Antibodies against KLK5

Abstract

This invention relates to antibodies that bind to and inhibit KLK5 and methods of using these antibodies to treat diseases caused by KLK5 imbalance. Specifically, this invention relates to inhibitory antibodies that bind to KLK5 and their use in the treatment of Natherton's disease, atopic dermatitis, and cancer.

Description

Invention Field

[0001] This invention relates to antibodies that bind to and inhibit KLK5 and methods of using such antibodies to treat diseases caused by KLK5 dysregulation. Specifically, this invention relates to anti-KLK5 antibodies and their use in the treatment of Natherton's disease, ichthyosis such as congenital ichthyosis, atopic dermatitis, and cancer. Background of the Invention

[0003] Kallikrein-associated peptidases (KLK) constitute a family of 15 highly conserved trypsin or chymotrypsin-like serine proteases encoded by the largest uninterrupted protease-coding gene cluster in the human genome (chromosome 19q13.4) (Sotiropoulou G. et al., 2009; JBC 284:48,32989-94).

[0004] KLK is synthesized in an inactive pre-pro-form, which is then proteolytically processed to secrete an inactive pro-form. This pro-form is subsequently activated into a mature peptidase by removing its N-terminal propeptide through other KLKs or endopeptidases, or by its own catalytic cleavage of specific proteases such as kallikrein 5 (KLK5).

[0005] KLK5 is present in several tissues, but is most abundantly expressed in the skin. Along with KLK7, KLK5 is expressed in the stratum spinosum and stratum granulosum of the skin, where keratinocytes undergo terminal differentiation and transform into keratinocytes that build the stratum corneum. The stratum corneum acts as a barrier against the external environment and is maintained by the continuous replacement of keratinocytes shed during the desquamation process. Because KLK5 can activate pre-KLK7 and other kallikreinases, its role in desquamation is essential.

[0006] Upon activation, the endogenous inhibitor LEKTI, encoded by the SPINK5 gene, inactivates mature KLK5 (Chavans P et al., 2005; Nat Genet 37, 56-65). LEKTI contains a 15-domain serine protease inhibitor domain, which forms a tight complex with KLK5. Changes in pH control this tight interaction with acidic pH, releasing active KLK5 from the complex (Deraison C et al., 2007; Mol Biol Cell 183607-19).

[0007] Loss-of-function mutations in the SPINK5 gene lead to Natherton syndrome, a rare autosomal recessive skin disorder characterized by ichthyosis, severe inflammation, desquamation, elevated IgE levels, and persistent allergic reactions (Hovnanian A. 2013; Cell Tissue Res 351 289-300). Secondary to overactivity of epidermal proteases, a deficiency of LEKTI results in keratinization caused by KLK5 activity against desmosomes and desmosomes, which in turn facilitates increased permeability to various allergens, leading to atopic dermatitis-like lesions. KLK5 activity against KLK7 also results in a defective skin barrier, allowing for the penetration of allergens and microorganisms, as well as the production of IL-1β.

[0008] SPINK5 - / - Mice reproduced a phenotype highly reminiscent of Natherton syndrome, replicating the skin and inflammatory aspects of the disease (Yant T et al.; 2004, Genes Dev 18 2354-58). SPINK5 from patients with Natherton syndrome... - / - The epidermis exhibits unopposed KLK5 and KLK7 protease activity, which appears to maintain the activation of pro-inflammatory and pro-signaling pathways, including the KLK5-PAR2-TSLP (thymic stromal lymphopoietin) axis.

[0009] In SPINK5 - / - and KLK5 - / - In mice, KLK5 knockout was sufficient to correct the skin manifestations of LEKTI knockout, demonstrating the crucial role of KLK5 in skin homeostasis.

[0010] In recent years, several studies have reported a genetic association between atopic dermatitis (AD) and LEKTI polymorphisms in which abnormal variants of LEKTI are expressed (Hovnanian A. 2013; Cell Tissue Res 351 289-300).

[0011] To date, only alternatives to LEKTI have been sought, including autologous grafts of patient keratinocytes via gene addition and gene correction using SPINK5 lentiviral or adenoviral vectors (Di WL.Et al.; 2011, Mol Ther19 408-16).

[0012] Therefore, there is still a need for anti-KLK5 therapies, such as passive immunotherapy designed to inhibit KLK5, which can play a therapeutic role in diseases associated with or caused by KLK5 dysregulation. Invention Overview

[0014] The present invention addresses the above-mentioned needs by providing an inhibitory anti-KLK5 antibody according to the following embodiments.

[0015] Implementation Scheme 1: An antibody that binds to kallikrein 5 (KLK5), wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0016] a. The variable light chain comprises CDR-L1 containing SEQ ID NO:1 or SEQ ID NO:62 or SEQ ID NO:63, CDR-L2 containing SEQ ID NO:2, and CDR-L3 containing SEQ ID NO:3; and

[0017] b. The variable heavy chain contains CDR-H1 containing SEQ ID NO:4, CDR-H2 containing SEQ ID NO:5, and CDR-H3 containing SEQ ID NO:6.

[0018] Implementation Scheme 2: The antibody according to Implementation Scheme 1, wherein:

[0019] a. The variable light chain comprises CDR-L1 containing SEQ ID NO:1, CDR-L2 containing SEQ ID NO:2, and CDR-L3 containing SEQ ID NO:3; and

[0020] 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.

[0021] Implementation Scheme 3: An antibody that binds to kallikrein 5 (KLK5), wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0022] Implementation Scheme 4: The antibody according to Implementation Scheme 3, wherein the epitope is characterized by X-ray crystallography.

[0023] Implementation Scheme 5: An antibody according to any one of Implementation Schemes 1 to 4, wherein the antibody inhibits or reduces the protease activity of KLK5.

[0024] Implementation Scheme 6: An antibody according to any one of Implementation Schemes 1 to 5, wherein the antibody binds to KLK5 when KLK5 binds to LEKTI or a LEKTI fragment.

[0025] Implementation Scheme 7: An antibody according to any one of Implementation Schemes 1 to 6, wherein the antibody does not compete with LEKTI or the LEKTI fragment for binding to KLK5.

[0026] Implementation Scheme 8: An antibody according to any one of Implementation Schemes 1 to 7, wherein the antibody forms a complex with KLK5 bound to LEKTI or a LEKTI fragment.

[0027] Implementation Scheme 9: An antibody according to any one of Implementation Schemes 6 to 8, wherein the LEKTI fragment is a human LEKTI domain 5 comprising amino acids 1 to 64 of SEQ ID NO: 54 or a LEKTI domain 8 comprising amino acids 1 to 71 of SEQ ID NO: 61.

[0028] Implementation Scheme 10: An antibody according to any one of the preceding implementation schemes, wherein the antibody binds to human KLK5 and cynomolgus monkey (cyno) KLK5, wherein the human KLK5 is preferably human KLK5 containing SEQ ID NO:53, and the cynomolgus monkey KLK5 is preferably cynomolgus monkey KLK5 containing SEQ ID NO:60.

[0029] Implementation Scheme 11: An antibody according to any one of the preceding implementation schemes, 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] Implementation Scheme 12: An antibody according to any one of Implementation Schemes 3 to 11, wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0031] a. The variable light chain comprises a CDR-L1 containing SEQ ID NO:1, SEQ ID NO:62, or SEQ ID NO:63, preferably a CDR-L1 containing SEQ ID NO:1, a CDR-L2 containing SEQ ID NO:2, and a CDR-L3 containing SEQ ID NO:3; and

[0032] b. The variable heavy chain contains CDR-H1 containing SEQ ID NO:4, CDR-H2 containing SEQ ID NO:5, and CDR-H3 containing SEQ ID NO:6.

[0033] Implementation Scheme 13: An antibody according to any one of the preceding implementation schemes, wherein the antibody is a chimeric antibody or a humanized antibody.

[0034] Implementation Scheme 14: The antibody according to any one of the preceding implementation schemes, wherein the antibody is a full-length antibody.

[0035] Implementation Scheme 15: The antibody according to Implementation Scheme 13, wherein the full-length antibody is selected from IgG1, IgG4 or IgG4P.

[0036] Implementation Scheme 16: An antibody according to any one of Implementation Schemes 1 to 13, wherein the antibody is selected from Fab, Fab', F(ab')2, scFv, dAb, or V. HH .

[0037] Implementation Scheme 17: An antibody according to any one of Implementation Schemes 1 to 16, wherein the antibody comprises:

[0038] a. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and / or

[0039] b. A variable heavy chain containing SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43.

[0040] Implementation Scheme 18: An antibody according to any one of Implementation Schemes 1 to 15 or 17, wherein the antibody comprises:

[0041] a. A light chain containing SEQ ID NO: 13, 17, 21, or 25; and

[0042] b. Heavy chains containing SEQ ID NO:29 or 33 or 37 or 41 or 45.

[0043] Implementation Scheme 19: The antibody according to Implementation Scheme 17 or 18, wherein the amino acid residue glutamine (Gln; Q) at position 24 of L-CDR1, referring to SEQ ID NO: 15 or 17, is replaced by arginine (Arg; R) or lysine (Lys; K).

[0044] Implementation Scheme 20: An antibody according to any one of the preceding implementation schemes, wherein KLK5 is human KLK5 containing SEQ ID NO: 51 or 52 or 53 or cynomolgus monkey KLK5 containing SEQ ID NO: 60.

[0045] Implementation Scheme 21: An antibody, wherein:

[0046] a. Competing for binding to KLK5 with any of the antibodies described in any one of embodiments 1 to 20; and / or

[0047] b. Cross-blocking of the binding of the antibody to KLK5 by any one of embodiments 1 to 20 or cross-blocking of the binding of the antibody to KLK5 by any one of embodiments 1 to 20; and / or

[0048] c. Bind to KLK5 at the same epitope as the antibody described in any one of embodiments 1 to 20; and / or

[0049] d. Containing a heavy chain variable region having at least 90% identity or similarity to the sequence according to SEQ ID NO: 29, 33, 37, 41, or 45; and / or

[0050] e. Contains a light chain variable region that has at least 90% identity or similarity to the sequence according to SEQ ID NO:13 or 17 or 21 or 25.

[0051] Implementation Scheme 22: An isolated polynucleotide encoding an antibody according to any one of Implementation Schemes 1 to 20.

[0052] Implementation Scheme 23: The isolated polynucleotide according to Implementation Scheme 22, wherein the polynucleotide encodes:

[0053] a. Light chain variable region, wherein the polynucleotide:

[0054] i. At least 90% identical to SEQ ID NO:8 (or nucleotides 1 to 330 of SEQ ID NO:8) or 12 (or nucleotides 1 to 330 of SEQ ID NO:12) or 16 or 20 or 24 or 64 or 66; or

[0055] ii. Containing SEQ ID NO:8 (or nucleotides 1 to 330 of SEQ ID NO:8) or 12 (or nucleotides 1 to 330 of SEQ ID NO:12) or 16 or 20 or 24 or 64 or 66; or

[0056] iii. Consisting essentially of SEQ ID NO:8 (or nucleotides 1 to 330 of SEQ ID NO:8) or 12 (or nucleotides 1 to 330 of SEQ ID NO:12) or 16 or 20 or 24 or 64 or 66; or

[0057] b. Heavy chain variable region, wherein the polynucleotide:

[0058] i. At least 90% identical to SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44; or

[0059] ii. Contains SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44; or

[0060] iii. Basically composed of SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44; or

[0061] c. The light chain, wherein the polynucleotide is:

[0062] i. At least 90% identical to SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104; or

[0063] ii. Containing SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104; or

[0064] iii. Basically composed of SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 101 or 102 or 103 or 104; or

[0065] d. Heavy chain, wherein the polynucleotide is:

[0066] i. At least 90% identical to SEQ ID NO: 30 or 34 or 38 or 42 or 46; or

[0067] ii. Contains SEQ ID NO: 30 or 34 or 38 or 42 or 46; or

[0068] iii. It is basically composed of SEQ ID NO: 30 or 34 or 38 or 42 or 46.

[0069] Implementation Scheme 24: A cloning or expression vector comprising one or more polynucleotides according to any one of Implementation Schemes 22 or 23.

[0070] Implementation Scheme 25: A host cell comprising:

[0071] a. One or more polynucleotides according to any one of embodiments 22 or 23, or

[0072] b. One or more expression carriers according to implementation scheme 24.

[0073] Implementation Scheme 26: A method for producing an antibody according to any one of Implementation Schemes 1 to 20, comprising culturing a host cell according to Implementation Scheme 25 under conditions suitable for producing the antibody and isolating the antibody produced by the host cell.

[0074] Implementation Scheme 27: A pharmaceutical composition comprising an antibody according to any one of Implementation Schemes 1 to 20 and one or more pharmaceutically acceptable carriers, excipients or diluents.

[0075] Implementation Scheme 28: An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 20 or a pharmaceutical composition according to Implementation Scheme 27, used for treatment.

[0076] Implementation Scheme 29: An antibody according to any one of Implementation Schemes 1 to 20 or a pharmaceutical composition according to Implementation Scheme 27, for treating a disease characterized by KLK5 dysregulation or KLK5 inhibition dysregulation.

[0077] Implementation Scheme 30: The antibody used according to Implementation Scheme 29, wherein the disease is selected from Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or a combination thereof.

[0078] Implementation Scheme 31: The antibody used according to Implementation Scheme 30, wherein the disease is Netherton syndrome.

[0079] Implementation Scheme 32: The antibody used according to Implementation Scheme 30, wherein the disease is atopic dermatitis.

[0080] Implementation Scheme 33: A method of treating a disease characterized by KLK5 dysregulation or KLK5 inhibition dysregulation in a patient, comprising administering to the patient a therapeutically effective amount of an antibody according to any one of Implementation Schemes 1 to 20 or a pharmaceutical composition according to Implementation Scheme 27.

[0081] Implementation Scheme 34: The method according to Implementation Scheme 33, wherein the disease is selected from Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer.

[0082] Implementation Scheme 35: The antibody used according to Implementation Scheme 34, wherein the disease is Netherton syndrome.

[0083] Implementation Scheme 36: The antibody used according to Implementation Scheme 34, wherein the disease is atopic dermatitis.

[0084] Brief description of the attached figures

[0085] Figure 1Size exclusion chromatography (SEC). Figures A and B show the elution curves of human KLK5 alone (solid line, far right), rabbit Fab antibody 10236 alone (dashed line), human KLK5+LEKTI D5 (long dashed line, Figure A) or human KLK5+LEKTI D8 (long dashed line, Figure B) and human KLK5+LEKTI D5+rabbit Fab antibody 10236 (short dashed line, far left, Figure A) or human KLK5+LEKTI D8+rabbit Fab antibody 10236 (short dashed line, far left, Figure B).

[0086] Figure 2 From such Figure 1 SDS-PAGE of the peak fractions of SEC shown in A and 1B. Lane 1, MW label. Lane 2, peak fraction of the binary complex human KLK5+LEKTI D5. Lane 3, peak fraction of the ternary complex KLK5+LEKTI D5+rabbit Fab antibody 10236. Lane 4, peak fraction of the binary complex KLK5+LEKTI D8. Lane 5, peak fraction of the ternary complex KLK5+LEKTI D8+rabbit Fab antibody 10236.

[0087] Figure 3 SDS-PAGE of KLK5 produced for X-ray crystallography studies. Lanes M and MW are labeled. Lane 1, human KLK5 purified from a culture grown in the presence of kifunensine (kif). Lane 2, human KLK5 purified from a kifunensine culture and treated with endoglycosidase H (Endo H).

[0088] Figure 4Schematic diagram of the complexation of human KLK5 epitope with rabbit Fab antibody 10236. A) Fab heavy chain (dark gray) and light chain (light gray) are shown in a cartoon and transparent surface. KLK5 is shown as a black band. Residues of KLK5 that are part of the epitope on human KLK5 that binds to antibody 10236 are depicted as black bars. B) Leucopeptide (surface and bars) modeled on the crystal structure (bands) of KLK5 bound to rabbit Fab10236 (surface rendering). Fab10236 is in contact with the 99-ring on KLK5. C) Superposition of crystal structures 2PSX (white, zinc-free) and 2PSY (gray, zinc-containing), highlighting the movement of the 99-ring and the side chain positions of His147 and His150 on KLK5 in the presence of zinc. Leucopeptide is shown as a white surface and bars. D) Superposition of the 2PSX crystal structure (KLK5 bound to leucopeptide) on the KLK5 crystal structure complexed with Fab10236. The movement of the 99-ring and the side chain positions of His147 and His150 is highlighted after comparing the two structures. The conformations of the ring and His residues in the 2PSX crystal structure and the Fab10236 crystal structure bound to KLK5 are shown in white and black, respectively. The white dashed rectangle around His147 (2PSX crystal structure) indicates that this conformation conflicts with Fab10236 (gray surface). His147 has a different conformation in the KLK5-Fab10236 structure. The white dashed circle around His150 (black, as observed in the KLK5 / Fab10236 complex) indicates that it points to the S2 pocket of the KLK5 active site, where substrates such as leucopeptide will bind. Leucinogen peptide (gray surface and bar) from crystal structure 2PSX shows the binding site of the expected substrate in the KLK5 active site.

[0089] Figure 5 Two orientations of the human KLK5 crystal structure complexed with rabbit Fab antibodies 10236 and 10273. Human KLK5 is shown as a banded structure, while rabbit Fab antibodies 10236 and 10273 are shown as a solid surface.

[0090] Figure 6 Humanization of the rabbit variable light chain sequence of antibody 10236. Grafts 10236gL5, gL6, gL7, and gL8 are humanized grafts of the rabbit variable light chain of antibody 10236 using the IGKV1-6 human line as the recipient framework. Donor residues are shown in bold / italic with gray shading: Y2, D3, and K63. CDRs are shown in bold / underlined. Mutations increasing pI in CDRL1 are shown in bold / underlined and highlighted: Q24R or Q24K.

[0091] Figure 7Humanization of the rabbit variable heavy chain sequence of antibody 10236. Graft 10236gH9,gH10,gH11,gH12, andgH14 are humanized grafts of the rabbit variable heavy chain of antibody 10236 using the IGHV4-4 human line as the recipient framework. CDRs are shown in bold / underline. Donor residues are shown in bold / italic with gray shading: F67, Q71, S73, T76, and V78.

[0092] Figure 8 Inhibitory activity of antibody 10236 gL6gH12 against kallikrein and inhibitory activity of LEKTI D5 rabbit Fc against human and cynomolgus monkey KLK5.

[0093] Figure 9 Inhibitory effect of antibody 10236 gL6gH12 on IP-1 release in HaCat cells. IP-1 release was stimulated by adding KLK5 to HaCat cells. Antibody 10236 gL6gH12 almost completely inhibited IP-1, reaching levels comparable to the reference LEKTI D5 rabbit Fc protein. A33 Hu IgG4 was used as an isotype control.

[0094] Figure 10 The mechanism of action of antibody 10236 gL6gH12 (A) and parental rabbit antibody (B). K obs Values ​​were plotted against substrate concentrations of antibody 10236 and LEKTI D5 rabbit Fc protein (the latter only in (A)). The data shown are for 10 nM antibody 10236 and 2 nM LEKTI D5 rabbit Fc. The slopes indicate that antibody 10236 is a non-competitive inhibitor, while LEKTI protein is a competitive inhibitor.

[0095] Figure 11 Hematoxylin and eosin staining revealed the skin structure and stratum corneum integrity in the reconstructed human skin epidermal model. The effects of culture medium and MC903 with and without antibody 10236 gL6gH12 IgG4P (Ab 10236) or isotype control (hIgG4P) were investigated.

[0096] Figure 12 In situ enzyme protease assays showed the serine protease activity in skin sections of atopic dermatitis treated with control buffer (A) or antibody 10236 gL6gH12 IgGP4 (B).

[0097] Figure 13 A stress study of antibody 10236 gL6gH12 IgG4P (named 10236gL6gH12) was conducted to assess the deamidation tendency of the Asn(94)Ser motif on the light chain CDR3. Invention Details

[0099] The present disclosure will now be described with reference to specific non-limiting aspects and implementations thereof, and with reference to certain accompanying drawings and embodiments.

[0100] Unless otherwise stated, technical terms are used in accordance with common sense. If a term conveys a specific meaning, its definition will be given in the context in which it is used.

[0101] When the term "comprising / including" is used in this specification and claims, it does not exclude other elements. For the purposes of this disclosure, the term "consisting of..." is considered a preferred embodiment of the term "comprising / including...".

[0102] Unless otherwise explicitly stated, use the indefinite or definite article, such as "a", "an" or "the", when referring to singular nouns, including the plural form of the noun.

[0103] As used herein, the terms “treatment”, “treating,” etc., refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in relation to the complete or partial prevention of a disease or its symptoms, and / or therapeutic in relation to the partial or complete cure of a disease and / or side effects attributable to it. Therefore, treatment encompasses any treatment of diseases in mammals, particularly humans, including: (a) preventing the occurrence of the disease in subjects who may be susceptible to it but have not yet been diagnosed with it; (b) suppressing the disease, i.e., halting its development; and (c) alleviating the disease, i.e., causing its remission.

[0104] "Therapeutic effective dose" refers to the amount of KLK5 antibody that, when administered to a mammal or other subject to treat a disease, would be sufficient to produce therapeutic effect. Therapeutic effective dose will vary depending on the subject's anti-KLK5 antibody levels, the severity and severity of the disease, as well as age, weight, etc.

[0105] Throughout the instruction manual, the term "isolated" refers to antibodies or polynucleotides, depending on the circumstances, existing in a physical environment different from that in which they might occur in nature.

[0106] In a first aspect of the invention, an antibody is provided that binds to kallikrein 5 (KLK5), wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0107] a. The variable light chain comprises CDR-L1 containing SEQ ID NO:1 or SEQ ID NO:62 or SEQ ID NO:63, CDR-L2 containing SEQ ID NO:2, and CDR-L3 containing SEQ ID NO:3; and

[0108] b. The variable heavy chain contains CDR-H1 containing SEQ ID NO:4, CDR-H2 containing SEQ ID NO:5, and CDR-H3 containing SEQ ID NO:6.

[0109] Preferably, the antibody that binds to kallikrein 5 (KLK5) and contains a variable light chain contains CDR-L1 comprising SEQ ID NO:1.

[0110] Therefore, in a preferred embodiment of the present invention, the antibody comprising a variable light chain and a variable heavy chain that binds kallikrein 5 (KLK5) is characterized by comprising a variable light chain comprising CDR-L1 of SEQ ID NO:1, CDR-L2 of SEQ ID NO:2 and CDR-L3 of SEQ ID NO:3; and a variable heavy chain comprising CDR-H1 of SEQ ID NO:4, CDR-H2 of SEQ ID NO:5 and CDR-H3 of SEQ ID NO:6.

[0111] Kallikrein 5 (KLK5, KLK-L2, SCTE, or any other known synonym) possesses trypsin-like activity. It is expressed as a precursor, comprising a 29-amino acid signal peptide according to the bioinformatics tool SignalP 5.0 (http: / / www.cbs.dtu.dk / services / SignalP / index.php), followed by a 37-amino acid propeptide sequence. Cleavage of the propeptide yields an active mature enzyme consisting of 237 amino acids, possessing an active site with a catalytic triplet of typical serine protease residues (Michael IP et al., 2005; JBC 280:15, 14628-35).

[0112] Unless otherwise stated, the term KLK5 refers to any natural pre- and pro-form (i.e., unprocessed KLK5 containing a signal sequence and an activating peptide), alternatively spliced ​​or natural variants, mutants, and KLK5 from other species (mice, cynomolgus monkeys, etc.) and active KLK5 (caused by autocleavage or other causes). When human KLK5 is specified, human KLK5 includes the sequence given in SEQ ID NO:53 (active human KLK5). Other KLK5 sequences mentioned herein include SEQ ID NO:52 (human KLK5 pre-form lacking the signal sequence) or SEQ ID NO:51 (full-length human KLK5 with both signal and propeptide sequences), sequences corresponding to Uniprot Q9Y337, or natural variants containing mutations at positions 55 and 153 (refer to SEQ ID NO:51). Examples of these mutations include human KLK5 containing residues 23 to 293 according to SEQ ID NO:51, which has a Gly to Arg change (G55R) at residue 55 and / or an Asp to Asn change (D153N) at residue 153.

[0113] The antibody according to the invention comprises complementarity-determining regions (CDRs), three from the heavy chain and three from the light chain. Typically, the CDRs are within a single frame and together form a variable region. By convention, the CDRs in the heavy chain variable region of the antibody or its antigen-binding fragment are designated CDR-H1, CDR-H2, and CDR-H3, and the CDRs in the light chain variable region are designated CDR-L1, CDR-L2, and CDR-L3. They are numbered sequentially from the N-terminus to the C-terminus of each chain.

[0114] CDRs are typically based on a numbering system devised by Kabat et al., 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 will be used throughout this specification unless otherwise stated.

[0115] Kabat residue names do not always correspond directly to linear amino acid residue numbers. The actual linear amino acid sequence can contain fewer or more amino acids than a strict Kabat number, corresponding to shortening or insertion of structural components (whether framework or complementarity-determining regions (CDRs)) in a basic variable domain structure. The correct Kabat residue number of an antibody can be determined by comparing a given antibody sequence with homologous residues in a “standard” Kabat-numbered sequence.

[0116] According to the Kabat numbering system, the CDR of the variable domain in the heavy chain is located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3). However, according to Chothia (Chothia, C. and Lesk, AM J Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Therefore, unless otherwise stated, as used herein, 'CDR-H1' is intended to refer to residues 26 to 35 as described by the combination of the Kabat numbering system and Chothia's definition of the topological loop.

[0117] According to the Kabat numbering system, the CDRs of the variable domains in the light chain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2), and residues 89-97 (CDR-L3).

[0118] In addition to the CDR rings, a fourth ring, formed by frame 3 (FR3), exists between CDR-2 (CDR-L2 or CDR-H2) and CDR-3 (CDR-L3 or CDR-H3). The Kabat numbering system defines frame 3 as positions 66-94 in the heavy chain and positions 57-88 in the light chain.

[0119] In a preferred embodiment, the antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDR-L1 comprising SEQ ID NO:1, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3, and the heavy chain variable region comprising CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6.

[0120] In another embodiment, the antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR-L1 comprising SEQ ID NO:62; CDR-L2 comprising SEQ ID NO:2 and CDR-L3 comprising SEQ ID NO:3; and the heavy chain variable region comprises CDR-H1 comprising SEQ ID NO:4; CDR-H2 comprising SEQ ID NO:5 and CDR-H3 comprising SEQ ID NO:6.

[0121] In another embodiment, the antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR-L1 comprising SEQ ID NO:63; CDR-L2 comprising SEQ ID NO:2 and CDR-L3 comprising SEQ ID NO:3; and the heavy chain variable region comprises CDR-H1 comprising SEQ ID NO:4; CDR-H2 comprising SEQ ID NO:5 and CDR-H3 comprising SEQ ID NO:6.

[0122] Antibodies containing such CDR sequences are particularly inventive because they provide antibodies with high affinity for KLK5, preferably human KLK5, high inhibitory activity against KLK5 biological function, and high stability crucial for manufacturability. For example, antibodies containing SEQ ID NO:3(QQGYT NS The mutation of the motif “NS” to “ND” (see SEQ ID NO:15) in CDR-L3 of NIINT (NIINT) results in a significant decrease in KLK5 affinity.

[0123] In a second aspect of the invention, an antibody is provided that binds to kallikrein 5 (KLK5), wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87 (36), Ala107 (56), Arg110 (59), Lys111 (60), Lys112 (61), Val113 (62), Val137 (86), Lys138 (87), Ser139 (88), Ile140 (89), Pro141 (90), His142 (91), Pro143 (92), Tyr145 (94), Ser146 (95), and His147 (96) as described in SEQ ID NO:51. Preferably, the epitope is characterized by X-ray crystallography. The numbers in parentheses correspond to protease nomenclature.

[0124] In a preferred embodiment, the antibody binds to kallikrein 5 (KLK5), wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51, and wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0125] a. The variable light chain comprises a CDR-L1 containing SEQ ID NO:1 or SEQ ID NO:62 or SEQ ID NO:63, preferably SEQ ID NO:1, a CDR-L2 containing SEQ ID NO:2, and a CDR-L3 containing SEQ ID NO:3; and

[0126] b. The variable heavy chain contains CDR-H1 containing SEQ ID NO:4, CDR-H2 containing SEQ ID NO:5, and CDR-H3 containing SEQ ID NO:6.

[0127] In this invention, the term "epitope" is used interchangeably for conformational epitopes and linear epitopes. A conformational epitope consists of a discontinuous portion of the primary amino acid sequence of an antigen, while a linear epitope is formed by a sequence of continuous amino acids.

[0128] Epitopes can be identified using any suitable epitope mapping method known in the art in conjunction with any antibody provided by this invention. Examples of such methods include screening peptides of varying lengths derived from full-length KLK5 to bind to the antibodies of this invention or fragments thereof, and identifying the smallest fragment of an antibody capable of specifically binding to an epitope sequence containing antibody recognition. KLK5 peptides can be synthesized or produced by proteolytic digestion of KLK5. Antibody-binding peptides can be identified, for example, by mass spectrometry. Methodologies such as NMR spectroscopy or X-ray crystallography can be used to identify antibody-bound epitopes. Typically, when epitope determination is performed by X-ray crystallography, the distance from the CDR is... The antigenic amino acid residues within the specified range are considered to be the amino acid residue portion of the epitope. Once identified, the epitope can be used to prepare fragments that bind to antibodies of the present invention, and, if desired, as immunogens to obtain additional antibodies that bind to the same epitope.

[0129] As indicated in the description of aspects and embodiments of the invention, the epitopes are preferably epitopes characterized by X-ray crystallography.

[0130] As used in the context of this disclosure, the term 'antibody' includes the complete antibody and its functionally active fragments, i.e., molecules containing antigen-binding domains that specifically bind to antigens, also referred to as antigen-binding fragments. Unless the context otherwise requires, the characteristics of antibodies described herein also apply to antigen-binding fragments. Antibodies can be (or derived from) monoclonal, multivalent, multispecific, bispecific, fully human, humanized, or chimeric.

[0131] Intact antibodies, also known as "immunoglobulins (Ig)," typically refer to complete or full-length antibodies, which are elements consisting of two heavy chains and two light chains linked together by disulfide bonds, assembling into a characteristic Y-shaped three-dimensional structure. Classical natural whole antibodies are monospecific because they bind to one type of antigen and bivalent because they have two separate antigen-binding domains. The terms "intact antibody," "full-length antibody," and "whole antibody" are used interchangeably to refer to monospecific bivalent antibodies with a structure similar to that of natural antibodies, including the Fc region as defined herein.

[0132] Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), which, depending on the Ig class, consists of three constant domains CH1, CH2, and CH3 or four constant domains CH1, CH2, CH3, and CH4. The “class” of an Ig or antibody refers to the type of constant region, including IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses such as IgG1, IgG2, IgG3, and IgG4. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0133] As used herein, the terms "constant region" or "constant domain" are used interchangeably and refer to the domain outside the variable region of an antibody. The constant domain is identical across all antibodies of the same isotype, but differs between isotypes. Typically, the constant region of the heavy chain is formed from the N-terminus to the C-terminus by a CH1-hinge-CH2-CH3-optionally CH4, and contains three or four constant domains.

[0134] The constant region domains of the antibody molecules of the present invention, if present, can be selected according to the proposed antibody function, particularly the effector function that may be required. For example, the constant region domains can be human IgA, IgD, IgE, IgG, or IgM domains. In particular, human IgG constant region domains, especially the IgG1 and IgG3 isoforms, can be used when the antibody is intended for therapeutic use and an antibody effector function is required. Alternatively, the IgG2 and IgG4 isoforms can be used when the antibody is used for therapeutic purposes and an antibody effector function is not required. It should be understood that sequence variants of these constant region domains can also be used. For example, IgG4, where the serine at position 241 (numbered according to the Kabat numbering system) has been replaced with proline, as described by Angal et al. (Angal et al., 1993). A single amino acid substitution (Mol Immunol 30, 105-108) can be used to eliminate the heterogeneity of chimeric mouse / human (IgG4) antibodies observed during SDS-PAGE analysis. This is referred to herein as IgG4P. This single amino acid substitution prevents the natural tendency of IgG4 molecules to undergo heavy chain exchange to produce chimeric molecules.

[0135] The terms "Fc region," "Fc fragment," or simply "Fc" are used interchangeably to refer to the C-terminal region of an antibody containing its constant domains, excluding the first constant domain, the 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, along with the N-terminus of the flexible hinge of these domains. In this paper, the Fc region of the human IgG1 heavy chain is defined as containing residue C226 at its C-terminus, where the numbering is based on the EU index as described in Kabat. In the case of human IgG1, according to the EU index as described in 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.

[0136] In the context of this disclosure, the constant region or Fc region, when present, can be native, as defined above, or can be modified in various ways, provided that it contains a functional FcR-binding domain, and preferably a functional FcRn-binding domain. Preferably, the modified constant region or Fc region results in improved function and / or pharmacokinetics. Modification may include the deletion of certain portions of the Fc fragment. Modification may also include various amino acid substitutions capable of affecting the biological properties of the antibody. Mutations that increase FcRn binding and thus increase the in vivo half-life may also be present. Modification may also include modifications to the antibody glycosylation profile. The native Fc fragment is glycosylated in the CH2 domain, and each of the two heavy chains contains an N-glycan that binds to an asparagine residue at position 297 (Asn297). In the context of this disclosure, the antibody may be glycomodified, i.e., engineered to have a specific glycosylation profile, which, for example, results in improved properties, such as improved effector function or improved serum half-life.

[0137] Antigen-binding fragments of antibodies 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 used in this invention include the Fab and Fab' fragments described in international patent applications WO2011 / 117648, WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171 (all incorporated herein by reference).

[0138] The methods for producing and manufacturing these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0139] As used herein, a typical “Fab’ fragment” or “Fab’” comprises a pair of heavy and light chains, wherein the heavy chain comprises a variable region VH, a constant structural domain CH1, and a natural or modified hinge region, and the light chain comprises a variable region VL and a constant region CL. Dimers of Fab’ according to this disclosure produce F(ab’)2, wherein dimerization, for example, can occur via hinges.

[0140] 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 or VL or VHH or V-NAR.

[0141] "Fv" refers to two variable structural domains, such as cooperative variable structural domains, cognate pairs, or affinity-mature variable structural domains, i.e., VH and VL pairs.

[0142] As used in this article, "single-chain variable fragment" or "scFv" refers to a single-chain variable fragment stabilized by a peptide linker between the VH and VL variable domains.

[0143] As used in this article, "disulfide-stable single-chain variable fragment" or "dsscFv" refers to a single-chain variable fragment stabilized by a peptide linker between the VH and VL variable domains and also includes the interdomain disulfide bond between the VH and VL domains (see, for example, Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012, WO2007109254).

[0144] The disulfide bond between the variable domains VH and VL is between the two residues listed below (unless the context otherwise indicates, Kabat numbering is used in the list below) (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. al (1990). Whenever Kabat number is referenced, the relevant reference is Kabat et al., 1991 (5th edition, Bethesda, Md.), in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.

[0145] • VH37+VL95C;

[0146] • VH44+VL100;

[0147] • VH44+VL105;

[0148] ·VH45+VL87;

[0149] • VH55+VL101;

[0150] • VH100+VL50;

[0151] ·VH100b+VL4

[0152] • VH98+VL 46;

[0153] ·VH101+VL46;

[0154] ·VH105+VL43,

[0155] • VH106+VL57;

[0156] And in the variable region pair located in the molecule, it corresponds to one or more positions of it.

[0157] As used herein, the term “antibody” also encompasses monovalent antibodies, which contain only one antigen-binding domain (e.g., a single-arm antibody containing an interconnected full-length heavy chain and a full-length light chain, also known as a “half-antibody”).

[0158] The term "antibody" also encompasses multivalent antibodies that contain multiple specificities, such as bispecific, trispecific, or multispecific antibodies.

[0159] As used herein, "multispecific" or "multispecific antibody" refers to an antibody as described herein that has at least two binding domains, i.e., two or more binding domains, such as two or three binding domains, wherein the at least two binding domains independently bind two different antigens or two different epitopes on the same antigen (also known as multiple complementary sites). Multispecific antibodies are typically monovalent for each specificity (antigen). The multispecific antibodies described herein encompass both monovalent and multivalent, such as bivalent, trivalent, and quadrivalent multispecific antibodies.

[0160] As used herein, an "antigen-binding domain" refers to a portion of an antibody that contains part or all of one or more interacting variable domains, such as part or all of a pair of variable domains VH and VL that specifically interact with a target antigen. Binding domains may comprise monovalent antibodies. In one embodiment, each binding domain is monovalent. Preferably, each binding domain contains no more than one VH and one VL.

[0161] Various forms of multispecific antibodies are known in the art. Different classifications have been proposed, but multispecific IgG antibody forms generally include bispecific IgG, additional IgG, multispecific (e.g., bispecific) antibody fragments, multispecific (e.g., bispecific) fusion proteins, and multispecific (e.g., bispecific) antibody conjugates, as described in Spiess et al., Mol Immunol. 67 (2015): 95-106.

[0162] Techniques for preparing bispecific antibodies include, but are not limited to, CrossMab technology (Klein et al., Methods 154 (2019) 21-31), Knobs-in-holes engineering (e.g., WO1996027011, WO1998050431), DuoBody technology (e.g., WO2011131746), and Azymetric technology (e.g., WO2012058768). For example, other techniques for manufacturing bispecific antibodies are described in Godar et al., 2018, Expert Opinion on Therapeutic Patents, 28:3, 251-276. Bispecific antibodies include, in particular, CrossMab antibodies, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, Knobs-in-holes conventional LC, Knobs-in-holes assemblies, Charge pairs, Fab arm exchanges, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body, and orthogonal Fab.

[0163] Added IgG typically includes full-length IgG modified by attaching additional antigen-binding domains or 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 forms specifically include DVD-IgG, lgG(H)-scFv, scFv-(H)lgG, lgG(L)-scFv, scFv-(L)lgG, lgG(L,H)-Fv, lgG(H)-V, V(H)-lgG, lgC(L)-V, V(L)-lgG, KIH IgG-scFab, 2scFv-lgG, lgG-2scFv, scFv4-lg, Zybody, and DVI-IgG (quadruple combination), as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67 (2015): 95-106.

[0164] Multispecific antibody fragments include nanobodies, nanobody-HAS, BiTE, dialoge, DART, TandAb, scDiabody, sc-Diabody-CH3, Diabody-CH3, Triple Body, Miniantibody; Minibody, Tri Biminibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc; and intracellular antibodies, such as those described, for example, Spiess et al., for bispecific antibodies. Mol Immunol. 67(2015):95-106.

[0165] Multispecific fusion proteins include Dock and Lock, ImmTAC, HSAbody, scDiabody-HAS, and Tandem scFv-Toxin. Multispecific antibody conjugates include IgG-IgG, Cov-X-Body, and scFv1-PEG-scFv2.

[0166] Other forms of multispecific antibodies have been described, for example, in Brinkmann and Kontermann, mAbs, 9:2, 182-212 (2017), particularly Figure 2 Examples include tandem scFv, triplebody, Fab-VHH, taFv-Fc, scFv4-Ig, scFv2-Fcab, and scFv4-IgG. For instance, bibody, tribody, and methods for producing them are disclosed in WO99 / 37791.

[0167] The attached IgG and attached Fab fragments respectively comprise whole IgG or Fab fragments engineered by attaching 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, VL, or VHH), scFv, dsscFv, or dsFv to the N-terminus and / or C-terminus of the heavy and / or light chains of the IgG or Fab, for example as described in WO2009 / 040562, WO2010035012, WO2011 / 030107, WO2011 / 061492, WO2011 / 061246, and WO2011 / 086091, all of which are incorporated herein by reference. Specifically, the Fab-Fv form was first disclosed in WO2009 / 040562, and its disulfide-stable form, Fab-dsFv, was first disclosed in WO2010 / 035012. The Fab-dsFv, which is connected to the 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 WO2014 / 096390, which is incorporated herein by reference. The addition of IgG to full-length IgG1, which is engineered by attaching dsFv to the C-terminus of the heavy or light chain of IgG, was first disclosed in WO2015 / 197789, which is incorporated herein by reference.

[0168] Alternatively, another multispecific form comprises a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binding to a therapeutic target, and another scFv or dsscFv increasing its half-life by binding, e.g., albumin). Such antibody fragments are described in WO2015 / 197772, which is incorporated herein by reference in its entirety. Another form comprises a Fab linked to only one scFv or dsscFv, as described, for example, in WO2013 / 068571 and Dave et al., Mabs, 8(7)1319-1335(2016), which are incorporated herein by reference.

[0169] Other well-known forms of multispecific antibodies include:

[0170] As used in this article, diabody refers to two Fv pairs with two Fv indirect headers, a first VH / VL pair and another VH / VL pair, such that the VH of the first Fv is connected to the VL of the second Fv and the VL of the first Fv is connected to the VH of the second Fv.

[0171] As used in this article, triabody refers to a form similar to diabody, which includes three Fvs and three Fv indirect headers.

[0172] As used in this article, a tetrabody refers to a form similar to a diabody, which includes four Fvs and four Fv indirect headers.

[0173] As used in this article, a series scFv refers to at least two scFvs connected by a single connector, such that there is a single Fv indirect connector.

[0174] As used in this paper, tandem scFv-Fc refers to at least two tandem scFvs, each of which is attached to the N-terminus of the CH2 domain via a hinge, for example, a constant region segment -CH2CH3.

[0175] As used herein, Fab-Fv refers to an Fv fragment having a variable region attached to the C-terminus of each of the following: CH1 of the heavy chain and CL of the light chain. This form can be provided as its PEGylated form.

[0176] As used in this paper, Fab'-Fv is similar to FabFv, where the Fab part is replaced by Fab'. This form can be provided as its PEGylated form.

[0177] As used in this paper, Fab-dsFv refers to a FabFv in which a disulfide bond within the Fv is stably attached to a C-terminal variable region. This form can be provided as its PEGylated form.

[0178] As used in this article, Fab-scFv refers to a Fab molecule with an additional scFv at the C-terminus of either the light or heavy chain.

[0179] As used in this article, Fab'-scFv refers to a Fab' molecule with an additional scFv at the C-terminus of either the light or heavy chain.

[0180] As used in this article, DiFab refers to two Fab molecules connected by the C-terminus of their heavy chains.

[0181] As used in this article, DiFab' refers to two Fab' molecules connected by one or more disulfide bonds in their hinge region.

[0182] As used in this article, scdiabody is a diabody containing an internal Fv connector, such that the molecule contains three connectors and forms a normal scFv, with its VH and VL ends each connected to one of the variable regions of another Fv pair.

[0183] The scdiabody-Fc used in this paper is one of two scdiabody types, each of which is attached to the N-terminus of the CH2 domain via a hinge, for example, a constant region segment -CH2CH3.

[0184] As used in this article, scFv-Fc-scFv refers to four scFvs, each of which is attached to the N-terminus and C-terminus of the heavy and light chains of the -CH2CH3 fragment.

[0185] As used in this article, scdiabody-CH3 refers to two scdiabody molecules that are each connected to the CH3 domain by, for example, a hinge.

[0186] The IgG-scFv used in this article is a full-length antibody with scFv at the C-terminus of each heavy chain or each light chain.

[0187] The scFv-IgG used in this article is a full-length antibody with scFv at the N-terminus of each heavy chain or each light chain.

[0188] The V-IgG used in this article is a full-length antibody with a variable domain at the N-terminus of each heavy chain or each light chain.

[0189] The IgG-V used in this article is a full-length antibody with a variable domain at the C-terminus of each heavy chain or each light chain.

[0190] DVD-Ig (also known as double-V domain IgG) is a full-length antibody with four additional variable domains, one at the N-terminus of each heavy chain and one at the N-terminus of each light chain.

[0191] In a preferred embodiment, the antibody binds to kallikrein 5 (KLK5), wherein the antibody inhibits or reduces the protease activity of KLK5 and wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0192] a. The variable light chain comprises CDR-L1 containing SEQ ID NO:1 or SEQ ID NO:62 or SEQ ID NO:63, CDR-L2 containing SEQ ID NO:2, and CDR-L3 containing SEQ ID NO:3; and

[0193] b. The variable heavy chain contains CDR-H1 containing SEQ ID NO:4, CDR-H2 containing SEQ ID NO:5, and CDR-H3 containing SEQ ID NO:6.

[0194] In another embodiment of the invention, the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 of reference SEQ ID NO:51 and inhibits or reduces the protease activity of KLK5.

[0195] In this invention, the term "inhibition" (and its grammatical variations) refers to the effect of the antibody according to the invention on the biological activity of KLK5. Preferably, the biological activity of KLK5 is protease activity, more preferably serine protease activity. This effect results in the complete or partial inhibition of the serine protease activity of KLK5.

[0196] Not wanting to be bound by theory, it is believed that the antibody according to the present invention binds to KLK5 and:

[0197] i) Inhibit (e.g., completely or partially) or reduce the protease activity (preferably serine protease activity) of KLK5; and / or

[0198] ii) Binding to KLK5 when KLK5 binds to LEKTI or LEKTI fragments; and / or

[0199] iii) Does not compete with LEKTI or LEKTI fragments for binding to KLK5; and / or

[0200] iv) Form a complex with KLK5 bound to LEKTI or the LEKTI fragment (i.e., form a complex comprising the antibody of the present invention, KLK5 and LEKTI or the LEKTI fragment).

[0201] In this invention, the term "LEKTI" refers to a Kazal-type lymphoepithelial inhibitor composed of 15 domains, which is cleaved into smaller functional fragments by a proprotein convertase such as furin to produce LEKTI fragments composed 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, also known as the serine protease inhibitor Kazal-type 5 (SPINK5), is a protein encoded by the SPINK5 gene in humans. In humans, three LEKTI mRNA splicing variants are generated, resulting in full-length, long, and short isoforms of the protein, differing only in their COOH terminal regions.

[0202] 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 term "LEKTI" in this invention.

[0203] The term "forming a complex" (and any of its grammatical variations) refers to the ability of the antibody according to the invention to bind to KLK5 when KLK5 is already bound to another protein such as LEKTI, or a LEKTI fragment, or another antibody or antibody fragment such as Fab.

[0204] The advantage associated with antibodies that can bind to KLK5 and inhibit KLK5 biological (i.e. protease) activity without competing with LEKTI or LEKTI fragments for binding to KLK5 is that they can inhibit KLK5 activity under conditions where LEKTI dissociates from the KLK5:LEKTI complex, such as in an environment that gradually becomes acidic from the basal layer to the stratum corneum of the epidermis.

[0205] Antibodies that “compete,” “cross-block,” “are cross-blocked,” or “bind to the same epitope on human KLK5” (and any of their grammatical variations) with the antibodies of the present invention refer to antibodies that cannot form complexes with KLK5 bound to the antibodies of the present invention.

[0206] In one embodiment of the invention, the LEKTI fragment is a human LEKTI domain 5 comprising amino acids 1 to 64 of SEQ ID NO: 54 or a LEKTI domain 8 comprising amino acids 1 to 71 of SEQ ID NO: 61.

[0207] Therefore, in a preferred embodiment, an antibody that binds to kallikrein 5 (KLK5) is used:

[0208] i. When KLK5 binds to LEKTI or LEKTI fragments, it binds to KLK5;

[0209] ii. Does not compete with LEKTI or LEKTI fragments for binding to KLK5; and / or

[0210] iii. Forms a complex with KLK5 bound to LEKTI or the LEKTI fragment;

[0211] The preferred LEKTI fragment is a human LEKTI domain 5 containing amino acids 1 to 64 of SEQ ID NO: 54 or a LEKTI domain 8 containing amino acids 1 to 71 of SEQ ID NO: 61, and the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0212] a. The variable light chain comprises CDR-L1 containing SEQ ID NO:1 or SEQ ID NO:62 or SEQ ID NO:63, CDR-L2 containing SEQ ID NO:2, and CDR-L3 containing SEQ ID NO:3; and

[0213] b. The variable heavy chain contains CDR-H1 containing SEQ ID NO:4, CDR-H2 containing SEQ ID NO:5, and CDR-H3 containing SEQ ID NO:6.

[0214] In another embodiment of the invention, the antibody binds to KLK5, preferably human KLK5, wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51, and:

[0215] i. When KLK5 binds to LEKTI or LEKTI fragments, it binds to KLK5;

[0216] ii. Does not compete with LEKTI or LEKTI fragments for binding to KLK5; and / or

[0217] iii. Forms a complex with KLK5 bound to LEKTI or the LEKTI fragment;

[0218] The preferred LEKTI fragment is the human LEKTI domain 5 containing amino acids 1 to 64 of SEQ ID NO:54 or the LEKTI domain 8 containing amino acids 1 to 71 of SEQ ID NO:61.

[0219] In another embodiment, the antibody according to the invention binds to human KLK5 and also to cynomolgus monkey KLK5, wherein the human KLK5 preferably comprises SEQ ID NO:53 and the cynomolgus monkey KLK5 preferably comprises the cynomolgus monkey KLK5 of SEQ ID NO:60.

[0220] In another embodiment, the antibody according to the invention does not bind to human or cynomolgus kallikrein 2 (KLK2); or human or cynomolgus kallikrein 4 (KLK4); or human or cynomolgus kallikrein 7 (KLK7). In other words, the antibody specifically targets KLK5, but not other kallikreinases.

[0221] As used herein, “specific” is intended to mean an antibody that recognizes only a specific antigen, or an antibody that has a significantly higher binding affinity for a specific antigen (e.g., KLK5) compared to binding to non-specific antigens (e.g., other kallikrein), such as at least 5, 6, 7, 8, 9, or 10 times the binding affinity.

[0222] In one embodiment, according to the invention, the binding of the antibody to KLK5 is characterized by a dissociation constant (KL) of about 500 pM or less, preferably about 172 pM. D ).

[0223] As used in this article, the term "K" D "" refers to the dissociation constant, which is determined by K d With K a The ratio (i.e., K) d / K a K is obtained and expressed as molar concentration (M). d and K a These refer to the dissociation rate and binding rate of a specific antigen-antibody (or its antigen-binding fragment) interaction, respectively. The K value of an antibody can be determined using methods well-established in the art. D Value. Used to determine antibody K. D The method involves using surface plasmon resonance, such as that described in the embodiments herein. The system uses recombinant KLK5 or a suitable fusion protein / peptide thereof. In one example, affinity is measured using recombinant KLK5 as described in the embodiments herein. For surface plasmon resonance, the target molecule is immobilized on a solid phase and exposed to a ligand in a mobile phase that flows along a flow cell. If the ligand binds to the immobilized target, the local refractive index changes, resulting in a change in the SPR angle, which can be monitored in real time by detecting changes in the intensity of reflected light. The rate of change of the SPR signal can be analyzed to generate the apparent rate constants of the binding and dissociation phases of the binding reaction. The ratio of these values ​​gives the apparent equilibrium constant (affinity) (see, for example, Wolff et al., Cancer Res. 53:2560-65 (1993)).

[0224] In one embodiment, the antibody according to the invention has a higher binding affinity for human KLK5 than for cynomolgus monkey or mouse KLK5 (i.e., a smaller KL). D The term "affinity" refers to the force of the interaction between the antibody and KLK5.

[0225] In one embodiment, the antibody according to the invention has an IC50 of less than 800 pM that blocks KLK5 protease activity. 50 Preferably, the antibody according to the invention has an IC50 of less than 18 pM blocking KLK5 protease activity in the in vitro assay described herein. 50 .

[0226] As used in this article, the term IC 50 IC50 refers to the half-maximal inhibitory concentration, which is a measure of the effectiveness of a substance, such as an antibody, in inhibiting a specific biological or biochemical function (in this invention, the protease activity of KLK5). 50 It is a quantitative measure that indicates how much of a specific substance is needed to inhibit a given biological process, function, or activity by half.

[0227] The antibody according to the invention may contain a frame region of the animal that produced the antibody. For example, if the antibody is produced in a rabbit, it will contain a CDR as defined above and a frame region of a rabbit antibody, such as an antibody containing a light chain variable region according to SEQ ID NO:7 (whose nucleotide sequence is shown in SEQ ID NO:8 or nucleotides 1 to 330 of SEQ ID NO:8) and a heavy chain variable region according to SEQ ID NO:9 (whose nucleotide sequence is shown in SEQ ID NO:10).

[0228] In one implementation, the antibody may be a chimeric or humanized antibody.

[0229] Chimeric antibodies are typically produced using recombinant DNA methods. DNA can be modified by replacing the coding sequences of the human L and H chain constant regions with the corresponding non-human (e.g., mouse or rabbit) H and L constant regions (Morrison; PNAS 81, 6851 (1984)).

[0230] If the variable region or full-length chain of an antibody is obtained from a system using a human germline immunoglobulin gene, the human antibody contains a heavy or light chain variable region or full-length heavy or light chain as a "product" or "derived from" a specific germline sequence. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with an antigen of interest or screening a library of human immunoglobulin genes displayed on a phage with an antigen of interest. Human antibodies or fragments thereof that are "products" or "derived from" human germline immunoglobulin sequences can be obtained by comparing the amino acid sequence of a human antibody with the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is most closely related to the human antibody sequence (i.e., has the highest percentage of identity). Compared to germline sequences, human antibodies that are "products" or "derived from" a specific human germline immunoglobulin sequence may contain amino acid differences, for example, due to naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, selected human antibodies typically share at least 90% amino acid sequence identity with the sequence encoded by human germline immunoglobulin genes and contain amino acid residues that identify the human antibody as human when compared to germline immunoglobulin sequences from other species (e.g., mouse germline sequences). In some 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 in amino acid sequence to the sequence encoded by germline immunoglobulin genes. Generally, human antibodies derived from a specific human germline sequence differ from the sequence encoded by human germline immunoglobulin genes by no more than 10 amino acids. In some cases, human antibodies may show no more than 5 amino acids, or even no more than 4, 3, 2, or 1 amino acid difference from the sequence encoded by germline immunoglobulin genes.

[0231] Human antibodies can be produced by many methods known to those skilled in the art. Human antibodies can be prepared using human myeloma or mouse-human heterologous myeloma cell lines via hybridoma methods (Kozbor, J Immunol; (1984) 133:3001; Brodeur, Monoclonal Isolated Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both of which utilize human variable region libraries (Winter G; (1994) Annu Rev Immunol 12:433-455, Green LL, (1999) J Immunol Methods 231:11-23).

[0232] In a preferred embodiment of the invention, the antibody according to the invention is humanized.

[0233] Antibodies according to the invention can be obtained using any suitable method known in the art. KLK5 includes its fusion protein, and cells expressing KLK5 (recombinant or natural) can be used to generate antibodies that specifically recognize KLK5. Various forms of KLK5 as described herein can be used.

[0234] In one embodiment, the antigen used is active KLK5, preferably generated as described in the examples below.

[0235] KLK5 or fragments thereof for use in immunizing a host can be prepared from genetically engineered host cells containing an expression system using methods well known in the art, or they can be recovered from natural biological sources. In some cases, KLK5 or fragments thereof may be part of a larger protein, such as a fusion protein fused to an affinity tag or the like.

[0236] When animal immunization is required, KLK5 can be administered to animals, preferably non-human animals, using well-known standard protocols to obtain antibodies against KLK5 produced according to the present invention (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.

[0237] 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).

[0238] Antibodies for use in this invention can also be generated by cloning and expressing immunoglobulin variable region cDNA using a single lymphocyte antibody method, wherein the immunoglobulin variable region cDNA is generated by single lymphocytes selected for the production of specific antibodies, for example by the methods described in Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-7848l; WO92 / 02551; WO2004 / 051268 and WO2004 / 106377.

[0239] Antibodies can be screened using assays that measure binding to KLK5 and / or assays that measure KLK5 bioactivity, preferably assays that measure inhibition of KLK5 protease activity.

[0240] In a preferred embodiment, the antibody binds to kallikrein 5 (KLK5), wherein the antibody is a chimeric or humanized antibody; preferably a humanized antibody; wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0241] a. The variable light chain comprises a CDR-L1 containing SEQ ID NO:1 or SEQ ID NO:62 or SEQ ID NO:63, preferably SEQ ID NO:1, a CDR-L2 containing SEQ ID NO:2, and a CDR-L3 containing SEQ ID NO:3; and

[0242] b. The variable heavy chain contains CDR-H1 containing SEQ ID NO:4, CDR-H2 containing SEQ ID NO:5, and CDR-H3 containing SEQ ID NO:6.

[0243] In another embodiment of the invention, the antibody binds to KLK5, wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51; wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0244] a. Light chain variable chain, which includes:

[0245] i. A CDR-L1 containing SEQ ID NO:1, SEQ ID NO:62, or SEQ ID NO:63, preferably SEQ ID NO:1;

[0246] ii. CDR-L2 containing SEQ ID NO:2; and

[0247] iii. CDR-L3 containing SEQ ID NO:3; and

[0248] b. Heavy chain variable region, which includes:

[0249] i. CDR-H1 containing SEQ ID NO:4;

[0250] ii. CDR-H2 containing SEQ ID NO:5; and

[0251] iii. CDR-H3 containing SEQ ID NO:6

[0252] The antibody is a chimeric or humanized antibody; preferably, the antibody is a humanized antibody.

[0253] In another preferred embodiment, the humanized antibody binding to KLK5 comprises a variable light chain and a variable heavy chain, wherein:

[0254] a. Light chain variable chain, which includes:

[0255] i. A CDR-L1 containing SEQ ID NO:1, SEQ ID NO:62, or SEQ ID NO:63, preferably SEQ ID NO:1;

[0256] ii. CDR-L2 containing SEQ ID NO:2; and

[0257] iii. CDR-L3 containing SEQ ID NO:3; and

[0258] b. Heavy chain variable region, which includes:

[0259] i. CDR-H1 containing SEQ ID NO:4;

[0260] ii. CDR-H2 containing SEQ ID NO:5; and

[0261] iii. CDR-H3 containing SEQ ID NO:6

[0262] The antibody, when KLK5 binds to LEKTI or a LEKTI fragment, inhibits or reduces the protease activity of KLK5 and / or binds to KLK5; and / or does not compete with LEKTI or a LEKTI fragment for binding to KLK5 and / or forms a complex with KLK5 bound to LEKTI or a LEKTI fragment.

[0263] More preferably, the humanized antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51; wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0264] a. Light chain variable chain, which includes:

[0265] i. A CDR-L1 containing SEQ ID NO:1, SEQ ID NO:62, or SEQ ID NO:63, preferably SEQ ID NO:1;

[0266] ii. CDR-L2 containing SEQ ID NO:2; and

[0267] iii. CDR-L3 containing SEQ ID NO:3; and

[0268] b. Heavy chain variable region, which includes:

[0269] i. CDR-H1 containing SEQ ID NO:4;

[0270] ii. CDR-H2 containing SEQ ID NO:5; and

[0271] iii. CDR-H3 containing SEQ ID NO:6

[0272] The antibody, when KLK5 binds to LEKTI or a LEKTI fragment, inhibits or reduces the protease activity of KLK5 and / or binds to KLK5; and / or does not compete with LEKTI or a LEKTI fragment for binding to KLK5 and / or forms a complex with KLK5 bound to LEKTI or a LEKTI fragment.

[0273] As used herein, the term "humanized" antibody refers to an antibody in which one or more CDRs (including one or more modified CDRs, if desired) from a donor antibody (e.g., a non-human antibody such as a mouse or rabbit monoclonal antibody) are grafted into the heavy chain variable region and / or light chain variable region framework of a recipient antibody (e.g., a human antibody). See Vaughan et al., Nature Biotechnology, 16, 535-539, 1998 for a review. In one embodiment, instead of transferring the entire CDR, only one or more specific determining residues from any of the CDRs described above are transferred into the human antibody framework (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only specific determining residues from one or more of the CDRs described above are transferred into the human antibody framework. In another embodiment, only specific determining residues from each of the aforementioned CDRs are transferred into the human antibody framework.

[0274] When CDRs are transplanted, the class / type of donor antibody from which the CDR is derived can be taken into account, including mouse, primate, and human frame regions, using any suitable receptor variable region frame sequence.

[0275] Preferably, the humanized antibody according to the invention has a variable domain comprising a human receptor framework region and one or more CDRs specifically provided herein. Thus, in one embodiment, a blocking humanized antibody binding to KLK5, preferably human KLK5, is provided, wherein the variable domain comprises a human receptor framework region and a non-human donor CDR.

[0276] Examples of human frames that can be used in this invention are KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al., ibid.). For example, KOL and NEWM can be used for the heavy chain, REI for the light chain, and EU, LAY, and POM for both the heavy and light chains. Alternatively, human germline sequences can be used; these are available at: http: / / www.imgt.org / . In the humanized antibodies according to the invention, the receptor heavy and light chains do not necessarily need to be derived from the same antibody, and if desired, a complex chain with frame regions derived from different chains can be included.

[0277] The appropriate framework region of the humanized antibody light chain according to the present invention is derived from the human lineage IGKV1-6 JK4 having SEQ ID NO:47, the nucleotide sequence of which is shown in SEQ ID NO:48.

[0278] The suitable framework region of the heavy chain of the humanized antibody or its antigen-binding fragment according to the present invention is derived from the human lineage IGHV4-4 JH4 having the sequence shown in SEQ ID NO:49, the nucleotide sequence of which is shown in SEQ ID NO:50.

[0279] Therefore, in one embodiment, a humanized antibody binding to KLK5 is provided, wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0280] a. Light chain variable chain, which includes:

[0281] i. A CDR-L1 containing SEQ ID NO:1, SEQ ID NO:62, or SEQ ID NO:63, preferably SEQ ID NO:1;

[0282] ii. CDR-L2 containing SEQ ID NO:2; and

[0283] iii. CDR-L3 containing SEQ ID NO:3; and

[0284] b. Heavy chain variable region, which includes:

[0285] i. CDR-H1 containing SEQ ID NO:4;

[0286] ii. CDR-H2 containing SEQ ID NO:5; and

[0287] iii. CDR-H3 containing SEQ ID NO:6

[0288] The light chain framework region is derived from the human lineage IGKV1-6 JK4 containing SEQ ID NO:47; and the heavy chain framework region is derived from the human lineage IGHV4-4 JH4 containing SEQ ID NO:49. Preferably, the antibody, when KLK5 binds to LEKTI or a LEKTI fragment, inhibits or reduces the protease activity of KLK5 and / or binds to KLK5; and / or does not compete with LEKTI or a LEKTI fragment for binding to KLK5 and / or forms a complex with KLK5 bound to LEKTI or a LEKTI fragment.

[0289] In another embodiment, a humanized antibody binding to KLK5 is provided, wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51; wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0290] a. Light chain variable chain, which includes:

[0291] i. A CDR-L1 containing SEQ ID NO:1, SEQ ID NO:62, or SEQ ID NO:63, preferably SEQ ID NO:1;

[0292] ii. CDR-L2 containing SEQ ID NO:2; and

[0293] iii. CDR-L3 containing SEQ ID NO:3; and

[0294] b. Heavy chain variable region, which includes:

[0295] i. CDR-H1 containing SEQ ID NO:4;

[0296] ii. CDR-H2 containing SEQ ID NO:5; and

[0297] iii. CDR-H3 containing SEQ ID NO:6

[0298] The light chain framework region is derived from the human lineage IGKV1-6 JK4 containing SEQ ID NO:47; and the heavy chain framework region is derived from the human lineage IGHV4-4 JH4 containing SEQ ID NO:49. Preferably, the antibody, when KLK5 binds to LEKTI or a LEKTI fragment, inhibits or reduces the protease activity of KLK5 and / or binds to KLK5; and / or does not compete with LEKTI or a LEKTI fragment for binding to KLK5 and / or forms a complex with KLK5 bound to LEKTI or a LEKTI fragment; and

[0299] In the humanized antibody according to the invention, the frame region may not have the exact same sequence as the receptor antibody. For example, for that receptor chain class or type, uncommon residues may be changed to more common residues. Alternatively, selected residues in the receptor frame region may be altered to correspond to residues at the same positions in the donor antibody (see Reichmannet al., 1998, Nature, 332, 323-324). Such alterations should be kept to the minimum necessary to restore affinity to the donor antibody. Schemes for selecting residues in the receptor frame region that may need to be altered are set forth in WO91 / 09967 (which is incorporated herein by reference).

[0300] Therefore, in one implementation, residues 1, 2, 3, 4, 5, 6, 7, or 8 in the framework are replaced with alternative amino acid residues.

[0301] Therefore, in one embodiment, a humanized antibody according to the invention is provided, wherein each residue at position 2 and / or 3 and / or 63 (refer to SEQ ID NO:47 or SEQ ID NO:15) of the variable light chain is a donor residue. Preferably, the residue at position 2 of the variable light chain is tyrosine, and the residue at position 3 of the variable light chain is aspartic acid. In some embodiments, the residue at position 63 of the variable light chain is lysine.

[0302] In another embodiment, a humanized antibody is provided, wherein at least each residue of the variable heavy chain at positions 68, 72, 74, 77 and 79 (refer to SEQ ID NO:49; or positions 67, 71, 73, 76 and 78 of SEQ ID NO:39) is a donor residue.

[0303] Preferably, the residue at position 72 of the variable heavy chain is glutamine, the residue at position 74 is serine, and the residue at position 79 is valine. In some embodiments, the residue at position 68 of the variable heavy chain is phenylalanine, and / or the residue at position 77 is threonine.

[0304] In a preferred embodiment, the humanized antibody binds to KLK5, wherein the humanized antibody comprises:

[0305] - A light chain variable region comprising SEQ ID NO: 11 or 15 or 19 or 23 and a heavy chain variable region comprising SEQ ID NO: 27 or 31 or 35 or 39 or 43; or

[0306] - Contains the light chain variable region of SEQ ID NO:15 (wherein the amino acid residue at position 24 is arginine (Arg; R) or lysine (Lys; K)) and the heavy chain variable region of SEQ ID NO:27, 31, 35, 39, or 43. Preferably, the antibody, when KLK5 binds to LEKTI or the LEKTI fragment, inhibits or reduces the protease activity of KLK5 and / or binds to KLK5; and / or does not compete with LEKTI or the LEKTI fragment for binding to KLK5 and / or forms a complex with KLK5 bound to LEKTI or the LEKTI fragment.

[0307] In another embodiment, a humanized antibody binding to KLK5 is provided, wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51; wherein the antibody comprises:

[0308] - A light chain variable region comprising SEQ ID NO: 11 or 15 or 19 or 23 and a heavy chain variable region comprising SEQ ID NO: 27 or 31 or 35 or 39 or 43; or

[0309] - Contains a light chain variable region of SEQ ID NO:15 (where the amino acid residue at position 24 is arginine (Arg;R) or lysine (Lys;K)) and a heavy chain variable region of SEQ ID NO:27 or 31 or 35 or 39 or 43.

[0310] Preferably, the antibody, when KLK5 binds to LEKTI or a LEKTI fragment, inhibits or reduces the protease activity of KLK5 and / or binds to KLK5; and / or does not compete with LEKTI or a LEKTI fragment for binding to KLK5 and / or forms a complex with KLK5 bound to LEKTI or a LEKTI fragment.

[0311] In a preferred embodiment of the invention, the antibody contains a light chain variable region comprising SEQ ID NO:15 and a heavy chain variable region comprising SEQ ID NO:39.

[0312] More preferably, the humanized antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51; wherein the antibody contains a light chain variable region comprising SEQ ID NO:15 and a heavy chain variable region comprising SEQ ID NO:39.

[0313] Even more preferably, the humanized antibody, when KLK5 binds to LEKTI or the LEKTI fragment, inhibits or reduces the protease activity of KLK5 and / or binds to KLK5; and / or does not compete with LEKTI or the LEKTI fragment for binding to KLK5 and / or forms a complex with KLK5 bound to LEKTI or the LEKTI fragment.

[0314] In one embodiment, the present invention provides an antibody comprising 80% or more of a sequence similar to or identical to the sequence disclosed herein, such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the related sequence, such as a variable domain sequence, a CDR sequence, or a variable domain sequence excluding a CDR. In one embodiment, the related sequence is SEQ ID NO:15. In one embodiment, the related sequence is SEQ ID NO:39.

[0315] In one embodiment, the antibody binding 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:15 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:39.

[0316] In one embodiment, the antibody binding to KLK5 comprises a light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar to the sequence given in SEQ ID NO:15, but wherein said antibody has a CDR-L1 of the sequence contained in SEQ ID NO:1 (or SEQ ID NO:62 or 63), a CDR-L2 of the sequence contained in SEQ ID NO:2, and a CDR-L3 of the sequence contained in SEQ ID NO:3.

[0317] As used herein, “identity,” “same,” or its grammatical variations indicate that the amino acid residues between sequences are identical at any specific position in the aligned sequences. As used herein, “similarity,” “resemblance,” or its grammatical variations indicate that the amino acid residues between sequences have a similar type at any specific position in the aligned sequences. For example, leucine can substitute for isoleucine or valine. Other amino acids that can frequently be substituted for each other include, but are not limited to:

[0318] - Phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains);

[0319] - Lysine, arginine, and histidine (amino acids with basic side chains);

[0320] - Aspartic acid and glutamic acid (amino acids with acidic side chains);

[0321] - Asparagine and glutamine (amino acids with amide side chains); and

[0322] - Cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0323] 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., Stockton Press, New York, 1991; BLAST). TM The software is available from NCBI (Altschul, SF et al., 1990, J. Mol. Biol. 215: 403-410; Gish, W. & States, DJ 1993, Nature Genet. 3: 266-272; Madden, T Let et al., 1996, Meth. Enzymol. 266: 131-141; Altschul, SF et al., 1997, Nucleic Acids Res. 25: 3389-3402; Zhang, J. & Madden, TL 1997, Genome Res. 7: 649-656).

[0324] In one embodiment, the antibody is a full-length antibody, preferably selected from IgG1 and IgG4 or IgG4P.

[0325] Therefore, the present invention provides a full-length humanized antibody that binds to KLK5 and comprises:

[0326] a. Light chain variable region, which includes:

[0327] i. A CDR-L1 containing SEQ ID NO:1;

[0328] ii. CDR-L2 containing SEQ ID NO:2; and

[0329] iii. CDR-L3 containing SEQ ID NO:3; and

[0330] b. Heavy chain variable region, which includes:

[0331] iv. CDR-H1 containing SEQ ID NO:4;

[0332] v. containing CDR-H2 of SEQ ID NO:5; and

[0333] vi. Contains CDR-H3 of SEQ ID NO:6.

[0334] Preferably, the full-length humanized antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referenced in SEQ ID NO:51, and wherein the antibody is an IgG4P isoform.

[0335] In another embodiment, a full-length humanized antibody is provided that binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referenced in SEQ ID NO:51, and wherein the antibody is an IgG4P isoform.

[0336] Those skilled in the art will also understand that antibodies can undergo various post-translational modifications. The type and extent of these modifications generally depend on the host cell line used to express the antibody and the culture conditions. Such modifications can include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A common modification is the loss of a carboxyl-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidase (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Therefore, the C-terminal lysine residue of the antibody heavy chain may be absent.

[0337] In one implementation, the C-terminal amino acid from the antibody is cleaved during post-translational modification.

[0338] In one implementation, the N-terminal amino acid from the antibody is cleaved during post-translational modification.

[0339] In a preferred embodiment, the antibody binding to KLK5 is a full-length antibody containing a light chain variable region comprising SEQ ID NO:15 and a heavy chain variable region comprising SEQ ID NO:39. Preferably, the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referenced in SEQ ID NO:51.

[0340] In another embodiment, the antibody binding to KLK5 is a full-length IgG4 antibody containing a light chain comprising SEQ ID NO:17 and a heavy chain comprising SEQ ID NO:41. Preferably, the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referenced in SEQ ID NO:51.

[0341] In yet another embodiment, the antibody is a Fab' fragment containing a light chain variable region comprising SEQ ID NO:15 and a heavy chain variable region comprising SEQ ID NO:39.

[0342] In another embodiment, the antibody comprises a light chain variable region comprising SEQ ID NO:15 and a heavy chain variable region comprising SEQ ID NO:27, 31, 35, 39, or 43. For example, the antibody is a full-length IgG4 antibody comprising a light chain variable region comprising SEQ ID NO:15 and a heavy chain variable region comprising SEQ ID NO:27, 31, 35, 39, or 43, preferably SEQ ID NO:39. In one embodiment, the amino acid glutamine 24 (Gln; Q) of reference SEQ ID NO:15 is arginine (Arg; R)Q24R or lysine (Lys; K)Q24K.

[0343] In another embodiment, the antibody is a full-length IgG4 antibody containing a light chain variable region comprising SEQ ID NO:11 and a heavy chain variable region comprising SEQ ID NO:27 or 31 or 35 or 39 or 43. In another embodiment, the antibody is a Fab' fragment containing a light chain variable region comprising SEQ ID NO:11 and a heavy chain variable region comprising SEQ ID NO:27 or 31 or 35 or 39 or 43.

[0344] In another embodiment, the antibody is a full-length IgG4 antibody containing a light chain variable region comprising SEQ ID NO:19 and a heavy chain variable region comprising SEQ ID NO:27, 31, 35, 39, or 43. In another embodiment, the antibody is a Fab' fragment containing a light chain variable region comprising SEQ ID NO:19 and a heavy chain variable region comprising SEQ ID NO:27, 31, 35, 39, or 43.

[0345] In another embodiment, the antibody is a full-length IgG4 antibody containing a light chain variable region according to SEQ ID NO:23 and a heavy chain variable region containing SEQ ID NO:27 or 31 or 35 or 39 or 43.

[0346] In another embodiment, the antibody is a Fab' fragment containing a light chain variable region according to SEQ ID NO:23 and a heavy chain variable region containing SEQ ID NO:27 or 31 or 35 or 39 or 43.

[0347] In another embodiment, the antibody binding to KLK5 is a full-length IgG4 antibody, which comprises:

[0348] 1. A light chain comprising SEQ ID NO:13 and a heavy chain comprising SEQ ID NO:29, 33, 37, 41, or 45; or

[0349] 2. A light chain comprising SEQ ID NO:17 and a heavy chain comprising SEQ ID NO:29, 33, 37, 41, or 45; or

[0350] 3. A light chain comprising SEQ ID NO:17 (wherein glutamine 24 (Gln; Q) is arginine (Arg; R) Q24R or lysine (Lys; K) Q24K) and a heavy chain comprising SEQ ID NO:29 or 33 or 37 or 41 or 45; or

[0351] 4. A light chain containing SEQ ID NO:21 and a heavy chain containing SEQ ID NO:29, 33, 37, 41, or 45; or

[0352] 5. A light chain containing SEQ ID NO:25 and a heavy chain containing SEQ ID NO:29 or 33 or 37 or 41 or 45.

[0353] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0354] In a preferred embodiment, the antibody binding to KLK5 is a full-length IgG4 antibody containing a light chain comprising SEQ ID NO:17 and a heavy chain comprising SEQ ID NO:41; wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referenced in SEQ ID NO:51.

[0355] The present invention also provides an antibody as described above forming a complex with KLK5, preferably human KLK5, bound to another antibody, wherein the other antibody comprises:

[0356] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:68, CDR-L2 containing SEQ ID NO:69, and CDR-L3 containing SEQ ID NO:70; and the variable heavy chain comprises CDR-H1 containing SEQ ID NO:71, CDR-H2 containing SEQ ID NO:72, and CDR-H3 containing SEQ ID NO:73; and / or

[0357] 2. A variable light chain comprising SEQ ID NO:74 and a variable heavy chain comprising SEQ ID NO:76; and / or

[0358] 3. A variable light chain encoded by a nucleotide containing SEQ ID NO:75 and a variable heavy chain encoded by a nucleotide containing SEQ ID NO:77.

[0359] In one embodiment, the antibody binds to KLK5, wherein the antibody preferably binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51, and wherein the antibody comprises:

[0360] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0361] 2. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43;

[0362] The antibody forms a complex with KLK5, preferably human KLK5, bound to another antibody, wherein the other antibody comprises:

[0363] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:68, CDR-L2 containing SEQ ID NO:69, and CDR-L3 containing SEQ ID NO:70; and the variable heavy chain comprises CDR-H1 containing SEQ ID NO:71, CDR-H2 containing SEQ ID NO:72, and CDR-H3 containing SEQ ID NO:73; and / or

[0364] 2. A variable light chain comprising SEQ ID NO:74 and a variable heavy chain comprising SEQ ID NO:76; and / or

[0365] 3. A variable light chain encoded by a nucleotide containing SEQ ID NO:75 and a variable heavy chain encoded by a nucleotide containing SEQ ID NO:77.

[0366] Therefore, the present invention also provides an antibody that binds to KLK5, preferably human KLK5, comprising:

[0367] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 comprising SEQ ID NO:68, CDR-L2 comprising SEQ ID NO:69, and CDR-L3 comprising SEQ ID NO:70; wherein the variable heavy chain comprises CDR-H1 comprising SEQ ID NO:71, CDR-H2 comprising SEQ ID NO:72, and CDR-H3 comprising SEQ ID NO:73; wherein the antibody is optionally humanized; and / or

[0368] 2. A variable light chain comprising SEQ ID NO:74 and a variable heavy chain comprising SEQ ID NO:76; and / or

[0369] 3. A variable light chain encoded by a nucleotide containing SEQ ID NO:75 and a variable heavy chain encoded by a nucleotide containing SEQ ID NO:77.

[0370] Furthermore, the present invention also includes a KLK5-antibody complex comprising:

[0371] a.KLK5, Preferred Person KLK5; and

[0372] b. An antibody binding to KLK5, wherein the antibody preferably binds to an epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO: 51, and wherein the antibody comprises:

[0373] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0374] 2. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; and

[0375] c. Another type of antibody, which comprises:

[0376] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 comprising SEQ ID NO:68, CDR-L2 comprising SEQ ID NO:69, and CDR-L3 comprising SEQ ID NO:70; wherein the variable heavy chain comprises CDR-H1 comprising SEQ ID NO:71, CDR-H2 comprising SEQ ID NO:72, and CDR-H3 comprising SEQ ID NO:73; wherein the antibody is optionally humanized; and / or

[0377] 2. A variable light chain comprising SEQ ID NO:74 and a variable heavy chain comprising SEQ ID NO:76; and / or

[0378] 3. A variable light chain encoded by a nucleotide containing SEQ ID NO:75 and a variable heavy chain encoded by a nucleotide containing SEQ ID NO:77.

[0379] It should be understood that, as shown in the examples below, the so-called "other antibody" binds to an epitope that is different from the epitope described herein and does not overlap with the epitope described herein (and similarly contains the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146 and His147 of reference SEQ ID NO:51), preferably human KLK5.

[0380] Furthermore, the present invention provides an antibody that competitively binds to KLK5, preferably human KLK5, by cross-blocking or being cross-blocked by the following antibodies: the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51, and the antibody comprises:

[0381] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0382] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0383] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0384] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0385] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0386] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0387] In one embodiment, such competitive antibodies have a heavy chain variable region that is at least 80% identical or similar to a sequence containing SEQ ID NO: 29 or 33 or 37 or 41 or 45; and / or have a light chain variable region that is at least 80% identical or similar to a sequence containing SEQ ID NO: 13 or 17 or 21 or 25 or 30.

[0388] Competitive antibodies can be identified using any suitable method in the art, such as by using a competitive ELISA or BIAcore assay, wherein the competitive antibody prevents the binding of the antibody of the present invention by cross-blocking or by being cross-blocked to bind to KLK5, or vice versa. Such competitive assays can use isolated natural or recombinant KLK5 or suitable fusion proteins / peptides thereof. In one example, competition is measured using recombinant human active KLK5 (e.g., containing SEQ ID NO: 53).

[0389] Biomolecules, such as antibodies or fragments, contain acidic and / or basic functional groups, thereby giving the molecule a net positive or negative charge. The total "observed" charge will depend 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 value at which a particular molecule or its solvent-accessible surface does not carry a net charge. In one example, an antibody binding to KLK5 can be engineered to have a suitable isoelectric point that binds to an epitope of human KLK5 containing the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referenced in SEQ ID NO:51. This may result in antibodies with more robust properties, particularly suitable solubility and / or stability profiles and / or improved purification characteristics.

[0390] Therefore, in one aspect, the present invention provides an antibody that binds to KLK5, preferably binding to an epitope of human KLK5, the epitope comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51, wherein said antibody comprises:

[0391] a. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0392] b. Light chains comprising SEQ ID NO: 13 or 17 or 21 or 25; and heavy chains comprising SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0393] It was engineered to have an isoelectric point different from that of the originally identified antibody.

[0394] Antibodies can be engineered, for example, by replacing acidic amino acid residues with one or more basic amino acid residues. Alternatively, basic amino acid residues can be introduced or acidic amino acid residues can be removed. Alternatively, if the molecule has an unacceptably high pI value, acidic residues can be introduced as needed to lower the pI. It is important that care be taken to maintain the desired activity of the antibody or fragment when manipulating the pI. Therefore, in one embodiment, the engineered antibody has the same or substantially the same activity as the "unmodified" antibody or fragment.

[0395] 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) can be used to predict the isoelectric point of antibodies.

[0396] It should be understood that any suitable method known in the art can be used to modify the affinity of the antibodies provided by the present invention. Therefore, the present invention also relates to antibody variants with improved affinity for KLK5, particularly human KLK5. Such variants can be obtained through a number of affinity maturation protocols, including mutant CDR (Yang et al., J. Mol. Biol., 254, 392-403, 1995), strand shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), using E. coli mutants (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., Nature, 391, 288-291, 1998).

[0397] If desired, the antibodies of the present invention can be conjugated to one or more effector molecules. It should be understood that the effector molecule may comprise a single effector molecule or two or more such molecules, linked in this way to form a single portion capable of attaching to the antibody of the present invention. In cases where it is desired to obtain an antibody fragment conjugated to an effector molecule, this can be prepared by standard chemical or recombinant DNA procedures, wherein the antibody fragment is conjugated to the effector molecule directly or via a conjugating agent. Techniques for conjugating such effector molecules to antibodies are well 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 WO 93 / 06231, WO 92 / 22583, WO 89 / 00195, WO 89 / 01476, and WO 03 / 031581. Alternatively, when the effector molecule is a protein or polypeptide, ligation can be achieved using recombinant DNA procedures as described in, for example, WO 86 / 01533 and EP0392745.

[0398] As used herein, the term effector molecule includes, for example, antitumor agents, drugs, toxins, bioactive proteins such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and their fragments such as DNA, RNA and their fragments, radionuclides, especially radioiodides, radioisotopes, chelated metals, nanoparticles, and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy.

[0399] Examples of effector molecules can include cytotoxins or cytotoxic agents, including any agent that is harmful to cells (e.g., kills them). Examples include cobustatin, salicylate, epothilone, astrocytocin, maytansine, spongistatin, rhizoxin, roridin, hemiasterlin, paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, donoxacin, danomycin, dihydroxyanthracin dione, mitoxantrone, styromycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues.

[0400] 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., oxamylmusa, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatin(II)(DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly doxorubicin) and doxorubicin), antibiotics (e.g., dermatomycin (formerly actinomycin), bleomycin, photomycin, anthramycin (AMC), calicheamicin or duocarmycin)), and antimitotic agents (e.g., vincristine and vinblastine).

[0401] Other effector molecules may include chelated radionuclides such as 111In and 90Y, Lu177, bismuth-213, californium-252, iridium-192 and tungsten-188 / rhenium-188; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxanes and suramin.

[0402] Other effector molecules include proteins, peptides, and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, and transferases. Proteins, polypeptides, and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin, proteins such as insulin, tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, or tissue plasminogen activator, thrombotic agents or anti-angiogenic agents such as angiostatin or endothelial inhibitory protein, or biological response modulators such as 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.

[0403] Other effector molecules may include detectable substances that can be used, for example, for diagnostic purposes. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron-emitting metals (for positron emission tomography) and non-radioactive paramagnetic metal ions. For information on metal ions that can be conjugated with antibodies for diagnostic purposes, see U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazinylamine, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and jellyfish luminescent protein; and suitable radionuclides include 125I, 131I, 111In, and 99Tc.

[0404] In another instance, effector molecules can increase the half-life of antibodies in vivo and / or decrease their immunogenicity and / or enhance their delivery to the immune system across the epithelial barrier. Examples of suitable effector molecules of this type include polymers, albumins, albumin-binding proteins, or albumin-binding compounds, such as those described in WO05 / 117984.

[0405] When the effector molecule is a polymer, it can typically be a synthetic or naturally occurring polymer, such as optionally substituted linear or branched polyalkylene, polyolefin or polyoxyethylene polymers, or branched or unbranched polysaccharides, such as homopolysaccharides or heteropolysaccharides.

[0406] Specific optional substituents that may be present on the above-mentioned synthetic polymers include one or more hydroxyl, methyl, or methoxy groups.

[0407] Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), or derivatives thereof, especially optionally substituted poly(ethylene glycol) such as methoxylated poly(ethylene glycol) or derivatives thereof.

[0408] Specific naturally occurring polymers include lactose, amylose, dextran, glycogen, or their derivatives.

[0409] In one embodiment, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof.

[0410] As used herein, “derivative” is intended to include reactive derivatives, such as thiol-selective reactive groups, such as maleimide. Reactive groups can be linked to the polymer directly or via linker segments. It should be understood that residues of such groups may, in certain cases, serve as linking groups between the antibody fragment and the polymer, forming part of the product.

[0411] The size of polymers can be varied as needed, but typically the average molecular weight ranges from 500 Da to 50,000 Da, for example, 5,000 to 40,000 Da, or 20,000 to 40,000 Da. The polymer size can be selected specifically based on the intended use of the product, such as its ability to target certain tissues like tumors or to prolong the circulating half-life (see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545 for a review). Therefore, for example, in cases where the product is expected to leave the circulation and penetrate tissues, such as for the treatment of tumors, using a low molecular weight polymer, such as one with a molecular weight of about 5,000 Da, may be advantageous. For applications where the product remains in circulation, using a higher molecular weight polymer, such as one with a molecular weight in the range of 20,000 Da to 40,000 Da, may be advantageous.

[0412] Suitable polymers include polyalkylene polymers, such as poly(ethylene glycol) or, in particular, methoxy poly(ethylene glycol) or derivatives thereof, and especially have a molecular weight in the range of about 15,000 Da to about 40,000 Da.

[0413] In one example, the antibody according to the invention is attached to a poly(ethylene glycol) (PEG) moiety. In a specific embodiment, the antibody and PEG molecule according to the invention can be attached 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 engineered into the antibody using recombinant DNA methods (see, for example, US 5,219,996; US 5,667,425; WO98 / 25971, WO2008 / 038024). In one example, the antibody of the invention is a modified Fab fragment, wherein the 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. Suitably, the additional amino acid forms a modified hinge region containing one or more cysteine ​​residues to which the effector molecule can attach. Multiple sites can be used to attach two or more PEG molecules.

[0414] Suitable, PEG molecules are covalently linked via a thiol group located on at least one cysteine ​​residue in the antibody fragment. Each polymer molecule attached to the modified antibody fragment may be covalently linked to a sulfur atom on a cysteine ​​residue in the fragment. The covalent bond is typically a disulfide bond, or particularly a sulfur-carbon bond. When the thiol group is used as an attachment site for a suitably activated effector molecule, thiol-selective derivatives such as maleimide and cysteine ​​derivatives may be used, for example. As described above, the activated polymer can be used as a starting material for preparing polymer-modified antibody fragments. The activated polymer can be any polymer containing a thiol reactive group, such as α-halocarboxylic acids or esters, such as iodoacetamide, imides, such as maleimide, vinyl sulfone, or disulfides. Such starting materials are commercially available (e.g., from Nektar, formerly known as Shearwater Polymers Inc., Huntsville, AL, USA) or can be prepared from commercially available starting materials using conventional chemical procedures. Specific PEG molecules include 20K methoxy-PEG-amine (available from Nektar, Rapp Polymere, and SunBio, formerly known as Shearwater) and M-PEG-SPA (available from Nektar, formerly known as Shearwater).

[0415] In one embodiment, the antibody is a modified Fab fragment, Fab' fragment, or diFab that is PEGylated, i.e., has a PEG (poly(ethylene glycol)) covalently attached thereto, for example according to the methods disclosed in EP 0948544 or EP1090037 (see also "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; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545). In one example, PEG is attached to a cysteine ​​residue in the hinge region. In another example, the PEG-modified Fab fragment has a maleimide group covalently attached to a single thiol group in the modified hinge region. Lysine residues can be covalently linked to the maleimide group, and each amino group on the lysine residue can be attached to a methoxylated poly(ethylene glycol) polymer with a molecular weight of approximately 20,000 Da. Therefore, the total molecular weight of PEG attached to the Fab fragment can be approximately 40,000 Da.

[0416] Specific PEG molecules include 2-[3-(N-maleimide)propionamido]acetamide of N,N'-bis(methoxypoly(ethylene glycol)MW 20,000) modified lysine, also known as PEG2MAL40K (available from Nektar, formerly known as Shearwater).

[0417] Alternative sources for PEG connectors include GL2-400MA3 (where m is 5 in the following structures) and GL2-400MA (where m is 2) with NOF having an n of approximately 450:

[0418]

[0419] m is 2 or 5.

[0420] Therefore, in one embodiment, the PEG is 2,3-bis(methylpolyoxyethylene-oxy)-1-{[3-(6-maleimide-1-oxohexyl)amino]propoxy}hexane(2-arm branched PEG,-CH2)3NHCO(CH2)5-MAL, Mw 40,000, referred to as SUNBRIGHT GL2-400MA3.

[0421] Other alternative PEG-effect molecules of the following types are available from Dr. Reddy, NOF, and Jenkem:

[0422]

[0423] In one embodiment, the Fab or Fab' according to the invention is conjugated with a PEG molecule.

[0424] In one embodiment, this disclosure provides a Fab'PEG molecule comprising one or more PEG polymers, such as one or two polymers, such as one or more 40kDa polymers.

[0425] Fab'-PEG molecules according to this disclosure may be particularly advantageous because they have a half-life independent of the Fc fragment. In one embodiment, a Fab' conjugated to a polymer such as a PEG molecule, starch molecule, or albumin molecule is provided. In one embodiment, an scFv conjugated to a polymer such as a PEG molecule, starch molecule, or albumin molecule is provided. In one embodiment, a Fab or Fab' according to this disclosure is conjugated to human serum albumin. In one embodiment, an antibody or fragment is conjugated to a starch molecule, for example, to increase the half-life. A method of binding starch to a protein is described in US 8,017,739, which is incorporated herein by reference.

[0426] The present invention also provides isolated polynucleotides encoding antibodies according to the invention. The isolated polynucleotides according to the invention may comprise synthetic DNA, such as DNA produced by chemical processing, cDNA, genomic DNA, or any combination thereof.

[0427] Standard molecular biology techniques can be used to prepare the DNA sequence encoding the antibody of this invention. The desired DNA sequence can be synthesized completely or partially using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used, depending on the circumstances.

[0428] In one embodiment, the isolated polynucleotide encoding according to the present invention is:

[0429] a. Light chain variable region, wherein the polynucleotide:

[0430] i. At least 90% identical to SEQ ID NO:8 (or nucleotides 1 to 330 of SEQ ID NO:8) or 12 (or nucleotides 1 to 330 of SEQ ID NO:12) or 16 or 20 or 24 or 64 or 66; or

[0431] ii. Containing SEQ ID NO:8 (or nucleotides 1 to 330 of SEQ ID NO:8) or 12 (or nucleotides 1 to 330 of SEQ ID NO:12) or 16 or 20 or 24 or 64 or 66; or

[0432] iii. It is essentially composed of SEQ ID NO:8 (or nucleotides 1 to 330 of SEQ ID NO:8) or 12 (or nucleotides 1 to 330 of SEQ ID NO:12) or 16 or 20 or 24 or 64 or 66;

[0433] b. Heavy chain variable region, wherein the polynucleotide:

[0434] i. At least 90% identical to SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44; or

[0435] ii. Contains SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44; or

[0436] iii. Basically composed of SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44;

[0437] c. The light chain, wherein the polynucleotide is:

[0438] i. At least 90% identical to SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104; or

[0439] ii. Containing SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104; or

[0440] iii. Basically composed of SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104;

[0441] d. Heavy chain, wherein the polynucleotide is:

[0442] i. At least 90% identical to SEQ ID NO: 30 or 34 or 38 or 42 or 46; or

[0443] ii. Contains SEQ ID NO: 30 or 34 or 38 or 42 or 46; or

[0444] iii. It is basically composed of SEQ ID NO: 30 or 34 or 38 or 42 or 46.

[0445] In one embodiment, the present invention provides an isolated polynucleotide encoding the heavy chain of the antibody Fab' fragment of the present invention or the heavy chain of an IgG1 or IgG4 antibody, comprising the sequence given in SEQ ID NO: 10, 28, 32, 36, 40, or 44. An isolated polynucleotide is also provided encoding the light chain of the antibody Fab' fragment of the present invention or the light chain of an IgG1 or IgG4 antibody, comprising the sequence given in SEQ ID NO: 8 (or nucleotides 1 to 330 of SEQ ID NO: 8), 12 (or nucleotides 1 to 330 of SEQ ID NO: 12), 16, 20, 24, 64, or 66.

[0446] In another embodiment, the present invention provides an isolated polynucleotide encoding the heavy and light chains of the IgG4(P) antibody of the present invention, wherein the polynucleotide encoding the heavy chain is contained in the sequence given in SEQ ID NO: 30 or 34 or 38 or 42 or 46, and the polynucleotide encoding the light chain is contained in the sequence given in SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104.

[0447] The present invention also provides a cloning or expression vector comprising one or more polynucleotides described herein. In one example, the cloning or expression vector according to the present invention comprises one or more isolated polynucleotides comprising a sequence selected from SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104 or 30 or 34 or 38 or 42 or 46.

[0448] General methods for constructing vectors, transfection methods, and culture methods are well known to those skilled in the art. In this regard, see “Current Protocols in Molecular Biology”, 1999, FMAusubel (ed.), Wiley Interscience, New York, and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0449] A host cell is also provided, comprising one or more isolated polynucleotide sequences according to the invention, or one or more clones or expression vectors comprising one or more isolated polynucleotide sequences encoding antibodies of the invention. Any suitable host cell / vector system can be used to express the polynucleotide sequence encoding the antibody of the invention. Bacterial (e.g., *Escherichia coli*) and other microbial systems can be used, or eukaryotic (e.g., mammalian) host cell expression systems can also be used. Suitable mammalian host cells include CHO, myeloma, or hybridoma cells.

[0450] Suitable types of Chinese hamster ovaries (CHO cells) for use in this invention may include CHO and CHO-K1 cells, including dhfr-CHO cells, such as CHO-DG44 cells and CHO-DXB11 cells, which may be used with DHFR selective markers, or CHOK1-SV cells which may be used with glutamine synthetase selective markers. Other cell types for antibody expression include lymphocyte lines, such as NSO myeloma cells and SP2 cells, and COS cells. Host cells can be stably transformed or transfected using the isolated polynucleotide sequences or expression vectors according to this invention.

[0451] In one embodiment, the host cell according to the invention is a CHO-DG44 cell stably transfected with an expression vector comprising the isolated polynucleotide sequence of the invention, preferably comprising the isolated polynucleotide sequence of SEQ ID NO:8 (or nucleotides 1 to 330 of SEQ ID NO:8), 10, 12 (or nucleotides 1 to 330 of SEQ ID NO:12), 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 64, 65, 66, 67, 100, 101, 102, 103, or 104.

[0452] The present invention also provides a method for generating an antibody that binds to KLK5 according to the present invention, comprising culturing a host cell according to the present invention under conditions suitable for generating the antibody and isolating the resulting antibody.

[0453] Antibodies can contain only the heavy chain or the light chain, in which case only the polynucleotide sequence of the heavy chain or the light chain needs to be used to transfect the host cell. To generate antibodies containing both the heavy and light chains, the cell line can be transfected with two vectors, the first encoding the light chain and the second encoding the heavy chain. Alternatively, a single vector can be used, containing polynucleotide sequences encoding both the light and heavy chains.

[0454] Therefore, a method is provided for culturing host cells and expressing antibodies, isolating the latter, and optionally purifying them to provide isolated antibodies. Thus, in one embodiment, an isolated antibody binding to KLK5, preferably human KLK5, such as a humanized antibody, particularly the antibody according to the invention, is provided in a substantially purified form, particularly free of or substantially free of endotoxins and / or host cell proteins or DNA, wherein the antibody comprises:

[0455] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0456] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0457] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0458] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0459] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0460] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0461] In another embodiment, an isolated antibody, preferably human KLK5, is provided that binds to KLK5, such as a humanized antibody, particularly the antibody according to the invention, in a substantially purified form, particularly free of or substantially free of endotoxins and / or host cell proteins or DNA, wherein said antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51, and said antibody comprises:

[0462] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0463] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0464] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0465] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0466] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0467] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0468] Essentially free of endotoxins generally means that the endotoxin content is 1 EU or less per milligram of antibody product, such as 0.5 or 0.1 EU per milligram of product.

[0469] Essentially free of host cell protein or DNA usually means that the host cell protein and / or DNA content is 400 μg / mg for antibody products or less, such as 100 μg / mg or less, especially 20 μg / mg, depending on the case.

[0470] Since the antibodies of the present invention can be used to treat, diagnose, and / or prevent pathological conditions, the present invention also provides a pharmaceutical composition or diagnostic composition comprising an antibody according to the present invention in combination with one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0471] Preferably, the pharmaceutical composition or diagnostic composition comprises an antibody that binds to KLK5, preferably human KLK5, wherein the antibody comprises:

[0472] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0473] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0474] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0475] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0476] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0477] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0478] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0479] In one embodiment, the antibody according to the invention is the sole active ingredient. In another embodiment, the antibody according to the invention is combined with one or more additional active ingredients. Alternatively, the pharmaceutical composition comprises the antibody according to the invention as the sole active ingredient, and it can be administered to a patient alone in combination with other therapeutic agents, diagnostic agents, or palliative agents (e.g., simultaneously, sequentially, or separately).

[0480] In another embodiment, the pharmaceutical composition comprises an antibody that binds to KLK5, preferably human KLK5, wherein the antibody comprises:

[0481] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0482] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0483] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0484] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0485] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0486] 6. A light chain comprising SEQ ID NO: 13 or 17 or 21 or 25 and a heavy chain comprising SEQ ID NO: 29 or 33 or 37 or 41 or 45;

[0487] And one or more pharmaceutically acceptable carriers, excipients or diluents.

[0488] Preferably, the pharmaceutical composition comprises an antibody that binds to a KLK5 epitope, the antibody binding to the human KLK5 epitope comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51. More preferably, the pharmaceutical composition comprises an antibody that binds to KLK5, wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referenced in SEQ ID NO:51, and the antibody comprises the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:39.

[0489] The pharmaceutical compositions according to the invention can be suitably administered to patients to identify the desired therapeutically effective amount. As used herein, the term "therapeuticly effective amount" refers to the amount of therapeutic agent required to treat, improve, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect. For any antibody, the therapeutically effective amount can initially be estimated in cell culture assays or animal models, typically rodents, rabbits, dogs, pigs, or primates. Animal models can also be used to determine suitable concentration ranges and routes of administration. Such information can then be used to determine the useful dose and route of administration for human use.

[0490] The precise therapeutically effective dose for human subjects will depend on the severity of the disease state, the subject's general health condition, age, weight and sex, diet, administration time and frequency, drug combination, sensitivity to response, and tolerance / response to treatment. Generally, the therapeutically effective dose will be from 0.01 mg / kg to 500 mg / kg, for example from 0.1 mg / kg to 200 mg / kg, for example 100 mg / kg. The pharmaceutical composition can conveniently be present in unit doses, each containing a predetermined amount of the active agent of the present invention.

[0491] Pharmaceutically acceptable carriers in therapeutic compositions may additionally comprise liquids such as water, saline, glycerin, and ethanol. Furthermore, excipients, such as wetting agents, emulsifiers, or pH buffers, may be present in such compositions. Such carriers enable the formulation of pharmaceutical compositions into tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, slurries, and suspensions for patient ingestion.

[0492] Suitable forms of administration include those suitable for parenteral administration, such as by injection or infusion, for example by bolus or continuous infusion, in intravenous, aspirable, or subcutaneous form. When the product is intended for injection or infusion, it can be in the form of a suspension, solution, or emulsion in an oily or aqueous solvent, and it can contain formulation agents such as suspending agents, preservatives, stabilizers, and / or dispersants. Alternatively, the antibody according to the invention can be in a dry form for reconstitution with a suitable sterile liquid prior to use. A solid form suitable for dissolving or suspending in a liquid solvent prior to injection can also be prepared.

[0493] Once formulated, the compositions of the present invention can be directly administered to subjects. Therefore, this document provides for the use of antibodies according to the present invention in the manufacture of pharmaceuticals.

[0494] Preferably, the pharmaceutical composition according to the invention is suitable for administration to human subjects.

[0495] Therefore, in another aspect, the present invention provides an antibody that binds to KLK5, wherein the antibody or a pharmaceutical composition comprising the antibody is used for treatment, wherein the antibody comprises:

[0496] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0497] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0498] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0499] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0500] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0501] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0502] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0503] In a preferred embodiment, an antibody conjugated to KLK5 or a pharmaceutical composition containing said antibody is used for treatment, wherein said antibody comprises:

[0504] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0505] 2. A variable light chain comprising SEQ ID NO:15; and a variable heavy chain comprising SEQ ID NO:39; or

[0506] 3. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:41.

[0507] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0508] In another preferred embodiment, an antibody binding to KLK5, wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51, or a pharmaceutical composition comprising said antibody for treatment, wherein said antibody comprises:

[0509] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0510] 2. A variable light chain comprising SEQ ID NO:15; and a variable heavy chain comprising SEQ ID NO:39; or

[0511] 3. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:41

[0512] Specifically, it is used to treat one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation.

[0513] In another aspect, the present invention provides a method for treating one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation in a patient, the method comprising administering to the patient a therapeutically effective amount of an antibody binding to KLK5 or a pharmaceutical composition comprising the antibody, wherein the antibody comprises:

[0514] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0515] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0516] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0517] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0518] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0519] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0520] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0521] In another preferred embodiment, the present invention provides a method for treating one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation in a patient, the method comprising administering to the patient a therapeutically effective amount of an antibody or a pharmaceutical composition comprising the antibody, wherein the antibody comprises:

[0522] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0523] 2. A variable light chain comprising SEQ ID NO:15; and a variable heavy chain comprising SEQ ID NO:39; or

[0524] 3. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:41.

[0525] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0526] In another aspect, an antibody conjugating KLK5 or a pharmaceutical composition containing such an antibody, wherein the antibody is used to treat one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation, wherein the antibody comprises:

[0527] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0528] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0529] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0530] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0531] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0532] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0533] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0534] In another aspect, an antibody conjugating KLK5 or a pharmaceutical composition containing such an antibody, wherein the antibody is used to treat one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation, wherein the antibody comprises:

[0535] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0536] 2. A variable light chain comprising SEQ ID NO:15; and a variable heavy chain comprising SEQ ID NO:39; or

[0537] 3. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:41.

[0538] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0539] Preferably, one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation are selected from Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or combinations thereof.

[0540] Therefore, the present invention provides a method for treating Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or combinations thereof, in a patient, comprising administering to the patient a therapeutically effective amount of an antibody binding to KLK5 or a pharmaceutical composition comprising the antibody, wherein the antibody comprises:

[0541] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0542] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0543] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0544] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0545] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0546] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0547] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0548] More preferably, the method is used to treat Natherton syndrome and / or atopic dermatitis.

[0549] In another aspect, an antibody binding to KLK5 or a pharmaceutical composition comprising such an antibody is provided, wherein the antibody is used to treat Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or a combination thereof, wherein the antibody comprises:

[0550] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0551] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0552] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0553] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0554] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0555] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0556] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0557] More preferably, the antibody is used to treat Natherton syndrome and / or atopic dermatitis.

[0558] In another preferred embodiment, the present invention provides a method for treating Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or combinations thereof, in a patient, comprising administering to the patient a therapeutically effective amount of an antibody binding to KLK5 or a pharmaceutical composition comprising the antibody, wherein the antibody comprises:

[0559] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0560] 2. A variable light chain comprising SEQ ID NO:15; and a variable heavy chain comprising SEQ ID NO:39; or

[0561] 3. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:41.

[0562] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0563] More preferably, the method is used to treat Natherton syndrome and / or atopic dermatitis.

[0564] In another preferred embodiment, an antibody conjugated to KLK5 or a pharmaceutical composition containing the antibody is used to treat Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or a combination thereof, wherein the antibody comprises:

[0565] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0566] 2. A variable light chain comprising SEQ ID NO:15; and a variable heavy chain comprising SEQ ID NO:39; or

[0567] 3. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:41.

[0568] Preferably, the antibody binds to the epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0569] More preferably, the antibody is used to treat Natherton syndrome and / or atopic dermatitis.

[0570] The present invention also provides an antibody binding to KLK5 or a pharmaceutical composition comprising the antibody, wherein the antibody is used to treat Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or a combination thereof, wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51.

[0571] The present invention also provides a method for treating Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or combinations thereof, in a patient, comprising administering to the patient a therapeutically effective amount of an antibody binding to KLK5 or a pharmaceutical composition comprising the antibody, wherein the antibody binds to an epitope of human KLK5 and comprises the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51.

[0572] The present invention also provides the use of an antibody binding to KLK5 and a pharmaceutical composition comprising the antibody in the manufacture of a medicament for treating one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation, wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51, wherein such dysregulation is preferably Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or combinations thereof, more preferably Natherton syndrome and / or atopic dermatitis.

[0573] Specifically, the present invention also provides the use of an antibody binding to KLK5 or a pharmaceutical composition comprising such an antibody in the manufacture of a medicament for treating one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation, wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51, wherein such dysregulation is preferably Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or combinations thereof, more preferably Natherton syndrome and / or atopic dermatitis, wherein the antibody comprises:

[0574] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0575] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0576] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0577] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0578] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0579] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0580] The present invention also provides the use of KLK5-binding antibodies as diagnostic active agents or in diagnostic assays, such as for the diagnosis of Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or combinations thereof, wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as referred to in SEQ ID NO:51.

[0581] More preferably, the antibody comprises:

[0582] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0583] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0584] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0585] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0586] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0587] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0588] Diagnostic use of biological samples is preferred. "Biological samples" encompasses a wide range of sample types obtained from an individual and that can be used for diagnostic or monitoring assays. This definition includes blood, such as plasma and serum, as well as other biologically derived liquid samples, such as urine and saliva, cerebrospinal fluid, and solid tissue samples, such as biopsy specimens, such as skin biopsies or tissue cultures or cells derived therefrom and their progeny. The definition also includes samples that have been processed in any way after excision, such as by reagent treatment, dissolution or enrichment of certain components (such as polynucleotides).

[0589] Diagnostic tests that do not involve contact with human or animal bodies are preferred for biological samples. Such diagnostic tests are also known as in vitro tests. In vitro diagnostic tests may rely on in vitro methods for detecting KLK5 in biological samples obtained from an individual, including the following steps: i) contacting the biological sample with an antibody as described herein; ii) detecting the binding of the antibody to KLK5. By comparing the detected KLK5 level or the presence of a specific post-translational modification of KLK5 (including any precursors) with a suitable control, one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation can be identified. Therefore, such detection methods can be used to determine whether a subject (including an embryo or fetus) has or is at risk of developing a disease characterized by KLK5 dysregulation or KLK5 inhibition dysregulation.

[0590] Therefore, the present invention provides an antibody that binds to KLK5, wherein the antibody preferably binds to an epitope of human KLK5 and comprises the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51. This antibody is used to diagnose one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation, preferably for diagnosing Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or combinations thereof. The antibody comprises:

[0591] 1. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:1, 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 NO:6; or

[0592] 2. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:62, 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 NO:6; or

[0593] 3. A variable light chain and a variable heavy chain, wherein the variable light chain contains CDR-L1 comprising SEQ ID NO:63, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3; and the variable heavy chain contains CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6; or

[0594] 4. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and a variable heavy chain comprising SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43; or

[0595] 5. A variable light chain comprising SEQ ID NO:15 (wherein the reference amino acid residue glutamine 24 (Gln; Q) of SEQ ID NO:15 is arginine (Arg; R) or lysine (Lys; K)) and a variable heavy chain comprising SEQ ID NO:9 or 27 or 31 or 35 or 39 or 43; or

[0596] 6. Light chains containing SEQ ID NO: 13 or 17 or 21 or 25 and heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

[0597] Therefore, the present invention relates to the following embodiments:

[0598] Implementation Scheme 1: An antibody that binds to kallikrein 5 (KLK5), wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0599] a. The variable light chain comprises CDR-L1 containing SEQ ID NO:1 or SEQ ID NO:62 or SEQ ID NO:63, CDR-L2 containing SEQ ID NO:2, and CDR-L3 containing SEQ ID NO:3; and

[0600] b. The variable heavy chain contains CDR-H1 containing SEQ ID NO:4, CDR-H2 containing SEQ ID NO:5, and CDR-H3 containing SEQ ID NO:6.

[0601] Implementation Scheme 2: The antibody according to Implementation Scheme 1, wherein:

[0602] a. The variable light chain comprises CDR-L1 containing SEQ ID NO:1, CDR-L2 containing SEQ ID NO:2, and CDR-L3 containing SEQ ID NO:3; and

[0603] 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.

[0604] Implementation Scheme 3: An antibody that binds to kallikrein 5 (KLK5), wherein the antibody binds to an epitope of human KLK5 comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:51.

[0605] Implementation Scheme 4: The antibody according to Implementation Scheme 3, wherein the epitope is characterized by X-ray crystallography.

[0606] Implementation Scheme 5: An antibody according to any one of Implementation Schemes 1 to 4, wherein the antibody inhibits or reduces the protease activity of KLK5.

[0607] Implementation Scheme 6: An antibody according to any one of Implementation Schemes 1 to 5, wherein the antibody binds to KLK5 when KLK5 binds to LEKTI or a LEKTI fragment.

[0608] Implementation Scheme 7: An antibody according to any one of Implementation Schemes 1 to 6, wherein the antibody does not compete with LEKTI or the LEKTI fragment for binding to KLK5.

[0609] Implementation Scheme 8: An antibody according to any one of Implementation Schemes 1 to 7, wherein the antibody forms a complex with KLK5 bound to LEKTI or a LEKTI fragment.

[0610] Implementation Scheme 9: An antibody according to any one of Implementation Schemes 6 to 8, wherein the LEKTI fragment is a human LEKTI domain 5 comprising amino acids 1 to 64 of SEQ ID NO: 54 or a LEKTI domain 8 comprising amino acids 1 to 71 of SEQ ID NO: 61.

[0611] Implementation Scheme 10: An antibody according to any one of the preceding implementation schemes, wherein the antibody binds to human KLK5 and cynomolgus monkey (cyno) KLK5, wherein the human KLK5 is preferably human KLK5 containing SEQ ID NO:53, and the cynomolgus monkey KLK5 is preferably cynomolgus monkey KLK5 containing SEQ ID NO:60.

[0612] Implementation Scheme 11: An antibody according to any one of the preceding implementation schemes, 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).

[0613] Implementation Scheme 12: An antibody according to any one of Implementation Schemes 3 to 11, wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein:

[0614] a. The variable light chain comprises a CDR-L1 containing SEQ ID NO:1, SEQ ID NO:62, or SEQ ID NO:63, preferably a CDR-L1 containing SEQ ID NO:1, a CDR-L2 containing SEQ ID NO:2, and a CDR-L3 containing SEQ ID NO:3; and

[0615] b. The variable heavy chain contains CDR-H1 containing SEQ ID NO:4, CDR-H2 containing SEQ ID NO:5, and CDR-H3 containing SEQ ID NO:6.

[0616] Implementation Scheme 13: An antibody according to any one of the preceding implementation schemes, wherein the antibody is a chimeric antibody or a humanized antibody.

[0617] Implementation Scheme 14: The antibody according to any one of the preceding implementation schemes, wherein the antibody is a full-length antibody.

[0618] Implementation Scheme 15: The antibody according to Implementation Scheme 13, wherein the full-length antibody is selected from IgG1, IgG4 or IgG4P.

[0619] Implementation Scheme 16: An antibody according to any one of Implementation Schemes 1 to 13, wherein the antibody is selected from Fab, Fab', F(ab')2, scFv, dAb, or V. HH .

[0620] Implementation Scheme 17: An antibody according to any one of Implementation Schemes 1 to 16, wherein the antibody comprises:

[0621] a. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and / or

[0622] b. A variable heavy chain containing SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43.

[0623] Implementation Scheme 18: An antibody according to any one of Implementation Schemes 1 to 15 or 17, wherein the antibody comprises:

[0624] a. A light chain containing SEQ ID NO: 13, 17, 21, or 25; and

[0625] b. Heavy chains containing SEQ ID NO:29 or 33 or 37 or 41 or 45.

[0626] Implementation Scheme 19: The antibody according to Implementation Scheme 17 or 18, wherein the amino acid residue glutamine (Gln; Q) at position 24 of L-CDR1, referring to SEQ ID NO: 15 or 17, is replaced by arginine (Arg; R) or lysine (Lys; K).

[0627] Implementation Scheme 20: An antibody according to any one of the preceding implementation schemes, wherein KLK5 is human KLK5 containing SEQ ID NO: 51 or 52 or 53 or cynomolgus monkey KLK5 containing SEQ ID NO: 60.

[0628] Implementation Scheme 21: An antibody according to any one of the preceding embodiments, wherein the antibody forms a complex with KLK5, preferably human KLK5, bound to another antibody, wherein the other antibody comprises:

[0629] a. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:68, CDR-L2 containing SEQ ID NO:69, and CDR-L3 containing SEQ ID NO:70; and the variable heavy chain comprises CDR-H1 containing SEQ ID NO:71, CDR-H2 containing SEQ ID NO:72, and CDR-H3 containing SEQ ID NO:73; and / or

[0630] b. A variable light chain comprising SEQ ID NO:74 and a variable heavy chain comprising SEQ ID NO:76; and / or

[0631] c. A variable light chain encoded by a nucleotide containing SEQ ID NO:75 and a variable heavy chain encoded by a nucleotide containing SEQ ID NO:77.

[0632] Implementation Scheme 22: An antibody that binds to KLK5, preferably human KLK5, wherein the antibody comprises:

[0633] a. A variable light chain and a variable heavy chain, wherein the variable light chain comprises CDR-L1 containing SEQ ID NO:68, CDR-L2 containing SEQ ID NO:69, and CDR-L3 containing SEQ ID NO:70; and the variable heavy chain comprises CDR-H1 containing SEQ ID NO:71, CDR-H2 containing SEQ ID NO:72, and CDR-H3 containing SEQ ID NO:73; and / or

[0634] b. A variable light chain comprising SEQ ID NO:74 and a variable heavy chain comprising SEQ ID NO:76; and / or

[0635] c. A variable light chain encoded by a nucleotide containing SEQ ID NO:75 and a variable heavy chain encoded by a nucleotide containing SEQ ID NO:77.

[0636] Implementation Scheme 23: A KLK5-antibody complex comprising:

[0637] a.KLK5, Preferred Person KLK5

[0638] b. Antibodies according to any one of implementation schemes 1 to 18, and

[0639] c. Antibodies according to implementation plan 19 or implementation plan 20.

[0640] Implementation Scheme 24: An antibody, wherein:

[0641] a. Competing for binding to KLK5 with any of the antibodies described in any one of embodiments 1 to 20; and / or

[0642] b. Cross-blocking of the binding of the antibody to KLK5 by any one of embodiments 1 to 20 or cross-blocking of the binding of the antibody to KLK5 by any one of embodiments 1 to 20; and / or

[0643] c. Bind to KLK5 at the same epitope as the antibody described in any one of embodiments 1 to 20; and / or

[0644] d. Containing a heavy chain variable region having at least 90% identity or similarity to the sequence according to SEQ ID NO: 29, 33, 37, 41, or 45; and / or

[0645] e. Contains a light chain variable region that has at least 90% identity or similarity to the sequence according to SEQ ID NO:13 or 17 or 21 or 25.

[0646] Implementation Scheme 25: An isolated polynucleotide encoding an antibody according to any one of Implementation Schemes 1 to 20.

[0647] Implementation Scheme 26: The isolated polynucleotide according to Implementation Scheme 25, wherein the polynucleotide encodes:

[0648] a. Light chain variable region, wherein the polynucleotide:

[0649] i. At least 90% identical to SEQ ID NO:8 (or nucleotides 1 to 330 of SEQ ID NO:8) or 12 (or nucleotides 1 to 330 of SEQ ID NO:12) or 16 or 20 or 24 or 64 or 66; or

[0650] ii. Containing SEQ ID NO:8 (or nucleotides 1 to 330 of SEQ ID NO:8) or 12 (or nucleotides 1 to 330 of SEQ ID NO:12) or 16 or 20 or 24 or 64 or 66; or

[0651] iii. Consisting essentially of SEQ ID NO:8 (or nucleotides 1 to 330 of SEQ ID NO:8) or 12 (or nucleotides 1 to 330 of SEQ ID NO:12) or 16 or 20 or 24 or 64 or 66; or

[0652] b. Heavy chain variable region, wherein the polynucleotide:

[0653] i. At least 90% identical to SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44; or

[0654] ii. Contains SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44; or

[0655] iii. Basically composed of SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44; or

[0656] c. The light chain, wherein the polynucleotide is:

[0657] i. At least 90% identical to SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104; or

[0658] ii. Containing SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104; or

[0659] iii. Basically composed of SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104; or

[0660] d. Heavy chain, wherein the polynucleotide is:

[0661] i. At least 90% identical to SEQ ID NO: 30 or 34 or 38 or 42 or 46; or

[0662] ii. Contains SEQ ID NO: 30 or 34 or 38 or 42 or 46; or

[0663] iii. It is basically composed of SEQ ID NO: 30 or 34 or 38 or 42 or 46.

[0664] Implementation Scheme 27: A cloning or expression vector comprising one or more polynucleotides according to any one of Implementation Schemes 25 or 26.

[0665] Implementation Scheme 28: A host cell comprising:

[0666] a. One or more polynucleotides according to any one of embodiments 25 or 26, or

[0667] b. One or more expression carriers according to implementation scheme 27.

[0668] Implementation Scheme 29: A method for producing an antibody according to any one of Implementation Schemes 1 to 20, comprising culturing a host cell according to Implementation Scheme 28 under conditions suitable for producing the antibody and isolating the antibody produced by the host cell.

[0669] Implementation Scheme 30: A pharmaceutical composition comprising an antibody according to any one of Implementation Schemes 1 to 20 and one or more pharmaceutically acceptable carriers, excipients or diluents.

[0670] Implementation Scheme 31: An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 20, or a pharmaceutical composition according to Implementation Scheme 30, for use in treatment.

[0671] Implementation Scheme 32: An antibody according to any one of Implementation Schemes 1 to 20 or a pharmaceutical composition according to Implementation Scheme 30, for treating a disease characterized by KLK5 dysregulation or KLK5 inhibition dysregulation.

[0672] Implementation Scheme 33: The antibody used according to Implementation Scheme 32, wherein the disease is selected from Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or a combination thereof.

[0673] Implementation Scheme 34: The antibody used according to Implementation Scheme 33, wherein the disease is Netherton syndrome.

[0674] Implementation Scheme 35: The antibody used according to Implementation Scheme 33, wherein the disease is atopic dermatitis.

[0675] Implementation Scheme 36: A method of treating a disease characterized by KLK5 dysregulation or KLK5 inhibition dysregulation in a patient, comprising administering to the patient a therapeutically effective amount of an antibody according to any one of Implementation Schemes 1 to 20 or a pharmaceutical composition according to Implementation Scheme 30.

[0676] Implementation Scheme 37: The method according to Implementation Scheme 34, wherein the disease is selected from Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer such as ovarian cancer or bladder cancer.

[0677] Implementation Scheme 38: The antibody used according to Implementation Scheme 35, wherein the disease is Netherton syndrome.

[0678] Implementation Scheme 39: The antibody used according to Implementation Scheme 35, wherein the disease is atopic dermatitis.

[0679] Implementation Scheme 40: Use of the antibody according to any one of Implementation Schemes 1 to 20 or the pharmaceutical composition according to Implementation Scheme 30 in the manufacture of a medicament for treating one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation.

[0680] Implementation Scheme 41: An antibody manufactured according to Implementation Scheme 40, wherein the disease is selected from Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or a combination thereof.

[0681] Implementation Scheme 42: An antibody manufactured according to Implementation Scheme 40, wherein the disease is selected from Netherton syndrome or atopic dermatitis or a combination thereof.

[0682] Implementation Scheme 43: An antibody according to any one of Implementation Schemes 1 to 20, which is used as a diagnostic agent or for use in diagnostic assays or diagnostic kits for the diagnosis of one or more diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation.

[0683] Implementation Scheme 44: The antibody according to Implementation Scheme 43, wherein the disease is selected from Natherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer, such as ovarian cancer or bladder cancer, or combinations thereof.

[0684] The sequences included in this invention are shown in Table 1:

[0685] Table 1

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

[0706] The invention will now be further described by way of embodiments with reference to the accompanying drawings. Example

[0707] Example 1: Cloning, Expression, and Purification of Kallikrein Protein and LEKTI Domain

[0708] Using the HindIII / EcoRI site, an optimized nucleotide sequence encoding the protein according to SEQ ID NO:51 was cloned into an internal mammalian expression vector to produce a vector encoding the untagged human KLK5 protein.

[0709] Similarly, mouse and cynomolgus monkey (cynomolgus monkey) KLK5 sequences were cloned to produce active forms of proteins containing SEQ ID NO:59 and 60, respectively.

[0710] Domain 5 (D5) and domain 8 (D8) of human LEKTI (UniprotQ9NQ38), containing residues 292-353 and 490-558 (as numbered in Uniprot), were cloned and expressed, respectively, to serve as reference proteins in in vitro assays.

[0711] Both the nucleotide sequences of human LEKTI domain 5 and domain 8 were optimized for expression in mammalian cells. They were separately cloned into internal mammalian expression vectors encoding rabbit Fc tags using the HindIII / XhoI sites, generating vectors encoding either the sequence of LEKTI domain 5 with a C-terminal rabbit Fc tag (SEQ ID NO: 54) or the sequence of LEKTI domain 8 with a C-terminal rabbit Fc tag (SEQ ID NO: 61). The encoded proteins will be designated LEKTI D5 rabbit Fc and LEKTI D8 rabbit Fc, respectively.

[0712] By using Expi293 TM Expression Systems (Life Technologies) TM Transient transfection was performed according to the manufacturer's protocol to express the rabbit Fc fusion protein containing KLK5, LEKTI domain 5, and LEKTI domain 8. During expression, KLK5 self-activates to produce active KLK5 (containing residues I67-S293 of SEQ ID NO:53 or SEQ ID NO:51) in the supernatant. Cells were harvested 5 days post-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 a salt gradient generated with a total of 10 column volumes using buffer A (50 mM Tris pH 7.0, 50 mM NaCl) and buffer B (50 mM Tris pH 7.0, 1 M NaCl). Fractions containing purified human (or mouse or cynomolgus monkey) active KLK5 were combined, concentrated, and further purified by size exclusion chromatography on an S200 26 / 60 column equilibrated with a pH 7.2 buffer consisting of 20 mM Tris, 150 mM NaCl, and 5% glycerol. SDS-PAGE analysis showed that the protein underwent glycosylation during expression. The expected molecular weight was obtained by mass spectrometry. Protein A affinity chromatography was first performed on the supernatant containing the human LEKTI D5 rabbit Fc fusion protein (according to SEQ ID NO:54). The supernatant was loaded into 5 mL of Hitrap. TMProtein A column. The bound protein was eluted with 1M citrate buffer (pH 2.0), and the fraction was neutralized with 2M Tris-HCl (pH 8.5). The fractions containing the LEKTI D5 rabbit Fc fusion protein were combined, concentrated, and further purified by size exclusion chromatography using an S200 26 / 60 column equilibrated with PBS. The fractions containing the purified human LEKTI D5 rabbit Fc fusion protein were then combined and concentrated. Similarly, the LEKTI D8 rabbit Fc fusion protein (according to SEQ ID NO: 61) was purified from the supernatant of transfected cell culture.

[0713] The LEKTI D5 Fab fusion molecule was expressed and purified by cation exchange chromatography (according to SEQ ID NO: 94 and 95). The nucleotide sequence of the LEKTI domain 5, flanked at the 5' and 3' ends and encoding the Gly4Ser linker, was integrated into frame 3 of the albumin-specific Fab heavy chain sequence (as described in WO2020011868, which is incorporated herein by reference); a tag encoding the 10x His sequence was also placed at the 3' end of the Fab H chain. The LEKTI D5 Fab fusion heavy chain sequence was optimized for expression in mammalian cells, cloned into an internal expression vector, and co-transfected with an appropriate light chain also optimized for mammalian expression in CHO SXE cells. Transfected cells were cultured in fusible flasks at 32°C for 13 days. The supernatant was harvested, concentrated, and buffer-exchanged to 20 mM Tris, 50 mM NaCl, pH 7.0, and then loaded onto an SPSepharose HP column. The bound protein was eluted using a salt gradient generated with a total of 10 column volumes using buffer A (50 mM Tris pH 7.0, 50 mM NaCl) and buffer B (50 mM Tris pH 7.0, 1 M NaCl). Fractions containing the LEKTI D5 Fab fusion protein were combined and further purified by size exclusion chromatography using an S200 column equilibrated with PBS pH 7.4. The relevant fractions were then combined.

[0714] Human and cynomolgus monkey nucleotide sequences encoding the full-length KLK7 protein were used to generate precursor KLK7 (SEQ ID NO: 55 and 57, respectively) for expression in a manner similar to that of human KLK5. Unlike KLK5, KLK7 does not self-activate during expression; therefore, the precursor peptide sequence was cleaved from the respective purified proteins using a thermophilic protease to generate the active forms of human and cynomolgus monkey KLK7 (SEQ ID NO: 56 and 58, respectively). The human and cynomolgus monkey precursor KLK7 proteins (SEQ ID NO: 55 and 57) were diluted to 1 mg / ml with activation buffer (50 mM Tris pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij 35). Nucleotide sequences from Sigma-Aldrich were used to generate precursor KLK7 protein (SEQ ID NO: 55 and 57). TM The thermophilic protease (25 mg) was resuspended in 25 ml of digestion buffer (50 mM Tris pH 8.0, 0.5 mM CaCl2), and added to the precursor KLK7 protein at a ratio of 1:10. The mixture was incubated at 37°C for 45 minutes, and then reacted with anion-exchange DEAE resin (GE Life Sciences). TM The mixture was used to bind and remove thermophilic proteases. The flow-through was collected as active (human or cynomolgus monkey) KLK7, and the buffer was exchanged for 50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM EDTA, and concentrated to approximately 3.2 mg / ml. Mouse precursor KLK7 was similarly generated and cleaved to produce the active enzyme.

[0715] Human KLK2, an active protein, is derived from R&D Systems. TM (Catalogue No. 4104-SE-010).

[0716] The precursor form of human KLK4 originated from R&D Systems. TM (Catalogue No. 1719-SE) and activated as follows. Human precursor KLK4 was diluted to 200 μg / mL in 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5. Bacterial thermophilic protease was obtained from R&D Systems. TM (Catalogue No. 3097-ZN), and diluted to 2 μg / mL in the same buffer. Equal volumes of the precursor human KLK4 and thermophilic protease were mixed and incubated at room temperature for 10 minutes to allow activation. The reaction was terminated with EDTA to a final concentration of 10 mM.

[0717] Example 2: Generation of Antibodies by Immunization with KLK5

[0718] Female New Zealand white rabbits (>2kg) received an equal volume of complete Freund's adjuvant (Sigma).TM Animals were subcutaneously immunized with a mixture of 100 μg of 0.4 mg / mL human active KLK5 and human active KLK7 (expressed according to Example 1). Booster injections were given every 21 days, including an equal volume of incomplete Freund's adjuvant (Sigma). TM 100 μg of the same immunogen was mixed in the solution. The treatment was terminated 14 days after the last booster immunization, at which time single-cell suspensions of spleen, bone marrow and peripheral blood mononuclear cells (PBMCs) were prepared and frozen in 10% dimethyl sulfoxide (DMSO) in fetal bovine serum (FCS) at -80°C.

[0719] B cell cultures were prepared using a method similar to that described in Tickle et al., 2015 J Biomol Screen: 20(4), 492-497. Briefly, lymph node cells, spleen cells, or peripheral blood mononuclear cells (PBMCs) from immunized animals were cultured at a density of 2000 cells per well in wells supplemented with 10% FCS (Sigma Aldrich). TM ), 2% HEPES solution (Sigma Aldrich) TM ), 2% L-glutamine solution (Gibco) TM ), 1% penicillin / streptomycin solution (Gibco) TM 0.2% Normocin (Invivogen) TM ), 0.1% β-mercaptoethanol (Gibco) TM RPMI 1640 medium (Gibco) TM The culture was performed in barcode-coded 96-well tissue culture plates using feeder cells expressing CD40L and IL-2, with or without B cell stimulation supernatant (BSS). BSS was generated by culturing PBMCs for 6 days in the presence of the mitotic agents phorbol-12-myristate-13-acetate (PMA) and phytohemagglutinin-L (PHA-L), followed by harvesting the supernatant. The plates were incubated at 37°C and 5% CO2 for six days. Cultures were established using B cells from all immunized animals, with a total selection of approximately 1 x 102 cells. 9 One B cell.

[0720] Six days later, Sol-R2 streptavidin beads (TTP Labtech) coated with biotinylated human KLK5 as the target antigen were used. TM The binding of the supernatant to human KLK5 (as produced in Example 1) was screened by multiple homogeneous fluorescence-based binding assays, and Sol-R4 streptavidin beads (TTP Labtech) coated with the relevant KLK7 were selected. TMThis was used for counter-screening. Compared to the supplier's protocol, using a 5-fold molar excess of protein, Lightning-Link Rapid Biotin Type B (Expedeon) was employed. TM This was used to biotinylate proteins to avoid complete modification of all lysine residues. A total of 10 μL of supernatant from a barcoded 96-well tissue culture plate was transferred using an Agilent Bravo liquid processor to a barcoded 384-well black-walled assay plate containing biotinylated KLK-coated Sol-R beads and FITC-conjugated goat anti-rabbit Fc fragment specificity (Jackson ImmunoResearch). TM One hour later, in the mirror ball instrument (TTP-Labtech) TM Read the culture plate from the plate.

[0721] After initial screening, Beckman Coulter Biomek NXP was used. TM The hit-and-select robot consolidated the KLK5-binding supernatant onto a 96-well barcode master plate, and B cells in the cell culture plate were frozen at -80°C. First, the consolidated supernatant was re-screened using fluorescence microanalysis (FMAT) to confirm binding to human KLK5. Briefly, 10 μL of supernatant was transferred to a barcode-embedded black Greiner plate. 50 μL / plate of 10 μm superavidin (Bangs Beads) was also added. TM KLK5 with human biotinylated coating, and Alexa-647 TM Goat anti-rabbit IgG Fc fragment specificity (Jackson ImmunoResearch) TM Mix. Then add the supernatant and mix with Applied Biosystems. TM Reading plate on the 8200 cell detection system.

[0722] Many antibodies that bind to KLK5 were selected, and their ability to specifically inhibit KLK5 and their specificity relative to other kallikrein for KLK5 were investigated.

[0723] Example 3: Identification of KLK5 Inhibitory Antibodies

[0724] To identify antibodies that specifically inhibit KLK5 activity from proteases and protease inhibitors present in compound B cell supernatant, a screening assay was developed. This assay used 10 μg / mL of F(ab')2 fragment goat anti-rabbit IgG Fc fragment specificity in carbonate buffer (Jackson ImmunoResearch).TM Nunc Maxisorp black 384 (Sigma Aldrich) TM ), and placed overnight at 4°C. (In Biotek) TM The plate was washed three times with PBS / 0.1% Tween-20 using a plate washer, and then blocked for 1 hour at room temperature with 20 μL / well of PBS / 1% BSA. 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 wells as a positive control for inhibition. 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... TM Wash three times with PBS / 0.1% Tween-20 using a plate washer. Add 10 μL of 250 pM human KLK5 in assay buffer A to each well and incubate the plate overnight at room temperature to allow for complete binding. The Boc-VPR-AMC substrate (Cambridge Research Biochemicals) in assay buffer A will be added. TM Add ) to the wells to make a final concentration of 600 μM, and after 4 hours use PHERAStar FSX (BMG Labtech) TM ) ELISA reader measures fluorescence (λ) ex 380 nmλ em 430 nm).

[0725] The following equations were used to analyze the data to determine the percentage of KLK5 activity inhibition:

[0726]

[0727] The test measures the fluorescence value of the antibody. The positive result is the average fluorescence value of the positive control in the inhibition well, while the negative result is the average fluorescence value of the negative control in the inhibition well.

[0728] Supernatant showing >40% inhibition was considered a hit. This represents approximately 4% of all screened supernatants. These antibodies were selected for variable region recovery.

[0729] To identify specific antibody-secreting cells to allow for the recovery of antibody variable region genes from a heterologous population of activated B cells, a deconvolution step must be performed. Fluorescence focusing is used (Clargo et al., 2014). In short, streptavidin beads coated with biotinylated human KLK5 (New England Biolabs) are used. TM) and goat anti-rabbit Fc fragment specific FITC conjugate (Jackson ImmunoResearch) TM Antibody-secreting cells were statically incubated at 37°C for 1 hour in the presence of [a specific substance / condition]. Antigen-specific antibody-secreting cells were then identified by the fluorescence halo surrounding them. [The cells were then analyzed using Eppendorf gels / gels]. TM Micromanipulation was used to select numerous cells from these individual B-cell clones identified using an Olympus microscope and placed them into PCR tubes. cDNA from single cells was obtained via standard RT-PCR, followed by PCR of variable immunoglobulin sequences for both heavy and light chains using immunoglobulin gene-specific primers. Nested PCR with overlapping vector sites was then performed, allowing direct cloning of the variable regions into rabbit IgG (VH) or rabbit κ (VL) mammalian expression vectors. ExpiFectamine was used. TM (LifeTechnologies TM The heavy and light chain constructs were co-transfected into ExpiHEK-293 cells, and the recombinant antibody was administered in 30 ml of 125 ml Erlenmeyer globulin. TM Expression in culture flasks. After 5-7 days of culture, harvest the supernatant and purify the antibody using an AKTA pure chromatography system via protein A affinity capture. Add 1 ml of Protein A HiTrap MabSelect... TM Sure TM The column (GE Healthcare) was attached to the system and equilibrated in PBS at pH 7.4. Cell culture supernatant was then added to the column at a flow rate of 0.25 ml / min. The column was then washed with PBS at pH 7.4, eluted with sodium citrate at pH 3.4, and neutralized with an appropriate volume of 2M Tris-HCl at pH 8.5. The eluted fraction buffer was exchanged for PBS at pH 7.4 (Sigma) and passed through a 0.22 μm filter. The fractions were analyzed by A280 scanning, SE-UPLC (BEH200 method), and other methods. PTS TM The system analyzes endotoxins to determine the final purified material.

[0730] From this analysis, rabbit antibodies 10236 and 10273 showed effective inhibitory effects and were selected for further characterization.

[0731] Example 4: Identification of KLK5-Specific Inhibitory Antibodies

[0732] Purified rabbit antibodies 10236 and 10273 were then screened to confirm their inhibitory activity against KLK5, and specificity for KLK5 was determined using a group of other kallikrein family members, including KLK2, KLK4, and KLK7, as well as mouse and cynomolgus monkey KLK5 and KLK7. Beckman Coulter FX was used. TM Using a multidrop system, prepare a 10.5 logarithmic dilution series of each antibody ranging from 600 nM to 20 pM, and transfer 5 μL to a black 384-well assay plate (Corning). TM (Catalogue No. 3575). Add 15 μL of active recombinant human kallikrein protein to the relevant wells to achieve the following final assay concentrations: 60 pM KLK5, 250 pM KLK7, 500 pM KLK2, 30 pM KLK4, 30 pM cynomolgus monkey KLK5, 500 pM cynomolgus monkey KLK7, 30 pM mouse KLK5, or 10 nM mouse KLK7 in assay buffer A (50 mM Tris, 150 mM NaCl, 200 μM EDTA, 0.05% (v / v) Tween-20, pH 7.6). As a control with 0% activity, add only 20 μL of assay buffer A (without KLK protein) to the wells. LEKTI D5 rabbit Fc was used as a positive control for inhibition (tested at the same concentration range as the antibody), while 15 μL of each active human kallikrein protein was added to 5 μL of assay buffer A to obtain a 100% activity reference. The plates were incubated overnight at room temperature, and then the following peptide substrates were added using a multidrop apparatus: 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). TM KHLF-AMC for humans and cynomolgus monkeys (Cambridge Research Biochemicals) TM (90μM and 150μM respectively), for human KLK4 PFR-AMC (R&D Systems) TM (200μM) and Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (R&D Systems) for mouse KLK7 TM (150 μM). The sample was incubated for 4 hours and then readjusted using a Pherastar FSX microplate reader (BMG Labtech). TM On λ ex 380 nm and λ emBoc-VPR-AMC, PFR-AMC, and KHLF-AMC were read at 430 nm; at λ ex 320 nm and λ em Mca-RPKPVE-Nval-WRK(Dnp)-NH2 was read at 400 nm. Data were analyzed as described in Example 3 to determine the percentage of inhibition. The data were plotted against the concentration of the test antibody and a fitted 4-parameter sigmoid curve to determine the IC50 (Genedata Screener). TM ).

[0733] 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 form of a mouse Cκ vector containing the S171C mutation to reconstruct additional disulfide bonds present in the rabbit VK light chain but not in the mouse constant region (SEQ ID NO: 80 and 81 for rabbit antibody 10273 mIgG, and SEQ ID NO: 84 and 85 (or nucleotides 1-1314 of SEQ ID NO: 85)). For rabbit antibody 10236 mIgG, this yields antibodies containing SEQ ID NO: 82 and 83, and for rabbit antibody 10273 mIgG, this yields antibodies containing SEQ ID NO: 78 and 79.

[0734] Rabbit antibodies 10236 and 10237 mIgG showed potent inhibition of human KLK5, but no activity against other family members tested (human KLK2, 4, and 7) (i.e., below the 40% threshold of the selection criteria in Example 2). Potential inhibition of cynomolgus monkey KLK5, but not cynomolgus monkey KLK7, was also demonstrated. No significant inhibitory activity was observed against mouse KLK5 or KLK7. IC50 values ​​of rabbit antibodies 10236, 10273, and LEKTI D5 rabbit Fc were also shown. 50 The results are shown in Table 2.

[0735] Table 2

[0736]

[0737]

[0738] NI = No inhibition; Hu = Human; Cy = Cynomolgus monkey; Mu = Mouse; n / a = Not available

[0739] Example 5: Determination of the Affinity of KLK5-Specific Ab

[0740] Through surface plasmon resonance (Biacore T200, GE Life Sciences) TMThe binding kinetics of mouse IgG molecules to human KLK5 were evaluated at 25°C.

[0741] Goat anti-mouse IgG Fc-specific antibody (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip via amine coupling chemistry to a level of approximately 7000 RU. Each analytical cycle consisted of the following: anti-KLK5 IgG molecules were captured onto the anti-Fc surface, KLK5 analyte (internally prepared) was injected at a rate of 30 μL / min for 300 seconds, followed by dissociation for 600 seconds. At the end of each cycle, the surface was regenerated by injecting 50 mM HCl for 60 seconds, followed by injecting 5 mM NaOH for 30 seconds and then injecting 50 mM HCl for a final 60 seconds, at a flow rate of 10 μL / min. Human KLK5 was titrated from 20 nM to 0.25 nM (4 x 3-fold serial dilutions) in HBS-EP+ run buffer (GE Healthcare) replenished to a final concentration of 300 mM NaCl. Buffered blank injections were included to reduce instrument noise and drift.

[0742] The dynamic parameters were determined using the Biacore T200 evaluation software with a 1:1 combined model.

[0743] The affinities of rabbit antibodies 10236 and 10273 are shown in Table 3.

[0744] Table 3

[0745] Rabbit / Mouse Antibody ka (Ms^-1) kd (s^-1) KD (pM) 10236 3.00E+06 5.17E-04 172.3 10273 1.14E+06 1.84E-04 160.0

[0746] Example 6: Characterization of Antibody 10236

[0747] Binding of LEKTI to KLK5 in the Presence of Antibody 10236

[0748] Surface plasmon resonance (SPR) assays were performed to determine whether antibody 10236 competitively binds to human KLK5 with the LEKTI D5 protein. These assays enabled a comparison of the affinity of the LEKTI D5 Fab fusion protein for KLK5 protein alone and for human KLK5 complexed with antibody 10236.

[0749] Using Biacore T200 (GE Life Sciences) TM Kinetic measurements of the binding of the LEKTI D5 Fab fusion protein to human KLK5 were obtained. To prepare the surface, it was first subjected to 5 minutes of EDC / NHS (GE Life Sciences) [process / treatment]. TM Mixture injection (30 μL min) -1 Activate CM5 chip (GE Life Sciences) TMThen it was injected into a pH 5.0 acetate buffer (GE Life Sciences). TM 100 μg mL -1 LEKTI D5 Fab fusion protein (UCB) was used to immobilize LEKTI D5 Fab fusion protein on a chip surface at 80 RU. Finally, the surface was inactivated by injection of 1M ethanolamine hydrochloride-NaOH at pH 8.5. Then, it was injected in HBS-EP buffer (GE Life Sciences) using a single-cycle kinetic mode. TM The concentrations of human KLK5 ranged from 0.32 to 32 nM. Values ​​obtained from KLK5 injections were subtracted from those obtained from buffer-only injections, and the results were evaluated using BIAcore software (GE Life Sciences). TM The 1:1 combination model in the model determines the dynamics.

[0750] To determine whether human LEKTI could bind to human KLK5 when human KLK5 bound to rabbit antibody 10236, an antibody-capturing surface was prepared using a goat anti-rabbit Fc polyclonal antibody and Ab 10236 as described in Example 4. 20 nM of human KLK5 was then injected until the surface was saturated. LEKTI D5 Fab fusion protein (generated as described in Example 1) was then injected at concentrations from 30 pM to 100 nM. The values ​​obtained using the analyte were first subtracted from the values ​​obtained using only buffer injection, and then fitted to Biacore. TM Evaluation software (GE Life Sciences) TM The 1:1 combined dynamic model in )

[0751] As a 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 when it was already conjugated with rabbit antibody 10236 with an affinity of 120 nM (Table 4A). Although human LEKTI showed a higher affinity for human KLK5 (40 pM) in the absence of rabbit antibody 10236, this analysis suggests that rabbit antibody 10236 can provide additional inhibitory activity against human KLK5 by binding it in the presence or absence of human LEKTI.

[0752] Table 4A

[0753] ka (Ms^-1) kd (s^-1) KD (M) Only Human KLK5 5.70E+05 2.30E-05 4.00E-11 10236 + Human KLK5 5.90E+04 7.30E-03 1.20E-07

[0754] Formation of the LEKTI-KLK5-Antibody 10236 Complex

[0755] KLK5 is produced in HEK293 cells as a secretory protein with an N-terminal, TEV-cleavable 8xHis tag. It is first generated via Ni... 2+ Affinity chromatography was used to purify proteins from conditioned medium. Proteins containing KLK5 from Ni... 2+ The column was fractionated and digested with TEV protease to remove the His tag, followed by a second Ni affinity step to remove the TEV protease, allowing the cleaved KLK5 to flow through the column. The residue from the second Ni... 2+ The column flow-through fraction was concentrated and run on a size-resistance column in 50 mM Tris pH 7, 50 mM NaCl, 1 mM EDTA, and 5% glycerol. The KLK5 fraction from the SEC was combined and concentrated to approximately 10 mg / mL and stored at -80°C.

[0756] LEKTI domain 5 (LEKTI D5 Fc) according to SEQ ID NO:98 and LEKTI domain 8 (LEKTI D8 Fc) according to SEQ ID NO:99 were produced in HEK293 cells as secretory proteins with a C-terminal, TEV-cleavable Fc tag. These proteins were purified by passing conditioned medium through Protein A beads. Bound proteins were eluted with 0.1 M citrate at pH 2.0, and fractions were neutralized by adding 2 M Tris-HCl at pH 8.5. Fractions containing LEKTI domain 5 or domain 8 from the Protein A column were combined, 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 at pH 7.2. Fractions containing LEKTI were combined and concentrated to approximately 10 mg / ml and stored at -80°C.

[0757] Rabbit Fab antibody 10236 was expressed as a secretory protein in HEK293 cells. Expression constructs containing SEQ ID NO:87 and 89 were co-transfected at a 1:1 molar ratio. Secreted Fab (containing SEQ ID NO:86 and 88) was purified by passing conditioned medium through protein G beads and eluting with 0.1 M glycine at pH 2.7. The fraction was neutralized by adding 2 M Tris-HCl at pH 8.5. The protein was dialyzed into PBS at pH 7.2, concentrated to approximately 10 mg / mL, and stored at -80°C.

[0758] The complex of KLK5, LEKTI D5 or LEKTI D8 and rabbit Fab antibody 10236 was formed as follows: 25 μM KLK5 was incubated with 25 μM LEKTI D5 or LEKTI D8 on ice for 60 minutes, then 25 μM rabbit Fab antibody 10236 was added and incubation continued on ice for another 60 minutes. The mixture was then injected into a Superdex HPLC system equilibrated with PBS at pH 7.2. The peak fractions are collected on a size-enhanced column for analysis by SDS-PAGE. Figure 1 A and 1B show individual human KLK5 (solid line, far right), individual rabbit Fab antibody 10236 (dashed line), and binary complexes of human KLK+LEKTI D5 or D8 (respectively). Figure 1 A or 1B (underlined) and the ternary complex of KLK5+LEKTI D5 or D8+rabbit Fab antibody 10236 (respectively) Figure 3 SEC chromatogram (A or 3B, dashed, far left).

[0759] The molecular weight (MW) of the component in each peak was confirmed by SDS-PAGE, such as... Figure 2 As shown.

[0760] In general, complexes between KLK5, LEKTI D5, or LEKTI D8 and rabbit Fab antibody 10236 readily form when mixed in a 1:1:1 ratio by mixing human KLK5 with each LEKTI fragment separately, followed by incubation of the binary complex with rabbit Fab antibody 10236. Binary and ternary complexes were observed on SEC and analyzed by SDS-PAGE of peak fractions, indicating their stability and suitability for separation / purification from other substances.

[0761] Example 7: Crystallization of the KLK5 / Fab Antibody 10236 Complex According to the manufacturer's specifications, use Expi293. TM Expression Systems (Life Technologies) TM Human KLK5 was expressed via transient transfection, followed by the addition of kifunensine at a final concentration of 5 mM. kifunensine is a potent inhibitor of mannosidase I, primarily used in cell culture to produce high-mannoglycoproteins.

[0762] During KLK5 expression, the protein is self-activated to produce active KLK5 protein (residues I67-S293 of SEQ ID NO: 53 (UniProt Q9Y337)) in the supernatant. Cells were harvested 5 days after transfection, and the supernatant was used immediately for purification. The supernatant containing active 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 using a salt gradient generated by a total of 10 column volumes using buffer A (50 mM Tris pH 7.0, 50 mM NaCl) and buffer B (50 mM Tris pH 7.0, 1 M NaCl). Fractions containing purified human active KLK5 were combined, concentrated, and further purified by size exclusion chromatography on an S200 26 / 60 column equilibrated with 20 mM Tris and 150 mM NaCl at pH 7.2.

[0763] KLK5 was characterized by SDS-PAGE and migrated to a position on the gel consistent with the expected molecular weight (MW) of approximately 35-38 kDa for highly mannose-glycosylated proteins. Figure 3 ).

[0764] Human KLK5 protein was then treated with endoglycosidase H (Endo H) protein at a ratio of 1:100 and incubated overnight at 4°C to form a homogeneous deglycosylated KLK5 protein for structural studies. Endo H is a recombinant glycosidase cloned from *Streptomyces plicatus* and overexpressed in *Escherichia coli*. Endo H cleaves a high-mannose chitobiose core and a limited number of heterozygous oligosaccharides from N-linked glycoproteins. It does not cleave complex glycans. Enzymatic cleavage occurs between two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, leaving one N-acetylglucosamine residue on asparagine. This step was performed to ensure that homologous human KLK5 was available for crystallographic studies. KLK5 was analyzed by SDS-PAGE (…). Figure 3 The protein was characterized and migrated to a position on the gel that corresponds to the expected molecular weight (MW) of approximately 25 kDa for the deglycosylated protein.

[0765] Rabbit Fab antibody 10236 was expressed as described in Example 6. Rabbit Fab antibody 10236 (containing SEQ ID NO: 86 and 88) was expressed as described in Example 6. Briefly, expression constructs containing SEQ ID NO: 87 and 89 were co-transfected at a 1:1 molar ratio. Rabbit Fab antibody 10236 was purified by passing conditioned medium through protein G beads and eluting with 0.1 M glycine at pH 2.7. The fraction was neutralized by adding 2 M Tris-HCl at pH 8.5. The individual proteins were dialyzed into PBS at pH 7.2, concentrated to approximately 10 mg / ml, and stored frozen at -80°C.

[0766] A human KLK5 / rabbit Fab antibody 10236 mixture with a molar ratio of 1.5:1 was prepared, incubated overnight at 4°C, and purified by size exclusion chromatography (20 mM Tris, 150 mM NaCl, elution buffer at pH 7.2). The resulting complex was isolated and concentrated to approximately 10.0 mg / mL, and then crystallized.

[0767] The crystallization conditions for the human KLK5 / rabbit Fab antibody 10236 complex were identified using several commercially available crystallization screens. These were performed dropwise using a Swissci 96-well 2-drop MRC crystallization plate (from Molecular Dimensions, catalog MD11-00-100). First, the reservoir for each crystallization solution was filled into the screen using a Microlab STAR liquid handling system (Hamilton). Then, 300 nL of the human KLK5 / rabbit Fab antibody 10236 complex and 300 nL of the reservoir solution were dispensed into the wells of the crystallization plate using a Mosquito liquid processor (TTP LabTech). Single crystals were obtained under condition 88 (well H4) of the ProComplex Suite (Qiagen). This condition contained 1.4 M sodium malonate. The crystals were temporarily transferred to droplets containing 1.4 M sodium malonate and 25% glycerol. The crystal was 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 (Evans PR, Murshudov GN. How good are my data and what is the resolution? Acta Crystallogr D BiolCrystallogr. 2013; 69(Pt 7): 1204–1214). The structure of the human KLK5 / rabbit Fab antibody 10236 complex was resolved by molecular substitution using Phaser (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Storoni, LC, & Read, RJP) in the Phenix software suite (Adams PD, Afonine PV, Bunkóczi G, et al. The Phenix software for automated determination of macromolecular structures. Methods. 2011; 55(1): 94–106). J. Appl. Cryst. (2007). 40, 658-674.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, RWGrosse-Kunstleve, N. Echols, JJ Headd, NW Moriarty, M. Mustyakimov, TCTerwilliger, A. Urzhumtsev, PHZwart, and PDAdams. Acta Crystallogr D Biol Crystallogr 68, 352-67 (2012)) were used in the following manual model completion and refinement cycles. Table 4B shows the refinement statistics when the invention was first described.

[0768] Table 4B

[0769]

[0770] A single human KLK5 / rabbit Fab antibody 10236 complex was observed in a crystalline asymmetric unit. Figure 4 B shows that the antibody-binding site of KLK5 differs from its substrate-binding site. NCONT in the CCP4 software suite is used to define the epitopes on KLK5 recognized by the Fab10236 molecule. KLK5 amino acid numbering is based on UnitProtKB entry Q9Y337, and the standard protease numbering is based on chymotrypsinogen (in parentheses).

[0771] Rabbit Fab antibody 10236 in The human KLK5 epitope binding at the contact distance consists of residues Arg87(36), Ala107(56), Arg110(59), Lys111(60), Lys112(61), Val113(62), Val137(86), Lys138(87), Ser139(88), Ile140(89), Pro141(90), His142(91), Pro143(92), Tyr145(94), Ser146(95), and His147(96) from reference SEQ ID NO:51, where the numbers in parentheses correspond to protease nomenclature. Figure 4The binding sites are shown in more detail in section A.

[0772] To visualize the binding site of Fab10236 relative to the KLK5 active site, a structural overlay diagram of the KLK5-Fab10236 structure presented in this paper and the published KLK5 structure was created, in which the leucopeptide peptide binds to the active site (2PSX). Figure 4 B). The overlay image shows the core location of the active site where Fab10236 does not bind KLK5, as indicated by the position of the leucopeptide peptide. Figure 4 B), but the light chain of Fab10236 contacts the 99 ring, which forms a partial active site slit on KLK5. The KLK5-Fab10236 structure presented herein is compared with previously published KLK5 structures (e.g., those disclosed as PDB ID 2PSX and 2PSY). The 2PSX crystal structure shows that KLK5 binds to the peptidase inhibitor leucopeptide at its active site, while the 2PSY crystal structure shows that KLK5 binds to the leucopeptide and a zinc ion adjacent to the active site. Zinc is known to inhibit KLK5 in a non-competitive manner. Comparison of the 2PSX and 2PSY structures shows that zinc affects protease activity by inducing a conformational shift in the 99 ring main chain, accompanied by significant positional changes in the His147 (96) and His150 (99) side chains. The shift of the main chain 99 ring is shown in the structural overlay diagram ( Figure 4 C, white (zinc-free) and black (zinc-added) and the movement of histidine side chains, especially His147 (99), from the outward position in the absence of zinc to the inward position when zinc is bound. Figure 4 C, the dashed arrow indicates the movement of the histidine side chain.

[0773] The structural overlay diagram of KLK5-Fab10236 and the zinc-free KLK leucosterol peptide structure (2PSX) highlights the main chain movement of ring 99 and the side chain movements of His147 (96) and His150 (99). Figure 4 D, showing the 99 rings and histidine side chains in detail—black represents the KLK5-Fab10236 structure, and white represents the zinc-free structure of the KLK5-leucinogen protease peptide.

[0774] After Fab10236 is bonded to KLK5, His147(96) located in the 99 ring cannot adopt the conformation previously observed in the 2PSX crystal structure because this would result in a reaction with the Fab light chain (dashed square). Figure 4 D) Spatial conflict. To accommodate the bonding of Fab10236, ring 99 adopts a different conformation, with His147(96) swinging from an outward position to an inward position (similar to what is observed with zinc bonding); Figure 4D, the dashed arrow indicates that His147(96) has moved. Simultaneously, the side chain of His150(99) changes position, shifting it to the S2 pocket, where it may prevent substrate binding. (As shown...) Figure 4 As shown in D, the S2 pocket is occupied by the modeled leucopeptide peptide, and the conflict between the His150(99) side chain and the leucopeptide peptide is highlighted by a white dashed circle.

[0775] Example 8: Crystallization of the KLK5 / Fab Antibody 10236 / Fab Antibody 10273 Complex

[0776] As in Example 7, human KLK5 was expressed, except that once the supernatant was loaded onto a HiTrap SP HP cation exchange column, the bound protein was eluted with a gradient of 10 column volumes using buffer B (50 mM Tris, pH 7.0, 1 M NaCl). Further purification, endoglucosidase treatment, and analytical characterization were performed as in Example 7.

[0777] Rabbit Fab antibody 10236 was expressed as in Example 6. A cloned rabbit Fab antibody 10273 (containing SEQ ID NO: 90 and 92) was also described for rabbit Fab antibody 10236. Briefly, expression constructs containing SEQ ID NO: 91 and 93 were co-transformed at a 1:1 molar ratio. The secreted protein was purified by passing conditioned medium through protein G beads and eluting with 0.1 M glycine at pH 2.7. The fraction was neutralized by adding 2 M Tris-HCl at pH 8.5. The protein was dialyzed into PBS at pH 7.2, then concentrated to approximately 10 mg / ml and stored frozen at -80°C.

[0778] A 1:1.5:1.5 human KLK5 / rabbit Fab antibody 10273 / rabbit Fab antibody 10236 complex was prepared, incubated overnight at 4°C, and purified by size exclusion chromatography (20 mM Tris, 150 mM NaCl, elution buffer at pH 7.2). The single peak containing the complex was concentrated to approximately 10.8 mg / mL and then crystallized.

[0779] The crystallization conditions for the human KLK5 / rabbit Fab antibody 10273 / rabbit Fab antibody 10236 complex were identified using several commercially available crystallization screens. These were performed dropwise using a Swissci 96-well 2-drop MRC crystallization plate (from Molecular Dimensions, catalog MD11-00-100). First, a reservoir of 75 μL of each crystallization solution was filled into the screen using a Microlab STAR liquid handling system (Hamilton). Then, 300 nL of the human KLK5 / rabbit Fab antibody 10273 / rabbit Fab antibody 10236 complex and 300 nL of the reservoir solution were dispensed into the wells of the crystallization plate using a Mosquito liquid processor (TTP LabTech). Single crystals were obtained under condition 16 (well B4) on a MIDAS+HT-96 screen (Molecular Dimensions, catalog MD1-107). The conditions consisted of 45% v / v pentaerythritol propoxylate (5 / 4), 0.2 M NaCl, and 0.1 M MES monohydrate at pH 6.0. Crystals were rapidly frozen in liquid nitrogen, and diffraction data were collected at beamline I03 (Diamond LightSource, UK). The data were indexed and integrated using XDS (Kabsch, W. XDS. Acta Cryst. D66, 125-132 (2010)), and then scaled using (2. Evans PR, Murshudov GN. How good are my data and what is the resolution? Acta Crystallogr D Biol Crystallogr. 2013; 69(Pt 7): 1204–1214). The human KLK5 / rabbit Fab antibody 10273 / rabbit Fab antibody 10236 complex structure was resolved by molecular substitution using Phaser (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Storoni, LC, & Read, RJP) in the Phenix software suite (Adams PD, Afonine PV, Bunkóczi G, et al. The Phenix software for automated determination of macromolecular structures. Methods. 2011; 55(1): 94–106). J. Appl. Cryst. (2007). 40, 658-674.In this process, 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 the proprietary Fab model were used as molecular substitution templates. 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, RWGrosse-Kunstleve, N. Echols, JJ Headd, NW Moriarty, M. Mustyakimov, TCTerwilliger, A. Urzhumtsev, PHZwart, and P.D. Adams. Acta Crystallogr D Biol Crystallogr 68, 352–67 (2012)) were used in the following manual model completion and revision cycles until acceptable Rwork, Rfree, and Ramachandran statistics were obtained (as by Molprobity (Williams et al. (2018) MolProbity: More and better reference data for improved)). (Analysis of all-atom structure validation. Protein Science 27:293-315).

[0780] The human KLK5 / rabbit Fab antibody 10273 / rabbit Fab antibody 10236 complex was observed in asymmetric crystal units. NCONT from the CCP4 software suite was used to identify epitopes on KLK5 recognized by Fab10273 and Fab10236 molecules. KLK5 amino acid numbers are based on UnitProtKB entry Q9Y337, and standard protease numbers are based on chymotrypsinogen (in parentheses). Table 4C shows the revised statistics at the time of the initial description of this invention.

[0781] Table 4C

[0782]

[0783] exist At the contact distance, the human KLK5 epitope bound by rabbit Fab antibody 10236 consists of residues from reference SEQ ID NO:51: Arg87(36), Ala107(56), Arg110(59), Lys111(60), Lys112(61), Val113(62), Val137(86), Lys138(87), Ser139(88), Ile140(89), Pro141(90), His142(91), Pro143(92), Tyr145(94), Ser146(95), and His147(96), while the numbers in parentheses correspond to protease nomenclature. Figure 4 The binding sites are shown in more detail in D.

[0784] like Figure 5 As shown, antibodies 10236 and 10273 have very different, non-overlapping binding sites and bind to different epitopes on human KLK5.

[0785] Example 8: Humanization and Characterization of Antibody 10236

[0786] Humanization of Ab 10236

[0787] Rabbit antibody 10236 was humanized by transplanting a CDR from the rabbit V region onto the human germline antibody V region framework. To restore antibody activity, many framework residues from the rabbit V region were also retained in the humanized sequence. These residues were selected using the protocol outlined by Adair et al. (1991) (Humanized antibodies.WO91 / 09967). Figure 6 and 7 The alignment of the rabbit antibody (donor) V region sequence with the human (recipient) V region sequence and the designed humanized sequence is shown. The CDR from donor to recipient sequence is defined by Kabat (Kabat et al., 1987), except for CDR-H1, which uses the Chothia / Kabat combination definition (see Adair et al., 1991 Humanized antibodies.WO91 / 09967).

[0788] For antibody 10236, the human V region IGKV1-6 plus JK4 J region (IMGT, http: / / www.imgt.org / ) was selected as the receptor for the light chain CDR. All framework residues in the humanized graft of the 10236 light chain are derived from human germline genes, except for one or more residues from groups containing residues 2, 3, and 63, where the donor residues tyrosine (Y2), aspartic acid (D3), and lysine (K63) relative to SEQ ID NO:15 were retained respectively. Figure 6 The retention of donor residues Y2 and D3 is crucial for binding with the highest affinity of human KLK-5.

[0789] The human V region IGHV4-4 plus JH4 J region (IMGT, http: / / www.imgt.org / ) was selected as the receptor for the heavy chain CDR of antibody 10236. Like many rabbit antibodies, the VH gene of antibody 10236 is shorter than the selected human receptor. When aligned with the human receptor sequence, the VH region frame 1 of antibody 10236 lacks an N-terminal residue, which is retained in the humanized antibody. Figure 7 The framework 3 of the 10236 rabbit VH region also lacks two residues (75 and 76) in the loop between D and E of the β-sheet: in humanized grafts, the vacancies are replaced by corresponding residues from the selected human receptor sequence (lysine 75, K75; asparagine 76, N76). Figure 7 Alternatively, lysine and threonine (lysine 75, K75; threonine 76, T76) can be used for filling. The framework residues in the humanized graft of the 10236 heavy chain are all derived from human germline gene sequences, except for one or more residues from the group containing residues 67, 71, 73, and 78, where the donor residues phenylalanine (F67), glutamine (Q71), serine (S73), and valine (V78) relative to SEQ ID NO:39 are retained, respectively. The retention of donor residues Q71, S73, and V78 is crucial for binding with the highest affinity for human KLK-5. The glutamine residue at human framework position 1 is replaced with glutamate (E1) to provide homogenized product expression and purification: the conversion of N-terminal glutamine to pyroglutamate in antibodies and antibody fragments has been widely reported. The theoretical pI of the humanized 10236 antibody is approximately 6.3 to 6.6. To facilitate the removal of impurities by ion exchange chromatography during downstream processing, pI is increased by mutating residue 24 of CDRL1 in graft gL6 from glutamine (Q) to arginine (R) or lysine (K) residues.

[0790] Rabbit / human antibody 10236 was used to test humanized grafts to assess whether their affinity was affected by the humanization process. Rabbit / human antibody 10236 was cloned into a modified form of a human Cκ vector containing the S171C mutation to reconstruct additional disulfide bonds absent in the human constant region but found in the rabbit VK light chain, thereby producing rabbit / human antibody 10236 according to SEQ ID NO: 96 and 97.

[0791] Measurement of the Affinity of the Humanized Graft of Ab 10236.

[0792] Goat anti-human IgG Fc-specific antibody (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip using amine coupling chemistry, achieving a level of approximately 6000 RU. Each analytical cycle consisted of: capturing anti-KLK5 IgG from the supernatant onto the anti-Fc surface, injecting KLK5 analyte (internally prepared) at a rate of 30 μL / min for 180 seconds, followed by dissociation for 600 seconds. At the end of each cycle, the surface was regenerated by injecting 50 mM HCl for 60 seconds, followed by 5 mM NaOH for 30 seconds, and finally 50 mM HCl for a final 60 seconds, at a flow rate of 10 μL / min. The final concentration was replenished with HBS-EP+ run buffer (GE) to a final concentration of 300 mM NaCl. The supernatant from the sample was titrated with human KLK5 from 20 nM to 0.08 nM (5 x 3 serial dilutions). Buffer blank injections were included to reduce instrument noise and drift. Kinetic parameters were determined using a 1:1 binding model with Biacore T200 evaluation software (version 3.0). Experiments were conducted at 25 °C.

[0793] Rabbit / human chimeric antibodies were analyzed at the start and end of the assays and showed good accuracy. High-quality data were generated for all samples, as summarized in Table 5.

[0794] Table 5

[0795]

[0796] * As described in Example 4 and Table 3; $ Average of two runs (Table 15)

[0797] As shown in Table 5, regardless of the presence or absence of the Q24R / K mutation, the graft 10236gL6gH12 maintains a high affinity for human KLK5.

[0798] Analysis of the Humanized Antibody

[0799] KLK5 Selectivity

[0800] A series of studies were conducted to ensure that the humanization of rabbit antibody 10236 did not alter the selectivity of KLK5 relative to other kallikreinases, nor reduce affinity or inhibitory activity. The purified antibodies were then screened according to the method described in Example 4 to confirm their inhibitory activity against KLK5. A series of 10.5 logarithmic dilutions ranging from 600 nM to 20 pM were used to test the antibodies. A Beckman Coulter FX was used. TM Using a multidrop system, 5 μL of each antibody was transferred to a black 384-well assay plate (Corning). TM (Catalogue No. 3575) and add 15 μL of the selected active recombinant kallikrein in assay buffer A (150 mM NaCl, 50 mM Tris, 200 μM EDTA, 0.05% (v / v) Tween-20, pH 7.6) to the appropriate wells to achieve the following final assay concentrations: 60 pM human KLK5 (UCB), 250 pM KLK7 (UCB), 500 pM KLK2 (R&D Systems) TM ), 30 pM MKLK4 (UCB), 30 pM cynomolgus monkey KLK5 (UCB), 500 pM cynomolgus monkey KLK7, 30 pM mouse KLK5 (UCB), and 5 nM mouse KLK7 (UCB). The enzymes prepared in UCB are shown in parentheses and prepared as described in Example 1 above. The sources of commercially available enzymes are shown in parentheses.

[0801] 20 μL of assay buffer A was added alone to each well as a 0% activity level. LEKTI D5 rabbit Fc (prepared by UCB, as described above) was used as a reference for inhibiting activity; 5 μL of LEKTI D5 rabbit Fc (with the same concentration range used for the 10236 antibody) was added to 15 μL of kallikrein. 15 μL of human KLK5 was added to 5 μL of assay buffer A as a 100% activity level reference.

[0802] The antibody and kallikrein were incubated overnight at room temperature. The following peptide substrates were added using a multi-dropper: 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). TM KHLF-AMC for humans and cynomolgus monkeys (Cambridge Research Biochemicals) TM (90μM and 150μM respectively), for human KLK4 PFR-AMC (R&D Systems) TM(200μM) and Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (R&D Systems) for mouse KLK7 TM (150 μM). The sample was incubated for 4 hours and then readjusted using a Pherastar FSX microplate reader (BMG Labtech). TM On λ ex 380 nm and λ em Boc-VPR-AMC, PFR-AMC, and KHLF-AMC were read at 430 nm; and at λ ex 320 nm and λ em Mca-RPKPVE-Nval-WRK(Dnp)-NH2 was read at 400 nm. Data were analyzed as described in Example 3 to determine the percentage of inhibition. The data were plotted against the concentration of the test antibody and a fitted 4-parameter sigmoid curve to determine the IC50 (Genedata Screener). TM ).

[0803] Humanized grafts of Ab 10236 retained specific inhibitory activity against KLK5 and showed little or no inhibitory activity against other members of the KLK family tested. Ab10236 gL6gH12 is a potent inhibitor of human and cynomolgus monkey KLK5 (Table 6, each value is a separate measurement), retaining similar potency to the parental non-humanized antibody. No significant activity was observed against human KLK2, 4, and 7, cynomolgus monkey KLK7, mouse KLK5, or mouse KLK7 (i.e., below the 40% threshold according to the selection criteria in Example 2). Figure 8 ).

[0804] Table 6

[0805]

[0806] n / a = Not available

[0807] Binding of LEKTI to KLK5 in the Presence of the Humanized Antibody 10236

[0808] As with the parent rabbit antibody 10236, a surface plasmon resonance (SPR) experiment as described in Example 5 was performed to determine the affinity of LEKTI for KLK5 complexed with humanized antibody 10236gL6gH12.

[0809] Except for the preparation of the anti-human Fc chip surface, the experimental conditions described in Example 5 were used, and the results are reported in Table 6.

[0810] Similar to the parent rabbit 10236 antibody, the LEKTI D5 Fab fusion protein can bind to KLK5 that has been complexed with antibody 10236gL6gH12, but the affinity is lower than that of human KLK5 alone (Table 7, 540 nM vs. 40 pM).

[0811] Table 7

[0812] ka (Ms^-1) kd (s^-1) KD (M) Only Human KLK5 5.70E+05 2.30E-05 4.00E-11 10236 gL6gH12 + Human KLK5 2.90E+04 1.60E-03 5.40E-07

[0813] KLK5-PAR2 Cell Assay

[0814] KLK5 has been shown to activate the proteinase-activated receptor 2 (PAR2) receptor on the surface of keratinocytes (K. Oikonomopoulou et al. Kallikrein-mediated cell signaling: targeting proteinase-activated receptors (PARs). Biol Chem, 387 (2006), pp. 817-824). This leads to an NF-κB-driven inflammatory cascade and the release of related cytokines such as TSLP.

[0815] Since PAR2 is a Gq-coupled G protein-coupled receptor (GPCR), its activation leads to phospholipase signaling and the production of inositol monophosphate (IP-1). The activation of endogenous PAR2 expressed on HaCat keratinocytes via KLK5 exposure was monitored using an assay kit from Cisbio to detect IP1.

[0816] Harvest the confluent HaCat cells and seed them at 10,000 cells / well in 384 Fluoblock plates (Corning). TM In 37°C, 5% CO2 DMEM medium + 10% FBS + 2mM L-glutamine + Pen / Strep (Life Technologies) TM Incubate overnight in the solution, then perform assays according to the IP-One Gq assay protocol (Cisbio). TM They were processed. The test antibodies (antibody 10236 gL6gH12 and negative human IgG4 A33) were prepared in 1x stimulation buffer B (IP-One Gq assay kit, Cisbio). TM The antibody / KLK5 mixture was serially diluted from the highest concentration of 2 μM and incubated at 37°C for 1 hour in the presence of 200 nM human KLK5. The antibody / KLK5 mixture was added to HaCat cells, and inositol 1-phosphate (IP1) was detected according to the IP-One Gq assay protocol, with fluorescence read at 665 nM and 620 nM on a Synergy Neo microplate reader.

[0817] Antibody 10236 gL6gH12 was able to almost completely inhibit IP1 release from KLK5-treated HaCat cells. Figure 9 It showed maximum inhibition of IP1 release similar to that of the LEKTI D5 rabbit Fc protein.

[0818] Mechanism of Action of Antibody 10236

[0819] Experiments were conducted to determine the mechanism of action of inhibitory antibodies. Non-competitive enzyme inhibitors reduce enzyme activity but can bind to the enzyme equally well in the presence or absence of a substrate. Both the inhibitor and substrate can bind to the enzyme simultaneously, but no product can be formed, resulting in the enzyme-substrate-inhibitor complex being degraded into either the enzyme-substrate or the enzyme-inhibitor complex. When using non-competitive inhibitors, the inhibition rate is not affected by increasing substrate concentration.

[0820] Antibody 10236 gL6gH12 or LEKTI D5 rabbit 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 300, 30, or 3 times the IC50 against human KLK5. 10 μL of antibody 10236 gL6gH12 was added to a Corning low-binding black low-flange 384-well assay plate. 10 μL of 30 mM–300 μM Boc-VPR-AMC (Cambridge Research Biochemicals) was serially diluted in 5-point series. TM Add to the plate. A Pherastar FSX microplate reader (BMG Labtech™) is used to simultaneously initiate the reaction by injecting 10 μL of 1.8 nM human KLK5 (Boc-VPR-AMC < 1 mM) or 180 pM KLK5 (Boc-VPR-AMC > 1 mM) and monitor fluorescence every 30 seconds (λex 380 nm λem 430 nm). The final reaction conditions include 100, 10, or 1-fold of antibody (10236 gL 6gH12 or LEKTI D5 rabbit Fc protein) with a determined IC50 of human KLK5, serial dilutions of Boc-VPR-AMC between 10 mM and 100 μM, and 60 or 600 pM human KLK5. Use assay buffer instead of antibody to set up an uninhibited control, and use buffer instead of enzyme to set up a background control.

[0821] The data were analyzed by subtracting the background fluorescence at each time point and plotting the fluorescence over time. The data conformed to the following equation (GraphPad). GraphPad Software):

[0822]

[0823] This makes it possible to determine k obs That is, the observed time-dependent inhibition rate, where v i It is the initial reaction rate, v s That is the final velocity. Let k obs The values ​​are plotted against substrate concentration to determine the inhibition mechanism.

[0824] Antibody 10236 gL6gH12 ( Figure 10 A) and rabbit antibody 10236 ( Figure 10 B) The inhibition rate of human KLK5 changed with increasing substrate concentration, indicating that antibody 10236gL6gH12 is a non-competitive inhibitor of human KLK5. In contrast, LEKTI D5 rabbit Fc protein showed a decreasing inhibition rate with increasing substrate concentration, indicating that it is a competitive inhibitor of human KLK5.

[0825] Example 9: In Vitro Skin System Study

[0826] EpiDermFT (MatTek) is a full-thickness skin system for the human body. TM Company; Morizane, Shin et al. “Kallikrein expression and cathelicidin processing are independently controlled in keratinocytes by calcium, vitamin D(3), and retinoic acid.”The Journal of Investigative Dermatology vol.130,5(2010):1297-306.doi:10.1038 / jid.2009.435) was used to demonstrate the effect of antibody 10236gL6gH12 IgG4P on human KLK5. Functional Impact .

[0827] A vitamin D3 analogue, MC903, was used to treat the in vitro skin system because it induces an atopic dermatitis-like phenotype in vivo (Naidoo, Karmella et al. “Eosinophils Determine Dermal Thickening and Water Loss in an MC903 Model of Atopic Dermatitis.” The Journal of Investigative Dermatology vol. 138, 12(2018): 2606-2616. doi:10.1016 / j.jid.2018.06.168).

[0828] EpiDermFT TM Full-thickness reconstructed skin tissue measured on EFT-400-ASY medium (MatTek Corporation) TM The culture medium was equilibrated overnight at 37°C and 5% CO2. On day 0, the medium was removed from the wells and replaced with 2.5 ml of EFT-400-ASY medium. Tissues were locally treated with 25 μl of the following: medium only; MC903 (Tocris) diluted in EFT-400-ASY medium. The final concentration was 2 nmol. MC903 (2 nmol) was diluted in culture medium, and 10 μg / ml of antibody 10236 gL6gH12 IgG4P or hIgG4P isotype control (internal production) was added. The plates were incubated at 37°C and 5% CO2. The basal medium was changed daily, and local treatments were performed daily. The experiment was stopped on day 4.

[0829] From transwell (Costar Snapwell) TM The tissue was removed from the culture dish, bisected using a scalpel, and placed in the OCT tissue embedding matrix (Cellpath). TM In preparation for histological analysis, 6 μm sections were cut and stained to assess structural integrity using hematoxylin and eosin, KLK5 was detected by immunofluorescence, and protease activity was assessed by in situ zymography.

[0830] For KLK5 immunofluorescence staining, sections were air-dried at room temperature for 10 minutes, washed three times with 0.1% Tween 20 in PBS, and then washed once in PBS. The sections were then blocked in 5% BSA in PBS for 10 minutes. Sections were circled using a PAP pen and coated with 10 μg / ml mouse anti-huKLK5 antibody. Incubate at 37°C for 1 hour in a humidified chamber. After antibody incubation, wash the sections again and fix in 4% PFA for 10 minutes. Then, inoculate the sections with secondary goat anti-mouse IgG Alexa546 (Life) Incubate at 37°C for 1 hour in a humidified chamber. Wash the sections and use a solution containing DAPI (Vector Labs). TM Sections were mounted with a sealing agent. Fluorescence images were obtained at 20x magnification on a Zeiss Axio Scan.

[0831] For in situ enzyme profiling, sections were air-dried at room temperature for 10 minutes, washed once in 2% Tween PBS, and then washed three times in PBS. The sections were then coated with 10 μg / ml casein-BODIPY-FL fluorescent substrate. Incubate at 37°C for 3 hours in a humidified chamber. Wash sections three times with PBS and use a solution containing DAPI (Vector Labs). TM The sections were mounted with a sealing agent. Fluorescence images were immediately acquired at 20x magnification on a Zeiss Axio Scan.

[0832] A comparison of tissue structures after MC903 treatment with and without antibody 10236 gL6gH12 IgG4P showed that KLK inhibition could prevent stratum corneum destruction in a human skin model. Figure 11 ).

[0833] Furthermore, although KLK5 expression remained unchanged, serine protease activity was reduced in skin systems treated with antibody 10236 gL6gH12IgG4P (data not shown).

[0834] Example 10: In Situ Zymography in Atopic Dermatitis Samples

[0835] Skin biopsies from patients with moderate to severe atopic dermatitis (National Bioservice Russia) were tested to evaluate the inhibitory effect of anti-KLK5 antibodies. Biopsies (4 mm) were embedded in an OCT tissue embedding matrix (Cellpath). TM The tissue sections were stored at -80°C. Tissue sections were cut (6 μm) and used for in situ enzyme profiling analysis, using the fluorescence quenching substrate [5-FAM]-FVNRSYPP-Lys(Dabcyl)-amide instead of casein-BODIPY-FL, with a final concentration determined at 20 μm, as previously described in Example 9. Cutting of the substrate in the tissue sections produced fluorescence, which was detected by Zeiss Axio Scan. TM The image was detected as a fluorescence image at a magnification of 20x.

[0836] Data showed that, compared with untreated sections, pre-incubation of atopic dermatitis tissue sections with antibody 10236 gL6gH12 IgG4P reduced the level of serine protease activity. Figure 12 As shown by the significant reduction in white staining in the stratum corneum (the outermost layer of the epidermis) and the stratum granulosum (immediately below the stratum corneum).

[0837] Example 11: Biophysical Characterization of the Humanized Antibody

[0838] The biophysical properties of 10236 gL6gH12 (as an isotype of IgG4P and IgG1) were determined to assess its exploitability. This included thermal stability (Tm), experimental pI, apparent hydrophobicity, solubility (PEG precipitation assay), and assessment of self-interactions (aggregation tendency) by AC-SINS.

[0839] In addition, antibody 10236 gL6N94D gH12 mutant was tested to assess chemical stability, i.e., the tendency of Asn(94)Ser motif (ref. SEQ ID NO:15) in light chain CDR3 (Table 8).

[0840] Table 8

[0841]

[0842] Mass Spectrometry Characterization

[0843] The identities of antibodies 10236IgG1 and IgG4P were determined by using the following methods: The complete mass of the heavy and light chains was confirmed by LC-MS measurement using a Waters ACQUITY UPLC system on a G2 Q-ToF mass spectrometer. The sample (approximately 5 μg) was reduced for 40 min at 37 °C with a 150 mM ammonium acetate solution of 5 mM tris(2-carboxyethyl)phosphine (TCEP). The LC column was a Waters BioResolve column. TM RP mAb Polyphenyl, Equilibrate at 2.7 μm and 80 °C 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. Elute the protein with a gradient of 5% to 50% solvent B over 8.8 min, then wash with 95% solvent B and reequilibrate. UV data were obtained at 280 nm. MS conditions were as follows: ion mode: ESI positive ion, resolution mode, mass range: 400–5000 m / z, external calibration with NaI. Waters MassLynx was used. TM Analyze data with MaxEnt software.

[0844] If determined by whole-body mass spectrometry, no difference was observed between the expected and observed molecular weights (Table 9).

[0845] Table 9

[0846]

[0847] Measurement of Thermal Stability (Tm)

[0848] The thermal displacement measurement is used to determine the unwinding temperature (Tm) or the temperature at the midpoint of the unfold.

[0849] For this determination, fluorescent dye Orange is used to monitor protein unfolding by binding to hydrophobic regions that become exposed as temperature increases. The reaction mixture contains 5 μL of 30x Orange Protein Gel Stain (Thermofisher Scientific, S6651) was prepared by diluting the concentrate from 5000x with test buffer. 45 μL of 0.2 mg / mL sample in PBS at pH 7.4 or 50 mM sodium acetate / 125 mM sodium chloride at pH 5.0 was added to the dye and mixed (as a commonly used pre-prepared buffer). 10 μL of this solution was quadrupled into 384 PCR optical plates and tested on a QuantStudio 7 real-time PCR system (Thermofisher). TM The PCR system was run on a PCR platform. The heating element was set at 20°C and increased to 99°C at a rate of 1.1°C / min. A charge-coupled device (CCD) was used to monitor fluorescence changes in the wells. A fluorescence intensity increase graph was plotted, and the inflection point of the slope was used to generate the apparent midpoint temperature (Tm). The data are shown in Table 10.

[0850] Two unfolding transitions were observed in 10236gL6gH12 (IgG4P). The first was attributable to the CH2 domain, and the second to the average Tm of the Fab unfolded domain and the CH3 domain. For the IgG1 molecule, one unfolded domain was observed to be attributable to the average Tm of the CH2 and Fab domains. This is consistent with the literature (Garber E. Demerest SJ. Biochem Biophys Res Commun. 2007 Apr; 355(3):751-7).

[0851] Table 10

[0852]

[0853] Measurement of Experimental Isoelectric Point (pI)

[0854] iCE3 TM A whole-capillary imaging capillary isoelectric focusing (cIEF) system (ProteinSimple) was used to experimentally determine pI. Samples were prepared by mixing the following substances: 30 μL sample (1 mg / mL stock solution from HPLC-grade water), 35 μL of 1% methylcellulose solution (ProteinSimple, 101876), 4 μL of pharmalyte at pH 3–10 (ProteinSimple, 042–848), 0.5 μL of 4.65 and 0.5 μL of 9.77 synthetic pI labels (ProteinSimple, 102223 and 102219), and 12.5 μL of 8M urea solution (Sigma). The final volume was brought to 100 μl using HPLC-grade water. The sample was focused at 1.5 kV for 1 min, then at 3 kV for 5 min, and capillary images were captured at 280 nm using Protein Simple software. The obtained electrophoresis images were analyzed using iCE3 software, and pI values ​​(linear relationships between pI labels) were assigned. The data are summarized in Table 11.

[0855] The pI of 10236gL6gH12 (hIgG4P) was found to be lower than that of the corresponding IgG1 molecule. It can be considered that neither molecule presents any development / manufacturing issues, and the pI is higher than the buffer solution typically used for formulation (approximately pH 5).

[0856] Table 11

[0857]

[0858] Hydrophobic Interaction Chromatography (HIC)

[0859] Hydrophobic interaction chromatography (HIC) separates molecules in order of increasing hydrophobicity. In the presence of high concentrations of polar salts, molecules are immobilized by hydrophobic bonds and desorbed into the mobile phase as the salt concentration decreases. Longer retention times correspond to greater hydrophobicity.

[0860] Two isoforms (IgG4P and IgG1) of 10236 g / mL 6gH12 were diluted 1:2 with 1.6M ammonium sulfate and PBS (pH 7.4) at a ratio of 1:2. 10 μg (10 μL) of the sample was injected into a Dionex ProPac Agilent 1200 binary HPLC system equipped with a fluorescence detector. TMThe column was chromatographically mounted on an HIC-10 column (100 mm x 4.6 mm). Separation was monitored by intrinsic fluorescence (excitation and emission wavelengths of 280 nm and 340 nm, respectively). Samples were analyzed by elution using the following gradient with buffer A (0.8 M ammonium sulfate, 100 mM phosphate, pH 7.4) and buffer B (100 mM phosphate, pH 7.4): (i) hold in 0% B for 2 min, (ii) a linear gradient from 0% to 100% B over 30 min (0.8 mL / min), and (iii) wash the column with 100% B for 2 min, followed by reequilibration in 0% B for 10 min before the next sample injection. The column temperature was maintained at 20 °C. Retention times (in minutes) are shown in Table 12.

[0861] Table 12

[0862]

[0863] A slight difference in retention time was observed between the two molecules, with 10236 gL6gH12 (IgG4P) exhibiting slightly higher apparent hydrophobicity than the corresponding IgG1 form. Based on results from other commercial antibodies, both molecules are expected to have an average aggregation tendency. (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).

[0864] Solubility Measurement Using Polyethylene Glycol (PEG)

[0865] Colloidal stability (solubility) can be understood by examining the effect of polyethylene glycol (PEG) precipitation. By increasing the concentration (w / v) of PEG and measuring the amount of protein remaining in the solution, PEG is used to reduce protein solubility in a quantitatively definable manner. This assay is used to simulate the effect of high concentration solubility without using conventional concentration methods.

[0866] A 40% (w / v) PEG 3350 (Merck, 202444) stock solution was prepared in PBS at pH 7.4 with 50 mM sodium acetate, 125 mM sodium chloride at pH 5.0 (a common pre-preparation storage buffer) and 50 mM histidine, 250 mM proline at pH 5.5 (a common preparation buffer). Serial titrations were performed using an Assist Plus liquid handling robot (INTEGRA, 4505) to obtain 40% to 15.4% PEG 3350. To minimize non-equilibrium precipitation, sample preparation involved mixing protein and PEG solution at a 1:1 volume ratio. 35 μL of the PEG 3350 stock solution was added to a 96-well v bottom PCR plate (A1 to H1) using the liquid handling robot. 35 μL of a 2 mg / mL sample solution was added to the PEG stock solution to obtain a 1 mg / mL test concentration. The solution was automatically and slowly mixed by repeated pipetting and incubated at 37°C for 0.5 h to redissolve any non-equilibrium aggregates. The sample was then incubated at 20°C for 24 h. The sample plate was subsequently centrifuged at 4000 x g for 1 h at 20°C. 50 μL of the supernatant was allocated to… Half area, 96 holes In microplates (Greiner, 675801). Used Protein concentration was determined by UV spectrophotometry at 280 nm using an Omega multi-detection microplate reader (BMG LABTECH). Values ​​were plotted using Graphpad Prism version 7.04, with the PEG midpoint (PEGmdpnt) score derived from the midpoint of the sigmoid dose-response (variable slope) fit.

[0867] The data are shown in Table 13. The higher the PEG midpoint (%), the greater the likelihood of high concentration stability / solubility.

[0868] Differences were observed between the two isoforms depending on the buffer conditions. In PBS at pH 7.4, 10236gL6gH12 (IgG4P) showed greater solubility than 10236gL6gH12 (IgG1); while the opposite was observed in 50mM sodium acetate, 125mM sodium chloride at pH 5. The solubility of 10236gL6gH12 (IgG4P) can be improved by using a more typical preparation buffer, namely 50mM histidine, 250mM proline at pH 5.5.

[0869] Table 13

[0870]

[0871] Assessment of Self-Interaction Using AC-SINS (Affinity Capture Self-Interaction Nanoparticle Spectroscopy).

[0872] AC-SINS assay (Liu Y. MAbs. 2014 Mar-Apr; 6(2):483-92) was used to assess the exploitability of 10236 g L6 g H12 by determining its self-interaction tendency, and thus inform its potential aggregation stability. This was performed in PBS at pH 7.4.

[0873] The goat anti-human Fcγ specific capture antibody (Jackson ImmunoResearch) buffer was replaced with 20 mM sodium acetate at 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) and incubated overnight at room temperature. The conjugated nanoparticles were blocked with 55 μL of PEG-thiol for 1 h, centrifuged at 21,000 x g for 6 min, the supernatant was removed, and the nanoparticles were resuspended in 20 mM sodium acetate at pH 4.3 to a final volume of 150 μL.

[0874] 10236 g L6g H12 (IgG4P and IgG1) was diluted to 22 μg / mL in 200 μL of PBS at pH 7.4, then added to an equal volume of nonspecific whole IgG (Jackson ImmunoResearch), briefly vortexed, and then 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 on a BMG microplate reader, Lorenzian curves (RShiny) were fitted, and PBS-only values ​​were subtracted from the sample to obtain Δλmax. Data are summarized in Table 14.

[0875] Both 10236gL6gH12, as isotypes of IgG4P and IgG1, showed low λmax and Δλmax (from PBS background), indicating a low tendency for self-interaction and a low risk of aggregation in PBS at pH 7.4.

[0876] Table 14

[0877]

[0878] Accelerated Stress Study for Evaluating the Deamidation Propensity of Asn(94) (Light Chain CDR3)

[0879] The deamidation motif Asn(94)Ser is located on the light chain CDR3 of 10236gL6gH12. The tendency / rate of deamidation is unpredictable as it depends on the primary sequence and 3D structure as well as solution properties (RC Stephenson and S. Clarke (1989); K. Diepold et al. (2012); Jasmin F. Sydow et al. (2014); NE Robinson et al. (2004)). Therefore, an accelerated stress study was set up to determine the deamidation tendency of 10236gL6gH12 on Asn(94). This was performed only on 10236gL6gH12 (IgG4P); since the deamidation motif is located in the variable region, the deamidation rate of IgG1 was assumed to be the same.

[0880] The baseline deamidation level (non-stressed sample) was also measured; low levels indicate low deamidation sensitivity, but these may vary depending on different manufacturing batches / conditions.

[0881] The antibody 10236 gL6gH12 (IgG4P) buffer was exchanged for the following conditions: (i) a buffer known to favor the deamidation of Asn(N) residues (Tris pH 8.0 / 125mM NaCl, 37°C) and (ii) a control buffer (acetate, pH 5.0 / 125mM sodium chloride, 37°C). The final concentration of the sample in each buffer was 5.9 mg / mL at pH 8.0 and 6.6 mg / mL at pH 5.0. The sample was then aliquoted into two equal aliquots, one stored at 4°C and the other at 37°C for up to 2 weeks. The aliquots were removed immediately (T0) and at 2 weeks and stored at -20°C.

[0882] Basic deamidation was obtained by analyzing a stock sample that had been stored in PBS at -20°C and pH 7.4.

[0883] All samples were thawed using the following method and analyzed by peptide mapping using mass spectrometry (MS):

[0884] In a solution containing 0.1% w / v Rapigest TM Stress protein samples were reduced with TCEP and alkylated with chloroacetic acid in Tris-HCl buffer (pH 8.0) with detergent. A trypsin / LysC mixture (1:50 w / w) was added, and the samples were digested at 37°C for 1 hour, followed by the addition of chymotrypsin (1:50 w / w) and continued digestion overnight at room temperature. Proteolysis was stopped by adding formic acid to 1% v / v, and the samples were diluted to 0.5 mg / ml and centrifuged to remove the rapid precipitate. TMThe obtained peptide library was separated and analyzed on a Waters BEH C18 column connected to a Thermo Fusion mass spectrometer, running a positive ion, data-dependent orbitrap-orbitrap method with CID fragmentation. Thermo Xcalibur was used. TM and Pepfinder TM Software analysis of LC-MS data.

[0885] The baseline deamidation level of KLK5 10236L-CDR3 at Asn94 was 0.7% (using Pepfinder). TM (Calculation). For samples incubated at 37°C for 2 weeks in Tris pH 8.0, this value is increased to a maximum of 10% ( Figure 13 ).

[0886] The tendency to deamide is low and can be controlled by minimizing the use of high pH buffers during manufacturing, storage and formulation.

[0887] Measurement of the Affinity of the Fully Deamidated Product: 10236 gL6-N94DgH12 (Mutation of NS to DS on Light Chain CDR3)

[0888] The light chain of 10236gL6gH12 was mutated to replace Asn94 with Asn94 (Asn94Asp) to generate a surrogate molecule of the fully deamidated product of 10236gL6gH12.

[0889] Through surface plasmon resonance (Biacore T200, GE Life Sciences) TM The binding kinetics of two antibodies, 10236gL6gH12 and 10236gL6-N94DgH12, to human KLK5 were evaluated at 25°C to assess the effect of 100% deamidation.

[0890] Goat anti-human IgG Fc-specific antibody (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip using amine coupling chemistry to achieve a level of approximately 6000 RU. Each analytical cycle consisted of: capturing anti-KLK5 IgG molecules onto the anti-Fc surface, injecting KLK5 analyte (internally prepared) at a rate of 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 a 60-second injection of 50 mM HCl, followed by a 30-second injection of 5 mM NaOH and a final 60-second injection of 50 mM HCl. Human KLK5 was titrated from 20 nM to 0.03 nM (6 x 3-fold serial dilutions) in HBS-EP+ run buffer (GE Healthcare) supplemented to a final concentration of 300 mM NaCl. Buffer blank injections were included to reduce instrument noise and drift. Kinetic parameters were determined using a 1:1 binding model with Biacore T200 evaluation software (version 3.0). The data is summarized in Table 15.

[0891] Two replicates of 10236gL6gH12 were included to demonstrate experimental reproducibility. The Asn94Asp mutation in the light chain CDR3 reduced affinity for KLK5 by 4.5-fold.

[0892] Therefore, without monitoring and control of manufacturing, storage, and formulation conditions, adequate deamidation may affect the efficacy of 10236gL6gH12. Accelerated stress experiments showed a low tendency for deamidation of the Asn94 residue, thus reducing the risk to the molecule.

[0893] Table 15

[0894] Antibody Description ka (1 / Ms) kd (1 / s) KD (pM) <![CDATA[10236gL6gH12 Run 1 $ > 2.26E+06 7.11E-04 314.3 <![CDATA[10236gL6gH12 Running 2 $ > 2.33E+06 6.58E-04 283.0 10236gL6N94DgH12 3.89E+06 4.86E-03 1249.2

[0895] $ The average of Run 1 and Run 2 = 299pM

[0896] Viscosity Evaluation of Antibody 10236 gL6gH12 (hIgG4P) at Different Concentrations

[0897] Low viscosity at high antibody concentrations is important for the subcutaneous administration of therapeutic molecules. The viscosity behavior was investigated by measuring the viscosity of antibody 10236 gL6gH12 at increasing concentrations in a standard pre-prepared buffer of 50 mM histidine / 250 mM proline at pH 5.5.

[0898] This study was conducted by (i) the initial concentration of the sample and (ii) the viscosity measurements detailed below.

[0899] (i) Concentration

[0900] use A total of 23 mL of antibody (10236 gL 6gH12 (hIgG4P)) was concentrated at 4000 x g using a 20 MWCO 30kD centrifuge filter (Z14637, Sigma-Aldrich) at 20 °C until the lysate volume was approximately 950 μL. The lysate solution was recovered and treated with NanoDrop. TM The final concentration of antibody 10236 gL6gH12 (hIgG4P) was determined by measuring UV absorbance at 280 nm and extinction coefficient of 1.46 mL / (mg cm⁻¹) using a 1000 instrument. The concentrated sample was determined to be 185 mg / mL (average of three replicates), and the theoretical recovery rate of the concentrated sample was 84%. The loss was within the expected range for this method.

[0901] The antibody samples were then diluted with 50 mM histidine and 250 mM proline at pH 5.5 to provide a concentration range suitable for viscosity measurements. The diluted sample concentrations of 159.8 mg / mL, 63.6 mg / mL, and 33.2 mg / mL were confirmed by UV absorbance measurements at 280 nm.

[0902] (ii) Viscosity measurement

[0903] Viscosities at each concentration were measured using a Discovery Hybrid Rheometer-1 (DHR-1, TA Instruments) with a Peltier plate, a liquid cooling system for temperature control, and a 20 mm stainless steel parallel plate geometry for measurement. Samples (80 μL) at different concentrations (33.2 mg / mL, 63.6 mg / mL, and 159.8 mg / mL) were placed at the center of a Peltier plate, and a steady-state flow scan program was used at 20 °C to measure viscosity from 2.87918 to 287.918 s. -1 Viscosities (mPa·s or cP) were measured at different shear rates. The measured viscosities were averaged when the value at each shear rate point was constant (SD ± 5%). The viscosities of 10236 gL6gH12 (IgG4P) at different concentrations are summarized in Table 16.

[0904] An increasing trend in the viscosity coefficient of antibody 10236 gL6gH12 (hIgG4P) was observed between concentration and concentration. Viscosity increased from 2.8 to 6.4 cP within a concentration range from 33.2 mg / mL to 159.8 mg / mL. All these samples exhibited a constant viscosity coefficient (variability less than 5%) at different shear rates. The study suggests that antibody 10236 gL6gH12 (hIgG4P) exhibits low viscosity at high concentrations (approximately 150 mg / mL) at 50 mM histidine / 250 mM proline pH 5.5, thus indicating its suitability for subcutaneous administration.

[0905] Table 16

[0906] Concentration (mg / mL) Average Viscosity (cP) SD (cP) %RSD 33.2 2.8 0.08 2.99 63.6 3.6 0.08 2.29 159.8 6.4 0.16 2.54 sequence list <110> UCB Biopharmaceuticals LLC <120> Antibodies against KLK5 <130> PF0196-WO <150> GB2001447.8 <151> 2020-02-03 <160> 104 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CDR-L1 <400> 1 Gln Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDR-L2 <400> 2 Leu Ala Ser Thr Leu Ala Ser 1 5 <210> 3 <211> 12 <212> PRT <213> Artificial sequence <220> <223> CDR-L3 <400> 3 Gln Gln Gly Tyr Thr Asn Ser Asn Ile Ile Asn Thr 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR-H1 <400> 4 Gly Phe Pro Leu Ser Asn Tyr Ala Met Ser 1 5 10 <210> 5 <211> 16 <212> PRT <213> Artificial sequence <220> <223> CDR-H2 <400> 5 Asp Ile Tyr Pro Ser Asp Ile Ile Asp Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR-H3 <400> 6 Asp Asn Asn Asp Tyr Gly Leu Asp Ile 1 5 <210> 7 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Rabbit VL <400> 7 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Asn Ser Asn 85 90 95 Ile Ile Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 8 <211> 336 <212> DNA <213> Synthetic Sequence <220> <223> Rabbit VL Nucleotide <400> 8 gcctatgata tgacccagac tccagcctct gtggaggtag ctgtgggagg cacagtcacc 60 atcaagtgcc aggccagtca gagcattagc agttggttag cctggtatca gcagaaacca 120 ggtcagcctc ccaagctcct gatctatctg gcatccactc tggcatctgg ggtctcatcg 180 cggttcaaag gcagtggatc tgggacacag ttcactctca ccatcagcgg cgtggagtgt 240 gccgatgctg ccacttacta ctgtcaacag ggttatacta atagtaatat tattaatact 300 ttcggcggag ggaccgaggt ggtggtcaaa cgtacg 336 <210> 9 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Rabbit VH <400> 9 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Pro Leu Ser Asn Tyr Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Asp Ile Tyr Pro Ser Asp Ile Ile Asp Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Gln Thr Ser Thr Thr Val Glu Leu Lys Ile Thr 65 70 75 80 Gly Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Asn 85 90 95 Asn Asp Tyr Gly Leu Asp Ile Trp Gly Pro Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 10 <211> 342 <212> DNA <213> Artificial sequence <220> <223> Rabbit VH nucleotide <400> 10 cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcaccgtct ctgggttccc cctcagtaat tatgcaatga gctgggtccg ccaggctcca 120 gggaaggggc tggaatggat cggagacatt tatcctagtg atatcataga ctacgcgagc 180 tgggcgaaag gccgattcac catctcccaa acctcgacca cggtggagct gaaaatcacg 240 ggtccgacaa ccgaggacac ggccacctat ttctgtgcca gagacaacaa tgactatggt 300 ctggacatct ggggcccagg caccctggtc accgtctcga gt 342 <210> 11 <211> 110 <212> PRT <213> Artificial sequence <220> <223> 10236 gL5 VL <400> 11 Ala Tyr Asp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Asn Ser Asn 85 90 95 Ile Ile Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 12 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 10236 gL5 VL nucleotides <400> 12 gcctacgaca tgactcagtc cccatcctcc ctgtccgcat ccgtggggga tagagtcacc 60 atcacctgtc aagccagcca gtcaattagc tcgtggctgg cctggtatca gcagaagccg 120 ggaaaggctc ccaagttgct gatctacctg gcctcaacgc tcgcgtcggg agtgcctagc 180 cgctttaagg gttccggatc tggcaccgac ttcactctca ccatttcgag ccttcaaccg 240 gaggacttcg ccacttacta ctgccagcag ggttacacca actccaacat catcaacacc 300 ttcggcggag ggaccaaagt ggaaatcaag cgtacg 336 <210> 13 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> 10236 gL5 light chain <400> 13 Ala Tyr Asp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Asn Ser Asn 85 90 95 Ile Ile Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 14 <211> 657 <212> DNA <213> Artificial Sequence <220> <223> 10236 gL5 light chain nucleotide <400> 14 gcctacgaca tgactcagtc cccatcctcc ctgtccgcat ccgtggggga tagagtcacc 60 atcacctgtc aagccagcca gtcaattagc tcgtggctgg cctggtatca gcagaagccg 120 ggaaaggctc ccaagttgct gatctacctg gcctcaacgc tcgcgtcggg agtgcctagc 180 cgctttaagg gttccggatc tggcaccgac ttcactctca ccatttcgag ccttcaaccg 240 gaggacttcg ccacttacta ctgccagcag ggttacacca actccaacat catcaacacc 300 ttcggcggag ggaccaaagt ggaaatcaag cgtacgcgta cggtggccgc tccctccgtg 360 ttcatcttcc caccctccga cgagcagctg aagtccggca ccgcctccgt cgtgtgcctg 420 ctgaacaact tctacccccg cgaggccaag gtgcagtgga aggtggacaa cgccctgcag 480 tccggcaact cccaggaatc cgtcaccgag caggactcca aggacagcac ctactccctg 540 tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgaa 600 gtgacccacc agggcctgtc cagccccgtg accaagtcct tcaaccgggg cgagtgc 657 <210> 15 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> 10236 gL6 VL <400> 15 Ala Tyr Asp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Asn Ser Asn 85 90 95 Ile Ile Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 16 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> 10236 gL6 VL Nucleotides <400> 16 gcctacgaca tgactcagtc cccatcctcc ctgtccgcat ccgtggggga tagagtcacc 60 atcacctgtc aagccagcca gtcaattagc tcgtggctgg cctggtatca gcagaagccg 120 atcacctgtc aagccagcca gtcaattagc tcgtggctgg cctggtatca gcagaagccg 120 ggaaaggctc ccaagttgct gatctacctg gcctcaacgc tcgcgtcggg agtgcctagc 180 ggaaaggctc ccaagttgct gatctacctg gcctcaacgc tcgcgtcggg agtgcctagc 180 cgcttttccg gttccggatc tggcaccgac ttcactctca ccatttcgag ccttcaaccg 240 cgcttttccg gttccggatc tggcaccgac ttcactctca ccatttcgag ccttcaaccg 240 gaggacttcg ccacttacta ctgccagcag ggttacacca actccaacat catcaacacc 300 gaggacttcg ccacttacta ctgccagcag ggttacacca actccaacat catcaacacc 300 ttcggcggag ggaccaaagt ggaaatcaag 330 ttcggcggag ggaccaaagt ggaaatcaag 330 <210> 17<210> 17 <211> 217<211> 217 <212> PRT<212> PRT <213> Artificial Sequence<213> Artificial Sequence <220><220> <223> 10236 gL6 light chain<223> 10236 gL6 light chain <400> 17<400> 17 Ala Tyr Asp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Ala Tyr Asp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Asn Ser Asn 85 90 95 Ile Ile Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 18 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> 10236 gL6 light chain nucleotide <400> 18 gcctacgaca tgactcagtc cccatcctcc ctgtccgcat ccgtggggga tagagtcacc 60 atcacctgtc aagccagcca gtcaattagc tcgtggctgg cctggtatca gcagaagccg 120 ggaaaggctc ccaagttgct gatctacctg gcctcaacgc tcgcgtcggg agtgcctagc 180 cgcttttccg gttccggatc tggcaccgac ttcactctca ccatttcgag ccttcaaccg 240 gaggacttcg ccacttacta ctgccagcag ggttacacca actccaacat catcaacacc 300 ttcggcggag ggaccaaagt ggaaatcaag cgtacggtgg ccgctccctc cgtgttcatc 360 ttcccaccct ccgacgagca gctgaagtcc ggcaccgcct ccgtcgtgtg cctgctgaac 420 aacttctacc cccgcgaggc caaggtgcag tggaaggtgg acaacgccct gcagtccggc 480 aactcccagg aatccgtcac cgagcaggac tccaaggaca gcacctactc cctgtcctcc 540 accctgaccc tgtccaaggc cgactacgag aagcacaagg tgtacgcctg cgaagtgacc 600 caccagggcc tgtccagccc cgtgaccaag tccttcaacc ggggcgagtg c 651 <210> 19 <211> 110 <212> PRT <213> Artificial sequence <220> <223> 10236 gL7 VL <400> 19 Ala Ile Asp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Asn Ser Asn 85 90 95 Ile Ile Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 20 <211> 330 <212> DNA <213> Artificial sequence <220> <223> 10236 gL7 VL nucleotides <400> 20 gcgatcgaca tgactcagag cccgtccagc ctgtccgcgt ccgtgggaga tcgcgtgact 60 gcgatcgaca tgactcagag cccgtccagc ctgtccgcgt ccgtgggaga tcgcgtgact 60 atcacgtgtc aggcctcaca atccattagc tcctggctgg cctggtacca gcagaagcca 120 atcacgtgtc aggcctcaca atccattagc tcctggctgg cctggtacca gcagaagcca 120 gggaaggctc cgaagctgct gatctacctg gcctccaccc ttgcctccgg cgtgccttca 180 gggaaggctc cgaagctgct gatctacctg gcctccaccc ttgcctccgg cgtgccttca 180 cggttttctg gatccggctc gggaaccgac ttcaccctca ccatctcgtc gctccaaccc 240 cggttttctg gatccggctc gggaaccgac ttcaccctca ccatctcgtc gctccaaccc 240 gaggacttcg caacctacta ctgccaacag gggtatacca acagcaacat catcaacacc 300 gaggacttcg caacctacta ctgccaacag gggtatacca acagcaacat catcaacacc 300 ttcggtggcg gaactaaggt cgaaatcaag 330 ttcggtggcg gaactaaggt cgaaatcaag 330 <210> 21<210> 21 <211> 217<211> 217 <212> PRT<212> PRT <213> 人工序列<213> Artificial sequence <220><220> <223> 10236 gL7轻链<223> 10236 gL7 light chain <400> 21 <400> 21 Ala Ile Asp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Ala Ile Asp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Asn Ser Asn 85 90 95 Ile Ile Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 22 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> 10236 gL7 Light Chain Nucleotides <400> 22 gcgatcgaca tgactcagag cccgtccagc ctgtccgcgt ccgtgggaga tcgcgtgact 60 atcacgtgtc aggcctcaca atccattagc tcctggctgg cctggtacca gcagaagcca 120 gggaaggctc cgaagctgct gatctacctg gcctccaccc ttgcctccgg cgtgccttca 180 cggttttctg gatccggctc gggaaccgac ttcaccctca ccatctcgtc gctccaaccc 240 gaggacttcg caacctacta ctgccaacag gggtatacca acagcaacat catcaacacc 300 ttcggtggcg gaactaaggt cgaaatcaag gtggccgctc cctccgtgtt catcttccca 360 ccctccgacg agcagctgaa gtccggcacc gcctccgtcg tgtgcctgct gaacaacttc 420 tacccccgcg aggccaaggt gcagtggaag gtggacaacg ccctgcagtc cggcaactcc 480 caggaatccg tcaccgagca ggactccaag gacagcacct actccctgtc ctccaccctg 540 accctgtcca aggccgacta cgagaagcac aaggtgtacg cctgcgaagt gacccaccag 600 ggcctgtcca gccccgtgac caagtcctt aaccggggcg agtgc 645 <210> 23 <211> 110 <212> PRT <213> artificial sequence <220> <223> 10236 gL8 VL <400> 23 Ala Tyr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Asn Ser Asn 85 90 95 Ile Ile Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 24 <211> 330 <212> DNA <213> artificial sequence <220> <223> 10236 gL8 VL nucleotide <400> 24 gcgtatcaga tgactcagag cccgtccagc ctgtccgcgt ccgtgggaga tcgcgtgact 60 atcacgtgtc aggcctcaca atccattagc tcctggctgg cctggtacca gcagaagcca 120 gggaaggctc cgaagctgct gatctacctg gcctccaccc ttgcctccgg cgtgccttca 180 cggttttctg gatccggctc gggaaccgac ttcaccctca ccatctcgtc gctccaaccc 240 gaggacttcg caacctacta ctgccaacag gggtatacca acagcaacat catcaacacc 300 ttcggtggcg gaactaaggt cgaaatcaag 330 <210> 25 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> 10236 gL8 light chain <400> 25 Ala Tyr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Asn Ser Asn 85 90 95 Ile Ile Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 26 <211> 645 <212> DNA <213> Artificial sequence <220> <223> 10236 gL8 light chain nucleotide <400> 26 gcgtatcaga tgactcagag cccgtccagc ctgtccgcgt ccgtgggaga tcgcgtgact 60 atcacgtgtc aggcctcaca atccattagc tcctggctgg cctggtacca gcagaagcca 120 gggaaggctc cgaagctgct gatctacctg gcctccaccc ttgcctccgg cgtgccttca 180 cggttttctg gatccggctc gggaaccgac ttcaccctca ccatctcgtc gctccaaccc 240 gaggacttcg caacctacta ctgccaacag gggtatacca acagcaacat catcaacacc 300 ttcggtggcg gaactaaggt cgaaatcaag gtggccgctc cctccgtgtt catcttccca 360 ccctccgacg agcagctgaa gtccggcacc gcctccgtcg tgtgcctgct gaacaacttc 420 tacccccgcg aggccaaggt gcagtggaag gtggacaacg ccctgcagtc cggcaactcc 480 caggaatccg tcaccgagca ggactccaag gacagcacct actccctgtc ctccaccctg 540 accctgtcca aggccgacta cgagaagcac aaggtgtacg cctgcgaagt gacccaccag 600 ggcctgtcca gccccgtgac caagtccttc aaccggggcg agtgc 645 <210> 27 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 10236 gH9 VH <400> 27 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Pro Leu Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Ser Asp Ile Ile Asp Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Val Thr Ile Ser Gln Asp Ser Ser Lys Thr Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asn Asn Asp Tyr Gly Leu Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 28 <211> 351 <212> DNA <213> Artificial sequence <220> <223> 10236 gH9 VH nucleotide <400> 28 gaagtgcagc tgcaagagtc aggaccgggc ttggtcaagc ccagcggaac cctgtccctg 60 acttgtgccg tgtcggggtt cccgctgtcg aactacgcga tgtcctgggt cagacagcct 120 cccggaaagg gccttgaatg gatcggcgac atctacccaa gcgacattat tgattacgca 180 tcctgggcca agggacgcgt gaccatctcc caggactctt ccaagaccca agtgtccctc 240 aagctgtcca gcgtgaccgc tgccgacact gccgtgtact attgcgcgcg ggataacaac 300 gactacgggc tggacatctg gggccagggt accctcgtga ctgtctcgag c 351 <210> 29 <211> 444 <212> PRT <213> Artificial sequence <220> <223> 10236 gH9 heavy chain <400> 29 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Pro Leu Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Ser Asp Ile Ile Asp Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Val Thr Ile Ser Gln Asp Ser Ser Lys Thr Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asn Asn Asp Tyr Gly Leu Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 30 <211> 1332 <212> DNA <213> Artificial sequence <220> <223> 10236 gH9 heavy chain nucleotide <400> 30 gaagtgcagc tgcaagagtc aggaccgggc ttggtcaagc ccagcggaac cctgtccctg 60 acttgtgccg tgtcggggtt cccgctgtcg aactacgcga tgtcctgggt cagacagcct 120 cccggaaagg gccttgaatg gatcggcgac atctacccaa gcgacattat tgattacgca 180 tcctgggcca agggacgct gaccatctcc caggactctt ccaagaccca agtgtccctc 240 aagctgtcca gcgtgaccgc tgccgacact gccgtgtact attgcgcgcg ggataacaac 300 gactacgggc tggacatctg gggccagggt accctcgtga ctgtctcgag cgcttctaca 360 aagggcccct ccgtgttccc tctggcccct tgctccccggt ccacctccga gtctaccgcc 420 gctctgggct gcctggtcaa ggactacttc cccgagcccg tgacagtgtc ctggaactct 480 ggcgccctga cctccggcgt gcacaccttc cctgccgtgc tgcagtcctc cggcctgtac 540 tccctgtcct ccgtcgtgac cgtgccctcc tccagcctgg gcaccaagac ctacacctgt 600 aacgtggacc acaagccctc caacaccaag gtggacaagc gggtggaatc tagtacggc 660 cctccctgcc ccccctgccc tgcccctgaa tttctgggcg gaccttccgt gttcctgttc 720 cccccaaagc ccaagggac cctgatgatc tcccggaccc ccgaagtgac ctgcgtggtg 780 gtggacgtgt cccaggaaga tcccgaggtc cagttcaatt ggtacgtgga cggcgtggaa 840 gtggacgtgt cccaggaaga tcccgaggtc cagttcaatt ggtacgtgga cggcgtggaa 840 gtgcacaatg ccaagaccaa gcccagagag gaacagttca actccaccta ccgggtggtg 900 gtgcacaatg ccaagaccaa gcccagagag gaacagttca actccaccta ccgggtggtg 900 tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960 tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960 tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020 tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020 cgcgagcccc aggtgtacac cctgccccct agccaggaag agatgaccaa gaaccaggtg 1080 cgcgagcccc aggtgtacac cctgccccct agccaggaag agatgaccaa gaaccaggtg 1080 tccctgacct gtctggtcaa gggcttctac ccctccgaca ttgccgtgga atgggagtcc 1140 tccctgacct gtctggtcaa gggcttctac ccctccgaca ttgccgtgga atgggagtcc 1140 aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggctcc 1200 aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggctcc 1200 ttcttcctgt actctcggct gaccgtggac aagtcccggt ggcaggaagg caacgtcttc 1260 ttcttcctgt actctcggct gaccgtggac aagtcccggt ggcaggaagg caacgtcttc 1260 tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320 tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320 agcctgggca ag 1332 agcctgggca ag 1332 <210> 31<210> 31 <211> 117 <211> 117 <212> PRT <212> PRT <213> 人工序列 <213> Artificial sequence <220> <220> <223> 10236 gH10 VH核苷酸 <223> 10236 gH10 VH nucleotide <400> 31 <400> 31 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Pro Leu Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Ser Asp Ile Ile Asp Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Val Thr Ile Ser Val Asp Ser Ser Lys Thr Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asn Asn Asp Tyr Gly Leu Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 32 <211> 351 <212> DNA <213> Artificial sequence <220> <223> 10236 gH10 VH nucleotide <400> 32 gaagtgcagc tgcaagagtc aggaccgggc ttggtcaagc ccagcggaac cctgtccctg 60 acttgtgccg tgtcggggtt cccgctgtcg aactacgcga tgtcctgggt cagacagcct 120 cccggaaagg gccttgaatg gatcggcgac atctacccaa gcgacattat tgattacgca 180 tcctgggcca agggacgcgt gaccatctcc gtggactctt ccaagaccca agtgtccctc 240 aagctgtcca gcgtgaccgc tgccgacact gccgtgtact attgcgcgcg ggataacaac 300 gactacgggc tggacatctg gggccagggt accctcgtga ctgtctcgag c 351 <210> 33 <211> 444 <212> PRT <213> Artificial sequence <220> <223> 10236 gH10 heavy chain <400> 33 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Pro Leu Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Ser Asp Ile Ile Asp Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Val Thr Ile Ser Val Asp Ser Ser Lys Thr Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asn Asn Asp Tyr Gly Leu Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 34 <211> 1332 <212> DNA <213> Artificial Sequence <220> <223> 10236 gH10 heavy chain nucleotide <400> 34 gaagtgcagc tgcaagagtc aggaccgggc ttggtcaagc ccagcggaac cctgtccctg 60 acttgtgccg tgtcggggtt cccgctgtcg aactacgcga tgtcctgggt cagacagcct 120 cccggaaagg gccttgaatg gatcggcgac atctacccaa gcgacattat tgattacgca 180 tcctgggcca agggacgcgt gaccatctcc gtggactctt ccaagaccca agtgtccctc 240 aagctgtcca gcgtgaccgc tgccgacact gccgtgtact attgcgcgcg ggataacaac 300 gactacgggc tggacatctg gggccagggt accctcgtga ctgtctcgag cgcttctaca 360 aagggcccct ccgtgttccc tctggcccct tgctccccggt ccacctccga gtctaccgcc 420 gctctgggct gcctggtcaa ggactacttc cccgagcccg tgacagtgtc ctggaactct 480 ggcgccctga cctccggcgt gcacaccttc cctgccgtgc tgcagtcctc cggcctgtac 540 tccctgtcct ccgtcgtgac cgtgccctcc tccagcctgg gcaccaagac ctacacctgt 600 aacgtggacc acaagccctc caacaccaag gtggacaagc gggtggaatc tagtacggc 660 cctccctgcc ccccctgccc tgcccctgaa tttctgggcg gaccttccgt gttcctgttc 720 cccccaaagc ccaagggac cctgatgatc tcccggaccc ccgaagtgac ctgcgtggtg 780 840 gtgcacaatg ccaagaccaa gcccagagag gaacagttca actccaccta ccgggtggtg 900 tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960 tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020 cgcgagcccc aggtgtacac cctgccccct agccaggaag agatgaccaa gaaccaggtg 1080 tccctgacct gtctggtcaa gggcttctac ccctccgaca ttgccgtgga atgggagtcc 1140 aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggctcc 1200 ttcttcctgt actctcggct gaccgtggac aagtcccggt ggcaggaagg caacgtcttc 1260 tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320 agcctgggca ag 1332 <210> 35 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 10236 gH11 VH Nucleotide <400> 35 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Pro Leu Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Ser Asp Ile Ile Asp Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Val Thr Ile Ser Gln Asp Lys Ser Lys Thr Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asn Asn Asp Tyr Gly Leu Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 36 <211> 351 <212> DNA <213> Artificial sequence <220> <223> 10236 gH11 VH nucleotide <400> 36 gaagtgcagc tgcaagagtc aggaccgggc ttggtcaagc ccagcggaac cctgtccctg 60 acttgtgccg tgtcggggtt cccgctgtcg aactacgcga tgtcctgggt cagacagcct 120 cccggaaagg gccttgaatg gatcggcgac atctacccaa gcgacattat tgattacgca 180 tcctgggcca agggacgcgt gaccatctcc caggacaagt ccaagaccca agtgtccctc 240 aagctgtcca gcgtgaccgc tgccgacact gccgtgtact attgcgcgcg ggataacaac 300 gactacgggc tggacatctg gggccagggt accctcgtga ctgtctcgag c 351 <210> 37 <211> 444 <212> PRT <213> Artificial sequence <220> <223> 10236 hG11 heavy chain <400> 37 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Pro Leu Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Ser Asp Ile Ile Asp Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Val Thr Ile Ser Gln Asp Lys Ser Lys Thr Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asn Asn Asp Tyr Gly Leu Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 38 <211> 1332 <212> DNA <213> Artificial sequence <220> <223> 10236 gH11 heavy chain nucleotide <400> 38 gaagtgcagc tgcaagagtc aggaccgggc ttggtcaagc ccagcggaac cctgtccctg 60 acttgtgccg tgtcggggtt cccgctgtcg aactacgcga tgtcctgggt cagacagcct 120 cccggaaagg gccttgaatg gatcggcgac atctacccaa gcgacattat tgattacgca 180 tcctgggcca agggacgcgt gaccatctcc caggacaagt ccaagaccca agtgtccctc 240 aagctgtcca gcgtgaccgc tgccgacact gccgtgtact attgcgcgcg ggataacaac 300 gactacgggc tggacatctg gggccagggt accctcgtga ctgtctcgag cgcttctaca 360 aagggcccct ccgtgttccc tctggcccct tgctcccggt ccacctccga gtctaccgcc 420 gctctgggct gcctggtcaa ggactacttc cccgagcccg tgacagtgtc ctggaactct 480 ggcgccctga cctccggcgt gcacaccttc cctgccgtgc tgcagtcctc cggcctgtac 540 tccctgtcct ccgtcgtgac cgtgccctcc tccagcctgg gcaccaagac ctacacctgt 600 aacgtggacc acaagccctc caacaccaag gtggacaagc gggtggaatc taagtacggc 660 cctccctgcc ccccctgcc tgcccctgaa tttctgggcg gaccttccgt gttcctgttc 720 cccccaaagc ccaaggacac cctgatgatc tcccggaccc ccgaagtgac ctgcgtggtg 780 gtggacgtgt cccaggaaga tcccgaggtc cagttcaatt ggtacgtgga cggcgtggaa 840 gtgcacaatg ccaagaccaa gcccagagag gaacagttca actccaccta ccgggtggtg 900 tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960 tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020 cgcgagcccc aggtgtacac cctgccccct agccaggaag agatgaccaa gaaccaggtg 1080 tcctgacct gtctggtcaa gggcttctac ccctccgaca ttgccgtgga atgggagtcc 1140 aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggctcc 1200 ttcttcctgt actctcggct gaccgtggac aagtcccggt ggcaggaagg caacgtcttc 1260 tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320 agcctgggca ag 1332 <210> 39 <211> 117 <212> PRT <213> Artificial sequence <220> <223> 10236 gH12 VH <400> 39 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Pro Leu Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Ser Asp Ile Ile Asp Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Val Thr Ile Ser Gln Asp Ser Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asn Asn Asp Tyr Gly Leu Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 40 <211> 351 <212> DNA <213> Artificial sequence <220> <223> 10236 gH12 VH nucleotides <400> 40 gaggtgcagc ttcaggaatc cggacccggt ctggtcaagc cgagcggaac cctgtcactg 60 gaggtgcagc ttcaggaatc cggacccggt ctggtcaagc cgagcggaac cctgtcactg 60 acttgcgcgg tgtcgggctt ccccctgtcc aattacgcca tgtcatgggt ccggcaacca 120 acttgcgcgg tgtcgggctt ccccctgtcc aattacgcca tgtcatgggt ccggcaacca 120 cctgggaaag ggttggagtg gattggcgac atctacccga gcgacatcat tgattacgcc 180 cctgggaaag ggttggagtg gattggcgac atctacccga gcgacatcat tgattacgcc 180 tcgtgggcca agggtagagt gaccatcagc caggactcct ccaagaacca agtgtcgctg 240 tcgtgggcca agggtagagt gaccatcagc caggactcct ccaagaacca agtgtcgctg 240 aagctctcct ccgtgaccgc agccgatacc gctgtgtact attgtgcccg cgacaacaac 300 aagctctcct ccgtgaccgc agccgatacc gctgtgtact attgtgcccg cgacaacaac 300 gactacggcc tggatatctg gggacaggga accctcgtga ctgtctcgag c 351 gactacggcc tggatatctg gggacaggga accctcgtga ctgtctcgag c 351 <210> 41<210> 41 <211> 444<211> 444 <212> PRT<212> PRT <213> 人工序列<213> Artificial sequence <220><220> <223> 10236 gH12重链 <223> 10236 gH12 heavy chain <400> 41 <400> 41 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 1 5 10 15 Thr Leu Ser ...

Claims

1. An antibody that binds to kallikrein 5 (KLK5), wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein: a. The variable light chain comprises CDR-L1 consisting of SEQ ID NO: 1, SEQ ID NO: 62, or SEQ ID NO: 63, CDR-L2 consisting of SEQ ID NO: 2, and CDR-L3 consisting of SEQ ID NO: 3; and b. The variable heavy chain comprises CDR-H1 consisting of SEQ ID NO: 4, CDR-H2 consisting of SEQ ID NO: 5, and CDR-H3 consisting of SEQ ID NO:

6.

2. The antibody according to claim 1, wherein: a. The variable light chain comprises CDR-L1 consisting of SEQ ID NO: 1, CDR-L2 consisting of SEQ ID NO: 2, and CDR-L3 consisting of SEQ ID NO: 3; and b. The variable heavy chain comprises CDR-H1 consisting of SEQ ID NO: 4, CDR-H2 consisting of SEQ ID NO: 5, and CDR-H3 consisting of SEQ ID NO:

6.

3. The antibody according to claim 1, wherein the antibody binds to an epitope of human KLK5, comprising the amino acid residues Arg87, Ala107, Arg110, Lys111, Lys112, Val113, Val137, Lys138, Ser139, Ile140, Pro141, His142, Pro143, Tyr145, Ser146, and His147 as described in SEQ ID NO:

51.

4. The antibody according to claim 3, wherein the epitope is characterized by X-ray crystallography.

5. The antibody according to any one of claims 1 to 4, wherein the antibody inhibits or reduces the protease activity of KLK5.

6. The antibody according to any one of claims 1 to 4, wherein the antibody binds to KLK5 when KLK5 binds to LEKTI or a LEKTI fragment.

7. The antibody according to any one of claims 1 to 4, wherein the antibody does not compete with LEKTI or the LEKTI fragment for binding to KLK5.

8. The antibody according to any one of claims 1 to 4, wherein the antibody forms a complex with KLK5 bound to LEKTI or the LEKTI fragment.

9. The antibody according to claim 6, wherein the LEKTI fragment is a human LEKTI domain 5 comprising amino acids 1 to 64 of SEQ ID NO: 54 or a LEKTI domain 8 comprising amino acids 1 to 71 of SEQ ID NO:

61.

10. The antibody according to any one of claims 1 to 4, wherein the antibody binds to human KLK5 and cynomolgus monkey KLK5.

11. The antibody of claim 10, wherein the antibody binds to human KLK5 comprising SEQ ID NO: 53 and cynomolgus monkey KLK5 comprising SEQ ID NO:

60.

12. The antibody according to any one of claims 1 to 4, 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).

13. The antibody according to claim 3 or 4, wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein: a. The variable light chain comprises CDR-L1 consisting of SEQ ID NO: 1, SEQ ID NO: 62, or SEQ ID NO: 63, CDR-L2 consisting of SEQ ID NO: 2, and CDR-L3 consisting of SEQ ID NO: 3; and b. The variable heavy chain comprises CDR-H1 consisting of SEQ ID NO: 4, CDR-H2 consisting of SEQ ID NO: 5, and CDR-H3 consisting of SEQ ID NO:

6.

14. The antibody of claim 13, wherein the antibody comprises a variable light chain and a variable heavy chain, and wherein: a. The variable light chain comprises CDR-L1 consisting of SEQ ID NO: 1, CDR-L2 consisting of SEQ ID NO: 2, and CDR-L3 consisting of SEQ ID NO: 3; and b. The variable heavy chain comprises CDR-H1 consisting of SEQ ID NO: 4, CDR-H2 consisting of SEQ ID NO: 5, and CDR-H3 consisting of SEQ ID NO:

6.

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

16. The antibody according to any one of claims 1 to 4, wherein the antibody is a full-length antibody.

17. The antibody of claim 16, wherein the full-length antibody is selected from IgG1, IgG4 or IgG4P.

18. The antibody according to any one of claims 1 to 4, wherein the antibody is selected from Fab, Fab', F(ab')2, scFv, dAb, or V. HH .

19. The antibody according to any one of claims 1 to 4, wherein the antibody comprises: a. A variable light chain comprising SEQ ID NO: 7 or 11 or 15 or 19 or 23; and / or b. A variable heavy chain containing SEQ ID NO: 9 or 27 or 31 or 35 or 39 or 43.

20. The antibody according to any one of claims 1 to 4, wherein the antibody comprises: Contains the light chain variable region of SEQ ID NO: 15; and Contains the heavy chain variable region of SEQ ID NO:

39.

21. The antibody according to any one of claims 1 to 4, wherein the antibody comprises: a. A light chain containing SEQ ID NO: 13 or 17 or 21 or 25; and b. Heavy chains containing SEQ ID NO: 29 or 33 or 37 or 41 or 45.

22. The antibody according to any one of claims 1 to 4, wherein the antibody comprises: A light chain containing SEQ ID NO: 17; and The heavy chain containing SEQ ID NO:

41.

23. The antibody according to claim 19, wherein the amino acid residue glutamine (Gln; Q) at position 24 of L-CDR1, referring to SEQ ID NO: 15 or 17, is replaced by arginine (Arg; R) or lysine (Lys; K).

24. The antibody according to any one of claims 1 to 4, wherein KLK5 is human KLK5 comprising SEQ ID NO: 51 or 52 or 53 or cynomolgus monkey KLK5 comprising SEQ ID NO:

60.

25. The antibody according to any one of claims 1 to 4, wherein the antibody comprises: a. Heavy chains having at least 95% identity or similarity to sequences according to SEQ ID NO: 29, 33, 37, 41, or 45; and / or b. A light chain having at least 95% identity or similarity to the sequence according to SEQ ID NO: 13 or 17 or 21 or 25.

26. An isolated polynucleotide encoding the antibody according to any one of claims 1 to 4.

27. The isolated polynucleotide of claim 26, wherein the polynucleotide encodes: a. Light chain variable region, wherein the polynucleotide: i. At least 90% identical to nucleotides 1 to 330 of SEQ ID NO: 8 or SEQ ID NO: 8, or nucleotides 1 to 330 of SEQ ID NO: 12 or SEQ ID NO: 12, or SEQ ID NO: 16, or SEQ ID NO: 20, or SEQ ID NO: 24, or SEQ ID NO: 64, or SEQ ID NO: 66; or ii. Nucleotides 1 to 330 containing SEQ ID NO: 8 or SEQ ID NO: 8, or nucleotides 1 to 330 containing SEQ ID NO: 12 or SEQ ID NO: 12, or SEQ ID NO: 16, or SEQ ID NO: 20, or SEQ ID NO: 24, or SEQ ID NO: 64, or SEQ ID NO: 66; or iii. Consistent essentially of nucleotides 1 to 330 of SEQ ID NO: 8 or SEQ ID NO: 8, or nucleotides 1 to 330 of SEQ ID NO: 12 or SEQ ID NO: 12, or SEQ ID NO: 16, or SEQ ID NO: 20, or SEQ ID NO: 24, or SEQ ID NO: 64, or SEQ ID NO: 66; or b. Heavy chain variable region, wherein the polynucleotide: i. At least 90% identical to SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44; or ii. Containing SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44; or iii. Basically composed of SEQ ID NO: 10 or 28 or 32 or 36 or 40 or 44; or c. The light chain, wherein the polynucleotide is: i. At least 90% identical to SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104; or ii. Containing SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104; or iii. Basically composed of SEQ ID NO: 14 or 18 or 22 or 26 or 65 or 67 or 100 or 101 or 102 or 103 or 104; or d. Heavy chain, wherein the polynucleotide is: i. At least 90% identical to SEQ ID NO: 30 or 34 or 38 or 42 or 46; or ii. Containing SEQ ID NO: 30 or 34 or 38 or 42 or 46; or iii. Basically composed of SEQ ID NO: 30 or 34 or 38 or 42 or 46.

28. A cloning or expression vector comprising one or more of the polynucleotides of claim 26.

29. A host cell comprising: a. One or more polynucleotides as described in claim 26, or b. One or more expression vectors of claim 28.

30. A method for producing an antibody according to any one of claims 1 to 4, comprising culturing a host cell according to claim 29 under conditions suitable for producing the antibody and isolating the antibody produced by the host cell.

31. A pharmaceutical composition comprising the antibody of any one of claims 1 to 4 and one or more pharmaceutically acceptable carriers, excipients or diluents.

32. The antibody according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 31, characterized in that, It is used for treatment.

33. The antibody according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 31, characterized in that, It is used to treat diseases characterized by KLK5 dysregulation or KLK5 inhibition dysregulation.

34. The antibody used according to claim 33, wherein the disease is selected from Netherton syndrome, atopic dermatitis, ichthyosis, rosacea, asthma, or cancer.

35. The antibody used according to claim 34, wherein the disease is selected from ovarian cancer or bladder cancer or a combination thereof.

36. The antibody used according to claim 34, wherein the disease is Netherton syndrome.

37. The antibody used according to claim 34, wherein the disease is atopic dermatitis.

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