperature. 70 μL was transferred to a deep-well plate for analysis by automated circular dichroism (Chiroscan®: ACD, Applied Photophysics, Leatherhead, UK). Circular dichroism measurements were acquired in the far-UV region from 260–190 nm using a flow cell cuvette with a step size of 0.5 nm, a bandwidth of 1 nm, and a path length of 0.2 mm at 1 sec / time. The CD signal at 210 nm was fitted to a sigmodal dose-response curve of Origin 9b.

[0373] The tested 10273 antibody appeared to have a similar denaturation profile according to the chemical denaturation midpoint (Cm) (Table 13), exhibiting two apparent transitions and thus demonstrating similar robustness to the chemical denaturant. [Table 13]

[0374] Evaluation of self-interactions using AC-SINS (Affinity-Snatched Self-Interacting Nanoparticle Spectroscopy). We tested a set of 10273 antibodies using the AC-SINS assay (Liu Y.MAbs.2014 Mar-Apr;6(2):483-92), evaluated their self-interaction tendencies, and therefore reported on their aggregation stability.

[0375] Goat anti-human Fcγ specific capture antibody (Jackson ImmunoResearch) was buffered with 20 mM sodium acetate, pH 4.3, diluted to 0.4 mg / mL, and 50 μL was added to 450 μL of citrate-stabilized 20 nm gold nanoparticles (TedPella, USA). The mixture was left at room temperature overnight. The conjugated nanoparticles were blocked with 55 μL of PEG-thiol for 1 hour, centrifuged at 21,000 × g for 6 minutes, the supernatant was removed, and the mixture was resuspended in 20 mM sodium acetate, pH 4.3 to a final volume of 150 μL.

[0376] The 10273 antibody was diluted to 22 μg / mL in PBS, pH 7.4 (200 μL), added to an equal volume of nonspecific total IgG (Jackson ImmunoResearch), vortexed briefly, and 72 μL was added to a 96-well plate. 8 μL of nanoparticles were added to each well (n=4). Absorbance was read from 500-600 nm using a BMG plate reader, fitted to a Lorentz curve (RShiny), and PBS only was subtracted from the sample to obtain the Δλmax detailed in Table 14. A larger Δλmax indicates a greater tendency for self-interaction. The tested 10273 antibody showed low λmax and Δλmax (from PBS background), suggesting a low tendency for self-interaction. [Table 14] [Sequence Listing Free Text]

[0377] Sequence Listing 30 <223> Rabbit VL Sequence Listing 31 <223> Rabbit VL nucleotides Sequence Listing 32 <223> Rabbit VH Sequence Listing 33 <223> Rabbit VH nucleotide Sequence Listing 35 <223> 10273 gL2 VLnucleotide Sequence Listing 36 <223> 10273 gL2 light chain Sequence Listing 37 <223> 10273 gL2 light chain nucleotide Sequence Listing 39 <223> 10273 gL2 VL Q1R nucleotide Sequence Listing 40 <223> 10273 gL2 Light Chain Q1R Sequence Listing 41 <223> 10273 gL2 light chain Q1R nucleotide Sequence Listing 43 <223> 10273 gL2 L Q1K nucleotide Sequence Listing 44 <223> 10273 gL2 Light Chain Q1K Sequence Listing 45 <223> 10273 gL2 light chain Q1K nucleotide Sequence Listing 47 <223> 10273 gL2 VL Q1H nucleotide Sequence Listing 48 <223> 1027 gL2 light chain Q1H Sequence Listing 49 <223> 10273 gL2 light chain Q1H nucleotide Sequence Listing 51 <223> 10273 gH1 VH nucleotide Sequence Listing 52 <223> 10273 gH1 heavy chain Sequence Listing 53 <223> 10273 gH1 heavy chain nucleotide Sequence Listing 55 <223> 10273 gH1 VH D19N nucleotide Sequence Listing 56 <223> 10273 gH1 heavy chain D19N Sequence Listing 57 <223> 10273 gH1 heavy chain D19N nucleotide Sequence Listing 59 <223> 10273 gH1 VH D19E nucleotide Sequence Listing 60 <223> 10273 gH1 heavy chain D19E Sequence Listing 61 <223> 10273 gH1 heavy chain D19E nucleotide Sequence Listing 63 <223> 10273 gH4 VH nucleotide Sequence Listing 64 <223> 10273 gH4 heavy chain Sequence Listing 65 <223> 10273 gH4 heavy chain nucleotide Sequence Listing 67 <223> 10273 gH4 VH D19N nucleotide Sequence Listing 68 <223> 10273 gH4 heavy chain D19N Sequence Listing 69 <223> 10273 gH4 heavy chain D19N nucleotide Sequence Listing 71 <223> 10273 gH5 VH nucleotide Sequence Listing 72 <223> 10273 gH5 double chain Sequence Listing 73 <223> 10273 gH5 heavy chain nucleotide Sequence Listing 75 <223> 10273 gH5 VH D19N nucleotide Sequence Listing 76 <223> 10273 gH5 heavy chain D19N Sequence Listing 77 <223> 10273 gH5 heavy chain D19N nucleotide Sequence Listing 79 <223> 10273 gH8 VH nucleotide Sequence Listing 80 <223> 10273 gH8 heavy chain Sequence Listing 81 <223> 10273 gH8 heavy chain nucleotide Sequence Listing 83 <223> 10273 gH VH D19N nucleotide Sequence Listing 84 <223> 10273 gH8 heavy chain D19N Sequence Listing 85 <223> 10273 gH8 heavy chain D19N nucleotide Sequence Listing 86 <223> 10273 gH10 VH nucleotide Sequence Listing 87 <223> 10273 gH10 VH nucleotide Sequence Listing 88 <223> 10273 gH10 heavy chain Sequence Listing 89 <223> 10273 gH10 heavy chain nucleotide Sequence Listing 91 <223> 10273 gH10 VH D19N nucleotide Sequence Listing 92 <223> 10273 gH10 heavy chain D19N Sequence Listing 93 <223> 10273 gH10 heavy chain D19N nucleotide Sequence Listing 95 <223> 10273 gH11 VH nucleotide Sequence Listing 96 <223> 10273 gH11 heavy chain Sequence Listing 97 <223> 10273 gH11 heavy chain nucleotide Sequence Listing 99 <223> 10273 gH11 VH D19N nucleotide Sequence Listing 100 <223> 10273 gH 11-chain D19N Sequence Listing 101 <223> 10273 gH 11-stranded D19N nucleotide Sequence Listing 103 <223> 10273 gH1 VH Y4F D19N nucleotide Sequence Listing 104 <223> 10273 gH1 heavy chain Y4F D19N Sequence Listing 105 <223> 10273 gH1 heavy chain Y4F D19N nucleotide Sequence Listing 107 <223> 10273 gH1 VH Y6F D19N nucleotide Sequence Listing 108 <223> 10273 gH1 heavy chain Y6F D19N Sequence Listing 109 <223> 10273 gH1 heavy chain Y6F D19N nucleotide Sequence Listing 111 <223> 10273 gH1 VH Y9F D19N nucleotide Sequence Listing 112 <223> 10273 gH1 heavy chain Y9F D19N Sequence Listing 113 <223> 10273 gH1 heavy chain Y9F D19N nucleotide Sequence Listing 115 <223> 10273 gH1 VH Y12F D19N nucleotide Sequence Listing 116 <223> 10273 gH1 heavy chain Y12F D19N Sequence Listing 117 <223> 10273 gH1 heavy chain Y12F D19N nucleotide Sequence Listing 119 <223> 10273 gH1 VH Y15F D19N nucleotide Sequence Listing 120 <223> 10273 gH1 heavy chain Y15F D19N Sequence Listing 121 <223> 10273 gH1 heavy chain Y15F D19N nucleotide Sequence Listing 123 <223> 10273 gH1 VH Y16F D19N nucleotide Sequence Listing 124 <223> 10273 gH1 heavy chain Y16F D19N Sequence Listing 125 <223> 10273 gH1 heavy chain Y16F D19N nucleotide Sequence Listing 126 <223> 10273 gH1 VH T14N D19N nucleotide Sequence Listing 127 <223> 10273 gH1 VH T14N D19N nucleotide Sequence Listing 128 <223> 10273 gH1 heavy chain T14N D19N Sequence Listing 129 <223> 10273 gH1 heavy chain T14N D19N nucleotide Sequence Listing 131 <223> 10273 gH1 VH T14V D19N nucleotide Sequence Listing 132 <223> 10273 gH1 heavy chain T14V D19N Sequence Listing 133 <223> 10273 gH1 heavy chain T14V D19N nucleotide Sequence Listing 135 <223> 10273 gH1 VH T14S D19N nucleotide Sequence Listing 136 <223> 10273 gH1 heavy chain T14S D19N Sequence Listing 137 <223> 10273 gH1 heavy chain T14S D19N nucleotide Sequence Listing 138 <223> Human IGKV1D-13 JK4 Acceptor Framework Sequence Listing 139 <223> Human IGKV1D-13 JK4 Acceptor Framework Nucleotide Sequence Listing 140 <223> Human IGHV3-66 JH6 Acceptor Framework Sequence Listing 141 <223> Human IGHV3-66 JH6 Acceptor Framework Nucleotide Sequence Listing 142 <223> Human KLK5 (full-length with signal sequence) Sequence Listing 143 <223> Human KLK5 Proform Sequence Listing 144 <223> Activated human KLK5 Sequence Listing 145 <223> Human LEKTI D5, Rabbit Fc Sequence Listing 146 <223> Human KLK7 Proform Sequence Listing 147 <223> Activated human KLK7 Sequence Listing 148 <223> Crab-eating macaque KLK7 Proform Sequence Listing 149 <223> Active Crab-eating Monkey KLK7 Sequence Listing 150 <223> Active mouse KLK5 Sequence Listing 151 <223> Active Crab-eating Monkey KLK5 Sequence Listing 152 <223> Human LEKTI D8, Rabbit Fc Sequence Listing 153 <223> Rabbit 10273 mIgG light chain Sequence Listing 154 <223> Rabbit 10273 mIgG heavy chain Sequence Listing 155 <223> Rabbit 10273 mIgG light chain nucleotide Sequence Listing 156 <223> Rabbit 10273 mIgG heavy chain nucleotide Sequence Listing 157 <223> Rabbit 10236 mIgG light chain Sequence Listing 158 <223> Rabbit 10236 mIgG heavy chain Sequence Listing 159 <223> Rabbit 10236 mIgG light chain nucleotide Sequence Listing 160 <223> Rabbit 10236 mIgG heavy chain nucleotide Sequence Listing 161 <223> 10236 Light Chain Fab Sequence Listing 162 <223> 10236 Light Chain Fab Nucleotide Sequence Listing 163 <223> 10236 Heavy Chain Fab Sequence Listing 164 <223> 10236 Heavy Chain Fab Nucleotides Sequence Listing 165 <223> 10273 Light Chain Fabric Sequence Listing 166 <223> 10273 Light Chain Fab Nucleotide Sequence Listing 167 <223> 10273 Heavy Chain Fabric Sequence Listing 168 <223> 10273 Heavy Chain Fab Nucleotides Sequence Listing 169 <223> Human LEKTI D5 Fab H chain Sequence Listing 170 <223> Human LEKTI D5 Fab L chain Sequence Listing 171 <223> Rabbit / Human Chimera Light Chain (hCK S171C) 10236 Sequence Listing 172 <223> Rabbit / Human Chimera Heavy Chain 10236 Sequence Listing 181 <223> 10236 Rabbit VL Sequence Listing 182 <223> 10236 Rabbit VL Nucleotides Sequence Listing 183 <223> 10236 Rabbit VH Sequence Listing 184 <223> 10236 Rabbit VH Nucleotides Sequence Listing 185 <223> Mouse KLK7 Proform Sequence Listing 186 <223> Active mouse KLK7

Claims

1. A monoclonal antibody that binds to kallikrein 5 (KLK5), comprising a variable light chain and a variable heavy chain, and a. The variable light chain includes CDR-L1 containing SEQ ID NO: 1, 7, 8, or 9, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, and b. The variable heavy chain comprises the monoclonal antibody described above, which includes CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 6 or any one of SEQ ID NOs from 10 to 29.

2. The antibody according to claim 1, wherein the CDR-H3 comprises one of SEQ ID NOs: 10, 11, 13-16, 18, 20, 22-25, 27, and 29.

3. a. The variable light chain includes CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, b. The antibody according to claim 1 or 2, wherein the variable heavy chain comprises CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO: 10, 14, or 23.

4. a. The variable light chain includes CDR-L1 containing SEQ ID NO: 7, CDR-L2 containing SEQ ID NO: 2, and CDR-L3 containing SEQ ID NO: 3, b. The antibody according to any one of claims 1 to 3, wherein the variable heavy chain comprises CDR-H1 containing SEQ ID NO: 4, CDR-H2 containing SEQ ID NO: 5, and CDR-H3 containing SEQ ID NO:

23.

5. The antibody according to any one of claims 1 to 4, wherein the antibody is a chimeric antibody or a humanized antibody.

6. The antibody according to any one of claims 1 to 5, wherein the antibody is a full-length antibody.

7. The antibody according to claim 6, wherein the full-length antibody is selected from IgG1, IgG4, or IgG4P.

8. Antibodies are Fab, Fab', F(ab') ２ An antibody according to any one of claims 1 to 5, selected from scFv or dAb.

9. Antibodies, a. Variable light chains including sequence numbers 30, 34, 38, 42, or 46, and / or b. The antibody according to any one of claims 1 to 8, comprising a variable heavy chain including SEQ ID NOs. 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134.

10. The antibody according to claim 9, wherein the variable heavy chain comprises SEQ ID NOs. 32, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 106, 110, 114, 118, 126, or 134.

11. Antibodies, a. Variable light chains containing Sequence ID No. 38, and / or b. The antibody according to any one of claims 1 to 10, comprising a variable heavy chain including SEQ ID NO:

110.

12. Antibodies, a. Light chains containing sequence numbers 36, 40, 44, or 48, and b. The antibody according to any one of claims 1 to 7, 9, 10 and 11, comprising a heavy chain including SEQ ID NOs. 52 or 56 or 60 or 64 or 68 or 72 or 76 or 80 or 84 or 88 or 92 or 96 or 100 or 104 or 108 or 112 or 116 or 120 or 124 or 128 or 132 or 136.

13. The antibody according to claim 12, wherein the heavy chain comprises SEQ ID NOs. 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 108, 112, 116, 120, 128, or 136.

14. Antibodies, a. Light chain containing Sequence ID No. 40, and b. The antibody according to any one of claims 1 to 10, comprising a heavy chain containing SEQ ID NO:

112.

15. The antibody according to any one of claims 1 to 14, wherein KLK5 is human KLK5 containing SEQ ID NO: 142, 143, or 144, or cynomolgus monkey KLK5 containing SEQ ID NO:

151.

16. The antibody according to any one of claims 1 to 15, wherein the antibody binds to kallikrein 5 (KLK5) and binds to human KLK5 epitopes comprising Leu212, Ser213, Glun214, Lys215, Arg216, Glu218, Asp219, Ala220, Pro222, Gly233, Pro269, Asn270 and Pro272, with reference to SEQ ID NO:

142.

17. The antibody according to claim 16, wherein the epitope is characterized by X-ray crystallography.

18. Antibodies, a. Inhibit or reduce the protease activity of KLK5, and / or b. When KLK5 binds to a lymphoepithelial cazar type inhibitor (LEKTI) or a fragment of LEKTI, it binds to KLK5 and / or c. KLK5 binding does not compete with LEKTI or LEKTI fragments, and / or d. Forms a complex with LEKTI or KLK5 bound to a fragment of LEKTI, The antibody according to any one of claims 1 to 17, wherein the LEKTI fragment is a human LEKTI domain 5 containing amino acids 1 to 64 of SEQ ID NO: 145 or a LEKTI domain 8 containing amino acids 1 to 71 of SEQ ID NO:

152.

19. The antibody according to any one of claims 1 to 18, wherein the antibody binds to human KLK5 and cynomorgus monkey (cyno) KLK5.

20. The antibody according to claim 19, wherein the human KLK5 comprises SEQ ID NO: 144, and the cynomolgus monkey (cyno) KLK5 comprises SEQ ID NO:

151.

21. The antibody according to any one of claims 1 to 20, wherein the antibody does not bind to human or cynomolgus kallikrein 2 (KLK2), or human or cynomolgus kallikrein 4 (KLK4), or human or cynomolgus kallikrein 7 (KLK7).

22. The antibody described in any one of claims 1 to 21 competes with KLK5 for binding, and a. With respect to binding to KLK5, the antibody described in any one of claims 1 to 21 is cross-blocked, or the antibody described in any one of claims 1 to 21 is cross-blocked, b. An antibody that binds to KLK5 at the same epitope as the antibody described in any one of claims 1 to 21, The antibody comprises a heavy chain variable region having at least 90% identity with the sequence of Sequence ID No. 38, and a light chain variable region having at least 90% identity with the sequence of Sequence ID No.

110.

23. An isolated polynucleotide encoding an antibody according to any one of claims 1 to 21.

24. Polynucleotides a. A light chain variable region in which polynucleotides are i. At least 90% identical to sequence numbers 31, 35, 39, 43, or 47, or ii. Including sequence numbers 31, 35, 39, 43, or 47, or iii. The above light chain variable region consisting of sequence numbers 31, 35, 39, 43, or 47, and A heavy chain variable region in which polynucleotides i. At least 90% identical to code numbers 33, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, or 135, or 99. Including code 33 or 51 or 55 or 59 or 63 or 67 or 71 or 75 or 79 or 83 or 87 or 91 or 95 or 99 or 103 or 107 or 111 or 115 or 119 or 123 or 127 or 131 or 135, or iii. Codes the above heavy chain variable region consisting of number 33 or 51 or 55 or 59 or 63 or 67 or 71 or 75 or 79 or 83 or 87 or 91 or 95 or 99 or 103 or 107 or 111 or 115 or 119 or 123 or 127 or 131 or 135, or b. A light chain, which consists of polynucleotides, i. At least 90% identical to sequence number 37, 41, 45, or 49, or ii. Including sequence numbers 37, 41, 45, or 49, or iii. The above light chain consisting of sequence numbers 37, 41, 45, or 49, and It is a heavy chain, and polynucleotides, i. At least 90% identical to number 53 or 57 or 61 or 65 or 69 or 73 or 77 or 81 or 85 or 89 or 93 or 97 or 101 or 105 or 109 or 113 or 117 or 121 or 125 or 129 or 133 or 137, or ii. Including code numbers 53 or 57 or 61 or 65 or 69 or 73 or 77 or 81 or 85 or 89 or 93 or 97 or 101 or 105 or 109 or 113 or 117 or 121 or 125 or 129 or 133 or 137, or iii. The heavy chain consisting of number 53 or 57 or 61 or 65 or 69 or 73 or 77 or 81 or 85 or 89 or 93 or 97 or 101 or 105 or 109 or 113 or 117 or 121 or 125 or 129 or 133 or 137. To code The isolated polynucleotide according to claim 23.

25. A cloning vector or expression vector comprising one or more polynucleotides as described in claim 23 or 24.

26. It is a host cell, a. One or more polynucleotides according to claim 23 or 24, or b. A host cell comprising one or more expression vectors according to claim 25.

27. A method for producing an antibody according to any one of claims 1 to 21, comprising culturing a host cell according to claim 26 under conditions suitable for antibody production, and isolating an antibody produced by the host cell.

28. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 21 and one or more pharmaceutically acceptable carriers, excipients, or diluents.

29. A pharmaceutical composition according to claim 28 for use in treatment.

30. The pharmaceutical composition according to claim 28 for treating a disease characterized by dysregulation of KLK5 or dysregulation of KLK5 inhibition in a patient.

31. The pharmaceutical composition according to claim 30, wherein the disease is selected from Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer.

32. The pharmaceutical composition according to claim 31, wherein the cancer is ovarian cancer or bladder cancer.

33. The pharmaceutical composition according to claim 30, wherein the disease is Netherton syndrome.

34. The pharmaceutical composition according to claim 30, wherein the disease is atopic dermatitis.

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