Dual inhibitory kallikrein antibody and its use

Abstract

Aspects of this application provide dual inhibitory anti-KLK5 / KLK7 antibodies and methods of using them to promote barrier function, reduce inflammation, and treat conditions such as Netherton syndrome, eosinophilic esophagitis, and atopic dermatitis. Certain aspects of this disclosure relate to the recognition that a loss of balance between endogenous KLK proteases and associated protease inhibitors can lead to barrier dysfunction and induce inflammation (see, for example, Figure 1), which can result in inflammatory conditions, such as Netherton syndrome, eosinophilic esophagitis, and atopic dermatitis.

Description

[Technical Field]

[0001] Related applications This application claims priority under 35 United States Code § 119(e) to U.S. Provisional Patent Application, USSN 63 / 489,414 (filed 9 March 2023) and U.S. Provisional Patent Application, USSN 63 / 614,102 (filed 22 December 2023), the entire contents of each of which are incorporated herein by reference.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (A104770001WO00-SEQ-LJG.xml; size: 36,826 bytes; and creation date: March 8, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] Kallikrein (KLK) enzymes regulate desquamation and innate immunity, supporting skin homeostasis and wound healing. In healthy skin, the outermost layer of the epidermis is regularly shed through a KLK-driven proteolytic cascade, resulting in the degradation of corneodesmosomes and desquamation. KLK5 is understood to be the major activator of this proteolytic cascade. Self-activated KLK5 enzymatically converts pro-KLK7 and pro-KLK14 into their active forms, stimulating a positive feedback loop that leads to the production of more pro-KLK5 via KLK14. These KLK enzymes are suppressed by endogenous serine protease inhibitors, such as lymphoepithelial Cazal-type-associated inhibitors. Dysregulation of KLK, including KLK5 and KLK7, is associated with skin disorders, inflammatory diseases, and cancer. For example, hyperactive kallikrein 5 and 7 can cause both hereditary and spontaneous epidermal barrier disorders (e.g., Netherton syndrome, eosinophilic esophagitis, atopic dermatitis). [Overview of the project] [Means for solving the problem]

[0004] Certain aspects of this disclosure relate to the recognition that a loss of balance between endogenous KLK proteases and associated protease inhibitors can cause barrier dysfunction and induce inflammation (see, for example, Figure 1), which can lead to inflammatory conditions such as Netherton syndrome, eosinophilic esophagitis, and atopic dermatitis. Some embodiments provide methods and related compositions useful for inhibiting KLK5 and KLK7 for the purpose of restoring disease severity, improving barrier function, and reducing inflammation. Specifically, aspects of this disclosure provide dual inhibitory antibodies (referred to as anti-KLK5 / KLK7 antibodies) that target KLK5 and KLK7 and have high binding affinity and specificity to both KLK5 and KLK7 via a common individual antigen-specific binding site. Accordingly, in some embodiments, the present disclosure provides methods, antibodies for use in these methods, and related antibody compositions for treating conditions associated with KLK5 and KLK7 dysregulation, such as Netherton syndrome, atopic dermatitis (with and without filaggrin mutations), eosinophilic esophagitis, prurigo nodosa, chronic pruritus of unknown cause (CPUO), asthma (e.g., KLK5-associated asthma), and ichthyosis vulgaris.

[0005] In some embodiments, the Disclosure provides a dual inhibitory antibody that specifically binds to KLK5 and KLK7, the dual inhibitory antibody comprising HC CDR1, HC CDR2 and HC CDR3 heavy chain variable domains having the amino acid sequence of SEQ ID NO: 7, and LC CDR1, LC CDR2 and LC CDR3 light chain variable domains having the amino acid sequence of SEQ ID NO: 8.

[0006] In some embodiments, the Disclosure provides a dual inhibitory antibody that specifically binds to KLK5 and KLK7, the dual inhibitory antibody comprising HC CDR1, HC CDR2 and HC CDR3 heavy chain variable domains having the amino acid sequence of SEQ ID NO: 13, and LC CDR1, LC CDR2 and LC CDR3 light chain variable domains having the amino acid sequence of SEQ ID NO: 14.

[0007] In some embodiments, the present disclosure provides a dual inhibitory antibody that specifically binds to KLK5 and KLK7, and the dual inhibitory antibody comprises HC CDR1, HC CDR2, and HC CDR3 of a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 17, and LC CDR1, LC CDR2, and LC CDR3 of a light chain variable domain having the amino acid sequence of SEQ ID NO: 14.

[0008] In some embodiments, the present disclosure provides a dual inhibitory antibody that specifically binds to KLK5 and KLK7, and the dual inhibitory antibody comprises HC CDR1, HC CDR2, and HC CDR3 of a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 21, and LC CDR1, LC CDR2, and LC CDR3 of a light chain variable domain having the amino acid sequence of SEQ ID NO: 14.

[0009] In some embodiments, the dual inhibitory antibody comprises HC CDR1 having the amino acid sequence of SEQ ID NO: 1, HC CDR2 having the amino acid sequence of SEQ ID NO: 2, HC CDR3 having the amino acid sequence of SEQ ID NO: 3, LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0010] In some embodiments, the dual inhibitory antibody comprises HC CDR1 having the amino acid sequence of SEQ ID NO: 9, HC CDR2 having the amino acid sequence of SEQ ID NO: 10, HC CDR3 having the amino acid sequence of SEQ ID NO: 11, LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0011] In some embodiments, the dual inhibitory antibody comprises an HC CDR1 having the amino acid sequence of SEQ ID NO: 9, an HC CDR2 having the amino acid sequence of SEQ ID NO: 15, an HC CDR3 having the amino acid sequence of SEQ ID NO: 16, an LC CDR1 having the amino acid sequence of SEQ ID NO: 4, an LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and an LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0012] In some embodiments, the dual inhibitory antibody comprises an HC CDR1 having the amino acid sequence of SEQ ID NO: 18, an HC CDR2 having the amino acid sequence of SEQ ID NO: 19, an HC CDR3 having the amino acid sequence of SEQ ID NO: 20, an LC CDR1 having the amino acid sequence of SEQ ID NO: 4, an LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and an LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0013] In some embodiments, the dual inhibitory antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 8.

[0014] In some embodiments, the dual inhibitory antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 13 and a VL comprising the amino acid sequence of SEQ ID NO: 14.

[0015] In some embodiments, the dual inhibitory antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 14.

[0016] In some embodiments, the dual inhibitory antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 21 and a VL comprising the amino acid sequence of SEQ ID NO: 14.

[0017] In some aspects, the present disclosure provides a dual inhibitory antibody comprising any one of the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and / or LC CDR3 of the dual inhibitory antibodies listed in Tables 1a and 1b.

[0018] In some embodiments, the disclosure provides a dual inhibitory antibody comprising one of the dual inhibitory antibodies listed in Tables 1a and 1b, which is VH and / or VL.

[0019] In some embodiments, the dual inhibitory antibody binds to the active sites of KLK5 and KLK7.

[0020] In some embodiments, the dual inhibitory antibody competes with SPINK5 and / or leupeptin for binding to the KLK5 and KLK7 active sites.

[0021] In some embodiments, a dual inhibitory antibody binds to the active forms of KLK5 and KLK7, but not to the inactive forms of KLK5 or KLK7. In some embodiments, the antibody specifically binds to the active forms of KLK5 and KLK7, but not specifically binds to the inactive forms of KLK5 or KLK7. In some embodiments, the antibody detectably binds to the active forms of KLK5 and KLK7, but does not detectably bind to the inactive forms of KLK5 or KLK7 under the same or equivalent conditions.

[0022] In some embodiments, the dual inhibitory antibody inhibits the protease activity of KLK5 and KLK7.

[0023] In some embodiments, the antibody is not cleaved in the heavy chain by KLK5 or KLK7 upon binding to KLK5 or KLK7.

[0024] In some embodiments, the anti-KLK5 / KLK7 antibody is not a bispecific antigen-binding molecule in which KLK5 binding is conferred by one binding site within the antibody and KLK7 binding is conferred by a different binding site.

[0025] In some embodiments, the anti-KLK5 / KLK7 antibody is a multispecific antigen-binding molecule that further includes an antigen-binding domain that binds to an antigen other than KLK5 or KLK7.

[0026] In some embodiments, this disclosure provides compositions comprising a dual inhibitory antibody and an acceptable carrier as described herein.

[0027] In some embodiments, this disclosure provides nucleic acids encoding dual inhibitory antibodies as described herein.

[0028] In some embodiments, the Disclosure provides a method for treating a skin barrier defect, the method comprising administering to a subject an effective amount of a dual inhibitory antibody or composition thereof as described herein. In some embodiments, the skin barrier defect is associated with Netherton syndrome, atopic dermatitis, eosinophilic esophagitis, prurigo nodosa, chronic pruritus of unknown cause (CPUO), dry skin, asthma (particularly KLK5), ichthyosis vulgaris, or itching or chronic itching.

[0029] In some embodiments, the Disclosure provides dual inhibitory antibodies against KLK5 and KLK7 (i.e., anti-KLK5 / KLK7 antibodies) or compositions thereof for use in methods of treating skin barrier defects. In some embodiments, the anti-KLK5 / KLK7 antibodies or compositions thereof are for use in methods of treating Netherton syndrome, atopic dermatitis, eosinophilic esophagitis, prurigo nodosa, chronic pruritus of unknown cause (CPUO), dry skin, asthma (particularly KLK5), ichthyosis vulgaris, or skin barrier defects associated with itching or chronic itching.

[0030] The preceding and other aspects, embodiments, actions, functions, features, and embodiments of this instruction can be better understood from the following description in conjunction with the accompanying drawings.

[0031] The accompanying drawings, incorporated herein and constituting part thereof, illustrate certain embodiments and, together with the written description, are useful in providing non-limiting examples of certain aspects of the compositions and methods disclosed herein. [Brief explanation of the drawing]

[0032] [Figure 1]This chart shows abnormal protease activation (e.g., abnormal KLK5, KLK7, and KLK14) that can lead to skin barrier deficiency-related diseases.

[0033] [Figure 2] A shows the relative response curve of KLK5 / 7-dual-Ab4 and comparative antibody #1, which bind to either the active form of human KLK5 (huKLK5) or the proform of huKLK5. B shows the relative response curve of KLK5 / 7-dual-Ab4 and comparative antibody #1, which bind to either the active form or the proform of huKLK7. Comparative antibody #1 shows binding to both forms, but KLK5 / 7-dual-Ab4 specifically binds to active huKLK5.

[0034] [Figure 3A] These are SDS-PAGE results showing the antibodies of control anti-KLK5-Ab1, KLK5 / 7-dual-Ab1, KLK5 / 7-dual-Ab2, KLK5 / 7-dual-Ab3, KLK5 / 7-dual-Ab4, and KLK5 / 7-dual-Ab5, either alone or after incubation with KLK5. Control anti-KLK5-Ab1 is a positive control for KLK5 cleavage activity, as evidenced by two bands of approximately 38 kDa and 12 kDa weight (but not cleaved by KLK7). There is no cleavage of the other antibodies by either KLK5 or KLK7. [Figure 3B] These are SDS-PAGE results showing the control anti-KLK5-Ab1, KLK5 / 7-dual-Ab1, KLK5 / 7-dual-Ab2, KLK5 / 7-dual-Ab3, KLK5 / 7-dual-Ab4, and KLK5 / 7-dual-Ab5 antibodies alone or after incubation with KLK7. Control anti-KLK5-Ab1 is a positive control for KLK5 cleavage activity, as evidenced by two bands of approximately 38 kDa and 12 kDa weight (but not cleaved by KLK7). There is no cleavage of the other antibodies by either KLK5 or KLK7.

[0035] [Figure 4] The inhibitory activity of the dual-specific antibodies KLK5 / 7-dual-Ab2, KLK5 / 7-dual-Ab3, and KLK5 / 7-dual-Ab4 against other KLK family members and related proteases is shown compared to the isotype control. The test antibodies do not specifically inhibit non-KLK5 / 7 family members or related proteases because their relative activity is not higher than that of the isotype control.

[0036] [Figure 5A] The relative responses of antibodies KLK5 / 7-dual-Ab2 and KLK5 / 7-dual-Ab4 to huKLK5 and huKLK7, respectively, in the presence of serine protease inhibitors PMSF, leupeptin, or SPINK5 are shown. [Figure 5B] The relative responses of antibodies KLK5 / 7-dual-Ab2 and KLK5 / 7-dual-Ab4 to huKLK5 and huKLK7, respectively, in the presence of serine protease inhibitors PMSF, leupeptin, or SPINK5 are shown.

[0037] [Figure 6-1] The graphs show the competitive binding of KLK5 (top) and KLK7 (bottom) between anti-KLK5 / 7 antibodies and SPINK5, which bind to the active sites of KLK5 and KLK7. In the left graph, when the antibody KLK5 / 7-dual-Ab4 is bound to the tip and KLK5 or KLK7 is added, the binding curve increases. Adding a second anti-KLK5 / 7 antibody or SPINK5 (indicated in parentheses and "mAb#2") does not increase binding. In the right graph, when SPINK5 is bound to the tip and either KLK5 or KLK7 is added, the binding curve increases. Adding an anti-KLK5 / 7 antibody (indicated in parentheses and "mAb#2") does not increase binding because SPINK5 is already bound to the active site of KLK5 or KLK7. [Figure 6-2]The graphs show the competitive binding of KLK5 (top) and KLK7 (bottom) between anti-KLK5 / 7 antibodies and SPINK5, which bind to the active sites of KLK5 and KLK7. In the left graph, when the antibody KLK5 / 7-dual-Ab4 is bound to the tip and KLK5 or KLK7 is added, the binding curve increases. Adding a second anti-KLK5 / 7 antibody or SPINK5 (indicated in parentheses and "mAb#2") does not increase binding. In the right graph, when SPINK5 is bound to the tip and either KLK5 or KLK7 is added, the binding curve increases. Adding an anti-KLK5 / 7 antibody (indicated in parentheses and "mAb#2") does not increase binding because SPINK5 is already bound to the active site of KLK5 or KLK7.

[0038] [Figure 7] Image A shows the effect of treatment with 30 mg / kg of KLK5 / 7-dual-Ab4 on stratum corneum thickness in the MC903 atopic dermatitis mouse model. Treatment with KLK5 / 7-dual-Ab4 significantly reduced the thickness. Image B shows the effect of treatment with 3 mg / kg of KLK5 / 7-dual-Ab4 on stratum corneum thickness in the MC903 atopic dermatitis mouse model. Treatment with KLK5 / 7-dual-Ab4 significantly reduced the thickness.

[0039] [Figure 8A] This graph shows the therapeutic efficacy of anti-KLK5 / 7 antibodies against disease presentation in a mouse model of Nc / Nga atopic dermatitis, as measured by clinical scores. [Figure 8B] This graph shows the therapeutic efficacy of anti-KLK5 / 7 antibodies against disease presentation in a mouse model of Nc / Nga atopic dermatitis, as measured by stratum corneum thickness (ear thickness). [Figure 8C] This graph shows the therapeutic efficacy of anti-KLK5 / 7 antibodies against disease presentation in an Nc / Nga atopic dermatitis mouse model, as measured by itching. [Figure 8D]This graph shows the therapeutic efficacy of anti-KLK5 / 7 antibodies against disease presentation in a mouse model of Nc / Nga atopic dermatitis, as measured by histological scores. [Figure 8E] This graph shows the therapeutic efficacy of anti-KLK5 / 7 antibodies against disease presentation in a mouse model of Nc / Nga atopic dermatitis, as measured by epidermal area. [Figure 8F] This graph shows the therapeutic efficacy of anti-KLK5 / 7 antibodies against disease presentation in a mouse model of Nc / Nga atopic dermatitis, as measured by IgE antibody production. [Figure 8G] This graph shows the therapeutic efficacy of anti-KLK5 / 7 antibodies against disease presentation in a mouse model of Nc / Nga atopic dermatitis, as measured by histological scores. [Figure 8H] This graph shows the therapeutic efficacy of anti-KLK5 / 7 antibodies against disease presentation in a mouse model of Nc / Nga atopic dermatitis, as measured by stratum corneum thickness (ear thickness). [Figure 8I] This graph shows the therapeutic efficacy of anti-KLK5 / 7 antibodies against disease presentation in an Nc / Nga atopic dermatitis mouse model, as measured by itching.

[0040] [Figure 9A] The results of administering anti-KLK5 / 7 antibodies to a flaky-tail mouse model, measured by epidermal area, are shown. [Figure 9B] The results of administering anti-KLK5 / 7 antibodies to a flaky-tail mouse model, measured by parakeratosis (a type of keratinization), are shown. [Figure 9C] The results of administering anti-KLK5 / 7 antibodies to a flaky-tail mouse model, measured by spongiform changes (histological characteristics of the epidermis in eczema), are shown. [Figure 9D] The results of administering anti-KLK5 / 7 antibodies to a flaky-tail mouse model, measured by IL-4 production in the ear, are shown. [Figure 9E]The results of administering anti-KLK5 / 7 antibodies to a flaky-tail mouse model, measured by TNFα production in the ear, are shown.

[0041] [Figure 10A] Figure 10C shows representative histological images and summarized measurements of keratosis in disease-induced human epidermal equivalent gas-liquid interface cultures (MC903 models) treated with KLK5 / 7-dual-Ab4 (Figure 10C), comparative antibody #1 (Figure 10D), and comparative antibody #3 (Figure 10E), compared with controls without MC903 (Figure 10A) and with MC903 + control IgG (Figure 10B). A quantitative summary of stratum corneum thickness under each condition is shown in Figure 10F. [Figure 10B] Figure 10C shows representative histological images and summarized measurements of keratosis in disease-induced human epidermal equivalent gas-liquid interface cultures (MC903 models) treated with KLK5 / 7-dual-Ab4 (Figure 10C), comparative antibody #1 (Figure 10D), and comparative antibody #3 (Figure 10E), compared with controls without MC903 (Figure 10A) and with MC903 + control IgG (Figure 10B). A quantitative summary of stratum corneum thickness under each condition is shown in Figure 10F. [Figure 10C] Figure 10C shows representative histological images and summarized measurements of keratosis in disease-induced human epidermal equivalent gas-liquid interface cultures (MC903 models) treated with KLK5 / 7-dual-Ab4 (Figure 10C), comparative antibody #1 (Figure 10D), and comparative antibody #3 (Figure 10E), compared with controls without MC903 (Figure 10A) and with MC903 + control IgG (Figure 10B). A quantitative summary of stratum corneum thickness under each condition is shown in Figure 10F. [Figure 10D] Figure 10C shows representative histological images and summarized measurements of keratosis in disease-induced human epidermal equivalent gas-liquid interface cultures (MC903 models) treated with KLK5 / 7-dual-Ab4 (Figure 10C), comparative antibody #1 (Figure 10D), and comparative antibody #3 (Figure 10E), compared with controls without MC903 (Figure 10A) and with MC903 + control IgG (Figure 10B). A quantitative summary of stratum corneum thickness under each condition is shown in Figure 10F. [Figure 10E]Figure 10C shows representative histological images and summarized measurements of keratosis in disease-induced human epidermal equivalent gas-liquid interface cultures (MC903 models) treated with KLK5 / 7-dual-Ab4 (Figure 10C), comparative antibody #1 (Figure 10D), and comparative antibody #3 (Figure 10E), compared with controls without MC903 (Figure 10A) and with MC903 + control IgG (Figure 10B). A quantitative summary of stratum corneum thickness under each condition is shown in Figure 10F. [Figure 10F] Figure 10C shows representative histological images and summarized measurements of keratosis in disease-induced human epidermal equivalent gas-liquid interface cultures (MC903 models) treated with KLK5 / 7-dual-Ab4 (Figure 10C), comparative antibody #1 (Figure 10D), and comparative antibody #3 (Figure 10E), compared with controls without MC903 (Figure 10A) and with MC903 + control IgG (Figure 10B). A quantitative summary of stratum corneum thickness under each condition is shown in Figure 10F.

[0042] [Figure 11] This is the crystal structure of the KLK5 / 7-dual-Ab1 antibody Fab, which binds to the active site of the StoA variant of the human KLK7 antigen. The heavy chain is shaded in light gray, the Fab light chain in dark gray, and the antigen in black.

[0043] [Figure 12] The heavy chain CDR3 loop residue of KLK5 / 7-dual-Ab1 (dark gray) occupies the binding pocket of the active site of the human KLK7 antigen (light gray). The three catalytic residues of the antigen, as well as the tryptophan residue marking the floor of the S4 binding pocket, are shown in stick representation. [Modes for carrying out the invention]

[0044] This disclosure is at least in part based on the development of dual inhibitory antibodies and variants thereof targeting KLK5 and KLK7. These dual inhibitory antibodies target KLK5 and KLK7 via a common individual antigen-specific binding site. Such dual inhibitory antibodies have high binding affinity and specificity to KLK5 and KLK7 (anti-KLK5 / KLK7 antibodies). Also provided are methods for using anti-KLK5 / KLK7 antibodies and variants thereof in research, diagnostic / detection, and therapeutic applications, as well as anti-KLK5 / KLK7 antibodies for use in such methods.

[0045] The preceding and other aspects, embodiments, actions, functions, features, and embodiments of this instruction can be better understood from the following description in conjunction with the accompanying drawings.

[0046] I. Definition To administer: As used herein, the terms “to administer” or “to administer” mean to provide an antibody or a composition thereof to a subject in a physiologically and / or pharmacologically useful manner (for example, to treat a condition of the subject).

[0047] Affinity-matured antibodies: In this specification, the term “affinity-matured antibody” is used to refer to an antibody obtained by adding one or more modifications to one or more CDRs that result in improved affinity (e.g., KD, kd, or ka) of the antibody to a target antigen compared to an unmodified parent antibody. Exemplary affinity-matured antibodies may, in some embodiments, have nanomolar or even picomolar affinity to a target antigen. Various procedures are available for generating affinity-matured antibodies, including screening of combination antibody libraries prepared using biodisplay. For example, Marks et al., BioTechnology, 10:779-783 (1992) describes affinity maturation by shuffling of VH and VL domains. Random mutagenesis of CDRs and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); and Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis sites and selective mutations at contact sites with activity-enhancing amino acid residues or at hypermutagenic sites have been described in U.S. Patent No. 6,914,128B1.

[0048] Antibody: As used herein, the term “antibody” refers to a polypeptide comprising at least one immunoglobulin variable domain containing at least one individual antigen-specific binding site, or a portion of an immunoglobulin variable domain (a paratope or a portion thereof) containing at least one individual antigen-specific binding site. In some embodiments, the antibody comprises at least one individual antigen-specific binding site that specifically binds to the active site of an enzyme. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, an F(ab')2 fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody is a multispecific antibody (e.g., a bispecific antibody). In some embodiments, the antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a diabody. In some embodiments, the antibody comprises a framework having a human germline sequence. In another embodiment, the antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. In some embodiments, the antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and / or a light (L) chain variable region (abbreviated herein as VL). In some embodiments, the antibody comprises a constant domain, e.g., an Fc region. The immunoglobulin constant domain refers to either a heavy chain constant domain or a light chain constant domain. The amino acid sequences of the constant domains of human IgG heavy and light chains and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of the antibody described herein may be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chains of the antibodies described herein may include human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chains. In certain embodiments, the antibodies described herein include the CH1, CH2, and / or CH3 domains of human gamma 1. In some embodiments, V HThe amino acid sequence of the domain includes the amino acid sequence of the human gamma (γ) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences are described in the art; see, for example, U.S. Patent No. 5,693,780 and Kabat EA et al., (1991) cited above. In some embodiments, V HThe domains include an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation, and / or methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glyciation (GPI anchor attachment), and / or phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, or phospholipid units. In some embodiments, the antibody is a construct comprising a polypeptide containing one or more antigen-binding fragments of the present disclosure linked to a linker polypeptide or an immunoglobulin constant domain. The linker polypeptide contains two or more amino acid residues joined by a peptide bond and is used to link one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-11233). Furthermore, the antibody may be part of a larger immunoadhesion molecule formed by the covalent or non-covalent association of the antibody or antibody moiety with one or more other proteins or peptides.Examples of such immunoadhesion molecules include the use of streptavidin core regions to construct tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to construct divalent biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol.Immunol.31:1047-1058).

[0049] Approximately: As used herein, the terms “approximately” or “about” refer to values ​​similar to the described reference values ​​when applied to the values ​​of one or more subjects. In certain embodiments, unless otherwise stated or evident from the content (except where such numbers may exceed 100% of the possible values), the terms “approximately” or “about” refer to a range of values ​​that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1% or less in either direction (above or below) the described reference values.

[0050] Bispecific antibodies: As used herein, the term “bispecific antibodies” refers to an antibody containing two distinct antigen-specific binding sites, or, in combination, two antibodies (covalently or noncovalently) linked, each containing two distinct antigen-specific binding sites. Non-limiting examples of the format or structure of bispecific antibodies are provided in Labrijn, AF, et al., Bispecific antibodies: a mechanistic review of the pipeline, Nature Reviews Drug Discovery volume 18, pages 585-608 (2019) and Brinkmann U and Kontermann EE, The making of bispecific antibodies, MAbs. 2017 Feb / Mar;9(2):182-212, the full contents of each of these are incorporated herein by reference.

[0051] CDR: As used herein, the term “CDR” refers to the complementarity-determining region within the antibody variable sequence. A typical antibody molecule contains a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions also known as “complementarity-determining regions” (“CDR”) and more conserved regions known as “framework regions” (“FR”). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methodologies known in the art, such as the Kabat definition, IMGT definition, Chothia definition, AbM definition, and / or contact definition, all of which are well known in the art.For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark) http: / / www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res.,27:209-212(1999);Ruiz,M.et al.,Nucleic Acids Res.,28:219-221(2000);Lefranc,M.-P.,Nucleic Acids Res.,29:207-209(2001);Lefranc,M.-P.,Nucleic Acids Res.,31:307-310(2003);Lefranc,M.-P.et al.,In Silico Biol.,5,0006(2004)[[Epub]],5:45-60(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,33:D593-597(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,37:D1006-1012(2009);Lefranc,M.-P.et al.,Nucleic Acids Res.,43:D413-422(2015);Chothia et al.,(1989)Nature 342:877;Chothia,C.et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. See al(1997)J.Molec.Biol.273:927-948; and Almagro,J.Mol.Recognit.17:132-143(2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. As used herein, CDR may mean CDR as defined by any method known in the art.Two antibodies having the same CDR means that the amino acid sequences of the CDRs of the two antibodies are the same, as determined by the same method, for example, the IMGT definition.

[0052] In certain embodiments, three CDRs are present in each of the variable regions of the heavy and light chains, and these are designated as CDR1, CDR2, and CDR3 with respect to each of the variable regions. As used herein, the term “CDR set” refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently depending on the system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define three CDRs. These CDRs are sometimes called Kabat CDRs. The sub-parts of the CDRs may be designated as L1, L2, and L3, or H1, H2, and H3, where "L" and "H" represent the light chain region and heavy chain region, respectively. These regions are sometimes called Chothia CDRs and have boundaries that overlap with Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs are described by Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol This is described in 262(5):732-45(1996). Furthermore, other CDR boundary definitions may not strictly adhere to one of the above systems and, although they overlap with Kabat CDRs, they may be shortened or extended in consideration of predictions or experimental results that certain residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, but preferred embodiments use Kabat or Chothia-defined CDRs.

[0053] CDR-transplanted antibody: As used herein, the term "CDR-transplanted antibody" refers to an antibody that contains heavy chain variable region sequences and light chain variable region sequences derived from one species, but in which one or more sequences of the VH and / or VL CDR regions are replaced with CDR sequences from another species. For example, in an antibody having mouse heavy chain variable region and light chain variable region, one or more mouse CDRs (e.g., CDR3) are replaced with human CDR sequences.

[0054] Chimeric antibody: As used herein, the term "chimeric antibody" refers to an antibody that contains a heavy chain variable region sequence and a light chain variable region sequence derived from one species and a constant region sequence derived from another species, such as an antibody in which the mouse heavy chain variable region and light chain variable region are linked to the human constant region.

[0055] Complementary: As used herein, the term “complementary” refers to the ability of two nucleotides or two sets of nucleotides to form a precise pairing. Specifically, complementarity is a term that characterizes the degree of hydrogen bond pairing that results in a bond between two nucleotides or two sets of nucleotides. For example, if a base at one position of an oligonucleotide can form a hydrogen bond with a base at a corresponding position of a target nucleic acid (e.g., mRNA), then those bases are considered complementary to each other at that position. Base pairings can include both canonical Watson-Crick base pairings and non-Watson-Crick base pairings (e.g., Wobble base pairings and Hoogsteen base pairings). For example, in some embodiments, in the case of complementary base pairing, an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanosine-type base (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to any A, C, U, or T and are considered complementary to them. Inosine (I) is also considered a universal base in the art and is considered complementary to any of A, C, U, or T.

[0056] Conservative amino acid substitutions: As used herein, “conservative amino acid substitutions” refer to amino acid substitutions that do not alter the relative charge or size properties of the protein being substituted. Variants can be prepared according to methods of modifying polypeptide sequences known to those skilled in the art, for example, according to references that summarize such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or according to methods found in Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0057] Cross-reactivity: As used herein, the term “cross-reactivity” refers to the property of a drug to specifically bind to multiple antigens of similar type or class (e.g., multiple homologous, paralogous, or orthologous antigens) with similar affinity or binding strength. For example, in some embodiments, an antibody that cross-reactive to human and non-human primate antigens of similar type or class (e.g., human KLK5 and non-human primate KLK5, human KLK7 and non-human primate KLK7) can bind to human and non-human primate antigens with similar affinity or binding strength. In some embodiments, the antibody cross-reactive to human and rodent antigens of similar type or class. In some embodiments, the antibody cross-reactive to rodent antigens and non-human primate antigens of similar type or class. In some embodiments, the antibody cross-reactive to human, non-human primate, and rodent antigens of similar type or class.

[0058] Dual-inhibitory antibody: As used herein, the term “dual-inhibitory antibody” refers to an antibody that targets at least two (e.g., 2, 3) different antigens via a common individual antigen-specific binding site and inhibits the activity of those antigens. In some embodiments, a dual-inhibitory antibody targets at least two different proteins (e.g., expressed from two different genes (e.g., endogenous genes, e.g., homologs, paralogs)) via a common individual antigen-specific binding site and inhibits the activity of at least two different proteins (e.g., enzymes such as proteases). In some embodiments, a dual-inhibitory antibody targets at least two different proteases (e.g., expressed from two different endogenous genes, e.g., KLK5 and KLK7) via a common individual antigen-specific binding site and inhibits the activity of at least two different proteases. In some embodiments, the common individual antigen-specific binding site binds to similar (e.g., homologous) domains shared between or among at least two different antigens. For example, in some embodiments, the common individual antigen-specific binding site binds to similar (e.g., homologous) catalytic domains or substrate-binding sites shared between or among at least two different enzymes, e.g., proteases. In some embodiments, the common individual antigen-specific binding site of a dual inhibitory antibody includes amino acids in one or more complementarity-determining regions of the antibody. In some embodiments, the common individual antigen-specific binding site of a dual inhibitory antibody is located within the heavy chain variable region and / or light chain variable region of the antibody. In some embodiments, the common individual antigen-specific binding site of a dual inhibitory antibody includes one or more complementarity-determining regions in the heavy chain variable region and / or light chain variable region of the antibody. In some embodiments, the common individual antigen-specific binding site of a dual inhibitory antibody includes HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 in the heavy chain and light chain variable regions of the antibody. In some embodiments, the dual inhibitory antibody specifically binds to two different proteins (e.g., KLK5 and KLK7) expressed from two different genes.

[0059] Effective dose: As used herein, “effective dose” refers to the amount of each activator (e.g., anti-KLK5 / KLK7 antibody) required to deliver the desired effect (e.g., therapeutic effect on a subject), either alone or in combination with one or more other activators. In some embodiments, the therapeutic effect is a reduction in KLK5 and / or KLK7 activity and / or relief of a disease (e.g., Netherton syndrome, eosinophilic esophagitis, and atopic dermatitis) or related symptoms, such as an improvement in barrier function.

[0060] Framework: As used herein, the terms “framework” or “framework sequence” refer to the sequence remaining after removing the CDRs from the variable region. Since the precise definition of a CDR sequence can be determined by various systems, the meaning of the framework sequence is also subject to different interpretations accordingly. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain, and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) divide the framework regions of the light and heavy chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, with CDR1 located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. When a framework region is referred to by others without specifying a particular sub-region as FR1, FR2, FR3, or FR4, it represents a combination of FRs within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of four subregions, and multiple FRs represent two or more of the four subregions that constitute a framework region. Human heavy and light chain acceptor sequences are known in the art. In one embodiment, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0061] Human Antibodies: As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo) in the CDR, specifically CDR3. However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, has been transplanted into a human framework sequence.

[0062] Humanized antibody: As used herein, the term "humanized antibody" includes heavy chain variable region sequences and light chain variable region sequences derived from non-human species (e.g., mouse), but V H and / or V L This refers to antibodies in which at least a portion of the sequence has been modified to be more "human-like," that is, to more closely resemble human germline variable sequences. One type of humanized antibody is a CDR-transplant antibody in which a human CDR sequence is introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequence. In one embodiment, a humanized antibody is provided. Such an antibody may be produced by obtaining a mouse monoclonal antibody using conventional hybridoma technology and then humanizing it using in vitro genetic engineering, such as that disclosed in PCT publication number WO2005 / 123126 A2 by Kasaian et al.

[0063] A humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capabilities. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may include residues not found in the recipient antibody or the transferred CDR or framework sequence, but included to further improve and optimize the antibody's performance. Generally, a humanized antibody may include substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region corresponds to that of the human immunoglobulin consensus sequence. The humanized antibody also includes, optimally, at least a portion of the immunoglobulin constant region or domain (Fc), typically at least a portion of the constant region or domain (Fc) of the human immunoglobulin. Antibodies may have modified Fc regions as described in WO99 / 58572. Other forms of humanized antibodies have one or more modified CDRs (1, 2, 3, 4, 5, 6) relative to the original antibody, also called one or more CDRs derived from one or more CDRs of the original antibody. Humanized antibodies may also undergo affinity maturation.

[0064] In some embodiments, humanization involves using a CDR (e.g., one shown in Table 1a or 1b) to create a human variable domain (e.g., IGKV1-NL1). * 01 and IGHV1-3 * This is achieved by transplanting (01 human variable domain). In some embodiments, the antibodies of this disclosure are humanized variants comprising one or more amino acid substitutions (e.g., in the VH framework region) compared to any one of the VHs listed in Table 1a or 1b, and / or comprising one or more amino acid substitutions (e.g., in the VL framework region) compared to any one of the VLs listed in Table 1a or 1b.

[0065] Isolated Antibodies: As used herein, “isolated antibodies” are intended to refer to antibodies that substantially contain other antibodies with different antigen specificities (for example, isolated dual inhibitory antibodies that specifically bind to KLK5 and KLK7 substantially contain antibodies that specifically bind to antigens other than KLK5 and KLK7). However, isolated antibodies may cross-reactive to other antigens in some embodiments. Furthermore, isolated antibodies may substantially contain other cellular material and / or chemical substances.

[0066] Kabat Numbering: As used herein, the terms “Kabat Numbering,” “Kabat Definition,” and “Kabat Labeling” are interchangeable. These terms, as recognized in the Art, refer to a system for numbering amino acid residues that are more variable than other amino acid residues (i.e., hypervariable) in the heavy chain variable region and light chain variable region of an antibody, or in their antigen-binding regions (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable region ranges from amino acid positions 31-35 in CDR1, from amino acid positions 50-65 in CDR2, and from amino acid positions 95-102 in CDR3. In the case of the light chain variable region, the hypervariable region is located at amino acid positions 24-34 in CDR1, amino acid positions 50-56 in CDR2, and amino acid positions 89-97 in CDR3.

[0067] Multispecific antigen-binding molecule: As used herein, the term “multispecific antigen-binding molecule” refers to a molecule containing two or more antigen-specific binding sites. In some embodiments, the multispecific antigen-binding molecule is a multispecific antibody (e.g., a bispecific antibody).

[0068] Multispecific antibody: As used herein, the term “multispecific antibody” refers to an antibody comprising at least two individual antigen-specific binding sites, or, in combination, at least two (covalently or noncovalently) linked antibodies comprising at least two individual antigen-specific binding sites. In some embodiments, a multispecific antibody is a bispecific antibody. Non-limiting examples of the format or structure of multispecific antibodies are provided in Sawant MS, et al., Toward Drug-Like Multispecific Antibodies by Design, Int J Mol Sci. 2020 Oct 12;21(20):7496; Klein C, et al., The use of CrossMAb technology for the generation of bi- and multispecific antibodies, Mabs 2016 Aug-Sep;8(6):1010-20; and Brinkmann U and Kontermann EE, The making of bispecific antibodies, Mabs. 2017 Feb / Mar;9(2):182-212, the full contents of each of these are incorporated herein by reference.

[0069] Recombinant Antibody: As used herein, the term "recombinant antibody" is intended to include all antibodies prepared, expressed, created or isolated by recombinant means, such as an antibody expressed using a recombinant expression vector transfected into a host cell (described in more detail in the present disclosure), which includes, for example, an antibody isolated from a recombinant combinatorial human antibody library (Hoogenboom H.R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W.E., (2002) Clin. Biochem. 35:425-445; Gavilondo J.V., and Larrick J.W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), an antibody isolated from an animal (e.g., a mouse) into which a human immunoglobulin gene has been introduced (see, for example, Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L.L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or an antibody prepared, expressed, created or isolated by any other method involving splicing a human immunoglobulin gene sequence to another DNA sequence. In some embodiments, recombinant human antibodies are provided herein. In certain embodiments, such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if an animal into which a human Ig sequence has been introduced is used, in vivo somatic mutagenesis), and thus, the amino acid sequences of the V H region and V L region are the human germline V H sequence and V LThese are sequences that originate from and are associated with other sequences, but may not naturally exist within the in vivo human antibody germline repertoire. One embodiment of the present disclosure provides, for example, a fully human antibody capable of binding to human KLK5 or KLK7, which can be generated using a suitable technique, such as, but not limited to, a human Ig phage library disclosed in PCT publication number WO2005 / 007699 A2 by Jermutus et al.

[0070] Selective: As used herein, the terms “selective” or “selectively” refer to the ability of a molecule to produce an effect (e.g., inhibition, antagonism, agonization, etc.) on its target molecule compared to a reference molecule. For example, a molecule that selectively inhibits its target molecule means that this molecule can inhibit the target molecule to a degree that is distinguishable from the reference molecule in an inhibition assay or other inhibition situation. For example, with respect to an inhibitor, the term “selectively inhibiting” refers to the ability of the inhibitor to inhibit the target molecule to a degree that is distinguishable from a reference molecule that is substantially not inhibited in an inhibition assay, to a degree that enables selective inhibition of the target molecule, as described herein. After the reaction is complete, the signal produced by the inhibition of the target molecule can be measured. The maximum half-molecule inhibitor concentrations for the target molecule and the reference molecule can be calculated.

[0071] Specific binding: As used herein, the term “specifically binding” refers to the ability of a molecule to bind to a binding partner with an affinity or binding strength sufficient to enable the use of the molecule to distinguish the binding partner from a suitable control in a binding assay or other binding context. With respect to an antibody, the term “specifically binding” refers to the ability of an antibody to bind to a specific antigen with an affinity or binding strength sufficient to enable the use of the antibody to distinguish a particular antigen from others, as described herein, compared to a suitable reference antigen(s). In some embodiments, the antibody possesses K for binding to a target. D However, at least about 10 -4 M, 10 -5 M, 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 If the antibody is M or less, it binds specifically to the target. In some embodiments, the antibody binds specifically to KLK5 or KLK7.

[0072] Subject: As used herein, the term “subject” refers to mammals. In some embodiments, the subject is a non-human primate or rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient who has or is suspected of having a disease, e.g., a human patient.

[0073] Treatment: As used herein, the terms “to treat” or “treatment” refer to the application or administration of a composition comprising one or more activators (e.g., anti-KLK5 / KLK7 antibodies) to a subject with a predisposition to a target disease or disorder, symptoms of a disease / disorder, or a disease / disorder, with the aim of treating, curing, alleviating, reducing, modifying, resuscitating, restoring, improving, or influencing the disorder, symptoms of a disease, or predisposition to a disease or disorder. Alleviation of a target disease / disorder includes delaying or preventing the onset or progression of the disease, or reducing the severity of the disease. It will be understood that “to treat” or references to treatment may also refer to antibodies, including dual inhibitory antibodies, for use in such a manner.

[0074] II. Dual inhibitory antibodies targeting KLK5 and KLK7 (a) Anti-KLK5 / KLK7 antibody In some embodiments, a dual inhibitory antibody targeting KLK5 and KLK7 (referred to as an anti-KLK5 / KLK7 antibody) is an antibody specific to both kallikrein-5 (KLK5) and KLK7 via a common specific antigen-binding site. In some embodiments, antibodies are provided herein that bind with high specificity and affinity to KLK5 (e.g., human KLK5, or mouse KLK5) and KLK7 (e.g., human KLK7, or mouse KLK7) via a common antigen-binding site. In some embodiments, the anti-KLK5 / KLK7 antibody described herein specifically binds to the KLK5 epitope that is or will be exposed to the antibody, and to the KLK7 epitope that is or will be exposed to the antibody. In some embodiments, the anti-KLK5 / KLK7 antibody provided herein specifically binds to KLK5 derived from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-KLK5 antibody provided herein specifically binds to human KLK5. In some embodiments, the anti-KLK5 antibody provided herein specifically binds to mouse KLK5.

[0075] In some embodiments, the anti-KLK5 / KLK7 antibody is not a bispecific antibody or bispecific antigen-binding molecule in which KLK5 binding is conferred by one binding site within the antibody and KLK7 binding is conferred by a different binding site within the antibody.

[0076] In some embodiments, the anti-KLK5 / KLK7 antibodies described herein may be characterized by reference to certain functional properties. In some embodiments, the anti-KLK5 / KLK7 antibodies described herein specifically bind to KLK5 and KLK7. In some embodiments, the anti-KLK5 / KLK7 antibodies specifically bind to the active forms of KLK5 and KLK7. In some embodiments, the anti-KLK5 / KLK7 antibodies do not bind to the inactive forms (proforms) of KLK5 and KLK7. In some embodiments, the antibody specifically binds to the active forms of KLK5 and KLK7, but does not specifically bind to the inactive forms of KLK5 or KLK7. In some embodiments, the antibody detectably binds to the active forms of KLK5 and KLK7, but does not detectably bind to the inactive forms of KLK5 or KLK7 under the same or equivalent conditions. In some embodiments, anti-KLK5 / KLK7 antibodies inhibit KLK5 and KLK7 protease activity. In some embodiments, anti-KLK5 / KLK7 antibodies are not cleaved by KLK5 or KLK7 when bound to them. In some embodiments, anti-KLK5 / KLK7 antibodies compete with SPINK5 and / or leupeptin for binding to the active sites of KLK5 and KLK7. In some embodiments, anti-KLK5 / KLK7 antibodies reduce keratosis and desquamation. In some embodiments, anti-KLK5 / KLK7 antibodies reduce stratum corneum thickness. In some embodiments, anti-KLK5 / KLK7 antibodies reduce inflammation and epidermal activity. Kallikrein-5, also known as stratum corneum trypsinase (SCTE), is a serine protease expressed in the epidermis and is encoded by the KLK5 gene. The KLK5 gene is one of 15 kallikrein subfamily members located in a cluster on the chromosome. Its expression is upregulated by estrogen and progestin. KLK5 is expressed in the granular layer and stratum corneum. In some embodiments, KLK5 controls epidermal desquamation. In some embodiments, KLK5 controls epidermal desquamation in conjunction with other members of the kallikrein family proteases (e.g., KLK7 and / or KLK14).In some embodiments, KLK5 degrades proteins that form the epidermis (e.g., the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale). In some embodiments, KLK5 degrades proteins that form the stratum corneum and / or stratum granulosum (e.g., cornodesmosine (CDSN), desmoglein 1 (DSG1), and desmocolin 1 (DSC1)). In the epidermis (e.g., the stratum granulosum and stratum corneum), KLK5 is expressed as pro-KLK5, an inactive form (sometimes called proform or pro-form), which can self-activate itself. Once activated, KLK5 can convert both pro-KLK7 and pro-KLK14 to their active forms via proteolytic cleavage. Subsequently, active KLK14 can activate newly generated pro-KLK5, thus forming a positive feedback loop (see, e.g., Nauroy et al., Kallikreins: Essential epidermal messengers for regulation of the skin microenvironment during homeostasis, repair and disease, Matrix Biol Plus. 2019;6-7:100019). KLK7 and KLK14 also degrade proteins that form the stratum corneum and / or granular layer (e.g., cornodesmosine (CDSN), desmoglein 1 (DSG1), and desmocolin 1 (DSC1)). Structural proteins such as CDSN, DSG1, and DSC1 are adhesion proteins in the extracellular portion of the corneodesmosome, which is a junctional structure that mediates the adhesion of keratinocytes. Degradation of these proteins on the epidermal surface leads to desquamation, which can result in skin barrier defects (e.g., stratum corneum exfoliation, reduced permeability barrier, allergies, and inflammation).KLK5 and KLK7 are involved in this process (see, for example, Caubet et al., Degradation of Corneodesmosome Proteins by Two Serine Proteases of the Kallikrein Family, SCTE / KLK5 / hK5 and SCCE / KLK7 / hK7, Journal of Investigative Dermatology, Volume 122, Issue 5, May 2004, Pages 1235-1244). Inhibition of KLK5 and / or KLK7 promotes improved skin barrier integrity and reduced inflammation (see, for example, Chavarria-Smith et al., Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis, SCIENCE TRANSLATIONAL MEDICINE, 14 Dec 2022, Vol 14, Issue 675).

[0077] Kallikrein-7 is a serine protease encoded by the KLK7 gene in humans. KLK7 is characterized as a stratum corneum chymotriptic enzyme (SCCE). It is the seventh member of the human kallikrein family, which includes 15 homologous serine proteases located on chromosome 19. KLK7 is secreted as an inactive zymogen (e.g., in the granular layer of the epidermis) and requires proteolytic cleavage to be activated. In some embodiments, KLK5 or matryptase activates KLK7. Once activated, KLK7 can cleave proteins that form the stratum corneum and / or granular layer (e.g., corneodesmosine (CDSN), desmoglein 1 (DSG1), and desmocolin 1 (DSC1)) (see, e.g., Caubet et al. (May 2004). Degradation of corneodesmosome proteins by two serine proteases of the kallikrein family, SCTE / KLK5 / hK5 and SCCE / KLK7 / hK7. The Journal of Investigative Dermatology. 122(5):1235-1244). These proteins constitute the extracellular component of corneodesmosomes, which are intercellular adhesion structures that connect intermediate filaments of adjacent cells in the stratum corneum. In some embodiments, proteolysis of keratinodesmosomes leads to epidermal desquamation (i.e., the shedding of keratinocytes from the outer layer of the epidermis). In some embodiments, the combined roles of KLK5 and KLK7 suggest that the KLK cutaneous cascade is responsible for regulating desquamation. KLK7 is a chymotrypsin-like serine protease that cleaves proteins at tyrosine, phenylalanine, or leucine residues. In some embodiments, dysregulation of KLK7 is linked to several skin disorders, including atopic dermatitis, psoriasis, and Netherton syndrome. These diseases are characterized by excessively dry, scaly, and inflamed skin due to a breakdown of skin homeostasis and normal barrier function.

[0078] In some embodiments, the anti-KLK5 / KLK7 antibodies described herein specifically bind to epitopes on human KLK5. Exemplary amino acid sequences of human KLK5 are described in NCBI accession numbers NP_001070959.1, NP_001070960.1, or NP_036559.1, and UniProt accession numbers: Q8IU55, Q6S9W8, M0QXX2, Q9P0G3, A0A2I2MP48, or A0A2I2MP49, and these entire sequences are incorporated herein by reference.

[0079] In some embodiments, the anti-KLK5 / KLK7 antibodies described herein specifically bind to epitopes on mouse KLK5. Exemplary amino acid sequences of mouse KLK5 are described in NCBI accession numbers NP_081082.1, XP_006541213.1, XP_006541214.1, XP_006541215.1, XP_036009294.1, or XP_036009295.1, and UniProt accession number P15945, or Q9D140, and these entire sequences are incorporated herein by reference.

[0080] In some embodiments, the anti-KLK5 / KLK7 antibodies described herein specifically bind to an epitope on human KLK7 via the same antigen-binding site that binds to KLK5 (e.g., human KLK5 or mouse KLK5). Exemplary amino acid sequences of human KLK7 are described in NCBI accession numbers NP_001193982.1, NP_001230055.1, NP_005037.1, NP_644806.1, and UniProt accession numbers: M0QYU8, Q6DTY1, X2J289, X2J4X7, A0A024R4H6, P49862, A0A2H4GDB2, and A0A2H4GDB6, the entirety of which are incorporated herein by reference.

[0081] In some embodiments, the anti-KLK5 / KLK7 antibodies described herein specifically bind to an epitope on mouse KLK7 via the same antigen-binding site that binds to KLK5 (e.g., human KLK5 or mouse KLK5). Exemplary amino acid sequences of mouse KLK7 are described in NCBI accession number NP_036002.1 and UniProt accession number Q91VE3, and these entire sequences are incorporated herein by reference.

[0082] In some embodiments, the anti-KLK5 / KLK7 antibody described herein specifically binds to epitopes on KLK5 (e.g., catalytic domains / pockets of human KLK5 or mouse KLK5) and epitopes on KLK7 (e.g., catalytic domains / pockets of human KLK7 or mouse KLK7). In some embodiments, the anti-KLK5 / KLK7 antibody described herein prevents KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) from cleaving their substrates. In some embodiments, the anti-KLK5 antibody described herein binds to fragments of KLK5 (e.g., human or mouse KLK5) and fragments of KLK7 (e.g., human or mouse KLK7). Fragments of KLK5 and / or KLK7 (e.g., human or mouse) may be approximately 5 to 425 amino acids, 10 to 400 amino acids, 50 to 350 amino acids, 100 to 300 amino acids, 150 to 250 amino acids, 200 to 300 amino acids, 75 to 150 amino acids, 25 to 100 amino acids, or 10 to 30 amino acids in length. Without intending to be bound by any particular theory, in some embodiments, the heavy chain (HC) complementarity-determining region 3 (CDR3) of any one of the anti-KLK5 / KLK7 antibodies described herein inhibits KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) by binding to the catalytic domain / pocket of KLK5.

[0083] In some embodiments, the anti-KLK5 / KLK7 antibodies described herein inhibit KLK5 protease activity, KLK7 protease activity, or protease activity of both KLK5 and KLK7. In some embodiments, the anti-KLK5 / KLK7 antibodies inhibit KLK5 (e.g., human KLK5 or mouse KLK5) cleavage of BOC-Val-Pro-Arg-AMC with an IC50 of less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2.5 nM, less than 2 nM, or less than 1.5 nM, less than 1 nM, less than 0.5 nM, less than 0.3 nM, less than 0.25 nM, less than 0.2 nM, or less than 0.1 nM. In some embodiments, anti-KLK5 / KLK7 antibodies cleave KLK5 (e.g., human KLK5 or mouse KLK5) in BOC-Val-Pro-Arg-AMC at concentrations of 0.1nM to 30nM, 0.1nM to 20nM, 0.1nM to 10nM, 0.1nM to 5nM, 0.1nM to 2.5nM, 0.1nM to 2nM, 0.1nM to 1nM, 0.1nM to 0.5nM, 0.1nM to 0.25nM, and 0. 1nM~50nM, 0.1nM~40nM, 0.1nM~30nM, 0.1nM~20nM, 0.1nM~10nM, 0.1nM~5nM, 0.1nM~2.5nM, 0.1nM~2nM, 0.1 nM~1nM, 0.1nM~0.9nM, 0.1nM~0.8nM, 0.1nM~0.7nM, 0.1nM~0.6nM, 0.1nM~0.5nM, 0.1nM~0.4nM, 0.1nM~0.3 nM, 0.1nM~0.25nM, 0.1nM~0.2nM, 0.1nM~0.15nM, 0.15nM~0.2nM, 0.15nM~0.25nM, 0.15nM~0.3nM, 0.15nM~ 0.4nM, 0.15nM~0.5nM, 0.15nM~1nM, 0.2nM~30nM, 0.2nM~20nM, 0.2nM~10nM, 0.2nM~5nM, 0.2nM~2.5nM, 0.2 nM~2nM, 0.2nM~1nM, 0.2nM~0.5nM, 0.2nM~0.2nM, 0.2nM~50nM, 0.2nM~40nM, 0.2nM~30nM, 0.2nM~20nM, 0.2 nM~10nM, 0.2nM~5nM, 0.2nM~2.5nM, 0.2nM~2nM, 0.2nM~1nM, 0.2nM~0.9nM, 0.2nM~0.8nM, 0.2nM~0.7nM, 0.2nM~0.6nM, 0.2nM~0.5nM, 0.2nM~0.4nM, 0.2nM~0.3nM, 0.2nM~0.25nM, 1nM~30nM, 1nM~20nM, 1nM~10nM, 1 nM~5nM, 1nM~2.5nM, 1nM~2nM, 1nM~3nM, 1nM~5.5nM, 1.5nM~2nM, 1.5nM~3nM, 1.5nM~5.5nM, 2nM~5nM, 2nM~ 4nM, 2nM~5.5nM, 3nM~5.5nM, 4nM~5.5nM, 3nM~30nM, 3nM~20nM, 3nM~10nM, 3nM~5nM, 3nM~2.5nM, 3nM~4nM, 3nM~5.5nM, 5nM~30nM, 5nM~20nM, 5nM~10nM, 5nM~9nM, 5nM~8nM, 5nM~7nM, 5nM~6nM, 5nM~5.5nM, 10nM~30n M, 10nM~25nM, 10nM~20nM, 10nM~18nM, 10nM~15nM, 10nM~12nM, 12nM~20nM, 12nM~25nM, 12nM~16nM, 12nM~ 18nM, 12nM~20nM, 12nM~24nM, 12nM~28nM, 12nM~30nM, 15nM~30nM, 15nM~25nM, 15nM~20nM, 15nM~18nM, 18 Inhibition occurs in IC50 values ​​within the ranges of nM~30nM, 18nM~25nM, 18nM~20nM, 20nM~30nM, 20nM~25nM, 20nM~22nM, 20nM~24nM, 20nM~26nM, 20nM~28nM, 22nM~30nM, 22nM~25nM, 22nM~28nM, 24nM~30nM, 24nM~25nM, 24nM~26nM, or 24nM~28nM. In some embodiments, the anti-KLK5 / KLK7 antibody inhibits KLK7 cleavage of KHLF-AMC with an IC50 of less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2.5 nM, less than 2 nM, or less than 1.5 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, less than 0.16 nM, less than 0.1 nM, or less than 0.05 nM. In some embodiments, the anti-KLK5 / KLK7 antibody cleaves KLK7 of KHLF-AMC at concentrations of 0.1nM to 30nM, 0.1nM to 20nM, 0.1nM to 10nM, 0.1nM to 5nM, 0.1nM to 2.5nM, 0.1nM to 2nM, 0.1nM to 1nM, 0.1nM to 0.5nM, and 0.1nM to 0.25nM、0.1nM~50nM、0.1nM~40nM、0.1nM~30nM、0.1nM~20nM、0.1nM~10nM、0.1nM~5nM、0.1nM~2.5nM、0.1nM~2nM、0.1nM~1nM、0.1nM~0.9nM、0.1nM~0.8nM、0.1nM~0.7nM、0.1nM~0.6nM、0.1nM~0.5nM、0.1nM~0.4nM、0.1nM~0.3nM、0.1nM~0.25nM、0.1nM~0.2nM、0.1nM~0.15nM、0.15nM~0.2nM、0.15nM~0.25nM、0.15nM~0.3nM、0.15nM~0.4nM、0.15nM~0.5nM、0.15nM~1nM、0.2nM~30nM、0.2nM~20nM、0.2nM~10nM、0.2nM~5nM、0.2nM~2.5nM、0.2nM~2nM、0.2nM~1nM、0.2nM~0.5nM、0.2nM~0.2nM、0.2nM~50nM、0.2nM~40nM、0.2nM~30nM、0.2nM~20nM、0.2nM~10nM、0.2nM~5nM、0.2nM~2.5nM、0.2nM~2nM、0.2nM~1nM、0.2nM~0.9nM、0.2nM~0.8nM、0.2nM~0.7nM、0.2nM~0.6nM、0.2nM~0.5nM、0.2nM~0.4nM、0.2nM~0.3nM、0.2nM~0.25nM、1nM~30nM、1nM~20nM、1nM~10nM、1nM~5nM、1nM~2.5nM、1nM~2nM、1nM~3nM、1nM~5.5nM、1.5nM~2nM、1.5nM~3nM、1.5nM~5.5nM、2nM~5nM、2nM~4nM、2nM~5.5nM、3nM~5.5nM、4nM~5.5nM、3nM~30nM、3nM~20nM、3nM~10nM、3nM~5nM、3nM~2.5nM、3nM~4nM、3nM~5.5nM、5nM~30nM、5nM~20nM、5nM~10nM、5nM~9nM、5nM~8nM、5nM~7nM、5nM~6nM、5nM~5.5nM, 10nM~30nM, 10nM~25nM, 10nM~20nM, 10nM~18nM, 10nM~15nM, 10nM~12nM, 12nM~20nM, 12nM~25nM, 12nM~1 6nM, 12nM~18nM, 12nM~20nM, 12nM~24nM, 12nM~28nM, 12nM~30nM, 15nM~30nM, 15nM~25nM, 15nM~20nM, 15nM~1 Inhibition occurs in IC50 values ​​within the ranges of 8nM, 18nM-30nM, 18nM-25nM, 18nM-20nM, 20nM-30nM, 20nM-25nM, 20nM-22nM, 20nM-24nM, 20nM-26nM, 20nM-28nM, 22nM-30nM, 22nM-25nM, 22nM-28nM, 24nM-30nM, 24nM-25nM, 24nM-26nM, or 24nM-28nM.

[0084] In some embodiments, the anti-KLK5 / KLK7 antibody described herein specifically binds to KLK5, KLK7, or the active forms of KLK5 and KLK7. In some embodiments, the anti-KLK5 / KLK7 antibody described herein does not bind to KLK5, KLK7, or the inactive forms of KLK5 and KLK7. In some embodiments, the anti-KLK5 / KLK7 antibody described herein specifically binds to the active form of KLK5, the active form of KLK7, or the active forms of KLK5 and KLK7, but does not specifically bind to the inactive form of KLK5, the inactive form of KLK7, or the inactive forms of KLK5 and KLK7. In some embodiments, the anti-KLK5 / KLK7 antibodies described herein are detectably bound to the active form of KLK5, the active form of KLK7, or the active forms of both KLK5 and KLK7, but are not detectably bound to the inactive form of KLK5, the inactive form of KLK7, or the inactive forms of both KLK5 and KLK7 under the same or equivalent conditions. In some embodiments, the anti-KLK5 / KLK7 antibodies described herein specifically bind to the active sites of KLK5, KLK7, or KLK5 and KLK7. The active sites of KLK5 and / or KLK7 are the sites to which KLK5 and / or KLK7 substrate molecules bind and undergo cleavage. The active sites may also be known as catalytic domains or catalytic triresidues. In some embodiments, the active site of KLK5 or KLK7 (i.e., the catalytic domain or catalytic triresidue) consists of the amino acids Ser195, His57, and Asp102 of KLK5 or KLK7 (see, for example, Goettig et al., Natural and synthetic inhibitors of kallikrein-related peptidases (KLKs), Biochimie. 2010 Nov;92(11):1546-1567).

[0085] In some embodiments, the antibodies described herein are optimized versions (e.g., affinity-mature) of the parent antibody. In some embodiments, the antibodies described herein are KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) with approximately 10 -4 Less than M, 10 -5Less than M, 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11 Less than M, 10 -12 Less than M, 10 -13 Binding affinity less than M or less (e.g., K D It specifically binds to (as shown by). In some embodiments, the antibodies described herein bind to KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) at a rate of 1 × 10⁻¹⁶ -10 M~5×10 -9 M, 1×10 -10 M~1×10 -9 M, 5×10 -10 ~1 × 10 -9 M, 5×10 -11 ~1 × 10 -10 M, 1×10 -11 ~5×10 -10 M, or 5×10 -13 ~1 × 10 -12 Binding affinity of M (e.g., K DThe antibodies specifically bind to the KLK5 protein (e.g., human or mouse KLK5) and the KLK7 protein (e.g., human or mouse KLK7) with affinities of 1 pM to 500 nM, e.g., 50 pM to 100 nM, 500 pM to 50 nM, 1 pM to 100 pM, 10 pM to 100 pM, 50 pM to 100 pM, 100 pM to 500 pM, 500 pM to 1 nM, 1 nM to 5 nM, 1 nM to 10 nM, 5 nM to 25 nM, 10 nM to 50 nM, 50 nM to 100 nM, and 100 nM to 500 nM. This disclosure also includes antibodies that compete with any of the antibodies described herein for binding to KLK5 protein (e.g., human or mouse KLK5) and KLK7 protein (e.g., human or mouse KLK7) and have affinities of 100 nM or less (e.g., 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding reaction rate of the antibodies can be tested using any suitable method, including but not limited to biosensor technologies (e.g., OCTET or BIACORE). In some embodiments, the antibodies described herein have K in the sub-nanomole range. D It connects to KLK5 and KLK7.

[0086] Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance (SPR), fluorescence-activated cell sorting (FACS), or spectroscopy (e.g., using fluorescence assays). Exemplary conditions for evaluating binding affinity are HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P2O) and PBS buffer (10 mM PO4-3, 137 mM NaCl, and 2.7 mM KCl). Using these techniques, the concentration of the binding protein can be measured as a function of the target protein concentration. Generally, the concentration of the binding protein ([[binding]]) is given by the following formula: [[Bound]]=[[Free]] / (Kd+[[Free]]) This is related to the concentration of free target protein (free).

[0087] K A While it is not always necessary to make an accurate determination, obtaining a quantitative measure of affinity determined using methods such as ELISA or FACS analysis is useful, as it indicates that K A Since it is proportional to the activity, it may be sufficient, and therefore can be used for comparisons to determine whether a higher affinity is, for example, twice as high, to obtain a qualitative measurement of affinity, or to infer affinity by activity in a functional assay (e.g., an in vitro or in vivo assay).

[0088] Exemplary anti-KLK5 / KLK7 antibody sequences (e.g., heavy chain (HC) sequence and light chain (LC) sequence, heavy chain variable domain (VH) and light chain variable domain (VL), and CDR sequence) are provided in Tables 1a and 1b. [Table 1-1] [Table 1-2]

[0089] In some embodiments, certain amino acid positions in the antibodies described herein (e.g., amino acids in the VH / VL region and / or CDR region) are substitutable, and this substitution results in an antibody having substantially similar binding and biological activity (e.g., substantially similar binding affinity, binding specificity, protease inhibitory activity, anti-inflammatory activity, or a combination thereof) to the reference antibody. To identify substitutable positions in an antibody, the amino acid sequence of that antibody is compared to the sequences of other antibodies belonging to the same group as that antibody. If the identity of that amino acid varies among related antibodies of different groups at any particular position, that position is a substitutable position in the antibody. In other words, a substitutable position is a position where the identity of the amino acid varies among related antibodies. Positions containing constant amino acids are not substitutable positions.

[0090] In some embodiments, the above method may be used to provide a consensus antibody sequence. In such a consensus sequence, non-substitutable positions are indicated by the amino acids present at those positions, and substitutable positions are indicated by "X".

[0091] Depending on how the antibody is adopted, X may be a) any amino acid, b) any amino acid present at that position in any of the related antibodies within that group, or a conservatively substituted variant thereof, or c) any amino acid present at that position in any of the related antibodies within that group. Any antibody having a sequence encompassed by consensus should bind to the same antigen as any of the related antibodies.

[0092] In some embodiments, the methods described above may be employed in a method for designing and producing variants of a parent antibody that at least maintain (e.g., maintain or increase) the antigen-binding activity of the parent antibody. Antibodies containing substitutions at substitutable positions can be produced and tested, and therefore substitutions at those positions should not significantly reduce the antibody's binding activity. Generally, antibody variants of a parent antibody have an antigen-binding affinity of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% (e.g., at least 150%, at least 200%, at least 500%, at least 1000%, and typically up to at least 10,000%) of the parent antibody's binding affinity to a particular antigen.

[0093] In some embodiments, the substitutable positions of the parent antibody may be substituted by: a) any of 20 naturally occurring amino acids to produce a random substitution; b) an amino acid with biochemical properties similar to an amino acid already present at the substitutable position to produce a conservative substitution; c) an amino acid present at the same position in the related antibody to produce a designated substitution; or d) an amino acid present at the same position in a similar human antibody to produce a humanization substitution. Substitutions may occur in any portion of the antibody variable region, including any framework region or CDR. In certain embodiments, a single substitutable amino acid may be substituted. However, in other embodiments, multiple substitutable amino acids (e.g., up to about 5 or 10 or more) may be substituted. In certain embodiments, the type of substitution that can be performed at each substitutable position may be indicated by the type of amino acid present at that position in the related antibody. For example, if an unrelated amino acid (e.g., Ala, Gly, Cys, Glu, and Thr) is present at a particular position in the group of related antibodies, any amino acid can be substituted at that position without significantly reducing the antibody's binding activity. Exemplary amino acid substitutions of anti-KLK5 / KLK7 antibodies described herein are shown in Table 1b: [Table 1-3]

[0094] In some embodiments, the antibodies of this disclosure include: HC CDR1 containing the amino acid sequence GSISSX1DYYWX2 (SEQ ID NO: 28), where X1 is S, D, or L, and X2 is G or V; HC CDR2 containing the amino acid sequence SIX3YX4X5X6TYYX7PSLKS (SEQ ID NO: 29), where X3 is Y or D, X4 is S, F, or Y, X5 is G or A, X6 is S or D, or X7 is N or S; HC CDR3 containing the amino acid sequence ARGRPLGYGAX8HX9YYGMDV (SEQ ID NO: 30), where X8 is R or K, or X9 is Y or D; LC CDR1 containing the amino acid sequence of SEQ ID NO: 4; LC CDR2 containing the amino acid sequence of SEQ ID NO: 5; and / or QQSPX 10 LC CDR3 containing the amino acid sequence of FPPLT (SEQ ID NO: 31), where X 10 This includes the statement that it is either P or Y.

[0095] In some embodiments, the antibody of the Disclosure comprises one or more HC CDR (e.g., HC CDR1, HC CDR2, or HC CDR3) amino acid sequences from any one of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b. In some embodiments, the antibody of the Disclosure comprises an HC CDR3 amino acid sequence from any one of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b. In some embodiments, the antibody of the Disclosure comprises HC CDR1, HC CDR2, and HC CDR3 provided for any one of the (elected) antibodies selected from Tables 1a and 1b. In some embodiments, the antibody of the Disclosure comprises an LC CDR3 amino acid sequence from any one of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b. In some embodiments, the antibody of the Disclosure comprises one or more LC CDR (e.g., LC CDR1, LC CDR2, or LC CDR3) amino acid sequences from any one of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b. In some embodiments, the antibody of the Disclosure comprises LC CDR1, LC CDR2, and LC CDR3 provided for any one of the anti-KLK5 antibodies selected from Tables 1a and 1b.

[0096] In some embodiments, the antibodies of this disclosure include HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 provided for any one of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b. In some embodiments, the heavy chain CDR3 domain and / or light chain CDR3 domain of the antibody may play a particularly important role in the antibody's binding specificity / affinity to the antigen. Therefore, the antibodies of this disclosure may include at least the heavy chain CDR3 and / or light chain CDR3 of any one of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b.

[0097] Any variant of the exemplary anti-KLK5 / KLK7 antibody disclosed herein is also within the scope of this disclosure. The variant may contain one or more amino acid residue mutations in the VH and / or VL, or in one or more of the HC CDRs and / or one or more of the LC CDRs, compared to the reference antibody, but retain substantially similar binding activity and biological activity (e.g., substantially similar binding affinity, binding specificity, protease inhibitory activity, anti-inflammatory activity, or a combination thereof) to the reference antibody.

[0098] In some embodiments, the antibody of this disclosure has one or more CDR (e.g., HC CDR or LC CDR) sequences substantially similar to any of HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and / or LC CDR3 from one of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b. In some embodiments, the positions of one or more CDRs along the VH (e.g., HC CDR1, HC CDR2, or HC CDR3) and / or VL (e.g., LC CDR1, LC CDR2, or LC CDR3) regions of the antibodies described herein can be mutated by only 1, 2, 3, 4, 5, or 6 amino acid positions, as long as specific binding to KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the binding of the original antibody from which it is derived). For example, in some embodiments, the position defining the CDR of any antibody described herein can be mutated by shifting the N-terminal and / or C-terminal boundary of the CDR by 1, 2, 3, 4, 5, or 6 amino acids relative to any one of the CDR positions of the antibodies described herein, insofar as specific binding of KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the binding of the original antibody from which it originates).In another embodiment, the length of one or more CDRs along the VH (e.g., HC CDR1, HC CDR2, or HC CDR3) and / or VL (e.g., LC CDR1, LC CDR2, or LC CDR3) regions of the antibodies described herein may be mutated (e.g., shortened or lengthened) by only 1, 2, 3, 4, 5, or more amino acids, as long as immunospecific binding to KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the binding of the original antibody from which it is derived).

[0099] Therefore, in some embodiments, the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and / or LC CDR3 described herein may be 1, 2, 3, 4, or 5 amino acids shorter than one or more of the CDRs described herein (e.g., CDRs from any of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b), as long as specific binding to KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) is maintained (e.g., substantially maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and / or LC CDR3 described herein may be 1, 2, 3, 4, or 5 amino acids or longer than one or more of the CDRs described herein (e.g., CDRs from any of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b), provided that specific binding to KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) is maintained (e.g., substantially maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the amino portions of HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and / or LC CDR3 described herein can be extended by 1, 2, 3, 4, or 5 amino acids or more compared to one or more of the CDRs described herein (e.g., CDRs from any of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b), provided that specific binding to KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) is maintained (e.g., substantially maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the binding of the original antibody from which it is derived).In some embodiments, the carboxyl portions of HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and / or LC CDR3 described herein can be extended by 1, 2, 3, 4, or 5 amino acids or more compared to one or more of the CDRs described herein (e.g., CDRs from any of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b), provided that specific binding to KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) is maintained (e.g., substantially maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the amino portion of HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and / or LC CDR3 described herein can be shortened by 1, 2, 3, 4, or 5 amino acids or more compared to one or more of the CDRs described herein (e.g., CDRs from any of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b), as long as specific binding to KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) is maintained (e.g., substantially maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the carboxyl portions of HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and / or LC CDR3 described herein can be shortened by 1, 2, 3, 4, or 5 amino acids or more compared to one or more of the CDRs described herein (e.g., CDRs from any of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b), as long as specific binding to KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) is maintained (e.g., substantially maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the binding of the original antibody from which it is derived).To confirm whether specific binding to KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) is maintained, any method can be used, such as using the binding assays and conditions described in the Art.

[0100] In some examples, the antibodies of this disclosure have one or more CDR (e.g., HC CDR or LC CDR) sequences substantially similar to any one of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b. For example, the antibodies described herein may contain one or more CDR sequences from any of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b, containing up to 5, 4, 3, 2, or 1 amino acid residue mutations compared to the corresponding CDR region of any one of the CDRs provided herein (e.g., CDRs from any of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b), insofar as specific binding to KLK5 (e.g., human or mouse KLK5) and KLK7 (e.g., human or mouse KLK7) is maintained (e.g., substantially maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, any amino acid mutations in any of the CDRs provided herein may be conservative mutations. Conservative mutations can be introduced into a CDR at a position where the residue is unlikely to be involved in interaction with KLK5 (e.g., human or mouse KLK5) and / or KLK7 (e.g., human or mouse KLK7) when determined, for example, based on the crystal structure. Some aspects of this disclosure provide antibodies comprising one or more heavy chain variable (VH) domains and / or light chain variable (VL) domains provided herein. In some embodiments, any VH domain provided herein comprises one or more of the HC CDR sequences provided herein (e.g., HC CDR1, HC CDR2, and HC CDR3), which are, for example, any of the HC CDR sequences provided for any one of the anti-KLK5 / KLK7 selected from Tables 1a and 1b. In some embodiments, each VL domain provided herein comprises one or more of the LC CDR sequences provided herein (e.g., LC CDR1, LC CDR2, and LC CDR3), which are, for example, any of the LC CDR sequences provided for any one of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b.

[0101] In some embodiments, the antibodies of the Disclosure include any antibody comprising a heavy chain variable domain and / or light chain variable domain of any one anti-KLK5 / KLK7 antibody selected from Tables 1a and 1b, as well as variants thereof. In some embodiments, the antibodies of the Disclosure include any antibody comprising a pair of heavy chain variable and light chain variable regions of any anti-KLK5 / KLK7 antibody selected from Tables 1a and 1b.

[0102] Aspects of this disclosure provide antibodies comprising heavy chain variable (VH) domain amino acid sequences and / or light chain variable (VL) domain amino acid sequences homologous to any of the amino acid sequences described herein. In some embodiments, the antibody comprises a heavy chain variable sequence or a light chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of the heavy chain variable sequences and / or light chain variable sequences of an anti-KLK5 / KLK7 antibody selected from Tables 1a and 1b. In some embodiments, the homologous heavy chain variable amino acid sequences and / or light chain variable amino acid sequences do not mutate within any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence mutation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may occur within the heavy chain variable sequence and / or light chain variable sequence excluding any of the CDR sequences provided herein. In some embodiments, the antibodies provided herein include heavy-chain and light-chain variable sequences that include a framework sequence which is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequence of any anti-KLK5 / KLK7 antibody selected from Tables 1a and 1b.

[0103] In some embodiments, the antibody of the Disclosure is a humanized antibody (e.g., a humanized variant containing one or more CDRs from Tables 1a and 1b). In some embodiments, the antibody of the Disclosure comprises HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3, which are the same as HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 shown in Tables 1a and 1b, and includes a humanized heavy chain variable region and / or a humanized light chain variable region.

[0104] In some embodiments, the antibody of the Disclosure is a humanized antibody comprising a VH containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the VH of any of the anti-KLK5 / KLK7 antibodies listed in Tables 1a and 1b. Alternatively or additionally, the antibody of the Disclosure is a humanized antibody comprising a VL containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to any one of the VL of any of the anti-KLK5 / KLK7 antibodies listed in Tables 1a and 1b.

[0105] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure comprises heavy chain variable domains HC CDR1, HC CDR2, and HC CDR3 having the amino acid sequence of SEQ ID NO: 7. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the present disclosure comprises light chain variable domains LC CDR1, LC CDR2, and LC CDR3 having the amino acid sequence of SEQ ID NO: 8.

[0106] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure includes HC CDR1 having the amino acid sequence of SEQ ID NO: 1, HC CDR2 having the amino acid sequence of SEQ ID NO: 2, HC CDR3 having the amino acid sequence of SEQ ID NO: 3, LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0107] In some embodiments, the anti-KLK5 / KLK7 antibodies of the Disclosure comprise HC CDR1, HC CDR2, and HC CDR3, which collectively contain 5 or fewer amino acid mutations (e.g., 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR1 having the amino acid sequence of SEQ ID NO: 1, HC CDR2 having the amino acid sequence of SEQ ID NO: 2, and HC CDR3 having the amino acid sequence of SEQ ID NO: 3. Wherever used in the Disclosure, “collectively” means that the total number of amino acid mutations in all three heavy chain CDRs is within a defined range. Alternatively or additionally, the anti-KLK5 / KLK7 antibodies of the Disclosure comprise LC CDR1, LC CDR2, and LC CDR3, which collectively contain 5 or fewer amino acid mutations (e.g., 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0108] In some embodiments, the anti-KLK5 / KLK7 antibody of the Disclosure comprises HC CDR1, HC CDR2, and HC CDR3 that are collectively at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to HC CDR1 having the amino acid sequence of SEQ ID NO: 1, HC CDR2 having the amino acid sequence of SEQ ID NO: 2, and HC CDR3 having the amino acid sequence of SEQ ID NO: 3. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the Disclosure comprises LC CDR1, LC CDR2, and LC CDR3 that are collectively at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0109] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure includes HC CDR1 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR1 having the amino acid sequence of SEQ ID NO: 1; HC CDR2 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR2 having the amino acid sequence of SEQ ID NO: 2; and / or HC CDR3 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR3 having the amino acid sequence of SEQ ID NO: 3. Alternatively or additionally, the anti-KLK5 / KLK7 antibodies of this disclosure include LC CDR1 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR1 having the amino acid sequence of SEQ ID NO: 4; LC CDR2 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR2 having the amino acid sequence of SEQ ID NO: 5; and / or LC CDR3 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0110] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 7. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the present disclosure comprises VL, which contains the amino acid sequence of SEQ ID NO: 8.

[0111] In some embodiments, the anti-KLK5 / KLK7 antibody of this disclosure includes a VH containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the VH described in SEQ ID NO: 7. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of this disclosure includes a VL containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the VL described in SEQ ID NO: 8.

[0112] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure comprises a VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH described in SEQ ID NO: 7. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the present disclosure comprises a VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VL described in SEQ ID NO: 8.

[0113] In some embodiments, the anti-KLK5 / KLK7 antibody of this disclosure comprises HC CDR1, HC CDR2, and HC CDR3 heavy chain variable domains having the amino acid sequence of SEQ ID NO: 13. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of this disclosure comprises LC CDR1, LC CDR2, and LC CDR3 light chain variable domains having the amino acid sequence of SEQ ID NO: 14.

[0114] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure includes HC CDR1 having the amino acid sequence of SEQ ID NO: 9, HC CDR2 having the amino acid sequence of SEQ ID NO: 10, HC CDR3 having the amino acid sequence of SEQ ID NO: 11, LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0115] In some embodiments, the anti-KLK5 / KLK7 antibodies of this disclosure comprise HC CDR1, HC CDR2, and HC CDR3, which collectively contain 5 or fewer amino acid mutations (e.g., 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR1 having the amino acid sequence of SEQ ID NO: 9, HC CDR2 having the amino acid sequence of SEQ ID NO: 10, and HC CDR3 having the amino acid sequence of SEQ ID NO: 11. As used anywhere in this disclosure, “collectively” means that the total number of amino acid mutations in all three heavy chain CDRs is within a defined range. Alternatively or additionally, the anti-KLK5 / KLK7 antibodies of this disclosure comprise LC CDR1, LC CDR2, and LC CDR3, which collectively contain 5 or fewer amino acid mutations (e.g., 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0116] In some embodiments, the anti-KLK5 / KLK7 antibody of the Disclosure comprises HC CDR1, HC CDR2, and HC CDR3 that are collectively at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to HC CDR1 having the amino acid sequence of SEQ ID NO: 9, HC CDR2 having the amino acid sequence of SEQ ID NO: 10, and HC CDR3 having the amino acid sequence of SEQ ID NO: 11. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the Disclosure comprises LC CDR1, LC CDR2, and LC CDR3 that are collectively at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0117] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure includes HC CDR1 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR1 having the amino acid sequence of SEQ ID NO. 9; HC CDR2 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR2 having the amino acid sequence of SEQ ID NO. 10; and / or HC CDR3 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR3 having the amino acid sequence of SEQ ID NO. 11. Alternatively or additionally, the anti-KLK5 / KLK7 antibodies of this disclosure include LC CDR1 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR1 having the amino acid sequence of SEQ ID NO: 4; LC CDR2 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR2 having the amino acid sequence of SEQ ID NO: 5; and / or LC CDR3 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0118] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 13. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the present disclosure comprises VL, which contains the amino acid sequence of SEQ ID NO: 14.

[0119] In some embodiments, the anti-KLK5 / KLK7 antibody of this disclosure includes a VH containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the VH described in SEQ ID NO. 13. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of this disclosure includes a VL containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the VL described in SEQ ID NO. 14.

[0120] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure comprises a VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH described in SEQ ID NO: 13. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the present disclosure comprises a VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VL described in SEQ ID NO: 14.

[0121] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure comprises HC CDR1, HC CDR2, and HC CDR3 heavy chain variable domains having the amino acid sequence of SEQ ID NO: 17. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the present disclosure comprises LC CDR1, LC CDR2, and LC CDR3 light chain variable domains having the amino acid sequence of SEQ ID NO: 14.

[0122] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure includes HC CDR1 having the amino acid sequence of SEQ ID NO: 9, HC CDR2 having the amino acid sequence of SEQ ID NO: 15, HC CDR3 having the amino acid sequence of SEQ ID NO: 16, LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0123] In some embodiments, the anti-KLK5 / KLK7 antibodies of this disclosure comprise HC CDR1, HC CDR2, and HC CDR3, which collectively contain 5 or fewer amino acid mutations (e.g., 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR1 having the amino acid sequence of SEQ ID NO: 9, HC CDR2 having the amino acid sequence of SEQ ID NO: 15, and HC CDR3 having the amino acid sequence of SEQ ID NO: 16. As used anywhere in this disclosure, “collectively” means that the total number of amino acid mutations in all three heavy chain CDRs is within a defined range. Alternatively or additionally, the anti-KLK5 / KLK7 antibodies of this disclosure comprise LC CDR1, LC CDR2, and LC CDR3, which collectively contain 5 or fewer amino acid mutations (e.g., 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0124] In some embodiments, the anti-KLK5 / KLK7 antibody of the Disclosure comprises HC CDR1, HC CDR2, and HC CDR3 that are collectively at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to HC CDR1 having the amino acid sequence of SEQ ID NO: 9, HC CDR2 having the amino acid sequence of SEQ ID NO: 15, and HC CDR3 having the amino acid sequence of SEQ ID NO: 16. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the Disclosure comprises LC CDR1, LC CDR2, and LC CDR3 that are collectively at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0125] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure includes HC CDR1 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR1 having the amino acid sequence of SEQ ID NO. 9; HC CDR2 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR2 having the amino acid sequence of SEQ ID NO. 15; and / or HC CDR3 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR3 having the amino acid sequence of SEQ ID NO. 16. Alternatively or additionally, the anti-KLK5 / KLK7 antibodies of this disclosure include LC CDR1 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR1 having the amino acid sequence of SEQ ID NO: 4; LC CDR2 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR2 having the amino acid sequence of SEQ ID NO: 5; and / or LC CDR3 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0126] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 17. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the present disclosure comprises VL, which contains the amino acid sequence of SEQ ID NO: 14.

[0127] In some embodiments, the anti-KLK5 / KLK7 antibody of this disclosure includes a VH containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the VH described in SEQ ID NO. 17. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of this disclosure includes a VL containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the VL described in SEQ ID NO. 14.

[0128] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure comprises a VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH described in SEQ ID NO: 17. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the present disclosure comprises a VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VL described in SEQ ID NO: 14.

[0129] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure comprises heavy chain variable domains HC CDR1, HC CDR2, and HC CDR3 having the amino acid sequence of SEQ ID NO: 21. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the present disclosure comprises light chain variable domains LC CDR1, LC CDR2, and LC CDR3 having the amino acid sequence of SEQ ID NO: 14.

[0130] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure includes HC CDR1 having the amino acid sequence of SEQ ID NO: 18, HC CDR2 having the amino acid sequence of SEQ ID NO: 19, HC CDR3 having the amino acid sequence of SEQ ID NO: 20, LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0131] In some embodiments, the anti-KLK5 / KLK7 antibodies of this disclosure comprise HC CDR1, HC CDR2, and HC CDR3, which collectively contain 5 or fewer amino acid mutations (e.g., 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR1 having the amino acid sequence of SEQ ID NO: 18, HC CDR2 having the amino acid sequence of SEQ ID NO: 19, and HC CDR3 having the amino acid sequence of SEQ ID NO: 20. As used anywhere in this disclosure, “collectively” means that the total number of amino acid mutations in all three heavy chain CDRs is within a defined range. Alternatively or additionally, the anti-KLK5 / KLK7 antibodies of this disclosure comprise LC CDR1, LC CDR2, and LC CDR3, which collectively contain 5 or fewer amino acid mutations (e.g., 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0132] In some embodiments, the anti-KLK5 / KLK7 antibody of the Disclosure comprises HC CDR1, HC CDR2, and HC CDR3 that are collectively at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to HC CDR1 having the amino acid sequence of SEQ ID NO: 18, HC CDR2 having the amino acid sequence of SEQ ID NO: 19, and HC CDR3 having the amino acid sequence of SEQ ID NO: 20. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the Disclosure comprises LC CDR1, LC CDR2, and LC CDR3 that are collectively at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0133] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure includes HC CDR1 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR1 having the amino acid sequence of SEQ ID NO. 18; HC CDR2 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR2 having the amino acid sequence of SEQ ID NO. 19; and / or HC CDR3 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to HC CDR3 having the amino acid sequence of SEQ ID NO. 20. Alternatively or additionally, the anti-KLK5 / KLK7 antibodies of this disclosure include LC CDR1 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR1 having the amino acid sequence of SEQ ID NO: 4; LC CDR2 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR2 having the amino acid sequence of SEQ ID NO: 5; and / or LC CDR3 having 3 or fewer amino acid mutations (e.g., 3, 2, or 1 or fewer amino acid mutations) compared to LC CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0134] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 21. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the present disclosure comprises VL, which contains the amino acid sequence of SEQ ID NO: 14.

[0135] In some embodiments, the anti-KLK5 / KLK7 antibody of this disclosure includes a VH containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the VH described in SEQ ID NO. 21. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of this disclosure includes a VL containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the VL described in SEQ ID NO. 14.

[0136] In some embodiments, the anti-KLK5 / KLK7 antibody of the present disclosure comprises a VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH described in SEQ ID NO: 21. Alternatively or additionally, the anti-KLK5 / KLK7 antibody of the present disclosure comprises a VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VL described in SEQ ID NO: 14.

[0137] The antibodies described herein may be in any antibody form, including but not limited to intact (i.e., full-length) antibodies, their antigen-binding fragments (Fab, F(ab'), F(ab')2, Fv, etc.), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the anti-KLK5 / KLK7 antibody described herein is scFv. In some embodiments, the anti-KLK5 / KLK7 antibody described herein is scFv-Fab (e.g., scFv fused to a portion of the constant region).

[0138] In some embodiments, the anti-KLK5 / KLK7 antibody of this disclosure is a chimeric antibody, which may include a heavy chain constant region and a light chain constant region derived from a human antibody. A chimeric antibody refers to an antibody having a variable region or a portion of a variable region derived from a first species and a constant region derived from a second species. Typically, in these chimeric antibodies, both the light chain and heavy chain variable regions mimic the variable regions of an antibody derived from one species of mammal (e.g., a non-human mammal such as a mouse, rabbit, and rat), while the constant region is homologous to the sequence in an antibody derived from another mammal, such as a human. In some embodiments, amino acid modifications may occur in the variable region and / or the constant region.

[0139] In some embodiments, the antibodies of this disclosure comprise the VL domain and / or VH domain of any one of the anti-KLK5 / KLK7 antibodies selected from Tables 1a and 1b, and include a constant region comprising the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) immunoglobulin molecule. Non-limiting examples of human constant regions have been described in the Art; see, for example, Kabat EA et al., (1991), cited above.

[0140] In some embodiments, the light chain of any of the anti-KLK5 / KLK7 antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the Art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain.

[0141] Other antibody heavy chain constant regions and light chain constant regions are well known in the art and are provided, for example, in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0142] In some embodiments, conservative mutations may be introduced into the antibody sequence (e.g., CDR or framework sequence) at a position where the residue is unlikely to be involved in interaction with the target antigen (e.g., human or mouse KLK5 and / or human or mouse KLK7), for example, if determined based on the crystal structure. In some embodiments, one, two or more mutations (e.g., amino acid substitutions) may be introduced into the Fc region (numbered according to the Kabat numbering system (e.g., the EU index in Kabat), for example, the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1) and / or the hinge region) of the anti-KLK5 / KLK7 antibody described herein to modify one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cytotoxicity.

[0143] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc domain (CH1 domain) to modify (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region, as described, for example, in U.S. Patent No. 5,677,425. Modifying the number of cysteine ​​residues in the hinge region of the CH1 domain can, for example, facilitate the assembly of light and heavy chains, or modify (e.g., increase or decrease) the stability of the antibody, or facilitate linker conjugation.

[0144] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the antibodies described herein (numbered according to the Kabat numbering system (e.g., the EU index in Kabat), for example, the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1) and / or the hinge region) to increase or decrease the affinity of the antibody to Fc receptors (e.g., activated Fc receptors) on the surface of effector cells. Mutations in the Fc region of antibodies that decrease or increase the affinity of an antibody to Fc receptors, and techniques for introducing such mutations into Fc receptors or fragments thereof, are known to those skilled in the art. Examples of antibody mutations at the Fc receptor that can be used to alter the affinity of an antibody to the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO02 / 060919; WO98 / 23289; and WO97 / 34631, which are incorporated herein by reference.

[0145] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably an Fc or hinge-Fc domain fragment) to modify (e.g., decrease or increase) the in vivo half-life of the antibody. See, for example, International Publication Nos. WO02 / 060919; WO98 / 23289; and WO97 / 34631; and U.S. Patents 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for examples of mutations that modify (e.g., decrease or increase) the in vivo half-life of the antibody.

[0146] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably an Fc or hinge-Fc domain fragment) to reduce the half-life of the anti-KLK5 / KLK7 antibody in vivo. In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably an Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in a second constant (CH2) domain (residues 231-340 of human IgG1) and / or a third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the EU index in Kabat (Kabat EA et al., (1991), cited above). In some embodiments, the constant region of IgG1 of the antibodies described herein, numbered according to the EU index as set forth in Kabat, includes a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG is called a “YTE mutant” and has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody includes an IgG constant domain that contains one, two, three or more amino acid substitutions of amino acid residues at positions 251–257, 285–290, 308–314, 385–389, and 428–436, numbered according to the EU index as found in Kabat.

[0147] In some embodiments, the antibody includes an Fc region that has been modified, for example, by introducing substitutions of M428L and / or N434A, for the purpose of extending the half-life. Non-limiting examples of such Fc variants that affect the circulating half-life are provided in Saunders KO, Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life, Front Immunol. 2019;10:1296, which is incorporated herein by reference.

[0148] In some embodiments, the effector function(s) of an anti-KLK5 / KLK7 antibody is modified by introducing one, two, or more amino acid substitutions into the IgG constant domain Fc region, for example, by introducing Leu234Ala and Leu235Ala mutations (commonly referred to as LALA mutations). The effector ligand whose affinity is modified may be, for example, the Fc receptor or the C1 component of complement. This technique is described in U.S. Patents No. 5,624,821 and No. 5,648,260. In some embodiments, deletion or inactivation of the constant domain (via point mutation or other means) can reduce the binding of the circulating antibody to the Fc receptor, thereby increasing tumor localization. For a description of mutations that delete or inactivate the constant domain and thereby increase tumor localization, see, for example, U.S. Patents No. 5,585,097 and No. 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibodies described herein to remove potential glycosylation sites on the Fc region, thereby reducing Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0149] In some embodiments, one or more amino acids in the constant region of the anti-KLK5 / KLK7 antibody described herein can be replaced with different amino acid residues, thereby giving the antibody modified C1q binding and / or reduced or ineffective complement-dependent cell-mediated cytotoxicity (CDC). This technique is described in detail in U.S. Patent No. 6,194,551 (Idusogie et al). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are modified, thereby altering the antibody's ability to immobilize complement. This technique is further described in International Publication No. WO94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or increase the antibody's affinity for the Fcγ receptor. This technique is further described in International Publication No. WO00 / 42072.

[0150] In some embodiments, the antibody comprises an Fc variant containing amino acid substitutions L234A, L235E, and P329G, in which case the numbering follows the EU index. In some embodiments, the antibody comprising the Fc variant exhibits reduced affinity for one or more or each of FcγRJ, FcγRIIA, FcγRIIIA, and Clq compared to the antibody comprising the wild-type human Fc region. Examples of such Fc variants are provided in International Patent Application Publication No. WO2021 / 055669, title, FC VARIANTS WITH REDUCED EFFECTOR FUNCTION, published March 25, 2021; and U.S. Patent Application Publication No. US2021-0087271, title, FC VARIANTS WITH REDUCED EFFECTOR FUNCTION, published March 25, 2021, the contents of which are incorporated herein by reference.

[0151] In some embodiments, as described elsewhere herein, variable domain(s) sequences(s) of the heavy and / or light chains of the antibodies provided herein can be used to generate, for example, CDR-implanted, chimeric, humanized, or compound human antibodies or antigen-binding fragments. As will be understood by those skilled in the art, any variant, CDR-implanted, chimeric, humanized, or compound antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and the variant, CDR-implanted, chimeric, humanized, or compound antibody will maintain the ability to specifically bind to KLK5 and KLK7 such that it has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more binding to KLK5 and KLK7 compared to the original antibody from which it is derived.

[0152] In some embodiments, the antibodies provided herein include mutations that confer desirable properties to the antibody. For example, to avoid potential complications from Fab-arm exchange, which are known to occur with natural IgG4 mAbs, the antibodies provided herein may include a stabilizing "Adair" mutation (Angal S., et al., “A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody,” Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. Thus, any of the antibodies may include a stabilizing "Adair" mutation.

[0153] In some embodiments, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation, and / or methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glyciation (GPI anchor attachment), and / or phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, there are about 1–10, about 1–5, about 5–10, about 1–4, about 1–3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is completely or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or enzymatic means. In some embodiments, the antibody is glycosylated in vitro or intracellularly, which may optionally be deficient in enzymes of the N- or O-glycosylation pathway, such as glycosyltransferase. In some embodiments, the antibody is functionalized with sugar molecules or carbohydrate molecules, as described in International Patent Application Publication No. WO2014065661, published May 1, 2014, titled "Modified antibody, antibody-conjugate and process for the preparation thereof".

[0154] In some embodiments, any one of the anti-KLK5 / KLK7 antibodies described herein may include a signal peptide (e.g., an N-terminal signal peptide) in the heavy chain sequence and / or light chain sequence. In some embodiments, the anti-KLK5 / KLK7 antibody described herein includes either a VH sequence or a VL sequence, either an IgG heavy chain sequence or a light chain sequence, or either an F(ab') heavy chain sequence or a light chain sequence described herein, and further includes a signal peptide (e.g., an N-terminal signal peptide).

[0155] (b) Multispecific antibody In some embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). For example, in some embodiments, one or more anti-KLK5 / KLK7 antibodies may be combined with one or more different anti-KLK5 / KLK7 antibodies to produce multispecific or bispecific anti-KLK5 / KLK7 antibodies. For example, one or more anti-KLK5 / KLK7 antibodies described herein (Tables 1a and 1b) can be combined with one or more different anti-KLK5 / KLK7 antibodies described herein (Tables 1a and 1b) to produce multispecific antibodies.

[0156] In some embodiments, one or more anti-KLK5 / KLK7 antibodies may be combined with any other suitable therapeutic antibody to generate multispecific or bispecific anti-KLK5 / KLK7 / additional target antibodies. For example, the anti-KLK5 / KLK7 antibodies described herein (Tables 1a and 1b) can be combined with any suitable antibody to generate bispecific antibodies. Such additional therapeutic antibodies include, but are not limited to, anti-IL4R antibodies (e.g., dupilumab), anti-IL-13 antibodies, TNF inhibitors (e.g., anti-TNF antibodies), IL-12 / 23 antibodies, IL-17 antibodies, adalimumab, infliximab, golimumab, ustekinumab, secukinumab, ixekizumab, brodalumab, abatacept, tildrakizumab-asmn, risankizumab-rzaa, and guselkumab.

[0157] In some embodiments, one or more anti-KLK5 / KLK7 antibodies may be combined with any other suitable anti-KLK7 antibody to produce multispecific or bispecific anti-KLK5 / KLK7 antibodies. For example, the anti-KLK5 / KLK7 antibodies described herein (Tables 1a and 1b) can be combined with any other suitable anti-KLK7 antibody to produce bispecific antibodies. Non-limiting examples of suitable anti-KLK7 antibodies include U.S. Patent Application Publication No. 2021-0130492, titled "ANTI-KLK7 ANTIBODIES, ANTI-KLK5 ANTIBODIES, MULTISPECIFIC ANTI-KLK5 / KLK7 ANTIBODIES, AND METHODS OF USE," published May 6, 2021; International Patent Application Publication No. WO2021226695, titled "RECOMBINANT HUMAN ANTIBODIES FOR INHIBITING HUMAN TISSUE KALLIKREIN 7 (KLK7) AND USE IN DISEASES RELATED TO THE PROCESS OF SKIN DESQUAMATION," published November 18, 2021; and International Patent Application Publication No. WO2005075667, titled "DIAGNOSTICS AND THERAPEUTICS FOR DISEASES Associated with Kallikrein 7 (KLK7), released on August 18, 2005, the contents of which are incorporated herein by reference.

[0158] In some embodiments, one or more anti-KLK5 / KLK7 antibodies may be combined with any suitable anti-KLK5 antibody to produce multispecific or bispecific anti-KLK5 / KLK7 antibodies. For example, the KLK5 / KLK7 antibodies described herein (Tables 1a and 1b) can be combined with any suitable anti-KLK5 antibody. Non-limiting examples of anti-KLK5 antibodies include: U.S. Patent No. 11,292,828, titled "KLK5 Inhibitory Peptide," filed April 5, 2022; U.S. Patent Publication No. 2022-0306725, titled "KLK5 Inhibitory Peptide," published September 29, 2022; U.S. Patent Publication No. 2019-0078160, titled "Use of KLK5 Antagonists for Treatment of a Disease," published March 14, 2019; International Patent Publication No. WO2021156171, titled "Antibodies Against KLK5," published August 12, 2021; International Patent Publication No. WO2021156170, titled "Antibodies Against KLK5" The information is provided in U.S. Patent Application Publication No. 2021-0301032, titled "ANTI-KLK5 ANTIBODIES AND METHODS OF USE", published on August 12, 2021; U.S. Patent Application Publication No. 2021-0130492, titled "ANTI-KLK7 ANTIBODIES, ANTI-KLK5 ANTIBODIES, MULTISPECIFIC ANTI-KLK5 / KLK7 ANTIBODIES, AND METHODS OF USE", published on May 6, 2021, and the contents of these are incorporated herein by reference.

[0159] In some embodiments, the multispecific antibody includes 3, 4, 5, 6, 7, 8, or more individual antigen-specific binding sites. In some embodiments, each individual antigen-specific binding site of the multispecific antibody targets a different antigen. In some embodiments, each individual antigen-specific binding site of the multispecific antibody targets a different region of the same antigen. In some embodiments, the multispecific antibody includes individual antigen-specific binding sites that target different antigens and / or individual antigen-specific binding sites that target different regions of the same antigen. In some embodiments, the multispecific antibody includes at least one antigen-specific binding site that targets a first antigen and at least one antigen-specific binding site that targets a second antigen. In some embodiments, the multispecific antibody includes two or more antigen-specific binding sites that target different regions of the first antigen and / or two or more antigen-specific binding sites that target different regions of the second antigen.

[0160] In some embodiments, the multispecific antibody targets two antigens and contains one antigen-specific binding site (1+1) for each antigen. In some embodiments, the multispecific antibody targets two antigens and contains two antigen-specific binding sites (2+2) for each antigen. In some embodiments, the multispecific antibody targets two antigens and contains one antigen-specific binding site for one antigen and two antigen-specific binding sites (1+2) for the other antigen. In some embodiments, the multispecific antibody targets two antigens and contains two antigen-specific binding sites for one antigen and three antigen-specific binding sites (2+3) for the other antigen. In some embodiments, the multispecific antibody targets two antigens and contains three antigen-specific binding sites for one antigen and three antigen-specific binding sites (3+3) for the other antigen.

[0161] In some embodiments, multispecific antibodies lack Fc-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement fixation, and FcRn-mediated recycling. However, in some embodiments, multispecific antibodies include one or more Fc regions that support Fc-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement fixation, and FcRn-mediated recycling.

[0162] In some embodiments, the antibodies provided herein are bispecific antibodies. In some embodiments, the bispecific antibody comprises at least two different Fv regions. In some embodiments, the bispecific antibody comprises two different heavy chains and two different light chains. In some embodiments, the bispecific antibody comprises one or more IgG molecules. In some embodiments, the bispecific antibody comprises one or more IgG molecules containing additional antigen-specific binding sites, for example, IgG molecules containing an added or modified Ig-like structure.

[0163] In some embodiments, the bispecific antibody comprises two single-stranded variable fragments (scFv) linked via a linker. In some embodiments, the bispecific antibody comprises two single-domain antibodies, such as VH or VL domains, VHH, VNAR, or nanobodies, linked via a linker (e.g., a flexible glycine-rich linker such as a (G4S)3¬ linker). In some embodiments, the bispecific antibody is in diabodies format, for example, as described in P. Holliger, T. Prospero, and G. Winter, “Diabodies”: small bivalent and bispecific antibody fragments, Proc Natl Acad Sci US A. 1993 Jul 15;90(14):6444-6448, the entire content of which is incorporated herein by reference. In some embodiments, the bispecific antibody is a Fab fusion protein, such as a Fab-Fab fusion protein, a Fab-scFv fusion protein, or a Fab-Fv fusion protein. In some embodiments, the bispecific antibody includes an antigen-binding site, e.g., scFv, which is modified to contain a second individual antigen-specific binding site, e.g., scFv, as an integral part of the antibody.

[0164] In some embodiments, the bispecific antibody is in a fragment-based format, a symmetric format, or an asymmetric format. In some embodiments, the fragment-based format bispecific antibody does not contain an Fc region. In some embodiments, the bispecific antibody is in a tandem VHH, tandem scFv, DART, diabody, F(ab)2, scFv-Fab, tandem VHH, (scFv)2-Fab, or tandem diabody format. In some embodiments, the bispecific antibody is in an asymmetric format selected from rat (arat)-mouse hybrid IgG, hetero H HL exchange and / or assembled IgG, hetero H forced HL IgG, cH IgG, hetero H CrossMab, scFv-Fab IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, and F(ab)3CrossMab format. In some embodiments, the bispecific antibodies are symmetrical formats selected from IgG-(scFv)2, Bs4Ab, DVD-Ig, tetravalent DART-Fc, (scFV)4-Fc, CODV-Ig, two-in-one, mAb2, F(ab)4CrossMab, and tandem VHH-Fc formats.

[0165] In some embodiments, bispecific antibodies are manipulated to facilitate formation via knobs-into-holes techniques, for example, to promote heterodimerization. Using knobs-into-holes technology, in some embodiments, bispecific IgG molecules, trivalent Ig-like antibodies, bispecific Fc and CH3 fusion proteins, and other formats may be generated, as described in Ridgway JB, et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 1996;9:617-21; Atwell S, et al., Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J Mol Biol 1997;270:26-35; and Merchant AM, et al., An efficient route to human bispecific IgG, Nat Biotechnol 1998;16:677-681, the entire contents of each are incorporated herein by reference.

[0166] In some embodiments, bispecific antibodies lack Fc-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement fixation, and / or FcRn-mediated recycling. However, in some embodiments, bispecific antibodies include one or more Fc regions that support Fc-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement fixation, and FcRn-mediated recycling.

[0167] III. Preparation of anti-KLK5 / KLK7 antibodies The antibodies described herein can be prepared by any method known in the art. For example, see Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0168] In some embodiments, antibodies specific to a target antigen (e.g., KLK5 and / or KLK7) can be produced by conventional hybridoma techniques. Using a full-length target antigen or a fragment thereof, optionally coupled to a carrier protein such as KLH, a host animal can be immunized to produce antibodies that bind to that antigen. The routes and schedules of immunization of the host animal are generally consistent with established conventional techniques for antibody stimulation and production, as further described herein. Common techniques for mouse, humanization, and human antibody production are known in the art and described herein. Any mammalian subject, including human or human-derived antibody-producing cells, is intended to be manipulated to serve as a basis for the production of mammalian, e.g., human hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, plantarly, and / or intradermally with a certain amount of immunogen, including those described herein.

[0169] If desired, the antibody of interest (monoclonal or polyclonal) (e.g., produced by a hybridoma) may be sequenced, and the polynucleotide sequence may then be cloned into a vector for expression or proliferation. The sequence encoding the antibody of interest may be maintained in a vector within host cells, and the host cells may then be grown and frozen for future use. Alternatively, the polynucleotide sequence may be used for genetic engineering to "humanize" the antibody, or to improve the antibody's affinity (affinity maturation) or other properties. For example, when the antibody is to be used in clinical trials and human treatments, the constant region may be manipulated to more closely resemble the human constant region in order to avoid an immune response. Genetic engineering of the antibody sequence may be desirable to obtain higher affinity and higher efficacy against the target antigen. It will be apparent to those skilled in the art that one or more polynucleotide changes can be made to an antibody while still maintaining its binding specificity to the target antigen.

[0170] In other embodiments, fully human antibodies can be obtained by using commercially available mice engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response can also be used to produce humanized antibodies or human antibodies. Examples of such techniques include Xenomouse® from Amgen, Inc. (Fremont, CA), HuMAb-Mouse® and TC mice® from Medarex, Inc. (Princeton, NJ), or H2L2 mice from Harbor Antibodies BV (Holland). Alternatively, antibodies can be recombinantly produced by phage display or yeast technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to generate human antibodies and antibody fragments in vitro from a gene repertoire of immunoglobulin variable (V) domains derived from non-immunized donors.

[0171] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by conventional methods. For example, the F(ab')2 fragment can be produced by pepsin digestion of the antibody molecule, and the Fab fragment can be produced by reducing the disulfide crosslinks of the F(ab')2 fragment. Genetically modified antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced, for example, by conventional recombination techniques. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. After isolation, the DNA may be placed in one or more expression vectors, which are then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as E. coli cells, Simian COS cells, Chinese hamster ovary (CHO) cells, human HEK293 cells, or myeloma cells, to synthesize monoclonal antibodies in the recombinant host cells. See, for example, PCT publication number WO87 / 04462. The DNA can then be modified, for example, by substituting the coding sequences of the constant domains of human heavy and light chains for homologous mouse sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently binding all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this way, genetically modified antibodies, such as “chimeric” or “hybrid” antibodies, which have binding specificity to target antigens, can be prepared.

[0172] Single-chain antibodies can be prepared via recombinant techniques by linking nucleotide sequences encoding a heavy chain variable region and a light chain variable region. Preferably, a movable linker is incorporated between the two variable regions.

[0173] Antibodies obtained according to methods known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitopes to which the antigen binds, or "epitope mapping." There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including analysis of the crystalline structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, for example, Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In one example, epitope mapping can be achieved using H / D-Ex (hydrogen-deuterium exchange) combined with proteolysis and mass spectrometry. In an additional example, epitope mapping can be used to determine the sequences to which the antibody binds. Epitopes can be linear epitopes, i.e., epitopes contained within a sequence of amino acids, or they can be structural epitopes formed by three-dimensional interactions of amino acids that are not necessarily contained within a sequence (a linear sequence of primary structure). Peptides of various lengths (e.g., at least 4-6 amino acid lengths) can be isolated or synthesized (e.g., by recombination) and used in antibody binding assays. In another example, the epitope to which the antibody binds can be determined in systematic screening by using duplicate peptides derived from the target antigen sequence and determining antibody binding. In gene fragment expression assays, an open reading frame encoding the target antigen is fragmented either randomly or by a specific gene construct, and the reactivity of the expressed antigen fragments with the antibody being tested is determined. The gene fragments can be generated, for example, by PCR, and then transcribed in vitro in the presence of radioactive amino acids and translated into proteins. The binding of antibodies to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis.A particular epitope can also be identified by using a large library of random peptide sequences presented on the surface of phage particles (a phage library). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In further examples, antigen-binding domain mutagenesis, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify the sufficient and / or required residues for epitope binding. Alternatively, a competitive assay can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as other antibodies. Competitive assays are well known to those skilled in the art.

[0174] In some cases, the antibodies described herein are prepared by recombinant techniques as illustrated below. The nucleic acids encoding the heavy and light chains of the antibodies described herein can be cloned into a single expression vector, with each nucleotide sequence operably ligated to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operably ligated to a separate promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably ligated to a single promoter so that both the heavy and light chains are expressed from the same promoter. If necessary, an intra-sequence ribosome entry site (IRES) can be inserted between the heavy chain coding sequence and the light chain coding sequence.

[0175] In some cases, the nucleotide sequences encoding the two chains of an antibody can be cloned into two vectors, and these can be introduced into the same or different cells. If these two chains are expressed in different cells, each of them can be isolated from the host cell expressing such a chain, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to enable antibody formation.

[0176] Generally, nucleic acid sequences encoding one or all of an antibody chain can be cloned into a suitable expression vector in an operable linked state with a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector can be contacted with restriction enzymes under suitable conditions to create complementary ends on each molecule that can pair with each other and be ligated together. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell to be used for antibody production.

[0177] Various promoters can be used for the expression of the antibodies described herein, but are not limited to, the initial promoter of cytomegalovirus (CMV), viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Simian virus 40 (SV40) initial promoter, E. coli lac UV5 promoter, and herpes simplex virus tk promoter.

[0178] Controllable promoters can also be used. Such controllable promoters include those that use an E. coli-derived lac repressor as a transcription modulator to control transcription from lac operator-containing mammalian cell promoters [[Brown, M. et al., Cell, 49:603-612 (1987)]] and those that use a tetracycline repressor (tetR) [[Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-555115 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]]. Other systems include FK506 dimers, VP16 or p65 using estradiol, RU486, diphenol murislerone, or rapamycin. Among these, derivable systems are available from Invitrogen, Clontech, and Ariad.

[0179] A controllable promoter containing a repressor can be used with the operon. In one embodiment, a lac repressor derived from E. coli can function as a transcription modulator that controls transcription from a mammalian cell promoter containing a lac operator [[M. Brown et al., Cell, 49:603-612 (1987)]]; Gossen and Bujard (1992); [[M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)]]. Combining a tetracycline repressor (tetR) with a transcription activator (VP16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP16), and combining it with a minimal promoter containing tetO derived from a human cytomegalovirus (hCMV) promoter, created a tetR-tet operator system for controlling gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. Tetracycline repressor (tetR) alone can function as a potent trans-regulator for controlling gene expression in mammalian cells, more so than tetR-mammalian cell transcription factor fusion derivatives, provided the tetracycline operator is properly positioned downstream of the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transcription activators or repressor fusion proteins, which can sometimes be toxic to cells, to achieve its controllable effect (Gossen 5 et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0180] Furthermore, the vector may contain, for example, some or all of the following: selection marker genes, e.g., neomycin genes for selection of stable or transient transtransferants in mammalian cells; enhancer / promoter sequences from the pre-early genes of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyomatous replication origin and ColE1 for proper episomal replication; intrasequence ribosome binding sites (IRESs), versatile multiplexing sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNAs. Appropriate vectors and methods for generating vectors containing transgenes are well known and available in the art. Examples of polyadenylation signals useful for carrying out the methods described herein include, but are not limited to, human collagen I polyadenylation signals, human collagen II polyadenylation signals, and SV40 polyadenylation signals.

[0181] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies (e.g., nucleic acid coding sequences listed in Table 3) can be introduced into suitable host cells for antibody production. Non-limiting examples of host cells include Chinese hamster ovary (CHO) cells, dhfr-CHO cells, human embryonic kidney (HEK)-293 cells, verda reno (VERO) cells, non-secretory null (NS0) cells, human embryonic retina (PER.C6) cells, Sp2 / 0 cells, baby hamster kidney (BHK) cells, Madin-Darby canine kidney (MDCK) cells, Madin-Darby bovine kidney (MDBK) cells, and SV40-transformed monkey kidney CV1 (COS) cells. In some embodiments, the host cells expressing the antibodies described herein are CHO cells. The host cells can be cultured under conditions suitable for the expression of the antibody or any polypeptide chain thereof. Such antibodies or their polypeptide chains can be recovered by cultured cells (e.g., from cells or culture supernatant) via conventional methods, such as affinity purification. If necessary, the polypeptide chains of the antibodies can be incubated under suitable conditions for a suitable period of time that allows for antibody production. In some embodiments, the host cells contain nucleic acids encoding the heavy chain of the antibodies described herein. In some embodiments, the host cells contain nucleic acids encoding the light chain of the antibodies described herein. In some embodiments, the host cells contain nucleic acids encoding both the heavy chain and the light chain.

[0182] In some embodiments, the method for preparing the antibodies described herein involves a recombinant expression vector encoding both the heavy and light chains of the antibodies described herein, as also described herein. The recombinant expression vector can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Selected positive transformant host cells can be cultured under suitable conditions that allow expression of the two polypeptide chains that form the antibody, and these can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cells can be incubated under suitable conditions that allow antibody formation.

[0183] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an antibody and the other encoding the light chain of an antibody. Both recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection.

[0184] Alternatively, each expression vector can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow expression of the antibody polypeptide chain. If two expression vectors are introduced into the same host cell, the antibody produced in the cell can be recovered from the host cell or culture medium. If necessary, the polypeptide chain can be recovered from the host cell or culture medium and then incubated under suitable conditions that allow antibody formation. If two expression vectors are introduced into different host cells, each can be recovered from the corresponding host cell or corresponding culture medium. The two polypeptide chains can then be incubated under suitable conditions for antibody formation.

[0185] Using standard molecular biology techniques, recombinant expression vectors are prepared, host cells are transfected, transformants are selected, host cells are cultured, and antibodies are recovered from the culture medium. Some antibodies can be isolated, for example, by affinity chromatography using a protein A or protein G binding matrix.

[0186] nucleic acids encoding the heavy chain, light chain, or both of the antibodies described herein (e.g., those provided in Table 3), vectors containing them (e.g., expression vectors), and host cells containing the vectors are all within the scope of this disclosure. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0187] In some embodiments, the Disclosure provides isolated nucleic acids containing sequences that are at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs. In some embodiments, the Disclosure provides isolated nucleic acids containing sequences that are at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs.

[0188] In some embodiments, this disclosure provides an expression vector encoding an anti-KLK5 / KLK7 antibody as described herein. In some embodiments, the expression vector comprises an isolated nucleic acid that is at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs. 23, 25, 32, or 33.

[0189] In some embodiments, the anti-KLK5 / KLK7 antibodies described herein are produced in recombinant cells by: (i) expressing isolated nucleic acids that are at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs. 22, 24, 26, or 27; and / or (ii) expressing isolated nucleic acids that are at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs. 23, 25, 32, or 33.

[0190] In some embodiments, the anti-KLK5 / KLK7 antibodies described herein are produced in recombinant cells by expressing an expression vector containing: (i) an isolated nucleic acid that is at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs. 22, 24, 26, or 27; and / or (ii) an isolated nucleic acid that is at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs. 23, 25, 32, or 33.

[0191] In some embodiments, the Disclosure provides isolated nucleic acids containing a sequence that is at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 22, and / or isolated nucleic acids that are at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 23.

[0192] In some embodiments, the Disclosure provides isolated nucleic acids containing a sequence that is at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 24, and / or isolated nucleic acids that are at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 25.

[0193] In some embodiments, the Disclosure provides isolated nucleic acids containing a sequence that is at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 26, and / or isolated nucleic acids that are at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 32.

[0194] In some embodiments, the Disclosure provides isolated nucleic acids containing a sequence that is at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 27, and / or isolated nucleic acids that are at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 33.

[0195] In some embodiments, the Disclosure provides an expression vector comprising an isolated nucleic acid having a sequence identical to at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 22, and / or an isolated nucleic acid having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 23.

[0196] In some embodiments, the Disclosure provides an expression vector comprising an isolated nucleic acid having a sequence identical to SEQ ID NO: 24 by at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), and / or an isolated nucleic acid having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), to SEQ ID NO: 25.

[0197] In some embodiments, the Disclosure provides an expression vector comprising an isolated nucleic acid having a sequence identical to at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 26, and / or an isolated nucleic acid having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 32.

[0198] In some embodiments, the Disclosure provides an expression vector comprising an isolated nucleic acid containing a sequence identical to SEQ ID NO: 27 by at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), and / or an isolated nucleic acid identical to SEQ ID NO: 33 by at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0199] In some embodiments, the antibodies described herein are used to modulate the activity or function of at least one gene, protein, and / or nucleic acid. In some embodiments, the molecular payload is responsible for the modulation of the gene, protein, and / or nucleic acid. The molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity capable of modulating the activity or function of an intracellular gene, protein, and / or nucleic acid.

[0200] In some embodiments, the multispecific antibody includes the direct fusion or linkage of different antigen-specific binding sites. In some embodiments, the multispecific antibody includes an immunoglobulin-derived heteromerized domain for generating the multispecific antibody. In some embodiments, the multispecific antibody may be formed by the co-expression of different heavy chains and two different light chains. In some embodiments, the multispecific antibody may be formed by the co-expression of different heavy chains and a common light chain. In some embodiments, the multispecific antibody includes, for example, an engineered CH1 domain (the first constant Ig domain of the heavy chain) that promotes appropriate heavy-light chain pairing in such co-expression systems, as disclosed, for example, in International Patent Application Publication No. WO2021067404, "CH1 DOMAIN VARIANTS ENGINEERED FOR PREFERENTIAL LIGHT CHAIN ​​PAIRING AND MULTISPECIFIC ANTIBODIES COMPRISING THE SAME," published April 8, 2021, which is incorporated herein by reference.

[0201] In some embodiments, the multispecific antibody includes a variant CH1 domain that pairs (e.g., preferentially pairs) with a particular variant CL domain. For example, in some embodiments, the multispecific antibody includes a heavy chain containing a variant CH1 domain that preferentially pairs with a variant CLK or CLλ domain. Non-limiting examples of such multispecific antibody configurations are provided in International Patent Application Publication No. WO2022150787, “VARIANT CH1 DOMAINS AND VARIANT CL DOMAINS ENGINEERED FOR PREFERENTIAL CHAIN ​​PAIRING AND MULTI-SPECIFIC ANTIBODIES COMPRISING THE SAME,” published July 14, 2022, the contents of which are incorporated herein by reference in their entirety.

[0202] In some embodiments, multispecific antibodies include a variant CH3 domain that preferentially forms CH3-CH3 heterodimers over CH3-CH3 homodimers. Incorporation of such a variant CH3 domain promotes, for example, heterodimerization of different antibodies to form multispecific antibodies. Non-limiting examples of such multispecific antibody configurations are provided in International Patent Application Publication No. WO2022150785, “VARIANT CH3 DOMAINS ENGINEERED FOR PREFERENTIAL CH3 HETERODIMERIZATION, MULTI-SPECIFIC ANTIBODIES COMPRISING THE SAME, AND METHODS OF MAKING THEREOF”; published July 14, 2022, the contents of which are incorporated herein by reference in their entirety.

[0203] In some embodiments, bispecific antibodies involve the direct fusion or linkage of different antigen-specific binding sites. In some embodiments, bispecific antibodies include an immunoglobulin-derived heterodimerizing domain for generating bispecific antibodies. For example, in some embodiments, bispecific antibodies may be formed by the co-expression of two different heavy chains and two different light chains. In some embodiments, bispecific antibodies may be formed by the co-expression of different heavy chains and a common light chain. In some embodiments, the fusion of two antibody-producing cell lines allows for the combination of heavy and light chains of two different antibodies, resulting in a bispecific antibody containing the heavy and light chains of a first antibody and the heavy and light chains of a second antibody. In some embodiments, the heavy chain constant region and the light chain constant region are of the same isotype. In some embodiments, the heavy chain constant region and the light chain constant region are of different isotypes.

[0204] In some embodiments, the bispecific antibody includes variant heavy and / or light chains that force correct assembly between the two heavy chains and the homologous heavy and light chains, or facilitate the purification of the correctly assembled bispecific antibody (see, for example, Figures 3 and 4 and Table 1 in Brinkmann U and Kontermann EE, The making of bispecific antibodies, MAbs. 2017 Feb / Mar;9(2):182-212). In some embodiments, the bispecific antibody is formed using, for example, a knobs-into-holes technique to facilitate heterodimerization. Using knobs-into-holes technology, in some embodiments, bispecific IgG molecules, trivalent Ig-like antibodies, bispecific Fc and CH3 fusion proteins, and other formats may be generated, as described in Ridgway JB, et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 1996;9:617-21; Atwell S, et al., Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J Mol Biol 1997;270:26-35; and Merchant AM, et al., An efficient route to human bispecific IgG, Nat Biotechnol 1998;16:677-681, the entire contents of each are incorporated herein by reference.

[0205] In some embodiments, the bispecific antibody includes, for example, an engineered CH1 domain (the first constant Ig domain of the heavy chain) that promotes appropriate heavy-light chain pairing in such co-expression systems, as disclosed, for example, in International Patent Application Publication No. WO2021067404, "CH1 DOMAIN VARIANTS ENGINEERED FOR PREFERENTIAL LIGHT CHAIN ​​PAIRING AND MULTISPECIFIC ANTIBODIES COMPRISING THE SAME," published on April 8, 2021, which is incorporated herein by reference.

[0206] In some embodiments, the bispecific antibody includes a variant CH1 domain that pairs (e.g., preferentially pairs) with a particular variant CL domain. For example, in some embodiments, the bispecific antibody includes a heavy chain containing a variant CH1 domain that preferentially pairs with a variant CLK or CLλ domain. Non-limiting examples of such bispecific antibody configurations are provided in International Patent Application Publication No. WO2022150787, “VARIANT CH1 DOMAINS AND VARIANT CL DOMAINS ENGINEERED FOR PREFERENTIAL CHAIN ​​PAIRING AND MULTI-SPECIFIC ANTIBODIES COMPRISING THE SAME,” published July 14, 2022, the contents of which are incorporated herein by reference in their entirety.

[0207] In some embodiments, bispecific antibodies include a variant CH3 domain that preferentially forms CH3-CH3 heterodimers over CH3-CH3 homodimers. Incorporation of such a variant CH3 domain promotes, for example, heterodimerization of different antibodies to form bispecific antibodies. Non-limiting examples of such bispecific antibody configurations are provided in International Patent Application Publication No. WO2022150785, “VARIANT CH3 DOMAINS ENGINEERED FOR PREFERENTIAL CH3 HETERODIMERIZATION, MULTI-SPECIFIC ANTIBODIES COMPRISING THE SAME, AND METHODS OF MAKING THEREOF”; published July 14, 2022, the contents of which are incorporated herein by reference in their entirety.

[0208] In some embodiments, bispecific antibodies may be formed using non-immunoglobulin heterodimerization modules to combine different antigen-specific binding sites in a non-covalent or covalent manner. For example, in some embodiments, bispecific antibodies are formed via a dock-and-lock method (DNL) that utilizes a heterodimer assembly of a regulatory subunit of cAMP-dependent protein kinase (PKA) and the anchoring domain (AD) of an A kinase anchor protein (AKAP). In some embodiments, bispecific antibodies may be formed using non-immunoglobulin heterodimerization modules such as the Balanase-Bulster system, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of three protein syntaxins, synaptobrevin and SNAP25, to combine different antigen-specific binding sites.

[0209] IV. Pharmaceutical Compositions The antibodies described herein, as well as the encoded nucleic acids or nucleic acid sets, vectors containing them, or host cells containing the vectors, can be mixed with pharmaceutically acceptable carriers (excipients) to form pharmaceutical compositions for use in the treatment of target diseases. “Acceptable” means that the carrier must be compatible with (and preferably capable of stabilizing) the active ingredient of the composition and must not be harmful to the subject being treated. pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000), Lippincott Williams and Wilkins, Ed. KE. Hoover.

[0210] The pharmaceutical compositions containing anti-KLK5 / KLK7 antibodies disclosed herein may further comprise suitable buffers. A buffer is a weak acid or base used to maintain the pH of a solution near a selected value after the addition of another acid or base. In some examples, the buffers disclosed herein may be buffers capable of maintaining physiological pH regardless of changes in carbon dioxide concentration (produced by cellular respiration). Exemplary buffers include, but are not limited to, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Dulbecco's phosphate-buffered saline (DPBS) buffer, or phosphate-buffered saline (PBS) buffer. Such buffers may comprise disodium hydrogen phosphate and sodium chloride, or potassium dihydrogen phosphate and potassium chloride.

[0211] The pharmaceutical compositions described herein comprise one or more suitable salts. Salts are ionic compounds that can be formed by the neutralization reaction of acids and bases. (Skoog, DA; West, DM; Holler, JF; Crouch, SR (2004). “Chapters 14-16”. Fundamentals of Analytical Chemistry (8th ed.)). Salts consist of a related number of cations (positively charged ions) and anions (negative ions) such that the product is electrically neutral (has no net charge).

[0212] In some embodiments, the pharmaceutical composition may contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE. Hoover). In some embodiments, the pharmaceutical composition may be formulated for intravenous injection. In some embodiments, the pharmaceutical composition may be formulated for subcutaneous injection.

[0213] Pharmaceutical compositions used for in vivo administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane. Therapeutic antibody compositions are generally placed in containers with sterile access ports, such as intravenous or subcutaneous solution bags or vials with a stopper that can be punctured with a subcutaneous needle.

[0214] V. How to use In certain embodiments, the Disclosure provides methods and related compositions for treating conditions associated with KLK5 and KLK7-related dysregulation, including, for example, Netherton syndrome, atopic dermatitis (with and without filaggrin mutations), eosinophilic esophagitis, prurigo nodosa, chronic pruritus of unknown cause (CPUO), dry skin, asthma (e.g., specifically KLK5-related asthma), ichthyosis vulgaris, and itching of the skin.

[0215] Aspects of this disclosure relate to methods and compositions (e.g., anti-KLK5 / KLK7 dual inhibitory antibodies) useful for promoting proper barrier function (e.g., epidermal barrier function). Overactive kallikrein 5 / 7 is the cause of both genetic and spontaneous disruption of epidermal barrier function and is associated with related disorders such as Netherton syndrome, eosinophilic esophagitis, and atopic dermatitis. Accordingly, in some embodiments, the methods provided herein involve administering an effective amount of one or more anti-KLK5 / KLK7 antibodies provided herein to a subject, which is aimed at restoring the epidermal barrier in the subject requiring such administration. In other embodiments, methods are provided for addressing one or more aspects of modified barrier function. For example, in some embodiments, the methods provided herein involve administering an effective amount of one or more anti-KLK5 / KLK7 antibodies provided herein to a subject, which is aimed at reducing dermal infiltration in the subject requiring such administration. In some embodiments, the method provided herein comprises administering an effective amount of one or more anti-KLK5 / KLK7 antibodies provided herein to a subject, with the aim of reducing epidermal inflammation in the subject requiring such administration. In some embodiments, the method provided herein comprises administering an effective amount of one or more anti-KLK5 / KLK7 antibodies provided herein to a subject, with the aim of reducing epidermal permeability in the subject requiring such administration. In some embodiments, the method provided herein comprises administering an effective amount of one or more anti-KLK5 / KLK7 antibodies provided herein to a subject, with the aim of reducing parakeratosis in the subject requiring such administration. In some embodiments, the method provided herein comprises administering an effective amount of one or more anti-KLK5 / KLK7 antibodies provided herein to a subject, with the aim of reducing skin inflammatory cytokines in the subject requiring such administration. In some embodiments, the method provided herein comprises administering an effective amount of one or more anti-KLK5 / KLK7 antibodies provided herein to a subject, with the aim of reducing transepidermal water loss in the subject requiring such administration.

[0216] Further aspects of this disclosure relate to methods and compositions (e.g., anti-KLK5 / KLK7 antibodies) useful for treating atopic dermatitis. Atopic dermatitis (AD), also known as eczema, is a common chronic pruritic inflammatory skin disease. In some embodiments, atopic dermatitis develops in subjects during infancy or early childhood (e.g., 2 years of age or about 2 years of age). Therefore, in some embodiments, the methods provided herein are useful for treating subjects having atopic dermatitis who are 2 years of age or older. Atopic dermatitis may be associated with elevated total serum IgE levels. Therefore, in some embodiments, the methods provided herein are useful for treating subjects having atopic dermatitis whose total serum IgE levels are elevated (e.g., compared to normal IgE levels in subjects without atopic dermatitis or related conditions). In some embodiments, atopic dermatitis is associated with chronic recurrent skin inflammation, impaired epidermal barrier function (e.g., leading to dry skin), and / or IgE-mediated sensitization to allergens such as food and environmental allergens. Accordingly, in some embodiments, a method for treating a subject having atopic dermatitis is provided herein, the method comprising administering an effective amount of anti-KLK5 / KLK7 antibody to the subject. Furthermore, in some embodiments, the subject to be treated exhibits chronic recurrent skin inflammation, impaired epidermal barrier function, and / or IgE-mediated sensitization to allergens.

[0217] Further embodiments of this disclosure relate to methods and compositions (e.g., anti-KLK5 / KLK7 antibodies) useful for treating subjects having Netherton syndrome. In some embodiments, methods for treating subjects having Netherton syndrome are provided herein, the methods comprising administering an effective amount of one or more anti-KLK5 / KLK7 antibodies to the subject. Netherton syndrome is a rare and severe autosomal recessive skin disorder. In some embodiments, Netherton syndrome is associated with atopic findings accompanied by congenital erythroderma, specific hair shaft abnormalities, and / or elevated IgE levels (e.g., compared to normal IgE levels in subjects without Netherton syndrome or related conditions). In some embodiments, subjects having Netherton syndrome exhibit atopic findings including eczematous rash, atopic dermatitis, pruritus, hay fever, angioedema, urticaria, elevated serum IgE levels, and / or eosinophilia. In some embodiments, Netherton syndrome is caused by a mutation in the serine protease inhibitor Cazal type 5 (SPINK5) gene, which encodes a protease inhibitor lymphoepithelial Cazal type-related inhibitor. In some embodiments, the absence of this protease inhibitor leads to hyperactivity of epidermal proteases, followed by stratum corneum exfoliation. Therefore, in some embodiments, the method provided herein comprises administering an effective amount of one or more anti-KLK5 / KLK7 antibodies provided herein to a target, with the aim of improving one or more aspects or symptoms associated with Netherton syndrome (e.g., atopic findings, e.g., cutaneous rash, scaling, stratum corneum exfoliation).

[0218] Further embodiments of this disclosure relate to methods and compositions (e.g., anti-KLK5 / KLK7 antibodies) useful for treating eosinophilic esophagitis. In some embodiments, symptoms of eosinophilic esophagitis include difficulty eating, growth retardation, vomiting, epigastric or chest pain, dysphagia, and food impaction. In some embodiments, subjects with eosinophilic esophagitis are young males with a relatively high predisposition to atopic diseases. In some embodiments, subjects with eosinophilic esophagitis are diagnosed by endoscopic and / or biopsy findings of isolated eosinophils in the esophagus. In some embodiments, eosinophilic esophagitis is histologically defined by the presence of proliferative changes, which in some embodiments include thickening of the basal epithelial layer and / or elongation of the papillae, at least 24 eosinophils per high-magnification field of view of the distal esophagus, and / or absence of eosinophilia in any other intestinal segment evaluated. In some embodiments, subjects with eosinophilic esophagitis have low levels or deficiencies of a specific serine protease inhibitor belonging to the lymphoepithelial Cazal-type inhibitor protein family, such as SPINK7, as observed in esophageal biopsy, for example. In some embodiments, eosinophilic esophagitis is distinguished from reflux esophagitis based on the degree of mucosal eosinophilia and lack of response to acid suppression. In some embodiments, a method for treating subjects with eosinophilic esophagitis is provided herein, comprising administering an effective dose of one or more anti-KLK5 / KLK7 antibodies to the subject.

[0219] Further aspects of this disclosure relate to methods and compositions (e.g., anti-KLK5 / KLK7 antibodies) useful for treating prurigo nodosa. Prurigo nodosa is a chronic inflammatory skin disease characterized by symmetrically distributed, extremely itchy rashes, most commonly appearing on the arms, legs, upper back, and / or abdomen. In some embodiments, prurigo nodosa appears alone. However, in some embodiments, prurigo nodosa is associated with other skin diseases or medical conditions, such as cancer, diabetes, chronic kidney disease, or AIDS. In some embodiments, alterations in the function of the immune and nerve systems in the skin are thought to be associated with an increased sensation of itchiness (pruritus) leading to frequent scratching behavior; such frequent scratching and picking of the skin contributes to further thickening and formation of the lesions. Accordingly, in some embodiments, methods for treating subjects having prurigo nodosa are provided herein, the methods comprising administering an effective amount of one or more anti-KLK5 / KLK7 antibodies to the subject.

[0220] Further embodiments of this disclosure relate to methods and compositions (e.g., anti-KLK5 / KLK7 antibodies) useful for treating chronic pruritus. In some embodiments, chronic pruritus is associated with itching lasting more than 6 weeks (e.g., up to 3 months, up to 6 months, up to 1 year or longer). In some embodiments, chronic pruritus occurs in association with chronic kidney disease, hepatobiliary disease, and potentially unrelated diseases, including neuropathic entities such as brachioradial pruritus and dysalgesia. In some embodiments, chronic pruritus of unknown etiology (CPUO) is established when the underlying cause of the itching cannot be identified. In some embodiments, chronic pruritus is associated with severe itching and significant scratching lesions. In some embodiments, methods for treating subjects having chronic pruritus including CPUO are provided herein, the methods comprising administering an effective amount of one or more anti-KLK5 / KLK7 antibodies to the subject.

[0221] Further embodiments of this disclosure relate to methods and compositions useful for treating ichthyosis vulgaris (e.g., anti-KLK5 / KLK7 antibodies). In some embodiments, ichthyosis vulgaris is caused by heterozygous mutations in the filaggrin gene. In some embodiments, subjects having homozygous or compound heterozygous mutations in this gene have a more severe phenotype. In some embodiments, ichthyosis vulgaris is histologically characterized by the absence or reduction of keratohyalin granules in the epidermis and mild keratosis. Keratohyalin contains a histidine-rich protein that is a precursor form (profilaggrin) of filaggrin, which is a keratin filament aggregate protein. In some embodiments, profilaggrin and filaggrin are reduced or absent in subjects having ichthyosis vulgaris. In some embodiments, ichthyosis vulgaris includes increased dermal texture of the palms, keratosis pilaris, and fine scaling most prominent on the lower abdomen, arms, and legs. In some embodiments, subjects may exhibit significant scaling. In some embodiments, subjects may exhibit enhanced palmar dermal texture, keratosis pilaris, and possibly microscaling. Accordingly, in some embodiments, methods for treating subjects having ichthyosis vulgaris or one or more symptoms or phenotypic features are provided herein, the methods comprising administering an effective amount of one or more anti-KLK5 / KLK7 antibodies to the subject.

[0222] Further embodiments of this disclosure relate to methods and compositions useful for treating psoriasis (e.g., anti-KLK5 / KLK7 antibodies). Psoriasis (or psoriasis vulgaris) is a chronic inflammatory dermatitis. In some embodiments, psoriasis is characterized by red, scaly skin patches that may be found on the scalp, elbows, and / or knees of the subject. In some embodiments, psoriasis is associated with severe arthritis in the subject. In some embodiments, psoriasis-related lesions are caused by abnormal keratinocyte proliferation and infiltration of inflammatory cells into the dermis and epidermis. In some embodiments, the subject experiences the onset of psoriasis between the ages of 15 and 30. In some embodiments, psoriatic lesions are characterized by induration, scaling, and / or erythema of the skin, which may be accompanied by histological evidence of inflammation, abnormal keratinocyte proliferation / terminal differentiation, and / or dermal angiogenesis. In some embodiments, psoriatic inflammatory infiltration may be prominent at the dermal-epidermal junction and may include activated T cells and antigen-presenting cells (APCs). In some embodiments, the presence of activated T cells and APCs in such infiltrates precedes the development of epidermal hyperplasia. In some embodiments, an increase in inflammatory cytokines is detectable in lesions of psoriatic epidermis, which may lead to enhanced T cell activation and accelerated keratinocyte hyperplasia and differentiation. In some embodiments, a method for treating subjects having psoriasis is provided herein, the method comprising administering an effective amount of one or more anti-KLK5 / KLK7 antibodies to the subjects. In some embodiments, the subjects are treated before the onset of epidermal hyperplasia. However, in some embodiments, the subjects are treated after the onset of epidermal hyperplasia and accelerated keratinocyte differentiation.

[0223] Further embodiments of this disclosure relate to methods and compositions (e.g., anti-KLK5 / KLK7 antibodies) useful for treating rosacea. Rosacea is an inflammatory disease characterized by erythema, pustular papules, and / or telangiectasia. In some embodiments, subjects with rosacea express abnormally high levels of cathelicidine in their facial skin. In some embodiments, the proteolytic forms of cathelicidine peptides found in rosacea differ from those present in normal subjects. In some embodiments, methods for treating subjects with rosacea are provided herein, the methods comprising administering an effective amount of one or more anti-KLK5 / KLK7 antibodies to the subject.

[0224] Further aspects of this disclosure relate to a method for treating a subject having asthma, which is provided herein, comprising administering an effective amount of one or more antibodies disclosed herein (e.g., anti-KLK5 / KLK7 antibodies) to the subject. In some embodiments, a method for treating a subject having asthma is provided herein, which comprises administering an effective amount of one or more anti-KLK5 / KLK7 antibodies. In some embodiments, the subject has asthma (e.g., a subject to be treated with the KLK5-targeted antibody provided herein). In some embodiments, asthma is selected from allergic asthma, aspirin-sensitive / exacerbating asthma, smoking-induced asthma, asthma uncontrolled with corticosteroids or other chronic asthma control agents, atopic asthma, bronchial obstruction-related asthma, etiology-related asthma, chronic asthma, corticosteroid-naive asthma, corticosteroid-refractory asthma, corticosteroid-resistant asthma, high eosinophil asthma, eosinophilic asthma, low eosinophil asthma, exercise-induced asthma, mild asthma, moderate to severe asthma, Netherton syndrome asthma, newly diagnosed and / or untreated asthma, non-allergic asthma, non-Th2-driven asthma, high periostin asthma, low periostin asthma, low Th2 asthma, type 2 (T2)-driven asthma, and low type 2 inflammatory asthma. In some embodiments, the subjects have atopic asthma or allergic asthma. In some embodiments, the subjects have aspirin-sensitive asthma or aspirin-exacerbated asthma. In some embodiments, the subjects have asthma associated with nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, in some embodiments, the subjects have asthma induced by aspirin or a similar NSAID (e.g., recently ingested aspirin or a similar NSAID). In some embodiments, the subjects have bronchospasm that may or may not be characterized as asthma. For example, in some embodiments, the subjects have exercise-induced bronchospasm.

[0225] In some embodiments, the subjects have eosinophilic asthma. In some embodiments, the subjects have eosinophil-positive (EIP) asthma. In some embodiments, the subjects have eosinophil-negative (EIN) asthma. In some embodiments, the subjects have high eosinophil asthma (e.g., at least about 150, 200, 250, 300, 350, or 400 eosinophils / ml in the blood). In some embodiments, the subjects have low eosinophil asthma (e.g., less than about 150 eosinophils / μL in the blood, or less than about 100 eosinophils / μL). These and further examples of asthma-related conditions treatable with the compositions provided herein (e.g., anti-KLK5 / KLK7 antibodies) are disclosed in WO2015 / 061441, METHODS OF DIAGNOSING AND TREATING EOSINOPHILIC DISORDERS, published April 30, 2015, and the relevant content of that publication is incorporated herein by reference.

[0226] In some embodiments, subjects have exercise-induced asthma, intermittent or exercise-induced, mild asthma, mild or corticosteroid-naive asthma, moderate to severe asthma, Netherton syndrome asthma, newly diagnosed untreated asthma, or severe asthma. In some embodiments, subjects have asthma that has never previously required or used the use of inhaled topical or systemic steroids (e.g., chronic use) to manage or control symptoms (e.g., cough, wheezing, shortness of breath / dyspnea, and chest pain).

[0227] In some embodiments, subjects have low-periostin asthma (e.g., serum periostin levels less than approximately 20 ng / mL). In some embodiments, subjects have high-periostin asthma (e.g., serum periostin levels of at least approximately 20 ng / mL, 25 ng / mL, or 50 ng / mL). In some embodiments, subjects have non-allergic asthma (e.g., induced by, for example, infection with respiratory viruses (e.g., influenza, coronavirus, parainfluenza, rhinovirus, human metapneumovirus, and respiratory fusion viruses), or inhalation of irritants (air pollutants, smog, combustion particles (e.g., diesel particles), volatile chemicals, indoor and outdoor gases), or relatively cool, dry air, or not). In some embodiments, subjects have asthma as a result of acute or chronic primary or indirect exposure to smoke (tobacco, cigars, pipes, or other combustion products), or inhalation or use of e-cigarettes (nicotine, cannabis, or other similar substances). In some embodiments, the subjects have persistent chronic severe asthma accompanied by acute events of potentially life-threatening symptom exacerbations (worsening or relapses).

[0228] In some embodiments, the subject has a type 2 (Th2) T helper lymphocyte or type 2 (Th2) high-level asthma condition. In some embodiments, the subject has Th2-induced asthma. For example, in some embodiments, Th2 cells and / or their secreted effector molecules mediate an immune response to an allergen, which is triggered by exposure to a specific allergen that results in allergic asthma in the subject. In some embodiments, the subject has activated Th2 cell-mediated asthma, which may be triggered in part by the secretion of interleukins, e.g., IL-4, IL-5, and IL-13. Therefore, in some embodiments, the KLK5 antibody (or other antibody) may be combined individually or in a multispecific antibody format with one or more antibodies targeting cytokines such as IL-13, IL-17, IL-5, and IL-4, as well as allergy-related targets such as IgE. Examples of such antibodies for the treatment of asthma include, but are not limited to, omalizumab (XOLAIR®) (targeting soluble IgE); lebrikizumab (targeting IL-13); mepolizumab (targeting IL-5); and kilizumab (targeting membrane-bound IgE).

[0229] In other embodiments, the antibodies described herein are used to treat inflammatory disorders, infectious diseases, allergic diseases, and autoimmune disorders, but are not limited to those described below. In some embodiments, inflammatory disorders are selected from, but are not limited to, rosacea, prurigo nodosa, Crohn's disease, ankylosing spondylitis, ulcerative colitis, hidradenitis suppurativa, and uveitis, or one or more symptoms related to their barrier function. In some embodiments, allergic diseases are selected from, but are not limited to, eczema, atopic dermatitis, asthma, sinusitis, and eosinophilic esophagitis. In some embodiments, autoimmune diseases are selected from, but are not limited to, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, Behçet's disease, and psoriasis vulgaris, or one or more symptoms related to their barrier function.

[0230] Determining whether a certain amount of antibody (e.g., anti-KLK5 / KLK7 antibody) has achieved a therapeutic effect will be obvious to those skilled in the art based on the teachings provided herein. The effective dose will vary depending on the specific condition being treated, the severity of the condition, the parameters of the individual patient, such as age, physical condition, size, sex, and weight, the duration of treatment, the nature of any concurrent treatments, the specific route of administration, and similar factors within the scope of the knowledge and expertise of the healthcare professional, as recognized by those skilled in the art. The specific administration plan used in the methods described herein, i.e., dose, timing, and repetitions, will depend on the specific subject and their medical history, as discussed herein.

[0231] Generally, empirical considerations, such as the time to maximum effect, half-life, and / or time above a certain concentration, will contribute to the determination of the dosage. For example, antibodies that are compatible with the human immune system, such as humanized or fully human antibodies, may be used to extend the antibody half-life and prevent the antibody from being attacked by the host immune system. Other reasons for adjusting the dose include differences in sex, age, individual response, polymorphism of the antibody target, and / or pharmacokinetic or pharmacodynamic responses driven by receptors involved in antibody clearance. The frequency of administration may be determined and adjusted throughout the course of treatment, generally, but not necessarily, based on the treatment and / or suppression and / or improvement and / or delay of the target disease / impairment. Alternatively, a sustained-release formulation of the antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0232] The frequency of administration may vary according to the claimed method. In some embodiments, the composition may be administered once. In some embodiments, the composition will be administered multiple times. In some embodiments, the frequency of administration may be weekly, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, or every ten weeks; or once a month, every two months, or every three months, or at longer intervals. In some embodiments, the composition may be administered daily, every other week, weekly, every other month, monthly, or at any time interval that provides suitable (e.g., maximum) efficacy while minimizing safety risks to the subject. In general, efficacy and risks of treatment and safety may be monitored throughout the course of treatment.

[0233] In some embodiments, a subject may be administered the composition provided herein (e.g., an anti-KLK5 / KLK7 antibody) at one or more intervals over a set period. In some cases, the periods during which the subject is administered the composition at one or more intervals may be separated by periods during which the subject is not administered the composition. In some embodiments, the relative duration of each period may depend on the subject's response to the treatment, the severity of the disease, or both, and / or may be determined based on the judgment of the physician administering the treatment.

[0234] In some embodiments, antibodies can be administered parenterally. For example, the parenterally administered composition may be administered topically, transmucosally, subcutaneously, intradermally, intravenously, intraperitoneally, intratumorally, intramuscularly, intraarticularly, intraarterially, or by infusion techniques.

[0235] In some embodiments, the antibody (e.g., anti-KLK5 / KLK7 antibody) is administered intravenously. In some embodiments, the antibody (e.g., anti-KLK5 / KLK7 antibody) is administered subcutaneously or topically.

[0236] For intravenous injection, water-soluble antibodies can be administered by drip infusion, thereby injecting a pharmaceutical preparation containing the antibody and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Other injectable compositions may contain various carriers, such as vegetable oil, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). In some cases, preparations, such as sterile preparations in a suitable soluble salt form of the antibody, can be dissolved and administered in pharmaceutical excipients such as water for injection, 0.9% saline, or 5% glucose solution.

[0237] In one embodiment, the antibody is administered via site-specific or targeted local delivery technology. Examples of site-specific or targeted local delivery technologies include various implantable, transdermal, or transmucosal depot sources of the antibody or local delivery system.

[0238] Anti-KLK5 / KLK7 antibodies and the treatment methods involved herein may be used in combination with other types of treatments for the target diseases or disorders disclosed herein. In this regard, the antibody compositions and therapeutic agents may be administered simultaneously or sequentially. Such treatments may be administered simultaneously or sequentially (in any order) with the treatments according to this disclosure.

[0239] Accordingly, aspects of this disclosure relate to methods and compositions (e.g., anti-KLK5 / KLK7 antibodies). In some embodiments, the antibodies described herein may be administered as combination therapy (simultaneously or sequentially, e.g., over time). In some embodiments, combination therapy involves administering one or more of the antibodies described herein (e.g., anti-KLK5 / KLK7 antibodies) and at least one additional therapeutic agent (e.g., therapeutic agents 1, 2, 3, 4, 5, 6, or 7). In some embodiments, one or more of the antibodies described herein and at least one additional therapeutic agent (e.g., therapeutic agents 1, 2, 3, 4, 5, 6, or 7) are administered together. In some embodiments, one or more of the antibodies described herein and at least one additional therapeutic agent (e.g., therapeutic agents 1, 2, 3, 4, 5, 6, or 7) are administered individually.

[0240] In some embodiments, the additional therapeutic agent is an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is selected from, but is not limited to, low-dose antibiotics, steroids, corticosteroids, tacrolimus, anti-IL4R antibodies (e.g., dupilumab), anti-IL-13 antibodies, TNF inhibitors (e.g., anti-TNF), IL-12 / 23 inhibitors, IL-17 inhibitors, and IL-4 receptor inhibitors, doxycycline, methotrexate, prednisone, cyclosporine, mycophenolate mofetil, dupilumab, certolizumab pegol, etanercept, adalimumab, infliximab, golimumab, ustekinumab, secukinumab, ixekizumab, brodalumab, abatacept, tildrakizumab-asmn, risankizumab-rzaa, and guselkumab. In some embodiments, the anti-inflammatory agent is administered orally. In some embodiments, the anti-inflammatory agent is administered topically. In some embodiments, the anti-inflammatory agent is administered by injection (e.g., intravenously, subcutaneously, or intramuscularly).

[0241] In some embodiments, the therapeutic combination includes one or more of the antibodies described herein (e.g., anti-KLK5 / KLK7 antibodies) delivered together with one or more additional antibodies or fragments thereof (e.g., antibodies 1, 2, 3, 4, 5, 6, or 7). In some embodiments, the therapeutic combination including one or more of the antibodies described herein and one or more additional antibodies or fragments thereof (e.g., antibodies 1, 2, 3, 4, 5, 6, or 7) is delivered separately. In some embodiments, the therapeutic combination including one or more of the antibodies described herein and one or more additional antibodies or fragments thereof (e.g., antibodies 1, 2, 3, 4, 5, 6, or 7) is delivered together.

[0242] In some embodiments, a therapeutic combination comprising one or more antibodies described herein and one or more additional antibodies or fragments thereof (e.g., 1, 2, 3, 4, 5, 6, or 7 antibodies) is a multispecific antibody combination. In some embodiments, a multispecific antibody combination comprises a KLK5 / KLK7 antigen-binding site and one or more additional individual antigen-binding sites from one or more additional antibodies. In some embodiments, a multispecific antibody comprises direct fusion or linkage of different antigen-specific binding sites. In some embodiments, additional antibodies or fragments thereof may be selected from, but are not limited to, anti-IL4R antibodies, anti-IL-13 antibodies, anti-TNF antibodies, anti-IL-12 / 23 antibodies, anti-IL-17 antibodies, doxycycline, dupilumab, certolizumab pegol, etanercept, adalimumab, infliximab, golimumab, ustekinumab, secukinumab, ixekizumab, brodalumab, abatacept, tildrakizumab-asmn, risankizumab-rzaa, and / or guselkumab.

[0243] In some embodiments, in non-limiting examples, in the treatment of rosacea, additional therapeutic agents may be selected from topical steroids; oral methotrexate; oral cyclosporine; and / or TNF inhibitors. In some embodiments, in non-limiting examples, in the treatment of atopic dermatitis, additional therapeutic agents may be selected from subcutaneous dupilumab and / or topical steroids.

[0244] Any of the anti-KLK5 / KLK7 antibodies disclosed in this specification can be used to detect the presence of KLK5 and / or KLK7 in vitro or in vivo. The results obtained from such detection methods can be used for diagnostic purposes (e.g., diagnosis of diseases related to KLK5 and / or KLK7) or scientific research purposes (e.g., identification of new KLK5-secreting cell types, research on the biological activity and / or regulation of secreted KLK5 and / or KLK7). In assay applications such as diagnostic uses, the anti-KLK5 / KLK7 antibodies described in this specification may be conjugated with a detectable label (e.g., an imaging agent such as a contrast agent) for detecting the presence of KLK5 and / or KLK7 either in vivo or in vitro.

[0245] As used herein, "conjugated" or "attached" means that two entities are associated with a sufficient affinity such that the therapeutic / diagnostic benefit of the association between the two entities is realized. The association between the two entities may occur directly or through a linker such as a polymeric linker. Conjugated or attached can include covalent or non-covalent bonds, and other forms of binding, such as entrapment where one entity is on or within another entity, or one or both of the entities are on or within a third entity, such as micelles.

[0246] In other embodiments, the anti-KLK5 / KLK7 antibodies described in this specification can be attached to a detectable label, which is a compound capable of emitting a detectable signal directly or indirectly, whereby the aptamer can be detected, measured, and / or identified in vitro or in vivo. Examples of such "detectable labels" are intended to include, but are not limited to, fluorescent labels, chemiluminescent labels, colorimetric labels, enzyme markers, radioisotopes, and affinity tags such as biotin. Such labels can be conjugated to the aptamer directly or indirectly by conventional methods.

[0247] The reporter agent may also be a dye, such as a fluorophore, that is useful for detecting diseases mediated by KLK5 and / or KLK7-expressing cells in tissue samples.

[0248] For in vitro diagnostic assays, anti-KLK5 / KLK7 antibodies can be brought into contact with a sample suspected of containing KLK5 and / or KLK7, such as KLK5-expressing cells or a disease microenvironment containing soluble KLK5. The antibody and sample can be incubated for a suitable period under suitable conditions that allow the antibody to bind to the KLK5 antigen. Such interactions can then be detected by conventional methods, such as ELISA, histological staining, or FACS. For in vivo diagnostic assays, a suitable amount of anti-KLK5 / KLK7 antibody can be conjugated with a label (e.g., an imaging agent or contrast agent) and administered to the subject requiring testing. The presence of the labeled antibody can be detected by conventional methods based on the signal released from the label.

[0249] Anti-KLK5 / KLK7 antibodies can be used to perform scientific research assays to study the biological activity of KLK5 and / or KLK7, detect the presence of intracellular or extracellular KLK5 and / or KLK7, and / or control the effects of KLK5. For example, a suitable amount of anti-KLK5 / KLK7 can be contacted with a sample suspected of producing KLK5 and / or KLK7 (e.g., a novel cell type not previously identified as a KLK5 and / or KLK7-producing cell). These cells are permeabilized before contact with the anti-KLK5 / KLK7 antibody. The antibody and sample can be incubated for a suitable period of time under suitable conditions that allow the antibody to bind to the KLK5 antigen. Such interactions can then be detected by conventional methods, such as ELISA, histological staining, or FACS.

[0250] VI. Kits for therapeutic and diagnostic use This disclosure also provides kits for therapeutic and diagnostic applications disclosed herein. Such kits may comprise one or more containers containing antibodies, for example, any of those described herein.

[0251] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administering antibodies to treat, delay the onset of, or mitigate target diseases as described herein. The kit may further include instructions for selecting individuals suitable for treatment based on whether or not the individual has a target disease. In other embodiments, the instructions may include instructions for administering antibodies to individuals at risk of a target disease.

[0252] Instructions for the use of the described antibody generally include information regarding the dosage, administration plan, and route of administration for the intended treatment. Containers may be unit doses, multi-dose packages (e.g., multi-dose packages) or sub-unit doses. Instructions provided in the kit of the present invention are typically written instructions on a label or accompanying document (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions stored on a magnetic or optical storage disk) are also acceptable.

[0253] The label or accompanying information indicates that the composition is used to treat, delay the onset of, and / or alleviate, a disease or disorder. Instructions for carrying out any of the methods described herein may be provided.

[0254] The kit of the present invention is contained in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth bottles, and flexible packaging (e.g., Shield Mylar or plastic bags).

[0255] Packaging for use in combination with specific devices, such as injection devices, such as minipumps, is also intended. The kit may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous needle). The container may also have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous needle). At least one activator in the composition is an antibody described herein.

[0256] The kit may optionally provide further components such as buffers and interpretation information. Typically, the kit includes a container and a label or accompanying information sheet on or near the container. In some embodiments, the present invention provides a pharmaceutical product comprising the contents of the kit described above.

[0257] Furthermore, this specification provides kits for use in detecting target proteins (e.g., KLK5 and / or KLK7) in a sample. Such kits may include any of the antibodies described herein. In some examples, the antibodies may be conjugated with detectable labels as described herein. As used herein, “conjugated” or “attached” means that two entities are associated, preferably with sufficient affinity to realize the therapeutic / diagnostic benefit of the association between the two entities. The association between the two entities may occur directly or via a linker such as a polymer linker. Conjugated or attached can include covalent or non-covalent bonds, and other forms of binding, such as entrapments, e.g., micelles, where one entity is on or inside another, or one or both entities are on or inside a third entity.

[0258] Alternatively or additionally, the kit may include a second antibody that can conjugate to the antibody described herein. The kit may further include instructions for using the antibody to detect a target protein (e.g., KLK5 and / or KLK7). [Examples]

[0259] Example 1: Generation and selection of anti-KLK5 and anti-KLK7 antibodies. (i) Affinity KLK5 and KLK7 are mediators of pathological conditions observed in cases of protease activation abnormalities (Figure 1). Antibody binding reaction kinetics via Biacore were used to screen for anti-KLK5 / KLK7 antibodies with high affinity for each target for use in inhibiting KLK5 / 7 activity and improving or eliminating symptoms associated with subsequent protease activation abnormalities. All screening assays were performed at 25°C. The running buffer used was 20 mM HEPES, 300 mM NaCl, 0.01% Tween®-20, pH 7.5. Antibodies (1 ug / mL) were captured on Fc2-4 of a Series S Protein A sensor chip (Cytiva) at a flow rate of 10 uL / min for 30 seconds. Reaction kinetics were performed in single-cycle reaction mode using a series of 4-5 concentrations at maximum concentrations of 25-100 nM and 4-fold serial dilutions. The addition time was typically 300 seconds, and the dissociation time ranged from 1800 to 3600 seconds. A typical flow rate of 30 μL / min was used. The sensor tip was regenerated using 10 mM glycine, pH 1.5, with an addition time of 30 seconds and a flow rate of 50 μL / min. The results show that the dual inhibitory antibodies listed in Table 1a exhibited binding specificity for both KLK5 and KLK7. A table of binding reaction rates for the anti-KLK5 antibody is provided in Table 2 below. [Table 2]

[0260] (ii) Protease inhibition assay To evaluate the ability of the antibodies listed in Table 1a to inhibit KLK5 and / or KLK7 protease activity, 1.5 nM human or mouse KLK5 or 0.5 nM human or mouse KLK7 were prepared in assay buffer (0.1 M NaH2PO4 pH=7.5 for KLK5, and 50 mM Tris, 150 mM NaCl, pH7.5 for KLK7). The antibodies, diluted to the test concentration in PBS, were added. Then, the substrates were added (50 μM BOC-Val-Pro-Arg-AMC for KLK5, and 30 μM KHLF-AMC for KLK7). The fluorescence signal was then measured minute by minute at room temperature for 30 minutes.

[0261] These results, as shown in Table 2, demonstrate that anti-KLK5 / KLK7 antibodies can inhibit the protease activity of KLK5 and KLK7.

[0262] Example 2. An anti-KLK5 / KLK7 antibody that specifically binds to the active form of human KLK5. KLK5 / 7-Dual-Ab4 or Comparative Antibody #1 (bispecific antibody having a first arm that binds to KLK5 and a second arm that binds to KLK7) was captured on a Series S Protein A sensor chip (Cytiva) at a concentration of 1 μg / ml. Either the active form or proform of huKLK5 or huKLK7 was flowed at 50 μL / min, at concentrations of 0.156 nM, 0.625 nM, 2.5 nM, 10 nM or 40 nM, with an injection time of 5 minutes and a dissociation time of 1 hour. The sensor chip was regenerated using 10 mM glycine, pH 1.5, with an injection time of 30 seconds and a flow rate of 50 μL / min. KLK5 / 7-Dual-Ab4 was found to bind to active huKLK5 (Figure 2A) and active huKLK7 (Figure 2B), but not to either proform. Comparative Antibody #1 binds to both the proform and active form of KLK5 (Figure 2A) and KLK7 (Figure 2B) with similar potency. These data indicate that the appropriate structure of the active site is not present in the proform of the protease, as KLK5 / 7-Dual-Ab4 binds to the active sites of huKLK5 and huKLK7. Comparative Antibody #1 binds to allosteric sites present in both the active form and proform, but not to the active site of the enzyme.

[0263] Example 3. Anti-KLK5 / KLK7 antibodies are not cleaved upon binding to their targets. A concern in the development of antibodies that bind to proteases is that the proteolytic activity of the target protein of the antibody can cause cleavage of the antibody once bound, eliminating the activity of the antibody. To determine whether KLK5 or KLK7 can cleave anti-KLK5 / KLK7 antibodies, stoichiometric amounts of KLK and mAb were incubated at 37 °C for 18 hours. 40 pmol of KLK5 or KLK7 per response was used and mixed with 20 pmol of each test antibody to a final volume of 20 μL. After overnight incubation, each response was analyzed by reducing an SDS-PAGE gel.

[0264] Cleavage of the antibody heavy chain (HC) in complementarity-determining region 3 (CDR3) yields 12kDa and 38kDa fragments. SDS-PAGE analysis demonstrated that co-incubation of active KLK5 with a control anti-KLK5 antibody (referred to as "control anti-KLK5 antibody" in this example), which binds to the active site of KLK5 and is cleaved by KLK5, produced 12kDa and 38kDa fragments, whereas incubation with anti-KLK5 / 7 antibody and active KLK5 did not produce any fragments, indicating that anti-KLK5 / 7 antibody is not cleaved by KLK5 (Figure 3A). SDS-PAGE analysis of co-incubation with active KLK7 and anti-KLK5 / 7 antibody demonstrated that neither anti-KLK5 / 7 antibody was cleaved by KLK7 (Figure 3B).

[0265] Recent crystal structures of control anti-KLK5-Ab1 Fab and KLK5 / 7-dual-Ab1 Fab show that HC CDR3s from these anti-KLK5 / 7 antibodies are inserted into their respective KLK active sites (Figure 11). While the control anti-KLK5-Ab1 HC CDR3 is inserted into the register (N→C), the KLK5 / 7-dual-Ab1 CDR3 is inserted into the register (C→N) (Figure 12). This reversed register insertion is likely the reason why anti-KLK5 / 7 antibodies are not cleaved by their targets.

[0266] Example 4. The anti-KLK5 / KLK7 antibody is specific to KLK5 and KLK7 and does not bind to other KLK family members. Members of the kallikrein family and kallikrein-like families are quite similar at the genetic and protein levels. Therefore, developing antibodies that specifically bind to selected members(s) of the KLK family is challenging. To determine whether anti-KLK5 / 7 antibodies bound to other KLK family members or related proteases, 10 ug / mL of anti-KLK5 / 7 antibody was tested for inhibition of plasma KLK, KLK1, KLK2, KLK4, KLK6, KLK12, KLK14, trypsin, chymotrypsin, and urokinase, using substrate cleavage as the read.

[0267] The substrates used for the protease assay were as follows: plasma KLK, KLK1, KLK2 (Pro-Phe-Arg-AMC); KLK4, KLK12, KLK13, and KLK14 (Boc-VPR-AMC); KLK6 (Boc-QAR-AMC); trypsin (MCA-RPKPVG-NVAL(DNP)-NH2); chymotrypsin (Suc-AAPF-AMC); and urokinase (Z-Gly-Gly-Arg-AMC).

[0268] The following enzymes were prepared in assay buffer at the following concentrations: plasma KLK (10 nM); KLK1 (10 nM); KLK2 (10 nM); KLK4 (5 nM); KLK6 (5 nM), KLK12 (2.5 nM), KLK14 (2.5 nM), trypsin (0.25 nM), chymotrypsin (17 nM), and urokinase (20 nM).

[0269] The assay buffers used were as follows: KLK1 and 2 (50mM Tris, 150mM NaCl, 10mM CaCl2, 0.05% (w / v) Brij(registered trademark)-35, pH 7.5); KLK3 (50mM Tris, 1M NaCl, pH 8.0); KLK4 (50mM Tris, 1M NaCl, pH 8.0); KLK6 (50mM Tris, 1M sodium citrate, pH 7.5); KLK12 and 13 (100mM Tris, 150mM NaCl, 10mM CaCl2, 0.05% (w / v) Brij-35, pH 7.5); KLK14 (50mM Tris, 150mM NaCl, 0.05% (w / v) Brij-35, pH 8.0), trypsin (1×HBS + 1mM CaCl2 + 0.05% Tween20, pH 7.4); chymotrypsin (25 mM Tris, 0.5 mM CaCl2, pH 8.0); urokinase (50 mM Tris + 0.01% Tween20, pH 8.5); and plasma kallikrein KLKb1 (50 mM Tris, 250 mM NaCl, pH 7.5).

[0270] The antibody was diluted in PBS to the test concentration, and then the substrate was added. Fluorescence was then measured every minute for 30 minutes at room temperature.

[0271] The following KLK family members were screened for affinity using Biacore: hKLK3, hKLK8, hKLK9, hKLK10, hKLK11, hKLK12, hKLK13, and hKLK15. All screening assays were performed at 25°C. The running buffer used was 20 mM HEPES, 300 mM NaCl, 0.01% Tween-20, pH 7.5. Antibodies (1 μg / mL) were captured on Fc2-4 of a Series S Protein A sensor chip (Cytiva) at a flow rate of 10 μL / min for 30 seconds. Reaction kinetics were performed in single-cycle reaction mode using 25 nM and 100 nM analytes. The addition time was 120 seconds, and dissociation was 300 seconds. A flow rate of 30 μL / min was typical. The Protein A sensor chip was regenerated using 10 mM glycine, pH 1.5, with an addition time of 30 seconds and a flow rate of 50 μL / min.

[0272] Since the anti-KLK5 / 7 antibody demonstrated some binding / inhibitory activity for KLK6, KLK14, and KLK13, follow-up studies were conducted on these antibodies. All IC50 curves were at least 100 times higher than those for KLK5 or KLK7, demonstrating that the anti-KLK5 / 7 antibody is highly specific to KLK5 and KLK7, and not specific to other KLK family members or related proteases (Figure 4).

[0273] Example 5. The anti-KLK5 / KLK7 antibody binds to the active sites of KLK5 and KLK7. The inhibitory ability of an antibody is related to its ability to compete with the target substrate for binding to the active site of its target. The binding activity of anti-KLK5 / 7 antibodies to KLK5 and KLK7 was tested in the presence of small molecule inhibitors known to bind to the active sites of KLK5 and KLK7.

[0274] Binning was performed by immobilizing dual-specific KLK5 / 7 antibody (2 ug / mL) onto the surface of a Protein A chip. The flow rate was 30 uL / min and the addition time was 30 seconds. Next, huKLK5 (Figure 5A) or huKLK7 (Figure 5B) was pre-incubated with an inhibitor and then applied to the chip. 20 nM KLK5 or KLK7 was incubated with 0.35 mg / mL of the small molecule inhibitor PMSF (2 mM final concentration, prepared from 100-fold stock), 1 mg / mL of the peptide inhibitor leupeptin (1 mM final concentration), or 25 ug / mL SPINK5. The flow rate was 30 uL / min, the addition time was 300 seconds, and the dissociation time was 120 seconds.

[0275] When KLK5 or KLK7 was pre-incubated with various inhibitors that bind to the active site, little to no binding of anti-KLK5 / 7 antibodies to KLK5 (Figure 5A) or KLK7 (Figure 5B) was observed. This indicates that anti-KLK5 / 7 antibodies cannot bind if the active site is already bound by another molecule, thus demonstrating that anti-KLK5 / 7 antibodies bind to the active sites of KLK5 and KLK7.

[0276] Example 6. The anti-KLK5 / KLK7 antibody binds to the same KLK5 and KLK7 epitopes as SPINK5. The binding of anti-KLK5 / 7 antibodies to the active sites of KLK5 and KLK7 was further analyzed in this example. Binning was performed in a sandwich format. In the first experiment, the anti-KLK5 / 7 antibody KLK5 / 7-dual-Ab4 was covalently bound to the CM5 chip surface, followed by huKLK5 or huKLK7. Next, a second anti-KLK5 / 7 antibody was flowed onto the chip surface at a concentration of 10 μg / mL (Figure 6, left graph). In the second experiment, recombinant human SPINK5 was covalently bound to the CM5 chip surface, followed by huKLK5 or huKLK7. Next, the anti-KLK5 / 7 antibody was flowed onto the surface (Figure 6, right graph). The running buffer was 20 mM HEPES, 300 mM NaCl, 0.05% Tween-20, pH 7.5. Biacore was performed at a flow rate of 50 μL / min. For each antibody or protein, the addition time was 60 seconds, and the dissociation time was 60 seconds.

[0277] These data demonstrate that the anti-KLK5 / 7 antibody KLK5 / 7-dual-Ab4 competes with human SPINK5 for binding to huKLK5 and huKLK7. Since SPINK5 binds to the active sites of both KLK5 and KLK7, this further indicates that the anti-KLK5 / 7 antibody KLK5 / 7-dual-Ab4 also binds to the active sites of KLK5 and KLK7, and that neither SPINK5 nor the inhibitory antibody can bind to either KLK5 or KLK7 simultaneously (Figure 6).

[0278] Example 7. Anti-KLK5 / 7 dual antibody treatment reduces barrier defects in the MC903 atopic dermatitis mouse model. Mouse atopic dermatitis was induced in C57 / B6 mice by topically applying 40 μL of MC903 (Sigma, calcipotriol hydrate, 2 ng / 10 μL in ethanol) to the shaved area of ​​the neck once daily from day 1 to day 8.

[0279] Mice were pre-treated by intraperitoneal administration of either an MC903-free vehicle control, control IgG (non-specific antibody), KLK5 / 7-dual-Ab4, or comparative antibody #2 (anti-mouse IL-4R antibody) at a dose of 30 mg / kg, three times per week starting from day -6. Alternatively, control IgG was administered subcutaneously (SC) at a dose of 30 mg / kg via Q3D starting from day -6; KLK5 / 7-dual-Ab4 was administered via SC at a dose of 3 mg / kg via Q3D, Q7D, or Q14D starting from day -6; and comparative antibody #2 was administered intravenously (IP) at a dose of 30 mg / kg via Q3D starting from day -6.

[0280] On day 9, dorsal skin samples were excised from each mouse and preserved in 10% neutral buffered formalin. The skin was trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Stratum corneum thickness was measured at 800 μM intervals according to a grid set up in Qupath and then averaged for each animal (Figures 7A-7B).

[0281] Treatment with KLK5 / 7-dual-Ab4 significantly reduced keratosis and desquamation, as measured by stratum corneum thickness, compared to the comparative antibody #2. The minimum effective subcutaneous dose was 3 mg / kg every 7 days.

[0282] Example 8. Treatment with anti-KLK5 / 7 dual antibodies reduces disease in a mouse model of Nc / Nga atopic dermatitis. Mouse atopic dermatitis was induced in 8-10 week old Nc / Nga mice (a strain with endogenous barrier deficiency) via topical administration of dust mite (HDM) allergen. A total of 480 mg per mouse was applied to the bilateral ears and dorsal skin area (including the neck) of Biostir-AD ointment (120 mg / mouse) containing HDM allergen derived from Dermatophagoides farina, twice weekly for two weeks. Two hours prior to the second Biostir-AD treatment (day 4), 150 μL of sodium dodecyl sulfate (SDS) was applied.

[0283] Mice were pre-treated by intraperitoneal injection of control IgG, KLK5 / 7-dual-Ab4, or comparative antibody #2 at a dose of 30 mg / kg, three times per week for a total of nine doses, starting from day -6. Tacrolimus, a steroid compound used to treat atopic dermatitis but which cannot be used long-term due to severe withdrawal effects, was applied topically once daily for a total of 21 days, starting from day -6, one hour after each application of Biostir-AD.

[0284] To evaluate the effectiveness of the treatment, mice were assessed on day 15 for scratching behavior (Figure 8C), macroscopic skin lesions, and ear thickness (Figure 8B). To assess scratching behavior, mice were housed individually and allowed to acclimate for 30 minutes before the start of evaluation. Scratching behavior was recorded by visual observation for 60 minutes on day 0 and for 30 minutes after administration on day 15. Ear thickness was measured using a Dyer model micrometer gauge. The severity of skin lesions was assessed using four parameters: erythema, hemorrhage, edema, exfoliation / erosion, and scaling / dryness. Skin lesion parameters were assessed on the ear, neck, and dorsal skin. The total clinical skin severity score was defined as the sum of the individual scores (0: none, 1: mild, 2: moderate, 3: severe) (Figure 8A).

[0285] From the dorsal skin of each mouse, 1 cm 2 The tissue was excised and preserved in 10% neutral buffered formalin. The skin was trimmed, embedded in paraffin, sectioned, and stained with H&E. Epidermal area (Figure 8E) was analyzed and measured using Qupath software, and stratum corneum thickness measurements were obtained every 800 μM based on a Qupath grid and then averaged to generate values ​​for each animal. The samples were microscopically examined by a pathologist and scored for inflammation, necrosis, hypertrophy, and keratosis (Figure 8D). Serum was also analyzed for the presence of HDM-specific IgE antibodies (Figure 8F).

[0286] Treatment with KLK5 / 7-dual-Ab4 reduced ear thickness, clinical skin lesions, and itching to a similar degree as treatment with comparative antibody #2 or tacrolimus. Histological analysis showed that the reduction in keratosis and desquamation, as measured by stratum corneum thickness, was significantly greater after treatment with KLK5 / 7-dual-Ab4 compared to treatment with tacrolimus or comparative antibody #2, suggesting that KLK5 / 7-dual-Ab4 is a potent inhibitor of atopic dermatitis.

[0287] When the above experiment was repeated using KLK5 / 7-dual-Ab2, it was found that itching (Figure 8I), stratum corneum thickness (Figure 8H), and histological score (Figure 8G) were reduced compared to control IgG, which is comparable to the effect of competing antibody #2 (Figure 8B).

[0288] Example 9. Treatment with anti-KLK5 / 7 antibody reduces disease in a mouse model of flaky tail atopic dermatitis. Mouse atopic dermatitis was induced in male flaky-tail mice (flaky-tail mice have a unique barrier defect caused by loss of function of filaggrin, filament-related proteins, and the transmembrane protein matrin) via topical application of HDM allergens. The necks of all mice were shaved before administration of HDM allergens. The disease-free control group received topical application of petrolatum to the shaved area of ​​the neck and both ears three times a week for six weeks. All other treatment groups received topical application of Biostir cream to the shaved area of ​​the neck and both ears three times a week for six weeks. Approximately 100 mg of Biostir cream was used per mouse for each application. Control IgG or KLK5 / 7-dual-Ab4 was administered intraperitoneally to the control and test groups at a dose of 30 mg / kg three times a week starting from day 0.

[0289] From the dorsal skin of each mouse, 1 cm 2The tissue was excised and preserved in 10% neutral buffered formalin. The skin was trimmed, embedded in paraffin, sectioned, and stained with H&E. Epidermal area (Figure 9A) was measured by analysis using Qupath software. The samples were microscopically examined by a pathologist and scored for parakeratosis (Figure 9B) and spongiform changes (Figure 9C). Cytokine expression was measured from lysed ear tissue.

[0290] Histological analysis revealed that KLK5 / 7-dual-Ab4 treatment for atopic dermatitis reduced epidermal thickness, parakeratosis, and spongiform changes. Cytokine analysis showed reductions in the allergy-related cytokine interleukin (IL)-4 (Figure 9D) and the inflammatory cytokine tumor necrosis factor alpha (TNFα) (Figure 9E). These data demonstrate that inhibition of KLK5 / 7 can reduce inflammation and epidermal activity in a mouse model of atopic dermatitis.

[0291] Example 10. Treatment with anti-KLK5 / 7 antibody reduces keratosis in disease-induced human epidermal equivalent gas-liquid interface cultures. EpiDermFT cultures consisted of primary human keratinocytes superimposed on a cortex seeded with fibroblasts, differentiated to produce epidermis in transwells, and incubated overnight in assay buffer. 80 μM MC903 treatment was applied to the upper part of the transwell on day 0 to induce injury and keratosis. The cultures were then placed in a medium containing 10 μg / mL of control IgG (Figure 10B, control without MC903, Figure 10A), KLK5 / 7-dual-Ab4 (Figure 10C), comparative antibody #1 (Figure 10D), or comparative antibody #3 (anti-KLK5 monospecific antibody that does not bind to the KLK5 active site) (Figure 10E) in the upper and lower wells, for a total of 20 μg / mL. Antibody treatment was started on day 0 and continued for 5 days. Each condition was tested in six separate cell culture inserts. Histogel was added to the top of the cell insert, the insert was fixed with formalin, embedded in paraffin, sectioned, and stained with H&E. Stratum corneum thickness was measured at 100 μM intervals using Qupath image analysis software.

[0292] Measurements of stratum corneum thickness (Figure 10F) showed that MC903 treatment induced keratosis in human EpiDermFT cultures. Treatment with either KLK5 / 7-dual-Ab4 or comparative antibody #1 bispecificity mitigates this keratosis because it inhibits both KLK5 and KLK7. Treatment with comparative antibody #3, which inhibits only KLK5, shows some effect on keratosis, but to a lesser degree. These data suggest that inhibition of both KLK5 and KLK7 is necessary for complete relief of epidermal dysfunction.

[0293] Example 11. Crystal structure of KLK5 / 7-dual-Ab1. The crystallization conditions for the complex between KLK5 / 7-dual-Ab1 and human KLK7 (StoA variant) were 25% (w / v) polyethylene glycol, 0.1 M PCTP buffer (pH 8), 20°C, and a protein-reservoir ratio of 1:1. The structure was fully refined using autoBUSTER and validated with MolProbity. KLK5 / 7-dual-Ab1 and its derivatives are characterized by a longer-than-average heavy chain CDR3 loop (indicated by arrows) occupying the antigen's active site (Figure 11). Inhibition of the catalytic activity of the KLK enzyme was achieved through competitive inhibition. The endogenous KLK7 substrate occupied the S4-S1 active site from the N-terminus to the C-terminus. In contrast, KLK5 / 7-dual-Ab1 Fab occupies its active site from the C-terminus to the N-terminus (indicated by the arrow) (Figure 12), thereby protecting the Fab from proteolysis through occupancy in the reverse register. Tyrosine occupies a crucial S1 binding pocket, anchored through a polar interaction between the hydroxyl group of the Tyr residue and the side chain of the Asn residue located behind the pocket.

[0294] Other Embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise expressly indicated, each disclosed feature is merely an example of a general set of equivalent or similar features.

[0295] From the above description, those skilled in the art will readily grasp the essential features of the present invention and will be able to adapt it to various uses and circumstances by making various changes and modifications without departing from the spirit and scope of this disclosure. Therefore, other embodiments are also described below.

[0296] Equivalents and Scope In the claims, articles such as “a,” “an,” and “the” may mean one or more unless otherwise indicated or the context makes it clear. A claim or description containing “or” between one or more members of a group is deemed satisfied unless otherwise indicated or the context makes it clear that one, more or all of the members of that group are present, adopted or otherwise related in a given product or process. The present invention includes embodiments in which exactly one member of a group is present, adopted or otherwise related in a given product or process. The present invention includes embodiments in which more or all of the members of a group are present, adopted or otherwise related in a given product or process.

[0297] Furthermore, the present invention encompasses all variations, combinations, and rearrangements in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Where elements are presented as a list, for example in Markush group format, each subgroup of the elements is also disclosed, and any element(s) may be removed from that group. In general, where the present invention or an aspect of the present invention is referred to as including certain elements and / or features, it should be understood that a particular embodiment or aspect of the present invention consists of, or is essentially derived from, such elements and / or features. For brevity, these embodiments are not specifically described herein in these terms.

[0298] As used herein and in the claims, the phrase “and / or” should be understood to mean “either or both” of the thus combined elements, i.e., elements that exist associatively in some cases and dissociatively in others. Multiple elements listed with “and / or” should be interpreted in the same manner, i.e., “one or more” of the thus combined elements. Other elements other than those specifically identified by the “and / or” clause may exist at their discretion, whether or not they are related to the specifically identified elements. Thus, as a non-restrictive example, when used in conjunction with non-restrictive language such as “comprising,” a reference to “A and / or B” may refer to A only in one embodiment (optionally including elements other than B); B only in another embodiment (optionally including elements other than A); and still in yet another embodiment to both A and B (optionally including other elements).

[0299] Where used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted as inclusive, that is, including at least one of several elements or lists of elements and additional items not listed by choice, but also including multiple. Only terms that indicate the exact opposite, such as “one of” or “exactly one of” or, where used in the claims, “consisting of,” would refer to including exactly one element of several elements or lists of elements. In general, where used herein, the term “or” should be interpreted as indicating exclusive substitutes (i.e., “one or the other, but not both”) only when preceded by exclusive terms such as “either,” “one of,” “one of” or “exactly one of.” Where used in the claims, “essentially consisting of” should have the usual meaning as used in the field of patent law.

[0300] When used herein and in the claims, the phrase “at least one” when referring to a list of one or more elements means at least one element selected from any one or more elements in the list of elements, and does not necessarily have to include at least one of every element specifically enumerated in the list of elements, nor does it exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase “at least one” refers, whether or not they are related to the specifically identified elements. Therefore, as a non-restrictive example, “at least one of A and B” (or equivalently, “at least one of A or B” or equivalently, “at least one of A and / or B”) could, in one embodiment, refer to at least one A that optionally includes two or more and B is absent (optionally including elements other than B); in another embodiment, refer to at least one B that optionally includes two or more and A is absent (optionally including elements other than A); and in yet another embodiment, refer to at least one A that optionally includes two or more, and at least one B that optionally includes two or more (optionally including other elements); and so on.

[0301] Furthermore, unless explicitly stated otherwise, it should be understood that in any method claimed herein that involves multiple steps or actions, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described.

[0302] As with the above specification, all transitional phrases in the claims, such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and “composing,” should be understood to be non-restrictive, meaning they include it but are not limited to it. Only the transitional phrases “essentially from” and “essentially from” are considered restrictive or semi-restrictive transitional phrases, respectively, as described in Section 2111.03 of the U.S. Patent and Trademark Office's Examination Manual. Embodiments described herein using non-restrictive transitional phrases (e.g., “including”) should be understood to also be construed in alternative embodiments as features “~consisting of” and “~essentially from” as described by non-restrictive transitional phrases. For example, if this application describes “a composition comprising A and B,” this application also construes alternative embodiments “a composition comprising A and B” and “a composition essentially from A and B.”

[0303] If a range is given, it includes the endpoints. Furthermore, unless otherwise indicated or otherwise evident from the context and the understanding of those skilled in the art, the values ​​expressed as a range may be any specific value or subrange within the range specified in various embodiments of the invention, up to one-tenth of the lower limit of the range, unless explicitly indicated otherwise in the context.

[0304] This application references various published patents, published patent applications, academic papers, and other publications, all of which are incorporated herein by reference. In the event of any conflict between any of the incorporated references and this specification, this specification shall prevail. In addition, any particular embodiment of the present invention that falls within the scope of prior art may be clearly excluded from any one or more claims. Such embodiments are considered to be known to those skilled in the art and may be excluded even if such exclusion is not expressly stated herein. Any particular embodiment of the present invention may be excluded from any claim for any reason, whether or not it relates to the existence of prior art.

[0305] Those skilled in the art will be able to recognize or confirm many equivalents to the specific embodiments described herein by simply using conventional experiments. The scope of the embodiments of the present invention described herein is not intended to be limited to the embodiments for carrying out the invention described above, but is as set out in the appended claims. Those skilled in the art will understand that various changes and modifications may be made to this specification without departing from the spirit or scope of the invention as defined in the following claims.

[0306] The enumeration of lists of chemical groups in any definition of a variable herein includes the definition of the variable as any single group or as a combination of the enumerated groups. The enumeration of embodiments relating to a variable herein includes embodiments as any single embodiment or embodiments in combination with any other embodiments or parts thereof. The enumeration of embodiments herein includes embodiments as any single embodiment or embodiments in combination with any other embodiments or parts thereof.

Claims

1. A dual inhibitory antibody that specifically binds to KLK5 and KLK7, (a) HC CDR1, HC CDR2, and HC CDR3 heavy chain variable domains having the amino acid sequence of SEQ ID NO: 7, and LC CDR1, LC CDR2, and LC CDR3 light chain variable domains having the amino acid sequence of SEQ ID NO: 8; (b) HC CDR1, HC CDR2, and HC CDR3 heavy chain variable domains having the amino acid sequence of SEQ ID NO: 13, and LC CDR1, LC CDR2, and LC CDR3 light chain variable domains having the amino acid sequence of SEQ ID NO: 14; (c) HC CDR1, HC CDR2 and HC CDR3 heavy chain variable domains having the amino acid sequence of SEQ ID NO: 17, and LC CDR1, LC CDR2 and LC CDR3 light chain variable domains having the amino acid sequence of SEQ ID NO: 14; or (d) The dual inhibitory antibody comprising HC CDR1, HC CDR2, and HC CDR3 heavy chain variable domains having the amino acid sequence of SEQ ID NO: 21, and LC CDR1, LC CDR2, and LC CDR3 light chain variable domains having the amino acid sequence of SEQ ID NO:

14.

2. The aforementioned dual inhibitory antibody (a) HC CDR1 having the amino acid sequence of SEQ ID NO: 1, HC CDR2 having the amino acid sequence of SEQ ID NO: 2, HC CDR3 having the amino acid sequence of SEQ ID NO: 3, LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 6; (b) HC CDR1 having the amino acid sequence of SEQ ID NO: 9, HC CDR2 having the amino acid sequence of SEQ ID NO: 10, HC CDR3 having the amino acid sequence of SEQ ID NO: 11, LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12; (c) HC CDR1 having the amino acid sequence of SEQ ID NO: 9, HC CDR2 having the amino acid sequence of SEQ ID NO: 15, HC CDR3 having the amino acid sequence of SEQ ID NO: 16, LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO: 12; or (d) A dual inhibitory antibody according to claim 1, comprising HC CDR1 having the amino acid sequence of SEQ ID NO: 18, HC CDR2 having the amino acid sequence of SEQ ID NO: 19, HC CDR3 having the amino acid sequence of SEQ ID NO: 20, LC CDR1 having the amino acid sequence of SEQ ID NO: 4, LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC CDR3 having the amino acid sequence of SEQ ID NO:

12.

3. The aforementioned antibody (a) VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 8; (b) VH containing the amino acid sequence of SEQ ID NO: 13, and VL containing the amino acid sequence of SEQ ID NO: 14, (c) VH containing the amino acid sequence of SEQ ID NO: 17 and VL containing the amino acid sequence of SEQ ID NO: 14; or (d) The dual inhibitory antibody according to claim 1 or 2, comprising VH containing the amino acid sequence of SEQ ID NO: 21 and VL containing the amino acid sequence of SEQ ID NO:

14.

4. A dual inhibitory antibody comprising one of the dual inhibitory antibodies listed in Tables 1a and 1b: HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and / or LC CDR3.

5. A dual inhibitory antibody comprising one of the dual inhibitory antibodies listed in Tables 1a and 1b, specifically the VH and / or VL.

6. The dual inhibitory antibody according to any one of claims 1 to 5, wherein the dual inhibitory antibody binds to the active site of KLK5 and the active site of KLK7.

7. The dual inhibitory antibody according to any one of claims 1 to 6, wherein the dual inhibitory antibody competes with SPINK5 and / or leupeptin for binding to the KLK5 active site and the KLK7 active site.

8. The dual inhibitory antibody according to any one of claims 1 to 7, wherein the dual inhibitory antibody binds to the active form of KLK5 and the active form of KLK7, but does not bind to the inactive form of KLK5 or the inactive form of KLK7.

9. The dual inhibitory antibody according to any one of claims 1 to 8, wherein the dual inhibitory antibody inhibits the protease activity of KLK5 and KLK7.

10. The dual inhibitory antibody according to any one of claims 1 to 9, wherein the antibody is not cleaved in the heavy chain by KLK5 or KLK7 upon binding to KLK5 or KLK7.

11. A dual inhibitory antibody according to any one of claims 1 to 10, wherein the antibody is not a bispecific antigen-binding molecule in which KLK5 binding is conjugated by one binding site within the antibody and KLK7 binding is conjugated by a different binding site.

12. The dual inhibitory antibody according to any one of claims 1 to 11, wherein the antibody is a multispecific antigen-binding molecule further comprising an antigen-binding domain that binds to an antigen other than KLK5 or KLK7.

13. A composition comprising a dual inhibitory antibody and an acceptable carrier according to any one of claims 1 to 12.

14. A nucleic acid encoding a dual inhibitory antibody according to any one of claims 1 to 12.

15. A method for treating a skin barrier defect, comprising administering to a subject an effective amount of a dual inhibitory antibody according to any one of claims 1 to 2, or the composition according to claim 13.

16. The method according to claim 15, wherein the skin barrier defect is associated with Netherton syndrome, atopic dermatitis, eosinophilic esophagitis, prurigo nodularis, chronic pruritus of unknown cause (CPUO), dry skin, asthma (especially KLK5), ichthyosis vulgaris, or itching or chronic itching.

17. A dual inhibitory antibody according to any one of claims 1 to 12 or a composition according to claim 13, for use in a method for treating skin barrier defects.

18. A dual inhibitory antibody for use according to claim 17, wherein the skin barrier defect is associated with Netherton syndrome, atopic dermatitis, eosinophilic esophagitis, prurigo nodosa, chronic pruritus of unknown cause (CPUO), dry skin, asthma (particularly KLK5), ichthyosis vulgaris, or itching or chronic itching.

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