CN117396506A

CN117396506A - Bispecific antibodies for use in the treatment of hidradenitis suppurativa

Info

Publication number: CN117396506A
Application number: CN202280036127.1A
Authority: CN
Inventors: S·贝克; M·基夫; J·科瓦里克; C·洛舍; M·罗德里格斯; R·斯坦; 唐宇; F·沃尔德隆-林奇
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2021-06-22
Filing date: 2022-06-20
Publication date: 2024-01-12

Abstract

The present invention relates to a bivalent, bispecific monoclonal antibody (bbmAb) or variant thereof for use in treating or alleviating symptoms of hidradenitis suppurativa in a subject.

Description

Bispecific antibodies for use in the treatment of hidradenitis suppurativa

Technical Field

The present invention relates to a bivalent bispecific monoclonal antibody (bbmAb) or variant thereof for use in treating a patient suffering from hidradenitis suppurativa. The disclosure also relates to methods and treatment regimens for treating hidradenitis suppurativa by employing bispecific antibodies that target both IL-1 beta and IL-18 simultaneously.

Background

Hidradenitis Suppurativa (HS), also known as "abnormal acne" or "maladie de Verneuilh", is a chronic, recurrent and debilitating inflammatory skin condition that is commonly manifested in deep, inflammatory, painful lesions of body parts with apocrine glands. The most common areas of infringement are the axillary, inguinal and anogenital areas (Jemec 2012; fimmel and Zouboulis 2010).

Currently, HS is considered an inflammatory disease of the pilo-sebum with a potential immune system imbalance that occurs in genetically susceptible individuals (Kelly et al 2014). While HS is considered to be a disease primarily caused by hair follicle blockage, HS is an inflammatory skin disease characterized by the presence of large numbers of neutrophils and macrophages in inflammatory lesions (Lima et al 2016, shah et al 2017). Although the pathophysiology of HS is still unknown, the benefits of tumor necrosis factor alpha (tnfa) blocking have been described in greater studies (kimdall et al 2016).

anti-IL 1 therapy (Tzannetakou et al 2016) and blocking IL-17A (Thorlaius et al 2017, schuch et al 2018, giuseppe et al 2018,2018 et al) or anti-IL-23 therapy (Sharon et al 2012, blok et al 2016) was also observed in smaller studies and/or case reports. Recently, research methods using the oral PDE4 inhibitor apremilast (Weber et al 2017) or anti-complement 5a compounds (Kanni et al 2018) have been described.

The disease begins after puberty and women are more common than men (3:1). Risk factors include obesity and smoking. Although epidemiological prevalence estimates vary widely (0.03% -4.3%; jemec 2012, jemec and Kimball 2015) and there are geographic differences, prevalence generally accepted by the scientific community is about 0.1% -1% (Garg et al 2018).

The clinical manifestations of HS are heterogeneous, but the disease tends to manifest as chronic recurrent, deep, painful, inflammatory skin lesions, mainly inflammatory nodules and abscesses, leading to possible drainage and suppuration. During disease progression, the formation and fistulation of the sinus tract complicates inflammatory lesions and may lead to hypertrophic scarring that can have an impact on functional use.

HS is associated with pain, malodorous secretions and scarring on wounds, and does frequently have damaging psychological effects. HS is a severely debilitating disease that has a significant negative impact on quality of life (QoL) and several studies have demonstrated that its impact is greater than that seen with other skin disorders (Deckers and kimdall 2016). HS patients also often suffer from depression, social isolation, impaired sexual health, and may have difficulty fulfilling their work duties (espann and Jemec 2011, fimmel and Zouboulis 2010, janse et al 2017).

HS is difficult to treat. European official guidelines for treatment were established only in 2015 and advice should be provided to patients for adjuvant, pharmaceutical and surgical therapies (Zouboulis et al 2015).

Although topical antibiotics may be used in mild cases, moderate to severe HS are preferably treated with multiple systemic antibiotics over a long period (typically with tetracycline or a combination of clindamycin and rifampin) and may be subsequently maintained for months or even years by chronic antibiotic treatment (Bettoli et al 2016, desiniti et al 2016, zouboulis et al 2015).

However, it is well known that HS is a chronic inflammatory disease, not an infectious disease (Jemec 2012). Thus, anti-inflammatory agents are an alternative and potentially more appropriate approach than antibiotics, or may be supplements to antibiotics. Over time, the result of chronic, recurrent, inadequately treated inflammation is irreversible fibrosis, manifested by scarring and channels or sinus passages that are generally unresponsive to drug therapy. Once a persistent anatomical change occurs, the only therapeutic option to reduce the fibrotic tissue volume and improve the function of the affected skin area is surgery (Andersen and Jemec 2017). One of the future therapeutic goals should be to reduce persistent scarring and avoid surgery, which may be achieved by preventing inflammatory lesions, or may require specific treatment methods.

In 2015, adalimumab was a recombinant human monoclonal immunoglobulin G1 (IgG 1) antibody directed against soluble and membrane-bound TNF- αRegulatory approval for treatment of moderate to severe HS was obtained. Efficacy of adalimumab has been observed with a response rate of hissc (clinical response to hidradenitis suppurativa) exceeding 16% (41.8% adalimumab to 26% placebo) and 31% (58.9% adalimumab to 27.6% placebo) of placebo according to that reported in the PIONEER I and II studies, respectively (Kimball et al 2016). As captured in adalimumab tags, adalimumab is associated with an increased risk of safety for severe infections including tuberculosis, invasive fungal infections and other opportunistic infections. The use of adalimumab also reports an increased incidence of malignancy.

Thus, there is an unmet need for systemic therapies that are effective in reducing inflammation while having beneficial safety for patients with moderate to severe HS.

Disclosure of Invention

In HS, both cytokines IL-1 beta and IL-18 are upregulated (Kelly et al 2015) and thus may play a role in the pathogenesis of HS. IL-1β characteristics are present in HS lesions and can be reversed by the application of IL-1 receptor antagonists (Witte-Handel et al 2019). The recombinant IL-1R antagonist anakinra showed promising clinical efficacy results in small studies relative to placebo (Tzannetakou et al 2016), while case reports confirmed these findings (Leslie et al 2014, zarchi et al 2013, andre et al 2019). Case reports indicate that anti-IL-1 β antibody kana Ji Nushan anti-blocking IL-1 β alone may be beneficial for moderate to severe HS (Houriet et al 2017) or related syndromes such as PASH (pyoderma gangrenosum, acne and hidradenitis suppurativa (Jaeger et al 2013)). However, some cases of failure in HS were observed with kana Ji Nushan antibody (Sun et al 2017, tekin et al 2017) and anakinra (van der Zee and preds 2013, russo and Alikhan 2016), which may suggest the following facts: only a subset may respond to anti-IL-1 blockade, or such blockade alone may be insufficient to achieve results in most patients.

Accordingly, it is an object of the present disclosure to target the inflammatory somatic effector cytokines IL-1 beta and IL-18, which may therefore provide excellent clinical efficacy in (self) inflammatory disorders or in cases where both IL-1 beta and IL-18 independently contribute to the pathophysiology of the disease (such as HS). It is a further object of the present disclosure that dual specific anti-IL-1 beta/18 antagonists can rapidly neutralize both the inflammatory body-dependent and inflammatory body-independent sources of IL-1 beta and IL-18.

Thus, any antagonist capable of inhibiting both IL-1β and IL-18 (e.g., a bispecific anti-IL-1β/18 antibody or fragment thereof) is suitable for use in the treatment of HS.

Bispecific antibodies or functional fragments thereof that target both IL-1 beta and IL-18 simultaneously for use in preventing or treating Hidradenitis Suppurativa (HS) in a subject are described herein. In a preferred embodiment, the anti-IL-1 β/18 antibody comprises a heavy chain CH3 mutation that silences ADCC activity, such as a so-called LALA mutant comprising L234A and L235A mutations in the lgG1 Fc amino acid sequence. In preferred embodiments, the anti-IL-1β/18 antibody comprises a complementary heavy chain CH3 knob mutation, e.g., a first heavy chain having S354C and T366W knob mutations (according to EU numbering), and a second heavy chain having Y349C, T366S, L368A, Y407V knob mutation. In preferred embodiments, the anti-IL-1 β/18 antibody comprises a first light chain that preferentially associates with the first heavy chain and comprises a kappa light chain (e.g., vk 6) and a second light chain that preferentially associates with the second heavy chain and comprises a lambda light chain (e.g., vλ1). In yet more preferred embodiments, the anti-IL-1β/18 antibody comprises a combination of: i) One or more ADCC silent mutations (e.g., LALA), ii) one or more knob and socket heavy chain modifications, iii) and/or kappa and lambda light chains. In still more preferred embodiments, the anti-IL-1β/18 antibody comprises all of the aforementioned features i) -iii), preferably wherein the antibody comprises a Vk6 and a V.lamda.1 light chain.

Also described herein are methods of preventing or treating Hidradenitis Suppurativa (HS) by administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody that simultaneously targets both IL-1 beta and IL-18.

Further provided herein are specific dosing regimens for the methods or uses of bispecific antibodies (e.g., bbmAb 1) described herein that simultaneously target both IL-1 beta and IL-18.

Further described herein are pharmaceutical combinations and compositions comprising a) a bispecific antibody that simultaneously targets both IL-1 beta and IL-18 (e.g., bbmAb 1), and b) at least one additional therapeutic agent, optionally in the presence of a pharmaceutically acceptable carrier, for use in the treatment or prevention of HS. Additional features and advantages of the methods and uses will become apparent from the detailed description that follows.

In a first aspect, the present disclosure relates to a method for treating or preventing HS in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody, wherein the antibody comprises

a. A first moiety which is an immunoglobulin having a first variable light chain (VL 1) and a first variable heavy chain (VH 1) and a first constant heavy chain (CH 1) with a heterodimerization modification, the VH1 specifically binding to IL-1 beta, and

b. A second moiety that is an immunoglobulin having a second variable light chain (VL 2) and a second variable heavy chain (VH 2), the VH2 specifically binding to IL-18, and a second constant heavy chain (CH 2) with a heterodimerization modification complementary to the heterodimerization modification of the first constant heavy chain.

In a second aspect, the disclosure relates to a method for slowing, arresting, or reducing the development of HS in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody, wherein the antibody comprises

In a particular embodiment of the first and second aspects of the disclosure, the first and second constant heavy chains of the bispecific antibody are human IgA, igD, igE, igG or IgM, preferably IgD, igE or IgG, such as human IgG1, igG2, igG3 or IgG4, preferably IgG1.

In another embodiment of the disclosure, the first and second constant heavy chains of the bispecific antibody are IgG1, and

a. the first constant heavy chain has a point mutation that produces a pestle structure, and the second constant heavy chain has a point mutation that produces a mortar structure, or

b. The first constant heavy chain has a point mutation that produces a mortar structure and the second constant heavy chain has a point mutation that produces a pestle structure, and optionally

c. The first and second constant heavy chains have mutations that result in disulfide bridges.

In a particularly preferred embodiment of the disclosure comprising the first and second aspects, the first immunoglobulin VH1 domain of the bispecific antibody comprises:

i. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO 76, said CDR2 having the amino acid sequence SEQ ID NO 77 and said CDR3 having the amino acid sequence SEQ ID NO 78; or alternatively

Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having 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. 81; and

the first immunoglobulin VL1 domain of the bispecific antibody comprises:

hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence of SEQ ID NO:92, said CDR2 having the amino acid sequence of SEQ ID NO:93 and said CDR3 having the amino acid sequence of SEQ ID NO:94 or

Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID No. 95, said CDR2 having the amino acid sequence SEQ ID No. 96 and said CDR3 having the amino acid sequence SEQ ID No. 97; and

the second immunoglobulin VH2 domain of the bispecific antibody comprises:

v. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO 44, said CDR2 having the amino acid sequence SEQ ID NO 45 and said CDR3 having the amino acid sequence SEQ ID NO 46; or alternatively

Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID No. 47, said CDR2 having the amino acid sequence SEQ ID No. 48 and said CDR3 having the amino acid sequence SEQ ID No. 49; and the second immunoglobulin VL2 domain of the bispecific antibody comprises:

hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:60, said CDR2 having the amino acid sequence SEQ ID NO:61 and said CDR3 having the amino acid sequence SEQ ID NO:62 or

The hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:63, said CDR2 having the amino acid sequence SEQ ID NO:64 and said CDR3 having the amino acid sequence SEQ ID NO:65.

In another preferred embodiment of the present disclosure, the antibodies used in the methods, compositions and uses described herein comprise:

a. a first immunoglobulin VH1 domain of amino acid sequence SEQ ID NO. 85,

b. a first immunoglobulin VL1 domain of amino acid sequence SEQ ID NO. 101,

c. a second immunoglobulin VH2 domain of amino acid sequence SEQ ID NO. 53

d. A second immunoglobulin VL2 domain of amino acid sequence SEQ ID NO. 69.

In another preferred embodiment of the present disclosure, an antibody for use in a method, composition or use described herein comprises:

e. a first immunoglobulin heavy chain of amino acid sequence SEQ ID NO. 87,

f. a first immunoglobulin light chain of amino acid sequence SEQ ID NO. 103,

g. a second immunoglobulin heavy chain of amino acid sequence SEQ ID NO. 55

h. A second immunoglobulin light chain of amino acid sequence SEQ ID NO. 71.

In particularly preferred embodiments of the present disclosure, the subject being treated suffers from HS.

In a third aspect, the disclosure relates to a bispecific antibody comprising

b. A second moiety which is an immunoglobulin having a second variable light chain (VL 2) and a second variable heavy chain (VH 2), the VH2 specifically binding to IL-18 for use in treating or preventing HS in a subject in need thereof, and a second constant heavy chain (CH 2) with a heterodimerization modification complementary to the heterodimerization modification of the first constant heavy chain.

In a fourth aspect, the disclosure relates to the methods and treatments of the first, second, and third aspects, wherein about 1mg/kg to about 35mg/kg of a bispecific antibody that simultaneously targets both IL-1 β and IL-18 is administered to the subject. In a preferred embodiment of the fourth aspect, about 10mg/kg of bispecific antibody is administered to the treated subject.

In one aspect of the disclosure, bispecific antibodies that target both IL-1 beta and IL-18 are administered to the subject intravenously or subcutaneously. In one embodiment, the route of administration is a subcutaneous or intravenous combination, such as an intravenous loading dose followed by a subcutaneous maintenance dose.

In embodiments, bispecific antibodies that target both IL-1 beta and IL-18 are administered intravenously, e.g., to a treated subject at a dose of about 1.5mg to about 15mg active ingredient per kilogram of human subject. In embodiments, bispecific antibodies that target both IL-1β and IL-18 are administered intravenously, e.g., to a treated subject at a dose of about 2, 4, or 5mg active ingredient per kilogram of human subject. In embodiments, bispecific antibodies that target both IL-1 beta and IL-18 are administered to a treated subject, e.g., intravenously, at a dose of about 4mg active ingredient per kilogram of human subject. The dose of active ingredient per kilogram of human subject, for example, between about 1.5 and about 15mg, may be administered weekly, biweekly, or weekly, or combinations thereof. In preferred embodiments, a dose of the active ingredient per kilogram of human subject, for example, between about 1.5 to about 15mg, may be administered every two weeks or every four weeks, or a combination thereof.

In one embodiment, the dosage is about 75mg to about 600mg of the active ingredient, preferably about 150mg to about 300mg of the active ingredient, or about 150mg or 300mg of the active ingredient, preferably 300mg of the active ingredient. The dose may be administered weekly, biweekly, or weekly, or a combination thereof. In preferred embodiments, a fixed dose (e.g., a non-weight based dose) is administered subcutaneously or intramuscularly (preferably subcutaneously).

In one embodiment, the dose is about 300mg of active ingredient.

In a preferred embodiment, the dose is 150mg of active ingredient. In another preferred embodiment, the dose is 300mg of active ingredient. In yet another preferred embodiment, the dose is 600mg of active ingredient. In yet another embodiment, the dosage is from about 150mg of active ingredient to about 600mg of active ingredient.

In one embodiment, the antibody is administered by loading and maintenance administration. In one embodiment, the loading dose is administered via one or more intravenous injections of a first dose and the maintenance dose is administered via subcutaneous injections of a second dose. In one embodiment, the loading dose is administered via a subcutaneous injection of a first dose and the maintenance dose is administered via a subcutaneous injection of a second dose.

In one embodiment, the loading dose is administered via a subcutaneous injection first dose regimen and the maintenance dose is administered via a subcutaneous injection second dose regimen. The amount of the first dose regimen may be the same as or higher than the amount of the second dose regimen. The first dosage regimen period may be the same as or more frequent than the second dosage regimen period.

In one embodiment, the first dose is between about 150mg and about 600mg of the active ingredient, such as about 300mg of the active ingredient, and the second dose is between about 150mg and about 600mg of the active ingredient, such as about 300mg of the active ingredient.

In one embodiment, the first dose is 150mg, 300mg or 600mg of active ingredient and the second dose is 150mg, 300mg or 600mg of active ingredient. In one embodiment, the first dose is 300mg of active ingredient and the second dose is 300mg of active ingredient.

In one embodiment, loading administration comprises at least two administrations and maintenance administration consists of weekly (Q1W), preferably biweekly (Q2W), or monthly (Q4W) administrations. In one embodiment, the loading dose consists of two administrations. In another embodiment, the loading dose consists of three administrations. In another embodiment, the loading dose consists of four administrations. In some cases, the loading dose is a once-weekly loading dose consisting of 3 doses from day 1 to day 29.

In one embodiment, the loading administration comprises at least two subcutaneous injections, preferably three subcutaneous injections, and the maintenance administration consists of once weekly (Q1W), once every two weeks (Q2W), or preferably once monthly (Q4W) subcutaneous injections. In one embodiment, the loading dose consists of two subcutaneous injections. In another embodiment, the loading administration consists of three subcutaneous injections, for example, 3 subcutaneous injections of 150mg or 300mg once every two weeks (Q2W). In another embodiment, the loading dose consists of four subcutaneous injections. In some cases, the loading dose is a once-weekly loading dose consisting of 3 subcutaneous injections from day 1 to day 29. In some cases, the maintenance dosing is a once monthly (Q4W) maintenance dose comprising a subcutaneous Q4W injection dose from day 57, e.g., after three subcutaneous injections of 150mg or 300mg once every two weeks (Q2W). In some cases, loading doses are once every two weeks consisting of 3 subcutaneous injections on days 1, 15, and 29, and maintenance doses are once monthly (Q4W) maintenance doses comprising subcutaneous Q4W injections starting on day 57. In some cases, the loading dose is a once every two weeks (e.g., 150mg or 300 mg) loading dose consisting of 3 subcutaneous injections doses on days 1, 15, and 29, and the maintenance dose is a once monthly (Q4W) (e.g., 150mg or 300 mg) maintenance dose comprising a subcutaneous Q4W injection dose from day 57.

In one embodiment, the loading dose is a weekly loading dose consisting of 3 doses from day 1 to day 29, wherein the amount of the dose is from about 1.5mg to about 15mg per kilogram of active ingredient, e.g., wherein the loading dose is administered intravenously, subcutaneously, or in a combination of intravenous or subcutaneous. In one embodiment, the loading dose is a weekly loading dose consisting of 3 doses from day 1 to day 29, wherein the amount of the dose is about 4 mg/kg of active ingredient, e.g., wherein the loading dose is administered intravenously, subcutaneously, or in a combination of intravenous or subcutaneous.

In one embodiment, the loading dose is a weekly loading dose consisting of 3 doses from day 1 to day 29, wherein the amount of the dose is about 150mg or about 300mg of active ingredient, preferably 300mg of active ingredient, e.g., wherein the loading dose is administered subcutaneously, or in intravenous or subcutaneous combination. In one embodiment, the loading dose is a once-every-two-week loading dose consisting of 3 doses from day 1 to day 29, wherein the amount of the dose is about 300mg of the active ingredient, wherein the loading dose is administered subcutaneously.

In some embodiments, the loading dose is a dose selected or predicted to achieve a plasma concentration of at least about 20 μg/mL to about 60 μg/mL during the loading dose period (e.g., within 1, 2, or 3 weeks or after 1, 2, or 3 loading dose injections). In some embodiments, the maintenance dose is a dose selected or predicted to provide a sustained plasma concentration of from about 20 μg/mL to about 60 μg/mL during the maintenance dosing period or a substantial portion of the maintenance dosing period (e.g., from at least about day 29 to about day 85).

In one embodiment, the multiple subcutaneous injections of the loading dose have different doses. In another embodiment, the multiple subcutaneous injections of the loading dose have the same dose.

The HS patient may be selected according to one of the following criteria:

the patient suffers from moderate to severe HS;

the patient is an adult;

the patient is adolescent;

the patient has an HS-PGA score of > 3 prior to treatment with IL-1 beta and IL-18 antagonists (e.g., bispecific antibodies that target both IL-1 beta and IL-18);

the patient has at least 3 inflammatory lesions prior to treatment with an IL-1 beta and an IL-18 antagonist (e.g., a bispecific antibody that targets both IL-1 beta and IL-18); or alternatively

The patient was free of extensive scarring (< 10 fistulae) caused by HS prior to treatment with IL-1 beta and IL-18 antagonists (e.g., bispecific antibodies that target both IL-1 beta and IL-18).

In one embodiment, by week 16 of treatment, the HS patient achieves at least one of:

simplified hissc;

reduction of HS reddening and swelling;

NRS30；

a decrease of 6 or less as measured by DLQI; and/or

Improvement of DLQI.

In one embodiment, at least 40% of the patients achieve simplified hissc by week 16 of treatment; or at least 25% of the patients achieve NRS30 responses; or less than 15% of the patients experience HS red bumps.

In one embodiment, the patient has at least one of the following at least one week after the first dose of the IL-1 β and IL-18 antagonist:

pain measured by VAS or NRS is rapidly reduced, and

CRP measured using the standard hsCRP assay decreased rapidly.

In one embodiment, the patient achieves a sustained response 3 months after the end of treatment, as measured by inflammatory lesions count, HS clinical response (hissc), numerical Rating Scale (NRS), modified sartorius HS score, hidradenitis suppurativa-physician global assessment (HS-PGA), or dermatological quality of life index (DLQI).

In one embodiment, the patient achieves a sustained response 3 months after the end of treatment, as measured by simplified hissc (shissc).

According to a second aspect, there is provided a method of treating HS in a human subject, the method comprising administering to the subject a therapeutically effective dose of IL-1 β and an IL-18 antagonist.

In another embodiment of the disclosure, bispecific antibodies targeting both IL-1 beta and IL-18 are administered in combination with at least one additional therapeutic agent.

In another aspect, the disclosure relates to the use of a bispecific antibody (e.g., bbmAb 1) that targets both IL-1 beta and IL-18 for the manufacture of a medicament for slowing, arresting, or reducing the development of HS in a subject.

In one embodiment, provided herein is a method of treating HS in a subject in need thereof, the method comprising administering an effective amount of a bispecific antibody that targets both IL-1β and IL-18, such as bbmAb1. In one embodiment, provided herein are bispecific antibodies, such as bbmAb1, that target both IL-1 beta and IL-18 for use in treating HS in a subject in need thereof. In some embodiments, provided herein is the use of a bispecific antibody (e.g., bbmAb 1) that targets both IL-1 beta and IL-18 for the manufacture of a medicament for the treatment of HS.

In one embodiment, provided herein is a method of treating, slowing, arresting, or reducing the development of HS in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody that targets both IL-1 beta and IL-18, such as 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 comprises the amino acid sequence SEQ ID NO. 85,

b. the first immunoglobulin VL1 domain of the bispecific antibody comprises the amino acid sequence SEQ ID NO. 101,

c. The second immunoglobulin VH2 domain of the bispecific antibody comprises the amino acid sequence SEQ ID NO. 53, an

d. The second immunoglobulin VL2 domain of the bispecific antibody comprises the amino acid sequence SEQ ID NO. 69.

According to another aspect, the present disclosure provides a liquid pharmaceutical composition comprising a bispecific antibody that specifically targets both IL-18 and IL-1β, wherein the liquid pharmaceutical formulation comprises a buffer, a stabilizer, and a solubilizing agent, a method or use of the liquid pharmaceutical composition. In one embodiment, the present disclosure provides or further provides means for administering or subcutaneously administering bispecific antibodies to patients with HS. In one embodiment, the use is for the manufacture of a medicament for the treatment of HS.

In a particularly preferred embodiment of the present disclosure, the first immunoglobulin VH1 domain of the bispecific antibody of the liquid pharmaceutical composition comprises:

The first immunoglobulin VL1 domain of the bispecific antibody comprises:

the second immunoglobulin VH2 domain of the bispecific antibody comprises:

In another preferred embodiment of the present disclosure, an antibody of a liquid pharmaceutical composition described herein comprises:

a. a first immunoglobulin VH1 domain of amino acid sequence SEQ ID NO. 85,

b. a first immunoglobulin VL1 domain of amino acid sequence SEQ ID NO. 101,

c. a second immunoglobulin VH2 domain of amino acid sequence SEQ ID NO. 53

d. A second immunoglobulin VL2 domain of amino acid sequence SEQ ID NO. 69.

e. a first immunoglobulin heavy chain of amino acid sequence SEQ ID NO. 87,

f. a first immunoglobulin light chain of amino acid sequence SEQ ID NO. 103,

g. a second immunoglobulin heavy chain of amino acid sequence SEQ ID NO. 55

h. A second immunoglobulin light chain of amino acid sequence SEQ ID NO. 71.

In some embodiments, the liquid pharmaceutical composition comprises about 50mg/mL to about 120mg/mL of a bispecific antibody (e.g., bbmAb 1), a buffer, a stabilizer (e.g., a nonionic stabilizer such as a sugar), and a surfactant (e.g., polysorbate 20 or polysorbate 80). In some embodiments, the liquid pharmaceutical composition comprises about 50mg/mL to about 120mg/mL of the bispecific antibody in a buffer at a pH of about 5.5 to about 6.5, preferably at a pH of about 5.5 to about 6.0, preferably at a pH of about 6.0.

In some embodiments, the liquid pharmaceutical composition comprises about 100mg/mL of a bispecific antibody (e.g., bbmAb 1), a buffer, a stabilizer (e.g., a nonionic stabilizer such as a sugar), and a surfactant (e.g., polysorbate 20 or polysorbate 80). In some embodiments, the liquid pharmaceutical composition comprises about 50mg/mL to about 120mg/mL of the bispecific antibody (e.g., bbmAb 1), from about 1mM to about 50mM histidine/histidine chloride, from about 50mM to about 400mM sugar (e.g., sucrose, trehalose, or mannitol), from about 0.01% to about 0.5% of a surfactant (e.g., polysorbate, such as polysorbate 20), at a pH of from about 5.5 to about 6.5, preferably from about 5.5 to about 6.0, more preferably about 6.0. In some cases, the liquid pharmaceutical composition comprises about 50mg/mL to about 120mg/mL of bbmAb1 (preferably about 100mg/mL bbmAb 1), about 20mM histidine/histidine chloride, about 220mM sucrose, about 0.04% polysorbate 20 (w/v), and a pH of about 6.0.

Drawings

FIG. 1 is a graphical illustration of the mRNA levels of AIM2, NLRC4, NLRP7 and NLRP3 inflammasome in HS lesions compared to the levels of non-diseased or healthy tissue.

Fig. 2 is the following TIBCO spotfire heat map representation: average expression levels of genes/members or CRS signaling characteristics of IL-18, IL-12 and IL-1β (alone or in combination) in biopsies of healthy skin or HS patients, peri-lesions and diseased skin. Identification of genes/members of the corresponding IL-18, IL-12 and IL-1β signaling characteristics was performed in separate experiments using PBMCs from healthy donors stimulated for 6 hours (limited to the gene induced by the corresponding cytokine = UP), or from publications (kana Ji Nushan anti-response characteristics CRS; brachat et al 2017).

FIG. 3 shows the effect of incubation of bbmAb1 (100. Mu.g/ml) and adalimumab (100. Mu.g/ml) on the levels of analytes IFNγ, TNFα, IL-1. Beta. And IL-2 produced after 24 hours of culture of supernatants of HS skin biopsies in vitro, compared to untreated controls. Each point represents a single biopsy. The leftmost column for each analyte is untreated control, the middle column for each analyte is bbmAb1, and the rightmost column for each analyte is adalimumab. The following values obtained from 9 different HS patients are shown: single biopsies for ifnγ, IL-1β and tnfα, n=54, n=36 and n=26; and biopsies for IL-2, n=43, n=20, and n=10.

FIG. 4 is a graphical illustration of predicting the effect of bbmAb1 administration on IL-1β levels. The dashed line is the specified lower limit of the amount. The predictions were modeled assuming a 300mg subcutaneous dose on days 1, 15, 29, 57 and 85.

Figure 5 is a graphical illustration of the effect of predicting bbmAb1 dosing on IL-18 levels relative to baseline changes in healthy control subjects. The dashed line refers to the level of healthy control subjects. The predictions were modeled assuming a 300mg subcutaneous dose on days 1, 15, 29, 57 and 85.

Figure 6 is a graphical illustration of the predicted pharmacokinetics of subcutaneously administered bbmAb1 compared to a single dose of 10mg/kg i.v.. The predictions were modeled assuming a 300mg subcutaneous dose on days 1, 15, 29, 57 and 85.

Detailed Description

Described herein are IgG-like bispecific monoclonal antibodies (e.g., bbmAb 1) that contain a heterodimeric Fc portion that simultaneously neutralizes the key inflammatory body effector cytokines interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18). In certain aspects, bispecific antibodies have picomolar (pM) affinity for both cytokines and inhibit IL-1β and IL-18 signaling at sub-nanomolar (nM) concentrations in an in vitro cell assay. IL-1β and IL-18 are two pro-inflammatory cytokines that are secreted after activation of the inflammasome in response to recognition of both damage-associated molecular patterns (DAMP) and pathogen-associated molecular patterns (PAMP). As described herein, combined inhibition of IL-1 beta and IL-18 may result in more effective down-regulation of the inflammatory body-driven pro-inflammatory response than inhibition of IL-1 beta or IL-18 cytokines alone.

Thus, any antagonist capable of blocking both IL-1β and IL-18 activity (e.g., bispecific anti-IL-1β and anti-IL-18 antibodies with silent ADCC activity) is suitable for use in the treatment of HS.

Without wishing to be bound by theory, the inventors have identified that in conditions where IL-1 β and/or IL-18 levels are elevated, higher doses or more frequent regimens are required at the beginning of treatment, whereas loading regimens may be beneficial at least partially or fully saturating the bispecific antibody binding sites (IL-1 β and/or IL-18) in these patients at the beginning of treatment. Thus, the loading regimen provides rapid saturation of antigen at the beginning of treatment (2 to 3 weeks), and then the maintenance regimen provides sustained therapeutic plasma concentrations throughout the treatment, taking into account the therapeutic effects in cases where IL-1 β and/or IL-18 expression in the affected tissue is enhanced (severity of the disorder).

The appropriate dosage will vary according to: such as the particular bispecific IL-1 beta and IL-18 antagonist to be employed (e.g., bispecific anti-IL-1 beta and anti-IL-18 antibodies or antigen-binding fragments thereof (e.g., bbmAb 1)), the subject being treated, the mode of administration, and the nature and severity of the condition being treated, as well as the nature of the previous treatment that the patient has undergone. Ultimately, the primary healthcare provider will decide the amount of dual specific IL-1 beta and IL-18 antagonists to be used to treat each individual patient. In some embodiments, the attending healthcare provider may administer low or even single doses of dual specific IL-1 beta and IL-18 antagonists and observe the patient's response. In other embodiments, the patient is administered one or more initial doses of the dual specific IL-1 beta and IL-18 antagonist in high amounts and then titrated down until signs of relapse occur. Larger doses of the bispecific IL-1 beta and IL-18 antagonist may be administered until the optimal therapeutic effect for the patient is obtained, and the dose is typically not increased further.

In one embodiment, the disclosure relates to bispecific anti-IL-1 beta and anti-IL-18 antibodies or antigen-binding fragments thereof for use according to any aspect or embodiment of the disclosure as described above, wherein the loading dose and maintenance dose of the bispecific anti-IL-1 beta and anti-IL-18 antibodies or antigen-binding fragments thereof (e.g., bbmAb 1) are adjusted such that the plasma or serum concentration of the antibodies is at therapeutic levels.

In practicing some therapeutic methods or uses of the present disclosure, a therapeutically effective amount of a bispecific IL-1β and an IL-18 antagonist, e.g., a bispecific anti-IL-1β and an anti-IL-18 antibody or antigen-binding fragment thereof (e.g., bbmAb 1), is administered to a patient (e.g., a mammal (e.g., a human)). While it is understood that the disclosed methods provide for the treatment of HS patients with dual specificity IL-1 beta and IL-18 antagonists (e.g., bbmAb 1), this does not preclude such treatment from being monotherapy if the patient is to be ultimately treated with the antagonist. Indeed, if a patient is selected for dual specific IL-1 β and IL-18 antagonist treatment, that antagonist (e.g., bbmAb 1) may be administered alone or in combination with other agents and therapies according to the methods of the disclosure.

It will be appreciated that the protocol changes may be applicable to certain HS patients, for example patients with inadequate therapeutic response to treatment with bispecific IL-1 beta and IL-18 antagonists, such as bispecific antibodies or antigen binding fragments thereof (e.g., bbmAb 1). Thus, administration may be more frequent than once a month, for example once every two weeks (every two weeks) or once a week.

Some patients may benefit from a loading regimen (e.g., once weekly administration for several weeks [ e.g., 1 to 4 weeks, e.g., at week 0, week 1, week 2, and/or week 3, such as week 3, at week 1, week 2, and week 3 loading dosing regimen ]) followed by a maintenance regimen, e.g., beginning at week 5, week 6, or week 7, wherein the bispecific antibody (e.g., bbmAb 1) may be administered once weekly, once biweekly, or preferably every 4 weeks for at least several weeks.

For example, a suitable regimen for bbmAb1 may be once every two weeks for several weeks [ e.g., 1 to 5 weeks, e.g., dosing at week 1, week 3, and week 5 ], followed by a once monthly Q4W maintenance regimen, e.g., at week 8 or week 9, preferably week 9, wherein the bispecific antibody (e.g., bbmAb 1) may be administered for at least several weeks.

It should also be appreciated that administration may be less frequent than once a month, e.g., every 6 weeks, every 8 weeks (every two months), every quarter (every three months), etc.

It will be appreciated that, based on the severity of the disease, the dose escalation may be applicable to certain HS patients, for example, patients with inadequate therapeutic response to treatment with the bispecific antagonists described herein (e.g., bbmAb1 or antigen-binding fragment thereof). Thus, the subcutaneous (s.c.) dose (loading dose or maintenance dose) may be greater than about 150mg to about 900mg s.c., for example about 75mg, about 100mg, about 125mg, about 175mg, about 200mg, about 250mg, about 350mg, about 400mg, about 450mg, about 500mg, about 600mg, etc.; similarly, intravenous (i.v.) doses may be greater than about 5mg/kg or 10mg/kg, for example about 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 11mg/kg, 12mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 35mg/kg, etc. It will also be appreciated that dose reduction may also be applicable to certain HS patients, for example, patients exhibiting adverse events or adverse responses to treatment with a bispecific antagonist (e.g., bbmAb1 or antigen-binding fragment thereof). Thus, the dose of the antagonist may be less than about 150mg to about 900mg s.c., for example, about 25mg, about 50mg, about 75mg, about 100mg, about 125mg, about 175mg, about 200mg, about 250mg, about 350mg, about 400mg, about 450mg, about 500mg, about 600mg, etc.

In some embodiments, a bispecific antagonist (e.g., bbmAb1 or antigen-binding fragment thereof) can be administered to a patient at an initial dose of 300mg delivered s.c., and then, if desired, the dose can be adjusted to 150mg or 600mg delivered s.c., as determined by a physician.

The bispecific antibody or antigen-binding fragment thereof may be bbmAb1, a functional derivative thereof, or a biomimetic thereof.

As defined herein, a "unit dose" refers to an s.c. dose, which may be comprised between about 75mg to 900mg, such as about 150mg to about 600mg, such as about 300mg to about 600mg, or such as about 150mg to about 300mg. For example, the unit s.c. dose is about 75mg, about 150mg, about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 550mg, about 600mg.

The present disclosure is based on, inter alia, the following unexpected findings: certain antibodies that neutralize both IL-1 beta and IL-18 more strongly attenuate IFN-gamma (and other pro-inflammatory cytokines) production than IL-1 beta or IL-18 neutralization alone, which the inventors consider to be an effective treatment for HS.

1. Definition of the definition

For the purposes of explaining the present specification, the following definitions will apply, and where appropriate, terms used in the singular also include the plural, and vice versa. Additional definitions are set forth throughout the detailed description.

The term "about" in relation to the value x means, for example, +/-10%. The term "about" when used in front of a range of values or a list of numbers applies to each number in the series, e.g., the phrase "about 1-5" should be interpreted as "about 1-about 5", or, e.g., the phrase "about 1, 2, 3, 4" should be interpreted as "about 1, about 2, about 3, about 4, etc.

The word "substantially" does not exclude "complete", e.g., a composition that is "substantially free" of Y may be completely free of Y. The word "substantially" may be omitted from the definition of the present disclosure, if desired.

The term "comprising" encompasses "including" as well as "consisting of … …", e.g., a composition "comprising" X may consist of X alone or may include other substances, such as x+y.

The term "IL-18" is an IL-18 polypeptide, interleukin-18 polypeptideSynonyms for peptide, IFN-gamma or interferon-gamma or INF-gamma inducers. The term "IL-18" refers to human IL-18 unless otherwise indicated. IL-18 is well known to those skilled in the art and can be derived, for example, fromInternational company (/ -)>International Corporation) under product number #B001-5. Throughout the specification, the term IL-18 interchangeably encompasses pre-IL-18 (a precursor of mature IL-18 prior to cleavage by a protease) and mature IL-18 (after cleavage by a protease), unless specifically stated to mean a pre-or mature form. / >

The term "IL-1β" or "IL-1b" is synonymous with IL-1β polypeptide and interleukin-1β polypeptide. The term "IL-1β" refers to human IL-1β unless otherwise indicated. IL-1β is well known to those skilled in the art and is available, for example, from Yinqiao China (Sino Biological) under product number # 10139-HNAE-5.

The term "antibody" refers to an intact immunoglobulin or a functional fragment thereof. Naturally occurring antibodies typically comprise tetramers, which are generally composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (typically consisting of three domains (CH 1, CH2 and CH 3). The heavy chain may be of any isotype including IgG (IgG 1, igG2, igG3 and IgG4 subtypes), igA (IgA 1 and IgA2 subtypes), igM and IgE. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Light chains include kappa (kappa) chains and lambda (lambda) chains. The heavy and light chain variable regions are generally responsible for antigen recognition, while the heavy and light chain constant regions may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). VH and VL regions can be further subdivided into regions of higher variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

As used herein, the term "antigen-binding portion" (or simply "antigenic portion") of an antibody refers to a full-length antibody or one or more fragments of an antibody that retain the ability to specifically bind to IL-18 or IL-1 β antigen. It has been shown that fragments of full length antibodies can perform the antigen binding function of antibodies. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include Fab fragments, which are monovalent fragments consisting of VL, VH, CL and CH1 domains; a F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked at a hinge region by a disulfide bridge; fd fragment consisting of VH and CH1 domains; fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al, (1989) Nature [ Nature ]; 341:544-546) consisting of VH domains; and isolated Complementarity Determining Regions (CDRs).

Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a flexible linker that enables them to be formed as a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (known as a single chain Fv (scFv); see, e.g., bird et al, (1988) Science 242:423-426; and Huston et al, (1988) Proc Natl Acad Sc [ Proc. Natl. Acad. Sci. USA ]; 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for efficacy in the same manner as whole antibodies.

Throughout the specification, the term "isolated" means that the immunoglobulin, antibody or polynucleotide (as the case may be) is present in a physical environment different from that in the natural environment.

Throughout this specification, complementarity determining regions ("CDRs") are defined according to the Kabat (Kabat) definition, unless it is indicated that the CDRs are defined according to another definition. The exact amino acid sequence boundaries for a given CDR can be determined using any of a number of well-known schemes, including those described by: kabat et al (1991), "Sequences of Proteins of Immunological Interest" [ protein sequences of immunological importance ], 5 th edition, national institutes of health (National Institutes of Health), public health agency (Public Health Service), bethesda, md. (Bethesda, md.) ("kappa" numbering scheme); al-Lazikani et Al, (1997) JMB [ journal of microbiology and biotechnology ]273,927-948 ("Qiao Xiya (Chothia)" numbering scheme) and ImMunoGenTics (IMGT) (Lefranc, M. -P., the immunology [ Immunologist ],7,132-136 (1999); lefranc, M. -P.et Al, dev. Comp. Immunol. [ developmental immunology and comparative immunology ],27,55-77 (2003) ("IMGT" numbering scheme). For example, for classical forms, CDR amino acid residues in The heavy chain variable domain (VH) are numbered 31-35 (HCDR 1), 50-65 (HCDR 2) and 95-102 (HCDR 3) according to kappa, and CDR amino acid residues in The light chain variable domain (VL) are numbered 24-34 (LCDR 1), 50-56 (LCDR 2) and 89-97 (LCDR 3). According to Qiao Xiya, CDR amino acid residues in The VH are numbered 26-32 (HCDR 1), 52-56 (HCDR 2) and 95-102 (HCDR 3), and amino acid residues in The VL are numbered 26-32 (LCDR 1), 50-52 (DR 2) and 91-96 (LCDR 3) are defined by binding to CDR amino acid residues in The VL 84, and human being defined as amino acid residues 26-32 (HCDR 1), 50-52 (HCDR 2) and 95-102 (LCDR 3) according to kappa, and 95-42 (LCDR 3) are defined by binding to CDR amino acid residues in The VH 1 and 95-35, amino acid residues in The VH 1-50-32 (LCDR 2) and LCDR2 and 95-35, CDR amino acid residues in VH are numbered approximately 26-35 (CDR 1), 51-57 (CDR 2) and 93-102 (CDR 3), and CDR amino acid residues in VL are numbered approximately 27-32 (CDR 1), 50-52 (CDR 2) and 89-97 (CDR 3) (numbering according to "kappa"). According to IMGT, CDR regions of antibodies can be determined using the procedure IMGT/DomainGap alignment.

Conventionally, CDR regions in the heavy chain are commonly referred to as H-CDR1, H-CDR2 and H-CDR3 and CDR regions in the light chain are commonly referred to as L-CDR1, LCDR2 and L-CDR3. They are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of a single molecule composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences, e.g., human germline sequences or mutated forms of human germline sequences, or antibodies containing consensus framework sequences derived from human framework sequence analysis, e.g., as described in Knappik et al, (2000) J Mol Biol [ journal of molecular biology ]; 296:57-86).

The human antibodies of the invention may include amino acid residues that are not encoded by human sequences (e.g., mutations introduced by random mutagenesis or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

The term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity with variable regions, wherein both the framework and CDR regions are derived from human sequences.

The term "recombinant human antibody" as used herein includes all human antibodies produced, expressed, produced, or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or hybridomas made therefrom, antibodies isolated from host cells transformed to express human antibodies (e.g., from transfectomas), antibodies isolated from recombinant combinatorial human antibody libraries, and antibodies produced, expressed, produced, or isolated by any other means that involves splicing of all or part of human immunoglobulin genes. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals of transgenic human Ig sequences are used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences derived from and related to human germline VH and VL sequences, which may not naturally occur in the human antibody germline repertoire in vivo.

The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

As used herein, a binding molecule that "specifically binds IL-18" is intended to mean K at 100nM or less, 10nM or less, 1nM or less _D Binding molecules that bind human IL-18.

As used herein, a binding molecule that "specifically binds IL-1β" is intended to mean K at 100nM or less, 10nM or less, 1nM or less _D Binding molecules that bind human IL-1 beta.

As used herein, the term "antagonist" is intended to refer to a binding molecule that inhibits signaling activity in the presence of an activating compound. For example, in the case of IL-18, an IL-18 antagonist will be a binding molecule that inhibits signaling activity in human blood cells in the presence of IL-18 in a human cell assay, such as an IL-18 dependent interferon-gamma (IFN-gamma) production assay. Examples of IL-18 dependent IFN-gamma production assays in human blood cells are described in more detail in the examples below.

The term bivalent bispecific antibody or antibodies refers to antibodies that bind to two different targets (e.g., IL-18 and IL-1β). Such bivalent bispecific antibodies are also referred to herein as bispecific antibodies.

Bispecific antibodies are "heterodimers," meaning that one portion is from a first antibody that is specific for a first target and another portion is from a second antibody that is specific for a second target. "heterodimerization modification" is a modification of one or both portions of an antibody that forms a heterodimerization bispecific antibody, intended to facilitate such formation. Examples of heterodimerization modifications of the Fc domains of the two IgG1 portions intended to form a bispecific antibody are a "knob" with a large amino acid (aa) side chain in the first heavy chain (S354C, T366W) and a "mortar" with a small amino acid side chain introduced in the second heavy chain (Y349C, T366S, L368A, Y407V) and an additional disulfide bridge connecting the two heavy chains in the CH3 region (Merchant et al, nat. Biotechnol. [ Nature Biotechnology ],16:677-681 (1998), page 678, table 1).

The term "K", as used herein _D "is intended to mean the dissociation constant, which is obtained from K _d And K is equal to _a Ratio (i.e. K) _d /K _a ) And expressed as molar concentration (M). The K of an antibody can be determined using well established methods in the art _D Values. K for determining antibodies _D By using surface plasmon resonance, e.gThe system.

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

As used herein, the term "high affinity" for an antibody refers to an antibody with a KD of 1nM or less for the target antigen.

As used herein, the term "subject" includes any subject that receives dual specific anti-IL-18 and IL-1 β antagonists as presently described, which may present with symptoms of or risk for HS.

As used herein, the term "optimized nucleotide sequence" means that the nucleotide sequence has been changed to encode an amino acid sequence using codons that are preferred in a cell or organism that is produced, typically a eukaryotic cell, such as Pichia pastoris (Pichia pastoris), chinese Hamster Ovary (CHO) cell, or human cell. The optimized nucleotide sequence is engineered to fully retain the amino acid sequence originally encoded by the starting nucleotide sequence, also referred to as the "parent" sequence. The optimized sequences herein have been engineered to have codons preferred in CHO mammalian cells; however, optimized expression of these sequences in other eukaryotic cells is also contemplated herein.

The term "identity" refers to the similarity between at least two different sequences. This identity can be expressed as a percentage of identity and is determined by: standard alignment algorithms, such as basic local alignment tool (BLAST) (Altshul et al, (1990) J MoI Biol [ journal of molecular biology ]; 215:403-410); needleman et al, (1970) J MoI Biol [ journal of molecular biology ];48:444-453 algorithm or Meyers et al, (1988) Comput Appl Biosci [ computer application in bioscience ]; 4:11-17. One set of parameters may be Blosum 62 scoring matrix with gap penalty of 12, gap extension penalty of 4, and frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using E.Meyers and W.Miller, (1989) CABIOS [ computer applications in biosciences ] which have incorporated the ALIGN program (version 2.0); 4 (1) algorithm 1-17, determined using PAM 120 weight residue table, gap length penalty 12 and gap penalty 4. Percent identity is typically calculated by comparing sequences of similar length.

The term "immune response" refers to the action of lymphocytes, antigen presenting cells, phagocytes, granulocytes and soluble macromolecules (including antibodies, cytokines and complements) produced by, for example, the above-mentioned cells or liver, which result in selective damage, destruction or elimination of an invading pathogen, pathogen-infected cells or tissue, cancer cells (or in the case of autoimmune or pathological inflammation, normal human cells or tissue) from the human body.

"Signal transduction pathway" or "signaling activity" refers to biochemical causal relationships that are typically caused by protein-protein interactions (e.g., binding of growth factors to receptors), resulting in the transfer of signals from one portion of a cell to another portion of the cell. Typically, the delivery involves specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in a series of reactions that cause signal transduction. Penultimate processes typically involve nuclear events, resulting in changes in gene expression.

Throughout the specification, the term "neutralising" and grammatical variations thereof refers to the complete or partial reduction of the biological activity of a target, as the case may be, in the presence of a binding protein or antibody.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single or double stranded form, as well as polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known natural nucleotide analogs that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al, nucleic Acid Res. [ Nucleic acids Res. ]19:5081 (1991); ohtsuka et al, J.biol. Chem. [ J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, mol. Cell. Probes [ molecules and cell probes ]8:91-98 (1994)).

The nucleotides in a "polynucleotide" or "nucleic acid" may comprise modifications, including base modifications, such as bromouridine and inosine derivatives; ribose modifications such as phosphorothioates, phosphorodithioates, phosphoroselenos (phosphoselenoates), phosphorodiselenoate (phosphodis-enoates), anilino phosphorothioates (phosphoanilothioates), anilino phosphates (phosphoanildates) and phosphoramidates.

The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) suitable for transforming or transfecting a host cell and comprising a nucleic acid sequence that directs and/or controls (binds to) the expression of one or more heterologous coding regions operably linked thereto.

"conservative variant" of a sequence encoding a binding molecule, antibody, or fragment thereof refers to a sequence that comprises conservative amino acid modifications. "conservative amino acid modification" is intended to mean an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Conservative amino acid substitutions are amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Modifications may be introduced into the binding proteins of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions may also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. Non-naturally occurring amino acids include, but are not limited to, peptidomimetics (inverted or reverse forms of amino acid moieties).

The term "epitope" is a portion of an antigen recognized by the immune system (e.g., an antibody or fragment thereof). In the present specification, the term "epitope" is used interchangeably for conformational epitope and linear epitope. Conformational epitopes are composed of discrete parts of the amino acid sequence of an antigen, whereas linear epitopes are formed by the amino acid sequence of an antigen.

A human antibody or fragment thereof may comprise a heavy or light chain variable region or full length heavy or light chain that is "the product of" or "derived from" a particular germline sequence if the variable region or full length chain of the antibody is obtained from a system using human germline immunoglobulin genes. Such systems include immunization of transgenic mice carrying human immunoglobulin genes with an antigen of interest or screening of libraries of human immunoglobulin genes displayed on phage with an antigen of interest.

The human antibody or fragment thereof, which is the "product of" or "derived from" human germline immunoglobulin sequences, may be identified as such by: the amino acid sequence of the human antibody is compared to the amino acid sequence of a human germline immunoglobulin and the human germline immunoglobulin sequence of the sequences that is closest to (i.e., the highest% identity to) the sequence of the human antibody is selected. A human antibody that is a "product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence due to, for example, naturally occurring somatic mutations or deliberate introduction of site-directed mutations.

However, the amino acid sequence of the selected human antibody is typically at least 90% identical to the amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as belonging to a human when compared to germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In certain instances, the amino acid sequence of a human antibody may be at least 60%, 70%, 80%, 90% or at least 95% or even at least 96%, 97%, 98% or 99% identical to the amino acid sequence encoded by a germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will exhibit no more than 10 amino acid differences from the amino acid sequence encoded by a human germline immunoglobulin gene. In certain instances, the human antibody may exhibit no more than 5 or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

Human antibodies can be produced by a number of methods known to those skilled in the art. Human antibodies can be prepared by the hybridoma method using human myeloma or mouse-human heteromyeloma cell lines (Kozbor, JImmunol J.Immunol.; (1984) 133:3001;Brodeur,Monoclonal Isolated Antibody Production Techniques and Applications [ monoclonal isolated antibody production techniques and uses ], pages 51-63, marssel Dekker Co., marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both using the human variable region library (Winter G; (1994) Annu Rev Immunol [ annual reviews of immunology ]12:433-455, green LL, (1999) J Immunol Methods [ journal of immunology methods ] 231:11-23).

Several transgenic mouse strains are now available in which their mouse immunoglobulin loci have been replaced by human immunoglobulin gene segments (Tomizuka K, (2000) Proc Natl Acad Sci [ Proc. Natl. Acad. Sci. USA ]]97:722-727; fishwild DM (1996) Nature Biotechnol [ Nature Biotechnology ]]14:845-851; mendez MJ, (1997) Nature Genetics [ Nature Genetics ]]15:146-156). Such mice are capable of producing a pool of human antibodies from which antibodies of interest can be selected after antigen challenge. Particularly notable is Trimera ^TM System (Eren R et al, (1988) Immunology [ Immunology ]]93:154-161), wherein human lymphocytes are transplanted into irradiated mice; selective lymphocyte separation antibody System (SLAM, babcook et al, proc Natl Acad Sci Proc. Natl. Acad. Sci. USA)](1996) 93:7843-7848) in which human (or other species) lymphocytes are effectively passed through a number of pooled in vitro isolation antibody production procedures, followed by deconvolution, limiting dilution and selection procedures, and Xenomouse ^TM (Abgenix Co.). Morphodoma can be used from Morphotek corporation ^TM The technology achieves an alternative approach.

Phage display technology can be used to produce human antibodies and fragments thereof (McCafferty, (1990) Nature [ Nature ],348:552-553 and Griffiths AD et al (1994) EMBO [ journal of European molecular biology ] 13:3245-3260). According to this technique, isolated antibody variable domain genes are cloned in-frame into the major or minor coat of the protein genes of filamentous phage (e.g., M13 or fd) and displayed as functionally isolated antibody fragments (typically with the aid of helper phage) on the surface of phage particles. Selection based on the functional properties of the isolated antibodies results in selection of genes encoding the isolated antibodies that exhibit these properties. Phage display technology can be used to select antigen-specific antibodies from libraries prepared from human B cells taken from individuals suffering from a disease or disorder, or alternatively from non-immunized human donors (Marks; J Mol Bio [ journal of molecular biology ] (1991) 222:581-591). When a fully human isolated antibody comprising an Fc domain is desired, the phage-displayed derived fragment must be recloned into a mammalian expression vector comprising the desired constant region and a stable expression cell line established.

Affinity maturation techniques (Marks; biotechnol [ Biotechnology ] (1992) 10:779-783) can be used to provide binding affinities wherein the affinity of a primary human isolated antibody is improved by sequential replacement of the H and L chain variable regions with naturally occurring variants and selection on the basis of improved binding affinities. Variants of this technique are also now provided, such as "epitope imprinting" (WO 93/06213;Waterhouse;Nucl Acids Res [ nucleic acids research ] (1993) 21:2265-2266).

The term "pure" when used in the context of purified bispecific antibodies relates to the purity and identity of the different bispecific antibody combinations and constructs after co-expression in selected cells under conditions in which the cells express the bispecific antibodies and after protein a purification using the complete UPLC-MS mass screening method. Pure or purity refers to the relative quantification of heterodimers and homodimers bbmabs formed. Using the method of the present invention, the correctly formed heterodimer bbmAb1 can be observed with a relative purity of over 85% based on the intact mass signal intensity.

As used herein, the term "inhibit" refers to a reduction or inhibition of a given condition, symptom or disorder, or disease, or a significant decrease in a baseline activity of a biological activity or process.

As used herein, the term "treating (treat, treating or treatment)" of any disease or disorder refers in one embodiment to ameliorating the disease or disorder (i.e., slowing or arresting or alleviating the development or progression of the disease or at least one clinical symptom thereof). In another embodiment, "treatment" refers to reducing or ameliorating at least one physical parameter, including those that are not discernible by the patient. In yet another embodiment, "treating (treat, treating or treatment)" refers to modulating a disease or disorder on the body (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. More specifically, the term "treating" disease HS refers to treating an inflammatory lesion (in terms of number or mass or reducing its volume and size) in an HS patient, and/or treating an abscess and inflammatory nodule and/or drainage fistula in an HS patient, and/or reducing the number of scarring and/or alleviating functional limitations associated with scarring. Treating disease HS also refers to alleviating pain, fatigue, and/or itch associated with HS, reducing pus release and reducing odors associated with pus release, and/or improving quality of life and/or reducing work disorders in HS patients.

As used herein, the term "prevent" refers to delaying the onset or progression of a disease or disorder. More specifically, the term "prevention" disease HS refers to the prevention of HS red tumors and or new lesions that will appear; prevention of scarring and prevention of functional limitations associated with scarring, and/or in particular prevention of surgical intervention against HS.

As used herein, a subject is "in need of" such treatment if such subject would benefit biologically, medically, or in quality of life from the treatment.

As used herein, a "therapeutically effective amount" refers to an amount of a bispecific antibody (e.g., bbmAb 1) or antigen-binding fragment thereof that simultaneously targets both IL-1 beta and IL-18 that is effective to treat, prevent, cure, delay, reduce the severity of, alleviate at least one symptom of, or extend the survival of a patient beyond that expected in the absence of such treatment when administered to a patient (e.g., a human) in a single dose or multiple doses. When applied to a single active ingredient (e.g., bbmAb 1) administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to the combined amounts of the active ingredients (whether administered sequentially or simultaneously in combination) that produce a therapeutic effect.

The phrase "treatment regimen" means a regimen for treating a disease, such as a dosing regimen used during HS treatment. The treatment regimen may include a loading regimen (or loading administration), followed by a maintenance regimen (or maintenance administration).

The phrase "loading regimen" or "loading period" refers to a treatment regimen (or portion of a treatment regimen) for the initial treatment of a disease. In some embodiments, the disclosed methods, uses, kits, procedures, and protocols (e.g., methods of treating HS) employ a loading protocol (or loading administration). In some cases, the loading period is a period of time until maximum efficacy is achieved. The overall goal of a loading regimen is to provide high levels of medication to the patient at the beginning of the treatment regimen. The loading regimen may include administering a higher dose of the drug than the physician would employ during the maintenance regimen, administering the drug more frequently than the physician would administer the drug during the maintenance regimen, or both. Dose escalation may occur during or after a loading regimen.

The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or portion of a treatment regimen) for maintaining a patient during treatment of a disease, for example, maintaining the patient in remission for a long period (months or years) after a loading regimen or loading period. In some embodiments, the disclosed methods, uses, and protocols use a maintenance protocol. The maintenance regimen may employ continuous therapy (e.g., administration of the drug at regular intervals, e.g., once weekly, biweekly or monthly (every 4 weeks), once annually, etc.) or intermittent therapy (e.g., discontinuing therapy, intermittent therapy, therapy upon recurrence, or therapy following achievement of certain predetermined criteria [ e.g., pain, disease manifestations, etc.). Dose escalation may occur during maintenance regimens.

The phrase "means for administering" is used to indicate any available means for systemically administering a drug to a patient, including but not limited to pre-filled syringes, vials and syringes, injection pens, auto-injectors, intravenous (i.v.) instillation and injection bags, pumps, patch pumps, and the like. With such articles, the patient may self-administer the drug (i.e., self-administer the drug) or the physician may administer the drug.

IL-18 antibodies

Particularly preferred IL-18 antibodies or antigen-binding fragments thereof for use in the disclosed methods are human antibodies.

For ease of reference, the hypervariable regions of a specific IL-18 antibody (called mAb 1) based on the kappa definition and the Qiao Xiya definition, and V are provided in Table 1 below _L And V _H Domains and the amino acid sequences of the complete heavy and light chains.

Table 1. Amino acid sequences of the hypervariable regions (CDRs), variable domains (VH and VL) and full chain of mabs 1. The DNA encoding VL of mAb1 is set forth in SEQ ID NO. 18. The DNA encoding the VH of mAb1 is set forth in SEQ ID NO. 8.

In one embodiment, the IL-18 antibody or antigen binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain (V _H ) The CDR1 has the amino acid sequence SEQ ID NO. 1, the CDR2 has the amino acid sequence SEQ ID NO. 2, and the CDR3 has the amino acid sequence SEQ ID NO. 3. In one embodiment, the IL-18 antibody or antigen binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain (V _H ) The CDR1 has the amino acid sequence SEQ ID NO. 4, the CDR2 has the amino acid sequence SEQ ID NO. 5, and the CDR3 has the amino acid sequence SEQ ID NO. 6.

In one embodiment, the IL-18 antibody or antigen binding fragment thereof comprises at least one immunoglobulin light chain variable domain (V _L ) The CDR1 has the amino acid sequence SEQ ID NO. 11, the CDR2 has the amino acid sequence SEQ ID NO. 12 and the CDR3 has the amino acid sequence SEQ ID NO. 13. In one embodiment, the IL-18 antibody or antigen binding fragment thereof comprises at least one immunoglobulin light chain variable domain (V _L ) The CDR1 has the amino acid sequence SEQ ID NO. 14, the CDR2 has the amino acid sequence SEQ ID NO. 15 and the CDR3 has the amino acid sequence SEQ ID NO. 16.

In one embodiment, the IL-18 antibody or antigen-binding fragment thereof comprises at least one immunoglobulin V _H Domain and at least one exemptionEpidemic globulin V _L Domain, wherein: a) The immunoglobulin V _H The domain comprises (e.g., in sequence): i) A hypervariable region CDR1, CDR2, and CDR3, said CDR1 having the amino acid sequence of SEQ ID No. 1, said CDR2 having the amino acid sequence of SEQ ID No. 2, and said CDR3 having the amino acid sequence of SEQ ID No. 3; or ii) a hypervariable region CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO. 4, said CDR2 having the amino acid sequence SEQ ID NO. 5 and said CDR3 having the amino acid sequence SEQ ID NO. 6; and b) the immunoglobulin V _L The domain comprises (e.g., in sequence): i) Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:11, said CDR2 having the amino acid sequence SEQ ID NO:12 and said CDR3 having the amino acid sequence SEQ ID NO:13 or ii) hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:14, said CDR2 having the amino acid sequence SEQ ID NO:15 and said CDR3 having the amino acid sequence SEQ ID NO:16.

In one embodiment, the IL-18 antibody or antigen-binding fragment thereof comprises: a) Immunoglobulin heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO. 7 (V _H ) The method comprises the steps of carrying out a first treatment on the surface of the b) An immunoglobulin light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO. 17 (V _L ) The method comprises the steps of carrying out a first treatment on the surface of the c) Immunoglobulin V comprising the amino acid sequence set forth in SEQ ID NO. 7 _H Domain and immunoglobulin V comprising the amino acid sequence set forth in SEQ ID No. 17 _L A domain; d) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 _H A domain; e) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13 _L A domain; f) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 _H A domain; g) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16 _L A domain; h) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 _H The domains and immunity comprising the hypervariable regions set forth in SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13Globulin V _L A domain; i) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 _H Domain and immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16 _L A domain; j) A light chain comprising SEQ ID NO. 19; k) A heavy chain comprising SEQ ID NO. 9; or l) a light chain comprising SEQ ID NO. 19 and a heavy chain comprising SEQ ID NO. 9.

In some embodiments, the IL-18 antibody or antigen binding fragment thereof (e.g., mAb 1) comprises the three CDRs of SEQ ID NO: 7. In other embodiments, the IL-18 antibody or antigen binding fragment thereof comprises the three CDRs of SEQ ID NO. 17. In other embodiments, the IL-18 antibody or antigen binding fragment thereof comprises three CDRs of SEQ ID NO. 7 and three CDRs of SEQ ID NO. 17. In some embodiments, the IL-18 antibody or antigen binding fragment thereof comprises the three CDRs of SEQ ID NO 9. In other embodiments, the IL-18 antibody or antigen binding fragment thereof comprises the three CDRs of SEQ ID NO. 19. In other embodiments, the IL-18 antibody or antigen binding fragment thereof comprises three CDRs of SEQ ID NO:9 and three CDRs of SEQ ID NO: 19.

In one embodiment, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb 1) is selected from the group consisting of a human IL-18 antibody comprising at least: a) An immunoglobulin heavy chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, and a constant portion of a human heavy chain or fragment thereof; the CDR1 has the amino acid sequence SEQ ID NO. 1, the CDR2 has the amino acid sequence SEQ ID NO. 2, and the CDR3 has the amino acid sequence SEQ ID NO. 3; and b) an immunoglobulin light chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:11, said CDR2 having the amino acid sequence SEQ ID NO:12 and said CDR3 having the amino acid sequence SEQ ID NO:13, and a constant portion of a human light chain or fragment thereof.

In one embodiment, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb 1) is selected from the group consisting of a human IL-18 antibody comprising at least: a) An immunoglobulin heavy chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, and a constant portion of a human heavy chain or fragment thereof; the CDR1 has the amino acid sequence SEQ ID NO. 4, the CDR2 has the amino acid sequence SEQ ID NO. 5, and the CDR3 has the amino acid sequence SEQ ID NO. 6; and b) an immunoglobulin light chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO 14, said CDR2 having the amino acid sequence SEQ ID NO 15 and said CDR3 having the amino acid sequence SEQ ID NO 16, and a constant portion of a human light chain or fragment thereof.

In one embodiment, the IL-18 antibody or antigen-binding fragment thereof is selected from a single chain antibody or antigen-binding fragment thereof comprising an antigen binding site comprising: a) A first domain comprising in sequence a hypervariable region CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID No. 1, said CDR2 having the amino acid sequence SEQ ID No. 2, and said CDR3 having the amino acid sequence SEQ ID No. 3; and b) a second domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO. 11, said CDR2 having the amino acid sequence SEQ ID NO. 12 and said CDR3 having the amino acid sequence SEQ ID NO. 13; and C) a peptide linker that binds the N-terminal end of the first domain and the C-terminal end of the second domain or binds the C-terminal end of the first domain and the N-terminal end of the second domain.

In one embodiment, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb 1) is selected from a single chain antibody or antigen-binding fragment thereof comprising an antigen-binding site comprising: a) A first domain comprising in sequence a hypervariable region CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID No. 4, said CDR2 having the amino acid sequence SEQ ID No. 5 and said CDR3 having the amino acid sequence SEQ ID No. 6; and b) a second domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO. 14, said CDR2 having the amino acid sequence SEQ ID NO. 15 and said CDR3 having the amino acid sequence SEQ ID NO. 16; and C) a peptide linker that binds the N-terminal end of the first domain and the C-terminal end of the second domain or binds the C-terminal end of the first domain and the N-terminal end of the second domain.

V of IL-18 antibodies or antigen-binding fragments thereof for use in the disclosed methods _H Or V _L The domains may have a sequence identical to the sequence set forth in SEQ ID NOs 7 and 17 _H Or V _L V with substantially identical domains _H And/or V _L A domain. The human IL-18 antibodies disclosed herein may comprise a heavy chain that is substantially identical to the heavy chain set forth in SEQ ID NO. 9 and/or a light chain that is substantially identical to the light chain set forth in SEQ ID NO. 19. The human IL-18 antibodies disclosed herein may comprise: heavy chain comprising SEQ ID NO. 9 and light chain comprising SEQ ID NO. 19. The human IL-18 antibodies disclosed herein may comprise: a) A heavy chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NO. 7 and a constant portion of a human heavy chain; and b) a light chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NO. 17 and a constant portion of a human light chain.

Other preferred IL-18 antagonists (e.g., antibodies) for use in the disclosed methods, kits and protocols are those listed below: U.S. patent No.: 9,376,489, which is incorporated herein by reference in its entirety.

IL-1 beta antibodies

Particularly preferred IL-1. Beta. Antibodies or antigen binding fragments thereof for use in the disclosed methods are human antibodies.

For ease of reference, the hypervariable regions of specific IL-1. Beta. Antibodies (called mAb 2) based on the kappa definition and the Qiao Xiya definition, and V are provided in Table 2 below _L And V _H Domains and the amino acid sequences of the complete heavy and light chains.

Table 2. Amino acid sequences of the hypervariable regions (CDRs), variable domains (VH and VL) and full chain of mabs 2. The DNA encoding VL of mAb2 is set forth in SEQ ID NO: 38. The DNA encoding the VH of mAb2 is set forth in SEQ ID NO 27.

In one embodiment, the IL-1β antibody or antigen binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain (V _H ) The CDR1 has the amino acid sequence SEQ ID NO. 21, the CDR2 has the amino acid sequence SEQ ID NO. 22, and the CDR3 has the amino acid sequence SEQ ID NO. 23. In one embodiment, the IL-1β antibody or antigen binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain (V _H ) The CDR1 has the amino acid sequence SEQ ID NO. 24, the CDR2 has the amino acid sequence SEQ ID NO. 25, and the CDR3 has the amino acid sequence SEQ ID NO. 26.

In one embodiment, the IL-1β antibody or antigen binding fragment thereof comprises at least one immunoglobulin light chain variable domain (V _L ) The CDR1 has the amino acid sequence SEQ ID NO. 31, the CDR2 has the amino acid sequence SEQ ID NO. 32 and the CDR3 has the amino acid sequence SEQ ID NO. 33. In one embodiment, the IL-1β antibody or antigen binding fragment thereof comprises at least one immunoglobulin light chain variable domain (V _L ) The CDR1 has the amino acid sequence SEQ ID NO 34, the CDR2 has the amino acid sequence SEQ ID NO 35 and the CDR3 has the amino acid sequence SEQ ID NO 36.

In one embodiment, the IL-1. Beta. Antibody or antigen binding fragment thereof comprises at least one immunoglobulin V _H Domain and at least one immunoglobulin V _L Domain, wherein: a) The immunoglobulin V _H The domain comprises (e.g., in sequence): i) Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO. 21, said CDR2 having the amino acid sequenceThe amino acid sequence SEQ ID NO. 22, and the CDR3 has the amino acid sequence SEQ ID NO. 23; or ii) a hypervariable region CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO. 24, said CDR2 having the amino acid sequence SEQ ID NO. 25 and said CDR3 having the amino acid sequence SEQ ID NO. 26; and b) the immunoglobulin V _L The domain comprises (e.g., in sequence): i) Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:31, said CDR2 having the amino acid sequence SEQ ID NO:32 and said CDR3 having the amino acid sequence SEQ ID NO:33 or ii) hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:34, said CDR2 having the amino acid sequence SEQ ID NO:35 and said CDR3 having the amino acid sequence SEQ ID NO:36.

In one embodiment, the IL-1 β antibody or antigen-binding fragment thereof comprises: a) An immunoglobulin heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO. 27 (V _H ) The method comprises the steps of carrying out a first treatment on the surface of the b) Immunoglobulin light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO. 37 (V _L ) The method comprises the steps of carrying out a first treatment on the surface of the c) Immunoglobulin V comprising the amino acid sequence set forth in SEQ ID NO. 27 _H Domain and immunoglobulin V comprising the amino acid sequence set forth in SEQ ID No. 37 _L A domain; d) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 21, SEQ ID NO. 22 and SEQ ID NO. 23 _H A domain; e) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 31, SEQ ID NO. 32 and SEQ ID NO. 33 _L A domain; f) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 24, SEQ ID NO. 25 and SEQ ID NO. 26 _H A domain; g) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 36 _L A domain; h) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 21, SEQ ID NO. 22 and SEQ ID NO. 23 _H Domain and immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 31, SEQ ID NO. 32 and SEQ ID NO. 33 _L A domain; i) Immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 24, SEQ ID NO. 25 and SEQ ID NO. 26 _H Domains and comprising SEQ ID NO 34, SEQ ID NO 35 andimmunoglobulin V of the hypervariable region set forth in SEQ ID NO. 36 _L A domain; j) A light chain comprising SEQ ID NO. 37; k) A heavy chain comprising SEQ ID NO. 29; or l) a light chain comprising SEQ ID NO. 39 and a heavy chain comprising SEQ ID NO. 29.

In some embodiments, the IL-1β antibody or antigen binding fragment thereof (e.g., mAb 2) comprises the three CDRs of SEQ ID NO: 37. In other embodiments, the IL-1β antibody or antigen binding fragment thereof comprises the three CDRs of SEQ ID NO 27. In other embodiments, the IL-1β antibody or antigen binding fragment thereof comprises three CDRs of SEQ ID NO. 37 and three CDRs of SEQ ID NO. 27. In some embodiments, the IL-1β antibody or antigen binding fragment thereof comprises the three CDRs of SEQ ID NO 39. In other embodiments, the IL-1β antibody or antigen binding fragment thereof comprises the three CDRs of SEQ ID NO. 29. In other embodiments, the IL-1β antibody or antigen binding fragment thereof comprises three CDRs of SEQ ID NO:39 and three CDRs of SEQ ID NO: 29.

In one embodiment, the IL-1β antibody or antigen-binding fragment thereof (e.g., mAb 2) is selected from the group consisting of a human IL-1β antibody comprising at least: a) An immunoglobulin heavy chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, and a constant portion of a human heavy chain or fragment thereof; the CDR1 has the amino acid sequence SEQ ID NO. 21, the CDR2 has the amino acid sequence SEQ ID NO. 22, and the CDR3 has the amino acid sequence SEQ ID NO. 23; and b) an immunoglobulin light chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:31, said CDR2 having the amino acid sequence SEQ ID NO:32 and said CDR3 having the amino acid sequence SEQ ID NO:33, and a constant portion of a human light chain or fragment thereof.

In one embodiment, the IL-1β antibody or antigen-binding fragment thereof (e.g., mAb 2) is selected from the group consisting of a human IL-1β antibody comprising at least: a) An immunoglobulin heavy chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, and a constant portion of a human heavy chain or fragment thereof; the CDR1 has the amino acid sequence SEQ ID NO. 24, the CDR2 has the amino acid sequence SEQ ID NO. 25, and the CDR3 has the amino acid sequence SEQ ID NO. 26; and b) an immunoglobulin light chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO 34, said CDR2 having the amino acid sequence SEQ ID NO 35 and said CDR3 having the amino acid sequence SEQ ID NO 36, and a constant portion of a human light chain or fragment thereof.

In one embodiment, the IL-1 β antibody or antigen-binding fragment thereof is selected from a single chain antibody or antigen-binding fragment thereof comprising an antigen binding site comprising: a) A first domain comprising in sequence a hypervariable region CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO. 21, said CDR2 having the amino acid sequence SEQ ID NO. 22 and said CDR3 having the amino acid sequence SEQ ID NO. 23; and b) a second domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO. 31, said CDR2 having the amino acid sequence SEQ ID NO. 32 and said CDR3 having the amino acid sequence SEQ ID NO. 33; and C) a peptide linker that binds the N-terminal end of the first domain and the C-terminal end of the second domain or binds the C-terminal end of the first domain and the N-terminal end of the second domain.

In one embodiment, the IL-1 β antibody or antigen-binding fragment thereof (e.g., mAb 2) is selected from a single chain antibody or antigen-binding fragment thereof comprising an antigen-binding site comprising: a) A first domain comprising in sequence a hypervariable region CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID No. 24, said CDR2 having the amino acid sequence SEQ ID No. 25, and said CDR3 having the amino acid sequence SEQ ID No. 26; and b) a second domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO 34, said CDR2 having the amino acid sequence SEQ ID NO 35 and said CDR3 having the amino acid sequence SEQ ID NO 36; and C) a peptide linker that binds the N-terminal end of the first domain and the C-terminal end of the second domain or binds the C-terminal end of the first domain and the N-terminal end of the second domain.

V of IL-1 beta antibodies or antigen binding fragments thereof for use in the disclosed methods _H Or V _L The domains may have a sequence identical to the sequence set forth in SEQ ID NOS 27 and 37 _H Or V _L V with substantially identical domains _H And/or V _L A domain. The human IL-1. Beta. Antibodies disclosed herein may comprise a heavy chain that is substantially identical to the heavy chain set forth in SEQ ID NO. 29 and/or a light chain that is substantially identical to the light chain set forth in SEQ ID NO. 39. The human IL-1 beta antibodies disclosed herein may comprise: heavy chain comprising SEQ ID NO. 29 and light chain comprising SEQ ID NO. 39. The human IL-1 β antibodies disclosed herein may comprise: a) A heavy chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NO. 27 and a constant portion of a human heavy chain; and b) a light chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NO. 37 and a constant portion of a human light chain.

Other preferred IL-1 beta antagonists (e.g., antibodies) for use in the disclosed methods, kits and protocols are those listed below: U.S. patent No.: 7,446,175 or 7,993,878 or 8,273,350, which are incorporated herein by reference in their entirety.

Fc modification

In addition to or as an alternative to modifications made within the framework or CDR regions, antibodies of the invention may be engineered to include modifications within the Fc region, typically in order to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, fc receptor binding, and/or antigen-dependent cytotoxicity. Furthermore, the antibodies of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation, thereby again altering one or more functional properties of the antibody. Each of these embodiments is described in more detail below. Residue numbering in the Fc region is that of the EU numbering scheme of Edelman et al, PNAS [ Proc. Natl. Acad. Sci. USA ], july. 1969, 63 (1): 78-85.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This method is further described in U.S. Pat. No. 5,677,425 to Bodmer et al. The number of cysteine residues in the CH1 hinge region is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment such that the antibody has impaired staphylococcal protein a (SpA) binding relative to native Fc hinge domain SpA binding. This method is described in further detail in U.S. Pat. No. 6,165,745 to Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. Various methods may be employed. For example, one or more of the following mutations may be introduced: such as T252L, T254S, T F described by Ward in U.S. patent No. 6,277,375. Alternatively, to increase biological half-life, antibodies can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from both loops of the CH2 domain of the Fc region of IgG, as described in U.S. Pat. nos. 5,869,046 and 6,121,022 to presa et al.

In still other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids may be substituted with different amino acid residues such that the antibody has an altered affinity for the effector ligand, but retains the antigen binding capacity of the parent antibody. The affinity-altering effector ligand may be, for example, an Fc receptor or the C1 component of complement. This method is described in further detail in Winter et al, U.S. Pat. Nos. 5,624,821 and 5,648,260.

In another embodiment, one or more amino acids selected from the group consisting of amino acid residues may be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or eliminated Complement Dependent Cytotoxicity (CDC). This method is described in further detail in U.S. Pat. No. 6,194,551 to Idusogie et al.

In another embodiment, one or more amino acid residues are altered, thereby altering the ability of the antibody to fix complement. This method is further described in PCT publication WO 94/29351 to Bodmer et al.

In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for fcγ receptors by modifying one or more amino acids. This process is further described by Presta in PCT publication WO 00/42072. Furthermore, the binding sites for FcgammaRl, fcgammaRII, fcgammaRIII and FcRn on human IgG1 have been mapped and variants with improved binding have been described (see Shields, R.L. et al, (2001) J.biol Chem [ journal of biochemistry ] 276:6591-6604).

In certain embodiments, an Fc domain of an IgG1 isotype is used. In some particular embodiments, mutant variants of IgG1 Fc fragments are used, such as silent IgG1 Fc, which may reduce or eliminate the ability of the fusion polypeptide to mediate Antibody Dependent Cellular Cytotoxicity (ADCC) and/or binding to fcγ receptors. Examples of IgG1 isotype silencing mutants, such as those described in Hezareh et al, J.Virol [ J.virol ] (2001); 75 (24) substitution of the leucine residue with the alanine residue at amino acid positions 234 and 235, as described in 12161-8.

In certain embodiments, the Fc domain is a mutant that prevents glycosylation at position 297 of the Fc domain. For example, the Fc domain contains an amino acid substitution of an asparagine residue at position 297. An example of such an amino acid substitution is the substitution of N297 with glycine or alanine.

Silent effector functions can be obtained by mutation of the Fc region of an antibody and have been described in the art: LALA and N297A (Strohl, W.,2009, curr. Opin. Biotechnol. [ current Biotechnology perspective ] volume 20 (6): 685-691); and D265A (Baudino et al, 2008, J.Immunol. [ J.Immunol. ]181:6664-69; strohl, W., supra); DAPA (D265A and P329A) (Shields RL., J Biol Chem [ journal of biochemistry ].2001;276 (9): 6591-604; U.S. patent publication No. US 2015/032580). Examples of silent Fc lgG1 antibodies comprise so-called LALA mutants comprising L234A and L235A mutations in the lgG 1Fc amino acid sequence. Another example of a silent lgG1 antibody comprises a D265A mutation. Another example of a silent lgG1 antibody is the so-called DAPA mutant, which comprises the D265A and P329A mutations in the amino acid sequence of IgG1 Fc. Another silent lgG1 antibody comprises a N297A mutation that results in an aglycosylated/non-glycosylated antibody. Additional Fc mutations for providing silent effector function are described in PCT publication No. WO 2014/145806 (e.g., in fig. 7 of WO 2014/145806), which is incorporated herein by reference in its entirety. One example of a silent IgG1 antibody from WO 2014/145806 comprises the E233P, L234V, L a and S267K mutations and the deletion of G236 (G236 del). Another example of a silent IgG1 antibody from WO 2014/145806 comprises the E233P, L V and L235A mutations, as well as the G236 deletion (G236 del). Another example of a silent IgG1 antibody from WO 2014/145806 comprises the S267K mutation.

In yet another embodiment, glycosylation of the antibody is modified. For example, an antibody that is aglycosylated (i.e., the antibody lacks glycosylation) may be prepared. Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such saccharide modification may be accomplished by: for example, one or more glycosylation sites within an antibody sequence are altered. For example, one or more amino acid substitutions may be made that result in elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. This aglycosylation can increase the affinity of the antibody for the antigen. Such a process is described in more detail in U.S. Pat. nos. 5,714,350 and 6,350,861 to Co et al.

Additionally or alternatively, antibodies with altered glycosylation patterns, such as low fucosylation antibodies with reduced fucosyl residues or antibodies with increased bisecting GlcNac structure, can be prepared. Such altered glycosylation patterns have been demonstrated to increase the ADCC capacity of antibodies. Such carbohydrate modification may be achieved, for example, by expressing the antibody in a host cell having an altered glycosylation mechanism. Cells having altered glycosylation machinery have been described in the art and can be used as host cells in which the recombinant antibodies of the invention are expressed, thereby producing antibodies having altered glycosylation. For example, EP 1,176,195 to Hang et al describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase such that antibodies expressed in such a cell line exhibit low fucosylation. Thus, in one embodiment, the antibodies of the invention are produced by recombinant expression in a cell line that exhibits a fucosylation pattern (e.g., a mammalian cell line that is deficient in the expression of the FUT8 gene encoding a fucosyltransferase). Presta, in PCT publication WO 03/035835, describes a variant CHO cell line Lecl3 cell with reduced ability to attach fucose to Asn (297) linked saccharides, also resulting in low fucosylation of antibodies expressed in the host cell (see also Shields, R.L. et al, 2002J.biol. Chem. [ J. Biochemistry ] 277:26733-26740). PCT publication WO 99/54342 to Umana et al describes cell lines engineered to express glycoprotein-modified glycosyltransferases (e.g., beta (1, 4) -N-acetylglucosaminyl transferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisected GlcNac structures that result in increased ADCC activity of the antibodies (see also Umana et al, 1999Nat. Biotech. [ Nature Biotechnology ] 17:176-180). Alternatively, the antibodies of the invention may be produced in a yeast or filamentous fungus which is engineered for a mammalian-like glycosylation pattern and which is capable of producing antibodies lacking fucose as the glycosylation pattern (see e.g. EP 1297172B 1).

Another modification of the antibodies herein contemplated by the present invention is pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG) (e.g., a reactive ester or aldehyde derivative of PEG) under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. PEGylation may be performed by either an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derive other proteins, such as mono (C1-C10) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods of pegylating proteins are known in the art and may be applied to the antibodies of the invention. See, for example, EP 0 154 316 to Nishimura et al and EP 0 401 384 to Ishikawa et al.

Another modification of the antibodies contemplated by the present invention is the conjugation or protein fusion of at least the antigen binding region of the antibodies of the present invention with a serum protein (such as human serum albumin or a fragment thereof) to increase the half-life of the resulting molecule. Such a method is described, for example, in ballancec et al EP 0322094.

Another modification of the antibodies contemplated by the present invention is one or more modifications to increase heterodimeric bispecific antibody formation. Various methods available in the art can be used to enhance dimerization of two heavy chain domains of a bispecific antibody, e.g., bbmAb, as disclosed, for example, in the following: EP 1870459A1; U.S. Pat. nos. 5,582,996; U.S. Pat. nos. 5,731,168; U.S. patent No. 5,910,573; U.S. patent No. 5,932,448; U.S. patent No. 6,833,441; U.S. patent No. 7,183,076; U.S. patent application publication No. 2006204493A1; and PCT publication No. WO 2009/089004A1, the contents of which are incorporated herein in their entirety.

For example, the use of a knob-and-socket structure to produce bispecific antibodies is disclosed in PCT publication Nos. WO 1996/027011, ridgway et al, (1996) and Merchant et al (1998).

In practicing some therapeutic methods or uses of the present disclosure, a therapeutically effective amount of a bispecific antibody that simultaneously targets both IL-1 beta and IL-18, such as bbmAb1, must be administered to a subject in need thereof. It should be appreciated that the regimen changes may be applicable to certain patients. Thus, administration (e.g., bbmAb 1) may be more frequent, such as once daily, once every two weeks, or once weekly.

Some patients may benefit from a loading regimen (e.g., once daily administration for days [ e.g., 1 to 4 days, e.g., on day 0, day 1, day 2, and/or day 3 ]) followed by a maintenance regimen, e.g., beginning at week 3 or week 4, wherein bbmAb1 may be administered once weekly, once every two weeks, or every 4 weeks for weeks. In some embodiments, the period of time for which bispecific antibodies (e.g., bbmAb 1) that target both IL-1 beta and IL-18 are administered lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days. In some embodiments, the period of time for which a bispecific antibody (e.g., bbmAb 1) that targets both IL-1 beta and IL-18 is administered lasts for 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 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 longer.

It will be appreciated that, based on the severity of the disease, the dose escalation may be applicable to certain patients (e.g., patients), such as patients with inadequate response to treatment with bbmAb 1. Thus, the dose (i.v.) may be greater than about 10mg/kg, such as about 11mg/kg, 12mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 35mg/kg, etc. Further, the subcutaneous (s.c.) dose (loading dose or maintenance dose) may be greater than about 50mg to about 900mg s.c., for example about 75mg, about 100mg, about 125mg, about 175mg, about 200mg, about 250mg, about 350mg, about 400mg, about 450mg, about 500mg, about 600mg, etc.;

It will also be appreciated that dose reduction may also be applicable to certain patients (e.g., patients), such as patients exhibiting adverse events or adverse responses to treatment with bbmAb1. Thus, the dosage may be less than about 10mg/kg, for example, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, or about 9mg/kg. In some embodiments, the bbmAB1 dose may be adjusted as determined by a physician.

In some embodiments, the bbmAB1 antibody can be administered to the patient in a single dose of 10mg/kg delivered i.v., where the dose can be adjusted to a higher or lower dose as determined by a physician, for example, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, or about 9mg/kg, or for example, about 11mg/kg, 12mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 35mg/kg, etc., if desired.

In some embodiments, the bbmAB1 antibody can be administered to the patient at an initial dose of 10mg/kg delivered i.v., and if desired, the dose can be adjusted to a higher or lower dose as determined by a physician.

In a particular embodiment, 10mg/kg of bbmAB1 is administered on day 1.

In particular embodiments, 10mg/kg bbmAB1 is administered on day 1 (D1) and on day 2 (D2), D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13 and/or D14

In another specific embodiment, 10mg/kg of bbmAB1 is administered i.v. on day 1.

Example 1: various inflammatory bodies may be involved in HS

Analysis of the patient's HS transcriptome showed increased mRNA levels of AIM2, NLRC4, NLRP7 and NLRP3 inflammasome in HS lesions compared to the levels of non-diseased or healthy tissue (fig. 1). All of these inflammatory body genes were expressed at significantly higher levels in HS lesion samples than in HS non-lesion samples and healthy samples, suggesting that a variety of inflammatory bodies may be involved in HS pathophysiology.

Example 2: both IL-1 beta and IL-18 signaling pathways are active in HS

(a) Transcriptomics of skin biopsies

Transcriptomic analysis of 18 lesion HS biopsies, 6 peri-lesion biopsies and 7 non-lesion biopsies relative to 8 biopsies from healthy skin donors was obtained as follows: from the quick frozen skin tissue, the homogenate was prepared using the recommended buffer from the Qiagen RNeasy mini kit. Total RNA from the cells was extracted according to the manufacturer's protocol. The cDNA of the sample was prepared from the same starting amount of RNA using a high-capacity cDNA reverse transcription kit (applied biosystems Co., ltd. (Applied Biosystem)). Samples were processed on an Affymetrix HG_U133_Plus2 microarray by CiToxLAB France. The RMA normalized data was analyzed using GeneSpring 11.5.1 (agilent technologies (Agilent Technologies), santa Clara, CA). Initially, the data were subjected to standard QC control by CiToxLAB and in GeneSpring (PCA, hybridization control). Subsequently, it was filtered at the expression level for probe sets above 20 percentile in 100% of the samples under either condition, prior to further analysis. The data set is stored in the NCBI GEO database (GSE 148027). The results were visualized using TIBCO Sporfire Analyst.

(b) Transcriptomics of cytokine stimulated PBMCs (from healthy donors)

Stimulation of PBMCs with recombinant cytokines was performed by wells of 7x106 PBMCs/12 well plate with a final volume of 1.5ml in RPMI medium. Recombinant cytokines were added at the following final concentrations: 10ng/ml recombinant IL-1 beta, 3nM recombinant IL-18, and 1ng/ml recombinant IL-12. Cells were harvested after 6 hours of stimulation in cell culture at 37 ℃ and 5% CO 2.

To isolate RNA, cells were pelleted and the pellet was lysed in 350 μl Kaiji RTL buffer with 2% beta-mercaptoethanol and frozen at-20℃or-80℃until all study samples were collected. RNA isolation was performed using the standard protocol of Kaijer. As described above, different RNA samples were processed by CiToxLAB France on Affymetrix HG_U133_Plu2 microarrays. The entity (probe set) was retained, wherein at least 100% of the samples had values above 20 percentile in any 1 experimental condition. Differentially Expressed Genes (DEG) were identified using the "volcanic pattern filter (filter on volcano plot)" function in GeneSpring. Using filtered genes with unpaired T-test (expressed between 20.0-100.0 percentiles), probe sets with corrected p-values below 0.05 and fold changes above 2.0 were considered differentially expressed. Where possible, benjamini-Hochberg multiplex test correction (Benjamini-Hochberg Multiple Testing Correction) was used.

The resulting gene list was used as a "cytokine signaling signature" and the expression levels of the defined signature were queried in the HS transcriptome dataset without using any correlation values in the original experiment.

(c) Generation of a spotfire transcriptome heatmap

Expression levels of differential upregulation genes obtained from cytokine-stimulated PBMCs were averaged for each individual skin biopsy, and increased color coding of color intensity was attributed to increased average expression levels of the corresponding PBMC features (fig. 2).

The following evidence is provided: both IL-1b signaling and IL-18 signaling are present and active in the diseased skin of HS patients, and both cytokines may play a role in HS pathophysiology.

Example 3:

the production of bbmAb1 is described in detail in examples 1 to 5 of patent application WO/2018/229612. Example 1 of WO/2018/229612 comprises (1) vector construction, (2) host cell lines and transfection, (3) cell selection and sorting, (4) cell expansion, (5) cloning stability, (6) manufacturing, (7) analytical characterization and purity assessment, (8) analytical results, which are incorporated herein by reference in their entirety.

bbmAb1 is a bispecific IgG1 with LALA silent mutations that bind to two different targets, IL-1β and IL-18. The antibody binds to two different antigen binding arms (Fab fragments), whereas Fab against IL-1β is based on mAb2 and comprises a kappa light chain (Vk 6). The Fab against IL-18 is based on mAb1 and consists of lambda light chain (V lambda 1). To drive heterodimerization of the Fc domain during expression, a "knob" with a large amino acid (aa) side chain (S354C and T366W) and a "hole" with a small aa side chain (Y349C, T366S, L368A, Y407V) were introduced into the mAb2 heavy chain.

For ease of reference, the bbmAb1 hypervariable region, based on the cabazite definition and the Qiao Xiya definition, and V are provided in Table 3 below _L And V _H Domains and the amino acid sequences of the complete heavy and light chains.

Table 3. Amino acid sequences of hypervariable regions (CDRs), variable domains (VH and VL) and full chain of bbmabs 1. The DNA encoding the first VL is set forth in SEQ ID NO. 102 and the DNA encoding the second VL is set forth in SEQ ID NO. 70. The DNA encoding the first VH is set forth in SEQ ID NO. 86 and the DNA encoding the second VH is set forth in SEQ ID NO. 54.

In one embodiment, an IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a first immunoglobulin heavy chain variable domain comprising hypervariable regions CDR1, CDR2 and CDR3 (V _H1 ) The 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:78. In one embodiment, an IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a first immunoglobulin heavy chain variable domain comprising hypervariable regions CDR1, CDR2 and CDR3 (V _H1 ) The CDR1 has the amino acid sequence SEQ ID NO:79, the CDR2 has the amino acid sequence SEQ ID NO:80, and the CDR3 has the amino acid sequence SEQ ID NO:81. In one embodiment, an IL-18/IL-1. Beta. Bispecific antibody for use in the disclosed treatment or prevention of HS of (i) comprises a first immunoglobulin heavy chain variable domain (V _H1 ) The CDR1 has the amino acid sequence SEQ ID NO. 82, the CDR2 has the amino acid sequence SEQ ID NO. 83, and the CDR3 has the amino acid sequence SEQ ID NO. 84.

In one embodiment, the IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a second immunoglobulin heavy chain variable domain comprising hypervariable regions CDR1, CDR2 and CDR3 (V _H2 ) The CDR1 has the amino acid sequence SEQ ID NO 44, the CDR2 has the amino acid sequence SEQ ID NO 45, and the CDR3 has the amino acid sequence SEQ ID NO 46. In one embodiment, the IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a second immunoglobulin heavy chain variable domain comprising hypervariable regions CDR1, CDR2 and CDR3 (V _H2 ) The CDR1 has the amino acid sequence SEQ ID NO. 47, the CDR2 has the amino acid sequence SEQ ID NO. 48, and the CDR3 has the amino acid sequence SEQ ID NO. 49. In one embodiment, use is made ofThe IL-18/IL-1 beta bispecific antibodies for use in the treatment or prophylaxis of HS comprise a second immunoglobulin heavy chain variable domain comprising hypervariable regions CDR1, CDR2 and CDR3 (V _H2 ) The CDR1 has the amino acid sequence SEQ ID NO:50, the CDR2 has the amino acid sequence SEQ ID NO:51, and the CDR3 has the amino acid sequence SEQ ID NO:52.

In one embodiment, an IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a first immunoglobulin light chain variable domain (V _L1 ) The CDR1 has the amino acid sequence SEQ ID NO:92, the CDR2 has the amino acid sequence SEQ ID NO:93, and the CDR3 has the amino acid sequence SEQ ID NO:94. In one embodiment, an IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a first immunoglobulin light chain variable domain (V _L1 ) The CDR1 has the amino acid sequence SEQ ID NO 95, the CDR2 has the amino acid sequence SEQ ID NO 96, and the CDR3 has the amino acid sequence SEQ ID NO 97. In one embodiment, an IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a first immunoglobulin light chain variable domain (V _L1 ) The CDR1 has the amino acid sequence SEQ ID NO. 98, the CDR2 has the amino acid sequence SEQ ID NO. 99, and the CDR3 has the amino acid sequence SEQ ID NO. 100.

In one embodiment, the IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a second immunoglobulin light chain variable domain (V _L2 ) The CDR1 has the amino acid sequence SEQ ID NO:60, the CDR2 has the amino acid sequence SEQ ID NO:61, and the CDR3 has the amino acid sequence SEQ ID NO:62. In one embodiment, the IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a second immunoglobulin light chain variable domain (V _L2 ) The CDR1 has the amino acid sequence SEQ ID NO:63, the CDR2 has the amino acid sequence SEQ ID NO:64, and the CDR3 hasThe amino acid sequence SEQ ID NO. 65. In one embodiment, the IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a second immunoglobulin light chain variable domain (V _L2 ) The CDR1 has the amino acid sequence SEQ ID NO:66, the CDR2 has the amino acid sequence SEQ ID NO:67, and the CDR3 has the amino acid sequence SEQ ID NO:68.

In one embodiment, the IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a first immunoglobulin V _H1 Domain and first immunoglobulin V _L1 Domain, wherein: a) The first immunoglobulin V _H1 The domain comprises (e.g., in sequence): i) Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO 76, said CDR2 having the amino acid sequence SEQ ID NO 77 and said CDR3 having the amino acid sequence SEQ ID NO 78; or ii) a hypervariable region CDR1, CDR2 and CDR3, said CDR1 having 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:81; or iii) the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO. 82, said CDR2 having the amino acid sequence SEQ ID NO. 83 and said CDR3 having the amino acid sequence SEQ ID NO. 84 and b) the first immunoglobulin V _L1 The domain comprises (e.g., in sequence): i) Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:92, said CDR2 having the amino acid sequence SEQ ID NO:93 and said CDR3 having the amino acid sequence SEQ ID NO:94 or ii) hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:95, said CDR2 having the amino acid sequence SEQ ID NO:96 and said CDR3 having the amino acid sequence SEQ ID NO:97 or iii) hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:98, said CDR2 having the amino acid sequence SEQ ID NO:99 and said CDR3 having the amino acid sequence SEQ ID NO:100.

In one embodiment, the IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises a second immunoglobulin V _H2 Domain and second immunoglobulin V _L2 The domain of the polypeptide,wherein: a) The second immunoglobulin V _H2 The domain comprises (e.g., in sequence): i) Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO 44, said CDR2 having the amino acid sequence SEQ ID NO 45 and said CDR3 having the amino acid sequence SEQ ID NO 46; or ii) a hypervariable region CDR1, CDR2 and CDR3, said CDR1 having an amino acid sequence SEQ ID NO. 47, said CDR2 having an amino acid sequence SEQ ID NO. 48 and said CDR3 having an amino acid sequence SEQ ID NO. 49; or iii) the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:50, said CDR2 having the amino acid sequence SEQ ID NO:51 and said CDR3 having the amino acid sequence SEQ ID NO:52 and b) the second immunoglobulin V _L2 The domain comprises (e.g., in sequence): i) Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:60, said CDR2 having the amino acid sequence SEQ ID NO:61 and said CDR3 having the amino acid sequence SEQ ID NO:62 or ii) hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:63, said CDR2 having the amino acid sequence SEQ ID NO:64 and said CDR3 having the amino acid sequence SEQ ID NO:65 or iii) hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:66, said CDR2 having the amino acid sequence SEQ ID NO:67 and said CDR3 having the amino acid sequence SEQ ID NO:68.

In one embodiment, an IL-18/IL-1 β bispecific antibody for use in the treatment or prevention of HS comprises: a) A first immunoglobulin heavy chain variable domain (VH 1) comprising an amino acid sequence set forth in SEQ ID No. 85; b) A first immunoglobulin light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO. 101 (V _L1 ) The method comprises the steps of carrying out a first treatment on the surface of the c) A first immunoglobulin V comprising an amino acid sequence set forth in SEQ ID NO. 85 _H1 Domain and first immunoglobulin V comprising the amino acid sequence set forth in SEQ ID NO. 101 _L1 A domain; d) First immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 76, SEQ ID NO. 77 and SEQ ID NO. 78 _H1 A domain; e) First immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 92, SEQ ID NO. 93 and SEQ ID NO. 94 _L1 A domain; f) Comprises SEQ ID NO 79,First immunoglobulin V of hypervariable regions set forth in SEQ ID NO. 80 and SEQ ID NO. 81 _H1 A domain; g) First immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 95, SEQ ID NO. 96 and SEQ ID NO. 97 _L1 A domain; h) First immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 76, SEQ ID NO. 77 and SEQ ID NO. 78 _H1 Domain and first immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 92, SEQ ID NO. 93 and SEQ ID NO. 94 _L1 A domain; i) First immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 79, SEQ ID NO. 80 and SEQ ID NO. 81 _H1 Domain and first immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 95, SEQ ID NO. 96 and SEQ ID NO. 97 _L1 A domain; j) A first light chain comprising SEQ ID NO. 103; 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.

In one embodiment, an IL-18/IL-1 β bispecific antibody for use in the treatment or prevention of HS comprises: a) A second immunoglobulin heavy chain variable domain (VH 2) comprising an amino acid sequence set forth in SEQ ID No. 53; b) A second immunoglobulin light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO. 69 (V _L2 ) The method comprises the steps of carrying out a first treatment on the surface of the c) A second immunoglobulin V comprising an amino acid sequence set forth in SEQ ID NO. 53 _H2 Domain and second immunoglobulin V comprising the amino acid sequence set forth in SEQ ID NO. 69 _L2 A domain; d) Second immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 44, SEQ ID NO. 45 and SEQ ID NO. 46 _H2 A domain; e) A second immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 60, SEQ ID NO. 61 and SEQ ID NO. 62 _L2 A domain; f) A second immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 47, SEQ ID NO. 48 and SEQ ID NO. 49 _H2 A domain; g) A second immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 63, SEQ ID NO. 64 and SEQ ID NO. 65 _L2 A domain; h) Second immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 44, SEQ ID NO. 45 and SEQ ID NO. 46 _H2 DomainAnd a second immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 60, SEQ ID NO. 61 and SEQ ID NO. 62 _L2 A domain; i) A second immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 47, SEQ ID NO. 48 and SEQ ID NO. 49 _H2 Domain and a second immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO. 63, SEQ ID NO. 64 and SEQ ID NO. 65 _L2 A domain; j) A second light chain comprising SEQ ID NO. 81; k) A second heavy chain comprising SEQ ID NO. 55; or l) a second light chain comprising SEQ ID NO. 81 and a second heavy chain comprising SEQ ID NO. 55.

In some embodiments, an IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises three CDRs of SEQ ID NO 53. In other embodiments, the IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises three CDRs of SEQ ID NO: 69. In other embodiments, an IL-18/IL-1β bispecific antibody for use in the treatment or prevention of HS comprises three CDRs of SEQ ID NO:53 and three CDRs of SEQ ID NO: 69. In some embodiments, the IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises three CDRs of SEQ ID NO. 85. In other embodiments, the IL-18/IL-1β bispecific antibody for use in treating or preventing HS comprises three CDRs of SEQ ID NO 101. In other embodiments, an IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises three CDRs of SEQ ID NO:85 and three CDRs of SEQ ID NO: 101.

In some embodiments, the IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises three CDRs of SEQ ID NO. 85. In other embodiments, the IL-18/IL-1β bispecific antibody for use in treating or preventing HS comprises three CDRs of SEQ ID NO 101. In other embodiments, an IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises three CDRs of SEQ ID NO:85 and three CDRs of SEQ ID NO: 101. In some embodiments, an IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises three CDRs of SEQ ID NO 53. In other embodiments, the IL-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises three CDRs of SEQ ID NO: 69. In other embodiments, an IL-18/IL-1β bispecific antibody for use in the treatment or prevention of HS comprises three CDRs of SEQ ID NO:53 and three CDRs of SEQ ID NO: 69. In an embodiment, an L-18/IL-1. Beta. Bispecific antibody for use in the treatment or prevention of HS comprises three CDRs of SEQ ID NO:85, three CDRs of SEQ ID NO:101, three CDRs of SEQ ID NO:53 and three CDRs of SEQ ID NO: 69.

In one embodiment, the first portion of the IL-18/IL-1 β bispecific antibody for use in the treatment or prevention of HS is selected from the group consisting of a human IL-18 antibody, which human IL-18 antibody comprises at least: a) An immunoglobulin heavy chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, and a constant portion of a human heavy chain or fragment thereof; the 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 78; and b) an immunoglobulin light chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:92, said CDR2 having the amino acid sequence SEQ ID NO:93 and said CDR3 having the amino acid sequence SEQ ID NO:94, and a constant portion of a human light chain or fragment thereof. Furthermore, the second part of the IL-18/IL-1β bispecific antibody is selected from the group consisting of a human IL-1β antibody comprising at least: a) An immunoglobulin heavy chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, and a constant portion of a human heavy chain or fragment thereof; the CDR1 has the amino acid sequence SEQ ID NO 44, the CDR2 has the amino acid sequence SEQ ID NO 45, and the CDR3 has the amino acid sequence SEQ ID NO 46; and b) an immunoglobulin light chain or fragment thereof comprising a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:60, said CDR2 having the amino acid sequence SEQ ID NO:61 and said CDR3 having the amino acid sequence SEQ ID NO:62, and a constant portion of a human light chain or fragment thereof.

First V of IL-18/IL-1 beta bispecific antibodies for use in the disclosed methods _H1 Or V _L1 The domains may have a sequence similar to the sequence set forth in SEQ ID NOS 85 and 101 _H Or V _L First V with substantially identical domains _H1 And/or a first V _L1 A domain. For use in therapy or prophylaxis as disclosed hereinThe IL-18/IL-1β bispecific antibody used in HS can comprise a first heavy chain that is substantially identical to the heavy chain set forth in SEQ ID NO. 87 and/or a first light chain that is substantially identical to the light chain set forth in SEQ ID NO. 103. The IL-18/IL-1β bispecific antibodies as disclosed herein for use in the treatment or prevention of HS can comprise a first heavy chain comprising SEQ ID NO. 87 and a first light chain comprising SEQ ID NO. 103. The IL-18/IL-1 beta bispecific antibody for use in the treatment or prevention of HS may comprise: a) A first heavy chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID No. 85 and a constant portion of a human heavy chain having a heterodimerization modification; and b) a first light chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NO. 101 and a constant portion of a human light chain. The constant portion of the human heavy chain may be IgG1. In one embodiment, the IgG1 is human IgG1 without effector mutations. In one embodiment, the human heavy chain IgG1 comprises the silent mutation N297A, D265A or a combination of L234A and L235A. In a specific embodiment, human heavy chain IgG1 comprises a silent mutation according to SEQ ID NO. 87 that is a combination of L234A and L235A.

Second V of IL-18/IL-1 beta bispecific antibodies for use in the disclosed methods _H2 Or V _L2 The domains may have a sequence identical to the sequence set forth in SEQ ID NOs 53 and 69 _H Or V _L Second V with substantially identical domains _H2 And/or a first V _L2 A domain. The IL-18/IL-1. Beta. Bispecific antibodies as disclosed herein for use in treating or preventing HS can comprise a second heavy chain that is substantially identical to the heavy chain set forth in SEQ ID NO. 55 and/or a second light chain that is substantially identical to the light chain set forth in SEQ ID NO. 71. The IL-18/IL-1β bispecific antibodies as disclosed herein for use in the treatment or prevention of HS can comprise a second heavy chain comprising SEQ ID NO. 53 and a second light chain comprising SEQ ID NO. 69. The IL-18/IL-1 beta bispecific antibodies as disclosed herein for use in the treatment or prevention of HS may comprise: a) A second heavy chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NO. 53 and havingA constant portion of a human heavy chain having a heterodimerization modification (which is complementary to the heterodimerization of the first heavy chain); and b) a second light chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NO. 69 and a constant portion of a human light chain. The constant portion of the human heavy chain may be IgG1. In one embodiment, the IgG1 is human IgG1 without effector mutations. In one embodiment, the human heavy chain IgG1 comprises the silent mutation N297A, D265A or a combination of L234A and L235A. In a specific embodiment, human heavy chain IgG1 comprises a silent mutation according to SEQ ID NO. 55 that is a combination of L234A and L235A.

Other preferred IL-18 antagonists (e.g., antibodies) for use as the first part of the bispecific antibodies in the disclosed methods, kits and protocols are those listed below: U.S. patent No.: 9,376,489, which is incorporated herein by reference in its entirety.

Other preferred IL-1 beta antagonists (e.g., antibodies) for use as the second part of the bispecific antibodies in the disclosed methods, kits and protocols are those listed below: U.S. patent No.: 7,446,175 or 7,993,878 or 8,273,350, which are incorporated herein by reference in their entirety.

Example 4: in vitro Activity of bbmAb1

The binding activity of bbmAb1 was tested in a variety of different cell assays.

(1) Materials and methods

(a) For Solution Equilibrium Titration (SET) determination

The following materials were used:

biotinylated recombinant human IL-18 (BTP 25828)

Recombinant cynomolgus monkey IL-1 beta (Novartis) is available from Noval corporation

SULFO-TAG labeled anti-human IgG antibody (miniprep.discovery Co., ltd. (Meso Scale discovery, MSD) #R32AJ-5). Goat anti-human Fab specific antibody conjugated with MSD SULFO-TAG NHS ester (Jackson immune research Co (Jackson Immuno Research) #109-005-097, MSD#R91AN-1), BSA (Sigma) # A-9647)

MSD read buffer T with surfactant (MSD#R92 TC-1)

Phosphate Buffered Saline (PBS) 10x (Teknova) #P0195) Tris buffered saline pH 7.5 (TBS) 10x (Teknova #T1680) Tween-20 (Fluka) #93773

Polypropylene microtiter plate (MTP) (Greiner) No. 781280

384 well plate, standard (MSD#L21XA)

(b) For cell assays and SET assays

mAb2 as described in the IL-1 beta antibody section.

mAb1 as described in the IL-18 antibody section.

bbmAb1 as described in example 1.

Recombinant human IL-18 (BTP 25829) (#B001-5) from MBL International Inc. (MBL Int. Corp.)

Recombinant marmoset IL-1 beta (North China)

Recombinant marmoset IL-18 (Norhua Co.)

Recombinant human IL-12 (# 573008) was purchased from Bosch company (bioleged) KG-1 cell line (ATCC #CCL-246)

Normal human dermal fibroblasts (#CC-2509) were purchased from Lonsha Inc. (Lonza)

Marmoset skin fibroblast (# 42637F (510))

HEK-Blue ^TM IL-18/IL-1. Beta. Cells (#hkb-IL 18) were purchased from InvivoGen corporation

Isolation of PBMC from buffy coat (from Blutspendezentrum Bern Co)

Marmoset blood is obtained from SILABE, niedelhausbergen

IL-6ELISA: human (bosch, # 430503); marmoset (U-CyTech bioscience Co., ltd. (U-CyTech biosciences), CT 974-5)

Ifnγ ELISA: human (BD 555142) and marmoset (U-CyTech bioscience Co. # CT 340A)

QUANTIBlue for detecting SEAP ^TM Assay (#rep-qb 1) was purchased from InvivoGen Co

Cell culture medium: RPMI 1640 (Invitrogen) # 31870) supplemented with 10% fetal bovine serum (Inje # 10108-157), 1% L-glutamine (Inje # 25030-03), 1% penicillin/streptomycin (Inje # 15140-148), 10. Mu.M 2-mercaptoethanol (Gibco # 31350-010), 5mM Hepes (Gibco # 15630-080)

96-well plate for round bottom tissue culture treatment (Costar # 3799)

96-well plate for flat bottom tissue culture treatment (Costar # 3596)

Ficoll-Pacque ^TM Plus (GE medical health life sciences Co. (GE Healthcare Life Sciences) # 17-1440-02) PBS1X, does not contain calcium and magnesium (Gibco Co. # 14190094)

Leucoep tube with porous barrier, 50ml, polypropylene (Greiner bio-one Co. # 227290) Falcon 15ml Polypropylene conical tube (BD# 352096)

Falcon 50ml polypropylene conical tube (BD company # 352070)

(c) Affinity measurement by SET

SET single target binding assay

22 consecutive 1.6n dilutions of antigen (highest concentration: huIL-18,5nM; marIL-18, 10nM; huIL-1β,0.5nM; marIL-1β,0.5 nM) were prepared in sample buffer (PBS containing 0.5% Bovine Serum Albumin (BSA) and 0.02% Tween-20) and a constant concentration of antibody (10 pM for IL-18 readings 1pM for IL-1β) was added. Each antigen-antibody mixture was dispensed in duplicate into 384-well polypropylene microtiter plates (MTPs) at a volume of 60 μl/well. Sample buffer was used as negative control, and samples containing only antibodies were used as positive control (maximum electrochemiluminescence signal without antigen, B _max ). Plates were sealed and incubated overnight (o/n, at least 16 hours) on a shaker at Room Temperature (RT).

IL-18 reading: streptavidin-coated 384-well MSD array MTP was coated with 30. Mu.l/well biotinylated huIL-18 (0.1. Mu.g/ml, PBS) and incubated on a shaker for 1h at room temperature.

IL-1. Beta. Reading: standard 384-well MSD array MTPs were coated with 30. Mu.l/well huIL-1 (3. Mu.g/ml, PBS) (as capture agent) diluted in PBS and incubated overnight at 4 ℃.

The plate was blocked with 50. Mu.l/well of blocking buffer (5% BSA in PBS) for 1 hour (h) at Room Temperature (RT). After washing (TBST, TBS with 0.05% Tween 20), the equilibrated antigen-antibody mixture was transferred from polypropylene MTP to coated MSD plates in a volume of 30. Mu.l/well and incubated for 20min at room temperature. After an additional washing step, 30 μl of sulfo-labeled anti-IgG detection antibody (0.5 μg/ml) diluted in sample buffer was added to each well and incubated on a shaker for 30min at room temperature. The MSD plates were washed and 35. Mu.l/well MSD read buffer was added and incubated for 5min at room temperature. Electrochemiluminescence (ECL) signals were generated and measured by MSD Sector Imager 6000.

SET simultaneous target binding assay

Except for assay a, SET assays were performed as described above: the equilibration process (antibody/antigen mixture) was performed in the presence of an excess of one target (500 pM IL18 or IL-1. Beta.) while evaluating K for the other target _D 。

Measurement B: the equilibration process (antibody/antigen mixture) was performed simultaneously in one mixture with serial dilutions of the two targets (constant concentration of antibody 10pM, highest antigen concentration, see above). The same mixture was then analyzed for free antibody concentration on IL18 and IL-1 beta coated plates as described above.

The SET data has been exported to the MS Excel loader software Xlfit. The average ECL signal was calculated from the repeated measurements in each assay. Baseline adjustments were made to the data by subtracting the minimum from all data points and plotted against the corresponding antigen concentrations to generate titration curves. K (K) _D The values were determined by fitting the graph to:

1:2 binding model of monospecific Ab

1:1 binding model of pestle-mortar structure bispecific Ab

Wherein the method comprises the steps of

y: blank subtracted ECL signal

B _max : maximum ECL signal at zero antigen concentration

[ IgG ]: concentration of antibody used

[ Fab ]: total Fab concentration applied

K _D : dissociation equilibrium constant

x: concentration of antigen used

(d) Cell culture

KG-1 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 1% L-glutamine and 1% penicillin/streptomycin at a density of 2X 10 ⁵ Up to 1 x 10 ⁶ Each living cell/mL.

Normal human fibroblasts and marmoset fibroblasts were grown in FBM (Clonetics Co., ltd., CC-3131) comprising bFGF (1 ng/ml, CC-4065), insulin (5. Mu.g/ml, CC-4021) and 2% FCS (CC-4101). Fibroblast basal medium (Dragon's Corp. # CC-3131) was used as starvation medium.

HEK-Blue ^TM IL-18/IL-1. Beta. Cells in growth Medium (DMEM, 4.5g/l glucose, 10% (v/v) fetal bovine serum, 50U/ml penicillin, 50mg/ml streptomycin, 100mg/ml Normocin) ^TM 2mM L-glutamine supplemented with blasticidin at 30. Mu.g/ml, hygroGold at 200. Mu.g/ml ^TM And 100 μg/ml Zeocin ^TM ) And (3) growing in the middle.

Human Peripheral Blood Mononuclear Cells (PBMCs) were freshly isolated from the buffy coat using a LeucoSep tube according to the manufacturer's instructions. Briefly, 13ml of Ficoll-Paque was preloaded into 14ml LeucoSep tubes by centrifugation at 1,000Xg for 30 s. Heparinized whole blood samples were diluted with an equal volume of PBS and 25ml of diluted blood was added to the LeucoSep tube. The cell separation tube was centrifuged at 800 Xg for 15min without interruption at room temperature. The cell suspension layer was collected, the cells were washed twice in PBS (640×g and 470×g for 10min, respectively, two consecutive washes) and resuspended in medium, and then counted.

Marmoset blood was collected in heparinized tubes and filtered using a 70 μm cell filter (BD Biosciences) # 352350)

(e) IL-1 beta neutralization assay

IL-1. Beta. Induced IL-6 production assays in fibroblasts were performed essentially as described (Gram 2000) with only minor modifications. Briefly, fibroblasts were seeded at a density of 5 x 103 cells/well (in 100 μl) in 96-well flat bottom tissue culture plates. The following day, cells were starved for 5h in starvation medium before adding the recombinant IL-1 β/compound solution mixture (IL-1 β concentrations shown in the table). The IL-1. Beta. -compound solution mixture was prepared in advance by incubating recombinant IL-1. Beta. -with a compound in a concentration range at 37℃for 30 min. Cell supernatants were collected after o/n incubation at 37℃and the amount of IL-6 released was determined by ELISA. The IL-1. Beta. Induced IL-6 production assay in PBMC was performed as follows. PBMC were pooled at 3X 10 ⁵ Individual cells/wells (in 100 μl) were seeded in 96-well tissue culture plates and incubated with the recombinant IL-1β/compound solution mixture at 37 ℃ for 24h (IL-1β concentrations shown in the table). The IL-1. Beta. -compound solution mixture was prepared in advance by incubating recombinant IL-1. Beta. -with a compound in a concentration range at 37℃for 30 min. Cell supernatants were collected 24h after stimulation and the amount of released IL-6 was determined by ELISA.

(f) IL-18 neutralization assay

The determination is carried out essentially as follows. Will have a density of 3 x 10 ⁵ KG-1 cells/wells (1 h starved in PBS+1% FCS beforehand) or PBMC were inoculated into round bottom 96-well cell culture plates and incubated with a solution mixture of recombinant IL-18/IL-12 and a range of compounds (IL-18/IL-12 concentrations shown in the table). After 24 hours incubation at 37 ℃, the supernatant was collected and the amount of ifnγ released was determined by ELISA. For the assay with marmoset blood, 85 μl blood/well was used.

(g)HEK-Blue ^TM Dual IL1 beta/IL-18 neutralization in cells

The determination is performed essentially as described in the manufacturer's procedures. Briefly, HEK-Blue ^TM Cells at 4X 10 ⁴ Density of wells/wells was inoculated into 96-well cell culture plates and incubated with a solution mixture of recombinant IL-1 beta and IL-18 (to generate a 1:1seap signal) and a range of concentration of compounds. After incubation at 37℃for 24h, the supernatant was collected and rooted Use of QUANTI-Blue according to manufacturer's instructions ^TM The method determines the amount of SEAP released.

All data were exported to EXCEL software and IC50 values were calculated by plotting dose response curves for the logical curve fitting function using EXCEL/XLfit4 or GraphPad Prism software.

(2) Results

(a) Affinity for recombinant human and marmoset IL1 beta and IL-18

Binding affinity of bbmAb1 to human and marmoset recombinant IL-1 beta and IL-18 proteins was measured by Solution Equilibrium Titration (SET) titration and the resulting K was used _D Value of K binding to mAb2 to IL-1 beta and mAb1 to IL-18 _D The values are compared.

Binding affinities were compared in a single target binding assay, bbmAb1 showed similar average KD for both pairs compared to mAb1 for human and marmoset IL-18 (table 7). For human IL-1β binding, the average KD value of bbmAb1 (2.6 pM) was slightly higher compared to mAb2 (0.6 pM), but still within the same low pM range. Subsequent measurements in simultaneous dual target binding assays (table 8) confirm that the binding KD values of bbmAb1 to IL-1β are similar to mAb2 values in the case of preclinical as well as clinical grade materials. Thus, bbmAb1 has binding affinity for both human and marmoset targets, which is similar to mAb2 and mAb1, respectively.

TABLE 7 affinity for recombinant human (hu) and marmoset (mar) IL-1. Beta. And IL-18 as measured by SET (Single target binding assay)

In addition to single target binding results, K binding was assessed by application _D Simultaneous dual target binding affinities of bbmAb1 were studied either in excess of one target relative to the other (assay a) or by applying a mixture of two targets in serial dilutions (assay B) (table 8). Simultaneous IL-1 beta/IL-18 affinity assays showed no significant difference between assay A (one antigen in excess) and assay B (in serial dilutions of a mixture of two antigens), demonstrating that the two targets bind simultaneously without ghostingIn response to binding of another target. In addition, K obtained by simultaneous dual binding assay _D Value and standard determination of K _D The values were similar (Table 7; in the absence of the second antigen), demonstrating that bbmAb1 can bind both antigens independently. Thus, bbmAb1 binds both human IL-1β and IL-18 simultaneously and independently and fully cross-reacts with the corresponding marmoset proteins.

TABLE 8 affinity for recombinant human (hu) and marmoset (mar) IL-1. Beta. And IL-18 (simultaneous target binding assay) as measured by SET

(b) Neutralizing Activity of bbmAb1 in human and marmoset cell assays

The neutralizing activity of bbmAb1 on two cytokines (IL 1 beta and IL-18) was evaluated (mAb 2mAb 1). In addition, the efficacy of bbmAb1 in neutralizing marmoset IL-1. Beta. And IL-18 using a marmoset cell assay system was evaluated (see section d).

(c) Separate and simultaneous IL-1 beta and IL-18 neutralization in human cells

The neutralizing activity of bbmAb1 on IL-1β was evaluated by inhibiting recombinant IL-1β -induced IL-6 production in human skin fibroblasts (IL-1β used at 6 pM) and human PBMC (IL-1β used at 60 pM). The neutralizing activity of bbmAb1 on IL-18 was measured by inhibiting recombinant IL-18-induced IFN-gamma production in KG-1 cells and human PBMC (both cells activated by 3nM recombinant human IL-18 and 1ng/ml recombinant human IL-12). The inhibition potency of bbmAb1 on IL-1β and IL-18 was consistently compared to mAb2 or mAb1, respectively. Depending on the assay, the average IC50 values of bbmAb1 were in the sub-nM or single digit nM range, but directly up to 2 to 4 fold higher than mAb2 (for IL-1β) and mAb1 (for IL-18), respectively (tables 9 and 10). The monovalent form of bbmAb1, but possibly also the KiH mutation, is responsible for the subtle differences in potency of bbmAb1 compared to the bivalent form of mAb2/mAb 1.

Table 9 average IC50 values for bbmAb1 and IL-1β compared to mAb2 in human skin fibroblasts and human PBMC. * Inhibition of IL-6 production in human skin fibroblasts stimulated by recombinant human IL-1 beta (6 pM for skin fibroblasts and 60pM for PBMC) or PBMC. Mean ± SEM (n=3 PBMC and n=6 human skin fibroblasts) are shown

Table 10 average IC50 values for bbmAb1 and IL-18 compared to mAb1 in KG-1 cells and human PBMC. * Inhibition of IFNγ production in KG-1 cells or PBMC stimulated with recombinant human IL-18 (3 nM) and human IL-12 (1 ng/ml). Mean ± SEM (n=3kg-1 and n=4pbmc) are shown

bbmAb1 is capable of neutralizing both IL-1 beta and IL-18 bioactivity, e.g., HEK Blue ^TM Report cells (which produced SEAP in response to 1+1 stimulation by recombinant IL-1 beta and IL-18) are shown (table 11). Similar inhibition of SEAP in this assay system could only be achieved by the combination of mAb2 and mAb1, but not by the use of a single antibody.

TABLE 11 Blue according to HEK ^TM SEAP reporter activity in cells, and at the same time, the average IC50 values of IL-1 beta and IL-18. Shown is the mean ± SEM of n=5 experiments.

(d) Neutralizing Activity of bbmAb1 on marmoset IL-1 beta and marmoset IL-18 in marmoset cell assay

To demonstrate the inhibitory activity of bbmAb1 in marmosets, similar in vitro assays were performed with marmoset cells and human cells, but stimulated with recombinant marmoset IL-1. Beta. And IL-18. When evaluated for inhibition of recombinant marmoset IL-1 beta-induced IL-6 production in marmoset skin fibroblasts, bbmAb1 exhibited sub-nM potency with IC50 values 2 to 3-fold higher than mAb2 (table 12). The human skin fibroblasts stimulated with marmoset IL-1. Beta. Were tested for inhibition profile of bbmAb1 production similar to human IL-6.

TABLE 12 inhibition of IL-1. Beta. Induced IL-6 production by recombinant marmoset in marmoset and human fibroblasts by bbmAb 1. * Inhibition of IL-6 production in recombinant marmoset IL-1 beta (18 pM) stimulated marmoset or human skin fibroblasts. The results of 3 separate experiments (A, B and C) are shown.

The single to two-digit nM IC50 values of bbmAb1 confirm the neutralizing activity of bbmAb1 on marmoset IL-18 tested in the IFNgamma production assay with marmoset blood cells (Table 13). When measuring the production of human ifnγ, testing bbmAb1 with marmoset IL-18 stimulated human PBMC produced a similar inhibition profile.

Thus, bbmAb1 showed complete cross-reactivity with marmoset IL-1. Beta. And marmoset IL-18 in a functional assay using marmoset responder cells.

Table 13 mean IC50 values for inhibition of IL-18 induced IFNγ production in whole marmoset blood or in human PBMC. * Inhibition of ifnγ production in marmoset whole blood (each compound/condition n=3) or human PBMC (n=6) stimulated with recombinant marmoset IL-18 (indicated concentration) and human IL-12 (10 ng/ml). Mean ± SEM are shown

It has been demonstrated that bbmAb1 (KiH-type IL-1β/IL-18 bispecific mAb) retains high affinity binding to two separate targets IL-1β and IL-18, as well as cytokine neutralization potency, in a variety of different cellular assays, as compared to the original mAb (mAb 2 and mAb 1). Not only the dual IL-1 beta and IL-18 neutralization properties of bbmAb1 were demonstrated for human cytokines/cells, but also for the corresponding marmoset cytokines/cells, thus facilitating appropriate toxicology studies. The up to 2 to 4-fold higher IC50 values produced in some cell assays directed to IL-1 beta and IL-18 neutralization may be the result of bbmAb1 monovalent binding rather than mAb2 and mAb1 divalent binding, respectively. However, dual neutralization of cytokines by bbmAb1 may result in additive or synergistic inhibitory activity in vivo, which may not be fully demonstrated in our in vitro cell system.

Example 5: IL-1 beta and IL-18 combination stimulates and blocks effects in PBMC

The inflammatory body activation-dependent cleavage of the effector cytokines IL-1 beta and IL-18 results in the induction of secondary pro-inflammatory mediators that promote immune cell recruitment/activation not only systemically but also at the site of inflammation. In two different models of lethal systemic inflammation mice (a) an LPS injection model and (b) FCAS mice (activating missense mutations in NLRP 3), the simultaneous absence/inhibition of both IL-1β and IL-18 is more protective against lethality than the absence/inhibition of either IL-1β alone or IL-18 alone, demonstrating additive or synergistic mechanisms of immune activation (Brydges 2013,van den Berghe2014). bbmAb1 is a human/marmoset IL-1 beta/IL-18 reactive bispecific mAb, which is free of rodent cross-reactivity and therefore cannot be tested in a mouse model. Thus, we stimulated human PBMC using LPS/IL-12 in vitro mimicking the activation of the inflammatory body-dependent pathway to reveal additive or synergistic inhibition of bbmAb1 neutralization-combined IL-1β/IL-18, and performed unbiased gene expression analysis using microarrays. As a complementary activity we also compared the gene expression profile of PBMCs from different donors stimulated with a combination of recombinant IL-1 beta and recombinant IL-18 or single cytokines alone.

(3) Materials and methods

(a) Cell culture and ELISA

RPMI 1640 (Enjer #31870 or Gibco # 61870-010) supplemented with 10% fetal bovine serum (Enjer # 10108-157), 1% L-glutamine (Enjer # 25030-03), 1% penicillin/streptomycin (Enjer # 15140-148), 10. Mu.M 2-mercaptoethanol (Gibco # 31350-010), 5mM Hepes (Gibco # 15630-080)

Recombinant human IL-1. Beta. Was purchased from Yiqiao Shenzhou Co (# 10139-HNAE-5)

Recombinant human IL-18 was purchased from MBL (#B001-5)

Recombinant human IL-12 was purchased from Boqi company (# 573008)

Ifnγ ELISA: MAX standard suite, bosch, #430103 or BD OptEIA human ifnγ ELISA suite, BD #555142

IL-6ELISA: MAX standard suite, boqi inc, #430503

IL-26ELISA: cloud Clone Corp- # SEB695Hu

mAb2 as described in the IL-1 beta antibody section.

mAb1 as described in the IL-18 antibody section.

bbmAb1 as described in example 1.

LPS is derived from salmonella enteritidis serotype salmonella enteritidis, sigma #L7770

Isolation of PBMC from buffy coat (from Blutspendezentrum Bern Co)

96 well plate treated with round bottom tissue culture (Costar Co. # 3799) 96 well plate treated with flat bottom tissue culture (Costar Co. # 3596) Ficoll-Pacque ^TM Plus (GE medical health life sciences # 17-1440-02) PBS1X, free of calcium and magnesium (Gibco Corp # 14190094)

Falcon 15ml polypropylene conical tube (BD company # 352096) Falcon 50ml polypropylene conical tube (BD company # 352070)

Leucoep tube with porous barrier, 50ml, greiner bio-one #227290

Cell filter 70. Mu.M, BD bioscience Co #352350

Trypan blue, sigma # T8154

RNA isolation, quantitative and quality measurements and qPCR:

nuclease-free water, ambion #AM9938

Rnase Zap, ambion company #am9780

1.5ml Eppendorf tube, sterile, ribonuclease-free and deoxyribonuclease

RLT buffer, kaijia #1015762

Rneasy mini kit, kaijia #74104

Ribonuclease-free deoxyribonuclease set, kaijer #79254

Agilent company RNA 6000 Nano kit, agilent company #5067-1511

Chip initiating station, agilent company #5065-4401

IKA vortex mixer

Ambion #9780

Agilent 2100 biological analyzer

High capacity cDNA reverse transcription kit, applied biosystems, # PN4374966

Nase-free thin-walled capped 0.2ml PCR tube, ambion #AM12225

MicroAmp Optical 384-well reaction plate, applied biosystems #4309849

TaqMan GenEx premix, applied biosystems #4369514

PCR primer (applied biosystems company)

Target(s)	Determination of ID Taqman	Color/quencher
			IFNγ	Hs00989291_m1	FAM-MGB
IL-26	Hs00218189_m1	FAM-MGB
			RPL27	Hs03044961_g1	FAM-MGB
HPRT1	Hs02800695_m1	FAM-MGB

PBMC preparation: PBMCs were isolated from buffy coats by Ficoll-Paque gradient centrifugation in a leucoep tube according to the manufacturer's instructions. Briefly, 15mL of Histopaque was placed in a 50mL Leucoep tube and centrifuged at 1300rpm for 30 seconds at room temperature. 30mL of the diluted suspension of buffy coat was added to the top of the Histopaque solution with a pipette and centrifuged at 1000g for 15min without interruption at room temperature. Plasma (about 20 ml) was discarded, interface loops (=human PBMC) were collected and transferred to 50ml falcon tubes. The tube was filled with 50mL of sterile PBS and centrifuged at 1200rpm for 5min at room temperature. The centrifugation was repeated 2 times. The supernatant was gently discarded and the cells were resuspended in 50mL of PBS containing 2% FCS and 2mM EDTA. The cell suspension was filtered using a 70 μm cell filter and the cells were counted using trypan blue staining (500 μl trypan blue +200 μl cells +300 μl LPBS).

LPS/IL-12 stimulated PBMC: cytokine production in the supernatant was prepared according to the following method. 250' 000 cells/well (final volume 100 μl) were dispensed into 96-well round bottom plates. LPS was used at a concentration between 0.3. Mu.g/ml and 3000. Mu.g/ml, together with 10ng/ml recombinant IL-12. At 37℃and 10% CO ₂ Supernatant was harvested after 24h down.

RNA extraction from cell pellet was performed according to the following. Will be 3x 10 ⁶ Individual cells/well were dispensed into flat bottom 24-well plates in a final volume of 1000 μl. 3. Mu.g/ml LPS was used together with 10ng/ml recombinant IL-12. At 37℃and 10% CO ₂ After 24h down, the cells were harvested.

Stimulation of PBMCs with recombinant cytokines: 7x 10 per well of a 12 well plate ⁶ The PBMCs were used in 1.5ml final complete RPMI medium. Recombinant cytokines were added at the following final concentrations: 10ng/ml recombinant IL-1 beta, 3nM recombinant IL-18, 1ng/ml weightGroup IL-12. At 37℃and 10% CO ₂ Supernatant and cells were collected after 4 hours and 24 hours.

RNA isolation, number and quality assessment: cells were pelleted and the pellet was lysed in 350 μl of Kaiji RTL buffer containing 2% beta-mercaptoethanol and frozen at-20℃or-80℃until all study samples were collected. RNA isolation was performed using the standard protocol of Kaijer. Briefly, 350. Mu.l of 70% ethanol was added to all samples, which were then transferred to an RNeasy centrifuge column and centrifuged at 8000g for 15s. After discarding the flow-through, 350. Mu.l of buffer RW1 was added, and the column was centrifuged at 8000g for 15s to wash the column membrane. The DNase I incubation mixture was prepared according to the manufacturer's instructions and added to the RNeasy spin column and incubated for 15min at room temperature. After washing with 350. Mu.l and 500. Mu.l of buffer RW1, the RNeasy cartridge was placed in a new 2ml collection tube and centrifuged at full speed for 1min. Finally, RNA was collected by adding 35. Mu.l of water free of ribonuclease directly to the spin column membrane and centrifuging at 8000g for 1min to elute RNA. The amount of RNA was measured using Nanodrop ND-1000 and RNA was stored at-20 ℃. RIN measurements were performed according to the manufacturer's instructions to assess RNA quality. Briefly, 1 μl of RNA or gradient was pipetted into an agilent RNA 6000Nano chip and measured using an agilent 2100 bioanalyzer.

Cytokine gene expression was analyzed by qPCR:

the method is performed according to the manufacturer's instructions. Briefly, 400ng of RNA was reverse transcribed using a high capacity cDNA reverse transcription kit according to the instructions. The cDNA solution was diluted 1/10 in RNA/DNA free water, then 1. Mu.l cDNA was transferred to 384 well reaction plates, then 1. Mu.l 20XGene expression assay target FAM Gene and 10 μl 2 xThe gene expression premix was mixed with 10. Mu.l of RNA/DNA free water. Loading boards to application biosystems ViiA ^TM 7 on a real-time PCR System, and useThe following instrument settings: />

Housekeeping genes used in this study were HPRT1 and RLP27. The relative expression level of the target gene was calculated using the following formula:

1) Ct [ reference ] = (Ct [ HPRT1] + Ct [ RLP27 ])/-2

2) dCt [ reference ] =40-Ct [ reference ]

3) dCt [ target ] =ct [ target ] -Ct [ reference ]

4) ddct=dct [ reference ] -dCt [ target ]

5) Expression of the relative target gene = 2 ddct

The microarray was performed according to the following method. Samples were processed on an Affymetrix HG_U133_Plus2 microarray by CiToxLAB France. They were RMA normalized and analyzed in GeneSpring 11.5.1 (agilent technologies, inc., santa clara, california). Pathway analysis was performed using the inventive pathway analysis (Ingenuity Pathway Analysis, IPA) and Nextbio (enomilna corporation (Illumina)). The two data sets are processed independently.

Initially, data were subjected to standard Quality Control (QC) by CiToxLAB, internal QC using R script (ma_affyqc.r) in Rstudio kit and GeneSpring (PCA, hybridization control). It was then filtered to eliminate unreliable expression levels: the entity (probe set) was retained, wherein at least 100% of the samples had values above 20 percentile in any 1 experimental condition.

Differentially Expressed Genes (DEG) were identified using the "volcanic pattern filter (filter on volcano plot)" function in GeneSpring. Using filtered genes with unpaired T-test (expressed between 20.0-100.0 percentiles), probe sets with corrected p-values below 0.05 and fold changes above 2.0 were considered differentially expressed. Where possible, i.e., in the study of LPS (NUID-0000-0202-4150), benjamini-Hochberg multiplex test corrections were used.

For cytokine stimulation experiments, synergy was calculated using the following formula: signal A+B/(Signal A+Signal B-control) is not less than 1.5.

The respective feature (or DEG list) is used to calculate a p-value for the Fisher's exact test case, which represents the statistical significance of observing the overlap between the feature and the "disease gene list" (lesion versus non-lesion) of the public dataset. To this end, the list is uploaded into the enomilna company basic spatial correlation engine (formerly Nextbio) and compared using Meta-Analysis (Meta-Analysis) functionality with keyword searches for disease.

All data were exported to EXCEL software and IC was calculated by plotting dose response curves for a logical curve fit function using EXCEL/XLfit4 or GraphPad Prism software ₅₀ Values. Differences between treatment groups were analyzed by one-way anova using GraphPad Prism software followed by multiple Dunnett (Dunnett) comparisons and the results were considered at p<At 0.05 there was statistical significance.

(4) Results

(a) bbmAb1 is very effective in inhibiting LPS/IL-12-induced IFNγ production in whole blood

Exposure of human whole blood to LPS supplemented with 10ng/ml IL-12 results in an IFNγ response that is largely (but not entirely) dependent on the "native" IL-18 produced by blood cells. The addition of IL-12 may enhance the LPS-induced IFN gamma response by upregulating the IL-18 receptor on the responsive cells.

Under the experimental conditions used, IL-18 neutralization with mAb1 resulted in only incomplete inhibition of ifnγ production, whereas IL-1β blocking (with mAb 2) had only a small effect on ifnγ response. Interestingly, the combined inhibition of IL-1β and IL-18 by bbmAb1 or a combination of mAb2 and mAb1 inhibited IFNγ production more profoundly and completely than single cytokine neutralization. Inhibition of LPS (0.3 μg/ml)/IL-12 induced IFNγ in whole blood by bbmAb1, mAb2, mAb1 or a combination of mAb2 and mAb1 (Combo).

In our cellular assays, no other cytokines tested (IL-2, -4, -6, -8, -10, -13, and TNFα) other than IFNγ were inhibited by the combined neutralization of IL-1β and IL-18. The potency of bbmAb1 is in the same range as the combination of mAb2 and mAb1 (combo), considering the monovalent form of the bispecific molecule.

(b) In LPS/IL-12 activated human PBMC, IFNγ is inhibited additively by bbmAb1 (i.e., combined IL-1. Beta./IL-18 inhibition) compared to either IL-1. Beta. Or IL-18 inhibition alone

Non-deflected transcriptomic assessment is required in order to reveal additional additive effects (except ifnγ) by combined IL-1β/IL-18 inhibition using bbmAb 1. Since whole blood is not the optimal material for transcriptomic analysis, we adapted the LPS/IL-12 stimulation assay conditions (as described in the materials and methods section above) to human PBMC samples. By using PBMCs from a total of 9 donors, we can demonstrate that bbmAb1 additively inhibits ifnγ protein secretion into PBMC supernatant. Ifnγ production was inhibited compared to whole blood experiments, approximately 10-fold lower concentration than each mAb used. Importantly, similar inhibition patterns were shown at the mRNA level of ifnγ, confirming that the samples were suitable for unbiased microarray-based gene expression analysis. It was demonstrated that bbmAb1, mAb2 and mAb1 (at 10nM concentration each) inhibited LPS (0.3. Mu.g/ml)/IL-12-induced IFN gamma protein production and IFN gamma gene expression in human PBMC.

Affymetrix microarrays were performed with n=5 individual donors from PBMC, which were sampled from the LPS/IL-12 stimulation experiments described in the materials and methods section above. Unfortunately, overall evaluation of gene expression profiles demonstrated strong LPS/IL-12 stimulation, PCA showed clusters per donor, rather than compounds within stimulated or unstimulated groups. However, comparing LPS/IL-12 stimulated samples with bbmAb1 for differentially expressed genes revealed a list of genes down-regulated by IL-1β/IL-18 blockade using the combination of bbmAb1 (Table 14). In addition to the strong downregulation of the ifnγ gene (which reconfirms our microarray data), the IL-26 gene is also another cytokine gene that is additively inhibited by bbmAb1, compared to either IL-1β inhibition alone (by mAb 2) or IL-18 inhibition (by mAb 1).

Table 14. Genes differentially expressed (genes down-regulated only between bbmAb1 and control in LPS/IL-12 stimulated samples). FC = fold change.

(c) IL-26 is another pro-inflammatory cytokine that is inhibited by bbmAb1 accumulation in LPS/IL-12 stimulated PBMC

To further demonstrate that the inhibition of LPS/IL-12 driven IL-26 gene expression and protein production by combined IL-1 β/IL-18 blockade using bbmAb1 was most effective, the study was extended to a total of n=9 PBMC donors and IL-26 gene expression was studied by qPCR and IL-26 protein production was studied by ELISA. ELISA largely confirmed the inhibition of IL-26 gene expression obtained by microarray methods. Interestingly, by adding mAb, the level of IL-26 protein in the supernatant was only partially reduced at 24 h. The reasons for this difference are not clear, but may be related to the kinetic differences between IL-26 gene expression and protein production and the differences in IL-26 consumption compared to IFNγ. However, bbmAb1 shows an advantage in reducing IL-26 protein levels in PBMC supernatants compared to mAb2 and mAb 1.

(d) IL1 beta/IL 18 signaling characteristics associated with disease

Previously established PBMC culture conditions in which recombinant IL-1 beta stimulation resulted in IL-6 production or recombinant IL-18/IL-12 stimulation resulted in ifnγ production were combined to reveal additive or synergistic downstream target genes or characteristics (data not shown). PBMCs from n=4 donors were sampled at two different time points (6 h and 24 h) and subjected to Affymetrix microarray evaluation for unbiased evaluation of gene expression profiles. Genes that were synergistically upregulated at 6h and 24h under stimulation with a combination of IL-1 beta and IL-18 were disclosed (data not shown). The addition of IL-12 to the IL-1 beta/IL-18 combination greatly enhances the synergy of a series of upregulated genes. The signaling characteristics generated by stimulation of the IL-1 beta/IL-18 pathway (up-regulated genes alone) alone or in combination are used to interrogate datasets throughout several autoimmune disease patients. For example, a correlation with a public sarcoidosis dataset is identified. P-values (calculated by Fisher's exact test) showed significant correlation with several public studies comparing healthy tissue and diseased tissue of patients with sarcoidosis. Tissues include skin, lung, lacrimal gland and anterior orbital part. In all data sets, the combination of IL1 beta/IL 18 signaling showed the best correlation with disease, followed by IL-1 beta and IL-18. IL-1β/IL-18 differential upregulation genes (DEG) in PBMC compared to 5 different sarcoidosis tissue "diseased relatively healthy" DEG.

(e) Conclusion(s)

LPS and recombinant IL-12 were used to mimic pathogen-associated molecular pattern (PAMP) -dependent NLRP3 inflammasome activation in the first 24h of in vitro culture. It was demonstrated that by using bbmAb1, the combined inhibition of IL-1 beta and IL-18 acts additively to reduce/inhibit IFNγ production in LPS/IL-12 stimulated PBMC. IL-12 and IL-18 synergistic effects to induce IFNγ production in T, B, NK cells, macrophages and dendritic cells were previously described (as reviewed in Nakanishi, 2001), but it can now be demonstrated that IL-1 β produces additional stimulation of IFNγ under the experimental conditions used. Thus, co-incubation of PBMC with LPS/IL-12 effectively driven the production of "native" IL-1. Beta. And IL-18 (both of which contribute to a strong IFNγ response). By using unbiased microarray transcriptomics, additional genes were identified that were down-regulated by combined IL-1β/IL-18 neutralization relative to single IL-1β or IL-18 blocking. Including IL-26, which is a member of the IL-20 cytokine subfamily (IL-19, IL-20, IL-22, IL-24, and IL-26), IL-26 is conserved in most vertebrate species, but absent in most rodent strains, including mice and rats (Donnely 2010). It transmits signals through a heterodimeric receptor complex consisting of IL-20R1 and IL-10R2 chains. IL-26 receptors are expressed primarily on non-hematopoietic cell types, particularly on epithelial cells. IL-26 levels are reported to be elevated in serum and especially in synovial fluid of RA patients, where IL-26 may act as a factor that promotes Th17 cell growth and differentiation. Unfortunately, the strong effect of LPS/IL-12 stimulation of PBMC samples prevented the discovery of additional genes/pathways induced by the combined blockade of IL-1β and IL-18. However, both ifnγ and IL-26, as well as IL-22, are also to some extent in genes that are synergistically upregulated by stimulation of the combination of recombinant IL-1 β and IL-18 in PBMCs, confirming that these two factors are downstream effectors of this activation pathway. Thus, the IL-20 subfamily of cytokines (including IL-26 and IL-22) appears to be strongly dependent on simultaneous signals from IL-1β and IL-18. These comparisons help to show that each pathway is active in sarcoidosis and the like, given sufficient attention paid to the selectivity of individual signal features and the potential efficacy of blocking.

Example 6: inhibition of spontaneous IFNγ, TNF α and IL-2 production by bbmAb1

A punch biopsy (2 mm) was taken from surgically excised skin of 9 different HS patients and incubated for 24 hours in 80. Mu.L of Medium (Iscove's Modified Dulbecco's Medium) supplemented with 10% knockout serum replacement (Gibco Co.) and 1% penicillin-streptomycin in Dulbecco's Medium modified by Iscove's Modified Dulbecco's Medium) under the following conditions: in 96-well cell culture plates (flat bottom, tissue culture treated; costar corporation) at 37 ℃ and 5% CO2, in medium as untreated control (fig. 3, leftmost column) or in the presence of bbmAb1 (fig. 3, middle column) or adalimumab (fig. 3, rightmost column) at a concentration of 100 ug/ml. After plate centrifugation, supernatants were collected from individual HS biopsies and multiplex MSD (Meso Scale Discovery platform) cytokine pro-inflammatory series 1 (protein) arrays were performed using an MSD plate reader according to the manufacturer's protocol. Data were normalized with single biopsy weight and exported to GraphPad Prims software for blot. Figure 3 shows that bbmAb1 reduces spontaneous ifnγ, tnfα and IL-2 production in HS biopsy supernatants.

Example 7: toxicity study

When up to 100mg/kg of the bispecific antibody bbmAb1 was administered twice weekly for 26 circumferential marmoset s.c. this bispecific antibody bbmAb1 was well tolerated (no level of action (NOEL) 100mg/kg, cmax, ss 3,110 μg/mL, AUC0-72h, ss 218,000 μ·h/mL) was observed, and did not show any safe pharmacology (central nervous, cardiovascular and respiratory), toxicology (including male reproductive and sperm motility) or local tolerization. No effect was seen after 26 weeks of intravenous (i.v.) administration at 100mg/kg twice a week (Cmax, ss: 4570. Mu.g/mL, AUC0-72h, ss: 261,000. Mu.g.h/mL). Furthermore, no therapeutic-related effects were found for the primary and secondary humoral immune responses following immune cell and foreign antigen challenge in peripheral blood. In a Single Ascending Dose (SAD) study of bbmAb1, the data (A1 to B1) for the first six groups of 0.1, 0.3, 1, 3, 10mg/kg i.v. and 100mg s.c. with respect to dose increase in a total of 48 subjects (12 of which are placebo-treated subjects) bbmAb1 is generally well tolerated at doses up to 10 mg/kg. The Cmax and AUC of bbmAb1 increased in a dose-proportional manner over the entire range of i.v. administration (0.1 mg/kg-10 mg/kg). The average half-life of bbmAb1 was about 21 to 26 days. The bioavailability of the s.c. dose was estimated to be 70%.

Example 8: therapeutic use

Randomized, subject and researcher double-blind, placebo-controlled and multicenter platform studies to assess efficacy and safety of bbmAb1 in moderate to severe suppurative sweat gland patients

Detailed clinical trial designs are provided below to demonstrate the efficacy of the bispecific anti-IL-1 beta anti-IL-18 antibody bbmAb 1.

Double-blind subjects and researchers allow for unbiased assessment of subjective readings, such as lesion counts of HS or overall HS-PGA scores, as well as adverse events.

A randomized, subject and researcher double-blind, placebo-controlled, multicenter and parallel group study was performed to assess the efficacy, safety and tolerability of several active therapeutic compounds (e.g., bispecific anti-IL-1β anti-IL-18 antibody bbmAb 1) in moderate to severe HS subjects. After a screening period of about 5 weeks, a treatment period of 16 weeks was planned, followed by a safety follow-up of about 12 weeks. Subjects were given bbmAb1, 300mg (3 injections, 1.5mL each; once every two weeks, on days 1, 15 and 29, then once monthly (Q4W) on days 57 and 85) s.c., or their corresponding placebo (2 x 1.5 mL) s.c.

The study included subjects were adult male and female subjects aged 18 to 65 years (inclusive) who exhibited a diagnosis of moderate to severe HS with recurrent inflammatory lesions for at least 12 months. At randomization (day 1 prior to dosing), the subject is required to have at least 5 inflammatory lesions (abscesses and nodules) in at least 2 anatomical regions to be included. The group and the corresponding group will be randomized using a centralized Interactive Response Technology (IRT) system.

After 16 weeks of treatment, the primary clinical endpoint was simplified hissc (clinical response to hidradenitis suppurativa).

On day 113 (week 17), after safety and other evaluations, all subjects will enter the follow-up period and will not receive any additional study drug administration. If medically sound and potential safety hazards are not identified (after discussion with the sponsor), the subject may receive previously prohibited medications during this follow-up.

Safety and efficacy assessments will be performed in subsequent follow-up visits as specified in the assessment schedule. Pharmacokinetic (PK), pharmacodynamic (PD) and biomarker samples will also be collected. End of study (EOS) visit will occur on day 197 (week 29) and will include completion of study evaluation and then release from the study. The investigator and subject will remain blind until the study is completed.

Approximately 40 subjects were randomized; 30 subjects will receive study treatment and 10 subjects will receive matched placebo.

On day 1, 300mg of bbmAb1 or its corresponding placebo (2 injections of 1.5mL each) will be administered by subcutaneous injection (s.c.) by trained field staff. Clinical assessment will be performed as well as PK, target engagement, immunogenicity, pathway and disease biomarkers, and safety assessment. Subjects will be discharged from the site on the same day as all evaluations are completed, provided that there are no safety issues. After the first administration, the subject should be observed in situ for at least one hour, or more (as determined by the researcher), of the immediate injection site response.

Subjects will return to the study center during the loading phase of the study (from day 1 (week 1) to day 29 (week 5)) receiving bbmAb1 (Q2W; 3 doses) at intervals s.c.

Then, during the maintenance phase of the study (from day 29 (week 5) to day 85 (week 13)), bbmAb1 (Q4W; 2 doses) will be administered every four weeks at 300mg s.c.

Safety and selection efficacy assessments will be performed during these visits, and PK, target engagement, immunogenicity, and pathway/disease biomarker samples will be collected.

The main objective was to show the primary efficacy of bispecific antibody bbmAb1 treatment in HS subjects after 16 weeks of treatment compared to placebo. After a 16 week treatment period, a 12 week follow-up period was performed to observe that the sustainability of the effect could be maintained or improved after 16 weeks of treatment.

For this study, simplified HiSCR (modified from Kimball 2014) was chosen as the primary endpoint. Simplified hissc was defined as 50% reduction in abscess plus inflammatory nodule total, while drainage fistulae did not increase.

Inflammatory lesions of HS will be counted as single lesions (inflammatory nodules, abscesses, and drainage fistulae) in a typical anatomical region. In addition to the counts, an overall assessment scale (hidradenitis suppurativa-physician overall assessment or HS-PGA) and a composite score (hidradenitis suppurativa score severity assessment or SAHS) will be used.

The outcome of several patient reports will be used, including dermatological quality of life index (DLQI). Finally, skin-related pain is the most important symptom, as from the perspective of the subject, and thus includes a Numerical Rating Scale (NRS) for pain.

Additional information about clinical assessment:

HS-PGA (hidradenitis suppurativa-physician overall evaluation): this score will be used as an exploratory goal for assessing HS and is used and described in Kimball AB, kerdel F, adams D et al (2012).

The SAHS score is a composite score (Hessam S, scholl L, sand M et al (2018)) and will be derived from the collected inflammatory lesions count, fistula count, and NRS pain information. In addition, anatomical areas and new or red existing furuncles will be collected in both groups.

Skin pain-NRS (numerical rating scale for pain): NRS for skin-related pain was used in adalimumab studies (kimbal et al (2016)) and used as a skin-or HS-related pain, which is one of the most burdensome to patients (Matusiak et al (2017)). The average and worst values of pain associated with HS over the last 24 hours (over the last 24 hours) will be recorded.

The outcomes (PRO) reported by other patients will include itch, fatigue and work disorders as well as aspects of dermatological quality of life index (DLQI) and dermatological quality of life (QoL) tools with providing a validated score in many countries and languages. It includes an overall patient assessment.

Target and related endpoint

Key inclusion criteria:

male and female subjects aged 18 to 65 years (inclusive) clinically diagnosed with HS at least 12 months prior to screening

Patients with moderate to severe HS according to the evaluation at screening and randomization (day 1 pre-dosing):

a total of at least 5 inflammatory lesions, i.e. abscesses and/or inflammatory nodules, and

no more than 15 fistulae, and

HS lesions require involvement of at least two anatomical regions

The minimum weight at the time of screening was 50kg (inclusive)

Can communicate well with researchers and understand and follow the requirements of the study, and is capable and willing to follow the study schedule for study visits.

Key exclusion criteria:

other study drugs were used at screening, or within 30 days or 5 half-lives of randomization (whichever is longer); or if local regulations require longer use of other study drugs or use of immunosuppressants.

WOCBP (defined as all women who are physiologically pregnant) will be required to adhere to high performance contraception from at least 3 months prior to the first drug administration until 5 months after the last dose (day 225 to 253), at which point pregnancy tests will be conducted.

Pregnant or lactating (lactating) women at screening or randomization, wherein pregnancy is defined as the state of the woman after conception until pregnancy is terminated, confirmed by positive hCG laboratory tests.

Study treatment and duration

Patients assigned to bbmAb1 group will receive bbmAb1 (300 mg, s.c.) or matched placebo as follows: once every two weeks (Q2W) from day 1 (week 1) to day 29 (week 5), then once a month (Q4W) until day 85 (inclusive) (week 13)

Efficacy assessment

Simplified and original clinical response to hidradenitis suppurativa (HiSCR)

International sweat gland suppurative severity scoring System (IHS 4)

Suppurative sweat gland inflammation-physician global assessment (HS-PGA) score and responder rate

HS inflammatory lesions count

Severity Assessment of Hidradenitis Suppurativa (SAHS)

For bbmAb1, the dosage form of the provided drug is a "ready-to-use" buffered sterile aqueous solution. The solution contained 100mg/ml bbmAb1 and the excipients L-histidine, sucrose and polysorbate 20, pH 6.0. The placebo control selected for this study was a solution of a matched composition containing inactive excipients.

Predicted effect of bbmAb1 dose on IL-1β and free IL-18

The model for predicting the kinetics of anti-IL-1 β/IL-18 bispecific antibodies and their targets in serum consisted of: a general competitive binding model for the IL-18 group (Yan et al 2012) and a previously published model for the kana Ji Nushan antibody to bbmAb1 for the IL-1 β group (Chakraborty et al 2012), wherein the new model describes free and total IL-18 kinetics. To predict kinetics of IL-1 β response to bbmAb1 application, a model established in the clinical kana Ji Nushan resistance study (Chakraborty et al 2012) was used, and bbmAb1 specific PK parameters and binding affinity were updated accordingly. To modulate IL-1 β concentrations in CAPS patients, we used the synthesis and clearance parameters of this interleukin listed in the kana Ji Nushan anti-clinical study (Chakraborty et al 2012). The model is based on internal in vitro and published human data from free and total IL-18 serum concentrations throughout several autoimmune disease patients (Weiss et al 2018).

Based on this, it was predicted that the dosing schedule of 300mg Q2W/Q4 ws.c. would result in simultaneous reduction of both IL-1 beta and IL-18 levels in serum, and that efficient neutralization of IL-1 beta and IL-18 was expected (fig. 4 and 5).

Pharmacokinetics of bbmAb1

Up to 10mg/kg i.v. bbmAb1 was evaluated in healthy volunteers without any drug related SAE in a FiH single dose escalation study. The Pharmacokinetics (PK) of bbmAb1 in humans followed human predictions based on marmoset data, and as expected for binding of a typical IgG1 antibody to one or more soluble ligand cytokine targets. bbmAb1 showed a linear increase in exposure dose matched to predicted human PK (rated up to 10mg/kg i.v.). The predicted human PK parameters for bbmAb1 are: clearance (CL) =0.158L/d, distribution volume (V _d ) = 5.586L (for 70-kg human subjects) or 0.08L/kg; half-life (T) _1/2 ) =24.5 days. Preliminary analysis of PK profile from FiH study provided no evidence of accelerated clearance of bbmAb1 due to formation of anti-drug antibodies (ADA).

Based on recent subcutaneous data from the FiH study, bioavailability and absorption constants were adjusted for these findings and used to predict subcutaneous PK.

The 300mg dose of Q2W/Q4 ws.c. bbmAb1 was predicted to result in rapid and simultaneous neutralization of all systemic free IL-1 beta and IL-18. After a single dose, 300mg s.c. post exposure will exceed in vitro IC90 for IL-1 beta and IL-18 for more than 100 days (fig. 6).

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Hessam S,Scholl L,Sand M,et al(2018)A Novel Severity AssessmentScoring System for Hidradenitis Suppurativa.JAMA Dermatol；154(3):330-335.

Hessam S,Sand M,Gambichler T,et al(2015)Correlation ofinflammatory serum markers with disease severity in patients withhidradenitis suppurativa(HS).J Am Acad Dermatol.；73(6):998-1005.

Houriet C,Seyed Jafari SM,Thomi R,et al(2017)Canakinumab forSevere Hidradenitis Suppurativa:Preliminary Experience in 2 Cases.JAMADermatol.；1；153(11):1195-1197.

Hunger RE,Surovy AM,Hassan AS,et al(2008)Toll-like receptor 2 ishighly expressed in lesions of acne inversa and colocalizes with C-type lectinreceptor.Br.J.Dermatol.；158(4):691-7.

Ingram JR,Woo PN,Chua SL,et al(2016)Interventions forhidradenitis suppurativa:a Cochrane systematic review incorporatingGRADE assessment of evidence quality.Br.J.Dermatol.；174(5):.970-8.

International Council for Harmonization of Technical Requirements forPharmaceuticals for Human Use(ICH)(2016)ICH E6(R2)GCP Guideline(2016).

Jaeger T,Andres C,Grosber M,et al(2013)Pyoderma gangrenosumand concomitant hidradenitis suppurativa-rapid response to canakinumab(anti-IL-1β).Eur J Dermatol.；23(3):408-410.

Janse IC,Deckers IE,van der Maten AD,et al(2017)Sexual health andquality of life are impaired in hidradenitis suppurativa:a multicentrecross-sectional study.Br.J.Dermatol.；176(4):1042-1047.

Jemec GB(2012)Clinical practice.Hidradenitis suppurativa.N.Engl.J.Med.；366(2):158-64.

Jemec GB,Kimball AB(2015)Hidradenitis suppurativa:Epidemiology and scope of the problem.J.Am.Acad.Dermatol.；73(5 Suppl 1):S4-7.

AR,Yao Y,Thomsen SF(2018)Therapeutic Response to Secukinumab in a 36-Year-Old Woman with Hidradenitis Suppurativa.Case Rep Dermatol Med；(Apr 16):8685136..

Kanni T,Zenker O,Habel M,et al(2018)Complement activation in hidradenitis suppurativa:a new pathway of pathogenesisBr.J.Dermatol.；179(2):413-419

Kelly G,Sweeney CM,Tobin AM,et al(2014)Hidradenitis suppurativa:the role of immune dysregulation.Int.J.Dermatol.；53(10):1186-96.

Kelly G,Hughes R,McGarry T,et al(2015)Dysregulated cytokine expression in lesional and nonlesional skin in hidradenitis suppurativa.Br J Dermatol.；173(6):1431-1439.

Kimball AB,Ganguli A,Fleischer A(2018)Reliability of the hidradenitis suppurativa clinical response in the assessment of patients with hidradenitis suppurativa.J Eur Acad Dermatol Venereol.；32(12):2254-2256.

Kimball AB,Jemec GB,Yang M,et al(2014)Assessing the validity,responsiveness and meaningfulness of the Hidradenitis Suppurativa Clinical Response(HiSCR)as the clinical endpoint for hidradenitis suppurativa treatment.Br.J.Dermatol.；171(6):1434-42.

Kimball AB,Kerdel F,Adams D,et al(2012)Adalimumab for the treatment of moderate to severe Hidradenitis suppurativa:a parallel randomized trial.Ann.Intern.Med.；157:846-855.

Kimball AB,Okun MM,Williams DA,et al(2016)Two Phase 3 Trials of Adalimumab for Hidradenitis Suppurativa.N.Engl.J.Med.；375(5):422-34.

Leslie KS,Tripathi SV,Nguyen TV,et al(2014)An open-label study of anakinra for the treatment of moderate to severe hidradenitis suppurativa.J Am Acad Dermatol.；70(2):243-251.

Lima AL,Karl I,Giner T,et al(2016)Keratinocytes and neutrophils are important sources of proinflammatory molecules in hidradenitis suppurativa.Br.J.Dermatol.；174(3):514-21.

Ljungman P,Boeckh M,Hirsch HH,et al(2017)Disease Definitions Working Group of the Cytomegalovirus Drug Development Forum.Definitions of Cytomegalovirus Infection 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 Peripheral Blood and Skin of Patients with Hidradenitis Suppurativa.J Invest Dermatol；140(5):1091-1094 Penno CA,P,Laguerre C et al(2020)Lipidomics Profiling of Hidradenitis Suppurativa Skin Lesions Reveals Lipoxygenase Pathway Dysregulation and Accumulation of Pro-Inflammatory Leukotriene B4.J Invest Dermatol.；140(12):2421-2432..

Z,Fougerousse AC,Maccari F,et al(2020)Effectiveness of secukinumab in hidradenitis suppurativa:an open study(20 cases).J Eur Acad Dermatol Venereol.；34(11):e750-e751.

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Riis PT,K,Saunte DM,et al(2015)Patients with hidradenitis suppurativa carry a higher systemic inflammatory load than other dermatological patients.Arch Dermatol Res.；307(10):885-9.

Russo V,Alikhan A(2016).Failure of Anakinra in a Case of Severe Hidradenitis Suppurativa.J Drugs Dermatol.；15(6):772-774.

Schuch A,Fischer T,Boehner A,et al(2018)Successful Treatment ofSevere Recalcitrant Hidradenitis Suppurativa with the Interleukin-17AAntibody Secukinumab.Acta Derm.Venereol.；98(1):151-152.

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Shah A,Alhusayen R,Amini-Nik S(2017)The critical role ofmacrophages in the pathogenesis of hidradenitis suppurativa.Inflamm.Res.；66(11):931-945.

Sharon VR,Garcia MS,Bagheri S,et al(2012)Management ofrecalcitrant hidradenitis suppurativa with ustekinumab.Acta Derm.Venereol.；92(3):320-1.

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Sun NZ,Ro T,Jolly P,et al(2017)Non-response to Interleukin-1Antagonist Canakinumab in Two Patients with Refractory PyodermaGangrenosum and Hidradenitis Suppurativa.J Clin Aesthet Dermatol.；10(9):36-38.

Tekin B,Salman A,Ergun T(2017)Hidradenitis suppurativaunresponsive to canakinumab treatment:A case report.Indian J DermatolVenereol Leprol.；83(5):615-617.

Thorlacius L,Theut Riis P,Jemec GBE(2018)Severe hidradenitissuppurativa responding to treatment with secukinumab:a case report.Br.J.Dermatol.；179(1):182-185.

Tzanetakou V,Kanni T,Giatrakou S,et al(2016)Safety and Efficacyof Anakinra in Severe Hidradenitis Suppurativa:A Randomized ClinicalTrial.JAMA Dermatol；152(1):52-59.

Van der Zee HH,Laman JD,de Ruiter L,et al(2012)Adalimumab (antitumour necrosis factor-α)treatment of hidradenitis suppurativa ameliorates skin inflammation:an in situ and ex vivo study.Br.J.Dermatol；166(2):298-305.

van der Zee HH,Prens EP(2013)Failure of anti-interleukin-1 therapy in severe hidradenitis suppurativa:a case report.Dermatology；226(2):97-100.Weber P,Seyed Jafari SM,Yawalkar N,et al(2017)Apremilast in the treatment of moderate to severe hidradenitis suppurativa:A case series of 9 patients.J.Am.Acad.Dermatol.；76(6):1189-1191.

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E,Wolk K,Tsaousi A,et al(2019)The IL-1 Pathway Is Hyperactive in Hidradenitis Suppurativa and Contributes to Skin Infiltration and Destruction.J Invest Dermatol.；139(6):1294-1305.

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Zarchi K,Dufour DN,Jemec GB(2013)Successful treatment of severe hidradenitis suppurativa with anakinra.JAMA Dermatol.；149(10):1192-1194.

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Zouboulis CC,Desai N,Emtestam L,et al(2015)European S1 guideline for the treatment of hidradenitis suppurativa/acne inversa.J Eur Acad Dermatol Venereol；29(4):619-44.

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Example 9: development of high concentration formulations of bbmAb1 (100 to 120 mg/mL)

Eight different formulations were initially tested in a high-throughput plate-based assay.

F1 (120 mg/mL bbmAb1, 20mM sodium succinate, pH 5.0, 220mM sucrose, 0.04% polysorbate 20);

f2 (120 mg/mL bbmAb1, 20mM histidine/histidine-Cl, pH 5.0, 220mM sucrose, 0.04% polysorbate 20);

f3 (120 mg/mL bbmAb1, 20mM histidine/histidine-Cl, pH 5.5, 220mM sucrose, 0.04% polysorbate 20);

f4 (120 mg/mL bbmAb1, 20mM histidine/histidine-Cl, pH 6.0, 220mM sucrose, 0.04% polysorbate 20);

f5 (120 mg/mL bbmAb1, 20mM histidine/histidine-Cl, pH 6.5, 220mM sucrose, 0.04% polysorbate 20);

f6 (120 mg/mL bbmAb1, 20mM potassium phosphate, pH 7.0, 220mM sucrose, 0.04% polysorbate 20);

f7 (50 mg/mL bbmAb1, 20mM histidine/histidine-Cl, pH 5.5, 220mM sucrose, 0.04% polysorbate 20);

f8 (50 mg/mL bbmAb1, 20mM potassium phosphate, pH 7.0, 220mM sucrose, 0.04% polysorbate 20);

aggregate formation of formulations F1 to F8 was tested by size exclusion chromatography after 2 or 4 weeks at 40 ℃. The results show a trend of increasing aggregation as pH increases from 5 to 7, formulations F6 and F8 at pH 7.0 exhibited significant aggregation. See fig. 7.

The formation of degradation products of formulations F1 to F8 was also tested by size exclusion chromatography. The results show that the protein is stable in all formulations tested under the test conditions (25 ℃,4 weeks). However, labChip analysis under non-reducing conditions showed a clear trend of degradation product formation at lower pH values, with a formulation at pH 5.0 showing significant degradation product formation. See fig. 8.

Formulations F1 to F8 were tested for acidic and basic variant formation in response to heat stress (25 ℃, 4W) by charge zone band electrophoresis (CZE). Significant acidic variant formation was observed at high pH (F8) and significant basic variant formation was observed at low pH (F1 and F2). See fig. 9A and 9B.

The above formulations were also tested for resistance to freeze thawing and mechanical stress (agitation) and the results further supported successful stabilization of the formulation at pH 5.5, preferably 6.0, with 50 to 120mg/mL bbmAb1 (preferably 100mg/mL bbmAb 1) and excipients (e.g. sugars such as sucrose) and surfactants (e.g. polysorbates such as polysorbate 20).

Additional test modes include visual, turbidity (a 405 nm), dynamic light scattering, microfluidic imaging and LysC peptide mapping by liquid chromatography mass spectrometry.

Sequence listing

Amino acid and nucleotide sequences useful in the practice of the present invention are disclosed in table 15.

TABLE 15 sequence according to an embodiment of the invention

Throughout this application, if there is a difference between the specification text (e.g., table 15) and the sequence listing, the specification text is subject.

All patents and publications cited herein are hereby incorporated by reference in their entirety for all purposes.

Sequence listing

<110> North Co., ltd (NOVARTIS AG)

<120> bispecific antibodies for use in the treatment of hidradenitis suppurativa

<130> PAT059148

<140>

<141>

<150> 63/213686

<151> 2021-06-22

<150> 63/223479

<151> 2021-07-19

<160> 104

<170> patent In version 3.5

<210> 1

<211> 5

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 1

Ser Tyr Ala Ile Ser

1 5

<210> 2

<211> 17

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 2

Asn Ile Ile Pro Met Thr Gly Gln Thr Tyr Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 3

<211> 10

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 3

Ala Ala Tyr His Pro Leu Val Phe Asp Asn

1 5 10

<210> 4

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 4

Gly Gly Thr Phe Lys Ser Tyr

1 5

<210> 5

<211> 6

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 5

Ile Pro Met Thr Gly Gln

1 5

<210> 6

<211> 10

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 6

Ala Ala Tyr His Pro Leu Val Phe Asp Asn

1 5 10

<210> 7

<211> 119

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 7

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Lys Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Ile Pro Met Thr Gly Gln Thr Tyr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Ala Tyr His Pro Leu Val Phe Asp Asn Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 8

<211> 356

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 8

gaggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcagcag cgtgaaggtg 60

agctgcaagg ccagcggcgg caccttcaag agctacgcca tcagctgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcaac atcatcccca tgaccggcca gacctactac 180

gcccagaagt tccagggcag ggtgaccatc accgccgacg agagcaccag caccgcctac 240

atggagctga gcagcctgag gagcgaggac accgccgtgt actactgcgc cagggccgcc 300

taccaccccc tggtgttcga caactgggcc agggcaccct ggtgaccgtg agcagc 356

<210> 9

<211> 449

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 9

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Lys Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Ile Pro Met Thr Gly Gln Thr Tyr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Ala Tyr His Pro Leu Val Phe Asp Asn Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 10

<211> 1347

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 10

gaggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcagcag cgtgaaggtg 60

agctgcaagg ccagcggcgg caccttcaag agctacgcca tcagctgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcaac atcatcccca tgaccggcca gacctactac 180

gcccagaagt tccagggcag ggtgaccatc accgccgacg agagcaccag caccgcctac 240

atggagctga gcagcctgag gagcgaggac accgccgtgt actactgcgc cagggccgcc 300

taccaccccc tggtgttcga caactggggc cagggcaccc tggtgaccgt gagcagcgcc 360

agcaccaagg gccccagcgt gttccccctg gcccccagca gcaagagcac cagcggcggc 420

accgccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgagctgg 480

aacagcggcg ccctgaccag cggcgtgcac accttccccg ccgtgctgca gagcagcggc 540

ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 600

atctgcaacg tgaaccacaa gcccagcaac accaaggtgg acaagagggt ggagcccaag 660

agctgcgaca agacccacac ctgccccccc tgccccgccc ccgaggccgc cggcggcccc 720

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag gacccccgag 780

gtgacctgcg tggtggtgga cgtgagccac gaggaccccg aggtgaagtt caactggtac 840

gtggacggcg tggaggtgca caacgccaag accaagccca gggaggagca gtacaacagc 900

acctacaggg tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960

tacaagtgca aggtgagcaa caaggccctg cccgccccca tcgagaagac catcagcaag 1020

gccaagggcc agcccaggga gccccaggtg tacaccctgc cccccagcag ggaggagatg 1080

accaagaacc aggtgagcct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 1140

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccccgtgctg 1200

gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag caggtggcag 1260

cagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 1320

aagagcctga gcctgagccc cggcaag 1347

<210> 11

<211> 13

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 11

Ser Gly Ser Ser Ser Asn Ile Gly Asn His Tyr Val Asn

1 5 10

<210> 12

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 12

Arg Asn Asn His Arg Pro Ser

1 5

<210> 13

<211> 11

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 13

Gln Ser Trp Asp Tyr Ser Gly Phe Ser Thr Val

1 5 10

<210> 14

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 14

Ser Ser Ser Asn Ile Gly Asn His Tyr

1 5

<210> 15

<211> 3

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 15

Arg Asn Asn

1

<210> 16

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 16

Trp Asp Tyr Ser Gly Phe Ser Thr

1 5

<210> 17

<211> 110

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 17

Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn His

20 25 30

Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Tyr Ser Gly

85 90 95

Phe Ser Thr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 18

<211> 330

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 18

gatatcgtcc tgactcagcc ccctagcgtc agcggcgctc ccggtcagag agtgactatt 60

agctgtagcg gctctagctc taatatcggt aatcactacg tgaactggta tcagcagctg 120

cccggcaccg cccctaagct gctgatctat agaaacaatc accggcctag cggcgtgccc 180

gataggttta gcggatctaa gtcaggcact agcgctagtc tggctatcac cggactgcag 240

tcagaggacg aggccgacta ctactgtcag tcctgggact atagcggctt tagcaccgtg 300

ttcggcggag gcactaagct gaccgtgctg 330

<210> 19

<211> 216

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 19

Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn His

20 25 30

Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Tyr Ser Gly

85 90 95

Phe Ser Thr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 20

<211> 648

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 20

gatatcgtcc tgactcagcc ccctagcgtc agcggcgctc ccggtcagag agtgactatt 60

agctgtagcg gctctagctc taatatcggt aatcactacg tgaactggta tcagcagctg 120

cccggcaccg cccctaagct gctgatctat agaaacaatc accggcctag cggcgtgccc 180

gataggttta gcggatctaa gtcaggcact agcgctagtc tggctatcac cggactgcag 240

tcagaggacg aggccgacta ctactgtcag tcctgggact atagcggctt tagcaccgtg 300

ttcggcggag gcactaagct gaccgtgctg ggtcagccta aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggagctgcag gccaacaagg ccaccctggt gtgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgagggca gcaccgtgga aaagaccgtg gccccaaccg agtgcagc 648

<210> 21

<211> 5

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 21

Val Tyr Gly Met Asn

1 5

<210> 22

<211> 17

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 22

Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 23

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 23

Asp Leu Arg Thr Gly Pro Phe Asp Tyr

1 5

<210> 24

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 24

Gly Phe Thr Phe Ser Val Tyr

1 5

<210> 25

<211> 6

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 25

Trp Tyr Asp Gly Asp Asn

1 5

<210> 26

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 26

Asp Leu Arg Thr Gly Pro Phe Asp Tyr

1 5

<210> 27

<211> 118

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 27

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Arg Thr Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 28

<211> 354

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 28

caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggcaggag cctgaggctg 60

agctgcgccg ccagcggctt caccttcagc gtgtacggca tgaactgggt gaggcaggcc 120

cccggcaagg gcctggagtg ggtggccatc atctggtacg acggcgacaa ccagtactac 180

gccgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240

ctgcagatga acggcctgag ggccgaggac accgccgtgt actactgcgc cagggacctg 300

aggaccggcc ccttcgacta ctggggccag ggcaccctgg tgaccgtgag cagc 354

<210> 29

<211> 448

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 29

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Arg Thr Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 30

<211> 1344

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 30

caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggcaggag cctgaggctg 60

agctgcgccg ccagcggctt caccttcagc gtgtacggca tgaactgggt gaggcaggcc 120

cccggcaagg gcctggagtg ggtggccatc atctggtacg acggcgacaa ccagtactac 180

gccgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240

ctgcagatga acggcctgag ggccgaggac accgccgtgt actactgcgc cagggacctg 300

aggaccggcc ccttcgacta ctggggccag ggcaccctgg tgaccgtgag cagcgccagc 360

accaagggcc ccagcgtgtt ccccctggcc cccagcagca agagcaccag cggcggcacc 420

gccgccctgg gctgcctggt gaaggactac ttccccgagc ccgtgaccgt gagctggaac 480

agcggcgccc tgaccagcgg cgtgcacacc ttccccgccg tgctgcagag cagcggcctg 540

tacagcctga gcagcgtggt gaccgtgccc agcagcagcc tgggcaccca gacctacatc 600

tgcaacgtga accacaagcc cagcaacacc aaggtggaca agagggtgga gcccaagagc 660

tgcgacaaga cccacacctg ccccccctgc cccgcccccg agctgctggg cggccccagc 720

gtgttcctgt tcccccccaa gcccaaggac accctgatga tcagcaggac ccccgaggtg 780

acctgcgtgg tggtggacgt gagccacgag gaccccgagg tgaagttcaa ctggtacgtg 840

gacggcgtgg aggtgcacaa cgccaagacc aagcccaggg aggagcagta caacagcacc 900

tacagggtgg tgagcgtgct gaccgtgctg caccaggact ggctgaacgg caaggagtac 960

aagtgcaagg tgagcaacaa ggccctgccc gcccccatcg agaagaccat cagcaaggcc 1020

aagggccagc ccagggagcc ccaggtgtac accctgcccc ccagcaggga ggagatgacc 1080

aagaaccagg tgagcctgac ctgcctggtg aagggcttct accccagcga catcgccgtg 1140

gagtgggaga gcaacggcca gcccgagaac aactacaaga ccaccccccc cgtgctggac 1200

agcgacggca gcttcttcct gtacagcaag ctgaccgtgg acaagagcag gtggcagcag 1260

ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag 1320

agcctgagcc tgagccccgg caag 1344

<210> 31

<211> 11

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 31

Arg Ala Ser Gln Ser Ile Gly Ser Ser Leu His

1 5 10

<210> 32

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 32

Tyr Ala Ser Gln Ser Phe Ser

1 5

<210> 33

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 33

His Gln Ser Ser Ser Leu Pro Phe Thr

1 5

<210> 34

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 34

Ser Gln Ser Ile Gly Ser Ser

1 5

<210> 35

<211> 3

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 35

Tyr Ala Ser

1

<210> 36

<211> 6

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 36

Ser Ser Ser Leu Pro Phe

1 5

<210> 37

<211> 107

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 37

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 38

<211> 321

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 38

gagatcgtgc tgacccagtc acccgacttt cagtcagtga cccctaaaga aaaagtgact 60

atcacctgta gggcctccca gtctatcggc tctagcctgc actggtatca gcagaagccc 120

gatcagtcac ctaagctgct gattaagtac gcctctcagt cctttagcgg cgtgccctct 180

aggtttagcg gctcaggctc aggcaccgac ttcaccctga ctatcaatag cctggaagcc 240

gaggacgccg ctgcctacta ctgtcatcag tcaagtagcc tgcccttcac cttcggccct 300

ggcactaaag tggatattaa g 321

<210> 39

<211> 214

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 39

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 40

<211> 642

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 40

gagatcgtgc tgacccagtc acccgacttt cagtcagtga cccctaaaga aaaagtgact 60

atcacctgta gggcctccca gtctatcggc tctagcctgc actggtatca gcagaagccc 120

gatcagtcac ctaagctgct gattaagtac gcctctcagt cctttagcgg cgtgccctct 180

aggtttagcg gctcaggctc aggcaccgac ttcaccctga ctatcaatag cctggaagcc 240

gaggacgccg ctgcctacta ctgtcatcag tcaagtagcc tgcccttcac cttcggccct 300

ggcactaaag tggatattaa gcgtacggtg gccgctccca gcgtgttcat cttccccccc 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420

ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcataag gtgtacgcct gcgaggtgac ccaccagggc 600

ctgtccagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642

<210> 41

<211> 10

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 41

Gly Phe Thr Phe Ser Val Tyr Gly Met Asn

1 5 10

<210> 42

<211> 17

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 42

Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 43

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 43

Asp Leu Arg Thr Gly Pro Phe Asp Tyr

1 5

<210> 44

<211> 5

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 44

Val Tyr Gly Met Asn

1 5

<210> 45

<211> 17

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 45

Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 46

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 46

Asp Leu Arg Thr Gly Pro Phe Asp Tyr

1 5

<210> 47

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 47

Gly Phe Thr Phe Ser Val Tyr

1 5

<210> 48

<211> 6

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 48

Trp Tyr Asp Gly Asp Asn

1 5

<210> 49

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 49

Asp Leu Arg Thr Gly Pro Phe Asp Tyr

1 5

<210> 50

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 50

Gly Phe Thr Phe Ser Val Tyr Gly

1 5

<210> 51

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 51

Ile Trp Tyr Asp Gly Asp Asn Gln

1 5

<210> 52

<211> 11

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 52

Ala Arg Asp Leu Arg Thr Gly Pro Phe Asp Tyr

1 5 10

<210> 53

<211> 118

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 53

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Arg Thr Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 54

<211> 354

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 54

caggtgcagc tggtggaatc aggcggcgga gtggtgcagc ctggtagatc actgagactg 60

agctgcgctg ctagtggctt cacctttagc gtctacggaa tgaactgggt ccgacaggcc 120

cctgggaaag gcctggagtg ggtggcaatt atctggtacg acggcgataa tcagtactac 180

gccgatagcg tgaagggacg gttcactatc tctagggata actctaagaa caccctgtac 240

ctgcagatga acggcctgag agccgaggac accgccgtct actactgcgc tagggacctg 300

agaaccggcc ccttcgacta ctggggacag ggcaccctgg tcaccgtgtc tagc 354

<210> 55

<211> 448

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 55

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Arg Thr Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys

355 360 365

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 56

<211> 1344

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 56

caggtgcagc tggtggaatc aggcggcgga gtggtgcagc ctggtagatc actgagactg 60

agctgcgctg ctagtggctt cacctttagc gtctacggaa tgaactgggt ccgacaggcc 120

cctgggaaag gcctggagtg ggtggcaatt atctggtacg acggcgataa tcagtactac 180

gccgatagcg tgaagggacg gttcactatc tctagggata actctaagaa caccctgtac 240

ctgcagatga acggcctgag agccgaggac accgccgtct actactgcgc tagggacctg 300

agaaccggcc ccttcgacta ctggggacag ggcaccctgg tcaccgtgtc tagcgcctct 360

actaagggcc caagcgtgtt ccccctggcc cctagctcta agtctactag cggaggcacc 420

gccgctctgg gctgcctggt caaggactac ttccccgagc ccgtgaccgt cagctggaat 480

agcggcgctc tgactagcgg agtgcacacc ttccccgccg tgctgcagtc tagcggcctg 540

tatagcctgt ctagcgtcgt gaccgtgcct agctctagcc tgggcactca gacctatatc 600

tgtaacgtga accacaagcc ctctaacact aaggtggaca agcgggtgga acctaagtcc 660

tgcgataaga ctcacacctg tcctccctgc cctgcccctg aggctgccgg aggacctagc 720

gtgttcctgt tcccacctaa gcctaaagac accctgatga tctctaggac ccccgaagtg 780

acctgcgtgg tggtggacgt ctcacacgag gaccctgaag tgaagtttaa ttggtacgtg 840

gacggcgtgg aagtgcacaa cgctaagact aagcctagag aggaacagta taactctacc 900

tatagggtcg tcagcgtgct gacagtgctg caccaggact ggctgaacgg gaaagagtat 960

aagtgtaaag tgtctaacaa ggccctgcca gcccctatcg aaaagactat ctctaaggct 1020

aaggggcagc ctagagaacc ccaagtgtgc actctgcccc ctagtagaga agagatgact 1080

aagaatcagg tgtcactgag ctgtgccgtg aagggcttct accctagcga tatcgccgtg 1140

gagtgggaga gcaacggcca gcccgagaac aactacaaga ccaccccccc agtgctggac 1200

agcgacggca gcttcttcct ggtgagcaag ctgaccgtgg acaagtccag gtggcagcag 1260

ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag 1320

tccctgagcc tgagccccgg caag 1344

<210> 57

<211> 11

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 57

Arg Ala Ser Gln Ser Ile Gly Ser Ser Leu His

1 5 10

<210> 58

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 58

Tyr Ala Ser Gln Ser Phe Ser

1 5

<210> 59

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 59

His Gln Ser Ser Ser Leu Pro Phe Thr

1 5

<210> 60

<211> 11

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 60

Arg Ala Ser Gln Ser Ile Gly Ser Ser Leu His

1 5 10

<210> 61

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 61

Tyr Ala Ser Gln Ser Phe Ser

1 5

<210> 62

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 62

His Gln Ser Ser Ser Leu Pro Phe Thr

1 5

<210> 63

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 63

Ser Gln Ser Ile Gly Ser Ser

1 5

<210> 64

<211> 3

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 64

Tyr Ala Ser

1

<210> 65

<211> 6

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 65

Ser Ser Ser Leu Pro Phe

1 5

<210> 66

<211> 6

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 66

Gln Ser Ile Gly Ser Ser

1 5

<210> 67

<211> 10

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 67

Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro

1 5 10

<210> 68

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 68

His Gln Ser Ser Ser Leu Pro Phe Thr

1 5

<210> 69

<211> 107

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 69

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 70

<211> 321

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 70

gagatcgtgc tgacccagtc acccgacttt cagtcagtga cccctaaaga aaaagtgact 60

atcacctgta gggcctccca gtctatcggc tctagcctgc actggtatca gcagaagccc 120

gatcagtcac ctaagctgct gattaagtac gcctctcagt cctttagcgg cgtgccctct 180

aggtttagcg gctcaggctc aggcaccgac ttcaccctga ctatcaatag cctggaagcc 240

gaggacgccg ctgcctacta ctgtcatcag tcaagtagcc tgcccttcac cttcggccct 300

ggcactaaag tggatattaa g 321

<210> 71

<211> 214

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 71

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 72

<211> 642

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 72

gagatcgtgc tgacccagtc acccgacttt cagtcagtga cccctaaaga aaaagtgact 60

atcacctgta gggcctccca gtctatcggc tctagcctgc actggtatca gcagaagccc 120

gatcagtcac ctaagctgct gattaagtac gcctctcagt cctttagcgg cgtgccctct 180

aggtttagcg gctcaggctc aggcaccgac ttcaccctga ctatcaatag cctggaagcc 240

gaggacgccg ctgcctacta ctgtcatcag tcaagtagcc tgcccttcac cttcggccct 300

ggcactaaag tggatattaa gcgtacggtg gccgctccca gcgtgttcat cttccccccc 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420

ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcataag gtgtacgcct gcgaggtgac ccaccagggc 600

ctgtccagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642

<210> 73

<211> 10

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 73

Gly Gly Thr Phe Lys Ser Tyr Ala Ile Ser

1 5 10

<210> 74

<211> 17

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 74

Asn Ile Ile Pro Met Thr Gly Gln Thr Tyr Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 75

<211> 10

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 75

Ala Ala Tyr His Pro Leu Val Phe Asp Asn

1 5 10

<210> 76

<211> 5

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 76

Ser Tyr Ala Ile Ser

1 5

<210> 77

<211> 17

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 77

Asn Ile Ile Pro Met Thr Gly Gln Thr Tyr Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 78

<211> 10

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 78

Ala Ala Tyr His Pro Leu Val Phe Asp Asn

1 5 10

<210> 79

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 79

Gly Gly Thr Phe Lys Ser Tyr

1 5

<210> 80

<211> 6

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 80

Ile Pro Met Thr Gly Gln

1 5

<210> 81

<211> 10

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 81

Ala Ala Tyr His Pro Leu Val Phe Asp Asn

1 5 10

<210> 82

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 82

Gly Gly Thr Phe Lys Ser Tyr Ala

1 5

<210> 83

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 83

Ile Ile Pro Met Thr Gly Gln Thr

1 5

<210> 84

<211> 12

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 84

Ala Arg Ala Ala Tyr His Pro Leu Val Phe Asp Asn

1 5 10

<210> 85

<211> 119

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 85

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Lys Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Ile Pro Met Thr Gly Gln Thr Tyr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Ala Tyr His Pro Leu Val Phe Asp Asn Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 86

<211> 357

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 86

gaggtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggctctag cgtgaaagtc 60

agctgtaaag ctagtggcgg caccttcaag tcctacgcta ttagctgggt cagacaggcc 120

ccaggtcagg gcctggagtg gatgggcaat attatcccta tgaccggtca gacctactac 180

gctcagaaat ttcagggtag agtgactatc accgccgacg agtctactag caccgcctat 240

atggaactgt ctagcctgag atcagaggac accgccgtct actactgcgc tagagccgcc 300

tatcaccccc tggtgttcga taactggggt cagggcaccc tggtcaccgt gtctagc 357

<210> 87

<211> 449

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 87

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Lys Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Ile Pro Met Thr Gly Gln Thr Tyr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Ala Tyr His Pro Leu Val Phe Asp Asn Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 88

<211> 1347

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 88

gaggtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggctctag cgtgaaagtc 60

agctgtaaag ctagtggcgg caccttcaag tcctacgcta ttagctgggt cagacaggcc 120

ccaggtcagg gcctggagtg gatgggcaat attatcccta tgaccggtca gacctactac 180

gctcagaaat ttcagggtag agtgactatc accgccgacg agtctactag caccgcctat 240

atggaactgt ctagcctgag atcagaggac accgccgtct actactgcgc tagagccgcc 300

tatcaccccc tggtgttcga taactggggt cagggcaccc tggtcaccgt gtctagcgct 360

agcactaagg gcccctcagt gttccccctg gcccctagct ctaagtctac tagcggcggc 420

accgccgctc tgggctgcct ggtgaaagac tacttccccg agcccgtgac cgtgtcatgg 480

aatagcggcg ctctgactag cggagtgcac accttccccg ccgtgctgca gtctagcggc 540

ctgtatagcc tgtctagcgt ggtgaccgtg cctagctcta gcctgggcac tcagacctac 600

atctgtaacg tgaaccacaa gccctctaac actaaggtgg acaagcgggt ggaacctaag 660

tcctgcgata agactcacac ctgtcccccc tgccctgccc ctgaggctgc cggaggacct 720

agcgtgttcc tgttcccacc taagcctaag gacaccctga tgatctctag gacccccgaa 780

gtgacctgcg tggtggtgga tgtgtctcac gaggaccctg aagtgaagtt caattggtac 840

gtggacggcg tggaagtgca caacgctaag actaagccta gagaggaaca gtataactcc 900

acctatagag tggtgtcagt gctgaccgtg ctgcatcagg actggctgaa cggcaaagag 960

tataagtgta aagtctctaa caaggccctg ccagccccta tcgaaaagac tatctctaag 1020

gctaagggcc agcctagaga acctcaggtg tacaccctgc ccccctgtag agaagagatg 1080

actaagaatc aggtgtccct gtggtgtctg gtgaaaggct tctaccctag cgatatcgcc 1140

gtggaatggg agtctaacgg ccagcccgag aacaactata agactacccc ccctgtgctg 1200

gatagcgacg gctcattctt cctgtactct aagctgaccg tggacaagtc taggtggcag 1260

cagggcaatg tgtttagctg tagcgtgatg cacgaggccc tgcataatca ctacactcag 1320

aagtcactga gcctgagccc cggcaag 1347

<210> 89

<211> 13

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 89

Ser Gly Ser Ser Ser Asn Ile Gly Asn His Tyr Val Asn

1 5 10

<210> 90

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 90

Arg Asn Asn His Arg Pro Ser

1 5

<210> 91

<211> 11

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 91

Gln Ser Trp Asp Tyr Ser Gly Phe Ser Thr Val

1 5 10

<210> 92

<211> 13

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 92

Ser Gly Ser Ser Ser Asn Ile Gly Asn His Tyr Val Asn

1 5 10

<210> 93

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 93

Arg Asn Asn His Arg Pro Ser

1 5

<210> 94

<211> 11

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 94

Gln Ser Trp Asp Tyr Ser Gly Phe Ser Thr Val

1 5 10

<210> 95

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 95

Ser Ser Ser Asn Ile Gly Asn His Tyr

1 5

<210> 96

<211> 3

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 96

Arg Asn Asn

1

<210> 97

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 97

Trp Asp Tyr Ser Gly Phe Ser Thr

1 5

<210> 98

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 98

Ser Ser Asn Ile Gly Asn His Tyr

1 5

<210> 99

<211> 3

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 99

Arg Asn Asn

1

<210> 100

<211> 11

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Peptide'

<400> 100

Gln Ser Trp Asp Tyr Ser Gly Phe Ser Thr Val

1 5 10

<210> 101

<211> 110

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 101

Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn His

20 25 30

Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Tyr Ser Gly

85 90 95

Phe Ser Thr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 102

<211> 330

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 102

gatatcgtcc tgactcagcc ccctagcgtc agcggcgctc ccggtcagag agtgactatt 60

agctgtagcg gctctagctc taatatcggt aatcactacg tgaactggta tcagcagctg 120

cccggcaccg cccctaagct gctgatctat agaaacaatc accggcctag cggcgtgccc 180

gataggttta gcggatctaa gtcaggcact agcgctagtc tggctatcac cggactgcag 240

tcagaggacg aggccgacta ctactgtcag tcctgggact atagcggctt tagcaccgtg 300

ttcggcggag gcactaagct gaccgtgctg 330

<210> 103

<211> 216

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polypeptide'

<400> 103

Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn His

20 25 30

Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Tyr Ser Gly

85 90 95

Phe Ser Thr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 104

<211> 648

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthesized

Polynucleotide'

<400> 104

gatatcgtcc tgactcagcc ccctagcgtc agcggcgctc ccggtcagag agtgactatt 60

agctgtagcg gctctagctc taatatcggt aatcactacg tgaactggta tcagcagctg 120

cccggcaccg cccctaagct gctgatctat agaaacaatc accggcctag cggcgtgccc 180

gataggttta gcggatctaa gtcaggcact agcgctagtc tggctatcac cggactgcag 240

tcagaggacg aggccgacta ctactgtcag tcctgggact atagcggctt tagcaccgtg 300

ttcggcggag gcactaagct gaccgtgctg ggtcagccta aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggagctgcag gccaacaagg ccaccctggt gtgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgagggca gcaccgtgga aaagaccgtg gccccaaccg agtgcagc 648

Claims

1. A method for treating or preventing Hidradenitis Suppurativa (HS) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody antagonist that specifically binds to IL-18 and IL-1 β and inhibits the activity thereof.

2. The method of claim 1, wherein the antibody comprises

a. A first moiety which is an immunoglobulin having a first variable light chain (VL 1) and a first variable heavy chain (VH 1) and a first constant heavy chain (CH 1) with a heterodimerization modification, said VH1 specifically binding to il1β, and

b. a second moiety that is an immunoglobulin having a second variable light chain (VL 2) and a second variable heavy chain (VH 2), and a second constant heavy chain (CH 2) with a heterodimerization modification complementary to the heterodimerization modification of the first constant heavy chain, the VH2 specifically binding IL-18.

3. A method for slowing, arresting, or reducing the progression of HS in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody antagonist that specifically binds to and inhibits the activity of IL-18 and IL-1β.

4. The method of claim 3, wherein the antibody comprises

5. The method of claim 2 or 4, wherein the first and second constant heavy chains of the bispecific antibody are human IgA, igD, igE, igG or IgM, preferably IgD, igE or IgG, such as human IgG1, igG2, igG3 or IgG4, preferably IgG1.

6. The method of claim 2 or 4, wherein the first and second constant heavy chains of the bispecific antibody are IgG1, and wherein

7. The method of claims 1-6, wherein:

a. the first immunoglobulin VH1 domain of the bispecific antibody comprises

b. the first immunoglobulin VL1 domain of the bispecific antibody comprises

c. the second immunoglobulin VH2 domain of the bispecific antibody comprises

Hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID No. 47, said CDR2 having the amino acid sequence SEQ ID No. 48 and said CDR3 having the amino acid sequence SEQ ID No. 49; and

d. the second immunoglobulin VL2 domain of the bispecific antibody comprises

8. The method of any of the preceding claims, wherein:

c. the second immunoglobulin VH2 domain of the bispecific antibody comprises the amino acid sequence SEQ ID NO. 53, and

9. The method of any of the preceding claims, wherein:

a. the first immunoglobulin heavy chain of the bispecific antibody comprises the amino acid sequence SEQ ID NO. 87,

b. the first immunoglobulin light chain of the bispecific antibody comprises the amino acid sequence SEQ ID NO. 103,

c. the second immunoglobulin heavy chain of the bispecific antibody comprises the amino acid sequence SEQ ID NO. 55, and

d. the second immunoglobulin light chain of the bispecific antibody comprises the amino acid sequence SEQ ID NO. 71.

10. The method of any one of the preceding claims, wherein the route of administration is subcutaneous or intravenous, or a combination of subcutaneous or intravenous.

11. The method of any one of the preceding claims, wherein the dose is about 1.5mg to about 15mg of active ingredient per kilogram of human subject.

12. The method of any one of the preceding claims, wherein the dose is about 5mg or 10mg of active ingredient per kilogram of human subject.

13. The method of any one of the preceding claims, wherein the dose is from about 150mg to about 600mg of active ingredient, such as about 300mg of active ingredient.

14. The method of any one of the preceding claims, wherein the antibody is administered by loading administration and maintenance administration.

15. The method of any one of the preceding claims, wherein the loading dose is administered via a subcutaneous injection of a first dose and the maintenance dose is administered via a subcutaneous injection of a second dose.

16. The method of claim 15, wherein the first dose is between about 150mg and about 600mg of active ingredient, such as about 300mg of active ingredient, and the second dose is between about 150mg and about 600mg of active ingredient, such as about 300mg of active ingredient.

17. The method of claim 15 or 16, wherein the first dose is 150mg, 300mg, or 600mg of active ingredient and the second dose is 150mg, 300mg, or 600mg of active ingredient.

18. The method of claim 17, wherein the loading administration comprises at least three subcutaneous injections every two weeks on days 1, 15, and 29, and the maintenance administration consists of one monthly (Q4W) subcutaneous injections starting on day 57.

19. The method of any one of the preceding claims, wherein the patient with hidradenitis suppurativa is selected according to one of the following criteria:

a. the patient suffers from moderate to severe HS;

b. the patient is an adult;

c. the patient is an adolescent;

d. the patient has an HS-PGA score of > 3 prior to treatment with the CD40 antagonist;

e. the patient has at least 3 inflammatory lesions prior to treatment with a CD40 antagonist; or alternatively

f. Prior to treatment with CD40 antagonists, the patient had no extensive scarring (< 10 fistulae) caused by HS.

20. The method of any one of claims 1-19, wherein by week 16 of treatment, the hidradenitis suppurativa patient achieves at least one of:

a. simplified hissc;

hs reddening and swelling mass reduction;

c.NRS30；

d. a decrease of 6 or less as measured by DLQI; and/or

Improvement of dlqi.

21. The method of any one of claims 1 to 19, wherein at least 40% of the patients achieve simplified hissc by week 16 of treatment; or at least 25% of the patients achieve NRS30 responses; or less than 15% of the patients experience HS red bumps.

22. The antibody for use of any one of claims 1-19, wherein the patient has at least one of the following at least one week after the first dose of the dual specific IL-18 and IL-1β antagonist:

a. pain measured by VAS or NRS is rapidly reduced, and

b. CRP measured using standard CRP assays decreased rapidly.

23. The method of any one of claims 1 to 19, wherein the patient achieves a sustained response 3 months after the end of treatment, as measured by inflammatory lesions count, clinical response to hidradenitis suppurativa (hissc), numerical Rating Scale (NRS), modified sartorius HS score, hidradenitis suppurativa-physician global assessment (HS-PGA), or dermatological quality of life index (DLQI).

24. The method of any one of claims 1 to 19, wherein the patient achieves a sustained response 3 months after the end of treatment, as measured by simplified hisscr (shisscr).

25. A pharmaceutical composition comprising a therapeutically effective amount of bispecific anti-IL-18 and anti-IL-1 beta antibody (e.g., bbmAb 1) and one or more pharmaceutically acceptable carriers.