Bispecific antibodies for use in treatment of hidradenitis suppurativa

Abstract

To provide a method and a pharmaceutical composition for the treatment or prevention of hidradenitis suppurativa (HS).SOLUTION: The present invention provides a method for the treatment or prevention of hidradenitis suppurativa (HS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody antagonist that specifically binds and inhibits the activity of IL-18 and IL-1β.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a bivalent, bispecific monoclonal antibody for use in treating patients suffering from hidradenitis suppurativa. The present disclosure relates to a clonal antibody (bbmAb) or variants thereof. By using a bispecific antibody that simultaneously targets both IL-18 and IL-19, It also relates to methods and treatment regimens for treating inflammation. [Background technology]

[0002] "Acne inversa" or "Maladie de Vergne" Hidradenitis suppurativa (HS), also known as hidradenitis suppurativa (HS), is a condition that generally affects the apocrine glands of the body. Chronic, recurrent and debilitating, presenting as deep, inflammatory and painful lesions in areas containing glands It is an inflammatory skin condition of the genital tract, anterior cruciate ligaments, and posterior cruciate ligaments. The most commonly affected areas are the armpits, groin, and anogenital area. Yes (Jemec 2012; Fimmel and Zouboulis 2010) .

[0003] HS is currently believed to be a hair follicle-mediated disease based on an imbalance in the immune system that occurs in genetically predisposed individuals. It is thought to be an inflammatory disease of the sebaceous follicles (Kelly et al 2014). While it is generally considered a disease caused primarily by hair follicle obstruction, HS is also thought to be a disease caused primarily by hair follicle obstruction. It is an inflammatory skin disease characterized by a large number of neutrophils and macrophages (Lima et al. The pathophysiology of HS remains unclear. However, there is a larger body of evidence that blocking tumor necrosis factor alpha (TNFα) is beneficial. This has been described in a recent study (Kimball et al. 2016). (Tzanetakou et al 2016) and IL-17A blockade (Thor Lacius et al 2017, Schuch et al 2018, Gius eppe et al 2018, Jorgensen et al 2018) or anti- IL-23 treatment (Sharon et al 2012, Blok et al 201 6) Evidence of efficacy is also available from smaller studies and / or case reports More recently, the oral PDE4 inhibitor apremilast (Weber et al. 2017) or research approaches using anti-complement 5a compounds (Kanni et al. 2018). is stated.

[0004] The disease begins after puberty and affects females more frequently than males (3:1). Risk factors include obesity and smoking. Epidemiological prevalence estimates vary widely ( 0.03~4.3%;Jemec 2012, Jemec and Kimball 2 015) Although geographical differences exist, the prevalence is estimated by the scientific community to be approximately 0.1-1%. It is accepted (Garg et al. 2018).

[0005] Although the clinical manifestations of HS are heterogeneous, the disease is characterized by a chronic, relapsing, deep-seated, painful inflammatory process. It tends to manifest with inflammatory skin lesions, which in most cases may lead to pus discharge and suppuration. Inflammatory lesions may develop into sinus tracts and fistulas during disease progression. This can lead to complicated hypertrophic scar formation that can have an impact on functional use.

[0006] HS is associated with pain, foul-smelling discharge from wounds and scarring, and has devastating psychosocial consequences. HS is a severe, debilitating disease that often affects quality of life. This has a significant negative impact on quality of life (QoL), and studies have shown that this impact is not seen in other skin diseases. It has been confirmed that the 2016). People with HS often also suffer from depression, social isolation, and poor sexual health. Patients may experience difficulty in carrying out their duties (Esmann and Jemec 2011, Fi mmel and Zouboulis 2010, Janse et al. 2017 ).

[0007] HS is difficult to treat. Official European treatment guidelines were only drawn up in 2015. that patients should be provided with adjuvant, medical and surgical treatment; has been suggested (Zouboulis et al. 2015).

[0008] Topical antibiotics may be used for mild cases, while topical antibiotics are recommended for moderate to severe HS. Generally, a combination of tetracycline or clindamycin and rifampicin is used. A prolonged course of multidrug systemic antimicrobial therapy with Maintenance with chronic antibiotic treatment for several years may follow (Bettoli et al. 2016, Dessinioti et al. 2016, Zouboulis et al. al 2015).

[0009] However, it is now widely recognized that HS is a chronic inflammatory state and not an infectious disease ( Jemec 2012). Anti-inflammatory drugs are therefore an alternative and perhaps better option than antibiotics. It may be a more appropriate approach or complementary to antibiotics. In addition, if chronic recurrent inflammation is inadequately treated, it can result in irreversible fibrosis. This manifests as scarring and tunnels or sinus tracts that do not respond to medical therapy. Persistent anatomical changes occur, reducing the volume of fibrous tissue and increasing the size of the affected skin. The only treatment option to improve function in the skin area is surgery (Ande rsen and Jemec 2017). One of the goals of future treatment is to reduce residual The goal should be to reduce scarring and avoid surgery, which is the prevention of inflammatory lesions. or may require specific treatment.

[0010] In 2015, a recombinant human monoclonal antibody against soluble and membrane-bound TNF-α was developed. Adalimumab (Humira®), an immunoglobulin G1 (IgG1) antibody, , which received regulatory approval for the treatment of moderate to severe HS. As reported in the PIONEER I and II studies, respectively, The SCR (hidradenitis suppurativa clinical response) rate was approximately 16% (41.8% adalimumab vs. 26% prolactin). placebo) and 31% (58.9% adalimumab vs. 27.6% placebo). The response rate was higher than that of adalimumab (Kimball et al 2016). When taken together, adalimumab has the potential to treat serious conditions including tuberculosis, invasive fungal infections and other opportunistic infections. Adalimumab is associated with increased safety risks for serious infections. Increases have also been reported.

[0011] Therefore, it has a favorable safety profile for patients with moderate to severe HS. There is an unmet need for systemic therapies that effectively reduce inflammation while retaining the immune system There is. Summary of the Invention

[0012] In HS, both cytokines IL-1β and IL-18 are upregulated ( Kelly et al. 2015), and thus may be involved in its pathogenesis. The IL-1β signature is present in HS lesions and is reversed by application of IL-1 receptor antagonists (Witte-Handel et al. 2019). Anakinra, an anti-cancer drug, has shown promising clinical results compared with placebo in small trials. The results of the bed effectiveness are presented (Tzanetakou et al. 2016), while Case reports confirm these findings (Leslie et al 2014 , Zarchi et al 2013, Andre et al 2019). Case report According to the report, blocking IL-1β using only the anti-IL-1β antibody canakinumab was effective in treating moderate to severe cases of in HS (Houriet et al 2017) or PASH (pyoderma gangrenosum) , and related syndromes such as acne and hidradenitis suppurativa (Jaeger et al. 201 3) may be beneficial. However, canakinumab (Sun et al. 2017, Tekin et al 2017) and anakinra (van der Ze e and Prens 2013, Russo and Alikhan 2016) It is likely that, because some of the studies have been unsuccessful in HS due to The fact that only a portion of the population may be responsive to anti-IL-1 blockade or this blockade alone The fact that the current treatment regimen may not be sufficient to achieve results in most patients suggests that To be tempted.

[0013] Accordingly, the object of the present disclosure is to detect and / or block the inflammasome effector cytokines, IL-1β and The aim of this study is to target both IL-18 and IL-19, thus preventing (auto)-inflammatory processes, e.g. HS. In some cases, IL-1β and IL-18 may contribute independently to the pathophysiology of the disease. It is a further object of the present disclosure to provide a method for the preparation of a bispecific anti-IL- IL-1β / 18 antagonist inhibits inflammasome-dependent and The key to these effects is their ability to rapidly neutralize inflammasome-independent and -dependent causes of inflammation.

[0014] Thus, the use of a bispecific anti-IL-1β / 18 antibody or fragment thereof, which is capable of binding to both IL-1β and IL-1 Any antagonist capable of inhibiting both 8 may be suitable for the treatment of HS.

[0015] IL-1β and The bispecific antibody or functional fragment thereof that simultaneously targets both IL-18 and IL-19 is described herein. In a preferred embodiment, the anti-IL-1β / 18 antibody is an IgG1 Fc amino acid sequence. The so-called LALA mutant contains the L234A and L235A mutations in the amino acid sequence. In a preferred embodiment, the anti-IL-1 antibody is an anti-IL-1 antibody. The β-1β / 18 antibody binds to the complementary heavy chain CH3 knob-in-hole e) a first heavy chain having mutations such as S354C and T366W (according to EU numbering) , knob-type mutations and Y349C, T366S, L368A, Y407V, hole-type mutations In a preferred embodiment, the anti-IL-1β / 18 antibody comprises a second heavy chain having a mutation. a first light chain that preferentially associates with a heavy chain of Vκ1, a kappa light chain, e.g., Vκ6, and a second It comprises a second light chain that preferentially associates with the heavy chain, and includes a lambda light chain, e.g., Vλ1. In a more preferred embodiment, the anti-IL-1β / 18 antibody comprises: i) one or more ADCC silencers; ii) one or more knob-in-hole mutations (e.g., LALA); iii) heavy chain modifications in-hole; and / or a combination of kappa and lambda light chains. In a more preferred embodiment, the anti-IL-1β / 18 antibody has the above-mentioned properties i) to ii). i), preferably the antibody comprises a Vκ6 and a Vλ1 light chain.

[0016] Administration of IL-1β and IL-18 to subjects in need of prevention or treatment of hidradenitis suppurativa (HS) suppurative sweat gland by administering a therapeutically effective amount of a bispecific antibody that simultaneously targets both Also described herein are methods for preventing or treating hepatitis (HS).

[0017] Bispecific antibodies that simultaneously target both IL-1β and IL-18 as described herein Specific dosing regimens for the methods or uses of (e.g., bbmAb1) are further described herein. will be provided.

[0018] For use in the treatment or prevention of HS, optionally in the presence of a pharma- ceutically acceptable carrier. a) a bispecific antibody that simultaneously targets both IL-1β and IL-18 (e.g., bb Pharmaceutical combinations and methods comprising administering to the patient a therapeutically effective amount of mAb 1) and b) at least one further therapeutic agent. Pharmaceutical compositions are further described herein. Further characteristics and uses of the described methods and uses Advantages will become apparent from the following detailed description.

[0019] In a first aspect, the present disclosure provides a method for administering to said subject a therapeutically effective amount of a bispecific antibody. The present invention relates to a method for treating or preventing HS in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of Regarding this antibody, a. A first variable light chain (VL1) and a first variable heavy chain (VH 1) and a first constant heavy chain (CH1) with a heterodimerization modification. a first portion which is b. A second variable light chain (VL2) and a second variable heavy chain (VL3) that specifically binds IL-18. H2) and a heterodimerization modification that is complementary to the heterodimerization modification of the first constant heavy chain. a second portion which is an immunoglobulin with a second constant heavy chain (CH2); Includes.

[0020] In a second aspect, the disclosure provides a method for administering to said subject a therapeutically effective amount of a bispecific antibody. In a subject in need of slowing, arresting, or reducing the onset of HS, For a method of slowing, inhibiting, or reducing the onset of HS, the antibody comprises: a. A first variable light chain (VL1) and a first variable heavy chain (VH 1) and a first constant heavy chain (CH1) with a heterodimerization modification. a first portion which is b. A second variable light chain (VL2) and a second variable heavy chain (VL3) that specifically binds IL-18. H2) and a heterodimerization modification that is complementary to the heterodimerization modification of the first constant heavy chain. a second portion which is an immunoglobulin with a second constant heavy chain (CH2); In certain embodiments of the first and second aspects of the disclosure, the first and second of the bispecific antibodies include The second constant heavy chain is selected from the group consisting of human IgA, IgD, IgE, IgG, and IgM, preferably IgD. , IgE or IgG, such as human IgG1, IgG2, IgG3 or IgG4, preferably is IgG1.

[0021] In another embodiment of the present disclosure, the first and second constant heavy chains of the bispecific antibody are IgG1. , a. the first constant heavy chain has a point mutation that generates a knob structure, and the second constant heavy chain has have a point mutation that creates a hole structure, or b. The first constant heavy chain has a point mutation that generates a hole structure, and the second constant heavy chain has Optionally, having a point mutation that generates a knob structure c. The first and second constant heavy chains have mutations that create disulfide bridges.

[0022] In a particularly preferred embodiment of the present disclosure, including the first and second aspects, the first The immunoglobulin VH1 domain is i. Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence said CDR2 having the amino acid sequence SEQ ID NO: 77; R3 has the amino acid sequence SEQ ID NO:78; or ii. Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence said CDR2 having the amino acid sequence SEQ ID NO: 80; DR3 has the amino acid sequence SEQ ID NO: 81 Includes; The first immunoglobulin VL1 domain of the bispecific antibody comprises iii. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 is 92, said CDR2 having the amino acid sequence SEQ ID NO: 93, CDR3 has the amino acid sequence SEQ ID NO: 94; or iv. Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence 95, said CDR2 having the amino acid sequence SEQ ID NO: 96, DR3 has the amino acid sequence SEQ ID NO: 97 Includes; The second immunoglobulin VH2 domain of the bispecific antibody comprises v. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence said CDR2 having the amino acid sequence SEQ ID NO: 45; R3 has the amino acid sequence SEQ ID NO:46; or vi. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence said CDR2 having the amino acid sequence SEQ ID NO: 48; DR3 has the amino acid sequence SEQ ID NO: 49 Includes; The second immunoglobulin VL2 domain of the bispecific antibody comprises vii. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 is 60, said CDR2 having the amino acid sequence SEQ ID NO: 61, CDR3 has the amino acid sequence SEQ ID NO: 62; or viii. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 is an amino acid the CDR2 has the amino acid sequence SEQ ID NO:64, wherein the CDR3 has the amino acid sequence SEQ ID NO: 65 Includes.

[0023] In another preferred embodiment of the present disclosure, the methods, compositions and uses described herein The antibodies used are a. a first immunoglobulin VH1 domain of the amino acid sequence SEQ ID NO: 85; b. A first immunoglobulin VL1 domain of the amino acid sequence SEQ ID NO: 101; c. a second immunoglobulin VH2 domain of the amino acid sequence SEQ ID NO:53; and d. A second immunoglobulin VL2 domain of the amino acid sequence SEQ ID NO:69 Includes.

[0024] In another preferred embodiment of the present disclosure, in the method, composition or use described herein The antibodies used are e. a first immunoglobulin heavy chain of the amino acid sequence SEQ ID NO: 87; f. a first immunoglobulin light chain of the amino acid sequence SEQ ID NO: 103; g. a second immunoglobulin heavy chain of the amino acid sequence SEQ ID NO:55; and h. A second immunoglobulin light chain having the amino acid sequence SEQ ID NO:71 Includes.

[0025] In a particularly preferred embodiment of the present disclosure, the subject being treated has HS.

[0026] In a third aspect, the present disclosure provides a method for treating HS in a subject in need of such treatment or prevention. or for prophylactic use, a. A first variable light chain (VL1) and a first variable heavy chain (VH 1) and a first constant heavy chain (CH1) with a heterodimerization modification. a first portion which is b. A second variable light chain (VL2) and a second variable heavy chain (VL3) that specifically binds IL-18. H2) and a heterodimerization modification that is complementary to the heterodimerization modification of the first constant heavy chain. a second portion which is an immunoglobulin with a second constant heavy chain (CH2); The present invention relates to a bispecific antibody comprising:

[0027] In a fourth aspect, the present disclosure provides a method for the treatment of atopic dermatitis, comprising administering to a patient a therapeutically effective amount of IL-1β or IL-2 to a patient in need thereof, the method ... A bispecific antibody that simultaneously targets both the first, second and third IL-18 is administered to the subject. In a preferred embodiment of the fourth aspect, the method and treatment is at about 10 mg / kg. The bispecific antibody of claim 1 is administered to the subject to be treated.

[0028] In one aspect of the disclosure, a bispecific antibody that simultaneously targets both IL-1β and IL-18 is The agent is administered to the subject intravenously or subcutaneously. In one embodiment, the route of administration is subcutaneous or intravenous. combination of drugs, e.g., an intravenous loading dose followed by subcutaneous maintenance doses. do.

[0029] In one embodiment, a bispecific antibody that simultaneously targets both IL-1β and IL-18 is , a dose of about 1.5 mg to about 15 mg of active ingredient per kilogram of human subject to be treated. In one embodiment, both IL-1β and IL-18 are administered at the same time, e.g., intravenously. Sometimes, the targeted bispecific antibodies are administered to the subject to be treated at a dose of about 2, 4, or 5 mg of active ingredient / ki. In one embodiment, the IL-1β and A bispecific antibody that simultaneously targets both IL-18 and IL-19 is administered to the treated subject at approximately 4 mg / kg. The dose is, for example, about 1.5 mg / kg of active ingredient per kilogram of human subject. ~About 15 mg of active ingredient / kilogram human subject dose weekly, every 2 weeks, or every 4 weeks or combinations thereof. In a preferred embodiment, for example, about 1. Dosage for humans is 5 to approximately 15 mg of active ingredient per kilogram every 2 weeks or every 4 weeks. or a combination thereof.

[0030] In one embodiment, the dosage is from about 75 mg to about 600 mg of active ingredient, preferably about 150 mg. mg to about 300 mg of active ingredient, or about 150 mg or 300 mg of active ingredient The dose is preferably 300 mg of active ingredient per minute. Dosage may be weekly, every two weeks, or every four weeks. In a preferred embodiment, the dose may be administered either in a fixed dose (e.g. Non-weight-based doses) are administered subcutaneously or intramuscularly, preferably subcutaneously.

[0031] In one embodiment, the dosage is about 300 mg of active ingredient.

[0032] In one preferred embodiment, the dosage is 150 mg of active ingredient. In another preferred embodiment, the dose is 60 mg of active ingredient. In another embodiment, the dosage is from about 150 mg of active ingredient to about 60 mg of active ingredient. 00mg of active ingredient.

[0033] In one embodiment, the antibody is administered via a loading dose and a maintenance dose. In this mode, the loading dose is administered via one or more intravenous injections of the first dose, followed by a maintenance dose. In one embodiment, the first loading dose is administered via subcutaneous injection of the second dose. The first dose is administered via subcutaneous injection, and the maintenance dose is administered via subcutaneous injection of the second dose. will be done.

[0034] In one embodiment, the loading dose is administered via subcutaneous injection of a first dose regimen, and the maintenance dose is The dose is administered via subcutaneous injection of the second dose regimen. The first dose regimen amount is The first dosing regimen may be the same as or greater than the second dosing regimen. The period is the same as the second dosing regimen period or is more frequent than the second dosing regimen period. obtain.

[0035] In one embodiment, the first dose is from about 150 mg to about 600 mg of active ingredient, for example about 3 00 mg of active ingredient, and the second dose is from about 150 mg to about 600 mg of active ingredient. , for example about 300 mg of active ingredient.

[0036] In one embodiment, the first dose contains 150 mg, 300 mg, or 600 mg of active ingredient. and the second dose is 150 mg, 300 mg or 600 mg of active ingredient. In the form, the first dose is 300 mg of active ingredient and the second dose is 300 mg of active ingredient. minutes.

[0037] In one embodiment, the loading dose comprises at least two doses and the maintenance doses are administered weekly ( In one embodiment, the dose is administered every 24 hours (Q1W), preferably every other week (Q2W) or monthly (Q4W). In one embodiment, the loading dose consists of two doses. In another embodiment, the loading dose consists of three doses. In another embodiment, the loading dose comprises four doses. The loading dose is a biweekly loading dose consisting of three doses on days 1 through 29. .

[0038] In one embodiment, the loading dose is administered in at least two subcutaneous injections, preferably three subcutaneous injections. Injections are administered weekly (Q1W), every other week (Q2W) or preferably monthly (Q4W). In one embodiment, the loading dose consists of two subcutaneous injections. In one embodiment, the loading dose consists of three subcutaneous injections, e.g., three times every other week (Q2W). In another embodiment, the loading dose is a single subcutaneous injection of 150 mg or 300 mg. It consists of four subcutaneous injections. In some cases, the loading dose is administered on days 1 through 29. A loading dose of three subcutaneous injections is given every other week. In some cases, a maintenance dose is given. Dosage is, for example, 150 mg or 300 mg subcutaneously every other week (Q2W) for three doses, followed by administration on day 57. The monthly (Q4W) maintenance dose is administered by subcutaneous injection starting at 0.5 mg / kg / month. In this study, the loading dose consisted of three subcutaneous injections administered on days 1, 15, and 29. The loading dose is every other week, and the maintenance dose is subcutaneous Q4W injections starting on day 57. In some cases, the loading dose is given on day 1. , biweekly (e.g., 150 mg or 30 mg) consisting of three subcutaneous injections on days 15 and 29. The initial loading dose was 100 mg / kg, and maintenance doses were administered subcutaneously Q4W starting on day 57. and a monthly (Q4W) (e.g., 150 mg or 300 mg) maintenance dose.

[0039] In one embodiment, the loading dose is a biweekly loading dose consisting of three doses on days 1 through 29. A loading dose, where the dosage is about 1.5 mg to about 15 mg per kilogram of active ingredient. For example, the loading dose may be administered intravenously, subcutaneously, or a combination of intravenously and subcutaneously. In one embodiment, the loading dose consists of three doses on days 1 through 29. This dose is approximately 4 mg / kilogram of active ingredient, e.g. For example, wherein the loading dose is administered intravenously, subcutaneously, or a combination of intravenously or subcutaneously. can be.

[0040] In one embodiment, the loading dose is a biweekly loading dose consisting of three doses on days 1 through 29. The loading dose is about 150 mg or about 300 mg of active ingredient, preferably about 300 mg. mg of active ingredient, e.g., the initial loading dose is administered subcutaneously or in combination with intravenous or subcutaneous administration. In one embodiment, the loading dose is administered in three doses on days 1 through 29. The loading dose is approximately 300 mg of active ingredient, and is administered subcutaneously.

[0041] In some embodiments, the loading dose is administered over a loading period (e.g., 1, 2 or Within 3 weeks or after 1, 2, or 3 initial loading dose injections) at least about 20 μg / mL to about The dose selected or expected to achieve a plasma concentration of 60 μg / mL. In some embodiments, the maintenance dose is from about 20 μg / mL to about 60 μg / mL during the maintenance administration period. mL or a substantial fraction thereof (e.g., at least about Day 29 to about Day 85) ) is a dose selected or expected to provide

[0042] In one embodiment, the initial loading subcutaneous injections are at different doses. The subcutaneous loading doses are the same doses.

[0043] HS patients may be selected according to one of the following criteria: The patient has moderate to severe HS; The patient is an adult; The patient is an adolescent; IL-1β and IL-18 antagonists (e.g., those that bind both IL-1β and IL-18) the patient had an HS-PGA score of ≥3 prior to treatment with the targeted bispecific antibody; IL-1β and IL-18 antagonists (e.g., those that bind both IL-1β and IL-18) The patient has at least three inflammatory lesions prior to treatment with the targeted bispecific antibody; or IL-1β and IL-18 antagonists (e.g., those that bind both IL-1β and IL-18) Prior to treatment with the HS-targeting bispecific antibody, the patient had no extensive scarring as a result of HS. (<10 fistulas).

[0044] In one embodiment, by week 16 of treatment, the HS patient achieves at least one of the following: R: Simple HiSCR; Reduction of HS erythema; NRS30; A decrease of ≦6 as measured by the DLQI; and / or Improved DLQI.

[0045] In one embodiment, by week 16 of treatment, at least 40% of said patients achieves an R; or at least 25% of said patients achieve an NRS30 response; or Fewer than 15% of such patients experience HS flares.

[0046] In one embodiment, the patient receives a short period of time from the first administration of the IL-1β and IL-18 antagonist. If not, then one week later, they have at least one of the following: Rapid relief of pain as measured by VAS or NRS, and A rapid decline in CRP as measured using a standard hsCRP assay.

[0047] In one embodiment, the patient is assessed for inflammatory lesion counts, HS clinical response (HiSCR), numerical rating scale (NMS), and / or inflammatory bowel disease. NRS, modified Sartorius HS score, hidradenitis suppurativa - physician global assessment ( HS-PGA or Dermatology Life Quality Index Three months after the end of treatment, as measured by the DLQI (Degree Level Quality Index) Achieve sustained response for months.

[0048] In one embodiment, the patient is in simplified HiSCR (sHiSCR) 3 months after the end of treatment. A sustained response is achieved when measured by

[0049] According to a second aspect, the therapeutic efficacy of an IL-1β and IL-18 antagonist is demonstrated in a human subject. A method of treating HS in a human subject is provided comprising administering an effective dose.

[0050] In another embodiment of the present disclosure, a bispecific antibody that targets both IL-1β and IL-18 is The body is administered in combination with at least one additional therapeutic agent.

[0051] In another aspect, the disclosure provides a method for treating, slowing, or arresting the onset of HS in a subject. and IL-18 for the manufacture of a medicament for preventing or reducing The present invention relates to the use of bispecific antibodies (e.g., bbmAb1) that

[0052] In one embodiment, a bispecific antibody that targets both IL-1β and IL-18, e.g. HS in a subject in need of treatment comprising administering an effective amount of bbmAb1. Provided herein is a method for treating HS. In one embodiment, a subject in need of treatment for HS is A dual inhibitor targeting both IL-1β and IL-18 for use in the treatment of HS in Specific antibodies, such as bbmAb1, are provided herein. In some embodiments and a combination of IL-1β and IL-18 targeting agents for the manufacture of a medicament for the treatment of HS. The use of specific antibodies, such as bbmAb1, is provided herein.

[0053] In one embodiment, the present invention relates to a method for treating, slowing, inhibiting, or reducing the onset of HS. Treating, slowing, arresting, or alleviating the onset of HS in a subject in need thereof Provided herein is a method for administering a therapeutically effective amount of IL-1β and IL-1β to a mammalian animal. administering to the subject a bispecific antibody that targets both IgG1 and IgG2-18, e.g., bbmAb1. In one embodiment, the therapeutically effective amount is an amount of a bispecific antibody comprising: a. The first immunoglobulin VH1 domain of the bispecific antibody has the amino acid sequence SEQ ID NO: Including 85, b. The first immunoglobulin VL1 domain of the bispecific antibody has the amino acid sequence SEQ ID NO: Including 101, c. The second immunoglobulin VH2 domain of the bispecific antibody has the amino acid sequence SEQ ID NO: Including 53, d. The second immunoglobulin VL2 domain of the bispecific antibody has the amino acid sequence SEQ ID NO: Includes 69.

[0054] In another aspect, the present disclosure provides a method for the preparation of a method for the treatment of IL-18 and IL-1β. The present invention provides a liquid pharmaceutical composition, a method or use of the liquid pharmaceutical composition, comprising a bispecific antibody, The formulation includes a buffer, a stabilizer, and a solubilizer. and providing a means for administering or subcutaneously administering the bispecific antibody to a patient having the same. In one embodiment, the use is for the manufacture of a medicament for the treatment of HS. This is the case.

[0055] In a particularly preferred embodiment of the present disclosure, the first immunoglobulin of the bispecific antibody of the liquid pharmaceutical composition The Brin VH1 domain is i. Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence said CDR2 having the amino acid sequence SEQ ID NO: 77; R3 has the amino acid sequence SEQ ID NO:78; or ii. Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence said CDR2 having the amino acid sequence SEQ ID NO: 80; DR3 has the amino acid sequence SEQ ID NO: 81 Includes; The first immunoglobulin VL1 domain of the bispecific antibody comprises iii. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 is 92, said CDR2 having the amino acid sequence SEQ ID NO: 93, CDR3 has the amino acid sequence SEQ ID NO: 94; or iv. Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence 95, said CDR2 having the amino acid sequence SEQ ID NO: 96, DR3 has the amino acid sequence SEQ ID NO: 97 Includes; The second immunoglobulin VH2 domain of the bispecific antibody comprises v. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence said CDR2 having the amino acid sequence SEQ ID NO: 45; R3 has the amino acid sequence SEQ ID NO:46; or vi. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence said CDR2 having the amino acid sequence SEQ ID NO: 48; DR3 has the amino acid sequence SEQ ID NO: 49 Includes; The second immunoglobulin VL2 domain of the bispecific antibody comprises vii. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 is 60, said CDR2 having the amino acid sequence SEQ ID NO: 61, CDR3 has the amino acid sequence SEQ ID NO: 62; or viii. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 is an amino acid the CDR2 has the amino acid sequence SEQ ID NO:64, wherein the CDR3 has the amino acid sequence SEQ ID NO: 65 Includes.

[0056] In another preferred embodiment of the present disclosure, the antibody of the liquid pharmaceutical composition described herein comprises: a. a first immunoglobulin VH1 domain of the amino acid sequence SEQ ID NO: 85; b. A first immunoglobulin VL1 domain of the amino acid sequence SEQ ID NO: 101; c. a second immunoglobulin VH2 domain of the amino acid sequence SEQ ID NO:53; and d. A second immunoglobulin VL2 domain of the amino acid sequence SEQ ID NO:69 Includes.

[0057] In another preferred embodiment of the present disclosure, the antibody of the liquid pharmaceutical composition described herein comprises: e. a first immunoglobulin heavy chain of the amino acid sequence SEQ ID NO: 87; f. a first immunoglobulin light chain of the amino acid sequence SEQ ID NO: 103; g. a second immunoglobulin heavy chain of the amino acid sequence SEQ ID NO:55; and h. A second immunoglobulin light chain having the amino acid sequence SEQ ID NO:71 Includes.

[0058] In some embodiments, the liquid pharmaceutical composition comprises from about 50 mg / mL to about 120 mg / mL A bispecific antibody (e.g., bbmAb1), a buffer, a stabilizer (e.g., a non-ionic stabilizer , such as sugars) and surfactants (e.g. polysorbate 20 or polysorbate 80). In some embodiments, the liquid pharmaceutical composition has a pH of about 5.5 to about 6.5, preferably or about 50 mg / mL in a buffer solution having a pH of about 5.5 to about 6.0, preferably about 6.0. The bispecific antibody is present in an amount of about 100 mg / mL to about 120 mg / mL.

[0059] In some embodiments, the liquid pharmaceutical composition contains about 100 mg / mL of a bispecific antibody (e.g., For example, bbmAb1), buffers, stabilizers (for example, non-ionic stabilizers, such as sugars, etc.), and and surfactants (e.g., polysorbate 20 or polysorbate 80). In an embodiment, the liquid pharmaceutical composition has a viscosity of about 5.5 to about 6.5, preferably about 5.5 to about 6.0 , more preferably at a pH of about 6.0, and at a bispecific concentration of about 50 mg / mL to about 120 mg / mL Antibodies (e.g., bbmAb1), from about 1 mM to about 50 mM histidine / histidine-chloride about 50 mM to about 400 mM sugar (e.g., sucrose, trehalose, or mannitol) ), about 0.01% to about 0.5% of a surfactant (e.g., polysorbate, In some cases, the liquid pharmaceutical composition comprises a pH of about 6.0. and about 50 mg / mL to about 120 mg / mL of bbmAb1 (preferably about 100 mg / mL). mL of bbmAb1), approx. 20 mM histidine / histidine-chloride, approx. 220 mM of sucrose, and about 0.04% polysorbate 20 (w / v). [Brief description of the drawings]

[0060] [Figure 1] FIG. 1 is a graphical representation of AIM2, NLRC4, NLRP7 and NLRP3 inflammasome mRNA levels in HS lesions compared to levels in non-lesioned or healthy tissue. [Diagram 2] Figure 2 is a TIBCO spotfire heatmap depiction of the mean expression levels of IL-18, IL-12 and IL-1β genes / members (alone or in combination) or CRS signaling signatures in healthy skin or lesional, perilesional and lesional skin biopsies from HS patients. Identification of genes / members of individual IL-18, IL-12 and IL-1β signaling signatures was performed in separate experiments using PBMCs from healthy donors stimulated for 6 h (restricted to genes induced by individual cytokines = UP) or adopted from publications (canakinumab response signature CRS; Brachat et al., 2017). [Diagram 3] FIG. 3 shows the effect of either bbmAb1 (100 μg / mL) or adalimumab (100 μg / mL) incubation compared to untreated controls on the levels of the analytes IFNγ, TNFα, IL-1β and IL-2 produced in the supernatants of HS skin biopsies after 24 hours of in vitro culture. Each dot represents one biopsy. The leftmost column for each specimen is the untreated control, the middle column for each specimen is bbmAb1, and the rightmost column for each specimen is adalimumab. Values ​​are shown from n=54, n=36 and n=26 individual biopsies for IFNγ, IL-1β and TNFα, and n=43, n=20 and n=10 biopsies for IL-2 from 9 different HS patients. [Figure 4] Figure 4 is a graphical representation of a prediction of the effect of bbmAb1 administration on IL-1β levels. The dotted line indicates the lower limit of quantification. The predictions are modeled assuming subcutaneous administration of 300 mg on days 1, 15, 29, 57 and 85. [Diagram 5]Figure 5 is a graphical representation of the prediction of the effect of bbmAb1 administration on IL-18 levels versus baseline change in healthy control subjects. The dotted line indicates the levels in healthy control subjects. Predictions are modeled assuming subcutaneous administration of 300 mg on days 1, 15, 29, 57 and 85. [Figure 6] Figure 6 is a graphical representation of the predicted pharmacokinetics of subcutaneously administered bbmAb1 compared to a single dose of 10 mg / kg iv. Predictions are modeled assuming subcutaneous administration of 300 mg on days 1, 15, 29, 57 and 85. [Figure 7] FIG. 7 shows the results of a test on aggregate formation. [Figure 8] FIG. 8 shows the results of a study of degradation product formation. [Figure 9] FIG. 9 shows the results of a study of acidic and basic variant formation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The key inflammasome effector cytokine interleukin-1 beta (I Heterodimeric F that simultaneously neutralizes interleukin-1β and interleukin-18 (IL-18) IgG-like bispecific monoclonal antibodies containing the c portion (e.g., bbmAb1) are disclosed herein. In certain embodiments, the bispecific antibody has picomolar binding activity against both cytokines. It has pM affinity and subnanomolar (nM) affinity in cellular in vitro assays. ) inhibits IL-1β and IL-18 signaling. IL-1β and IL-18 are Recognition of damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) These are two pro-inflammatory cytokines secreted in response to inflammasome activation. As described herein, the combined inhibition of IL-1β and IL-18 results in Inflammasome-driven proinflammatory responses compared to inhibition of either cytokine alone More effective down-regulation of sexual responses may occur.

[0062] Therefore, bispecific anti-IL-1β and anti-IL-18 antibodies with suppressed ADCC activity Any antagonist that can simultaneously block IL-1β and IL-18 activity may be useful in the treatment of HS. This may be appropriate for the location.

[0063] Without wishing to be bound by theory, the inventors believe that IL-1β and / or These patients may be at least partially or completely attributable to enhanced IL-18 levels. to completely saturate the bispecific antibody binding sites (IL-1β and / or IL-18). At the beginning of treatment, a loading dose regimen may be beneficial, with higher or more We identified the need for a more frequent dosing regimen. A loading regimen provides rapid saturation of the antigen, followed by a maintenance regimen for the entire treatment period. Conditions that promote expression of IL-1β and / or IL-18 in affected tissues throughout the body Providing sustained therapeutic plasma concentrations in patients with a range of symptoms (severity of symptoms) is considered for therapeutic efficacy. do.

[0064] Suitable dosages may be determined, for example, by administering to a patient a specific bispecific IL-1β and IL-18 antagonist, e.g. For example, the bispecific anti-IL-1β and anti-IL-18 antibodies or their antigen binding the nature of the fragment (e.g., bbmAb1), the subject of treatment, the mode of administration and the condition being treated; The severity and nature of previous treatments the patient has received will vary. A bispecific IL-1β and IL-18 antagonist for treating each individual patient is provided to a medical provider. In some embodiments, the attending healthcare provider will determine the amount of bispecific IL- A single dose of low or equal doses of IL-1β and IL-18 antagonists will be administered and patients' responses will be monitored. In another embodiment, the bispecific IL-1β and IL-18 antigens administered to the patient are The initial dose of the antagonist is high, then tapered until signs of relapse occur. Bispecific IL-1β and IL-18 antagonists are used until optimal therapeutic effect is achieved. Higher doses of the compound may be administered, and the dosage generally is not further increased.

[0065] In one embodiment, the present disclosure provides a method for the use according to any of the aspects or embodiments of the present disclosure as described above. The present invention relates to a bispecific anti-IL-1β and anti-IL-18 antibody or antigen-binding fragment thereof for use in Here, bispecific anti-IL-1β and anti-IL-18 antibodies or antigen-binding fragments thereof (e.g., bb The loading dose and maintenance dose of mAb1) are determined to achieve therapeutic plasma or serum concentrations of the antibody. It is adjusted as follows.

[0066] In some implementations of the disclosed methods of treatment or use, a therapeutically effective amount of a bispecific I L-1β and IL-18 antagonists, e.g., bispecific anti-IL-1β and anti-IL-18 The antibody or antigen-binding fragment thereof (e.g., bbmAb1) is administered to a patient, e.g., a mammal (e.g., a human The disclosed methods provide a bispecific IL-1β and IL-18 antagonist. It is understood that the treatment of HS patients using a serogroup (e.g., bbmAb1) is provided. On the other hand, this is important if the patient is ultimately to be treated with an antagonist. This does not exclude that such treatments are necessarily monotherapies. Indeed, bispecific IL-1β and if the patient is selected for treatment with an IL-18 antagonist, (e.g., bbmAb1), either alone or in combination with other drugs and treatments, It may be administered according to the methods of the present disclosure.

[0067] Certain HS patients, such as those who are treated with bispecific IL-1β and IL-18 antagonists, e.g. For example, a bispecific antibody or an antigen-binding fragment thereof (e.g., bbmAb1) is insufficient for treatment. It will be appreciated that modifications to the dosing regimen may be appropriate for patients who show a significant response. In some cases, administration may be more frequent than monthly, such as every other week (every 2 weeks) or weekly. It is possible.

[0068] Some patients may be administered a loading dose regimen (e.g., weekly dosing for several weeks [e.g., 1–4 weeks For example, administration at weeks 0, 1, 2 and / or 3, for example, administration at weeks 1, 2 and 3, for example, for 3 weeks loading regimen]) followed by at least several weeks of bispecific antibody (e.g. A maintenance dosing regimen in which bbmAb1) may be administered weekly, every other week, or preferably every 4 weeks; For example, there may be benefit from starting at week 5, 6 or 7.

[0069] For example, a suitable dosing regimen for bbmAb1 would be biweekly [e.g., 1-5 for example, at weeks 1, 3 and 5, followed by administration at week 8 or 9, preferably A monthly maintenance regimen of Q4W is followed in the first 9 weeks of treatment, and a bispecific antibody (e.g., bbmAb1) is administered. It may be administered for at least several weeks.

[0070] Dosing is less frequent than monthly, e.g., every 6 weeks, every 8 weeks (every 2 months), or yearly It will also be appreciated that the dose may be administered four times (every three months) or more.

[0071] Based on the severity of the disease, certain HS patients, such as those treated with the bispecific antigens described herein, may be treated with the bispecific antigens described herein. Inadequate response to treatment with an antagonist, such as bbmAb1 or an antigen-binding fragment thereof It will be appreciated that dose escalation may be appropriate for responsive patients. c) The dosage (loading or maintenance dose) is about 150 mg to about 900 mg sc. may exceed, for example, about 75 mg, about 100 mg, about 125 mg, about 175 mg, about 200 mg , about 250mg, about 350mg, about 400mg, about 450mg, about 500mg, about 600 mg, etc.; similarly, an intravenous (iv) dose may be about 5 mg / kg or 10 mg / kg, for example, about 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 11mg / kg, 12mg / kg, 15mg / kg, 20mg / kg, 25mg / kg, 30 mg / kg, 35 mg / kg, etc. In certain HS patients, e.g., bispecific Adverse reactions to treatment with an antagonist (e.g., bbmAb1 or an antigen-binding fragment thereof) It will also be appreciated that dose reductions may be appropriate for patients who experience an event or adverse reaction. Therefore, the dosage of the antagonist is about 150 mg to less than about 900 mg sc, e.g. Approximately 25 mg, approximately 50 mg, approximately 75 mg, approximately 100 mg, approximately 125 mg, approximately 175 mg, Approx. 200mg, approx. 250mg, approx. 350mg, approx. 400mg, approx. 450mg, approx. 500m g, about 600 mg, etc.

[0072] In some embodiments, the bispecific antagonist, e.g., bbmAb1 or its antigen, The binding fragment may be administered to a patient at an initial dose of 300 mg delivered sc, followed by subsequent doses of , 150 mg or 600 mg delivered sc as needed, as determined by physician The amount of the noise can be adjusted so that the noise is suppressed.

[0073] The bispecific antibody or antigen-binding fragment thereof may be bbmAb1, a functional derivative thereof or a biological derivative thereof. It may be an analogue.

[0074] As defined herein, a "unit dose" is from about 75 mg to 900 mg, for example about 150 mg to about 600 mg, for example, about 150 mg to about 600 mg, for example, about 300 mg to It refers to a sc dose that may contain about 600 mg or, for example, about 150 mg to about 300 mg. For example, unit sc doses are about 75 mg, about 150 mg, about 300 mg, about 350 mg, Approximately 400 mg, approximately 450 mg, approximately 500 mg, approximately 550 mg, approximately 600 mg.

[0075] The present disclosure provides, inter alia, IL-1β neutralization as compared to single IL-1β or IL-18 neutralization alone. Certain antibodies that simultaneously neutralize IL-18 and IFN-γ (and other proinflammatory cytokines) This is based on the unexpected finding that the compound more strongly attenuates cytokinin production. It is considered an effective treatment for HS by

[0076] 1.Definition For purposes of interpreting this specification, the following definitions shall apply and, whenever appropriate, the singular Terms used herein include the plural and vice versa. Further definitions are provided throughout the detailed description. will be done.

[0077] The term "about" in reference to a numerical value x means, for example, + / - 10%. When used before a list of numbers, the term "about" applies to every number in the series, e.g., The phrase "about 1 to 5" should be interpreted as "about 1 to about 5" or, for example, "about The phrase "1, 2, 3, 4" should be interpreted as "about 1, about 2, about 3, about 4, etc." be.

[0078] The term "substantially" does not exclude "completely", for example, "substantially free" of Y. The composition may be completely free of Y. Optionally, the term "substantially" may be used in the present disclosure. may be omitted from the definition.

[0079] The term "comprise" encompasses "comprising" as well as "consisting of", e.g., a composition When an item "comprises" X, it can consist exclusively of X or can include some additional For example, X+Y.

[0080] The term "IL-18" refers to the IL-18 polypeptide, interleukin-18 polypeptide, Peptides, IFN-gamma-inducing factor or interferon-gamma-inducing factor or INF The term "IL-18" is synonymous with IL-γ-inducing factor unless another species is specified. refers to human IL-18. IL-18 is well known to those skilled in the art and is available, for example, from product no. #B 001-5 MBL (registered trademark) International Corporation Throughout this application, the term IL-18 refers to the pro- or mature form. Unless otherwise specified, pro-IL-18 (mature IL-18 before protease cleavage) The term "IL-18" interchangeably encompasses both the precursor of IL-18 and mature IL-18 (following protease cleavage).

[0081] The terms "IL-1β" and "IL-1b" refer to the IL-1β polypeptide and The term "IL-1β" is a synonym of IL-1β polypeptide. Unless otherwise specified, the term refers to human IL-1β. IL-1β is well known to those skilled in the art and is, for example, For example, available from Sino Biological under product number #10139-HNAE-5 It is Noh.

[0082] The term "antibody" refers to an intact immunoglobulin or a functional fragment thereof. Naturally occurring antibodies generally comprise at least two heavy (H) chains and at least two light (L) chains. Each heavy chain comprises a tetramer composed of a heavy chain variable region (abbreviated herein as VH). The heavy chain constant region is usually composed of three domains (CH1, CH2, and CH3). The heavy chain is composed of IgG (IgG1, IgG2, IgG3 and IgG4 subunits). IgA (IgA1 and IgA2 subtypes), IgM, and IgE. Each light chain can be of the following isotype: and the light chain constant region (CL). The light chain consists of kappa (κ) chains and lambda (λ) chains. The heavy and light chain variable regions are generally responsible for antigen recognition, while the heavy and light chain constant regions are responsible for immune regulation. Various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq) The VH and VL regions, including the VH and VL regions, can mediate the binding of immunoglobulins to host tissues or factors. The complementarity determining regions are separated by more conserved regions called framework regions (FR). Each VH and VL can be further subdivided into regions of hypervariability called CDRs. , from amino terminus to carboxy terminus, in the following order: FR1, CDR1, FR2, CDR2 It is composed of three CDRs and four FRs arranged as follows: FR1, FR2, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0083] The term "antigen-binding portion" (or simply "antigen portion") of an antibody is used herein. When used in combination with other antibodies, the antibodies retain the ability to specifically bind to the IL-18 or IL-1β antigen. It refers to a full-length antibody or one or more fragments. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. It has been shown that binding fragments are encompassed within the term "antigen-binding portion" of an antibody. Examples of such fragments include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ab)2 fragment, containing two Fab fragments linked by a disulfide bridge at the hinge region a bivalent fragment consisting of the VH and CH1 domains; an Fd fragment consisting of the VL and V Fv fragment consisting of the H domain; dAb fragment consisting of the VH domain (Ward et al. .,(1989)Nature;341:544-546); and isolated complementation determiners Examples include CDRs.

[0084] Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes. Although there are many known antibodies that bind to VL and VH domains, they are not single proteins in which the VL and VH domains pair to form monovalent molecules. The flexible linkers allow them to be generated as protein chains. Alternatively, the Fv can be linked using recombinant techniques (known as single chain Fv (scFv); e.g., Bir d et al., (1988) Science 242:423-426; and Hus ton et al.,(1988)Proc Natl Acad Sc;.85:5 Such single chain antibodies are also included within the term "antigen-binding portion" of an antibody. These antibody fragments can be produced using conventional techniques known to those of skill in the art. The fragments are then screened for utility in the same way as intact antibodies. do.

[0085] The term "isolated" is used throughout this application to refer to an immunoglobulin, antibody or polynucleotide. that a peptide may, in some cases, exist in physical environments different from those in which it may occur in nature; This means:

[0086] Throughout this application, the term "complementarity determining region ("CDR") refers to a CDR according to different definitions. Unless otherwise indicated, the exact amino acid sequence of a given CDR is defined according to the Kabat definition. The sequence boundaries are determined by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,”5th Ed.Public Health Service,National Institute utes of Health,Bethesda,MD(“Kabat” numbe ring scheme), Al-Lazikani et al., (1997) JM B 273,927-948(“Chothia” numbering scheme ) and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P.,The Immunologist,7,132-136(1999);L efranc,M.-P.et al.,Dev.Comp.Immunol.,27, 55-77(2003) ("IMGT" numbering scheme) The amount of ion exchange can be determined using any of a number of well-known schemes, including the classical form In the formula, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered according to Kabat: 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) and the CDR amino acid residues in the light chain variable domain (VL) are numbers 24 to 34 (LCD R1), 50-56 (LCDR2) and 89-97 (LCDR3). Under a, the CDR amino acids in VH are numbered 26 to 32 (HCDR1), 52 to 56 (HC HCDR2) and 95-102 (HCDR3); the amino acid residues in VL are numbered 26- 32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). By combining both Kabat and Chothia CDR definitions, The amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and and 95-102 (HCDR3) and amino acid residues 24-34 (LCDR1) in human VL , 50-56 (LCDR2) and 89-97 (LCDR3). Under IMGT, V The CDR amino acid residues in H are approximately 26-35 (CDR1), 51-57 (CDR2), ) and 93-102 (CDR3), and the CDR amino acid residues in VL are approximately 27- 32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) ("K Under IMGT, the CDR regions of antibodies are numbered according to the program IMGT / D The alignment can be determined using the MainGap Align function.

[0087] By convention, the CDR regions in the heavy chain are generally designated H-CDR1, H-CDR2 and H-CDR3. In the light chain, they are called L-CDR1, LCDR2 and L-CDR3. They are numbered sequentially from the amino terminus to the carboxy terminus.

[0088] The terms "monoclonal antibody" or "monoclonal antibody composition" are used herein. As used herein, refers to a preparation of antibody molecules of single molecular composition. An antibody exhibits a single binding specificity and affinity for a particular epitope.

[0089] The term "human antibody" as used herein refers to a human antibody having framework and CDR The invention also includes antibodies having variable regions in which both sequences are derived from sequences of human origin. In the case where the antibody contains a constant region, the constant region may also include such a human sequence, e.g., Kna ppik, et al., (2000) J Mol Biol;296:57-86) As described above, for example, human germline sequences or mutant versions of human germline sequences or human fragments. The antibody is derived from an antibody that contains a consensus framework sequence derived from framework sequence analysis. .

[0090] The human antibodies of the invention may include amino acid residues that are not encoded by human sequences (e.g. By random or site-specific mutagenesis in vitro or by in vivo cell division However, the term "human antibody" is used herein as a When used in a human genome, CDR sequences derived from the germline of another mammalian species, such as mouse, may be used in place of the human genome. This does not include antibodies grafted onto a framework sequence.

[0091] The term "human monoclonal antibody" refers to an antibody in which both the framework and CDR regions are human. The term refers to antibodies displaying a single binding specificity which have variable regions derived from the same sequence.

[0092] The term "recombinant human antibody" as used herein refers to a recombinant human immunoglobulin. Animals that are transgenic or transchromosomal for the gene (e.g., mice) or antibodies isolated from hybridomas prepared therefrom, Any human antibody prepared, expressed, generated, or isolated from a human antibody, such as a tyrosine kinase inhibitor, isolated from host cells transformed to express human antibodies from fectomas Antibodies isolated from recombinant, combinatorial human antibody libraries and human immunoglobulins or by any other means, including splicing of all or part of the immunoglobulin gene. Such recombinant human antibodies include those produced, expressed, engineered, or isolated antibodies. The antibody has framework and CDR regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are transgenic for human Ig sequences in vitro When animals are used, they may be subjected to in vivo somatic mutagenesis, thus The amino acid sequences of the VH and VL regions of the recombinant antibody are derived from human germline VH and VL sequences. and related to, but not naturally present in the human antibody germline repertoire in vivo. This is an impossible arrangement.

[0093] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" mean "an antibody that specifically binds to an antigen." The term "antibody binding to the antibody" is used interchangeably herein.

[0094] As used herein, a binding molecule that "specifically binds to IL-18" is defined as a molecule that is K below nM, below 10 nM, below 1 nM D refers to a binding molecule that binds to human IL-18 It shall be so.

[0095] As used herein, a binding molecule that "specifically binds to IL-1β" is defined as a molecule that is K below nM, below 10 nM, below 1 nM D This refers to a binding molecule that binds to human IL-1β. It shall be so.

[0096] As used herein, the term "antagonist" refers to an antagonist that inhibits the activity of an activating compound. For example, in the case of IL-18, IL-18 antagonist inhibits IL-18-dependent interferon activation in human blood cells - The presence of IL-18 in human cell assays, such as the gamma (IFN-γ) production assay It is a binding molecule that inhibits signaling activity under IL-18-dependent conditions in human blood cells. An example of an assay for specific IFN-γ production is detailed in the Examples below.

[0097] The term bivalent bispecific antibody, or bivalent bispecific antibody (plural), refers to an antibody that binds IL-18 and I This refers to antibodies binding to two different targets, such as L-1β. are also referred to herein as bispecific antibodies.

[0098] Bispecific antibodies are "heterodimers" that have one portion specific for a first target. one portion is derived from a first antibody specific for a second target, and another portion is derived from a second antibody specific for a second target. "Heterodimerization modification" refers to the modification of one of the antibodies that forms a heterodimeric bispecific antibody. or both, which facilitate such formation. Example of heterodimerization modification of the Fc domains of two IgG1 moieties of an antibody intended to has bulky amino acid (aa) side chains (S354C, T366W) in the first heavy chain The “knob” and small aa side chains (Y349C, T366S, L368A, Y407V) A "hole" having a nucleotide sequence similar to that of the first heavy chain is introduced in the second heavy chain, and a further "hole" having a nucleotide sequence similar to that of the first heavy chain is introduced in the CH3 region. A disulfide bridge connects the heavy chains (Merchant et al., Na t.Biotechnol.,16:677-681(1998),page 678, table 1).

[0099] "K D The term "as used herein refers to the dissociation constant, which is K d and K. a (i.e., K d / K a ) and expressed as molar concentration (M). K for antibodies D Values ​​may be determined using methods well established in the art. K D The method for determining the .alpha.-amino acid sequence is carried out using a surface plasma such as a Biacore.RTM. system. By using Mont resonance.

[0100] As used herein, the term "affinity" refers to the affinity of a binding molecule at a single antigenic site. This refers to the strength of the interaction between a antibody and an antigen.

[0101] As used herein, the term "high affinity" for an antibody refers to an antibody that has high affinity for a target antigen. This refers to an antibody that has a KD of 1 nM or less against a target molecule.

[0102] As used herein, the term "subject" refers to a person who is capable of exhibiting symptoms of HS or Bispecific anti-IL-18 and anti-IL-1β as described herein may be a risk factor for HS. This includes any human subject that receives the antagonist.

[0103] As used herein, the term "optimized nucleotide sequence" refers to a nucleotide sequence that is optimized in a production cell or is an organism, typically a eukaryotic cell, such as Pichia pastoris is cells, Chinese hamster ovary cells (CHO) or human cells. The nucleotide sequence has been modified to code for the amino acid sequence using new codons. An optimized nucleotide sequence is a starting nucleotide sequence, also known as a "parent" sequence. The amino acid sequence originally encoded by the peptide sequence is completely retained. The optimized sequences in the specification have been modified to have codons preferred in CHO mammalian cells. however, optimized expression of these sequences in other eukaryotic cells is also contemplated herein. can be.

[0104] The term "identity" refers to the similarity between at least two different sequences. The identity can be expressed as a percentage and can be calculated using standard alignment algorithms, such as Bas ic Local Alignment Tool(BLAST)(Altshul e t al.,(1990)J MoI Biol;215:403-410);Need leman et al.,(1970)J MoI Biol;48:444-453 or Meyers et al., (1988) Comput Appl The algorithm is determined by a set of parameters. can be a Blosum 62 score matrix, with a gap penalty of 12 and gap extension The length penalty can be 4 and the frameshift gap penalty can be 5. The percent identity between acid or nucleotide sequences is also calculated using the PAM120 weighted residue table ( light residue table), 12 gap lengths and 4 gap penalties It has been incorporated into the ALIGN program (version 2.0) using the E. Meyers and W. Miller (1989) CABIOS;4(1):1-17 Percent identity can be determined using the algorithm of It is calculated by comparing

[0105] The term "immune response" refers to the immune response to an invading pathogen, a cell or tissue infected by a pathogen, a cancerous cell, or in the case of autoimmune or pathological inflammation, selective damage to normal human cells or tissues. causing harm, destruction or elimination from the human body, e.g., lymphocytes, antigen-presenting cells, Phagocytes, granulocytes and soluble macromolecules (antibodies, cytokines) produced by these cells or the liver. This refers to the action of the immune system (including kinekinin and complement).

[0106] A "signal transduction pathway" or "signal transduction activity" generally refers to the binding of a growth factor to a receptor. These interactions are initiated by protein-protein interactions, such as the binding of It refers to the biochemical causal relationship that results in the transmission of a signal from one part of a cell to another. , one or more proteins in a signal transduction that triggers a series of reactions It involves the specific phosphorylation of tyrosine, serine, or threonine residues. Typically, these events involve nuclear events that result in changes in gene expression.

[0107] The term "neutralize" and its grammatical variations are used throughout this application at times in conjunction with In the presence of a protein or antibody, the biological activity of the target is reduced, either completely or partially. This means that

[0108] The term "nucleic acid" or "polynucleotide" refers to any nucleic acid molecule in either single-stranded or double-stranded form. This refers to oxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers. Unless otherwise limited, the term refers to a nucleic acid that has similar binding properties to the reference nucleic acid and does not differ from naturally occurring nucleotides. It also includes nucleic acids containing known analogues of natural nucleotides that are metabolized similarly. Unless otherwise indicated, particular nucleic acid sequences also include conservatively modified variants thereof (e.g., degenerate codons). Substitutions), alleles, orthologs, SNPs and complementary sequences as well as explicitly indicated sequences are implicit Specifically, degenerate codon substitutions include substitutions of one or more selected (or all) codons. substituted with mixed base and / or deoxyinosine residues at the third position of This can be achieved by (Batzer et al., Nucleic Acid R es.19:5081(1991);Ohtsuka et al., J.Biol.C hem.260:2605-2608(1985); and Rossolini et al. l.,Mol.Cell.Probes 8:91-98(1994)).

[0109] The nucleotides in a "polynucleotide" or "nucleic acid" are bromouridine and ino Base modifications such as sine derivatives, phosphorothioates, phosphorodithioates, phosphorosele phosphoroate, phosphorodiselenoate, phosphoroanilothioate nilothioate, phosphoraniladat e) and modifications including ribose modifications such as phosphoramidates.

[0110] The term "vector" refers to a vector suitable for transformation or gene transfer into a host cell. Directs (in conjunction with a host cell) the expression of one or more heterologous coding sequences to which it is operably linked; and / or any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus).

[0111] "Conservative variants" of a sequence encoding a binding molecule, antibody or fragment thereof are "Conservative amino acid modifications" refers to a sequence that contains an amino acid modification. Refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the molecule. Such conservative modifications include amino acid substitutions, additions and deletions. Targeted amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, etc.). histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, polar side chains (e.g. alanine, valine, leucine, isoleucine, amine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine leucine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, triisopropyl ether, Site-directed mutagenesis and PCR-mediated mutagenesis Modifications can be introduced into the binding proteins of the invention by standard techniques known in the art, such as derivatization. Conservative amino acid substitutions are generally made by synthesis rather than by synthesis in biological systems. It may also include unnatural amino acid residues incorporated by chemical peptide synthesis. Acids include, but are not limited to, inverted or inverted peptidomimetics of the amino acid moiety. It won't be done.

[0112] The term "epitope" refers to an antigen that is recognized by the immune system, such as an antibody or a fragment thereof. Within this application, the term "epitope" refers to conformational epitopes and linear epitopes. Conformational epitopes are used interchangeably to refer to both conformational and conformational epitopes. A linear epitope is composed of a non-contiguous section of amino acid sequence from an antigen, whereas a linear epitope is composed of a non-contiguous section of amino acid sequence from an antigen. It is formed by a consecutive sequence of amino acids.

[0113] Human antibodies or fragments thereof are antibodies in which the variable regions or full-length chains of the antibody are derived from human germline immunoglobulins. When derived from a system that uses a specific germline sequence, the "product" of a specific germline sequence or a specific germline The sequences may include heavy or light chain variable regions "derived from" a sequence or full-length heavy or light chains. Such systems involve the introduction of transgenic mice carrying human immunoglobulin genes with an antigen of interest. Immunizing a non-human mouse with an antigen of interest or immunofluorescence assay using a human phage-displayed antigen The method includes screening a library of human immunoglobulin genes.

[0114] Human antibodies that are "the product of" or "derived from" human germline immunoglobulin sequences or fragments thereof that have the same amino acid sequence as a human germline immunoglobulin and the amino acid sequence of a human antibody. The sequences are compared and the human antibody sequence that has the closest sequence (i.e., the highest percent identity) to the human antibody sequence is determined. can be identified as such by selecting germline immunoglobulin sequences. Human immunoglobulins that are the "product" of or "derived from" specific human germline immunoglobulin sequences. A human antibody may be a mutant, e.g., antibody that is a direct replicator of a human antibody, e.g., due to natural somatic mutations or deliberate introduction of site-specific mutations. It may contain amino acid differences when compared to the breeding sequence.

[0115] However, the human antibody selected will generally be derived from human germline immunoglobulin genes. The amino acid sequence encoded by the nucleotide sequence of ... Human immunoglobulin amino acid sequences compared to germline immunoglobulin sequences (e.g., mouse germline sequences) Contains amino acid residues that identify a human antibody as being human. Antibodies are antibodies that are directed against amino acid sequences encoded by germline immunoglobulin genes. At least 60%, 70%, 80%, 90% or at least 95% or may further be at least 96%, 97%, 98% or 99% identical. Human antibodies derived from a particular human germline sequence are made by human germline immunoglobulin genes. The amino acid sequence differs from the encoded amino acid sequence by no more than 10 amino acids. In this regard, a human antibody has an amino acid sequence identical to that encoded by a germline immunoglobulin gene. , may show no more than 5, or even no more than 4, 3, 2 or 1 amino acid difference.

[0116] Human antibodies can be produced by a number of methods known to those of skill in the art. Myeloma or mouse-human heteromyeloma cell lines were used to generate hybridomas. (Kozbor, J Immunol; (1984) 133:3001; Brod eur, Monoclonal Isolated Antibody Product ion Techniques and Applications,pp51-63, Marcel Dekker Inc, 1987). An alternative method is to use phagilla Examples of such methods include the use of libraries or transgenic mice, both of which use human variable region replicators. Tree (Winter G; (1994) Annu Rev Immunol 12:433-455, Green LL, (1999) J Immunol Met. hods 231:11-23).

[0117] Mouse immunoglobulin loci replaced with human immunoglobulin gene segments Several lines of transgenic mice that have been ka K,(2000)Proc Natl Acad Sci,97:722-727 ;Fishwild DM (1996) Nature Biotechnol 14:8 45-851;Mendez MJ,(1997)Nature Genetics 1 5:146-156). Such mice are able to select for antibodies of interest upon antigen challenge. Of particular note is the ability of human lymphocytes to produce a repertoire of human antibodies that can be generated by irradiating human lymphocytes. The Trimera™ system (Eren R et al., (1 988)Immunology 93:154-161) A large pool of in vitro Human (or other species) lymphocytes are effectively recruited through an isolated antibody production procedure, followed by The selective lymphocyte isolation antibody system (Se) in which the deconvolution, limiting dilution and selection procedures are performed lected Lymphocyte Isolated antibody Syst em)(SLAM,Babcook et al,Proc Natl Acad Sc i(1996)93:7843-7848) and Xenomouse™ (Abge An alternative approach is to use the Morphodoma™ technology and available from Morphotek Inc.

[0118] Phage display technology can be used to generate human antibodies and fragments thereof ( McCafferty; (1990) Nature, 348:552-553 and Gri ffiths AD et al (1994) EMBO 13:3245-3260). This technique involves the transcription of protein genes from filamentous bacteriophages such as M13 or fd. The isolated antibody variable domain genes were cloned in-frame into either the large or small coat. and functionally isolate the phage on its surface (usually with the aid of a helper phage). Selection based on the functional properties of the isolated antibodies results in the selection of antibodies that provide these properties. A gene encoding an isolated antibody according to the present invention is selected. or alternatively, libraries generated from human B cells harvested from non-immunized human donors. Phage display technology can be used to select antigen-specific antibodies from rks; J Mol Bio (1991) 222:581-591). Intact human If it is desired that the isolated antibody contain an Fc domain, a mammalian gene containing the desired constant region can be used. Recloning the phage display-derived fragment into the current vector and generating stable expression cell lines It is necessary to establish

[0119] Affinity maturation techniques (Marks; Biotechnol (1992) 10:779-7 83) can be used to provide binding affinity, and the heavy and light chain variable regions can be transformed into naturally occurring variants. By successively replacing the primary human monoclonal antibody with The affinity of the isolated antibody is improved. Variations of this technique, such as "epitope imprinting," Also available now are WO 93 / 06213; Waterhouse et al. use;Nucl Acids Res (1993)21:2265-2266).

[0120] The term "pure" when used in reference to a purified bispecific antibody refers to cells that are expressing the bispecific antibody. After co-expression in selected cells under conditions of expressing the entire protein, and intact Different bispecific antibodies following Protein-A purification using a mass screening approach Purity or purity refers to the purity and identity of the combinations and constructs formed. This refers to the relative quantification of hetero- and homodimeric bbmAbs. The heterodimer bbmAb1 formed at 85% rDNA was 85% rDNA based on intact mass signal intensity. % relative purity can be observed.

[0121] As used herein, the terms "inhibit," "inhibit," or "inhibiting" The term refers to the alleviation or suppression of a given condition, symptom, disorder, or disease, or to the biological It refers to a significant decrease in the baseline activity of an activity or process.

[0122] As used herein, the term "treat" or "treatment of any disease or disorder" refers to The term "treatment" of any disease or disorder refers, in one embodiment, to treating the disease or disorder. ameliorate (i.e., slow the onset or progression of a disease or at least one of its clinical symptoms) In another embodiment, "treat" refers to "preventing, inhibiting, or reducing the "Doing" or "treatment" includes at least some things, including things that may not be discernible by the patient. The term "therapeutic agent" refers to the alleviation or amelioration of at least one physical parameter. In this embodiment, "treat," "treating," or "treatment" refers to a physically (e.g., identifiable) The disease may be improved either physiologically (e.g., stabilization of symptoms), physiologically (e.g., stabilization of physical parameters), or both. More specifically, the term "treating" the disease HS refers to modifying a disease or disorder. The present invention relates to treating inflammatory lesions (in number or quality, or their To reduce the volume and size of abscesses and inflammatory nodules and / or drain fistulas and / or reduce the amount of scarring in HS patients. Treating the disease HS It has also been shown to relieve pain, fatigue and / or itching associated with HS, as well as purulent discharge. and reducing the odor associated with purulent discharge, and / or It also refers to improving quality of life and / or reducing work disability for people with HS. .

[0123] As used herein, the term "prevention" refers to preventing or preventing the onset or development of a disease or disorder. refers to delaying the progression of the disease. More specifically, it is used to "prevent" the disease HS. The term refers to preventing HS erythema and / or the appearance of new lesions; preventing scarring and reducing scarring. Surgical intervention to prevent scarring and / or functional limitations specifically for HS This refers to preventing

[0124] As used herein, a subject is a person who has not yet undergone any of the above-mentioned treatments, and who is not aware of any of the above-mentioned treatments. A procedure is considered "necessary" if it provides a therapeutic, medical, or quality of life benefit. "Let us say."

[0125] As used herein, a "therapeutically effective amount" refers to an amount sufficient to treat at least a portion of a disorder or a recurring disorder. Treat, prevent, prevent the onset of, cure, delay, or cure another condition reduce or ameliorate the severity of, or reduce the severity of, what would be expected in the absence of such treatment For single or multiple administration to a patient (such as a human) to extend the patient's survival beyond A bispecific antibody ( For example, bbmAb1) or an antigen-binding fragment thereof. When applied to a component (e.g., bbmAb1), the term refers to that component only. When applied to a combination of agents, the term refers to agents administered in combination, sequentially or simultaneously. It refers to the combined amount of active ingredients that produces a therapeutic effect, regardless of the amount.

[0126] The phrase "therapeutic regimen" refers to a regimen used to treat a disease, e.g., the treatment of HS. A treatment regimen refers to a loading regimen (or initial This may include a loading dose followed by a maintenance dosing regimen (or maintenance dose).

[0127] The phrase "loading regimen" or "loading period" is used for the early treatment of a disease. In some embodiments, the disclosed The methods, uses, kits, processes and dosing regimens (e.g., methods for treating HS) include a loading dose In some cases, the loading period is The general goal of a loading regimen is to achieve the maximum efficacy of the treatment regimen. The goal of a loading regimen is to provide high levels of a drug to a patient during the initial period of A nurse may administer a higher dose of a drug than would be used during a maintenance regimen; The administration of the drug may include administering the drug more frequently than would be administered during a dosing regimen, or both. Dose escalation can occur during or after the loading regimen.

[0128] The phrase "maintenance regimen" or "maintenance period" refers to, for example, a period following a loading regimen or period. During the treatment of an illness, such as to keep a patient in good condition for an extended period of time (months or years) Refers to a treatment plan (or part of a treatment plan) used to maintain a patient. In some embodiments, the disclosed methods, uses and dosing regimens use a maintenance dosing regimen. The plan is to administer continuous treatment (e.g., at regular intervals, e.g., weekly, biweekly, or monthly (every 4 weeks), annually, etc. administering a drug in a short period of time (e.g., administering a drug in a short period of time, e ... treatment at the time of onset or when certain predefined criteria (e.g. pain, disease manifestations, etc.) are reached. Dose escalation may occur during the maintenance regimen.

[0129] The phrase "means for administering" includes prefilled syringes, vials and syringes, Includes injection pens, autoinjectors, IV drips and bags, pumps, patch pumps, etc. to refer to any available tool for systemically administering a drug to a patient, including but not limited to Such articles may be used to allow patients to self-administer drugs (i.e., administer them themselves). The patient may administer the drug to the patient) or a physician may administer the drug.

[0130] 2.IL-18 antibody Particularly preferred IL-18 antibodies or antigen-binding fragments thereof for use in the disclosed methods are human IL-18 antibodies or antigen-binding fragments thereof. It is an antibody.

[0131] For ease of reference, it is referred to as mAb1, based on the Kabat and Chothia definitions. The amino acid sequence of the hypervariable region of the specific IL-18 antibody and the V L and V H Domain and The complete heavy and light chains are provided in Table 1 below.

[0132] [Table 1]

[0133] In one embodiment, the IL-18 antibody, or antigen-binding fragment thereof, has CDR1 consisting of the amino acid sequence CDR2 has the amino acid sequence SEQ ID NO:2, and CDR3 has the amino acid sequence SEQ ID NO:3. The nucleic acid sequence of at least one of the nucleic acid sequences of SEQ ID NO:3 is SEQ ID NO:4. Each of them contains one immunoglobulin heavy chain variable domain (V H In one embodiment, IL The CDR1 of the antibody or antigen-binding fragment thereof has the amino acid sequence SEQ ID NO:4, DR2 has the amino acid sequence SEQ ID NO:5 and CDR3 has the amino acid sequence SEQ ID NO:6 at least one immunoglobulin complex comprising hypervariable regions CDR1, CDR2 and CDR3; Chain variable domain (V H ).

[0134] In one embodiment, the IL-18 antibody, or antigen-binding fragment thereof, has CDR1 consisting of the amino acid sequence CDR2 has the amino acid sequence SEQ ID NO:12, and CDR3 has the amino acid sequence SEQ ID NO:13. The hypervariable regions CDR1, CDR2 and CDR3 have the amino acid sequence SEQ ID NO: 13. At least one immunoglobulin light chain variable domain (V L In one embodiment, The IL-18 antibody or antigen-binding fragment thereof has CDR1 having the amino acid sequence of SEQ ID NO:14. CDR2 has the amino acid sequence SEQ ID NO: 15 and CDR3 has the amino acid sequence SEQ ID NO: At least one immunoglobulin having hypervariable regions CDR1, CDR2 and CDR3 having the sequence number 16. Globulin light chain variable domain (V L ).

[0135] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof is Epidemiological globulin V H domain and at least one immunoglobulin V L domain, a ) Immunoglobulin V H A domain can be (e.g., in a sequence): i) CDR1 is a sequence of amino acids 1, CDR2 has the amino acid sequence SEQ ID NO:2, and CDR3 has the amino acid sequence SEQ ID NO: or ii) the hypervariable regions CDR1, CDR2 and CDR3 having SEQ ID NO: 3 of CDR1 has the amino acid sequence SEQ ID NO:4, CDR2 has the amino acid sequence SEQ ID NO:5, The hypervariable regions CDR1, CDR2 and CDR3 have the amino acid sequence SEQ ID NO:6. 3; b) immunoglobulin V L A domain can be (e.g., in a sequence): i) CDR1 is amino CDR1 has the amino acid sequence SEQ ID NO:11, CDR2 has the amino acid sequence SEQ ID NO:12, hypervariable regions CDR1, CDR2 and CDR3 having the amino acid sequence SEQ ID NO: 13; or ii) CDR1 has the amino acid sequence SEQ ID NO: 14 and CDR2 has the amino acid sequence SEQ ID NO: Hypervariable region CDR having sequence number 15 and CDR3 having amino acid sequence SEQ ID NO: 16. 1, including CDR2 and CDR3.

[0136] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof has the structure: a) an IL-18 antibody having the sequence as set forth in SEQ ID NO:7; An immunoglobulin heavy chain variable domain (V) comprising the amino acid sequence shown H );b) sequence number 17, an immunoglobulin light chain variable domain (V L );c) Immunoglobulin V comprising the amino acid sequence shown as SEQ ID NO:7 H Domains and SEQ ID NOs Immunoglobulin V containing the amino acid sequence designated as 17 L domain; d) SEQ ID NO:1; Immunoglobulin V comprising the hypervariable regions shown as SEQ ID NO:2 and SEQ ID NO:3 H domain e) an immunoglobulin comprising the hypervariable regions shown as SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; Epidemiological globulin V L f) domains shown as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 Immunoglobulin V containing hypervariable regions H Domain; g) SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: Immunoglobulin V containing the hypervariable region shown as column number 16 L domain; h) sequence number 1, an immunoglobulin V comprising a hypervariable region shown as SEQ ID NO:2 and SEQ ID NO:3 H Domestic and the hypervariable regions shown as SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13. Immunoglobulin V L Domains; i) shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 Immunoglobulin V containing the hypervariable region H Domain and SEQ ID NO: 14, SEQ ID NO: 15 and Immunoglobulin V containing the hypervariable region shown as column number 16 L domain; j) sequence number 19; k) a heavy chain comprising SEQ ID NO: 9; or l) a light chain comprising SEQ ID NO: 19 and sequence Contains a heavy chain containing number 9.

[0137] In some embodiments, an IL-18 antibody or antigen-binding fragment thereof (e.g., a mAb 1) comprises the three CDRs of SEQ ID NO: 7. In another embodiment, the IL-18 antibody or The antigen-binding fragment comprises the three CDRs of SEQ ID NO: 17. The L-18 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:7 and the CDR of SEQ ID NO:17. In some embodiments, the IL-18 antibody or antigen-binding The fragment comprises the three CDRs of SEQ ID NO: 9. In another embodiment, the IL-18 antibody or The antigen-binding fragment comprises the three CDRs of SEQ ID NO: 19. The L-18 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:9 and the CDR of SEQ ID NO:19. It contains 3 CDRs.

[0138] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb1) a) CDR1 has the amino acid sequence SEQ ID NO: 1 and CDR2 has the amino acid sequence and hypervariable region CDR1 having the amino acid sequence SEQ ID NO: 2, and CDR3 having the amino acid sequence SEQ ID NO: 3. A variable domain containing CDR2 and CDR3 in its sequence and a constant portion of a human heavy chain or a fragment thereof and b) an immunoglobulin heavy chain or fragment thereof comprising the CDR1 of the amino acid sequence SEQ ID NO:1. 1, CDR2 has the amino acid sequence SEQ ID NO: 12, and CDR3 has the amino acid sequence SEQ ID NO: A variable domain having hypervariable regions CDR1, CDR2 and CDR3 in the sequence having sequence number 13. and an immunoglobulin light chain or a fragment thereof comprising an immunoglobulin and a constant portion of a human light chain or a fragment thereof. The antibody is selected from human IL-18 antibodies comprising

[0139] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb1) a) CDR1 has the amino acid sequence SEQ ID NO: 4 and CDR2 has the amino acid sequence and hypervariable region CDR1 having the amino acid sequence SEQ ID NO: 5, and CDR3 having the amino acid sequence SEQ ID NO: 6. A variable domain containing CDR2 and CDR3 in its sequence and a constant portion of a human heavy chain or a fragment thereof and b) an immunoglobulin heavy chain or fragment thereof comprising the CDR1 of the amino acid sequence SEQ ID NO:1. 4, CDR2 has the amino acid sequence SEQ ID NO: 15, and CDR3 has the amino acid sequence SEQ ID NO: A variable domain having hypervariable regions CDR1, CDR2 and CDR3 in the sequence having sequence number 16. and an immunoglobulin light chain or a fragment thereof comprising an immunoglobulin and a constant portion of a human light chain or a fragment thereof. The antibody is selected from human IL-18 antibodies comprising

[0140] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof has a CDR1 that is CDR2 has the amino acid sequence SEQ ID NO:2, and CDR3 has the amino acid sequence SEQ ID NO:3. The amino acid sequence SEQ ID NO:3 is a sequence of hypervariable regions CDR1, CDR2 and CDR3. and b) a first domain comprising CDR1 having the amino acid sequence SEQ ID NO: 11, CDR2 has the amino acid sequence SEQ ID NO:12, and CDR3 has the amino acid sequence SEQ ID NO:13. a second domain comprising in its sequence the hypervariable regions CDR1, CDR2 and CDR3; and c) The N-terminus of the first domain and the C-terminus of the second domain or the C-terminus of the first domain and A peptide linker linked to either the N-terminus of the domain of The antibody is selected from a single chain antibody or an antigen-binding fragment thereof, comprising

[0141] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb1) a) CDR1 has the amino acid sequence SEQ ID NO: 4 and CDR2 has the amino acid sequence SEQ ID NO: 5 and CDR3 has the amino acid sequence SEQ ID NO:6. and a) a first domain having CDR3 in its sequence, and b) CDR1 having the amino acid sequence SEQ ID NO: 14, CDR2 has the amino acid sequence of SEQ ID NO: 15, and CDR3 has the amino acid sequence of A second sequence comprising hypervariable regions CDR1, CDR2 and CDR3 having SEQ ID NO: 16. and c) an N-terminus of the first domain and a C-terminus of the second domain or an N-terminus of the first domain. and a peptide linker linked to either the C-terminus of the first domain or the N-terminus of the second domain. The antibody or antigen-binding fragment thereof is selected from the group consisting of a single chain antibody and an antigen-binding fragment thereof, the single chain antibody or antigen-binding fragment thereof comprising an antigen-binding site comprising

[0142] V of the IL-18 antibody or antigen-binding fragment thereof used in the disclosed methods H Or V L D The main sequence is V shown in SEQ ID NO: 7 and 17. H Or V L V, which is virtually identical to the domain H and / or V L The human IL-18 antibodies disclosed herein may have the following domains: A heavy chain substantially identical to that shown as SEQ ID NO: 9 and / or a heavy chain substantially identical to that shown as SEQ ID NO: 19 The human IL-1A and IL-1B may comprise a light chain substantially identical to that shown. The 18 antibody may comprise a heavy chain comprising SEQ ID NO:9 and a light chain comprising SEQ ID NO:19. The human IL-18 disclosed in the present application is a human IL-18 having a sequence substantially identical to that shown in SEQ ID NO:7. a) a heavy chain comprising a variable domain having the amino acid sequence of A variable domain and a human having an amino acid sequence substantially identical to that shown in SEQ ID NO:17. and one light chain comprising the constant portion of a human light chain.

[0143] Other preferred IL-18 antagonists for use in the disclosed methods, kits and treatment regimens include: Agonists (e.g., antibodies) are described in US Pat. This is shown in Patent No. 9,376,489.

[0144] 3.IL-1β antibody Particularly preferred IL-1β antibodies or antigen-binding fragments thereof for use in the disclosed methods are human IL-1β antibodies. It is an antibody.

[0145] For ease of reference, we will use the term mAb2 based on the Kabat and Chothia definitions. The hypervariable regions and V of specific IL-1β antibodies L and V H Domains and complete heavy chains and the amino acid sequences of the light chain are provided in Table 2 below.

[0146] [Table 2]

[0147] In one embodiment, the IL-1β antibody, or antigen-binding fragment thereof, has CDR1 consisting of the amino acid sequence CDR2 has the amino acid sequence SEQ ID NO:22, and CDR3 has the amino acid sequence SEQ ID NO:23. The hypervariable regions CDR1, CDR2 and CDR3 have the amino acid sequence SEQ ID NO:23. At least one immunoglobulin heavy chain variable domain (VH In one embodiment, The IL-1β antibody or antigen-binding fragment thereof has CDR1 having the amino acid sequence of SEQ ID NO:24. and CDR2 has the amino acid sequence SEQ ID NO: 25 and CDR3 has the amino acid sequence SEQ ID NO: At least one immunoglobulin having hypervariable regions CDR1, CDR2 and CDR3 having the sequence ID No. 26. Globulin heavy chain variable domain (V H ).

[0148] In one embodiment, the IL-1β antibody, or antigen-binding fragment thereof, has CDR1 consisting of the amino acid sequence CDR2 has the amino acid sequence SEQ ID NO: 32, and CDR3 has the amino acid sequence SEQ ID NO: 33. comprising hypervariable regions CDR1, CDR2 and CDR3 having the amino acid sequence SEQ ID NO:33 At least one immunoglobulin light chain variable domain (V L In one embodiment, The IL-1β antibody or antigen-binding fragment thereof has CDR1 having the amino acid sequence of SEQ ID NO:34. and CDR2 has the amino acid sequence SEQ ID NO: 35 and CDR3 has the amino acid sequence SEQ ID NO: At least one immunoglobulin having hypervariable regions CDR1, CDR2 and CDR3 having the sequence ID No. 36. Globulin light chain variable domain (V L ).

[0149] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof is Epidemiological globulin V H domain and at least one immunoglobulin V L domain, a ) Immunoglobulin V H A domain can be (e.g., in a sequence): i) CDR1 has the amino acid sequence SEQ ID NO:21, CDR2 has the amino acid sequence SEQ ID NO:22, and CDR3 has the amino acid sequence SEQ ID NO:23. hypervariable regions CDR1, CDR2 and CDR3 having the amino acid sequence SEQ ID NO: 23; or ii. CDR1 has the amino acid sequence SEQ ID NO:24 and CDR2 has the amino acid sequence SEQ ID NO:2 5, and CDR3 has the amino acid sequence SEQ ID NO: 26, R2 and CDR3; b) Immunoglobulin V L A domain can be (e.g., in a sequence): CDR1 has the amino acid sequence SEQ ID NO: 31 and CDR2 has the amino acid sequence SEQ ID NO: 3 2 and CDR3 has the amino acid sequence SEQ ID NO: 33; R2 and CDR3 or ii) CDR1 has the amino acid sequence SEQ ID NO: 34 and CDR2 has having the amino acid sequence SEQ ID NO: 35, and CDR3 having the amino acid sequence SEQ ID NO: 36; The hypervariable regions include CDR1, CDR2 and CDR3.

[0150] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof has: a) SEQ ID NO: 27 An immunoglobulin heavy chain variable domain (V H );b) Array An immunoglobulin light chain variable domain (V) comprising the amino acid sequence shown as number 37 L ); c) Immunoglobulin V comprising the amino acid sequence shown as SEQ ID NO: 27 H Domain and Distribution Immunoglobulin V containing the amino acid sequence shown as sequence number 37 L Domain; d) sequence number No. 21, an immunoglobulin comprising a hypervariable region as shown in SEQ ID NO: 22 and SEQ ID NO: 23 V H e) hypervariable domains shown as SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33 Immunoglobulin V containing domain L domain; f) SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO: Immunoglobulin V containing the hypervariable region designated as 26 H domain; g) SEQ ID NO: 34, Immunoglobulin V comprising the hypervariable regions shown as SEQ ID NO:35 and SEQ ID NO:36 L Domestic h) comprising the hypervariable regions shown as SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23; Immunoglobulin V H As domains and SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33 Immunoglobulin V containing the hypervariable regions shown L Domain; i) SEQ ID NO: 24, SEQ ID NO: 2 5 and an immunoglobulin V comprising the hypervariable region shown as SEQ ID NO:26 H Domain and Distribution SEQ ID NO: 34, an immunoglobulin comprising the hypervariable regions shown as SEQ ID NO: 35 and SEQ ID NO: 36 Rin V L domain; j) a light chain comprising SEQ ID NO: 37; k) a heavy chain comprising SEQ ID NO: 29; or l ) The light chain comprises SEQ ID NO:39 and the heavy chain comprises SEQ ID NO:29.

[0151] In some embodiments, an IL-1β antibody or antigen-binding fragment thereof (e.g., a mAb 2) comprises the three CDRs of SEQ ID NO: 37. In another embodiment, the IL-1β antibody or In another embodiment, the antigen-binding fragment comprises the three CDRs of SEQ ID NO: 27. The IL-1β antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:37 and the CDR of SEQ ID NO:2 In some embodiments, the IL-1β antibody or its antigen- The binding fragment comprises the three CDRs of SEQ ID NO: 39. The antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:29. The IL-1β antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:39 and SEQ ID NO: It contains the three CDRs of No. 29.

[0152] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof (e.g., mAb2) a) CDR1 has the amino acid sequence SEQ ID NO: 21 and CDR2 has the amino acid sequence SEQ ID NO: 22. and CDR3 has the amino acid sequence SEQ ID NO: 23. A variable domain comprising DR1, CDR2 and CDR3 in sequence and the constant part of a human heavy chain, or and b) an immunoglobulin heavy chain or a fragment thereof, the CDR1 of which has an amino acid sequence CDR2 has the amino acid sequence SEQ ID NO: 32; CDR3 has the amino acid sequence SEQ ID NO: 33; The sequence may include hypervariable regions CDR1, CDR2 and CDR3 having SEQ ID NO: 33. Immunoglobulin light chain or fragment thereof comprising a variable domain and a constant portion of a human light chain or fragment thereof and a human IL-1β antibody comprising:

[0153] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof (e.g., mAb2) a) CDR1 has the amino acid sequence SEQ ID NO: 24 and CDR2 has the amino acid sequence SEQ ID NO: 25. and CDR3 has the amino acid sequence SEQ ID NO: 26. A variable domain comprising DR1, CDR2 and CDR3 in sequence and the constant part of a human heavy chain, or and b) an immunoglobulin heavy chain or a fragment thereof, the CDR1 of which has an amino acid sequence CDR2 has the amino acid sequence SEQ ID NO: 35, and CDR3 has the amino acid sequence SEQ ID NO: 36. The sequence may include hypervariable regions CDR1, CDR2 and CDR3 having SEQ ID NO: 36. Immunoglobulin light chain or fragment thereof comprising a variable domain and a constant portion of a human light chain or fragment thereof and a human IL-1β antibody comprising:

[0154] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof has a CDR1 that is CDR1 has the amino acid sequence SEQ ID NO:21, CDR2 has the amino acid sequence SEQ ID NO:22, 3 comprises hypervariable regions CDR1, CDR2 and CDR3 having the amino acid sequence SEQ ID NO:23 a) a first domain comprising the amino acid sequence SEQ ID NO: 31, and b) a CDR1 having the amino acid sequence SEQ ID NO: 31, DR2 has the amino acid sequence SEQ ID NO:32 and CDR3 has the amino acid sequence SEQ ID NO:33 a second domain comprising hypervariable regions CDR1, CDR2 and CDR3 in the sequence thereof; ) the N-terminus of the first domain and the C-terminus of the second domain or the C-terminus of the first domain and A peptide linker linked to either the N-terminus of the second domain, and an antigen-binding site comprising the The antibody is selected from the group consisting of a single chain antibody and an antigen-binding fragment thereof.

[0155] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof (e.g., mAb2) a) CDR1 has the amino acid sequence SEQ ID NO: 24 and CDR2 has the amino acid sequence SEQ ID NO: 25, and CDR3 has the amino acid sequence SEQ ID NO: 26, a) a first domain having a sequence including CDR2 and CDR3; and b) a CDR1 having an amino acid sequence of CDR2 has the amino acid sequence SEQ ID NO: 35, and CDR3 has the amino acid sequence SEQ ID NO: 36. The sequence of the hypervariable regions CDR1, CDR2 and CDR3 having SEQ ID NO: 36 of the present invention is c) an N-terminus of the first domain and a C-terminus of the second domain or a peptide linker linked to either the C-terminus of the main domain or the N-terminus of the second domain; The antibody is selected from the group consisting of single chain antibodies and antigen-binding fragments thereof, each of which comprises an antigen-binding site comprising:

[0156] V of the IL-1β antibody used in the disclosed methods H Or V L Domain or its antigen The binding fragment is represented by SEQ ID NOs: 27 and 37. H Or V L Virtually identical to the domain A V H and / or V L The human IL-1β antibody disclosed herein may have a The antibody may comprise a heavy chain substantially identical to that shown as SEQ ID NO:29 and / or a nucleic acid sequence similar to that shown as SEQ ID NO:3. 9. The human IgG1A antibody disclosed herein may comprise a light chain substantially identical to that shown in The human IL-1β antibody can comprise a heavy chain comprising SEQ ID NO:29 and a light chain comprising SEQ ID NO:39. The human IL-1β antibody disclosed herein is: a) that shown in SEQ ID NO:27 A single antibody containing a variable domain and a constant portion of a human heavy chain having a qualitatively identical amino acid sequence. and b) a heavy chain having an amino acid sequence substantially identical to that set forth in SEQ ID NO:37. and one light chain comprising the variable domain and the constant portion of a human light chain.

[0157] Other preferred IL-1β antagonists for use in the disclosed methods, kits and treatment regimens include: Agonists (e.g., antibodies) are incorporated herein by reference in their entirety. U.S. Patent No. 7,446,175 or 7,993,878 or This is shown in specification 8,273,350.

[0158] 4.Fc modification In addition to, or instead of, modifications made within the framework or CDR regions, the present invention The antibodies generally contain modifications within the Fc region that improve serum half-life, complement binding, Fc receptor binding, and Alter one or more functional properties of an antibody, such as receptor binding and / or antigen-dependent cellular cytotoxicity. In addition, the antibodies of the present invention may be chemically modified (e.g., by one or more The above chemical moieties can be linked to the antibody, or its glycosylation can be altered, again to form the Each of these embodiments may be modified to change one or more functional properties. The numbering of residues in the Fc region is based on the Edelman et al., PNAS, 1969 May, 63(1):78-85 EU numbering This is a part of the G20 scheme.

[0159] In one embodiment, the number of cysteine ​​residues in the hinge region is altered, e.g., increased. The hinge region of CH1 is modified to increase or decrease the binding of CH1. This approach was For example, the light chain is further described in U.S. Pat. No. 5,677,425 to R. and CH1 hin to promote heavy chain assembly or to increase or decrease antibody stability. The number of cysteine ​​residues in the dimeric region is changed.

[0160] In another embodiment, mutations are made to the Fc hinge region of the antibody to improve the biology of the antibody. More specifically, it reduces the effective half-life of SpA binding relative to native Fc-hinge domain. Fc - one or more amino acid mutations are introduced into the CH2-CH3 domain boundary region of the hinge fragment This approach is described in U.S. Patent No. 6,165,745 by Ward et al. This is described in further detail in the specification.

[0161] In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. See, for example, U.S. Patent No. 6,277,333 to Ward. 75, one or more of the following mutations: T252L, T254S, T25 Alternatively, 6F may be introduced as described in U.S. Patent No. 5,869 by Presta et al. As described in US Pat. Nos. 6,121,022 and 6,046, the biological half-life To extend the CH1 or CL region of the Fc region of IgG, the antibody can be modified within the CH1 or CL region. May contain salvage receptor binding epitopes taken from two loops of two domains .

[0162] In yet another embodiment, at least one antibody is added to alter the effector function of the antibody. The Fc region is modified by replacing one amino acid residue with a different amino acid residue. For example, the affinity of an antibody for an effector ligand may be altered, but the antigen binding of the parent antibody may be preserved. One or more amino acids may be replaced with different amino acid residues so as to retain affinity. The effector ligand that alters the expression of the β-terminal domain may be, for example, an Fc receptor or the C1 component of complement. This approach is described in U.S. Patent No. 5,624 by Winter et al. This is described in further detail in US Pat. Nos. 5,648,260 and 5,821.

[0163] In another embodiment, the antibody has altered C1q binding and / or complement dependent cell proliferation. 1. Select from amino acid residues such that the toxicity (CDC) is reduced or eliminated. One or more amino acids may be replaced with different amino acid residues. This approach is As described in further detail in U.S. Pat. No. 6,194,551 to Gie et al. It has been done.

[0164] In another embodiment, one or more amino acid residues are altered to thereby enhance complement resistance. This approach was first reported in an international publication by Bodmer et al. This is further described in Publication No. 94 / 29351.

[0165] In yet another embodiment, the ability of an antibody to mediate antibody-dependent cellular cytotoxicity is improved. and / or by modifying one or more amino acids to target the Fcγ receptor. Fc region modification to improve affinity. This approach was first described by Presta et al. Further, FcγRII is a ribozyme that is capable of inhibiting FcγRII. The binding sites on human IgG1 for FcγRII, FcγRIII and FcRn are matched. The binding of the β-lactamases has been improved and mutants with improved binding have been described (Shields, RLett. al, (2001) J Biol Chem 276:6591-6604).

[0166] In certain embodiments, an Fc domain of the IgG1 isotype is used. In some specific embodiments, mutants of the IgG1 Fc fragment, e.g., antibody-dependent Fusion polypeptides that mediate anti-inflammatory cell cytotoxicity (ADCC) and / or bind to Fcγ receptors A silent IgG1 Fc is used that reduces or eliminates the ability of the amino acid IgG1 antibodies in which leucine residues at positions 234 and 235 are replaced by alanine residues An example of a type-silent mutant is described in Hezareh et al., J. Virol. 2001);75(24):12161-8.

[0167] In certain embodiments, the Fc domain comprises a glycosylated nucleotide at position 297 of the Fc domain. For example, the Fc domain has an asparagine residue at position 297 that prevents glycation. Examples of such amino acid substitutions are It is a replacement for the N297.

[0168] Mutations in the Fc region of an antibody can result in suppression of effector function, which is well known in the art. Described in the field: LALA and N297A (Strohl, W., 2009, C urr.Opin.Biotechnol.vol.20(6):685-691); and and D265A (Baudino et al., 2008, J.lmmunol.181 :6664-69; Strohl, W., supra); and DAPA (D265A and P329 A)(Shields RL.,J Biol Chem.2001;276(9):6 591-604; U.S. Patent Application Publication No. 2015 / 0320880). An example of an Fc IgG1 antibody is the so-called L23 in the IgG1 Fc amino acid sequence. and LALA mutants containing the L4A and L235A mutations. Silent IgG Another example of a silent IgG1 antibody contains a D265A mutation. The so-called DA, which contains the D265A and P329A mutations relative to the G1 Fc amino acid sequence Another silent IgG1 antibody contains the N297A mutation, This results in aglycosylated / non-glycosylated antibodies, which suppress effector functions. Further Fc mutations for WO 2014 / 145806 brochure (e.g. WO 2014 / 145 The silent IgG1 antibody is shown in Figure 7 of the International Publication No. Examples from the 2014 / 145806 brochure include E233P, L234V, and L23 Contains 5A and S267K mutations and a deletion of G236 (G236del). Silent Another example from WO 2014 / 145806 of an IgG1 antibody is E2 33P, L234V, and L235A mutations and deletion of G236 (G236del). Silent IgG1 antibody from WO 2014 / 145806 Another example includes the S267K mutation.

[0169] In yet another embodiment, the glycosylation of an antibody is modified. Glycosylated antibodies (i.e., antibodies lacking glycosylation) can be made. Such carbohydrate modifications can, for example, increase the affinity of an antibody for an antigen. This can be accomplished, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more variable region framework glycosylation sites may be eliminated, thereby One or more amino acid substitutions can be made to eliminate glycosylation at such sites. Glycosylation can increase the affinity of an antibody for an antigen. Nos. 5,714,350 and 6,350 to Co et al. This is described in further detail in US Pat. No. 5,861.

[0170] Additionally or alternatively, a hypofucosylated antibody or GlcN having a reduced amount of fucosyl residues. Generate antibodies with altered glycosylation types, including antibodies with increased ac bipartite structure Such altered glycosylation patterns can improve the ADCC ability of antibodies. Such carbohydrate modifications can occur in host cells that have altered glycosylation machinery, for example. This can be accomplished by expressing the antibody in host cells. The cells are then transformed with the recombinant antibodies of the invention as described in the art, thereby improving the glycosylation of the antibodies. The host cells can be used to produce antibodies with altered silencing. For example, Hange et al. European Patent No. 1,176,195 by et al. They describe a disrupted cell line that encodes a fucosyltransferase and Antibodies expressed in such cell lines will exhibit hypofucosylation. In one embodiment, the antibody of the present invention is, for example, FUT8, which encodes a fucosyltransferase. In cell lines that display low fucosylation patterns, such as mammalian cell lines where genes are poorly expressed The recombinant expression of the . Brochure shows that a mutant CHO cell line, Asn(297)-linked carbohydrate, is attached to fucose This also reduces the ability of the antibody to bind to the host cell, resulting in hypofucosylation of the antibody expressed in that host cell. The Lecl3 cells were described (Shields, RL et al., 2002 J (See also Biol. Chem. 277:26733-26740). Umana et al. WO 99 / 54342 by et al. discloses antibodies expressed in engineered cell lines. The body has the ability to modify the glycosylation of antibodies to exhibit increased bisecting GlcNac structures, which increases the ADCC activity of the antibody. Protein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosamine) It was engineered to express guanosine-glucosaminyltransferase III (GnTIII) The cell lines used are described below (Umana et al., 1999 Nat. Biotech. 17:176-180). Alternatively, it may be modified for a mammalian-like glycosylation pattern. The glycosylation pattern of the antibody is modified to produce a yeast or filamentous fungus that lacks fucose. The antibodies of the present invention can be produced in the above-mentioned manner (see, for example, European Patent No. 1297172B1). see).

[0171] Another modification of the antibodies herein that is contemplated by the invention is pegylation. The antibody may be pegylated to increase its biological (e.g., serum) half-life. In order to achieve this, typically one or more PEG groups are linked to the antibody or antibody fragment. Under certain conditions, polyethylene glycol (PEG), e.g., a reactive ester or aldehyde of PEG, may be formed. The hydride derivative is reacted with an antibody or a fragment thereof by acylation or alkylation. In this case, PEGylation can be carried out using a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to mono(C1-C 10) Alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol Cholesterol-maleimide, PEG, has been used to derivatize other proteins. In certain embodiments, the antibody to be pegylated is The antibody is an aglycosylated antibody. Methods for pegylating proteins are known in the art. and can be applied to the antibodies of the present invention. No. 0154316 by Ishikawa et al. See specification No. 0401384.

[0172] Another modification of the antibodies contemplated by the present invention is to enhance the half-life of the resulting molecule. In particular, the present invention provides an antibody against a serum protein such as human serum albumin or a fragment thereof. Such an approach is at least a conjugate or protein fusion of an antigen-binding domain. This approach is described, for example, in Ballance et al., European Patent No. 0322094. It has been done.

[0173] Another modification of the antibodies contemplated by the present invention is to enhance the formation of heterodimeric, bispecific antibodies. For example, the contents of the present invention in their entirety are one or more modifications to add to the scope of the present invention. European Patent No. 1870459A1; U.S. Patent No. 5,582, both of which are incorporated herein by reference. ,996; U.S. Pat. No. 5,731,168; U.S. Pat. No. 5,910,5 No. 73; U.S. Pat. No. 5,932,448; U.S. Pat. No. 6,833,441 No. 7,183,076; U.S. Patent Application Publication No. 2006204 493A1; and WO 2009 / 089004A1 The present invention also provides a method for promoting dimerization of the two heavy chain domains of a bispecific antibody, such as a bbmAb, as described herein. To advance the method, various approaches available in the art may be used.

[0174] Bispecifics using knobs-into-holes The antibodies can be produced, for example, by the method described in WO 1996 / 027011; (1996) and Merchant et al. (1998) As shown.

[0175] In some implementations of the disclosed methods of treatment or use, a therapeutically effective amount of IL-1β and Bispecific antibodies that simultaneously target both IL-18 and IL-19, such as bbmAb1, are needed. The drug should be administered to subjects who need it. Changes in the dosing schedule may be necessary for certain patients. It is understood that administration (e.g., of bbmAb1) may be appropriate, e.g., every It may be more frequent, such as daily, biweekly or weekly dosing.

[0176] Some patients may receive bbmAb1 weekly, every other week, or every four weeks for several weeks. loading dosing regimens (e.g., daily dosing for several days / [e.g., 1-4 days, e.g. for example, on days 0, 1, 2, and / or 3] followed by maintenance starting, for example, in week 3 or 4. In some embodiments, the administration of IL-1β and IL-18 may be beneficial. The duration of administration of a bispecific antibody that simultaneously targets both, such as bbmAb1, is 1 day, 2 days, or In some embodiments, the IL The administration of a bispecific antibody that simultaneously targets both IL-1β and IL-18, such as bbmAb1, The duration of the grant is 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 days, Weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months Or even more.

[0177] Dose escalation may be performed for certain patients, e.g., patients based on disease severity, e.g., bbmAb It will be appreciated that this may be appropriate for patients who have an inadequate response to treatment with 1. The dose (intravenous (iv)) is greater than about 10 mg / kg, e.g., about 11 mg / kg. g / kg, 12mg / kg, 15mg / kg, 20mg / kg, 25mg / kg, 30m g / kg, 35 mg / kg, etc. Additionally, the subcutaneous (sc) dose (loading dose) or maintenance dose) is from about 50 mg to more than about 900 mg sc, for example about 75 mg g, approx. 100mg, approx. 125mg, approx. 175mg, approx. 200mg, approx. 250mg, approx. 35 It may be about 0 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, etc.

[0178] Dose reductions may also be considered in certain patients, such as those with adverse events or adverse reactions to treatment with bbmAb1. It will be appreciated that the dosage may be appropriate for responsive patients, etc. Less than 10 mg / kg, for example, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg or about 9 mg / kg. In some embodiments, the bbmAB1 dose is determined by a physician. The time may be adjusted as desired.

[0179] In some embodiments, the bbmAB1 antibody is administered at 10 mg / kg delivered i.v. It may be administered to the patient as a single dose, which may be increased or decreased as needed as determined by the physician. Depending on the circumstances, higher or lower doses may be used, for example, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, or about 4 mg / kg. g / kg, approx. 4mg / kg, approx. 5mg / kg, approx. 6mg / kg, approx. 7mg / kg, approx. 8m g / kg or about 9 mg / kg, or for example about 11 mg / kg, 12 mg / kg, 15 mg / kg, 20mg / kg, 25mg / kg, 30mg / kg, 35mg / kg, etc. It can be done.

[0180] In some embodiments, the bbmAB1 antibody is administered at 10 mg / kg delivered i.v. An initial dose may be administered to the patient, followed by doses as needed, as determined by the physician. , may be adjusted to a higher or lower dose.

[0181] In a specific embodiment, 10 mg / kg bbmAB1 is administered on day 1.

[0182] In a specific embodiment, 10 mg / kg bbmAB1 is administered on day 1 (D1), and 2nd (D2), D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, Administered on D13 and / or D14.

[0183] In another specific embodiment, 10 mg / kg bbmAB1 is administered iv on day 1. can be. EXAMPLES

[0184] Example 1: Multiple inflammasomes appear to be involved in HS. Analysis of the HS transcriptome from patients revealed levels comparable to those in non-diseased or healthy tissues. In comparison, AIM2, NLRC4, NLRP7 and NLRP3 inflammasomes in HS lesions were significantly increased. Increased levels of inflammasome mRNA were shown (Figure 1). The expression levels of the genes were significantly higher in HS-lesioned samples than in HS-nonlesioned and healthy samples. , indicating that multiple inflammasomes appear to be involved in HS pathophysiology.

[0185] Example 2: Both IL-1β and IL-18 signaling pathways are active in HS. (a) Transcriptomics of skin biopsies Eighteen lesional HS biopsies, six lesional HS biopsies, and eight lesional HS biopsies from healthy skin donors were compared. Transcriptomic analysis of the perilesional biopsies and seven nonlesional biopsies was performed as follows: Use the recommended buffer from the Qiagen RNeasy mini kit and flash freeze. A homogenate was prepared from the skin tissue. Total cell count was determined according to the manufacturer's protocol. RNA was extracted using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems cDNA of the samples was prepared from the same starting amount of RNA using Affy CiToxLAB Fr on metrix HG_U133_Plus2 microarrays The sequences were processed using GeneSpring 11.5.1 (Agilent Technology). echnologies, Santa Clara, CA) to generate RMA normalized data. The data was analyzed first by CiToxLAB and then by GeneSpring (PCA The data were subjected to standard QC controls (hybridization controls). Afterwards, the 20th pass in 100% of the samples was performed under either one of the conditions before further analysis. This was filtered by expression level for probe sets above the centile. Deposit the dataset in the NCBI GEO database (GSE148027) Results were visualized using TIBCO Sporfire Analyst.

[0186] (b) Transcriptome of cytokine-stimulated PBMCs (from healthy donors). Kus 7x106 PBMCs / well (12 wells) in 1.5mL final volume in RPMI medium PBMCs were stimulated with recombinant cytokines at the following final concentrations in a 100-well plate: : 10ng / mL recombinant IL-1β, 3nM recombinant IL-18 and 1ng / mL Recombinant cytokines were added at 100 μg / ml of recombinant IL-12. Cells were harvested 6 hours after stimulation in cell culture.

[0187] For RNA isolation, cells were pelleted and incubated for 35 min with 2% β-mercaptoethanol. The pellet was dissolved in 100 μL of Qiagen RTL buffer and all test samples were collected. The samples were frozen at -20°C or -80°C until further notice. RNA isolation was performed using Affymetrix HG_U133_Plus2 as described above. Different RNA samples were processed by CiToxLAB France on the microarray. At least 100% of the samples in any one of the experimental conditions were in the 20th percentile. Entities (probe sets) with values ​​above the threshold were retained. Using the "Filter on Volcano Plot" feature, differentially expressed genes ( DEGs were identified. Genes (range 20.0–100) were filtered with an unpaired T-test. Using the .0th percentile expression, adjusted p-values ​​below 0.05 and fold changes were Probe sets with a ratio >2.0 were considered differentially expressed. , Benjamini-Hochberg multiple testing correction was used.

[0188] The resulting gene list was used as a "cytokine signaling signature" to The expression levels of the defined signatures were calculated without using any relevant values ​​from the original experiments. bell in the HS transcriptome dataset.

[0189] (c) Creation of spotfire transcriptome heatmap Differential responses obtained from cytokine-stimulated PBMCs for every single skin biopsy The average expression levels of genes that were upregulated in the individual samples were calculated, and color coding with increasing color intensity indicated individual This was due to increased mean expression levels of individual PBMC signatures (Figure 2).

[0190] Both IL-1b and IL-18 signaling are present in the lesional skin of HS patients. and that these two cytokines play a role in HS pathophysiology. Provide evidence of what you believe to be accomplished.

[0191] Example 3: The production of bbmAb1 is described in the patent application WO 2018 / 229612. (1) Vector construction, (2) Host cell line and (3) cell selection and sorting; (4) cell proliferation; (5) clonal stability; (6) (7) analytical characteristics and purity assessment; (8) analysis results; Example 1 of 9612 is incorporated herein by reference in its entirety. Be absorbed.

[0192] The resulting bbmAb1 is a bispecific IgG1 with a LALA silencing mutation. It binds simultaneously to two distinct targets, IL-1β and IL-18. Separate antigen-binding arms (Fab fragments) were combined, whereas the Fab against IL-1β Based on mAb2 and containing a kappa light chain (Vk6). Fab against IL-18 is mAb Based on b1, it is composed of lambda light chains (Vλ1). To promote merization, bulky amino acid (aa) side chains (S354C and and T366W) and a small aa side chain (Y349C, T366S, L36 A "hole" with the amino acid sequence (A, Y407V) was introduced in the mAb2 heavy chain.

[0193] For ease of reference, bbmAb is based on the Kabat and Chothia definitions. 1 hypervariable region and V L and V H The domains and complete heavy and light chain amino acid sequences are shown below. The results are provided in Table 3.

[0194] [Table 3]

[0195] [Table 4]

[0196] In one embodiment, an IL-18 / IL-1β bispecific antibody for use in the treatment or prevention of HS is provided. The antibody comprises a first immunoglobulin heavy chain comprising hypervariable regions CDR1, CDR2 and CDR3. Variable domain (V H1 ), wherein said CDR1 has the amino acid sequence SEQ ID NO: 76, The CDR2 has the amino acid sequence SEQ ID NO: 77, and the CDR3 has the amino acid sequence SEQ ID NO: In one embodiment, the IL-18 / I antibody for use in the treatment or prevention of HS is The L-1β bispecific antibody comprises a first immunoglobulin comprising hypervariable regions CDR1, CDR2 and CDR3. Globulin heavy chain variable domain (V H1 ), wherein the CDR1 comprises the amino acid sequence SEQ ID NO: 79, said CDR2 having the amino acid sequence SEQ ID NO: 80, and said CDR3 having the amino acid sequence SEQ ID NO: The nucleic acid sequence of SEQ ID NO: 81. In one embodiment, the disclosed treatment or prevention of (i) HS The IL-18 / IL-1β bispecific antibody for use in A first immunoglobulin heavy chain variable domain (V H1 ), and DR1 has the amino acid sequence SEQ ID NO: 82 and said CDR2 has the amino acid sequence SEQ ID NO: 8 3, said CDR3 having the amino acid sequence SEQ ID NO:84.

[0197] In one embodiment, an IL-18 / IL-1β antagonist for use in the treatment or prevention of HS is provided. The specific antibody has CDR1 having the amino acid sequence SEQ ID NO: 44 and CDR2 having the amino acid sequence and CDR3 has the amino acid sequence SEQ ID NO: 46. A second immunoglobulin heavy chain variable domain (V) comprising R1, CDR2 and CDR3 H2 )of In one embodiment, an IL-18 / IL-1 for use in the treatment or prevention of HS. The beta bispecific antibody has CDR1 having the amino acid sequence SEQ ID NO: 47 and CDR2 having the amino acid sequence The hypervariable region has the sequence SEQ ID NO: 48 and the CDR3 has the amino acid sequence SEQ ID NO: 49 A second immunoglobulin heavy chain variable domain (V H2 In one embodiment, the IL-18 / IL-13 complex for use in the treatment or prevention of HS is The CDR1 has the amino acid sequence SEQ ID NO:50 and the CDR2 has the amino acid sequence SEQ ID NO:51. and CDR3 has the amino acid sequence SEQ ID NO:52. A second immunoglobulin heavy chain variable domain (V) comprising the regions CDR1, CDR2 and CDR3. H2 ).

[0198] In one embodiment, an IL-18 / IL-1β antagonist for use in the treatment or prevention of HS is provided. The specific antibody has CDR1 having the amino acid sequence SEQ ID NO: 92 and CDR2 having the amino acid sequence and CDR3 has the amino acid sequence SEQ ID NO: 94. A first immunoglobulin light chain variable domain (V) comprising R1, CDR2 and CDR3 L1 )of In one embodiment, an IL-18 / IL-1 for use in the treatment or prevention of HS. The beta bispecific antibody has CDR1 having the amino acid sequence SEQ ID NO: 95 and CDR2 having the amino acid sequence The hypervariable region has the sequence SEQ ID NO: 96 and the CDR3 has the amino acid sequence SEQ ID NO: 97 A first immunoglobulin light chain variable domain (V L1 In one embodiment, the IL-18 / IL-13 antibody for use in the treatment or prevention of HS is The CDR1 has the amino acid sequence of SEQ ID NO: 98 and the CDR2 has the amino acid sequence of SEQ ID NO: 99. and CDR3 has the amino acid sequence of SEQ ID NO: 100. A first immunoglobulin light chain variable domain ( V L1 ).

[0199] In one embodiment, an IL-18 / IL-1β antagonist for use in the treatment or prevention of HS is provided. The specific antibody has CDR1 having the amino acid sequence SEQ ID NO: 60 and CDR2 having the amino acid sequence and CDR3 has the amino acid sequence SEQ ID NO: 62. A second immunoglobulin light chain variable domain (V) comprising R1, CDR2 and CDR3 L2 )of In one embodiment, an IL-18 / IL-1 for use in the treatment or prevention of HS. The beta bispecific antibody has CDR1 having the amino acid sequence SEQ ID NO:63 and CDR2 having the amino acid sequence The hypervariable region has the sequence SEQ ID NO: 64 and the CDR3 has the amino acid sequence SEQ ID NO: 65 A second immunoglobulin light chain variable domain (V L2 In one embodiment, the IL-18 / IL-13 antibody for use in the treatment or prevention of HS is The CDR1 has the amino acid sequence of SEQ ID NO:66 and the CDR2 has the amino acid sequence of SEQ ID NO:67. and CDR3 has the amino acid sequence SEQ ID NO:68. A second immunoglobulin light chain variable domain (V) comprising the regions CDR1, CDR2 and CDR3. L2 ).

[0200] In one embodiment, an IL-18 / IL-1β antagonist for use in the treatment or prevention of HS is provided. The specific antibody is the first immunoglobulin V H1 Domain and first immunoglobulin V L1 D a) a first immunoglobulin V H1 A domain can be ): i) CDR1 has the amino acid sequence SEQ ID NO: 76 and CDR2 has the amino acid sequence Hypervariable region CDR1 having the amino acid sequence SEQ ID NO: 77 and CDR3 having the amino acid sequence SEQ ID NO: 78 ii) CDR1 has the amino acid sequence SEQ ID NO: 79, DR2 has the amino acid sequence SEQ ID NO: 80 and CDR3 has the amino acid sequence SEQ ID NO: 81 or iii) hypervariable regions CDR1, CDR2 and CDR3 having the amino acid CDR2 has the amino acid sequence SEQ ID NO: 83, and CDR3 has the amino acid sequence SEQ ID NO: 84. The amino acid sequence of SEQ ID NO: 84 is included in the hypervariable regions CDR1, CDR2 and CDR3. b) the first immunoglobulin V L1 A domain can be (e.g., in a sequence): i) CDR1 is CDR1 has the amino acid sequence SEQ ID NO: 93, and CDR2 has the amino acid sequence SEQ ID NO: 94. The hypervariable regions CDR1, CDR2 and CDR3 have the amino acid sequence SEQ ID NO: 94. R3 or ii) CDR1 has the amino acid sequence SEQ ID NO: 95 and CDR2 has the amino acid sequence and CDR3 has the amino acid sequence SEQ ID NO: 97. or iii) CDR1 has the amino acid sequence SEQ ID NO: 98. and CDR2 has the amino acid sequence SEQ ID NO: 99 and CDR3 has the amino acid sequence SEQ ID NO: 100, and comprises hypervariable regions CDR1, CDR2 and CDR3.

[0201] In one embodiment, an IL-18 / IL-1β antagonist for use in the treatment or prevention of HS is provided. The specific antibody is a second immunoglobulin V H2 Domain and second immunoglobulin V L2 D a) a second immunoglobulin V H2 A domain can be ): i) CDR1 has the amino acid sequence SEQ ID NO: 44 and CDR2 has the amino acid sequence Hypervariable region CDR1 having the amino acid sequence SEQ ID NO: 45 and CDR3 having the amino acid sequence SEQ ID NO: 46 ii) CDR1 has the amino acid sequence SEQ ID NO: 47, DR2 has the amino acid sequence SEQ ID NO: 48 and CDR3 has the amino acid sequence SEQ ID NO: 49 or iii) hypervariable regions CDR1, CDR2 and CDR3 having the amino acid CDR2 has the amino acid sequence SEQ ID NO:51, and CDR3 has the amino acid sequence SEQ ID NO:52. The amino acid sequence of SEQ ID NO:52 is included in the hypervariable regions CDR1, CDR2 and CDR3. b) a second immunoglobulin V L2 A domain can be (e.g., in a sequence): i) CDR1 is CDR1 has the amino acid sequence SEQ ID NO: 61, and CDR2 has the amino acid sequence SEQ ID NO: 62. The hypervariable regions CDR1, CDR2 and CDR3 have the amino acid sequence SEQ ID NO: 62. R3 or ii) CDR1 has the amino acid sequence SEQ ID NO: 63 and CDR2 has the amino acid sequence and CDR3 has the amino acid sequence SEQ ID NO: 65. or iii) CDR1 has the amino acid sequence SEQ ID NO: 66. and CDR2 has the amino acid sequence SEQ ID NO: 67 and CDR3 has the amino acid sequence SEQ ID NO: 68, including hypervariable regions CDR1, CDR2 and CDR3.

[0202] In one embodiment, an IL-18 / IL-1β antagonist for use in the treatment or prevention of HS is provided. The specific antibody comprises: a) a first immunoglobulin comprising the amino acid sequence shown as SEQ ID NO: 85; Heavy chain variable domain (V H1 ); b) comprising the amino acid sequence set forth as SEQ ID NO: 101. The first immunoglobulin light chain variable domain (V L1 c) the sequence shown as SEQ ID NO: 85 The first immunoglobulin V containing the amino acid sequence H1 The domain and sequence shown as SEQ ID NO: 101 The first immunoglobulin V contains an amino acid sequence L1 Domain; d) SEQ ID NO: 76, SEQ ID NO: A first immunoglobulin V comprising a hypervariable region shown as SEQ ID NO: 77 and SEQ ID NO: 78. H1 D e) the hypervariable regions shown in SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94 The first immunoglobulin VL1 Domain; f) SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: The first immunoglobulin V containing the hypervariable region designated as No. 81 H1 Domain;g) sequence A first immunization group comprising hypervariable regions shown as SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97. Roblin V L1 domain; h) SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78 The first immunoglobulin V contains a hypervariable region that H1 Domain and SEQ ID NO: 92, SEQ ID NO: 93 and a first immunoglobulin V comprising a hypervariable region shown as SEQ ID NO: 94. L1 Domestic i) comprising the hypervariable regions shown as SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81; The first immunoglobulin V H1 Domain and SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:9 The first immunoglobulin V contains a hypervariable region designated 7. L1 Domain; j) SEQ ID NO: 10 3; k) a first heavy chain comprising SEQ ID NO: 87; or l) a first light chain comprising SEQ ID NO: 103. and a first heavy chain comprising SEQ ID NO:87.

[0203] In one embodiment, an IL-18 / IL-1β antagonist for use in the treatment or prevention of HS is provided. The specific antibody comprises: a) a second immunoglobulin comprising the amino acid sequence shown as SEQ ID NO:53; Heavy chain variable domain (V H2 ); b) a nucleic acid sequence comprising the amino acid sequence shown as SEQ ID NO: 69; 2 immunoglobulin light chain variable domain (V L2 c) the amino acid sequence shown as SEQ ID NO:53 A second immunoglobulin V containing the 5'-amino acid sequence H2 Domain and the a shown as SEQ ID NO: 69 A second immunoglobulin V containing the amino acid sequence L2Domain; d) SEQ ID NO: 44, SEQ ID NO: 4 5 and a second immunoglobulin V comprising a hypervariable region shown as SEQ ID NO:46. H2 Domain e) comprising the hypervariable regions set forth as SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62 Second Immunoglobulin V L2 domain; f) SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 4 A second immunoglobulin V containing a hypervariable region designated as 9 H2 domain; g) sequence number 63, a second immunoglobulin comprising the hypervariable regions shown as SEQ ID NO: 64 and SEQ ID NO: 65. Rin V L2 Domain; h) as shown in SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 A second immunoglobulin V that contains a hypervariable region H2 Domain and SEQ ID NO: 60, SEQ ID NO: 61 and a second immunoglobulin V comprising a hypervariable region set forth as SEQ ID NO:62. L2 domain i) a sequence comprising the hypervariable regions shown as SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49; 2 Immunoglobulin V H2 Domain and SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65 A second immunoglobulin V containing a hypervariable region designated L2 Domain; j) SEQ ID NO: 81 k) a second light chain comprising SEQ ID NO: 55; or l) a second heavy chain comprising SEQ ID NO: 81. The antibody comprises two light chains and a second heavy chain comprising SEQ ID NO:55.

[0204] In some embodiments, the present invention relates to an IL-18 / IL-1 The beta bispecific antibody comprises the three CDRs of SEQ ID NO: 53. In another embodiment, the method is for treating HS. or an IL-18 / IL-1β bispecific antibody for prophylactic use comprising the triplet of SEQ ID NO: 69 In another embodiment, the present invention relates to an IL-18 / IL-19 CDR for use in the treatment or prevention of HS. The IL-1β bispecific antibody comprises the three CDRs of SEQ ID NO:53 and the three CDRs of SEQ ID NO:69. In some embodiments, the present invention relates to an IL-18 / R inhibitor for use in the treatment or prevention of HS. The IL-1β bispecific antibody comprises the three CDRs of SEQ ID NO: 85. The IL-18 / IL-1β bispecific antibody for use in the treatment or prevention of S. In another embodiment, the present invention relates to a method for the treatment or prevention of HS comprising administering to a patient a therapeutically effective amount of I or Ib. The L-18 / IL-1β bispecific antibody comprises the three CDRs of SEQ ID NO: 85 and SEQ ID NO: 101. It contains three CDRs:

[0205] In some embodiments, the present invention relates to an IL-18 / IL-1 The beta bispecific antibody comprises the three CDRs of SEQ ID NO: 85. In another embodiment, the treatment of HS or an IL-18 / IL-1β bispecific antibody for prophylactic use having the structure of SEQ ID NO: 3 In another embodiment, the IL-18 polypeptide for use in the treatment or prevention of HS comprises one or more CDRs. / The IL-1β bispecific antibody comprises the three CDRs of SEQ ID NO: 85 and the three CDRs of SEQ ID NO: 101. In some embodiments, the present invention provides an IL-1 CDR for use in the treatment or prevention of HS. The IL-8 / IL-1β bispecific antibody comprises the three CDRs of SEQ ID NO: 53. The IL-18 / IL-1β bispecific antibody for use in the treatment or prevention of HS has the sequence In another embodiment, the present invention relates to a method for the treatment or prevention of HS comprising administering to a patient a therapeutically effective amount of The IL-18 / IL-1β bispecific antibody comprises the three CDRs of SEQ ID NO:53 and the CDR of SEQ ID NO:69. In one embodiment, the L-18 for use in the treatment or prevention of HS comprises the three CDRs: / The IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 85, three CDRs of SEQ ID NO: 101 53 and 3 CDRs of SEQ ID NO:69.

[0206] In one embodiment, an IL-18 / IL-1β antagonist for use in the treatment or prevention of HS is provided. The first portion of the specific antibody comprises at least: a) CDR1 having the amino acid sequence SEQ ID NO: 76 CDR2 has the amino acid sequence SEQ ID NO: 77 and CDR3 has the amino acid sequence SEQ ID NO: A variable domain comprising hypervariable regions CDR1, CDR2 and CDR3 having the sequence No. 78 and a) an immunoglobulin heavy chain or a fragment thereof comprising a constant portion of a human heavy chain or a fragment thereof; and DR1 has the amino acid sequence SEQ ID NO: 92 and CDR2 has the amino acid sequence SEQ ID NO: 93 and CDR3 has the amino acid sequence SEQ ID NO: 94. and a variable domain comprising CDR3 and a constant portion of a human light chain or a fragment thereof. and a human IL-18 antibody comprising an IL-18 globulin light chain or a fragment thereof. The second portion of the IL-18 / IL-1β bispecific antibody comprises at least: a) CDR1 having amino acid sequence similar to that of the IL-18 / IL-1β bispecific antibody; CDR2 has the amino acid sequence SEQ ID NO: 45, and CDR3 has the amino acid sequence SEQ ID NO: 46. having the amino acid sequence SEQ ID NO:46, and hypervariable regions CDR1, CDR2 and CDR3. An immunoglobulin heavy chain comprising a variable domain and a constant portion of a human heavy chain or a fragment thereof. or a fragment thereof, and b) CDR1 having the amino acid sequence SEQ ID NO: 60 and CDR2 having the amino acid sequence SEQ ID NO: 61. The hypervariable region has the amino acid sequence SEQ ID NO:61, and the CDR3 has the amino acid sequence SEQ ID NO:62. The variable domain and the constant part of the human light chain, which comprises the domains CDR1, CDR2 and CDR3 in sequence. or a fragment thereof, and a human IL-1β antibody comprising the immunoglobulin light chain or a fragment thereof. Be selected.

[0207] In one embodiment, an IL-18 / IL-1β antagonist for use in the treatment or prevention of HS is provided. The first portion of the specific antibody comprises at least: a) CDR1 having the amino acid sequence SEQ ID NO: 76 CDR2 has the amino acid sequence SEQ ID NO: 77 and CDR3 has the amino acid sequence SEQ ID NO: A variable domain comprising hypervariable regions CDR1, CDR2 and CDR3 having the sequence No. 78 and a) an immunoglobulin heavy chain or a fragment thereof comprising a constant portion of a human heavy chain or a fragment thereof; and DR1 has the amino acid sequence SEQ ID NO: 92 and CDR2 has the amino acid sequence SEQ ID NO: 93 and CDR3 has the amino acid sequence SEQ ID NO: 94. and a variable domain comprising CDR3 and a constant portion of a human light chain or a fragment thereof. and a human IL-18 antibody comprising an IL-18 globulin light chain or a fragment thereof. The second portion of the IL-18 / IL-1β bispecific antibody comprises at least: a) CDR1 having amino acid sequence similar to that of the IL-18 / IL-1β bispecific antibody; CDR2 has the amino acid sequence SEQ ID NO: 45, and CDR3 has the amino acid sequence SEQ ID NO: 46. having the amino acid sequence SEQ ID NO:46, and hypervariable regions CDR1, CDR2 and CDR3. An immunoglobulin heavy chain comprising a variable domain and a constant portion of a human heavy chain or a fragment thereof. or a fragment thereof, and b) CDR1 having the amino acid sequence SEQ ID NO: 60 and CDR2 having the amino acid sequence SEQ ID NO: 61. The hypervariable region has the amino acid sequence SEQ ID NO:61, and the CDR3 has the amino acid sequence SEQ ID NO:62. The variable domain and the constant part of the human light chain, which comprises the domains CDR1, CDR2 and CDR3 in sequence. or a fragment thereof, and a human IL-1β antibody comprising the immunoglobulin light chain or a fragment thereof. Be selected.

[0208] The first V of the IL-18 / IL-1β bispecific antibody used in the disclosed method H1 or V L1 The domain is represented by SEQ ID NOs: 85 and 101. H Or V L Domain and Virtually The first V is the same H1 and / or the first V L1 The present invention relates to a method for the preparation of a nucleic acid sequence comprising: As such, the present invention provides an IL-18 / IL-1β bispecific antibody for use in the treatment or prevention of HS. The antibody comprises a first heavy chain substantially identical to that shown as SEQ ID NO: 87 and / or a sequence The first light chain may be substantially identical to that shown as number 103. As disclosed in, the present application, an IL-18 / IL-1β bispecific antibody for use in the treatment or prevention of HS. The heterologous antibody comprises a first heavy chain comprising SEQ ID NO:87 and a first light chain comprising SEQ ID NO:103. An IL-18 / IL-1β bispecific antibody for use in the treatment or prevention of HS comprises: a) a variable domain having an amino acid sequence substantially identical to that set forth in SEQ ID NO:85 a) a first heavy chain comprising a constant portion of a human heavy chain having a heterodimerization modification and a second heavy chain having a sequence number of SEQ ID NO: A variable domain and a human having an amino acid sequence substantially identical to that shown in No. 101. and a first light chain comprising the constant portion of a human heavy chain, the constant portion of which is IgG1. In one embodiment, the IgG1 is a human IgG1 lacking effector mutations. In one embodiment, the human heavy chain IgG1 has a silencing mutation N297A, In a specific embodiment, the combination of D265A or L234A and L235A is included. In the present invention, the human heavy chain IgG1 is a combination of L234A and L235A according to SEQ ID NO: 87. These include silencing mutations that are

[0209] The second V of the IL-18 / IL-1β bispecific antibody used in the disclosed methods H2 or V L2 The domain is represented by SEQ ID NOs: 53 and 69. H Or V L Virtually the same as the domain The second V H2 and / or the first V L2 The present disclosure may include a domain. IL-18 / IL-1β bispecific antibody for use in the treatment or prevention of HS, as described herein. a second heavy chain and / or a sequence number substantially identical to that shown as SEQ ID NO: 55 The second light chain may be substantially identical to that shown in SEQ ID NO:71. IL-18 / IL-1β bispecific antibody for use in the treatment or prevention of HS as indicated. The antibody may comprise a second heavy chain comprising SEQ ID NO:53 and a second light chain comprising SEQ ID NO:69. IL-18 / IL-1 for use in the treatment or prevention of HS as disclosed herein A β-bispecific antibody has: a) an amino acid sequence substantially identical to that set forth in SEQ ID NO:53 and a heterodimerization modification that is complementary to the heterodimerization of the first heavy chain. and b) a second heavy chain comprising a constant portion of a human heavy chain having a motif as set forth in SEQ ID NO: 69. A second antibody comprising a variable domain and a constant portion of a human light chain having substantially identical amino acid sequences. and the constant portion of the human heavy chain may be IgG1. In one embodiment, In one embodiment, the IgG1 is a human IgG1 lacking effector mutations. , human heavy chain IgG1 silencing mutations N297A, D265A or L234A In a specific embodiment, the human heavy chain IgG1 is a silencing gene that is a combination of L234A and L235A according to SEQ ID NO:55 Including mutations.

[0210] Use as the first portion of a bispecific antibody in the disclosed methods, kits and dosing regimens Other preferred IL-18 antagonists (e.g., antibodies) for No. 9,376,489, which is incorporated herein by reference. That is why.

[0211] For use as a bispecific second moiety in the disclosed methods, kits and dosing regimens Other preferred IL-1β antagonists (e.g., antibodies) for use in the treatment of IL-1β infection are described herein in their entirety. No. 7,446,175 or 7,999, both of which are incorporated by reference herein. This is shown in the specifications of US Pat. No. 3,878 or US Pat. No. 8,273,350.

[0212] Example 4: In vitro activity of bbmAb1 The binding activity of bbmAb1 was tested in a variety of different cellular assays.

[0213] (1) Materials and methods (a) Solution equilibrium titration (SET) assay The following materials were used: Recombinant human IL-18, biotinylated (BTP25828) Recombinant cynomolgus IL-1β (Novartis) Anti-human IgG antibody, sulfo-TAG labelled (Meso Scale discovery (MSD)#R32AJ-5) Goat anti-human Fab specific, conjugated with MSD Sulfo-TAG NHS ester (Jack son Immuno Research#109-005-097, MSD#R91A N-1) BSA (Sigma #A-9647) MSD Lead Buffer with Detergent (MSD#R92TC-1) Phosphate-buffered saline (PBS) 10x (Teknova #P0195) Tris-buffered diet Saline, pH 7.5 (TBS) 10x (Teknova #T1680) Tween-20 ( Fluka #93773) Polypropylene microtiter plates (MTP) (Greiner #781280 ) 384-well plate, standard (MSD#L21XA)

[0214] (b) Cell assay and SET assay mAb2 as described in the IL-1β antibody section. mAb1 as described in the IL-18 antibody section. bbmAb1 as described in Example 1. Recombinant human IL-1 purchased from MBL Int. Corp. (#B001-5) 8(BTP25829) Recombinant marmoset IL-1β (Novartis) Recombinant marmoset IL-18 (Novartis) Recombinant human IL-12 (#573008) was purchased from Biolegend. Cell line (ATCC#CCL-246) Normal human dermal fibroblasts (#CC-2509) were purchased from Lonza. Marmoset skin fibroblasts (#42637F(510)) HEK-Blue(TM) IL-18 / IL-1β cells (#hkb-il18) are In Purchased from vivoGen PBMCs were isolated from the buffy coat and cultured in Blutspendezentrum B Obtained from ern Marmoset blood was obtained from SILABE, Niederhausbergen. IL-6 ELISA: human (BioLegend, #430503); marmoset (U-CyTech biosciences,CT974-5) IFNγ ELISA: human (BD555142) and marmoset (U-CyTec h biosciences#CT340A) QUANTI-Blue™ Assay for Detection of SEAP (#rep-qb1) was purchased from InvivoGen. Cell culture medium: 10% fetal bovine serum (Invitrogen #10108-157), 1% L-glutamine (Invitrogen #25030-03), 1% penicillin / streptomycin ptomycin (Invitrogen #15140-148), 10 μM 2-mercapto Triethanol (Gibco #31350-010), 5 mM Hepes (Gibco # RPMI 1640 (Invitrogen #31870 15630-080) supplemented with ) Round bottom, tissue culture treated 96 well plates (Costar #3799) Flat bottom, tissue culture treated 96 well plates (Costar #3596) Ficoll-Pacque(TM) Plus(GE Healthcare Lif eSciences#17-1440-02) PBS 1X, calcium and magnesium Umbilical cord free (Gibco #14190094) Leucosep tubes with porous septa, 50 mL, polypropylene (Grein er bio-one#227290) Falcon 15mL polypropylene conical tube (BD#352096) Falcon 50mL polypropylene conical tube (BD#352070)

[0215] (c) Affinity measurement by SET SET individual target binding assays Sample buffer (0.5% bovine serum albumin (BSA) and 0.02% Tween-20 22 serial 1.6n dilutions of antigen (maximum concentration: huIL-1 8, 5nM; marIL-18, 10nM; huIL-1β, 0.5nM; marIL- 1β, 0.5 nM) was prepared and a constant concentration of antibody was added (1 0 pM for IL-1β readout, 1 pM for IL-1β readout). 60 μL / well of each antigen-antibody mixture was added. Distribute the volume in duplicate into 384-well polypropylene microtiter plates (MTPs) Sample buffer was used as a negative control, and a sample containing only antibody was used as a positive control (no antigen). Maximum electrochemiluminescence signal, B max The plate is sealed and incubated at room temperature (RT) on a shaker. ) overnight (o / n, at least 16 hours).

[0216] IL-18 readout: streptavidin-coated 384-well MSD array MT Coat P with 30 μL / well of biotinylated huIL-18 (0.1 μg / mL, PBS). The plates were then plated with 100 ml of PBS and incubated for 1 hour at RT on a shaker.

[0217] IL-1β readout: Standard 384-well MSD array MTPs supplemented with PB Coat the plates with 30 μL / well of huIL-1 (3 μg / mL, PBS) diluted in S. The plates were then incubated at 4°C overnight.

[0218] Incubate at room temperature (RT) for 1 h with 50 μL / well of blocking buffer (containing 5% BSA). The plate was blocked with PBS containing 0.05% Tween 100 mM NaCl. After washing, the plate was blocked with 0.05% Tween 100 mM NaCl. Add 30 μL / well of the equilibrated antigen-antibody mixture to polypropylene The cells were transferred from the MTP to the coated MSD plate and incubated at RT for 20 min. After a washing step, 30 μL sulfo-tag-labeled anti-IgG detection antibody (0. 5 μg / mL) was added to each well and incubated for 30 min at RT on a shaker. The plates were washed and 35 μL / well MSD read buffer was added and incubated for 5 min at RT. Electrochemiluminescence (ECL) signals were generated and imaged using the MSD Sector Imager 600 0 was measured.

[0219] SET simultaneous target binding assay SET assays were performed as described above, except for assay A: K D Evaluate While evaluating the IL-1β antibody, an excess of one target (either IL-18 or IL-1β at 500 pM) was administered. Assay B: One mixture at a time was used for the equilibration step (antibody / antigen mixture). An equilibration step (antibody / antigen mixture) was performed with both targets in serial dilutions in (Concentration 10 pM, maximum antigen concentration, see above). Next, IL18 and IL-1β were The same mixture was analyzed for its free antibody concentration on a coating plate.

[0220] Export SET data to Xlfit, an MS Excel add-in software The mean ECL-signal was calculated from duplicate measurements within each assay. Data are presented as the lowest value. Baseline adjustment was performed by subtracting from all data points and the corresponding antigen concentration The titration curve was generated by plotting the titration curve using the following: By K D The values ​​were determined:

[0221] 1:2 binding model for monospecific Abs

[0222]

number

number

[0223] During the ceremony, y: ECL signal subtracted from blank B max : Maximum ECL signal at zero antigen concentration [IgG]: applied antibody concentration [Fab]: total Fab concentration applied K D :dissociation equilibrium constant x: antigen concentration applied

[0224] (d) Cell culture 2x10 5 ~1x10 6 At a density of 10 viable cells / mL, 10% fetal bovine serum, 1% L- K in RPMI 1640 supplemented with glutamine and 1% penicillin / streptomycin G-1 cells were grown.

[0225] bFGF (1 ng / mL, CC-4065), insulin (5 μg / mL, CC-40 21) and FBM (Clonetics, CC-3 Normal human fibroblasts and marmoset fibroblasts were grown in starvation medium and For this purpose, fibroblast basal medium (LONZA#CC-3131) was used.

[0226] 30 μg / mL Blasticidin, 200 μg / mL HygroGold™ and growth medium (DMEM, 4.5 g) supplemented with 100 μg / mL Zeocin™ / L glucose, 10% (v / v) fetal bovine serum, 50 U / mL penicillin, 50 mg / mL Streptomycin, 100 mg / mL Normocin™, 2 mM L- HEK-Blue™ IL-18 / IL-1β cells were grown in medium containing 100% glutamine.

[0227] Human peripheral blood mononuclear cells were isolated using LeucoSep tubes according to the manufacturer's instructions. (PBMCs) were freshly isolated from buffy coats. Briefly, they were cultured at 1,000 × g. 13 ml of lysed lysate was collected in a 14 mL LeucoSep tube by centrifugation for 30 seconds at 4°C. The heparinized whole blood samples were then diluted with an equal volume of PBS. The blood was diluted with 0.01% ethanol and 25 mL of the diluted blood was added to a LeucoSep tube. The cell separation tube was centrifuged at 800xg for 15 minutes. The cell suspension layer was collected and The cells were washed twice with PBS (2 consecutive washes at 640 and 470 x g for 10 min each) for a total The cells were resuspended in medium several times.

[0228] Marmoset blood was collected in a heparinized tube and strained through a 70 μm cell strainer (B The filter was filtered using a 50 mL filter paper (D Biosciences #352350).

[0229] (e) IL-1β neutralization assay The assay in fibroblasts was performed essentially as described (Gram 2000) with minor modifications. Briefly, an IL-1β-induced IL-6 production assay was performed using a 96-well flat-bottom plate. Fibroblast cells were seeded in a fibroblast culture plate at a density of 5x103 cells / well (in 100 μL). The next day, the cells were incubated in starvation medium for 5 hours before the addition of the recombinant IL-1β / compound solution mixture. The cells were then starved for 30 min at 37°C with IL-1β at the concentrations indicated in the table. Preincubation of recombinant IL-1β with a range of compounds After incubation at 37°C overnight, the cell supernatant was collected and the released The amount of IL-6 released was determined by ELISA. IL-6 production assay was performed. Cells 3x10 5 96 wells with 100μL per well PBMCs were seeded in tissue culture plates and incubated with recombinant IL-1β for 24 h at 37°C. / compound solution mixture (IL-1β concentrations are shown in the table). 30 min at 37°C. The I The L-1β / compound solution mixture was prepared. 24 hours after stimulation, the cell supernatant was collected and the released The amount of IL-6 produced was determined by ELISA.

[0230] (f) IL-18 neutralization assay The assay was essentially performed as follows: 3x10 5 KG-1 cells at a density of 100 μg / well (previous PBMCs were then cultured in a round-bottom 96-well plate and starved for 1 h in PBS + 1% FCS. Cells were seeded onto cell culture plates and incubated with recombinant IL-18 / IL-12 along with a range of compounds. (IL-18 / IL-12 concentrations are shown in the table). After 4 hours of incubation, supernatants were harvested and the amount of released IFNγ was determined by ELISA. For assays with marmoset blood, 85 μL of blood / well was used.

[0231] (g) Dual IL1β / IL-18 neutralization in HEK-Blue™ cells The assay was performed essentially as described in the manufacturer's operating instructions. 4x10 well cell culture plates 4 Seed HEK-Blue™ cells at a density of 1000 x g / well. The compounds were seeded with a solution mixture of recombinant IL-1β and IL-18 together with a range of concentrations of the compounds. (to generate a 1:1 SEAP signal). After 24 hours of incubation at 37°C The supernatant is collected and analyzed using the QUANTI-Blue™ method according to the manufacturer's instructions. The amount of released SEAP was determined by the above procedure.

[0232] All data was exported to EXCEL software and analyzed using EXCEL / XLfit4. or GraphPad Prism software to perform logarithmic curve fitting. IC50 values ​​were calculated by plotting dose-response curves against the binding function.

[0233] (2) Results (a) Affinity for recombinant human and marmoset IL1β and IL-18 Human and marmoset recombinant IL-1β and I were titrated by solution equilibrium titration (SET) The binding affinity of bbmAb1 to L-18 protein was measured and the resulting K D Value, I IL-1β was compared with mAb2 and IL-18 binding with mAb1.

[0234] Comparing binding affinities in individual target binding assays, bbmAb1 exhibited binding affinity to both human and It showed a similar average KD compared to mAb1 against marmoset IL-18 (Table 7). For human IL-1β binding, the average KD value was bb (0.6 pM) compared to mAb2 (0.6 pM). It was slightly higher than mAb1 (2.6 pM) but in the same low pM range. Subsequent measurements in a heavy target binding assay (Table 8) confirmed the binding of bbmAb1 to IL-1β. Combined KD values ​​were similar to those of mAb2 in preclinical and clinical grade material Thus, bbmAb1 has a human mAb similar to mAb2 and mAb1, respectively. and retains binding affinity for both targets in marmosets.

[0235] [Table 5]

[0236] In addition to the individual target binding results, the K D What is the effect of excess one target during the evaluation of value binding? by applying either one of the targets (Assay A) or a mixture of both targets in a dilution series. The simultaneous dual target binding affinity of bbmAb1 was examined by performing the same assay (Assay B) (Table 8 Simultaneous IL-1β / IL-18 affinity determinations revealed that assay A (excess of one antigen) and assay B (excess of one antigen) No significant difference was observed between Assay B (a mixture of both antigens in a serial dilution) and the results for the other target. We demonstrated that both targets can be bound simultaneously without affecting the binding of the two targets. K obtained by assay D Values ​​are the K values ​​obtained in standard assays. D The values ​​are similar to those in Table 7 ; in the absence of the second antigen), demonstrating that bbmAb1 can bind both antigens independently. Thus, bbmAb1 inhibits both human IL-1β and IL-18 simultaneously and independently. and fully cross-reacts with the corresponding marmoset protein.

[0237] [Table 6]

[0238] (b) Neutralizing activity of bbmAb1 in human and marmoset cell assays. The neutralizing activity of bbmAb1 against both cytokines (IL-1β and IL-18) was evaluated. Furthermore, the marmoset IL-1 was detected using a marmoset cell assay system. The potency of bbmAb1 for neutralization of IL-1β and IL-18 was evaluated (see section d). ).

[0239] (c) Individual and simultaneous IL-1β and IL-18 neutralization in human cells In human dermal fibroblasts (IL-1β at 6 pM) and in human PBMCs (60 pM Inhibition of recombinant IL-1β-induced IL-6 production in mice (IL-1β used in M The neutralizing activity of bbmAb1 on IL-1β was evaluated in KG-1 cells and human PBMCs. Inhibition of recombinant IL-18-induced IFN-γ production in The neutralizing activity of bmAb1 was measured (3 nM with 1 ng / mL of recombinant human IL-12). M Activation of both cells by recombinant human IL-18. BB on IL-1β and IL-18 The inhibitory potency of mAb1 was always compared to that of either mAb2 or mAb1, respectively. Depending on the assay, the average IC50 values ​​of bbmAb1 are in the sub-nM or single-digit nM range. and respectively, for direct comparison mAb2 (IL-1β) and mAb1 (IL-18) In the case of the mAb2 / mAb1 bivalent system, the mAb2 / mAb1 bivalent system was up to 2-4 times higher (Tables 9 and 10). The monovalent format of bbmAb1 compared to bbmAb1, and potentially the KiH mutation, This may be the reason for this slight difference in efficacy of 1.

[0240] [Table 7]

[0241] [Table 8]

[0242] bbmAb1 inhibits SEA in response to 1 + 1 stimulation with recombinant IL-1β and IL-18. As revealed by HEK Blue™ reporter cells producing IL- The assay was able to simultaneously neutralize the bioactivity of both IL-1β and IL-18 (Table 11). Similar inhibition of SEAP in the mouse system was observed only by the combination of mAb2 and mAb1. This was achievable, but not with the use of individual antibodies.

[0243] [Table 9]

[0244] (d) Marmoset IL-1β and marmoset IL- Neutralizing activity of bbmAb1 in 18 To determine the inhibitory activity of bbmAb1 in marmosets, we performed a similar experiment to human cells. However, we used recombinant marmoset IL-1β and IL-18 for stimulation. A similar in vitro assay was performed with marmoset cells. When inhibition of recombinant marmoset IL-1β-induced IL-6 production was evaluated in bbmAb1 showed sub-nM potency with IC50 values ​​2-3-fold higher than mAb2 ( Table 12. bbmAb1 using human skin fibroblasts stimulated with marmoset IL-1β Studies using this antibody yielded a similar inhibition profile to that achieved with human IL-6.

[0245] [Table 10]

[0246] From the single- to double-digit nM IC50 value of bbmAb1, I Neutralizing activity of bbmAb1 against marmoset IL-18 tested in the FNγ production assay The results confirmed that BBM was induced by human PBMC stimulated with marmoset IL-18 (Table 13). Ab1 assays yielded a similar inhibition profile as measured human IFNγ production .

[0247] Thus, bbmAb1 inhibited the responsiveness of marmoset cells in functional assays. , and was found to be completely cross-reactive with marmoset IL-1β and marmoset IL-18. was shown.

[0248] [Table 11]

[0249] When compared to mAb, mAb2 and mAb1 in a variety of different cellular assays: bbmAb1, a KiH-based IL-1β / IL-18 bispecific mAb, targets two distinct It retains high affinity binding and cytokine neutralizing potency for IL-1β and IL-18. The dual IL-1β and IL-18 neutralizing properties of bbmAb1 were Not only for target cytokines / cells, but also for corresponding markers to facilitate appropriate toxicology studies The effect of IL-1β and IL-18 neutralization was also demonstrated in mouse cytokine / cells. The up to 2- to 4-fold higher IC50 values ​​in some of the cell assays were obtained with mAb 2 and 2, respectively. This could be the result of monovalent binding of bbmAb1, as opposed to the bivalent binding of mAb1. Regardless, dual cytokine neutralization with bbmAb1 resulted in our in vitro cell line Additive or synergistic inhibitory activities in vivo may be obtained that cannot be precisely expressed by the NMR spectrum.

[0250] Example 5: Stimulatory and blocking effects of IL-1β and IL-18 combinations in PBMCs Fruit Dependence of effector cytokines IL-1β and IL-18 on inflammasome activation Cleavage leads to the induction of secondary proinflammatory mediators, which are not only systemic but also localized in the inflammatory area. Two different mice respond to lethal systemic inflammation Models: (a) LPS injection model and (b) FCAS mouse (NLRP3 activated mitochondria). In the case of IL-1β and IL-18 (sense mutation), the simultaneous absence / inhibition of both IL-1β and IL-18 was more protective against lethality than single IL-1β or single IL-18 deficiency / inhibition This may reveal additive or synergistic mechanisms for immune activation (Brydge s 2013, van den Berghe 2014). bbmAb1 is a rodent A human / marmoset IL-1β / IL-18-reactive bispecific mAb with no cross-reactivity with Therefore, it is not possible to test in mouse models. To clarify the additive or synergistic inhibitory effects of the IL-1β / IL-18 neutralization combination, To this end, the inventors performed an in vitro study of the effect of LPS / IL-12 on the stimulation of human PBMCs. Mimicking inflammasome-dependent pathway activation in mice and detecting unbiased genetic changes using microarrays Expression analysis was performed to determine the complementary activity and Therefore, the inventors have developed a combination or single subunit of recombinant IL-1β and recombinant IL-18. Gene expression profiles of PBMCs from different donors stimulated with either cytokines alone or We also compared the

[0251] (3) Materials and methods (a) Cell culture and ELISA 10% fetal bovine serum (Invitrogen #10108-157), 1% L-glutamate (Invitrogen #25030-03), 1% penicillin / streptomycin (Invitrogen #15140-148), 10 μM 2-mercaptoethanol (Gibco#31350-010), 5mM Hepes (Gibco#15630) -080) supplemented with RPMI 1640 (Invitrogen #31870 or Gib co#61870-010) Recombinant human IL-1β was purchased from Sino Biological Inc. (#1013 9-HNAE-5) Recombinant human IL-18 was purchased from MBL (#B001-5). Recombinant human IL-12 was purchased from Biolegend (#573008). IFNγ ELISA:MAX Standard Set, BioLegend, # 430103 or BD OptEIA Human IFNγ ELISA Set, BD#55 5142 IL-6 ELISA:MAX Standard Set, BioLegend, # 430503 IL-26 ELISA:Cloud Clone Corp #SEB695Hu mAb2 as described in the IL-1β antibody section. mAb1 as described in the IL-18 antibody section. bbmAb1 as described in Example 1. Salmonella enterica serovar Enteric LPS from L. enteritidis, Sigma #L7770 PBMCs were obtained from buffy coats from the Blutspendezentrum Bern. Isolated from Round-bottom tissue culture-treated 96-well plates (Costar #3799) Flat-bottom tissue culture-treated 96-well plate (Costar #3596) Ficoll-Pacque™ Plus(GE Healthcare Life Sciences#17-1440 -02) PBS 1X, calcium and magnesium free (Gibco #1419009 4) Falcon 15mL polypropylene conical tube (BD#352096) lcon 50mL polypropylene conical tube (BD#352070) LeucosepTM tubes with porous septum, 50mL, Greiner bio- one#227290 Cell strainer 70μM, BD Biosciences #352350 Trypan blue, Sigma #T8154 RNA isolation, quantification and quality determination and qPCR: Nuclease-free water, Ambion #AM9938 Rnase Zap, Ambion #AM9780 1.5mL Eppendorf tube, sterile, Rnase- and Dnase-free RLT buffer, Qiagen#1015762 Rneasy Mini Kit, Qiagen#74104 RNase-Free DNase Set, Qiagen#79254 Agilent RNA 6000 Nano Kit, Agilent#5067- 1511 Chip Priming Station, Agilent #5065-4401 IKA Vortex Mixer RNaseZAP®, Ambion #9780 Agilent 2100 Bioanalyzer High Capacity cDNA Reverse Transcription Kit, Applied Biosys tems, #PN4374966 Nase-free, thin wall, forsted lid 0.2mL PCR tubes, Ambion #AM12225 MicroAmp Optical 384-well reaction plate, Applied B iosystems#4309849 TaqMan GenEx Master Mix, Applied Biosyst ems#4369514 PCR primers (Applied Biosystems)

[0252] [Table 12]

[0253] PBMC preparation: Ficoll in Leucosep tubes according to the manufacturer's instructions. PBMCs were isolated from buffy coats by β-Paque gradient centrifugation. Put 15mL of Histopaque into a 50mL Leucosep™ tube. The mixture was centrifuged at 1300 rpm for 30 seconds at room temperature. Add 30 mL of the diluted suspension to the top of the Histopaque solution and discard without disrupting the mixture. Centrifuge at 1000 g for 15 min at RT. Discard the plasma (approximately 20 mL) and The circular portion of the PBMC was collected (=human PBMC) and transferred to a 50 mL Falcon tube. Fill the tube with 50 mL of sterile PBS and centrifuge once at 1200 rpm for 5 min at RT. This centrifugation was repeated twice. The supernatant was gently discarded and the cells were washed with 2% FCS and 2 The cells were resuspended in 50 mL of PBS containing 1 mM EDTA. Filter the cell suspension using a filter and stain with trypan blue (500 μL of trypan blue + 2 00 μL cells + 300 μL PBS) was used to count the cells.

[0254] LPS / IL-12 stimulation of PBMCs: Cytokine production in supernatants was determined as follows: 250,000 cells / well in 100 μL final volume in a 96-well round-bottom plate. The mixture was distributed from 0.3 μg / mL to 3000 μg / mL together with recombinant IL-12 at 10 ng / mL. LPS was used at a concentration of 1 μg / mL. After 24 h at 37°C and 10% CO2, the supernatant was circulated. It was recovered.

[0255] RNA extraction from cell pellets was performed as follows: 3x cells in 1000 μL final volume 10 6 10 ng / mL of recombinant IgG was distributed per well in a flat-bottom 24-well plate. LPS was used at 3 μg / mL together with IL-12. Cells were harvested after 4 hours.

[0256] Stimulation of PBMCs with recombinant cytokines: 1 mL of 1000 ng / ml in 1.5 mL of final complete RPMI medium. 7x10 per well of a 2-well plate 6 PBMCs were used at the following final concentrations: 1 0 ng / mL recombinant IL-1β, 3 nM recombinant IL-18, 1 ng / mL recombinant Recombinant cytokines were added with recombinant IL-12. The cells were incubated at 37°C and 10% CO2 for 4 hours. After 24 hours, both the supernatants and the cells were harvested.

[0257] RNA isolation, quantification and quality assessment: Pellet cells and hydrate with 2% β-mercaptoethanol Dissolve the pellet in 350 μL of Qiagen RTL buffer containing 100 μl of 10 ... The samples were frozen at -20°C or -80°C until harvest. Standard Qiagen protocols were used. Briefly, RNA was isolated using RNeasy spin columns for 35 min. 0 μL of 70% ethanol was added to all samples and centrifuged at 8000 g for 15 seconds. After discarding the flow-through, 350 μL of Buffer RW1 was added and the spin column membrane was To wash the column, centrifugation was performed at 8000 g for 15 seconds. Prepare DNase I incubation mix, add it to the RNeasy spin column, and incubate at RT for 15 min. After washing with 350 μL and 500 μL of buffer RW1, the RNeasy slides were Place the pin column into a new 2 mL collection tube and centrifuge at full speed for 1 minute. The RNA was finally purified by adding 35 μL RNase-free water directly to the column membrane. The RNA was eluted by centrifugation at 8000g for 1 minute. The amount of RNA was measured using -1000 and the RNA was stored at -20°C. RIN measurement was performed to assess RNA quality according to the protocol. Pipette A or ladder onto an Agilent RNA 6000 Nano chip. , measured by using an Agilent 2100 Bioanalyser.

[0258] Cytokine gene expression analysis by qPCR: The method was performed according to the manufacturer's instructions. 400 ng of RNA was reverse transcribed using the ty cDNA reverse transcription kit according to the manufacturer's instructions. Dilute the cDNA solution 1 / 10 in RNA / DNA-free water and add 1 μL of cDNA to 384 wells. Transfer the mixture to a well reaction plate and then add 1 μL of 20X TaqMan® Gene Expression Assay: Target FAM gene and 10 μL of 2x TaqMa n® Gene Expression Master Mix and 10 μL The plates were mixed with RNA / DNA-free water. Place on the ViiA™ 7 Real-Time PCR System and set up the following instruments: The following settings were used:

[0259] [Table 13]

[0260] The housekeeping genes used for this study were HPRT1 and RLP27. The following formula was used to calculate the relative expression level of the target genes: 1)Ct[Ref]=(Ct[HPRT1]+Ct[RLP27]) / 2 2) dCt[Ref]=40-Ct[Ref] 3)dCt[Target]=Ct[Target]-Ct[Ref] 4) ddCt = dCt[Ref] - dCt[Target] 5) Relative target gene expression = 2^ddCt

[0261] Microarray was performed as follows: Affymetrix HG_U133_Plu The samples were processed by CiToxLAB France on the s2 microarray. The results were normalized using RMA and analyzed using GeneSpring 11.5.1 (Agilent Tech The data were analyzed by Ingenuity Technologies, Santa Clara, CA. Pathway Analysis (IPA) and Nextbio (Illumina) Pathway analysis was performed using the 2 datasets were processed independently.

[0262] First, we used Rstudio suite and GeneSpring (PCA, hybridization, The R script (MA_AffyQC.R) was used for Therefore, CiToxLAB and our in-house QC control the data. ), which was then filtered to remove unreliable expression levels: At least 100 percent of the samples in any one of the experimental conditions were in the 20th percentile. Entities (probe sets) with values ​​above the tile were kept.

[0263] Using the "Filter on Volcano Plot" feature in GeneSpring , Differentially expressed genes (DEGs) were identified. Filtering with unpaired t-test The genes (20.0–100.0th percentile expression) were used to calculate the corrected p -Differentially expressed probe sets with values ​​below 0.05 and fold change above 2.0 When possible, i.e. LPS (NUID-0000-0202- 4150), the Benjamini-Hochberg multiple testing correction was used. Used.

[0264] For cytokine stimulation experiments, the following formula: Signal A + B / (Signal A + Signal B - Synergy was calculated using a mean ± SEM (control) of ≥ 1.5.

[0265] Using individual signatures (or DEG lists), we compare the signatures with public datasets. The statistical significance of observing overlap between the “disease gene list” of the patients (lesioned vs. non-lesioned) was shown. p values ​​were calculated using Fisher's exact test. To do so, Illumina Base Space Correlation Engine (formerly Nextb Upload the list to io) and use meta-analysis characteristics and keyword search for diseases. and compared.

[0266] All data can be exported to Excel software, Excel / XLfit4 or Logistic curve fitting using either GraphPad Prism software IC by plotting dose-response curves against fitting functions 50 Calculate the value One-way ANOVA, followed by Differences between treatment groups were analyzed by Dunnett's multiple comparisons and results were evaluated at p < 0.05. This was considered statistically significant.

[0267] (4) Results (a) bbmA in inhibiting LPS / IL-12-induced IFNγ production in whole blood B1 is very effective Exposure of human whole blood to LPS supplemented with 10 ng / mL IL-12 resulted in increased expression of IL-12 in blood cells. IFNγ is highly dependent on, but not exclusively on, “native” IL-18 produced by The addition of IL-12 probably upregulates the IL-18 receptor on the responding cells. Regulation of this pathway enhances LPS-induced IFNγ responses.

[0268] Under the experimental conditions used, IL-18 neutralization with mAb1 resulted in incomplete inhibition of IFNγ production. whereas IL-1β blockade (using mAb2) had little effect on the IFNγ response. Interestingly, the combination of bbmAb1 or mAb2 and mAb1 The combination of IL-1β and IL-18 inhibition by either a single cytokine Compared with neutralization of mAb1, bbmAb2 and mAb3 inhibited IFNγ production more profoundly and completely. L in whole blood by mAb2, mAb1, or combined mAb2 and mAb1 (Combo) Inhibition of PS (0.3 μg / mL) / IL-12-induced IFNγ.

[0269] Apart from IFNγ, the other cytokines tested in our cellular assays ( IL-2, -4, -6, -8, -10, -13 and TNFα), among which IL-1β and IL None of the neutralizing combinations of -18 were additively inhibited (bbmAb1 The potency was higher for the combination of mAb2 and mAb1 (CoA), considering the monovalent format of the bispecific molecule. The range was the same as that of the 1990s (Nbo).

[0270] (b) IFNγ inhibited the proliferation of single IL-1β or IL-1β in LPS / IL-12-activated human PBMCs. Compared with IL-18 inhibition, bbmAb1 (i.e., combination IL-1β / IL-18 inhibition) The effect was additively inhibited by Combined IL-1β / IL-18 inhibition with bbmAb1 provides additional benefit To clarify the specific effects of IFNγ, we conducted unbiased transcriptomics analysis. Because whole blood is not optimal for transcriptomics analysis, We performed the LPS / IL-1 antibody therapy described in the Materials and Methods section above on human PBMC samples. The PBMCs from a total of nine donors were used. By this, the inventors demonstrated that bbmAb1 inhibited IFNγ protein secretion into the supernatant of PBMCs. Compared to the whole blood experiment, the inhibitors were inhibited in an additive manner at approximately 10-fold lower concentrations. IFNγ production was inhibited using mAbs. Importantly, the mR A similar pattern of inhibition was evident at the NA level, which supports the notion of unbiased microarrays. The suitability of the samples for gene expression analysis based on BBM in human PBMCs was confirmed. LPS (0.3 μg / mL) with Ab1, mAb2 and mAb1 (each at 10 nM concentration) Inhibition of IL-12-induced IFNγ protein production and IFNγ gene expression was demonstrated.

[0271] P sampled from the LPS / IL-12 stimulation experiment described in the Materials and Methods section above Affymetrix microarrays were performed on n=5 donors from BMC. Global evaluation of gene expression profiles revealed a strong LPS / IL-12 stimulatory effect. It became clear that PCA clustered compounds by donor rather than by stimulated or unstimulated group. Nevertheless, the LPS / I association for differentially expressed genes was not significant. Comparing the L-12 stimulated sample with the stimulated + bbmAb1 sample, the IL-1β / I A shortlist of genes downregulated by combined L-18 blockade was revealed. The strong downregulation of the IFNγ gene revalidated our microarray data (Table 14). In addition to the IL-26 gene regulation, the IL-26 gene was also regulated by a single IL-1β inhibition (by mAb2) or I Additive inhibition by bbmAb1 compared to L-18 inhibition (by mAb1) The cytokine genes were:

[0272] [Table 14]

[0273] (c) IL-26 was enhanced by bbmAb1 in LPS / IL-12 stimulated PBMCs. Another pro-inflammatory cytokine that is selectively inhibited Using bbmAb1, we investigated LPS / IL-12-driven IL-26 gene expression and protein We found that cytokine production was most effectively inhibited by combined IL-1β / IL-18 blockade. To further confirm, the study was expanded to a total of n=9 PBMC donors and IL-26 gene expression and IL-26 protein production were examined by ELISA. LISA revealed that inhibition of IL-26 gene expression was obtained using a microarray approach. Interestingly, the IL-26 protein levels in the supernatant were significantly higher than those in the control group. The addition of b only partially reduced the levels at 24 h. The reason for this difference is unclear. Although it is clear that the dynamic differences between IL-26 gene expression and protein production, as well as the This may be related to the difference in IL-26 consumption compared to that of m Reduced IL-26 protein levels in PBMC supernatants compared to Ab2 and mAb1. It was excellent in that it allowed

[0274] (d) IL1β / IL18 signaling signature correlates with disease Stimulation with recombinant IL-1β results in the production of IL-6 or recombinant IL-18 / We combined previously established PBMC culture conditions in which IL-12 stimulation results in IFNγ production. In combination, additive or synergistic downstream target genes or signatures were identified (data not shown). Data not shown. n=4 donors sampled at two different time points (6 and 24 hours). Affy for unbiased evaluation of gene expression profiles using PBMCs from The combination of IL-1β and IL-18 stimulation was performed. We used a combination of the two to reveal genes that were synergistically upregulated at 6 and 24 hours (data not shown). (Data not shown). The addition of IL-12 to the IL-1β / IL-18 combination , and the synergistic effect on a series of upregulated genes was greatly improved. IL-1β, either alone or in combination, was used to examine patient-derived datasets across diseases. Generated signaling signature of IL-18 pathway stimulation (upregulated genes only) For example, correlations against public sarcoidosis data sets are identified. Values ​​(calculated by Fisher's exact test) were calculated for diseased tissue from sarcoidosis patients and healthy It shows significant correlations to several published studies comparing tissues. , skin, lungs, lacrimal gland and preorbital region. The combination of IL-1β / IL18 signaling showed the best correlation to disease, with IL-1β IL-1β / IL-18 was associated with five different sarcoid tumors. Differentially upregulated genes in PBMCs compared to DEGs in disease vs. healthy tissues (DEG).

[0275] (e) Conclusion Pathogen-associated molecular pattern (PAMP)-dependent N To mimic LRP3 inflammasome activation, we used LPS and recombinant IL-12. Combined inhibition of IL-1β and IL-18 using bbmAb1 Additively reduces IFNγ production in PBMCs stimulated with LPS / IL-12 It was revealed that IL-12 acts synergistically with IL-18 to inhibit the Using the above, T, B, and NK cells (as reviewed by Nakanishi, 2001) It has previously been described to induce IFNγ production in macrophages and dendritic cells, In this study, the additional stimulatory effect of IL-1β on IFNγ production was not evident under the experimental conditions used. Thus, co-incubation of PBMCs with LPS / IL-12 potently inhibited both Effectively inhibits the production of “native” IL-1β and IL-18, which contribute to a potent IFNγ response By using unbiased microarray transcriptomics, Combined IL-1β / IL-18 neutralization versus single IL-1β or IL-18 blockade Further genes that were additively downregulated by the addition of β-glucosamine were identified. The IL-20 gene is expressed in mice but is absent in most rodent lineages (including mice and rats). Tokine subfamily (IL-19, IL-20, IL-22, IL-24 and IL- 26), particularly IL-26 (Donnelly 2010). It is a heterodimeric receptor complex composed of the IL-20R1 and IL-10R2 chains. It signals throughout the body. The IL-26 receptor is primarily expressed on non-blood cell types, especially epithelial cells. Elevated levels of IL-26 have been reported in the serum and especially in the synovial fluid of RA patients, This may act as a factor to promote Th17 cell proliferation and differentiation. Expression of additional genes / pathways induced by combined blockade of IL-1β and IL-18 These findings were countered by the potent effect of LPS / IL-12 stimulation of PBMC samples. Nevertheless, both IFNγ and IL-26, and to some extent IL-22, were expressed in PBMCs. Synergistic upregulation of IL-1β and IL-18 stimulation in mice This suggests that these two factors play a key role in this activation pathway. Therefore, it is confirmed that IL-26 and IL-22 are inflammatory effectors. (i) The IL-20 subfamily of cytokines is a cascade of cytokines produced by IL-1β and IL-18. The selectivity and latency of individual signaling signatures appear to be highly signal-dependent. With due caution regarding the potential effects of blocking these pathways, these comparisons are based on the assumption that individual pathways are involved in sarcoid These compounds are useful for demonstrating activity in diseases such as bronchitis.

[0276] Example 6: Inhibition of spontaneous IFNγ, TNFα and IL-2 production by bbmAb1 Punch biopsies (2 mm) were taken from surgically excised skin from nine different HS patients. The untreated control was treated with either medium (Figure 3, leftmost column) or b at a concentration of 100 μg / mL. b Either mAb1 (Figure 3, center column) or adalimumab (Figure 3, rightmost column) 96-well cell culture plates (flat bottom, tissue culture treated; Costa r) at 37°C and 5% CO2 for 24 h, in 80 μL of medium (10% KnockOut medium). Isc supplemented with t serum replacer (Gibco) and 1% penicillin-streptomycin After centrifugation of the plates, individual HS biopsies were cultured in ove's modified Dulbecco's medium. The supernatant was collected from the wells and analyzed using an MSD plate reader according to the manufacturer's protocol. Multiplex MSD (Meso Scale Discovery Platform m) Cytokine Proinflammatory Panel 1 (protein) array was performed. Data was collected by individual biopsy weight. The data was normalized and the blotting plots were plotted using GraphPad Prims software. Figure 3 shows that bbmAb1 inhibited spontaneous IFN-γ expression in the supernatants of HS biopsies. It has been shown to reduce Nγ, TNFα and IL-2 production.

[0277] Example 7: Toxicity Testing The bispecific antibody bbmAb1 was administered twice weekly up to 100 mg / kg for 26 weeks. When administered sc to rhesus monkeys, it was well tolerated (no observed effect level (NOEL) of 1 00mg / kg; Cmax, 3,110μg / mL ss; AUC0-72h, 218, 000 μ·h / mL ss), safety pharmacology (central nervous system, cardiovascular system and respiratory system), toxicity No effects on sexology (including the male reproductive system and sperm motility) or local tolerability were noted. No effect was observed after 6 weeks of intravenous (iv) dosing at 100 mg / kg twice weekly. (Cmax, ss of 4570 μg / mL, AUC0-72h, 261,000 μ g·h / mL ss). Furthermore, in immune cells in peripheral blood and upon exposure to exogenous antigens, No treatment-related effects were observed on primary and secondary humoral immune responses. A total of 48 subjects (12 of whom were placebo-controlled) were enrolled in a single ascending dose (SAD) study. (subjects treated with sebo) at 0.1, 0.3, 1, 3, and 10 mg / kg iv and data from the first six cohorts (A1–B1) at escalating doses of 100 mg sc. In patients with bronchitis, bbmAb1 was generally well tolerated at doses up to 10 mg / kg. The Cmax and AUC of bbmAb1 were consistent across the entire range of iv administration (0.1 mg / kg to The mean half-life of bbmAb1 was approximately 21–10 mg / kg. The bioavailability of the sc dose was estimated to be 70%.

[0278] Example 8: Therapeutic Uses To evaluate the efficacy and safety of bbmAb1 in patients with moderate to severe hidradenitis suppurativa A randomized, subject- and investigator-blinded, placebo-controlled, and multicenter platform study to To clarify the efficacy of the bispecific anti-IL-1β anti-IL-18 antibody bbmAb1, Details of the clinical trial design are provided below.

[0279] The number of lesions or total HS-PGA score in the HS and the mean PGA score were calculated by blinding the subjects and the investigators. This allows for unbiased evaluation of subjective readings such as adverse events.

[0280] In subjects with moderate to severe HS, several active treatment compounds, such as bispecific anti-I To evaluate the efficacy, safety and tolerability of L-1β anti-IL-18 antibody bbmAb1, This will be a randomized, subject- and investigator-blinded, placebo-controlled, multicenter, parallel-group study. After a 5-week screening period, a 16-week treatment period was planned, after which approximately Safety follow-up will continue for 12 weeks. bbmAb1, 300 mg (1.5 mL x 3) injections; every other week on days 1, 15, and 29, then monthly (Q4W) on days 57 and 85. c. or its corresponding placebo (2 x 1.5 mL) sc.

[0281] Subjects included in this study were diagnosed with recurrent inflammatory lesions for at least 12 months. Adult males and females aged 18-65 years (inclusive) with moderate to severe HS At randomization (prior to dosing on Day 1), subjects were required to participate in at least two Patients must have at least five inflammatory lesions (abscesses and nodules) in the anatomical region. Centralized Interactive R Cohort and individual analysis using the Informatics Research Technology (IRT) system Randomization to different groups will be performed.

[0282] The primary clinical outcome measure was the simplified HiSCR (Hidradenitis Suppurativa Clinical Response) 16 weeks after treatment. do.

[0283] On Day 113 (Week 17), after safety and other evaluations were performed, all subjects were If medically justified and for safety reasons, If the above potential concerns have not been identified (after discussion with the sponsor), the subject may During the low-up period, patients may receive medication that was previously prohibited.

[0284] Safety and efficacy assessments will be performed at follow-up visits, if specified in the evaluation schedule. Pharmacokinetic (PK), pharmacodynamic (PD) and biomarker samples will also be collected. The End of Study (EOS) visit will occur on Day 197 (Week 29) and will include an end of study evaluation and subsequent This includes release from the study. Investigator and subject blinding will be maintained until study completion.

[0285] Approximately 40 subjects will be randomized; 30 subjects will receive the study treatment and 10 subjects will Receive a matching placebo. - On Day 1, 300mg bbmAb1 or its matching placebo (1.5mL 2 The drug is administered subcutaneously (sc) by trained medical staff. Floor evaluation, penalty kicks, target engagement nt), immunogenicity, pathway and disease biomarkers, and safety evaluations will be performed. If there are no concerns, patients will be discharged from the study site on the same day after completion of all evaluations. , subjects should be administered the drug for at least 1 hour or longer, at the discretion of the investigator. The center should be observed for immediate injection site reactions. - Subjects will receive bbmAb1 sc every other week (Q2W; 3 doses) Then, they will return to the study facility during the initial loading phase of the study (Day 1 (Week 1) to Day 29 (Week 5)). - Next, every 4 weeks during the maintenance phase of the study (Day 29 (Week 5) to Day 85 (Week 13)) bbmAb1 will be administered at 300 mg sc (Q4W; 2 doses).

[0286] During these visits, safety and selected efficacy assessments will be performed and PK, target engagement, and Target engagement, immunogenicity and pathway / disease biomarkers Collect a marker sample.

[0287] The primary objective is to evaluate the efficacy and safety of bispecific antibody b in HS subjects after 16 weeks of treatment compared to placebo. The objective of this study is to demonstrate the preliminary efficacy of treatment with bmAb1. After a 16-week treatment period, To observe whether the persistence of effects could be maintained or increased after 16 weeks, The follow-up period will be 12 weeks.

[0288] The primary endpoint of this study was the simplified HiSCR (Kimball 2014). The simplified HiSCR was chosen to evaluate the presence of abscesses and inflammation without any increase in draining fistulas. This is defined as a 50% reduction in the total number of sexual nodules.

[0289] Inflammatory lesions in HS are characterized by discrete lesions (inflammatory nodules, abscesses) in typical anatomical areas. and draining fistulas). In addition to the count, a comprehensive rating scale (hidradenitis suppurativa - Physician's Global Assessment or HS-PGA) and composite score (Hidradenitis Suppurativa Severity Assessment Score) or SAHS) is used.

[0290] Dermatology Life Quality Index Several patient-reported outcomes will be used, including the DLQI (Dementia Qualification Index). Finally, since skin-related pain was the most important symptom in the subjects' perspective, the number of pain-related Includes the Value Rating Scale (NRS).

[0291] For further information on clinical evaluation: HS-PGA (Hidradenitis Suppurativa - Physician Global Assessment): This score is used to assess HS. Used for exploratory purposes, Kimball AB, Kerdel F, Adams D, The method was used and described in (2012).

[0292] The SAHS score is a composite score (Hessam S, Scholl L, San d M, et al (2018)), inflammatory lesion count, fistula count, and NRS pain were collected. In addition, both anatomical regions and new or recurrent existing swellings were identified. Collected in cohorts.

[0293] Cutaneous Pain-NRS (Numerical Rating Scale for Pain): The NRS for skin-related pain is: It was used in the adalimumab trial (Kimball et al. (2016)) and was Skin or HS-related pain is one of the greatest burdens for patients (Matusiak et al. l (2017)), this is used. HS-related pain was The average and worst case scenario (in the last 24 hours) are recorded.

[0294] Other patient-reported outcomes (PROs) included aspects such as pruritus, fatigue and work disability, as well as The Dermatology Life Quality Index, a validated score available in many countries and languages, Dermatology Life Quality Index (DLQI) and and dermatology-related quality of life (QoL) tools. Includes a combined evaluation.

[0295] [Table 15]

[0296] [Table 16]

[0297] Selection criteria: Men and women with clinically diagnosed HS at least 12 months prior to screening Target age: 18-65 years old (inclusive) Moderate to severe H, as assessed at screening and randomization (pre-dose on Day 1) Patients with S: A total of at least 5 inflammatory lesions, i.e. abscesses and / or inflammatory nodules, and Not more than 15 fistulas, and At least two anatomical regions must contain HS lesions Minimum weight at screening: 50kg (inclusive) Able to communicate well with the investigator and understand and comply with the requirements of this study Yes, and able and willing to complete study visits according to the study schedule.

[0298] Exclusion criteria: At screening or within 30 days or 5 half-lives of randomization, whichever is longer duration; or the use of other investigational products for a longer period if required by local regulations. WOCBP (defined as any woman of physiological childbearing potential) is considered when a pregnancy test is performed. , at least 3 months before the first drug administration and up to 5 months after the last drug administration (days 225 to 253) JP) Highly effective contraception is urged to be consistently practiced. Women who are pregnant or breastfeeding at screening or randomization, where pregnancy Pregnancy is defined as the state of a woman after conception, confirmed by a positive clinical hCG test, and until the termination of pregnancy. It is defined as:

[0299] Study treatment and duration Patients assigned to the bbmAb1 group received bbmAb1, 300 mg, sc, or Matching placebo will be administered every other week from day 1 (Week 1) to day 29 (Week 5). (Q2W) and then every month (Q4W) until the 85th day (inclusive) (13th week).

[0300] Efficacy evaluation -Evaluation of the simplified and original Hidradenitis Suppurativa Clinical Response (HiSCR) International Hidra Suppurativa Severity Scoring System denitis Suppurativa Severity Score Syste m)(IHS4) Hidradenitis Suppurativa-Physician Global Assessment (HS-PGA) score and responder rate HS Inflammatory Lesion Count Hidradenitis Suppurativa Severity Rating (SAHS) In the case of bbmAb1, the drug formulation supplied is a "ready-to-use" buffered sterile aqueous solution. This solution contains 100 mg / mL bbmAb1 and the excipients L-histidine, sucrose, and and polysorbate 20, pH 6.0. The placebo control selected for this study The reference is a solution with a suitable composition of inert excipients.

[0301] Predicted effects of bbmAb1 administration on IL-1β and free IL-18 Predicting the dynamics of anti-IL-1β / IL-18 bispecific antibodies and their targets in serum The model used to determine the dynamics of free and total IL-18 is a novel model In addition, a general competitive binding model for IL-18 (Yan et al. 201 2) and the previously published results of canakinumab adjusted for bbmAb1 against the IL-1β group The model was developed based on the previously published (Chakraborty et al. 2012). To predict the dynamics of IL-1β in response to application of canakinumab in clinical trials, Using an empirically established model (Chakraborty et al. 2012), The PK parameters and binding affinity specific to bbmAb1 were updated. To modulate IL-1β concentrations in patients, we performed a canakinumab clinical trial The synthesis and clearance parameters of this interleukin were used as listed (Cha Kraborty et al. 2012). This model has been shown to be effective in treating several autoimmune diseases. In-house in vitro and published human IL-18 data from a range of patient-derived free and total IL-18 serum concentrations Based on data from the 2018 study (Weiss et al. 2018).

[0302] Based on this, the 300 mg Q2W / Q4W sc schedule resulted in It is expected that a simultaneous decrease in both IL-1β and IL-18 levels in the serum will occur. Effective neutralization of IL-1β as well as IL-18 is predicted (FIGS. 4 and 5).

[0303] Pharmacokinetics of bbmAb1 FiH monotherapy up to 10 mg / kg iv was administered in healthy volunteers without drug-related SAEs. bbmAb1 will be evaluated in an ascending dose study. The PK profile was determined based on the soluble ligand site, in keeping with human predictions based on marmoset monkey data. As expected for a typical IgG1 antibody binding to a kine target. Ab1 demonstrated a dose-linear increase in exposure (10 mg / mL) consistent with expected human PK. The predicted human PK parameters for bbmAb1 were evaluated at up to 100 mg / kg iv. CL = 0.158 L / d, distribution volume (V d )=5.586L(for a 70 kg person) for elephants) or 0.08L / kg; half-life (T 1 / 2 ) = 24.5 days. Preliminary analysis of the PK profile from this study indicates that BBM is associated with anti-drug antibody (ADA) formation. Provides no evidence of accelerated clearance of Ab1.

[0304] Based on recent subcutaneous data from the FiH study, these findings suggest that bioavailability The drug availability and absorption rate constants were adjusted and used to predict subcutaneous PK.

[0305] A bbmAb1 dose of 300 mg Q2W / Q4W sc reduced all systemic free I This is expected to result in rapid and simultaneous neutralization of IL-1β and IL-18. 300mg s. c. Subsequent exposure to IL-1β and IL-18 for >100 days after a single dose exceeds the in vitro IC90 for (Figure 6).

[0306] References Akdogan N, Dogan S, Incel-Uysal P, et al(20 20)Serum amyloid A and C-reactive protei n levels and erythrocyte sedimentation r ate are important indicators in hidraden itis suppurativa.Arch Dermatol Res.;312( 4):255-262. Andersen RK,Jemec GB(2017)Treatments for hidradenitis suppurativa.Clin.Dermatol. p.218-224. Andre R, Marescassier H, Gabay C, et al(201 9)Long-term therapy with anakinra in hid radenitis suppurativa in three patients. Int J Dermatol;58(11):e208-e209. Bettoli V, Zauli S, Virgili A (2016) Oral cl. indamycin and rifampicin in the treatmen t of hidradenitis suppurativa-acne inver sa:can some factors influence the respon se to the treatment? G Ital Dermatol Ven ereol p.216-8. Blok JL,Li K,Brodmerkel C,et al(2016)Ust ekinumab in hidradenitis suppurativa:cli nical results and a search for potential biomarkers in serum.Br.J.Dermatol.p.839 -46. Byrd AS,Kerns ML,Williams DW,et al(2018) Collagen deposition in chronic hidradeni tis suppurativa:potential role for CD163 + macrophages.Br.J.Dermatol.;179(3):792- 794. Boergeson E,Docherty NG,Murphy M,et al(2 011)Lipoxin A4 and benzo-lipoxin A4 atte nuate experimental renal fibrosis.FASEB J.p.2967-79. Casseres RG,Prussick L,Zancanaro P,et al (2020)Secukinumab in the treatment of mo derate to severe hidradenitis suppurativ a:Results of an open-label trial.J Am Ac ad Dermatol.;82(6):1524-1526 Celtaxsys Inc.Press-release(2018)Celtaxs ys announces results of phase 2 trial sh owing clinically meaningful improvement in pulmonary exacerbations in cystic fib rosis patients. Chakraborty,A,Tannenbaum,S,Rordorf,C.et al(2012)Pharmacokinetic and pharmacodyna mic properties of canakinumab,a human an ti-interleukin-1β monoclonal antibody.Cl inical pharmacokinetics;51(6):e1-e18. Christmas P,Fox JW,Ursino SR,et al(1999) Differential localization of 5- and 15-l ipoxygenases to the nuclear envelope in RAW macrophages.J.Biol.Chem.p.25594-8. Deckers IE,Kimball AB(2016)The Handicap of Hidradenitis Suppurativa.Dermatol Cli n p.17-22. Dessinioti C,Zisimou C,Tzanetakou V,et a l(2016)Oral clindamycin and rifampicin c ombination therapy for hidradenitis supp urativa:a prospective study and 1-year f ollow-up.Clin.Exp.Dermatol.p.852-857. Esmann S,Jemec GB(2011)Psychosocial impa ct of hidradenitis suppurativa:a qualita tive study.Acta Derm.Venereol.p.328-32. Fimmel S,Zouboulis CC(2010)Comorbidities of hidradenitis suppurativa(acne invers a).Dermatoendocrinol;2(1):9-16.. Finlay AY,Khan GK(1994)Dermatology Life Quality Index(DLQI)--a simple practical measure for routine clinical use.Clin.Ex p.Dermatol;19:210-216. Garg A,Wertenteil S,Baltz R,et al(2018)P revalence Estimates for Hidradenitis Sup purativa among Children and Adolescents in the United States:A Gender-and Age-Ad justed Population Analysis.J.Invest.Derm atol.;138(10):2152-2156. Giamarellos-Bourboulis EJ,Argyropoulou M ,Kanni T,et al(2020).Clinical efficacy o f complement C5a inhibition by IFX-1 in hidradenitis suppurativa:an open-label s ingle-arm trial in patients not eligible for adalimumab.Br J Dermatol.;183(1):17 6-178. Giuseppe P,Nicola P,Valentina C,et al(20 18)A Case of Moderate Hidradenitis Suppu rativa and Psoriasis Treated with Secuki numab.Ann Dermatol;30(4):462-464. Gottlieb A,Natsis NE,Kerdel F,et al(2020 )A Phase II Open-Label Study of Bermekim ab in Patients with Hidradenitis Suppura tiva Shows Resolution of Inflammatory Le sions and Pain.J Invest Dermatol;140(8): 1538-1545. Hessam S,Scholl L,Sand M,et al(2018)A No vel Severity Assessment Scoring System f or Hidradenitis Suppurativa.JAMA Dermato l;154(3):330-335. Hessam S,Sand M,Gambichler T,et al(2015) Correlation of inflammatory serum marker s with disease severity in patients with hidradenitis suppurativa(HS).J Am Acad Dermatol.;73(6):998-1005. Houriet C,Seyed Jafari SM,Thomi R,et al( 2017)Canakinumab for Severe Hidradenitis Suppurativa:Preliminary Experience in 2 Cases.JAMA Dermatol.;1;153(11):1195-119 7. Hunger RE,Surovy AM,Hassan AS,et al(2008 )Toll-like receptor 2 is highly expresse d in lesions of acne inversa and colocal izes with C-type lectin receptor.Br.J.De rmatol.;158(4):691-7. Ingram JR,Woo PN,Chua SL,et al(2016)Inte rventions for hidradenitis suppurativa:a Cochrane systematic review incorporatin g GRADE assessment of evidence quality.B r.J.Dermatol.;174(5):.970-8. International Council for Harmonization of Technical Requirements for Pharmaceut icals for Human Use(ICH)(2016)ICH E6(R2) GCP Guideline(2016). Jaeger T,Andres C,Grosber M,et al(2013)P yoderma gangrenosum and concomitant hidr adenitis suppurativa-rapid response to c anakinumab(anti-IL-1β).Eur J Dermatol.;2 3(3):408-410. Janse IC,Deckers IE,van der Maten AD,et al(2017)Sexual health and quality of lif e are impaired in hidradenitis suppurati va:a multicentre cross-sectional study.B r.J.Dermatol.;176(4):1042-1047. Jemec GB(2012)Clinical practice.Hidraden itis suppurativa.N.Engl.J.Med.;366(2):15 8-64. Jemec GB,Kimball AB(2015)Hidradenitis su ppurativa:Epidemiology and scope of the problem.J.Am.Acad.Dermatol.;73(5 Suppl 1 ):S4-7. Jorgensen AR,Yao Y,Thomsen SF(2018)Thera peutic Response to Secukinumab in a 36-Y ear-Old Woman with Hidradenitis Suppurat iva.Case Rep Dermatol Med;(Apr 16):86851 36.. Kanni T,Zenker O,Habel M,et al(2018)Comp lement activation in hidradenitis suppur ativa:a new pathway of pathogenesis? Br. J.Dermatol.;179(2):413-419 Kelly G,Sweeney CM,Tobin AM,et al(2014)H idradenitis suppurativa:the role of immu ne dysregulation.Int.J.Dermatol.;53(10): 1186-96. Kelly G,Hughes R,McGarry T,et al(2015)Dy sregulated cytokine expression in lesion al and nonlesional skin in hidradenitis suppurativa.Br J Dermatol.;173(6):1431-1 439. Kimball AB,Ganguli A,Fleischer A(2018)Re liability of the hidradenitis suppurativ a clinical response in the assessment of patients with hidradenitis suppurativa. J Eur Acad Dermatol Venereol.;32(12):225 4-2256. Kimball AB,Jemec GB,Yang M,et al(2014)As sessing the validity,responsiveness and meaningfulness of the Hidradenitis Suppu rativa Clinical Response(HiSCR)as the cl inical endpoint for hidradenitis suppura tiva treatment.Br.J.Dermatol.;171(6):143 4-42. Kimball AB,Kerdel F,Adams D,et al(2012)A dalimumab for the treatment of moderate to severe Hidradenitis suppurativa:a par allel randomized trial.Ann.Intern.Med.;1 57:846-855. Kimball AB,Okun MM,Williams DA,et al(201 6)Two Phase 3 Trials of Adalimumab for H idradenitis Suppurativa.N.Engl.J.Med.;37 5(5):422-34. Leslie KS,Tripathi SV,Nguyen TV,et al(20 14)An open-label study of anakinra for t he treatment of moderate to severe hidra denitis suppurativa.J Am Acad Dermatol.; 70(2):243-251. Lima AL,Karl I,Giner T,et al(2016)Kerati nocytes and neutrophils are important so urces of proinflammatory molecules in hi dradenitis suppurativa.Br.J.Dermatol.;17 4(3):514-21. Ljungman P,Boeckh M,Hirsch HH,et al(2017 )Disease Definitions Working Group of th e Cytomegalovirus Drug Development Forum .Definitions of Cytomegalovirus Infectio n and Disease in Transplant Patients for Use in Clinical Trials.Clin Infect Dis. ;64(1):87-91. Musilova J,Moran B,Sweeney CM,et al(2020 )Enrichment of Plasma Cells in the Perip heral Blood and Skin of Patients with Hi dradenitis Suppurativa.J Invest Dermatol ;140(5):1091-1094 Penno CA,Jager P,Lague rre C et al(2020)Lipidomics Profiling of Hidradenitis Suppurativa Skin Lesions R eveals Lipoxygenase Pathway Dysregulatio n and Accumulation of Pro-Inflammatory L eukotriene B4.J Invest Dermatol.;140(12) :2421-2432.. Reguiai Z,Fougerousse AC,Maccari F,et al (2020)Effectiveness of secukinumab in hi dradenitis suppurativa:an open study(20 cases).J Eur Acad Dermatol Venereol.;34( 11):e750-e751. Revuz J(2009)Hidradenitis suppurativa.J European Acad Dermatol Venereol;23:985-9 98. Riis PT,Soeby K,Saunte DM,et al(2015)Pat ients with hidradenitis suppurativa carr y a higher systemic inflammatory load th an other dermatological patients.Arch De rmatol Res.;307(10):885-9. Russo V,Alikhan A(2016).Failure of Anaki nra in a Case of Severe Hidradenitis Sup purativa.J Drugs Dermatol.;15(6):772-774 . Schuch A,Fischer T,Boehner A,et al(2018) Successful Treatment of Severe Recalcitr ant Hidradenitis Suppurativa with the In terleukin-17A Antibody Secukinumab.Acta Derm.Venereol.;98(1):151-152. Serhan CN,Chiang N,Van Dyke TE(2008)Reso lving inflammation:dual anti-inflammator y and pro-resolution lipid mediators.Nat .Rev.Immunol.;8(5):349-61. Shah A,Alhusayen R,Amini-Nik S(2017)The critical role of macrophages in the path ogenesis of hidradenitis suppurativa.Inf lamm.Res.;66(11):931-945. Sharon VR,Garcia MS,Bagheri S,et al(2012 )Management of recalcitrant hidradenitis suppurativa with ustekinumab.Acta Derm. Venereol.;92(3):320-1. Singer EA(2004)The necessity and the val ue of placebo.Sci Eng Ethics;10(1):51-6. Sun NZ,Ro T,Jolly P,et al(2017)Non-respo nse to Interleukin-1 Antagonist Canakinu mab in Two Patients with Refractory Pyod erma Gangrenosum and Hidradenitis Suppur ativa.J Clin Aesthet Dermatol.;10(9):36- 38. Tekin B,Salman A,Ergun T(2017)Hidradenit is suppurativa unresponsive to canakinum ab treatment:A case report.Indian J Derm atol Venereol Leprol.;83(5):615-617. Thorlacius L,Theut Riis P,Jemec GBE(2018 )Severe hidradenitis suppurativa respond ing to treatment with secukinumab:a case report.Br.J.Dermatol.;179(1):182-185. Tzanetakou V,Kanni T,Giatrakou S,et al(2 016)Safety and Efficacy of Anakinra in S evere Hidradenitis Suppurativa:A Randomi zed Clinical Trial.JAMA Dermatol;152(1); 52-59. 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[0307] Example 9: Development of a high concentration formulation (100-120 mg / mL) of bbmAb1 Eight different formulations were initially tested in a high-throughput plate-based assay. I tried it. F1 (120 mg / mL bbmAb1, 20 mM sodium succinate, pH 5.0 , 220 mM sucrose, 0.04% polysorbate 20); F2 (120 mg / mL bbmAb1, 20 mM histidine / histidine-Cl, pH 5.0, 220 mM sucrose, 0.04% polysorbate 20); F3 (120 mg / mL bbmAb1, 20 mM histidine / histidine-Cl, pH 5.5, 220 mM sucrose, 0.04% polysorbate 20); F4 (120 mg / mL bbmAb1, 20 mM histidine / histidine-Cl, pH 6.0, 220 mM sucrose, 0.04% polysorbate 20); F5 (120 mg / mL bbmAb1, 20 mM histidine / histidine-Cl, pH 6.5, 220 mM sucrose, 0.04% polysorbate 20); F6 (120 mg / mL bbmAb1, 20 mM potassium phosphate, pH 7.0, 2 20 mM sucrose, 0.04% polysorbate 20); F7 (50 mg / mL bbmAb1, 20 mM histidine / histidine-Cl, p H5.5, 220 mM sucrose, 0.04% polysorbate 20); F8 (50 mg / mL bbmAb1, 20 mM potassium phosphate, pH 7.0, 22 0 mM sucrose, 0.04% polysorbate 20).

[0308] After 2 or 4 weeks at 40°C, aggregate formation was monitored by size exclusion chromatography. Formulations F1 to F8 were tested for the effect of flocculation. The results showed that increasing the pH from 5 to 7 A trend towards increased aggregation was observed, with formulations F6 and F8 showing significant aggregation at pH 7.0. See FIG. It refers to lighting.

[0309] Formulations F1-F8 were also tested for degradation product formation by size exclusion chromatography. The results showed that under the test conditions (25°C, 4 weeks), all the formulations tested showed However, the protein was stable under non-reducing conditions. The analysis showed clear fluorescence of degradation product formation at lower pH and formulations at pH 5.0. showed significant degradation product formation, see FIG.

[0310] Acidification in response to heat stress (25°C, 4W) was determined by charge zone electrophoresis (CZE). Formulations F1-F8 were tested for acidic and basic variant formation. Variant formation was observed (F8), with significant basic variant formation observed at low pH. (F1 and F2). See Figures 9A and 9B.

[0311] The above formulations were also tested for resistance to freeze-thaw and mechanical stress (agitation). The results showed that excipients (e.g., sugars such as sucrose) and surfactants (e.g., polysorbate 50-120mg / mL bbmAb1 (preferred) with polysorbate 20 or other polysorbate. or 100 mg / mL bbmAb1), at pH 5.5, preferably 6.0. A good and stable formulation is further demonstrated.

[0312] Further test modalities include visual, turbidity (A405nm), and dynamic light scattering. , microflow imaging and liquid chromatography The results included LysC peptide mapping by fluorometric mass spectrometry.

[0313] Sequence Listing Amino acid and nucleotide sequences useful for practicing the present invention are disclosed in Table 15.

[0314] [Table 17]

[0315] [Table 18]

[0316] [Table 19]

[0317] [Table 20]

[0318] [Table 21]

[0319] [Table 22]

[0320] [Table 23]

[0321] [Table 24]

[0322]

Table 25

[0323]

Table 26

[0324]

Table 27

[0325]

Table 28

[0326]

Table 29

[0327]

Table 30

[0328]

Table 31

[0329]

Table 32

[0330]

Table 33

[0331]

Table 34

[0332] [Table 35]

[0333] [Table 36]

[0334] [Table 37]

[0335] [Table 38]

[0336] [Table 39]

[0337] Throughout the text of this application, there are discrepancies between the text of the specification (e.g., Table 15) and the Sequence Listing. In cases where there is, the text of this specification takes precedence.

[0338] All patents and publications referred to herein are hereby incorporated by reference in their entirety and for all purposes. and are hereby incorporated by reference.

Claims

1. Hidradenitis Suppurativa (HS) in a subject in need of treatment or prevention of HS The present invention relates to a method for the treatment or prevention of a disease comprising administering to a subject a therapeutically effective amount of an IL-18 and IL-1β specific administering to the subject a bispecific antibody antagonist which specifically binds to and inhibits its activity. and

2. The antibody, a. A first variable light chain (VL1) and a first variable heavy chain (VH) that specifically bind to IL1β 1) and a first constant heavy chain (CH1) with a heterodimerization modification. and a first portion, b. A second variable light chain (VL2) and a second variable heavy chain (VL3) that specifically binds IL-18. H2) and a heterodimerization modification complementary to the heterodimerization modification of the first constant heavy chain. a second portion which is an immunoglobulin with a second constant heavy chain (CH2) with a modification; The method of claim 1 , comprising:

3. A therapeutically effective amount of the compound that specifically binds to IL-18 and IL-1β and inhibits their activity. Slowing or preventing the onset of HS, comprising administering to said subject a specific antibody antagonist. or slowing, arresting or reducing the onset of HS in a subject in need of reduction. How to.

4. The antibody, a. A first variable light chain (VL1) and a first variable heavy chain (VH) that specifically bind to IL1β 1) and a first constant heavy chain (CH1) with a heterodimerization modification. and a first portion, b. A second variable light chain (VL2) and a second variable heavy chain (VL3) that specifically binds IL-18. H2) and a heterodimerization modification complementary to the heterodimerization modification of the first constant heavy chain. a second portion which is an immunoglobulin with a second constant heavy chain (CH2) with a modification; The method of claim 3 , comprising:

5. The first and second constant heavy chains of the bispecific antibody are selected from the group consisting of human IgA, IgD, IgE, IgF, IgG, IgH ... IgG or IgM, preferably IgD, IgE or IgG, e.g. human IgG1, IgG2 5. The method according to claim 2 or 4, wherein the antibody is IgG1, such as IgG2, IgG3 or IgG4. 。

6. wherein the first and second constant heavy chains of the bispecific antibody are IgG1; a. the first constant heavy chain has a point mutation that generates a knob structure, and the second constant heavy chain The strand has a point mutation that creates a hole structure, or b. The first constant heavy chain has a point mutation that generates a hole structure, and the second constant The heavy chain has a point mutation that generates a knob structure, and optionally c. the first and second constant heavy chains have a mutation that creates a disulfide bridge. 、 The method according to claim 2 or 4.

7. a. the first immunoglobulin VH1 domain of the bispecific antibody i. CDR1 has the amino acid sequence SEQ ID NO: 76 and CDR2 has the amino acid sequence SEQ ID NO: hypervariable region CDR1 having the amino acid sequence SEQ ID NO: 77 and CDR3 having the amino acid sequence SEQ ID NO: 78; CDR2 and CDR3; or ii. CDR1 has the amino acid sequence SEQ ID NO:79 and CDR2 has the amino acid sequence SEQ ID NO: Hypervariable region CDR1 having the amino acid sequence SEQ ID NO: 80 and CDR3 having the amino acid sequence SEQ ID NO: 81 , CDR2 and CDR3 Including; b. the first immunoglobulin VL1 domain of the bispecific antibody comprises: iii. CDR1 has the amino acid sequence SEQ ID NO: 92 and CDR2 has the amino acid sequence 93, and CDR3 has the amino acid sequence SEQ ID NO:

94.

1. CDR2 and CDR3 or iv. CDR1 has the amino acid sequence SEQ ID NO:95 and CDR2 has the amino acid sequence Hypervariable region CDR1 having the amino acid sequence SEQ ID NO: 96 and CDR3 having the amino acid sequence SEQ ID NO: 97 , CDR2 and CDR3 Including; c. the second immunoglobulin VH2 domain of the bispecific antibody v. CDR1 has the amino acid sequence SEQ ID NO: 44 and CDR2 has the amino acid sequence SEQ ID NO: hypervariable region CDR1 having the amino acid sequence SEQ ID NO: 45 and CDR3 having the amino acid sequence SEQ ID NO: 46; CDR2 and CDR3; or vi. CDR1 has the amino acid sequence SEQ ID NO: 47 and CDR2 has the amino acid sequence Hypervariable region CDR1 having the amino acid sequence SEQ ID NO: 48 and CDR3 having the amino acid sequence SEQ ID NO: 49 , CDR2 and CDR3 Including, d. the second immunoglobulin VL2 domain of the bispecific antibody comprises: vii. CDR1 has the amino acid sequence SEQ ID NO: 60 and CDR2 has the amino acid sequence and CDR3 has the amino acid sequence SEQ ID NO:

62.

1. CDR2 and CDR3 or viii. CDR1 has the amino acid sequence SEQ ID NO:63 and CDR2 has the amino acid sequence Hypervariable region CD3 has the amino acid sequence SEQ ID NO: 64 and CDR3 has the amino acid sequence SEQ ID NO:

65. R1, CDR2 and CDR3 The method according to any one of claims 1 to 6, comprising:

8. a. the first immunoglobulin VH1 domain of the bispecific antibody has the amino acid sequence comprising SEQ ID NO:85, b. the first immunoglobulin VL1 domain of the bispecific antibody has the amino acid sequence comprising SEQ ID NO: 101, c. the second immunoglobulin VH2 domain of the bispecific antibody has the amino acid sequence comprising SEQ ID NO:53, d. the second immunoglobulin VL2 domain of the bispecific antibody has the amino acid sequence The method of any one of claims 1 to 7, comprising SEQ ID NO:

69.

9. a. the first immunoglobulin heavy chain of the bispecific antibody has the amino acid sequence SEQ ID NO:8 7, b. the first immunoglobulin light chain of the bispecific antibody has the amino acid sequence SEQ ID NO:1 Including 03, c. the second immunoglobulin heavy chain of the bispecific antibody has the amino acid sequence SEQ ID NO:5 Including 5, d. the second immunoglobulin light chain of the bispecific antibody has the amino acid sequence SEQ ID NO:7 The method according to any one of claims 1 to 8, including 1.

10. The route of administration is subcutaneous or intravenous or a combination of subcutaneous and intravenous.

10. The method according to any one of 1 to 9.

11. 1. The method of claim 1, wherein the dosage is from about 1.5 mg to about 15 mg of active ingredient per kilogram of human subject.

11. The method according to any one of claims 1 to 10.

12. 1 to 5, wherein the dosage is about 5 mg or 10 mg of active ingredient per kilogram of human subject.

2. The method according to any one of claims 1 to 11.

13. Dosages of about 150 mg to about 600 mg of active ingredient, such as about 300 mg of active ingredient. The method according to any one of claims 1 to 12, wherein

14. The method according to any one of claims 1 to 13, wherein the antibody is administered via a loading dose and a maintenance dose.

2. The method according to claim 1.

15. The loading dose is administered via subcutaneous injection of a first dose and the maintenance dose is administered via subcutaneous injection of a second dose. The method according to any one of claims 1 to 14, wherein the therapeutic dose is administered via subcutaneous injection in an amount of 0.1 mg / kg.

16. The first dose may contain from about 150 mg to about 600 mg of active ingredient, for example about 300 mg of active ingredient. active ingredient, and the second dose is from about 150 mg to about 600 mg of active ingredient, e.g.

16. The method of claim 15, wherein the amount is about 300 mg of active ingredient.

17. the first dose is 150 mg, 300 mg, or 600 mg of active ingredient; 15 or 16, wherein the dose of 2 is 150 mg, 300 mg or 600 mg of active ingredient.

16. The method according to claim 16.

18. The loading dose comprises at least three biweekly subcutaneous injections on days 1, 15, and 29. and the maintenance administration consists of monthly (Q4W) subcutaneous injections beginning on day 57.

20. The method of claim 17.

19. The patient with hidradenitis suppurativa meets the following criteria: a. the patient has moderate to severe HS; b. the patient is an adult; c. the patient is an adolescent; d. the patient had an HS-PGA score of ≥ 3 prior to treatment with the CD40 antagonist; R; e. the patient has at least three inflammatory lesions prior to treatment with the CD40 antagonist. or f. Prior to treatment with a CD40 antagonist, the patient had extensive scarring as a result of HS. No fistulas (<10 fistulas) The method according to any one of claims 1 to 18, selected according to one of the following:

20. By the 16th week of treatment, the hidradenitis suppurativa patients had the following: a. Simple HiSCR; b. Reduction of HS erythema; c. NRS30; d. A decrease of ≦6 as measured by DLQI; and / or e. Improvement of DLQI The method according to any one of claims 1 to 19, which achieves at least one of the following:

21. By week 16 of treatment, at least 40% of said patients achieve a simple HiSCR; or at least 25% of said patients achieve an NRS30 response; or 15% of said patients 20. The method of any of claims 1-19, wherein less than one subject experiences HS erythema.

22. The patient is administered the bispecific IL-18 and IL-1β antagonist as early as the first administration. If not, after one week, a. Rapid pain relief as measured by VAS or NRS; and b. A rapid decline in CRP as measured using a standard CRP assay 20. The antibody for use according to any one of claims 1 to 19, comprising at least one of the following:

23. Number of inflammatory lesions, Hidradenitis Suppurativa Clinical Response (HiSCR), Numeric Rating Scale (NRS), Modified Sartorius HS score, Hidradenitis Suppurativa - Physician Global Assessment (HS-PGA) or is the Dermatology Life Quality Index (Dermatology Life Quality Index) The patient is at least 3 years of age at the end of treatment as measured by DLQI (Dysfunction Quality Index).

20. The method of any one of claims 1 to 19, wherein a sustained response is achieved after one month.

24. The patient has a high SCR at the end of treatment as measured by simple HiSCR (sHiSCR).

20. The method of any one of claims 1 to 19, wherein a sustained response is achieved three months after administration of the compound.

25. a therapeutically effective amount of a bispecific anti-IL-18 and anti-IL-1β antibody (e.g., bbmAb1); and one or more pharma- ceutically acceptable carriers.

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