EP4731653A1 - Bispecific antibodies for use in treatment of patients with xiap deficiency or cdc42 mutations - Google Patents

Abstract

The invention relates to bivalent bispecific monoclonal antibodies (bbmAb) or variants thereof for use in the treatment or for use in alleviating the symptoms of an CDC42 mutation or XIAP deficiency based autoinflammation or disease in a subject in need thereof.

Description

Bispecific Antibodies for use in treatment of patients with XIAP deficiency or CDC42 mutations.

TECHNICAL FIELD

The invention relates to treatments for bivalent bispecific monoclonal antibodies for the use in treatment of an XIAP deficiency based autoinflammation or disease or an CDC42 mutation based autoinflammation or disease in a patient in need thereof. The disclosure also relates to methods and treatment regimens for treating XIAP deficiency based autoinflammation or disease or an CDC42 mutation based autoinflammation or disease by employing bispecific antibodies targeting both I L-1 p and IL-18 simultaneously. In some cases, the e.g., bbmAb (or a variant thereof) is for the treatment of subjects harboring mutations in the genes encoding the XIAP gene or mutations in the CDC43 gene and suffering from e.g., hypogammaglobulinemia, cytopaenias, inflammatory bowel disease, abdominal pain and diarrhea, recurrent fevers, splenomegaly, haemophagocytic lymphohistiocytosis, neonatal-onset cytopenia, syndromic form of thrombocytopenia, Macrothrombocytopenia, dyshematopoiesis, autoinflammation, syndromic immune- deficiency or recurrent haemophagocytic lymphohistiocytosis (HLH), autoinflammation with infantile enterocolitis (AIFEC) or macrophage activation syndrome (MAS).

BACKGROUND OF THE DISCLOSURE

The inflammasomes are intracellular multiprotein complexes that are normally formed and activated in response to pathogen- or danger-associated molecular patterns (PAMPs/DAMPs). The inflammasomopathies are a group of mechanistically related diseases resulting from the over-activation of individual inflammasomes leading to differentiated clinical phenotypes dependent on the effector cytokines produced and the tissue-specific expression. X-linked inhibitor of apoptosis (XIAP) deficiency, also known as X-linked lymphoproliferative syndrome type 2 (XLP2), is an autoinflammation or inflammasomopathy and adaptive immune deficiency characterized by hyperinflammation and immune dysregulation (Geerlinks et al., Journal of Clinical Immunology (2022) 42:901-903). XIAP deficiency, first described in 2006, is caused by pathogenic variants in the XIAP/BIRC4 gene (identifications for XIAP Gene: HGNC: 592; NCBI Gene: 331 ; Ensembl: ENSG00000101966; OMIM®: 300079; UniProtKB/Swiss-Prot: P98170) leading to multiple clinical manifestations, most commonly hemophagocytic lymphohistiocytosis (HLH) inflammatory bowel disease and splenomegaly.

Cell division cycle 42 (CDC42) (identifications for CDC42 gene: HGNC: 1736 NCBI Gene: 998 Ensembl: ENSG00000070831 OMIM®: 116952 UniProtKB/Swiss-Prot: P60953) is an intracellular member of the Ras homology (Rho) GTPases, which control cell polarity by regulating the assembly of actin cytoskeleton structures. From an immunological point of view, CDC42 regulates multiple cellular processes such as migration, immunological synapse formation, and polarized cytokine secretion. CDC42 also has a critical role in cell proliferation and hematopoiesis (Miyazawa H and Wada T (2022) Immune-mediated inflammatory diseases with chronic excess of serum interleukin- 18. Front. Immunol. 13:930141).

Patients affected by an XIAP deficiency or an CDC42 mutation associated autoinflammatory disease generally have grossly elevated IL-1 p and/or IL-18 levels. These elevated effector cytokines drive the above listed associated clinical manifestations centered on neurodevelopment, hematopoiesis, and immune response (Takenouchi et al 2015, Asiri et al 2021 , Coppola et al 2022). The present inventors believe that only the combination of anti-IL-1 receptor (e.g., anakinra) and recombinant IL-18 binding protein (e.g., IL-18 BP) has been reported as clinically efficacious in a limited number of infant cases for XIAP deficiency.

There are no approved therapeutics that directly and specifically target the underlying I L-1 p and IL-18 driven autoinflammatory process to improve the overall clinical outcome for XIAP deficiency based autoinflammation or disease or CDC42 mutation based autoinflammation or disease in patients. Thus, there is a long-felt unmet need in the art for improved treatments of XIAP deficiencies or CDC42 mutation based autoinflammation or disease or inflammasomopathy.

SUMMARY OF THE DISCLOSURE

Described is a bispecific antibody or functional fragments thereof targeting both IL- 1 and IL-18 simultaneously, for use in preventing or treating an XIAP deficiencies or CDC42 mutation based autoinflammation or disease or inflammasomopathy in a subject. In some cases, the bispecific antibody or functional fragments thereof targeting both I L-1 and IL-18 simultaneously are for use in treating patients/methods of treating patients being XIAP deficient or harboring a CDC42 mutation showing clinical symptoms of or is suffering from hypogammaglobulinemia, cytopaenias, inflammatory bowel disease, abdominal pain and diarrhea, recurrent fevers, splenomegaly, haemophagocytic lymphohistiocytosis, neonatal-onset cytopenia, syndromic form of thrombocytopenia, Macrothrombocytopenia, dyshematopoiesis, autoinflammation, syndromic immune-deficiency or recurrent haemophagocytic lymphohistiocytosis (HLH). In some cases, the use for preventing or treating, an XIAP deficiencies or CDC42 mutation based autoinflammation or disease in a subject showing clinical symptoms of autoinflammation with infantile enterocolitis (AIFEC) or macrophage activation syndrome by administering the therapeutically effective amount of the bispecific antibody targeting both I L-1 p and IL-18 simultaneously. Described herein are also methods of preventing or treating an XIAP deficiencies or CDC42 mutation based autoinflammation or disease by administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody targeting both I L- 1 p and IL- 18 simultaneously. In some cases, the methods include treatment of autoinflammation with infantile enterocolitis (AIFEC) or macrophage activation syndrome (MAS) in patients being XIAP deficient or harboring a CDC42 mutation showing clinical disease symptoms by administering the therapeutically effective amount of the bispecific antibody targeting both I L-1 and IL-18 simultaneously.

Further provided herein are specific dosing regimens for the methods or use of a bispecific antibody targeting both I L-1 p and IL-18 simultaneously (e.g., bbmAb) described herein. In one embodiment, in the above describe methods or treatment of use of the bispecific antibody targeting both IL-1 p and IL-18 simultaneously for the treatment of a XIAP deficiencies or CDC42 mutation based autoinflammation or disease said methods or uses comprising administering about 1 mg/kg to about 35 mg/kg of the bispecific antibody to the subject in need thereof. In another embodiment, about 10 mg/kg of the bispecific antibody is administered to said subject, wherein the bispecific antibody is administered intravenously or subcutaneously. In a different embodiment, the 10 mg/kg of the bispecific antibody targeting both IL-i p and IL-18 simultaneously is administered intravenously, optionally wherein said antibody is administered every other week. In another embodiment, the dose of the administered bispecific I L-1 p and IL-18 antibody is about 50 mg to about 900 mg administered subcutaneously.

Additionally further herein described embodiments relate to pharmaceutical combinations and pharmaceutical compositions comprising a bispecific antibody targeting both IL-i p and IL-18 simultaneously (e.g., bbmAb) optionally in the presence of a pharmaceutically acceptable carrier, for use in the treatment or prevention of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease, such as for use in treatment of inflammatory bowel disease, abdominal pain and diarrhea, recurrent fevers, splenomegaly, haemophagocytic lymphohistiocytosis, neonatal-onset cytopenia, autoinflammation, recurrent haemophagocytic lymphohistiocytosis (HLH), AIFEC or macrophage activation syndrome (MAS). Further features and advantages of the described methods and uses will become apparent from the following detailed description In a first aspect the disclosure relates to a method for the treatment or prevention of symptoms of XIAP deficiencies or CDC42 mutation based autoinflammation or disease the method comprising administering to said subject a therapeutically effective amount of a bispecific antibody, wherein the antibody comprises a. a first part which is an immunoglobulin with a first variable light chain of (VL1) and a first variable heavy chain (VH1), that binds specifically to a IL1 p, and a first constant heavy chain (CH1) with a hetero-dimerization modification, and b. a second part which is an immunoglobulin with a second variable light chain (VL2) and a second variable heavy chain (VH2), that binds specifically to IL- 18 and a second constant heavy chain (CH2) with a hetero-dimerization modification which is complementary to the hetero-dimerization modification of the first constant heavy chain.

In a second aspect the disclosure relates to a method for slowing, arresting, or reducing the severity of symptoms of XIAP deficiencies or CDC42 mutation based autoinflammation or disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a bispecific antibody, wherein the antibody comprises a. a first part which is an immunoglobulin with a first variable light chain of (VL1) and a first variable heavy chain (VH1), that binds specifically to a IL1 p, and a first constant heavy chain (CH1) with a hetero-dimerization modification, and b. a second part which is an immunoglobulin with a second variable light chain (VL2) and a second variable heavy chain (VH2), that binds specifically to IL-18 and a second constant heavy chain (CH2) with a hetero-dimerization modification which is complementary to the hetero-dimerization modification of the first constant heavy chain.

In a third aspect, the disclosure relates to a bispecific antibody comprising a. a first part which is an immunoglobulin with a first variable light chain of (VL1) and a first variable heavy chain (VH1), that binds specifically to a IL1 p, and a first constant heavy chain (CH1) with a hetero-dimerization modification, and b. a second part which is an immunoglobulin with a second variable light chain (VL2) and a second variable heavy chain (VH2), that binds specifically to IL- 18 and a second constant heavy chain (CH2) with a hetero-dimerization modification which is complementary to the hetero-dimerization modification of the first constant heavy chain, for use in the treatment or prevention of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease, such as an in a subject in need thereof.

In a fourth aspect the disclosure relates to method and treatments of the first, second and third aspect, wherein about 1 mg/kg to about 35 mg/kg of the bispecific antibody targeting both I L-1 p and IL-18 simultaneously is administered to the subject. In a preferred embodiment of the fourth aspect, about 10 mg/kg of the bispecific antibody are administered to the treated subject.

In one aspect of the disclosure the bispecific antibody targeting both IL-1 p and IL- 18 simultaneously is administered to the subject intravenously or subcutaneously. In a furthermore preferred embodiment, the bispecific antibody targeting both IL- 1 P and IL-18 simultaneously is administered intravenously to the treated subject at a dose of about 10 mg/kg.

In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL- 1 P and IL-18 simultaneously is administered intravenously to the patient only once on day 1. In another embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL- 1 P and IL-18 simultaneously is administered intravenously to the patient on day 1 and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13 and/or day 14. In a further embodiment, the 10 mg/kg dose of the bispecific antibody targeting both I L-1 p and IL-18 simultaneously is administered intravenously to the patient on day 1 and day 14. In a further embodiment, the 10 mg/kg dose of the bispecific antibody targeting both I L-1 p and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks. In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both I L-1 p and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks in a period of up to 28 weeks. In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL-1 p and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks in a period of up to 24 weeks.

In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL- 1 P and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks in a period of up to 3 years. In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL-1 p and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks for 2 weeks. In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL-i p and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks for 8 weeks. In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL-1 p and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks for 14 weeks. In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL-1 p and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks for 24 weeks. In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL- 1 P and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks for at least 2 weeks. In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL-1 p and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks for at least 8 weeks. In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL-i p and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks for at least 14 weeks. In one embodiment, the 10 mg/kg dose of the bispecific antibody targeting both IL-1 p and IL-18 simultaneously is administered intravenously to the patient once every 2 weeks for at least 24 weeks.

In a preferred embodiment, a bispecific antibody targeting both IL-1 p and IL-18 simultaneously is administered to a patient every two weeks, e.g., intravenously, such as for example at a dose of, or of about, 10 mg/kg.

In another embodiment of the preceding aspects of the disclosure, the bispecific antibody targeting both IL-1 and IL- 18 is administered in combination with at least one further therapeutic agent.

In a particular embodiment of any one of the preceding 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 lgG1 , lgG2, lgG3, or lgG4, preferably lgG1.

In another embodiment of any one of the preceding aspects of the disclosure the first and second constant heavy chains of the bispecific antibody are IgG 1 , and a. the first constant heavy chain has point mutations generating a knob structure and the second constant heavy has point mutations generating a hole structure, or b. the first constant heavy chain has point mutations generating a hole structure and the second constant heavy has point mutations generating a knob structure, and optionally c. the first and second constant heavy chains have mutations resulting in a disulfide bridge.

In a specifically preferred embodiment of the first, second and third aspect of the herein describe methods and uses, 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 ii. 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 NQ:80, and said CDR3 having the amino acid sequence SEQ ID NO:81 ; and the first immunoglobulin VL1 domain of the bispecific antibody comprises: iii. 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 iv. 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 vi. 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: vii. hypervariable regions CDR1 , CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NQ: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 viii. 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 disclosure, the antibody used in the methods/treatments according to any of the preceding aspects comprises: a. the first immunoglobulin VH1 domain of amino acid sequence SEQ ID NO: 85, b. the first immunoglobulin VL1 domain of amino acid sequence SEQ ID NO: 101 , c. the second immunoglobulin VH2 domain of amino acid sequence SEQ ID NO: 53, and d. the second immunoglobulin VL2 domain of amino acid sequence SEQ ID NO: 69.

In another preferred embodiment of the disclosure, the antibody used in the methods/treatments according to any of the preceding aspects comprises e. the first immunoglobulin heavy chain of amino acid sequence SEQ ID NO: 87, f. the first immunoglobulin light chain of amino acid sequence SEQ ID NO: 103, g. the second immunoglobulin heavy chain of amino acid sequence SEQ ID NO: 55, and h. the second immunoglobulin light chain of amino acid sequence SEQ ID NO: 71.

In another embodiment of the preceding aspects of the disclosure, the treated subject has an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In another embodiment of the preceding aspects of the disclosure, the treated subject has a loss-of-function mutation in the XIAP/BIRC4 gene encoding the XIAP protein (as disclosed herein below) or a mutation in the CDC42 gene resulting in aberrant palmitoylation of CDC42 protein or missense mutation at the C-terminus of the CDC42 protein affecting localization of the CDC42 protein (as disclosed herein below).

In some cases, the subject in need of a XIAP deficiencies or CDC42 mutation based autoinflammation or disease treatment has hyper elevated levels of IL-18, I L-1 p, or hyper elevated serum levels of IL-18 and IL-1 as compared to a control population of subjects that do not exhibit an XIAP deficiencies or CDC42 mutation based autoinflammation or disease, wherein hyper-elevated levels of IL18 and/or I L1 p refers to a situation in which Patients having I L18 and/or I L1 levels significantly above the upper limit of a healthy person. In some cases, the subject in need of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease treatment has hyper-elevated serum levels IL-18 and I L-1 p as compared to a control population of subjects that do not exhibit an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In some cases, the hyper elevated serum IL-18 level is a hyper elevated total IL-18 serum level. In some cases, the hyper elevated serum IL-18 level is a hyper elevated free IL-18 serum level. In some cases, the subject has high serum C-reactive protein (CRP) levels as compared to a control population. In some cases, the subject has high serum ferritin levels as compared to a control.

In a preferred embodiment, the subject in need of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease treatment has hyper elevated total IL-18 serum level as compared to a control population of subjects that do not exhibit an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In some cases, the hyper elevated total IL-18 serum level is a level greater than 1000 pg/mL. In some cases, the hyper elevated total IL-18 serum level is a level greater than 5000 pg/mL. In some cases, the hyper elevated total IL-18 serum level is a level greater than 10,000 pg/mL. In some cases, the hyper elevated total IL-18 serum level is a level of between about 1000 pg/mL and about 20000 pg/mL. In some cases, the hyper elevated total IL-18 serum level is a level of between about 5000 pg/mL and about 20000 pg/mL. In some cases, the hyper elevated total IL-18 serum level is a level of between about 10000 pg/mL and about 20000 pg/mL. In some cases, the hyper elevated total IL-18 serum level is a level of between about 1000 pg/mL and about 25000 pg/mL. In some cases, the hyper elevated total IL-18 serum level is a level of between about 5000 pg/mL and about 25000 pg/mL. In some cases, the hyper elevated total IL-18 serum level is a level of between about 10000 pg/mL and about 25000 pg/mL.

In some cases, the subject in need of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease treatment has hyper elevated free I L-18 serum level as compared to a control population of subjects that do not exhibit an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In some cases, the hyper elevated free I L-18 serum level is a level greater than 5000 pg/mL.

In some cases, the hyper elevated serum IL-1 p level is a level greater than 5 pg/mL. In some cases, the hyper elevated serum IL-1 level is a level greater than 10 pg/mL. In some cases, the hyper elevated serum IL-1 p level is a level of between about 5 pg/mL and about 25 pg/mL. In some cases, the hyper elevated serum IL-1 p level is a level of between about 10 pg/mL and about 25 pg/mL. In some cases, the subject in need of an XI AP deficiencies or CDC42 mutation based autoinflammation or disease treatment is under the age of 17 and weighs at least 3 kg. In some cases, the subject in need of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease treatment is under the age of 10 and weighs at least 3 kg. In some cases, the subject in need of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease treatment is under the age of 5 and weighs at least 3 kg. In some cases, the subject in need of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease treatment is an infant weighing at least 3 kg, and wherein the subject has both infantile enterocolitis and hyper elevated total IL-18 serum level. In some cases, the subject in need of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease treatment is an infant weighing at least 3 kg, and wherein the subject has both infantile enterocolitis and hyper elevated free IL-18 serum level.

In a specifically preferred embodiment of the preceding aspects of the disclosure, the treated subject has AIFEC. In a further preferred embodiment, the treated subject has AIFEC and hyper elevated total IL-18 serum level. In another embodiment, the treated subject has AIFEC and hyper elevated free IL-18 serum level.

In a specifically preferred embodiment of the preceding aspects of the disclosure, the XIAP gene or the CDC42 gene of a treated subject comprises one or more point mutations or mutations including deletions (small or large exon deletions) and insertions resulting in nonsense and missense mutations, intronic or frameshift mutation. These mutations can result in an absence of a functionally expressed XIAP and CDC42 protein, truncated or dysfunctional protein, respectively. A detailed overview of 90 disease causing mutations in the XIAP gene is disclosed in Mudde et al., (2021) Evolution of Our Understanding of XIAP Deficiency. Front. Pediatr. 9:660520. The content of figure 1 of Mudde et al, is herewith incorporated by reference. As disclosed by Martinelli and coworkers (The American Journal of Human Genetics 102, 309-320, February 1 , 2018 ) nine different gene mutations have been identified for the CDC42 gene (two amino acid substitutions affected the N-terminal a helix (residues Ile21 and Tyr23), three involved adjacent residues within the switch II motif (Tyr64, Arg66, and Arg68), two mapped to the fourth b strand (Cys81 and Ser83), and the remaining two were located close to the C terminus (Ala159 and Glu171). Gernez and co-worker have described inflammatory and hematological phenotypes of 4 patients heterozygous for 3 variants affecting the CDC42 C-terminus (p.R186C, p.C188Y, and p.*192Cext*24) (Gernez Y, de Jesus AA, Alsaleem H, Macaubas C, Roy A, Lovell D, et al. Severe autoinflammation in 4 patients with C- terminal variants in cell division control protein 42 homolog (CDC42) successfully treated with IL-1 b inhibition. J Allergy Clin Immunol 2019;144:1122-5. e6). In one embodiment, the subject in need of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease treatment has a serum C-reactive protein (CRP) level higher than 20 mg/L. In another embodiment, the subject in need of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease treatment has a serum ferritin level higher than 600 pg /L. In a further preferred embodiment, the subject in need of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease treatment has a serum CRP level higher than 20 mg/L and a serum ferritin level higher than 600 pg /L.

In one embodiment of the preceding aspects, the treatment with the bispecific antibody targeting both IL-1 p and IL-18 increases the lifespan of the patient compared to standard of care treatment. In another embodiment, treatment with the bispecific antibody targeting both IL-ip and IL-18 reduces serum CRP and/or serum ferritin levels as compared with standard of care (SoC) after 14 days of treatment. In another embodiment, treatment with the bispecific antibody targeting both I L-1 and IL-18 reduces serum CRP and/or serum ferritin levels as compared with standard of care (SoC) after 28 days of treatment.

In another embodiment treatment with the bispecific antibody targeting both I L-1 p and IL-18 reduces the serum CRP level of patients after 7 days of treatment compared to standard of care. In another embodiment treatment with the bispecific antibody targeting both I L-1 p and IL-18 reduces the serum CRP level of patients after 14 days of treatment compared to standard of care. In another embodiment treatment with the bispecific antibody targeting both I L-1 p and IL-18 reduces the serum CRP level of patients after 28 days of treatment compared to standard of care. In another embodiment treatment with the bispecific antibody targeting both IL-1 p and IL-18 reduces the serum ferritin level of patients after 7 days of treatment compared to standard of care. In another embodiment treatment with the bispecific antibody targeting both IL-1 p and IL-18 reduces the serum ferritin level of patients after 14 days of treatment compared to standard of care. In another embodiment treatment with the bispecific antibody targeting both I L-1 p and IL-18 reduces the serum ferritin level of patients after 28 days of treatment compared to standard of care.

In one embodiment, provided herein is a bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for use in reducing the serum C-reactive protein (CRP) level in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In some embodiments, provided herein is the use of a bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb, for the manufacture of a medicament for reducing the serum C-reactive protein (CRP) level in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In some embodiments, the serum CRP level in the subject decreases by at least 1 mg/l, at least 2 mg/l, at least 3 mg/l, at least 4 mg/l or at least 5 mg/l. In some embodiments, the serum CRP level in subjects having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease and having received the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb decreases by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% from baseline compared to patients not having received the same treatment, e.g., patients having received standard of care (SOC). In some embodiments, the reduction in serum CRP levels occurs after 2 days, 3 days, 4 days, 5 days, 6 days or 7 days after administration of a bispecific antibody targeting both I L-1 p and IL-18, e.g., bbmAb. In some embodiments, the reduction in serum CRP levels occurs after 14 days, or 28 days after administration of a bispecific antibody targeting both I L-1 p and IL-18, e.g., bbmAb.

In one embodiment, provided herein is a method of reducing the serum ferritin level in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease, comprising administering to said subject a therapeutically effective amount of a bispecific antibody targeting both IL-ip and IL-18, e.g., bbmAb. In one embodiment, provided herein is a bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for use in reducing the serum ferritin level in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In some embodiments, provided herein is the use of a bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb, for the manufacture of a medicament for reducing the serum ferritin level in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In some embodiments, the serum ferritin level in the subject decreases by at least 100 ng/l, at least 200 ng/l, at least 300 ng/l, at least 400 ng/l or at least 500 ng/l. In some embodiments, the serum ferritin level in subjects having received the bispecific antibody targeting both I L-1 p and IL-18, e.g., bbmAb decreases by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% from baseline compared to patients not having received the same treatment, e.g., patients having received standard of care (SOC). In some embodiments, the reduction in serum ferritin levels occurs after 2 days, 3 days, 4 days, 5 days, 6 days or 7 days after administration of a bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb. In some embodiments, the reduction in serum ferritin levels occurs after 14 days or 28 days after administration of a bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb.

In one embodiment, provided herein is a method of reducing the serum level of a biomarker selected from the group consisting of CXCL9, CXCL10 (IP-10), IL-6, and slL2R in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease, comprising administering to said subject a therapeutically effective amount of a bispecific antibody targeting both IL-ip and IL-18, e.g., bbmAb. In one embodiment, provided herein is a bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for use in reducing the serum level of a biomarker selected from the group consisting of CXCL9, CXCL10 (IP-10), IL-6, and slL2R in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In some embodiments, provided herein is the use of a bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb, for the manufacture of a medicament for reducing the serum level of a biomarker selected from the group consisting of CXCL9, CXCL10 (IP-10), IL-6, and slL2R in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In some embodiments, the serum level of a biomarker selected from the group consisting of CXCL9, CXCL10 (I P-10), IL-6, and slL2R in subjects having received the bispecific antibody targeting both IL-1 and IL-18, e.g., bbmAb decreases by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% from baseline compared to patients not having received the same treatment, e.g., patients having received standard of care (SOC). In some embodiments, the reduction in the serum level of a biomarker selected from the group consisting of CXCL9, CXCL10 (IP-10), IL-6, and slL2R occurs after 2 days, 3 days, 4 days, 5 days, 6 days or 7 days after administration of a bispecific antibody targeting both I L-1 p and IL-18, e.g., bbmAb.

In an eighth aspect of the disclosure a method is provided for preventing or reducing the incidence or severity of fever in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease, comprising administering to said subject a therapeutically effective amount of a bispecific antibody targeting both I L-1 p and IL-18, e.g., bbmAb. In one embodiment, provided herein is a bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for use in preventing or reducing the incidence or severity of fever in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In some embodiments, provided herein is the use of a bispecific antibody targeting both I L-1 p and IL-18, e.g., bbmAb, for the manufacture of a medicament for preventing or reducing the incidence or severity of fever in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease.

In a ninth aspect of the disclosure a method is provided for preventing or reducing the incidence or severity of diarrhea in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease, comprising administering to said subject a therapeutically effective amount of a bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb. In one embodiment, provided herein is a bispecific antibody targeting both I L-1 p and IL-18, e.g., bbmAb for use in preventing or reducing the incidence or severity of diarrhea in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In some embodiments, provided herein is the use of a bispecific antibody targeting both I L-1 p and IL-18, e.g., bbmAb, for the manufacture of a medicament for preventing or reducing the incidence or severity of diarrhea in a subject having an XIAP deficiencies or CDC42 mutation based autoinflammation or disease. Various embodiment of the preceding aspects may be beneficially combined with the other treatments of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease in a subject in need thereof. Such therapies could be, for example, any known therapies for the disease, disorder, condition, or syndrome being treated. As a non-limiting set of examples, the at least one additional therapeutic agent may be selected from a list consisting of: a nonsteroidal anti-inflammatory, cyclosporin, a glucocorticoid, IL-18 binding protein (IL-18 BP), and combinations thereof.

In some cases, the treatment with a bispecific antibody described herein provides a reduction in, or elimination of, the maintenance dose of a glucocorticoid required to treat the subject. In some cases, the treatment includes administration of a bispecific antibody described herein and a discontinuation of a dose of a glucocorticoid. In some cases, the treatment includes administration of a bispecific antibody described herein and a discontinuation of a dose of cyclosporin.

In some cases, the treatment includes administration of a bispecific antibody described herein and a tapering of a dose of an additional therapeutic agent. In some cases, the treatment includes administration of a bispecific antibody described herein and a tapering of a dose of a glucocorticoid. In some embodiments, the treatment includes a reduction, taper, or discontinuation of glucocorticoid administration in a subject administered glucocorticoid and the bispecific antibody. In some cases, the treatment includes a reduction or taper to a dose of less than or equal to a dose equivalent to a 0.2 mg/kg/day dose of prednisone. In some cases, the treatment includes a reduction or taper to a dose of less than or equal to 0.2 mg/kg/day of prednisone. In some cases, the subject administered the treatment maintains at least a partial, or a complete, response to the bispecific antibody for at least two weeks after tapering to a dose equivalent to a 0.2 mg/kg/day dose of prednisone. In some embodiments, the treatment includes a reduction, taper, or discontinuation of glucocorticoid administration and a discontinuation of cyclosporin in a subject administered the bispecific antibody, glucocorticoid, and cyclosporin.

In some embodiments, the subject in need thereof has been, or is, administered cyclosporin, anti-TNFa, corticosteroids, anti-IFNy, anti-IL-1 p or an anti-IL-18 therapy, or a combination thereof. In some embodiments, the subject in need thereof has not achieved adequate control of the XIAP deficiencies or CDC42 mutation based autoinflammation or disease by administration of cyclosporin, anti-TNFa, corticosteroids, anti-IFNy, anti-IL-1 or an anti-IL-18 therapy, or a combination thereof. In some embodiments, XIAP deficiencies or CDC42 mutation based autoinflammation or disease in the subject in need thereof is resistant to cyclosporin, anti-TNFa, corticosteroids, anti-IFNy, anti-IL-1 p therapies as mono-therapy, or a combination thereof. In some cases, the XIAP deficiencies or CDC42 mutation based autoinflammation or disease does not respond to cyclosporin, anti-TNFa, corticosteroids, anti-IFNy, anti-IL-1 p or an anti-IL-18 therapy, or a combination thereof. In some cases, resistance and/or inadequate control is indicated by a failure to achieve a PPGA score as described herein of less than 2. In some cases, resistance and/or inadequate control is indicated by a failure to achieve a PPGA score as described herein of less than 1. In some cases, non-response is indicated by a failure to reduce a PPGA score as described herein.

In some embodiments, the treatment reduces or prevents occurrence of disease flare in the subject in need thereof compared with standard of care (SoC) after 7 or 14 or 21 or 29 days of treatment. In some embodiments, the reduction or prevention of occurrence of disease flare in the subject is a reduction or prevention for at least about a week, two weeks, or three weeks, or for about four weeks.

In some embodiments, the treatment reduces or prevents occurrence of one or more MAS features in the subject, wherein the MAS features are selected from the group consisting of fever, rash, tachycardia, cytopenia, liver dysfunction and coagulopathy. In some cases, the treatment reduces or prevents enterocolitis in the subject. In some cases, the treatment reduces or prevents severe, refractory, neonatal diarrhea in the subject. In some cases, the treatment reverses gastrointestinal pathology in the patient.

In some embodiments, the treatment increases the time to first flare in a patient, e.g., by about (or at least about) a week, a month, two months, three months, six months, or a year. In some embodiments, the treatment further comprises inducing serological remission in the patient. Serological remission can be indicated by full suppression of serum IL-18 to undetectable levels or to levels found in a healthy individual (e.g., less than 500 pg/mL, less than 1000 pg/mL, or less than 5000 pg/mL).

In some embodiments, the patient has an XIAP mutation in the XIAP gene resulting in a truncated or dysfunctional XIAP protein, wherein the mutation can be a point mutation, deletion (small or large exon deletions) or insertions resulting in nonsense and missense mutations, intronic or frameshift mutation.

In some embodiments, the patient has a CDC42 mutation in the CDC42 gene resulting in a truncated or dysfunctional CDC42 protein, wherein the mutation can be a point mutation, deletion (small or large exon deletions) or insertions resulting in nonsense and missense mutations, intronic or frameshift mutation. In particular the mutation can be an amino acid substitutions affecting the N-terminal alpha helix (residues Ile21 and Tyr23), or the adjacent residues within the switch II motif (Tyr64, Arg66, and Arg68), or in the fourth beta strand (Cys81 and Ser83), in the C terminus (Ala159 and Glu171) or the mutations p.R186C, p.C188Y, and p.*192Cext*24 as disclosed in Gernez Y, et al., J Allergy Clin Immunol 2019;144:1122-5.e6). In some embodiments, the patient is neonatal. In some embodiments, the patient is pediatric (< 18 yrs old). In some embodiments, the patient is less than 1 year old. In some embodiments, the patient is between 1 week and 1 year old. In some embodiments, the patient is between one month and 1 year old. In some embodiments, the patient is at least one week old. In some embodiments, the patient is at least one month old.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is a schematic depiction of a treatment protocol for a clinical study for treatment of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease with a bispecific antibody targeting both IL-ip and IL-18, e.g., bbmAb.

Fig. 2 is a schematic depiction of a treatment protocols of periods 1 (2A), 2 (2B) and 3 (2C).

2A: Period 1 is an Open-Label Treatment Period to identify responders to bbmAb treatment and to allow for these patients to taper their glucocorticoid dose and/or discontinue cyclosporin treatment. Period 1 is separated into 3 sub-parts (Period 1a, 1 b and 1c).

2B: Period 2 consists of a 24-week placebo-controlled, double-blind, randomized withdrawal period, primarily to assess the efficacy of bbmAb compared to placebo. At the beginning of Period 2, bbmAb responders (complete response to treatment at the end of the open label treatment in Period 1) will be randomized in a ratio of 1 :1 to bbmAb treatment (i.e. continue with 10 mg/kg) or placebo. The first scheduled blinded dosing following randomization in Period 2 will be 2 weeks after the last dose in Period 1c and will continue with dosing every 2 weeks until either disease flare occurs or 24 weeks has elapsed in Period 2.

2C: Period 3 consists of a 3-year long-term safety phase, with open-label bbmAb treatment (10 mg/kg). For Cohort 1 , the first scheduled dosing in Period 3 will be 2 weeks after the last dose in Period 1 or Period 2 and will continue with dosing separated by a minimum of 2 weeks. For Cohort 2, dosing will be scheduled following completion of the Screening and Baseline visits and confirmation that all cohort-specific eligibility criteria have been met. Dosing in Period 3 will continue approximately every 2 weeks, and protocol assessments have a reduced visit frequency as outlined in the assessment schedule

Fig. 3 depicts a profile of bbmAb serum concentration over time as predicted (lines) and as measured (data points) from a first in human healthy volunteer study.

Fig. 4 depicts predicted free IL-18 and IL-i p concentrations after administration of a bispecific antibody targeting both I L-1 p and IL-18, e.g., bbmAb, at 10 mg/kg i.v. relative to recombinant IL-18 BP and canakinumab treatment (grey shading). The dashed line refers to the lower limit of quantification (LLOQ).

DETAILED DESCRIPTION OF THE DISCLOSURE

X-linked inhibitor of apoptosis protein deficiency (XIAP, or XIAP deficiency) is a rare, inherited immunodeficiency that occurs almost exclusively in young males (Rigaud et al 2006). XIAP displays a pleiotropic function in cell survival, innate immunity, and inflammation. In particular, XIAP has been ascribed as an essential regulator of NLRP3 inflammasome activation in addition to its role in regulating caspase activity (Miyazawa and Wado 2022). The key features of XIAP deficiency include inflammatory bowel disease, often presenting as abdominal pain and diarrhea, recurrent fevers, splenomegaly and haemophagocytic lymphohistiocytosis (HLH). The latter manifestation is often triggered by infection with Epstein-Barr virus (EBV). Once diagnosed, the initial aim of therapy is to suppress inflammation, commonly using corticosteroids and biologic therapies. Inflammatory bowel disease may be treated with standard immunosuppressant medicines, although response rates are generally low. EBV infections may be treated with specific antiviral therapies and in some cases immunoglobulin therapy is used. At present the only potentially curative treatment for XIAP deficiency is hematopoietic stem cell transplant (HSCT). However, due to features of the underlying disease low intensity conditioning regimens are required and outcomes from HSCT are often poorer than the norm for patients of this age. Although the life expectancy of patients with XIAP has increased over the past decade, there is still significant morbidity and mortality in this condition (Mudde et al 2021). Loss of XIAP results in dysregulation of inflammasome activity, overproduction of inflammasome-activated cytokines, including IL-18, and cell death (Wada et al 2014). In patients with XIAP deficiency, IL-18 levels are severely elevated during HLH episodes, overwhelming the IL-18BP blocking capacity, and free IL-18 levels are detected in peripheral blood. The approach of combined IL-1 and IL-18 blockade has been utilized to alleviate clinical disease activity including HLH and gastrointestinal symptoms in one published case (Geerlinks et al 2022). An 8-year old male presented with recurrent fevers since infancy, and subsequent abdominal pain and diarrhea associated with transaminitis. The patient was hypersensitive to anakinra and was unable to wean off steroids in the context of multiple immune therapies including canakinumab, tocilizumab, colchicine, etanercept, infliximab, mesalamine, rituximab, and vedolizumab. After treatment with the recombinant IL-18 BP tadekinig alfa the patient was able to withdraw from oral steroids and had almost complete remission of symptoms apart from one episode of fever and diarrhea in over 2 years of treatment (Geerlinks et al 2022). Cell division control protein 42 (CDC42) belongs to the Rho family of small monomeric GTPases (Heasman et al 2008). It affects multiple cellular processes including cell division and migration and regulates the nervous, immune, and hematopoietic systems (Melendez et al 2011). Pathogenic mutations in CDC42 can variously affect protein function, leading to an increasingly appreciated diversity of disease phenotypes centered on neurodevelopment, hematopoiesis, and immune response (Takenouchi et al 2015, Asiri et al 2021 , Coppola et al 2022). In particular, it has been demonstrated that aberrant palmitoylation of CDC42R186C results in trapping of CDC42 within the golgi apparatus which can induce pyrin inflammasome overactivation and marked elevation of IL-1 p and IL18 (Coppola et al 2022). In 4 pediatric patients, a de novo missense mutation (p.R186C) at the C-terminus has been demonstrated to affect CDC42 localization and cause a specific constellation of neonatal-onset cytopenia, autoinflammation, and recurrent HLH (Lam et al 2019). The existence of chronic elevation of serum IL-18, even after clinical responsiveness to anti-IL-1 therapy, suggests that IL-18 is a promising therapeutic target for CDC42 C terminal disease (Lam et al 2019). In a single surviving patient in a case series of 4 patients identified with CDC42R186C mutations, ongoing HLH was associated with gross and prolonged elevation of IL18. Pharmacological intervention with glucocorticoid pulses, cyclosporine A, and high-dose anakinra (10 mg/kg/d) failed to prevent ongoing HLH and the episode only responded to emapalumab, a monoclonal antibody targeting IFN-y, a downstream mediator of IL18 activity. The patient eventually had a curative hematopoietic stem cell transplant, after which IL18 levels were normalized and the patient remained free of HLH flares (Lam et al 2019).

As used herein, the umbrella term XIAP deficiencies or CDC42 mutation based autoinflammation or disease will be used as it best reflects the underlying etiology of the disease.

XIAP deficiencies based autoinflammation or diseases or CDC42 mutation based autoinflammation or disease refer to/can results in the following clinical symptoms of hypogammaglobulinemia, cytopaenias, inflammatory bowel disease, abdominal pain and diarrhea, recurrent fevers, splenomegaly, haemophagocytic lymphohistiocytosis, neonatal-onset cytopenia, syndromic form of thrombocytopenia, Macrothrombocytopenia, dyshematopoiesis, autoinflammation, syndromic immunedeficiency, recurrent haemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome or AIFEC.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is hypogammaglobulinemia. In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is cytopaenias.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is inflammatory bowel disease.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is abdominal pain and diarrhea.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is recurrent fevers.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is splenomegaly.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is haemophagocytic lymphohistiocytosis.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is neonatal- onset cytopenia.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is syndromic form of thrombocytopenia.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is M acroth ro m bocy to pe n i a . In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is dyshematopoiesis.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is autoinflammation.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is syndromic immunodeficiency.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is recurrent haemophagocytic lymphohistiocytosis (HLH)

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is macrophage activation syndrome (MAS).

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an XIAP deficiencies based autoinflammation or disease, wherein the clinical symptom is AIFEC. CDC42 mutation based autoinflammation or diseases refer to/can results in the following clinical symptoms of hypogammaglobulinemia, cytopaenias, inflammatory bowel disease, abdominal pain and diarrhea, recurrent fevers, splenomegaly, haemophagocytic lymphohistiocytosis, neonatal-onset cytopenia, syndromic form of thrombocytopenia, Macrothrombocytopenia, dyshematopoiesis, autoinflammation, syndromic immune- deficiency or recurrent haemophagocytic lymphohistiocytosis (HLH). In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is hypogammaglobulinemia.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is cytopaenias. In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is inflammatory bowel disease.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is abdominal pain and diarrhea. In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is recurrent fevers.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is splenomegaly.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is haemophagocytic lymphohistiocytosis.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is neonatal-onset cytopenia.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is syndromic form of thrombocytopenia.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is Macrothrombocytopenia.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is dyshematopoiesis.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is autoinflammation.

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is syndromic immunodeficiency. In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is recurrent haemophagocytic lymphohistiocytosis (HLH)

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is macrophage activation syndrome (MAS).

In one embodiment, the disclosure relates to uses or methods of the bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb for the treatment of an CDC42 mutation based autoinflammation or disease, wherein the clinical symptom is AIFEC.

In subjects, e.g., infants, it is hypothesized that early treatment of the MAS-like features and enterocolitis can prevent progression of the disease to irreversible end organ damage that commonly results in a fatal outcome (Romberg et al 2014, Moghaddas et al 2018). In the critically ill pediatric population, the disease has been found to be resistant to cyclosporin, anti-TNFa treatments, systemic glucocorticoids and anti-IL-1 p therapies in combination or alone. Currently, apart from supportive medical care and immunosuppression that is of limited benefit in this population, there are no approved therapeutics that directly and specifically target the underlying IL-1 p and IL-18 driven autoinflammatory process to improve the overall clinical outcome for XlAP deficiencies or CDC42 mutation based autoinflammation or disease patients.

Herein are described methods of treating or preventing an XIAP deficiencies or CDC42 mutation based autoinflammation or disease by administering to a subject in need thereof an effective amount of a bispecific antibody targeting both IL-i p and IL-18 simultaneously (e.g., bbmAb) or functional fragments thereof. Accordingly, in one aspect provided is a method of preventing or treating AIFEC comprising administering to a subject in need thereof an effective amount of a bispecific antibodies targeting both I L-1 p and IL- 18 simultaneously (e.g., bbmAb).

The disclosure relates to bispecific monoclonal antibodies (bbmAb) or variants thereof, e.g., bispecific antibodies targeting both IL-i p and IL-18 simultaneously (e.g., bbmAb) for the use in treatment of XIAP deficiencies or CDC42 mutation based autoinflammation or disease patients having hyper elevated IL-i p and/or IL-18. The disclosure also relates to methods, treatment regimens, uses, kits and therapies for treating XIAP deficiencies or CDC42 mutation based autoinflammation or disease by employing bispecific antibodies targeting both IL-1 p and IL-18 simultaneously.

Data suggests, such as data described herein, that combined simultaneous I L-1 p and I L-18 neutralization could more potently attenuate IFN-y (and other pro-inflammatory cytokines) production compared to individual neutralization of IL-1 p or IL-18 by either anti- IL-1 or anti-IL-18 mAbs. Thus, the present disclosure is inter alia based on the unexpected finding that certain antibodies that simultaneously neutralize I L-1 and IL-18 more potently attenuate IFN-y (and other pro-inflammatory cytokines) production compared to single IL- 1 P or IL-18 neutralization alone, which is considered by the inventors to be an efficacious treatment of (i) XIAP deficiencies based autoinflammation or disease or (ii) CDC42 mutation based autoinflammation or disease or (iii) AIFEC, in patients having XIAP deficiencies or CDC42 mutation based autoinflammation or disease.

Moreover, the present inventors hypothesize that treatment with an antibody that simultaneously neutralizes IL-1 p and IL-18 (e.g., bbmAb), can allow for a significantly reduced dose frequency in XIAP deficiencies or CDC42 mutation based autoinflammation or disease patients, with administration every 2 weeks and treatment with a single agent compared to the more complex investigational combination requiring anti-IL-1 p (every two weeks for canakinumab or every day for anakinra) potentially with glucocorticoids, cyclosporin and IL-18BP (every two days).

1. Definitions

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. Additional definitions are set forth throughout the detailed description. All references, publications, patents, and database accession codes, including GenBank and OMIM and the contents therein, are herein incorporated by reference in the entirety and for all purposes.

The term “flare” in the context of a treatment of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease, refers to:

• an increase in disease activity from absent or minimal to greater than minimal in a physician global assessment of disease activity as described herein;

• a 60 % increase from normalized levels in treated patients of serum ferritin and/or C- reactive protein (CRP), wherein normalized levels indicate minimal or absent XIAP deficiencies or CDC42 mutation based autoinflammation or disease disease activity (e.g., CRP < 20 mg/L; ferritin < 600 ng/L); or

• an increase in serum ferritin levels of > 2500 ng/mL and/or elevation of CRP of > 20 mg/mL.

The term "I L-18" is synonym to I L-18 polypeptide, Interleukin- 18 polypeptide, IFN- gamma inducing factor or Interferon-gamma-inducing-factor or INF-y inducing factor. The term "IL-18" refers to human IL-18, unless another species is indicated. IL-18 is well known to a person skilled in the art, and for example obtainable from MBL® International Corporation under product reference #B001-5. Throughout this specification, the term IL- 18 encompasses both pro-IL-18 (precursor of mature IL-18 prior protease cleavage) and mature IL-18 (post protease cleavage) interchangeably unless it is specified that the pro- or mature form is meant.

The term “IL-1p” or “IL-1 b” is synonym to IL-1 polypeptide and lnterleukin-1 polypeptide. The term “IL-1 p” refers to human I L-1 p unless another species is indicated. IL-1 p is well known to a person skilled in the art, and for example obtainable from Sino Biological under product reference #10139-HNAE-5.

The term "antibody" refers to an intact immunoglobulin or a functional fragment thereof. Naturally occurring antibodies typically comprise a tetramer which is usually composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, usually comprised of three domains (CH1 , CH2 ad CH3). Heavy chains can be of any isotype, including IgG (lgG1 , lgG2, lgG3 and lgG4 subtypes), IgA (lgA1 and lgA2 subtypes), IgM and I g E. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Light chain includes kappa (K) chains and lambda (A) chains. The heavy and light chain variable region is typically responsible for antigen recognition, whilst the heavy and light chain constant region may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), 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 aminoterminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

The term "antigen-binding portion" of an antibody (or simply "antigen portion"), as used herein, refers to full length or one or more fragments of an antibody that retain the ability to specifically bind to IL-18 or IL-1 p antigen. It has been shown that the antigenbinding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a fragment comprising two monovalent Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH 1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., (1989) Nature; 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a flexible linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc Natl Acad Sc;. 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 of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The term “isolated” means throughout this specification, that the immunoglobulin, antibody or polynucleotide, as the case may be, exists in a physical milieu distinct from that in which it may occur in nature.

Throughout this specification, complementarity determining regions (“CDR”) are defined according to the Kabat definition unless specified that the CDR are defined according to another definition. The precise amino acid sequence boundaries of 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,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme) and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al. , Dev. Comp. Immunol., 27, 55-77 (2003) (“IMGT” numbering scheme). For example, for classic formats, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Under IMGT the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (numbering according to “Kabat”). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT/DomainGap Align. By convention, the CDR regions in the heavy chain are typically referred to as H- CDR1 , H-CDR2 and H-CDR3 and in the light chain as L-CDR1 , LCDR2 and L-CDR3. They are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

The term "human antibody", as used herein, 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 also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik, et al., (2000) J Mol Biol; 296:57-86).

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

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

The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created 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 a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

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

As used herein, a binding molecule that "specifically binds to I L-18"is intended to refer to a binding molecule that binds to human IL-18 with a KD of a 100nM or less, 10nM or less, 1nM or less.

As used herein, a binding molecule that "specifically binds to I L-1 p" is intended to refer to a binding molecule that binds to human IL-1 with a K_D of a 100nM or less, 10nM or less, 1nM or less.

As used herein, the term “antagonist” is intended to refer to a binding molecule that inhibits the signalling activity in the presence of activating compound. For example, in the case of IL-18, an IL-18 antagonist would be a binding molecule inhibiting the signalling activity in the presence of IL-18 in a human cell assay such as IL-18 dependent Interferongamma (IFN-y) production assay in human blood cells. Examples of an IL-18 dependent IFN-y production assay in human blood cells are described in more details in the examples below.

The term bivalent bispecific antibody or bivalent bispecific antibodies refer to antibodies binding to two different targets, such as IL-18 and I L-1 p. Typically, the bivalent bispecific antibody binds each target monovalently.

The bispecific antibodies are “hetero-dimers”, which means that one part comes from first antibody, specific for a first target, and another part comes from a second antibody, specific for a second target. A “hetero-dimerization modification” is a modification to one or both parts of the antibodies forming the hetero-dimeric bispecific antibody, intended to facilitate such formation. An example of hetero-dimerization modifications of the Fc domains of two I gG 1 parts of antibodies intended to form a bispecific is a “knob” with a bulky amino acid (aa) side chain (S354C, T366W) in the first heavy chain and a “hole” with small aa side chains (Y349C, T366S, L368A, Y407V) were introduced in the second heavy chain as well as an additional disulfide bridge in the CH3 region connecting both heavy chains (Merchant et al., Nat. Biotechnol., 16:677-681 (1998), page 678, table 1).

The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to K_a (i.e. Kd/K_a) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A method for determining the KD of an antibody is by using surface plasmon resonance, such as a Biacore® system.

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

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

As used herein, the term "subject" includes any human subjects receiving the bispecific antibody as presently described. The term subject can additionally or alternatively include any human subject that presents symptoms of or is at risk of an XIAP deficiencies or CDC42 mutation based autoinflammation or disease or an XIAP deficiencies or CDC42 mutations based inflammasomopathy, or AIFEC, e.g., as hereinabove defined.

The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

The nucleotide in the “polynucleotide” or “nucleic acid” may comprise modifications including base modifications such as bromouridine and inosine derivatives, ribose modification such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate and phosphoroamidate.

The term “treat”, “treating”, “treatment”, “prevent”, “preventing” or “prevention” includes therapeutic treatments, prophylactic treatments and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses the reduction of the symptoms or underlying risk factors.

The term “treating or preventing” includes the administration of a compound, e.g., the a bispecific antibody targeting both IL-1 p and IL-18, e.g., bbmAb optionally in combination with at least one further therapeutic agent, to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, condition, disorder, or syndrome e.g., XIAP deficiencies or CDC42 mutation based autoinflammation or disease, AIFEC), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, disorder, or syndrome. Treatment may be prophylactic (to prevent or delay the onset of the disease, condition, disorder, or syndrome, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease, condition, disorder, or syndrome.

As used herein, the term “prevent”, “preventing”, or “prevention” in connection to a disease, condition, disorder, or syndrome refers to the prophylactic treatment of a subject who is at risk of developing a condition {e.g., a specific disease, condition, disorder, or syndrome or clinical symptom thereof such as XIAP deficiencies or CDC42 mutation based autoinflammation or disease or AIFEC) resulting in a decrease in the probability that the subject will develop the condition.

The terms “treat”, “treating”, and “treatment” refer to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to ameliorate the disease, condition, disorder, or syndrome (/.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof) by alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. The terms “treat”, “treating”, or “treatment” also refer to modulating the disease or disorder, either physically, {e.g., stabilization of a discernible symptom), physiologically, {e.g., stabilization of a physical parameter), or both and/or to preventing or delaying the onset or development or progression of the disease or disorder.

For example, “treating XIAP deficiencies or CDC42 mutation based autoinflammation or disease may refer to ameliorating, alleviating or modulating at least one of the symptoms or pathological features associated with XIAP deficiencies or CDC42 mutation based autoinflammation or disease; e.g., an elevated serum inflammatory marker (such as one or more of serum CRP, serum ferritin, serum IL-18, serum total IL-18, serum free IL-18, serum IL-i p, serum total IL-i p, serum free IL-1 P), fever, diarrhea, rash, tachycardia, cytopenia, liver dysfunction and/or coagulopathy; e.g., may refer to slowing progression, reducing or stopping at least one of the symptoms or pathological features associated with XIAP deficiencies or CDC42 mutation based autoinflammation or disease; e.g., an elevated serum inflammatory marker (such as one or more of serum CRP, serum ferritin, serum IL-18), fever, diarrhea, rash, tachycardia, cytopenia, liver dysfunction and/or coagulopathy or hypogammaglobulinemia, cytopaenias, inflammatory bowel disease, abdominal pain and diarrhea, recurrent fevers, splenomegaly, haemophagocytic lymphohistiocytosis, neonatal-onset cytopenia, syndromic form of thrombocytopenia, Macrothrombocytopenia, dyshematopoiesis, autoinflammation, syndromic immune- deficiency or recurrent haemophagocytic lymphohistiocytosis (HLH), MAS or AIFEC. It may also refer to preventing or delaying one or more of the described symptoms, e.g., slow the progress of, halt, or reverse disease, condition, disorder, or syndrome progression and improve clinical outcomes {e.g., prevent fatal progression of AIFEC, and improve survival). Total IL-18 in serum can be measured by conjugating anti-human IL-18 antibody (e.g., clone 125-2H, MBL International) to Bio-plex Magnetic COOH beads (Bio-Rad, Inc.), detected using biotinylated anti-human IL-18 (clone 159-12B, MBL), and concentrations calculated using the IL-18 contained in the Group II cytokine standard curve (Bio-Rad, Inc.). Free IL-18 can be measured as described in Girard et al. Rheumatology (Oxford). 2016 Dec;55(12):2237-2247.

Serum IL-1 p can be measured using a commercially available ELISA kit (88-7261- 88, eBioscience) used in accordance with manufacturer instructions.

In some embodiments, the reduction in one or more elevated serum inflammatory marker by administration of a bispecific antibody or functional fragments thereof targeting both IL-1 and IL-18 simultaneously can be a reduction of at least 10% from baseline compared to patients not having received the same treatment, e.g., patients having received standard of care (SOO). In some embodiments, the reduction in one or more elevated serum inflammatory marker by administration of a bispecific antibody or functional fragments thereof targeting both I L-1 p and IL-18 simultaneously can be a reduction of at least 20% from baseline compared to patients not having received the same treatment, e.g., patients having received standard of care (SOO). In some embodiments, the reduction in one or more elevated serum inflammatory marker by administration of a bispecific antibody or functional fragments thereof targeting both IL-i p and IL-18 simultaneously can be a reduction of at least 30% from baseline compared to patients not having received the same treatment, e.g., patients having received standard of care (SOO). In some embodiments, the reduction in one or more elevated serum inflammatory marker by administration of a bispecific antibody or functional fragments thereof targeting both IL-1 p and IL-18 simultaneously can be a reduction of at least 40% from baseline compared to patients not having received the same treatment, e.g., patients having received standard of care (SOO). In some embodiments, the reduction in one or more elevated serum inflammatory marker by administration of a bispecific antibody or functional fragments thereof targeting both I L-1 p and IL-18 simultaneously can be a reduction of at least 50% from baseline compared to patients not having received the same treatment, e.g., patients having received standard of care (SOO).

As one example, the XIAP deficiencies or CDC42 mutation based autoinflammation or disease suitable for treatment with the compositions and methods described herein include those caused by or associated with point mutation, deletion (small or large exon deletions) or insertions resulting in nonsense and missense mutations, intronic or frameshift mutation.

In some embodiments, the suitable subject or subject in need thereof has a one of the 90 mutations in the XIAP gene disclosed in Mudde et al., ((2021) Evolution of Our Understanding of XIAP Deficiency. Front. Pediatr. 9:660520) or a mutation in the CDC42 gene, resulting in a truncated or dysfunctional CDC42 protein, wherein the mutation can be a point mutation, deletion (small or large exon deletions) or insertions resulting in nonsense and missense mutations, intronic or frameshift mutation, in particular a mutation in the N-terminal alpha helix (e.g., residues Ile21 and Tyr23), or the switch II motif (e.g., Tyr64, Arg66, and Arg68), or in the fourth beta strand (e.g., Cys81 and Ser83) or in the C terminus (e.g., Ala159 and Glu171 , or deletions) or the mutations in p.R186C, p.C188Y, and p.*192Cext*24 (as disclosed in Gernez Y, et al., J Allergy Clin Immunol 2019; 144:1122-5. e6).

Also “treating” may refer to slow the progress of, halt, or reverse disease, condition, disorder, or syndrome progression and improve clinical outcomes, e.g, moving from a higher number to a lower number on a 5-category ordinal scale as follows:

As used herein, the term “therapeutically effective amount” of the compound described herein refers to an amount of the compound that will elicit the biological or medical response of a subject, for example, ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, condition, disorder, or syndrome, etc. In one non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of the compound described herein that, when administered to a subject, is effective to at least partially alleviating, inhibiting, preventing and/or ameliorating XIAP deficiencies or CDC42 mutation based autoinflammation or disease. In one non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of the compound described herein that, when administered to a subject, is effective to at least partially alleviating, inhibiting, preventing and/or ameliorating XIAP deficiencies or CDC42 mutation based autoinflammation or disease presenting with elevated levels of IL-18 and I L-1 p. In one non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of the compound described herein that, when administered to a subject, is effective to at least partially alleviating, inhibiting, preventing and/or ameliorating AIFEC.

As used herein, a human antibody or a fragment thereof comprises heavy or light chain variable regions or full length heavy or light chains that are "the product of" or "derived from" a particular germline sequence if the variable regions or full length chains of the antibody are obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. A human antibody or fragment thereof that is "the product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e. , greatest % identity) to the sequence of the human antibody. A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences as compared to the germline sequence, due to, for example, naturally occurring somatic mutations or intentional introduction of site-directed mutation. However, a selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In certain cases, 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 in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

2. IL-18 antibody

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

For ease of reference, the amino acid sequences of the hypervariable regions of a specific IL-18 antibody, called mAb1 , based on the Kabat definition and the Chothia definition, as well as the VL and VH domains and full heavy and light chains are provided in Table 1 , below. Table 1. Amino acid sequences of the hypervariable regions (CDRs), variable domains (VH and VL) and full chains of mAb1. The DNA encoding the 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 ( H) comprising hypervariable regions 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. In one embodiment, the IL-18 antibody or antigen-binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain ( H) comprising hypervariable regions 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 N0:6.

In one embodiment, the IL-18 antibody or antigen-binding fragment thereof comprises at least one immunoglobulin light chain variable domain (VL) comprising 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. In one embodiment, the IL- 18 antibody or antigen-binding fragment thereof comprises at least one immunoglobulin light chain variable domain (VL) comprising 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 at least one immunoglobulin V_H domain and at least one immunoglobulin V_L domain, wherein: a) the immunoglobulin V_H domain comprises (e.g., in sequence): i) hypervariable regions 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; or ii) hypervariable regions 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 L 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) an immunoglobulin heavy chain variable domain (VH) comprising the amino acid sequence set forth as SEQ ID NO:7; b) an immunoglobulin light chain variable domain (VL) comprising the amino acid sequence set forth as SEQ ID NO: 17; c) an immunoglobulin VH domain comprising the amino acid sequence set forth as SEQ ID NO:7 and an immunoglobulin VL domain comprising the amino acid sequence set forth as SEQ ID NO:17; d) an immunoglobulin VH domain comprising the hypervariable regions set forth as SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3; e) an immunoglobulin VL domain comprising the hypervariable regions set forth as SEQ ID NO:11 , SEQ ID NO:12 and SEQ ID NO:13; f) an immunoglobulin VH domain comprising the hypervariable regions set forth as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; g) an immunoglobulin VL domain comprising the hypervariable regions set forth as SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO: 16; h) an immunoglobulin VH domain comprising the hypervariable regions set forth as SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3 and an immunoglobulin VL domain comprising the hypervariable regions set forth as SEQ ID NO:11 , SEQ ID NO:12 and SEQ ID NO:13; i) an immunoglobulin H domain comprising the hypervariable regions set forth as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 and an immunoglobulin V_L domain comprising the hypervariable regions set forth as SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO: 16; j) a light chain comprising SEQ ID NO: 19; k) a heavy chain comprising SEQ ID NO:9; or I) 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., mAb1) 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 I D NO: 17. In other embodiments, the IL-18 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:7 and the 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, 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 the three CDRs of SEQ ID NO:9 and the three CDRs of SEQ ID NO: 19.

In one embodiment, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb1) is selected from a human IL-18 antibody that comprises at least: a) an immunoglobulin heavy chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2 and CDR3 and the constant part or fragment thereof of a human heavy chain; 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) an immunoglobulin light chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2, and CDR3 and the constant part or fragment thereof of a human light chain, 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.

In one embodiment, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb1) is selected from a human IL-18 antibody that comprises at least: a) an immunoglobulin heavy chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2 and CDR3 and the constant part or fragment thereof of a human heavy chain; 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) an immunoglobulin light chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2, and CDR3 and the constant part or fragment thereof of a human light chain, 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 is selected from a single chain antibody or antigen-binding fragment thereof that comprises an antigen-binding site comprising: a) a first domain comprising, in sequence, the hypervariable regions 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 which is bound either to the N-terminal extremity of the first domain and to the C-terminal extremity of the second domain or to the C-terminal extremity of the first domain and to the N-terminal extremity of the second domain.

In one embodiment, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb1) is selected from a single chain antibody or antigen-binding fragment thereof that comprises an antigen-binding site comprising: a) a first domain comprising, in sequence, the hypervariable regions 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 which is bound either to the N-terminal extremity of the first domain and to the C-terminal extremity of the second domain or to the C-terminal extremity of the first domain and to the N-terminal extremity of the second domain.

The VH or VL domain of an IL-18 antibody or antigen-binding fragment thereof used in the disclosed methods may have VH and/or L domains that are substantially identical to the VH or VL domains set forth in SEQ ID NO:7 and 17. A human IL-18 antibody disclosed herein may comprise a heavy chain that is substantially identical to that set forth as SEQ ID NO:9 and/or a light chain that is substantially identical to that set forth as SEQ ID NO: 19. A human IL- 18 antibody disclosed herein may comprise a heavy chain that comprises SEQ ID NO:9 and a light chain that comprises SEQ ID NO: 19. A human IL-18 antibody disclosed herein may comprise: a) one heavy chain, comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO:7 and the constant part of a human heavy chain; and b) one light chain, comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO:17 and the constant part of a human light chain.

Other preferred IL-18 antagonists (e.g., antibodies) for use in the disclosed methods, kits and regimens are those set forth in US Patent No: 9,376,489, which is incorporated by reference herein in its entirety.

3. I L-ip antibody

Particularly preferred IL-1 p antibodies or antigen-binding fragments thereof used in the disclosed methods are human antibodies.

For ease of reference, the amino acid sequences of the hypervariable regions of a specific IL-i p antibody, called mAb2, based on the Kabat definition and the Chothia definition, as well as the V_L and V_H domains and full heavy and light chains are provided in Table 2, below.

Table 2. Amino acid sequences of the hypervariable regions (CDRs), variable domains (VH and VL) and full chains of mAb2. The DNA encoding the 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-i p antibody or antigen-binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain (VH) comprising hypervariable regions 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. In one embodiment, the I L-1 p antibody or antigen-binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain (VH) comprising hypervariable regions 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.

In one embodiment, the IL-i p antibody or antigen-binding fragment thereof comprises at least one immunoglobulin light chain variable domain (VL) comprising 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. In one embodiment, the IL- 1 P antibody or antigen-binding fragment thereof comprises at least one immunoglobulin light chain variable domain ( L) comprising 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-i p 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 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 sequence SEQ ID NO:22, and said CDR3 having the amino acid sequence SEQ ID NO:23; or ii) hypervariable regions 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 VL 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-ip antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable domain (V_H) comprising the amino acid sequence set forth as SEQ ID NO:27; b) an immunoglobulin light chain variable domain (V_L) comprising the amino acid sequence set forth as SEQ ID NO:37; c) an immunoglobulin VH domain comprising the amino acid sequence set forth as SEQ ID NO:27 and an immunoglobulin VL domain comprising the amino acid sequence set forth as SEQ ID NO:37; d) an immunoglobulin H domain comprising the hypervariable regions set forth as SEQ ID NO:21 , SEQ ID NO:22, and SEQ ID NO:23; e) an immunoglobulin VL domain comprising the hypervariable regions set forth as SEQ ID NO:31 , SEQ ID NO:32 and SEQ ID NO:33; f) an immunoglobulin VH domain comprising the hypervariable regions set forth as SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26; g) an immunoglobulin VL domain comprising the hypervariable regions set forth as SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36; h) an immunoglobulin VH domain comprising the hypervariable regions set forth as SEQ ID NO:21 , SEQ ID NO:22, and SEQ ID NO:23 and an immunoglobulin VL domain comprising the hypervariable regions set forth as SEQ ID NO:31 , SEQ ID NO:32 and SEQ ID NO:33; i) an immunoglobulin VH domain comprising the hypervariable regions set forth as SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26 and an immunoglobulin VL domain comprising the hypervariable regions set forth as SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36; j) a light chain comprising SEQ ID NO:37; k) a heavy chain comprising SEQ ID NO:29; or I) a light chain comprising SEQ ID NO:39 and a heavy chain comprising SEQ ID NO:29.

In some embodiments, the I L-1 antibody or antigen-binding fragment thereof (e.g., mAb2) comprises the three CDRs of SEQ ID NO:37. In other embodiments, the IL-1 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ I D NO:27. In other embodiments, the I L-1 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:37 and the three CDRs of SEQ ID NO:27. In some embodiments, the I L-1 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:39. In other embodiments, IL-i antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:29. In other embodiments, the I L-1 p antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:39 and the three CDRs of SEQ ID NO:29.

In one embodiment, the IL-1 antibody or antigen-binding fragment thereof (e.g., mAb2) is selected from a human IL-1 p antibody that comprises at least: a) an immunoglobulin heavy chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2 and CDR3 and the constant part or fragment thereof of a human heavy chain; 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) an immunoglobulin light chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2, and CDR3 and the constant part or fragment thereof of a human light chain, 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.

In one embodiment, the IL-1 p antibody or antigen-binding fragment thereof (e.g., mAb2) is selected from a human IL-1 p antibody that comprises at least: a) an immunoglobulin heavy chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2 and CDR3 and the constant part or fragment thereof of a human heavy chain; 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) an immunoglobulin light chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2, and CDR3 and the constant part or fragment thereof of a human light chain, 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-ip antibody or antigen-binding fragment thereof is selected from a single chain antibody or antigen-binding fragment thereof that comprises an antigen-binding site comprising: a) a first domain comprising, in sequence, the hypervariable regions 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 which is bound either to the N-terminal extremity of the first domain and to the C-terminal extremity of the second domain or to the C-terminal extremity of the first domain and to the N-terminal extremity of the second domain.

In one embodiment, the IL-1 p antibody or antigen-binding fragment thereof (e.g., mAb2) is selected from a single chain antibody or antigen-binding fragment thereof that comprises an antigen-binding site comprising: a) a first domain comprising, in sequence, the hypervariable regions 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 which is bound either to the N-terminal extremity of the first domain and to the C-terminal extremity of the second domain or to the C-terminal extremity of the first domain and to the N-terminal extremity of the second domain.

The V_H or V_L domain of an IL-i antibody or antigen-binding fragment thereof used in the disclosed methods may have V_H and/or V_L domains that are substantially identical to the V_H or V_L domains set forth in SEQ ID NO:27 and 37. A human IL-1 antibody disclosed herein may comprise a heavy chain that is substantially identical to that set forth as SEQ ID NO:29 and/or a light chain that is substantially identical to that set forth as SEQ ID NO:39. A human IL-1 p antibody disclosed herein may comprise a heavy chain that comprises SEQ ID NO:29 and a light chain that comprises SEQ ID NO:39. A human IL- 1 P antibody disclosed herein may comprise: a) one heavy chain, comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO:27 and the constant part of a human heavy chain; and b) one light chain, comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO:37 and the constant part of a human light chain.

Other preferred IL-i p antagonists (e.g., antibodies) for use in the disclosed methods, kits and regimens are those set forth in US Patent Nos: 7,446,175 or 7,993,878 or 8,273,350, which are incorporated by reference herein in their entirety.

4. Fc modifications

In addition or 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 to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the Ell numbering scheme of Edelman et al., PNAS, 1969 May, 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 approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered to, for example, 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 an antibody is mutated to decrease 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 approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase the biological half life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121 ,022 by Presta et al.

In yet 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 functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C1 q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al. In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by 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 an Fey receptor by modifying one or more amino acids. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human lgG1 for FcyRI, FcyRI I, FcyRI 11 and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et al, (2001) J Biol Chem 276:6591-6604).

In certain embodiments, the Fc domain of lgG1 isotype is used. In some specific embodiments, a mutant variant of lgG1 Fc fragment is used, e.g., a silent lgG1 Fc which reduces or eliminates the ability of the fusion polypeptide to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to bind to an Fey receptor. An example of an lgG1 isotype silent mutant wherein Leucine residue is replaced by Alanine residue at amino acid positions 234 and 235 as described by Hezareh et al, J. Virol (2001); 75(24):12161-8.

In certain embodiments, the Fc domain is a mutant preventing glycosylation at position 297 of Fc domain. For example, the Fc domain contains an amino acid substitution of asparagine residue at position 297. Example of such amino acid substitution is the replacement of N297 by a glycine or an alanine.

Silenced effector functions can be obtained by mutation in the Fc region of the antibodies and have been described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. 181 :6664-69; Strohl, W., supra); and DAPA (D265A and P329A) (Shields RL., J Biol Chem. 2001 ;276(9):6591-604; U.S. Patent Publication US2015/0320880). Examples of silent Fc lgG1 antibodies comprise the so-called LALA mutant comprising L234A and L235A mutation in the lgG1 Fc amino acid sequence. Another example of a silent lgG1 antibody comprises the D265A mutation. Another example of a silent IgG 1 antibody is the so-called DAPA mutant, comprising D265A and P329A mutations to the lgG1 Fc amino acid sequence. Another silent IgG 1 antibody comprises the N297A mutation, which results in aglycosylated/non-glycosylated antibodies. Additional Fc mutations for providing silenced effector function are described in PCT publication no. WO2014/145806 (e.g., in Figure 7 of WO2014/145806), herein incorporated by reference in its entirety. One example from WO2014/145806 of a silent lgG1 antibody comprises a E233P, L234V, L235A, and S267K mutation, and a deletion of G236 (G236del). Another example from WO2014/145806 of a silent lgG1 antibody comprises a E233P, L234V, and L235A mutation, and a deletion of G236 (G236del). Another example from WO2014/145806 of a silent lgG1 antibody comprises a S267K mutation.

In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished by; for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GIcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, EP 1 ,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. Therefore, in one embodiment, the antibodies of the invention are produced by recombinant expression in a cell line which exhibit hypofucosylation pattern, for example, a mammalian cell line with deficient expression of the FUT8 gene encoding fucosyltransferase. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al., 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1 ,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GIcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180). Alternatively, the antibodies of the invention can be produced in a yeast or a filamentous fungi engineered for mammalian-like glycosylation pattern, and capable of producing antibodies lacking fucose as glycosylation pattern (see for example EP1297172B1). Another modification of the antibodies herein that is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (01-010) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol- maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Another modification of the antibodies that is contemplated by the invention is a conjugate or a protein fusion of at least the antigen-binding region of the antibody of the invention to serum protein, such as human serum albumin or a fragment thereof to increase half-life of the resulting molecule. Such approach is for example described in Ballance et al. EP0322094.

Another modification of the antibodies that is contemplated by the invention is one or more modifications to increase formation of a heterodimeric bispecific antibody. A variety of approaches available in the art can be used in for enhancing dimerization of the two heavy chain domains of bispecific antibodies, e.g., bbmAbs, as disclosed in, for example, EP 1870459A1 ; U.S. Pat. No. 5,582,996; U.S. Pat. No. 5,731 ,168; U.S. Pat. No. 5,910,573; U.S. Pat. No. 5,932,448; U.S. Pat. No. 6,833,441 ; U.S. Pat. No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1 ; and PCT Publication No. W02009/089004A1 , the contents of which are incorporated herein in their entireties.

Generation of bispecific antibodies using knobs-into-holes is disclosed e.g., in PCT Publication No. W01996/027011 , Ridgway et al., (1996), and Merchant et al. (1998).

In practicing some of the methods of treatment or uses of the present disclosure, a therapeutically effective amount of a bispecific antibodies targeting both IL-1 and IL-18 simultaneously, e.g., bbmAb has to be administered to a subject in need thereof. It will be understood that regimen changes may be appropriate for certain patients. Thus, administration (e.g., of bbmAb) may be more frequent e.g., daily, bi-weekly dosing, or weekly dosing.

Some patients may benefit from a loading regimen (e.g., daily administrations for several days/ [e.g., 1 to 4 days e.g., dosing at day 0, 1 , 2, and/or 3] followed by a maintenance regimen starting e.g., at Week 3 or 4 where bbmAb may be administered weekly, bi-weekly or every 4 weeks for several weeks. In some embodiments, the period of administration of a a bispecific antibodies targeting both IL-1 p and IL-18 simultaneously, e.g., bbmAb is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days. In some embodiments, the period of administration of a a bispecific antibodies targeting both I L-1 and IL-18 simultaneously, e.g., bbmAb is 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, 1 1 months, 12 months, or more.

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

It will also be understood that dose reduction may also be appropriate for certain patients, such as patients, e.g., patients that display adverse events or an adverse response to treatment with the bbmAb. Thus, dosages of the may be less than about 10 mg/kg e.g., about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg or about 9 mg/kg. In some embodiments, the bbmABI dose may be adjusted as determined by a physician.

In some embodiments, the bbmABI antibody may be administered to the patient as a single dose of 10 mg/kg delivered i.v., wherein the dose may be adjusted to a higher or lower dose if needed, as determined by a physician, e.g., about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg or about 9 mg/kg or e.g., about 11 mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, etc.

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

In a specific embodiment, 10 mg/kg bbmABI is administered on day 1.

In a specific embodiment, 10 mg/kg bbmABI 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, 10 mg/kg bbmABI is administered i.v. on day 1. Example 1 :

The generation of bbmAb has been described in detail in the examples 1 to 5 of the patent application WO/2018/229612. The examples 1 of WO/2018/229612, comprising (1) vector construction, (2) Host cell line and transfection, (3) Cell selection and sorting, (4) Cell expansion, (5) Clone stability, (6) Manufacturing, (7) Analytical characterization and purity assessment, (8) Analytical Results are herewith incorporated by reference in their entirety.

The bbmAb, is a bispecific lgG1 , with LALA silencing mutations, simultaneously binding to two distinct targets, IL-1 p and IL-18. The antibody combines two distinct antigen binding arms (Fab fragments), whereas the Fab directed against IL-1 is based on mAb2 and contains a kappa light chain (Vk6). The Fab directed against IL-18 is based on mAb1 and is composed of a lambda light chain (VA1). In order to drive hetero-dimerization of the Fc domain during expression a “knob” with a bulky amino acid (aa) side chain (S354C and T366W) in the mAb1 heavy chain and a “hole” with small aa side chains (Y349C, T366S, L368A, Y407V) were introduced in the mAb2 heavy chain.

For ease of reference, the amino acid sequences of the hypervariable regions of bbmAb, based on the Kabat definition and the Chothia definition, as well as the V_L and V_H domains and full heavy and light chains are provided in Table 3, below.

Table 3. Amino acid sequences of the hypervariable regions (CDRs), variable domains (VH and VL) and full chains of bbmAb. The DNA encoding the first VL of 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, the I L-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a first immunoglobulin heavy chain variable domain (V_Hi) comprising 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. In one embodiment, I L-18/IL-1 bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a first immunoglobulin heavy chain variable domain (VHI) comprising 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 NQ:80, and said CDR3 having the amino acid sequence SEQ ID NO:81. In one embodiment, IL-18/IL-1P bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a first immunoglobulin heavy chain variable domain (VHI) comprising 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.

In one embodiment, the I L-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a second immunoglobulin heavy chain variable domain (V_H2) comprising 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. In one embodiment, the IL-18/IL-i bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a second immunoglobulin heavy chain variable domain (V_H2) comprising 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. In one embodiment, the IL-18/IL-i bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a second immunoglobulin heavy chain variable domain (VH2) comprising hypervariable regions CDR1 , CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NQ:50, said CDR2 having the amino acid sequence SEQ ID NO:51 , and said CDR3 having the amino acid sequence SEQ ID NO:52.

In one embodiment, the I L-18/IL-1 bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a first immunoglobulin light chain variable domain (Vi_i) comprising 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. In one embodiment, the IL-18/IL-i p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a first immunoglobulin light chain variable domain (VLI) comprising 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. In one embodiment, the IL-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a first immunoglobulin light chain variable domain (VLI) comprising 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 NQ:100.

In one embodiment, the I L-18/IL-1 bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a second immunoglobulin light chain variable domain (V_L2) comprising hypervariable regions CDR1 , CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NQ:60, said CDR2 having the amino acid sequence SEQ ID NO:61 and said CDR3 having the amino acid sequence SEQ ID NO:62. In one embodiment, the IL-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a second immunoglobulin light chain variable domain (V_L2) comprising 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 one embodiment, the IL-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a second immunoglobulin light chain variable domain (VL2) comprising 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, the I L-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a first immunoglobulin VHI domain and a first immunoglobulin VLI domain, wherein: a) the first immunoglobulin VHI 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) 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 N0:81 ; or iii) 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 VLI 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 NQ:100.

In one embodiment, the I L-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises a second immunoglobulin V_H2 domain and a second immunoglobulin V_L2 domain, wherein: a) the second immunoglobulin V_H2 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) 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; or iii) hypervariable regions CDR1 , CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NQ: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 VL2 domain comprises (e.g., in sequence): i) hypervariable regions CDR1 , CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NQ: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, the I L-18/IL-1 bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises: a) a first immunoglobulin heavy chain variable domain (VH1) comprising the amino acid sequence set forth as SEQ ID NO:85; b) a first immunoglobulin light chain variable domain (VLI) comprising the amino acid sequence set forth as SEQ ID NO: 101 ; c) a first immunoglobulin V_Hi domain comprising the amino acid sequence set forth as SEQ ID NO:85 and a first immunoglobulin VLI domain comprising the amino acid sequence set forth as SEQ ID NO: 101 ; d) a first immunoglobulin VHI domain comprising the hypervariable regions set forth as SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78; e) a first immunoglobulin V_Li domain comprising the hypervariable regions set forth as SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94; f) a first immunoglobulin V_Hi domain comprising the hypervariable regions set forth as SEQ ID NO:79, SEQ ID NQ:80 and SEQ ID NO:81 ; g) a first immunoglobulin V_Li domain comprising the hypervariable regions set forth as SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97; h) a first immunoglobulin V_Hi domain comprising the hypervariable regions set forth as SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78 and a first immunoglobulin V_Li domain comprising the hypervariable regions set forth as SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94; i) a first immunoglobulin V_Hi domain comprising the hypervariable regions set forth as SEQ ID NO:79, SEQ ID NQ:80, and SEQ ID NO:81 and a first immunoglobulin VLI domain comprising the hypervariable regions set forth as SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97; j) a first light chain comprising SEQ ID NQ:103; k) a first heavy chain comprising SEQ ID NO:87; or I) a first light chain comprising SEQ ID NQ:103 and a first heavy chain comprising SEQ ID NO:87.

In one embodiment, the I L-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises: a) a second immunoglobulin heavy chain variable domain (VH2) comprising the amino acid sequence set forth as SEQ ID NO:53; b) a second immunoglobulin light chain variable domain (V1.2) comprising the amino acid sequence set forth as SEQ ID NO:69; c) a second immunoglobulin VH2 domain comprising the amino acid sequence set forth as SEQ ID NO:53 and a second immunoglobulin VL2 domain comprising the amino acid sequence set forth as SEQ ID NO:69; d) a second immunoglobulin VH2 domain comprising the hypervariable regions set forth as SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46; e) a second immunoglobulin VL2 domain comprising the hypervariable regions set forth as SEQ ID NQ:60, SEQ ID NO:61 and SEQ ID NO:62; f) a second immunoglobulin VH2 domain comprising the hypervariable regions set forth as SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49; g) a second immunoglobulin VL2 domain comprising the hypervariable regions set forth as SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65; h) a second immunoglobulin VH2 domain comprising the hypervariable regions set forth as SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46 and a second immunoglobulin VL2 domain comprising the hypervariable regions set forth as SEQ ID NQ:60, SEQ ID NO:61 and SEQ ID NO:62; i) a second immunoglobulin V_H2 domain comprising the hypervariable regions set forth as SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49 and a second immunoglobulin VL2 domain comprising the hypervariable regions set forth as SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65; j) a second light chain comprising SEQ ID NO:81 ; k) a second heavy chain comprising SEQ ID NO:55; or I) a second light chain comprising SEQ ID NO:81 and a second heavy chain comprising SEQ ID NO:55.

In some embodiments, the I L-18/I L-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises three CDRs of SEQ ID NO:53. In other embodiments, the IL-18/IL-1 bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises the three CDRs of SEQ ID NO:69. In other embodiments, the IL-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises the three CDRs of SEQ ID NO:53 and the three CDRs of SEQ ID NO:69. In some embodiments, the I L-18/I L-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises the three CDRs of SEQ ID NO:85. In other embodiments, the IL-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises the three CDRs of SEQ ID NQ:101. In other embodiments, the I L-18/I L-1 bispecific antibody for use in (i) the disclosed treatment or prevention of cytokine release syndrome or cytokine storm syndrome or (ii) for use in the disclosed methods for the treatment or prevention of cytokine release syndrome or cytokine storm syndrome, comprises the three CDRs of SEQ ID NO:85 and the three CDRs of SEQ ID NQ:101. In some embodiments, the I L-18/I L-1 p bispecific antibody for use in (i) the disclosed treatment of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, comprises a the three CDRs of SEQ ID NO:85. In other embodiments, the IL-18/IL-1 bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, comprises the three CDRs of SEQ ID NO:101. In other embodiments, the IL-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, comprises the three CDRs of SEQ ID NO:85 and the three CDRs of SEQ ID NQ:101. In some embodiments, the IL-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, comprises the three CDRs of SEQ ID NO:53. In other embodiments, the IL-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, comprises the three CDRs of SEQ ID NO:69. In other embodiments, the IL-18/IL-1P bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, comprises the three CDRs of SEQ ID NO:53 and the three CDRs of SEQ ID NO:69. In an embodiment, the L-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, comprises the three CDRs of SEQ ID NO:85, the three CDRs of SEQ ID NQ:101 , the three CDRs of SEQ ID NO:53 and the three CDRs of SEQ ID NO:69.

In one embodiment, the first part of the I L-18/I L-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, is selected from a human IL-18 antibody that comprises at least: a) an immunoglobulin heavy chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2 and CDR3 and the constant part or fragment thereof of a human heavy chain; 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; and b) an immunoglobulin light chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2, and CDR3 and the constant part or fragment thereof of a human light chain, 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. Furthermore the second part of the IL-18/IL-1 p bispecific antibody is selected from a human IL-1 p antibody that comprises at least: a) an immunoglobulin heavy chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2 and CDR3 and the constant part or fragment thereof of a human heavy chain; 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; and b) an immunoglobulin light chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2, and CDR3 and the constant part or fragment thereof of a human light chain, said CDR1 having the amino acid sequence SEQ ID NQ:60, said CDR2 having the amino acid sequence SEQ ID NO:61 , and said CDR3 having the amino acid sequence SEQ ID NO:62.

In one embodiment, the first part of the I L-18/I L-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, is selected from a human IL-18 antibody that comprises at least: a) an immunoglobulin heavy chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2 and CDR3 and the constant part or fragment thereof of a human heavy chain; 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; and b) an immunoglobulin light chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2, and CDR3 and the constant part or fragment thereof of a human light chain, 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. Furthermore, the second part of the IL- 18/IL-1p bispecific antibody is selected from a human IL-1 antibody that comprises at least: a) an immunoglobulin heavy chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2 and CDR3 and the constant part or fragment thereof of a human heavy chain; 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; and b) an immunoglobulin light chain or fragment thereof which comprises a variable domain comprising, in sequence, the hypervariable regions CDR1 , CDR2, and CDR3 and the constant part or fragment thereof of a human light chain, said CDR1 having the amino acid sequence SEQ ID NQ:60, said CDR2 having the amino acid sequence SEQ ID NO:61 , and said CDR3 having the amino acid sequence SEQ ID NO:62.

The first VHI or VLI domain of an IL-18/IL-1 p bispecific antibody used in the disclosed methods may have a first VHI and/or first VLI domains that are substantially identical to the VH or VL domains set forth in SEQ ID NO:85 and 101. An IL-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, as disclosed herein may comprise a first heavy chain that is substantially identical to that set forth as SEQ ID NO:87 and/or a first light chain that is substantially identical to that set forth as SEQ ID NO:103. An IL- 18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, as disclosed herein may comprise a first heavy chain that comprises SEQ ID NO:87 and a first light chain that comprises SEQ ID NQ:103. An IL-18/I L-1 bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, as disclosed herein may comprise: a) a first heavy chain, comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO:85 and the constant part of a human heavy chain having a hetero-dimerization modification; and b) a first light chain, comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO: 101 and the constant part of a human light chain. The constant part of the human heavy chain may be lgG1. In one embodiment, the lgG1 is a human lgG1 without effector mutations. In one embodiment, the human heavy chain lgG1 comprising a silencing mutation N297A, D265A or a combination of L234A and L235A. In one specific embodiment, the human heavy chain lgG1 comprises the silencing mutation which is a combination of L234A and L235A, according to SEQ ID NO:87.

The second V_H2 or V_L2 domain of an IL-18/IL-1 p bispecific antibody used in the disclosed methods may have a second VH2 and/or first VL2 domains that are substantially identical to the VH or VL domains set forth in SEQ ID NO:53 and 69. An IL-18/IL-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, as disclosed herein may comprise a second heavy chain that is substantially identical to that set forth as SEQ ID NO:55 and/or a second light chain that is substantially identical to that set forth as SEQ ID NO:71. An IL-18/1 L-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, as disclosed herein may comprise a second heavy chain that comprises SEQ ID NO:53 and a second light chain that comprises SEQ ID NO:69. An I L-18/I L-1 p bispecific antibody for use in (i) the disclosed treatment or prevention of XIAP deficiencies or CDC42 mutation based autoinflammation or disease, as disclosed herein may comprise: a) a second heavy chain, comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO:53 and the constant part of a human heavy chain having a hetero-dimerization modification, which is complementary to the hetero-dimerization of the first heavy chain; and b) a second light chain, comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO:69 and the constant part of a human light chain. The constant part of the human heavy chain may be lgG1. In one embodiment, the lgG1 is a human lgG1 without effector mutations. In one embodiment, the human heavy chain I gG 1 comprising a silencing mutation N297A, D265A or a combination of L234A and L235A. In one specific embodiment, the human heavy chain IgG 1 comprises the silencing mutation which is a combination of L234A and L235A, according to SEQ ID NO:55.

Other preferred IL-18 antagonists (e.g., antibodies) for use as the first part of a bispecific antibody in the disclosed methods, kits and regimens are those set forth in US Patent No: 9,376,489, which is incorporated by reference herein in its entirety.

Other preferred I L- 1 p antagonists (e.g., antibodies) for use as the second part of a bispecific in the disclosed methods are those set forth in US Patent Nos: 7,446,175 or 7,993,878 or 8,273,350, which are incorporated by reference herein in their entirety.

Example 2: In vitro activity of bbmAb

Binding activity of bbmAb was tested in a variety of different cell assays.

(1) Materials and methods

(a) For solution equilibrium titration (SET) assays

The following material was used:

Recombinant human IL-18, biotinylated (BTP25828)

Recombinant Cynomolgus monkey I L-1 (Novartis)

Anti-human IgG antibody, SULFO-TAG labeled (Meso Scale discovery (MSD) # R32AJ- 5). Goat anti-human Fab specific, conjugated with MSD SULFO-TAG NHS Ester (Jackson Immuno Research # 109-005-097, MSD #R91AN-1) BSA (Sigma # A-9647) MSD read buffer T with surfactant (MSD #R92TC-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 #781280)

384-well plates, standard (MSD #L21XA)

(b) For cellular assays and SET assays mAb2 as described in section I L-1 p antibody. mAb1 as described in section IL-18 antibody. bbmAb as described in Example 1.

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

Recombinant marmoset I L-1 p (Novartis)

Recombinant marmoset IL-18 (Novartis) Recombinant human IL-12 (#573008) was purchased from Biolegend KG-1 cell line (ATCC #CCL-246)

Normal human dermal fibrobasts (#00-2509) were purchased from Lonza Marmoset skin fibroblasts (#42637F (510))

HEK-Blue™ I L-18/IL-1 p cells (#hkb-il18) were purchased from InvivoGen

PBMC were isolated from buffy coats were obtained from the Blutspendezentrum Bern Marmoset blood was obtained from SI LABE, Niederhausbergen

IL-6 ELISA: Human (BioLegend, #430503); Marmoset (U-CyTech biosciences, CT974-5) IFNy ELISA: Human (BD555142) and marmoset (U-CyTech biosciences #CT340A) QUANTI-Blue™ assays (#rep-qb1) for the detection of SEAP was purchased from InvivoGen

Cell medium: RPMI 1640 (Invitrogen #31870) supplemented with 10% Foetal Bovine Serum (Invitrogen #10108-157), 1% L-Glutamine (Invitrogen #25030-03), 1 % penicillin/ streptomycin (Invitrogen #15140-148), 10pM 2-Mercaptoethanol (Gibco #31350-010), 5mM Hepes (Gibco #15630-080)

Round-bottomed, tissue-culture treated 96-well plates (Costar #3799) Flat-bottomed, tissue-culture treated 96-well plates (Costar #3596) Ficoll-Pacque™ Plus (GE Healthcare Life Sciences #17-1440-02) PBS 1X, without Calcium & Magnesium (Gibco #14190094)

Leucosep tubes with porous barrier, 50ml, polypropylene (Greiner bio-one #227290) Falcon 15ml polypropylene conical tubes (BD#352096)

Falcon 50ml polypropylene conical tubes (BD #352070)

(c) Affinity measurements by SET

SET individual target binding assay

22 serial 1.6n dilutions of the antigens (highest cone.: hulL-18, 5 nM; marlL-18, 10 nM; hulL-1 p, 0.5 nM; marIL-i p, 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 was added (for IL-18 readout 10 pM, for IL-ip readout 1 pM). A volume of 60 pl/well of each antigen-antibody mix was distributed in duplicates to a 384-well polypropylene microtiter plate (MTP). Sample buffer served as negative control and a sample containing only antibody as positive control (Maximal electrochemiluminescence signal without antigen, B_max). The plate was sealed and incubated overnight (o/n, at least 16 h) at room temperature (RT) on a shaker.

IL-18 readout: A streptavidin coated 384-well MSD array MTP was coated with 30 pl/well biotinylated hulL-18 (0.1 pg/ml, PBS) and incubated for 1 h at RT on a shaker. I L-1 p readout: A standard 384-well MSD array MTP was coated with 30 pl/well of hulL-1 (3 pg/ml, PBS) diluted in PBS as capture agent and incubated overnight at 4°C.

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

SET simultaneous target binding assays

The SET assay was performed a described above, except for Assay A: The equilibration process (antibody/antigen mix) was performed in presence of an excess of one target (500 pM of either IL18 or I L-1 ) while assessing the K_D of the other target. Assay B: The equilibration process (antibody/antigen mix) was performed with both targets in serial dilutions in one mix simultaneously (constant concentration of antibody 10 pM, highest antigen cone, see above). The same mix was then analyzed for its free antibody concentration on IL18 and IL-1 p coated plates as described above.

The SET Data were exported to Xlfit, an MS Excel add-in software. Average ECL- signals were calculated from duplicate measurements within each assay. Data were baseline adjusted by subtracting the lowest value from all data points and plotted against the corresponding antigen concentration to generate titration curves. KD values were determined by fitting the plot with the following:

1 :2 binding model for the monospecific Ab

1 :1 binding model for the knob in hole bispecific Ab wherein y: blank subtracted ECL signal

Bmax: maximal ECL signal at zero antigen concentration

[IgG]: applied antibody concentration [Fab]: applied total Fab concentration KD: Dissociation equilibrium constant x: applied antigen concentration

(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 2x10⁵ to 1x10⁶ viable cells/mL.

Normal human fibroblasts and marmoset fibroblasts were grown in FBM (Clonetics, CC- 3131 ) including bFGF (1 ng/ml, CC-4065), insulin (5 pg/ml, CC-4021), and 2% FCS (CC- 4101). As starving medium, Fibroblast Basal Medium (LONZA # CC-3131) was used.

HEK-Blue™ I L-18/I L-1 p cells were grown in Growth Medium (DMEM, 4.5 g/l glucose, 10% (v/v) fetal bovine serum, 50 ll/rnl penicillin, 50 mg/ml streptomycin, 100 mg/ml Normocin™, 2 mM L-glutamine supplemented with 30 pg/ml of Blasticidin, 200 pg/ml of HygroGold™ and 100 pg/ml of Zeocin™.

Human peripheral blood mononuclear cells (PBMC) were freshly isolated from buffy coats using LeucoSep tubes according to the instructions of the manufacturer. In brief, 13 ml of Ficoll-Paque was preloaded in a 14 ml LeucoSep tube by centrifugation for 30 s at 1 ,000 x g. The heparinized whole- blood samples were diluted with equal volumes of PBS, and 25 ml of the diluted blood was added to a LeucoSep tube. The cell separation tubes were centrifuged for 15 min at 800 x g without break at room temperature. The cell suspension layer was collected, and the cells were washed twice in PBS (for 10 min at 640 and 470 x g, respectively, for the two successive washes) and re-suspended in culture medium before counting.

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

(e) IL-1 /3 neutralization assays

The IL-1 induced IL-6 production assay in fibroblasts was conducted essentially as described (Gram 2000) with only minor modifications. Briefly, fibroblasts were seeded at a density of 5x103 cells per well (in 10OpI) in a 96 well flat bottom tissue culture plate. The following day, cells were starved for 5h in starving medium before addition of the recombinant IL-ip/compound solution mix (IL-1 p concentration indicated in the table). The IL-ip/compound solution mix was prepared beforehand by incubating recombinant IL-i p with a concentration range of compound for 30min at 37°C. The cell supernatants were collected after o/n incubation at 37°C and the amount of released IL-6 determined by ELISA. The I L-1 p induced IL-6 production assay in PBMC was performed according to the following. PBMC were seeded at 3x10⁵ cells per well (in 10OpI) in a 96 well tissue culture plate and incubated with a recombinant IL-1 p/compound solution mix for 24h at 37°C (IL- 1 concentration indicated in the table). The I L-1 p/compound solution mix was prepared beforehand by incubating recombinant I L-1 p with a concentration range of compound for 30min at 37°C. The cell supernatants were collected after 24h of stimulation and the amount of released IL-6 determined by ELISA.

(f) I L-18 neutralization assays

The assay was conducted essentially according to the following. KG-1 cells (starved for 1 h in PBS + 1% FCS beforehand) or PBMC at a density of 3 x10⁵ per well were seeded into round bottom 96-well cell culture plates and incubated with a solution mix of recombinant IL-18/IL-12 together with a concentration range of compounds (IL- 18/IL-12 concentrations indicated in the table). After an incubation of 24h at 37°C, supernatants were collected and the amount of released IFNy determined by ELISA. For the assays with marmoset blood 85pl of blood per well were used.

(g) Dual IL 1/3/1 L- 18 neutralization in HEK-Blue ™ cells

The assay was conducted essentially as described in the manufacturer’s handling procedures. Briefly, the HEK-Blue™ cells were seeded at a density of 4 x10⁴ per well into 96-well cell culture plates and incubated with a solution mix of recombinant I L-1 p and IL- 18 (to produce a 1 :1 SEAP signal) together with a concentration range of compounds. After an incubation of 24h at 37°C, supernatants were collected and the amount of released SEAP determined by using the QUANTI-Blue™ method according to the manufacturer’s instructions.

All Data were exported to EXCEL software and IC50 values calculated by plotting dose-response curves for the logistic curve fitting functions using either EXCEL/XLfit4 or GraphPad Prism software.

(2) Results

(a) Affinities to recombinant human and marmoset IL 7/3 and IL-18

Binding affinities of bbmAb to human and marmoset recombinant I L-1 p and IL-18 proteins were measured by solution equilibrium titration (SET) titration and the K_D values generated were compared to those of mAb2 for IL-1 p and mAb1 for IL-18 binding. Comparing binding affinities in the individual target binding assay, bbmAb showed a similar mean KD compared to mAb1 for human and marmoset IL-18 (Table 4). For human I L-1 p binding the mean KD value was slightly higher for bbmAb (2.6 pM) compared to mAb2 (0.6 pM) but still in the same low pM range. Subsequent measurements in the simultaneous dual target binding assay (Table 5) confirmed that bbmAb binding KD values for IL-1 p were similar to values of mAb2 with the pre- clinical as well as with the clinical grade material. Thus, bbmAb possesses binding affinities for both targets in humans and marmosets that are in similar to mAb2 and mAb1, respectively.

Table 4. Affinities to recombinant human (hu) and marmoset (mar) IL-ip and IL-18 measured by SET (individual target binding determination)

In addition to the individual target binding results, simultaneous dual target binding affinities of bbmAb were investigated (Table 5) by applying either excess of one target during the assessment of the binding the KD values of the other target (Assay A) or by applying a mixture of both targets in serial dilutions (Assay B). Simultaneous IL-1 p/IL-18 affinity determination showed no significant difference between Assay A (excess of one antigen) and Assay B (mixture of both antigens in serial dilutions) which proved that both targets are bound simultaneously without affecting the binding of the other target. Furthermore, the KD values obtained with the simultaneous dual binding assays were similar to the KD values obtained with the standard assay (Table 4; in the absence of the second antigen) which proved that bbmAb can bind both antigens independently. Thus, bbmAb binds simultaneously and independently both human IL-ip and IL-18 and fully cross-reacts with the corresponding marmoset proteins.

Table 5. Affinities to recombinant human (hu) and marmoset (mar) IL-ip and IL-18 measured by SET (simultaneous target binding determination

(b) Neutralizing activity of bbmAb in human and marmoset cell assays

The neutralizing activity of bbmAb for both cytokines (IL1 p and IL- 18) was assessedmAb2mAb1). In addition, the potency of bbmAb for the neutralization of marmoset I L-1 p and IL-18 using marmoset cell assay systems was assessed (see section d).

(c) Individual and simultaneous IL-1/3 and IL-18 neutralization in human cells

The neutralizing activity of bbmAb on IL-ip was assessed by the inhibition of recombinant IL-ip-induced IL-6 production in human dermal fibroblasts (IL-1 used at 6pM) and in human PBMC (IL-1 p used at 60pM). The neutralizing activity of bbmAb on IL- 18 was measured by the inhibition of recombinant IL-18-induced IFN-y production in KG- 1 cells and human PBMC (both cells activated with 3nM recombinant human IL-18 together with 1 ng/ml of recombinant human IL-12). The inhibitory potency of bbmAb on I L-1 p and IL-18 was always compared to that of either mAb2 or mAb1 , respectively. Depending on the assays, the mean IC50 values of bbmAb were in sub-nM or single digit nM ranges and up to 2-to 4-fold higher in direct comparison against mAb2 (for IL-1 p) and mAb1 (for IL-18), respectively (Table and Table 77). The monovalent format of bbmAb as compared to the bivalent format of mAb2/mAb1 but also potentially the KiH mutations may be reasons for this slight difference in potency of bbmAb.

Table 6. Mean IC50 values for IL-1 p neutralization by bbmAb in comparison to mAb2 in human dermal fibroblasts and human PBMC. inhibition of IL-6 production in human dermal fibroblasts or PBMC stimulated with recombinant human IL-1 p (6pM for dermal fibroblasts and 60pM for PBMC). Shown are mean values ± SEM (n=3 PBMC and n=6 human dermal fibroblasts) Table 7. Mean IC50 values for IL-18 neutralization by bbmAb in comparison to mAb1 in KG-1 cells and human PBMC. ^Inhibition of IFNy production in KG-1 cells or PBMC stimulated with recombinant human IL-18 (3nM) and human IL-12 (1ng/ml). Shown are mean values ± SEM (n=3 KG-1 and n=4 PBMC) bbmAb was able to neutralize simultaneously the bioactivity of both I L-1 p and IL- 18 as demonstrated with the HEK Blue™ reporter cells producing SEAP in response to a 1+1 stimulation with recombinant IL-1 and IL-18 (Table 88). A similar inhibition of SEAP in this assay system was only achievable by the combination of mAb2 and mAb1 but not by the use of the individual antibodies.

Table 8. Mean IC50 values for simultaneous neutralization of IL-1 p and IL-18 on SEAP reporter activity in HEK Blue™ cells. Shown are means ± SEM of n=5 experiments.

(d) Neutralizing activity of bbmAb on marmoset IL-1/3 and marmoset IL-18 in marmoset cell assays

In order to demonstrate the inhibitory activity of bbmAbl in marmoset, similar in vitro assays were performed with marmoset cells as with human cells however using recombinant marmoset IL -ip and IL-18 for stimulation. When assessing the inhibition of recombinant marmoset IL-ip-induced IL-6 production in marmoset dermal fibroblasts, bbmAbl displayed sub-nM potency with 2-to 3-fold higher IC50 values compared to mAb2 (Table 99). Testing bbmAbl with human dermal fibroblasts stimulated with marmoset IL- 1P generated a similar inhibition profile as with human IL-6.

Table 9. Inhibition of recombinant marmoset IL-1 p induced IL-6 production in marmoset and human fibroblasts by bbmAbl. * Inhibition of IL-6 production in marmoset or human dermal fibroblasts stimulated with recombinant marmoset IL-1 p (18pM). Results of 3 individual experiments (A, B and C) are shown.

Single to double digit nM IC50 values of bbmAbl confirmed the neutralizing activity of bbmAbl for marmoset IL-18 tested in the IFNy production assay with marmoset blood cells (Table 4-7). Testing bbmAbl with human PBMC stimulated with marmoset IL-18 generated a similar inhibition profile when measuring the production of human IFNy.

Thus, bbmAbl was shown to be fully cross -reactive to marmoset IL-i p and marmoset IL- 18 in functional assays using marmoset responder cells.

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

It was demonstrated that bbmAbl , a KiH format IL-1 p/IL-18 bi-specific mAb retains the high affinity binding as well as the cytokine neutralizing potency to the two individual targets IL-1 and IL-18 when compared to the original mAbs, mAb2 and mAb1 , in a variety of different cell assays. The dual I L-1 p and IL-18 neutralizing properties of bbmAbl were not only demonstrated for the human cytokines/cells but also for the corresponding marmoset cytokines/cells, facilitating appropriate toxicology studies. The up to 2-to 4-fold higher IC50 values that were generated in some of the cellular assays for IL-1 p and IL-18 neutralization may be the consequence of the monovalent binding of bbmAbl as opposed to bi-valent binding of mAb2 and mAb1 , respectively. Nevertheless, the dual cytokine neutralization by bbmAbl may result in additive or synergistic inhibitory activities in vivo that may not be adequately represented in our in vitro cellular systems.

Example 3: Effects of combined IL-1 p and IL-18 stimulation and blockade in PBMC

Inflammasome activation-dependent cleavage of the effector cytokines I L-1 and IL-18 leads to the induction of secondary pro-inflammatory mediators and promotes immune cell recruitment/activation not only systemically but also at the site of inflammation. In two different mouse models for lethal systemic inflammation (a) LPS injection model and (b) FCAS mice (activating missense mutations in NLRP3), the simultaneous absence/inhibition of both I L-1 p and IL-18 was more protective from lethality compared to the single IL -ip or single IL-18 absence/inhibition, demonstrating additive or synergistic mechanisms for immune activation (Brydges 2013, van den Berghe 2014). bbmAbl is a human/marmoset IL-1 p/l L-18 reactive bi-specific mAb with no rodent crossreactivity and thus cannot be tested in mouse models. Therefore, we used LPS/IL-12 to mimic inflammasome-dependent pathway activation in vitro for the stimulation of human PBMC to reveal additive or synergistic inhibitory effects of combined I L-1 p/l L-18 neutralization by bbmAbl and performed a non-biased gene expression analysis using microarrays. As a complementary activity we also compared the gene expression profiles of PBMCs from different donors stimulated with either the combination of recombinant IL- 1 P and recombinant IL-18 or the single cytokines alone.

(3) Materials and methods

(a) Cell culture and ELISA

RPMI 1640 (Invitrogen #31870 or Gibco #61870-010) supplemented with 10% Foetal Bovine Serum (Invitrogen #10108-157), 1 % L-Glutamine (Invitrogen #25030-03), 1 % penicillin/ streptomycin (Invitrogen #15140-148), 10pM 2-Mercaptoethanol (Gibco #31350- 010), 5mM Hepes (Gibco #15630-080)

Recombinant Human I L-1 p was purchased from Sino Biological Inc. (#10139-HNAE-5) Recombinant human IL-18 was purchased from MBL (#B001-5)

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

IFNy ELISA: MAX Standard Set, BioLegend, #430103 or BD OptEIA human IFNy ELISA Set, BD #555142

IL-6 ELISA: MAX Standard Set, BioLegend, #430503

IL-26 ELISA: Cloud Clone Corp #SEB695Hu mAb2 as described in section I L-1 p antibody. mAb1 as described in section IL-18 antibody. bbmAbl as described in Example 1.

LPS from Salmonella enterica serotype enteritidis, Sigma #L7770

PBMC were isolated from buffy coats that were obtained from the Blutspendezentrum Bern Round-bottomed, tissue-culture treated 96-well plates (Costar #3799) Flat-bottomed, tissue-culture treated 96-well plates (Costar #3596) Ficoll-Pacque ™Plus (GE Healthcare Life Sciences #17-1440-02) PBS 1X, without Calcium & Magnesium (Gibco #14190094) Falcon 15ml polypropylene conical tubes (BD#352096) Falcon 50ml polypropylene conical tubes (BD #352070)

LeucosepTM tubes with porous barrier, 50ml, Greiner bio-one #227290

Cell strainer 70pM, BD Biosciences #352350

Trypanblue, Sigma # T8154

RNA isolation, quantity and quality measurements and qPCR:

Nuclease-free water, Ambion #AM9938

Rnase Zap, Ambion #AM9780

1.5ml Eppendorf tubes, sterile, Rnase & Dnase free

RLT buffer, Qiagen #1015762

Rneasy Mini Kit, Qiagen #74104

RNase-Free DNase Set, Qiagen #79254

Agilent RNA 6000 Nano Kit, Agilent #5067-1511

Chip priming station, Agilent #5065- 4401

I KA vortex mixer

RNaseZAP®, Ambion #9780

Agilent 2100 Bioanalyzer

High Capacity cDNA reverse transcription kit, Applied Biosystems, # PN4374966 Nase-free, Thin-Walled, forsted Lid 0.2ml PCR tubes, Ambion #AM 12225 MicroAmp Optical 384 well reaction plate, Applied Biosystems #4309849 TaqMan GenEx Master Mix, Applied Biosystems #4369514

PCR primer (Applied Biosystems)

PBMC preparation: PBMCs were isolated from buffy coat by means of Ficoll - Paque gradient centrifugations in Leucosep tubes according to the manufacturer’s instructions. Briefly, 15mL of Histopaque was put in 50mL LeucosepTM tubes and centrifuged for 30 sec at 1300rpm at RT. With a pipette, 30mL of a diluted suspension of the buffy coat was added on the top of the Histopaque solution and centrifuged during 15min at RT at 1000g without break. Plasma was discarded (approx. 20ml) and the interface ring collected (=human PBMC) and transferred in a 50 ml falcon tube. The tube was filled with 50mL of sterile PBS and centrifuged once at 1200rpm during 5min at RT. This centrifugation was repeated 2 times. The supernatant was gently discarded and cells re-suspended in 50mL of PBS with 2% FCS and 2mM EDTA. The cell suspension was filtered using a 70pm cell strainer and cells counted using trypan blue staining (500pL of trypan blue + 200pL of cells + 300pL of PBS).

LPS/IL-12 stimulation of PBMC: Cytokine production in supernatants was prepared according to the following. 250'000 cells/well in 100ul final volume were distributed in 96- well round bottom plates. LPS was used at concentrations between 0.3ug/ml and 3000ug/ml together with recombinant IL-12 at 10ng/ml. Supernatants were harvested after 24h at 37°C and 10% CO₂.

RNA extraction from cell pellets was performed according to the following. 3x10⁶ cells/well in 1000ul final volume were distributed in flat bottom 24-well plates. LPS was used at 3ug/ml together with recombinant IL-12 at 10ng/ml. Cells were harvested after 24h at 37°C and 10% CO₂.

Stimulation of PBMC with recombinant cytokines: 7x10⁶ PBMC per well of a 12- well plate were used in 1.5ml final of complete RPMI medium. Recombinant cytokines were added at the following final concentrations: 10ng/ml of recombinant IL-1 p, 3nM of recombinant IL-18, 1 ng/ml of recombinant IL-12. Both, supernatants as well as cells were collected after 4h and 24h at 37°C and 10% CO₂.

RNA isolation, quantity and quality assessments: Cells were pelleted and the pellet lysed in 350pl of Qiagen RTL buffer with 2% p-mercaptoethanol and frozen at -20°C or - 80°C until all samples of the study have been collected. The RNA isolation was performed using the Qiagen standard protocol. Briefly, 350pl of 70% Ethanol was added in all samples prior to the transfer to the RNeasy spin column and centrifuged for 15s at 8000g. After discarding the flow-through, 350pl of buffer RW1 was added and the column centrifuged for 15s at 8000g to wash the spin column membrane. DNase I incubation mix solution was prepared according to the manufacturer’s instructions and added to the RNeasy spin column and incubated for 15min at RT. After washes with 350pl and 500pl of buffer RW1 , the RNeasy spin column was placed in new 2 ml collection tube and centrifuged at full speed for 1min. RNA was finally collected by adding 35pl RNase-free water directly to the spin column membrane and a centrifugation for 1 min at 8000g to elute the RNA. The amount of RNA was measured using Nanodrop ND-1000 and the RNA was stored at -20°C. RIN measurements were performed for the RNA quality assessment according to manufacturer’s instructions. Briefly 1 l of RNA or ladder were pipetted into an Agilent RNA 6000 Nano chip and measured by using the Agilent 2100 Bioanalyser.

Cytokine gene expression analysis by qPCR:

The method was performed corresponding to the manufacturer’s instructions. Briefly, 400ng of RNA was reverse transcribed according to the instructions using the High -Capacity cDNA Reverse Transcription Kit. The cDNA solutions were diluted 1/10 in RNA/DNA free water and 1 pl cDNA was transferred into a 384-well reaction plate and then mixed with 1 pl of 20X TaqMan® Gene Expression Assay target FAM gene and 10pl of 2x TaqMan® Gene Expression Master Mix and 10pl RNA/DNA free water. The plate was loaded onto the Applied Biosystems ViiA™ 7 Real-Time PCR System and the following instrument settings were used:

The house keeping genes used for this study were HPRT1 and RLP27. The following formula was used to calculate the relative expression levels of target genes:

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

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

3) dCt [Target] = Ct [Target] - Ct [Ref]

4) ddCt = dCt [Ref] - dCt [Target]

5) Relative target gene expression = 2^AddCt

Microarrays was performed according to the following. Samples were processed by CiToxLAB France on Affymetrix HG_U133_Plus2 microarrays. They were RMA normalized and analyzed in GeneSpring 11.5.1 (Agilent Technologies, Santa Clara, CA). Pathway analysis was done using Ingenuity Pathway Analysis (I PA) and Nextbio (Illumina). The two datasets were treated independently.

Initially, the data were subject to standard quality control (QC) by CiToxLAB, inhouse QC by using an R script (MA_AffyQC.R) in Rstudio suite and in GeneSpring (PCA, hybridization controls). Subsequently, it was filtered to eliminate unreliable expression levels: Entities (probesets) were kept where at least 100 percent of samples in any 1 of the experimental conditions have values above the 20th percentile. Differentially expressed genes (DEG) were identified using the “filter on volcano plot” feature in GeneSpring. Using the filtered genes (expression between 20.0 - 100.0th percentiles) with an unpaired T-test, probesets with a corrected p-value below 0.05 and a fold change above 2.0 were considered differentially expressed. Where possible, i.e. in the study with LPS (NU ID-0000-0202-4150) a Benjamini-Hochberg Multiple Testing Correction was used.

For cytokine stimulation experiments, synergism was calculated using the following formula: Signal A+B I (Signal A + Signal B - Control) >1.5

The respective signatures (or DEG lists) were used to calculate p-values with a Fisher’s exact test which represent the statistical significance of observing an overlap between the signature and the ‘disease gene list’ (lesional vs non-lesional) of public datasets. To do so, the lists were uploaded into Illumina Base Space Correlation Engine (former Nextbio) and compared using the Meta-Analysis feature and keyword search for diseases.

All Data were exported to EXCEL software and IC50 values calculated by plotting dose- response curves for the logistic curve fitting functions using either EXCEL/XLfit4 or GraphPad Prism software. Differences between treatment groups were analyzed by oneway ANOVA followed by Dunnett's multiple comparison using GraphPad Prism software and results were considered statistically significant at p < 0.05.

(4) Results

(a) bbmAbl is highly efficacious in inhibiting LPS/IL-12 induced IFNy production in whole blood

Exposure of human whole blood to LP S supplemented with 10ng/ml IL- 12 results in an IFNy response that is largely but not exclusively dependent on the “native” IL -18 produced by the blood cells. The addition of IL-12 enhances the LPS induced IFNy responses, likely by up-regulating IL-18 receptors on responder cells.

In the experimental conditions used, IL-18 neutralization with mAb1 lead only to an incomplete inhibition of IFNy production whereas IL-1 p blockade (using mAb2) had only small effects on the IFNy response. Interestingly, the combined inhibition of I L-1 and IL- 18 either by bbmAbl or the combination of mAb2 and mAb1 was more profoundly and completely inhibiting IFNy production compared to the single cytokine neutralization.

Apart from IFNy, none of the other cytokines tested (IL-2,-4,-6,-8,-10,-13 and TNFa) were additively inhibited by the combined neutralization of IL-1 p and IL-18 in our cell assay (data not shown). The potency of bbmAbl was in the same range as the combination (combo) of mAb2 and mAb1 , considering the monovalent format of the bispecific molecule.

(b) IFNy is additively inhibited by bbmAbl (i.e. combined IL-1/3/IL-18 inhibition) compared to single IL-1/3 or IL-18 inhibition in LPS/IL-12 activated human PBMC An unbiased transcriptomics evaluation was required in order to reveal further additive effects (apart of IFNy) by combined IL-ip/IL-18 inhibition using bbmAbl. Since whole blood is not optimal for transcriptomics analysis we adapted the LPS/IL-12 stimulation assay conditions, described in the materials and method section above, to human PBMC samples. By using PBMCs from a total of 9 donors, we could confirm that bbmAbl additively inhibited IFNy protein secretion into the supernatants of the PBMCs. Compared to whole blood experiments, IFNy production was inhibited at approximatively 10-fold lower concentrations of the respective mAbs used. Importantly, a similar inhibition pattern was demonstrated at the mRNA level for IFNy which confirmed the suitability of the samples for a non -biased microarray based gene expression analysis. Data shows the inhibition of LPS (0.3pg/ml) /IL-12 induced IFNy protein production and IFNy gene expression by bbmAbl , mAb2 and mAb1 (at 10nM cone, each) in human PBMC.

The Affymetrix microarray was conducted with n=5 individual donors from PBMCs that were sampled from the LPS/IL-12 stimulation experiments described in the materials and method section above. Unfortunately, the overall assessment of the gene expression profiles evidenced a strong LPS/IL-12 stimulation effect and the PCA showed clustering per donor rather than compound within the stimulated or unstimulated groups. Nevertheless, comparing the LPS/IL-12 stimulated samples with the stimulated plus bbmAbl for differentially expressed genes revealed a shortlist of genes that are downregulated by the combined IL-ip/IL-18 blockade with bbmAbl (Table 11). Apart from the strong downregulation of the IFNy gene that re-validated our microarray data, also the IL-26 gene was a further cytokine gene additively inhibited by bbmAbl compared to the single IL-ip inhibition (by mAb2) or IL-18 inhibition (by mAb1). Microarray data derived gene expression levels for IFNy and IL-26 and the inhibition by bbmAbl , mAb2 and mAb1 (10nM each) in LPS (0.3ug/ml) I IL-12 stimulated PBMC at 24h was observed.

Table 11. Differentially expressed genes (downregulated genes only between the bbmAbl and control group in LPS/IL-12 stimulated samples). FC= fold change.

(c) IL-26 is another pro-inflammatory cytokine additively inhibited by bbmAbl in LPS/IL-12 stimulated PBMC

To further confirm that LPS/IL-12 driven IL-26 gene expression and protein production is most efficiently inhibited by combined IL-1 p/IL-18 blockade using bbmAbl , the study was extended to a total of n=9 PBMC donors and investigated IL-26 gene expression by qPCR and IL-26 protein production by ELISA. The results largely confirmed the inhibition of IL-26 gene expression obtained with the microarray approach. Interestingly, IL-26 protein levels in supernatants were only partly reduced at 24h by the addition of the mAbs. The reasons for this differences are unknown, could however be related to kinetic differences between IL-26 gene expression and protein production as well as differences in the consumption of IL-26 compared to IFNy. Nevertheless, bbmAbl was superior in reducing IL-26 protein levels in the PBMC supernatants compared to mAb2 and mAb1. Results demonstrated the inhibition of LPS (0.3ug/ml) /IL-12 induced IL-26 gene expression (by qPCR) and IL-26 protein levels by bbmAbl , mAb2 and mAb1 (10nM each) in human PBMC.

(d) IL1 /I L18 signaling signatures correlate with disease

Previously established PBMC culture conditions where recombinant IL-i p stimulation resulted in either IL-6 production or recombinant IL-18/IL-12 stimulation resulted in IFNy production was combined to reveal additive or synergistic downstream target genes or signatures (data not shown). With PBMCs from n=4 donors sampled at two different time points (6h and 24h) an Affymetrix microarray evaluation for unbiased assessment of the gene expression profiles was conducted. Genes that were synergistically upregulated at 6h and at 24h with the combined stimulation by IL-1 p and IL-18 were revealed (data not shown). The addition of IL-12 to the I L-1 p/IL-18 combination largely increased the synergy for a series of upregulated genes. The generated signalling signatures of single or combined IL -1 p/IL-18 pathway stimulation (UP-regulated genes only) were used to interrogate dataset from patients across several autoimmune diseases. For example, correlation to public sarcoidosis datasets was observed. P-values (calculated with a Fisher’s exact test) show a significant correlation to several public studies comparing healthy to diseased tissues from sarcoidosis patients. Tissues include skin as well as lung, lacrimal glands and anterior orbit. Across all datasets, the combination of IL1 p /IL18 signaling shows the best correlation to disease, followed by I L-1 p and IL-18. I L-1 p/IL-18 differentially up-regulated genes (DEG) in PBMC (x-axis) compared to 5 different sarcoidosis tissue ‘diseased vs healthy’ DEG. P- values (y-axis) represent the statistical significance of observing an overlap between the signature and the ‘disease gene list'. Black bar is skin from cutaneous sarcoidosis lesion vs skin from healthy patients. Light grey bar is skin from cutaneous sarcoidosis lesion vs non-lesioned skin. White bar is lacrimal glands from sarcoidosis patients vs normal. Dark grey bar is anterior orbit tissues from sarcoidosis patients vs normal. Striped bar is lung samples with progressive fibrotic, pulmonary sarcoidosis vs nodular self-limiting pulmonary sarcoidosis.

(e) Conclusion

LPS and recombinant IL-12 was used to mimic pathogen associated molecular pattern (PAMP)-dependent NLRP3 inflammasome activation within the first 24h of in vitro culture. It was demonstrated that combined inhibition of I L-1 p and IL-18, by using bbmAb, acts additively to decrease/inhibit IFNy production in PBMC stimulated with LPS/IL-12. IL- 12 was previously described to act synergistically with IL-18 to induce IFNy production in T, B, NK cells, macrophages and dendritic cells (as reviewed by Nakanishi, 2001) but now an additional stimulatory effect of IL-1 p on IFNy production could be demonstrated under the experimental conditions used. Thus, the co-incubation of PBMC with LPS/IL-12 drives efficiently the production of “native” IL-1 p and IL-18 which contribute both to a strong IFNy response. By using unbiased microarray transcriptomics, additional genes were identified that were additively down- regulated by combined IL-i p/IL-18 neutralization vs. single IL- 1 P or IL-18 blockade. Amongst those was IL-26, a member of the IL-20 cytokine subfamily (IL-19, IL-20, IL-22, IL-24, and IL-26), which is conserved in most vertebrate species but absent in most rodent strains (including mice and rats) (Donnelly 2010). It signals through a heterodimeric receptor complex composed of the IL-20R1 and IL-10R2 chains. IL-26 receptors are primarily expressed on non-hematopoietic cell types, particularly epithelial cells. Increased levels of IL-26 were reported in serum and particularly in the synovial fluid of RA patients where it could act as factor to promote Th17 cell growth and differentiation. Unfortunately, the discovery of further genes/pathways induced by the combined blockade of I L-1 p and IL-18 was hampered by the strong effect of the LPS/IL-12 stimulation of the PBMC samples. Nevertheless, both IFNy and IL-26 and to some extend also IL-22 were also among the genes that were synergistically upregulated by the combined stimulation with recombinant IL-i and IL-18 in PBMC, confirming that these two factors are downstream effectors in this activation pathway. Thus, the IL-20 subfamily of cytokines (including IL-26 and IL-22) seems to be strongly dependent on the simultaneous signals from IL-1 and IL-18. With all due caution about selectivity of the individual signalling signatures as well as potential efficacy of blocking, these comparisons are useful to show that the respective pathways are active in various inflammatory diseases.

Example 4: Therapeutic use

A three-period multicenter study, with a randomized-withdrawal, double-blind, placebo- controlled design to evaluate the clinical efficacy, safety and tolerability of bbmAb in XIAP deficiencies or CDC42 mutation based autoinflammation or disease.

Protocol summary

1. Introduction

1.1. Background

X-linked Inhibitor of Apoptosis Protein (XIAP) deficiency

X-linked inhibitor of apoptosis protein (XIAP) deficiency is a rare, inherited immunodeficiency that occurs almost exclusively in young males (Rigaud et al 2006). XIAP displays a pleiotropic function in cell survival, innate immunity, and inflammation. In particular, XIAP has been ascribed as an essential regulator of NLRP3 inflammasome activation in addition to its role in regulating caspase activity (Miyazawa and Wado 2022). The key features of XIAP deficiency include inflammatory bowel disease, often presenting as abdominal pain and diarrhea, recurrent fevers, splenomegaly and haemophagocytic lymphohistiocytosis (HLH). The latter manifestation is often triggered by infection with Epstein-Barr virus (EBV). Once diagnosed, the initial aim of therapy is to suppress inflammation, commonly using corticosteroids and biologic therapies. Inflammatory bowel disease may be treated with standard immunosuppressant medicines, although response rates are generally low. EBV infections may be treated with specific antiviral therapies and in some cases immunoglobulin therapy is used. At present the only potentially curative treatment for XIAP deficiency is hematopoietic stem cell transplant (HSCT). However, due to features of the underlying disease low intensity conditioning regimens are required and outcomes from HSCT are often poorer than the norm for patients of this age. Although the life expectancy of patients with XIAP has increased over the past decade, there is still significant morbidity and mortality in this condition (Mudde et al 2021).

CDC42 mutations

Cell division control protein 42 (CDC42) belongs to the Rho family of small monomeric GTPases (Heasman et al 2008). It affects multiple cellular processes including cell division and migration and regulates the nervous, immune, and hematopoietic systems (Melendez et al 2011). Pathogenic mutations in CDC42 can variously affect protein function, leading to an increasingly appreciated diversity of disease phenotypes centered on neurodevelopment, hematopoiesis, and immune response (Takenouchi et al 2015, Asiri et al 2021 , Coppola et al 2022). In particular, it has been demonstrated that aberrant palmitoylation of CDC42R186C results in trapping of CDC42 within the golgi apparatus which can induce pyrin inflammasome overactivation and marked elevation of IL-1 p and IL18 (Coppola et al 2022). In 4 pediatric patients, a de novo missense mutation (p.R186C) at the C-terminus has been demonstrated to affect CDC42 localization and cause a specific constellation of neonatal-onset cytopenia, autoinflammation, and recurrent HLH (Lam et al 2019). The existence of chronic elevation of serum IL-18, even after clinical responsiveness to anti-IL-1 therapy, suggests that IL-18 is a promising therapeutic target for CDC42 C terminal disease (Lam et al 2019).

Table 4 XIAP, CDC42 clinical phenotypes, cytokine profiles, and response to investigational treatments

XIAP CDC42 mutations Germline de novo and inherited Germline de novo mutations mutations

Clinical Early onset Inflammatory bowel disease very early onset of pancytopenia, with severe diarrhea, fevers, recurrent hepatosplenomegaly, rash,

HLH, cytopenia’s, splenomegaly, persistent fever, enterocolitis with hypogammaglobulinemia, recurrent diarrhea, fever, skin rash, infection and other less common hepatomegaly and recurrent complications uveitis, periodic fever, HLH/MAS granulomatous, lymphocytic interstitial lung disease, and fistulating skin disease

Cytokine Elevated IL-1 p and IL-18 Significantly elevated IL-18, profile elevated IL-1 p

Prognosis Limited survival to adulthood Limited survival to childhood

Investigational Immunosuppression (e.g., steroids, Anti-IL-1 therapies, glucocorticoid treatments azathioprine, etoposide) combined with pulses, cyclosporine combined

IL18BP. with emapalumab (anti-IFNg.

Definitive treatment HSCT - high risk 50- Definitive treatment HSCT - 75% survival with significant mortality single survivor reported due to conditioning and complications of GvHD

Summary bbmAb is a heterodimeric Fc, monovalent format bispecific IgG 1 monoclonal antibody (mAb) composed of Novartis clinical stage anti-IL-1 p and anti-IL-18 mAbs in a single molecule. By simultaneously targeting and neutralising both inflammasome effector cytokines I L-1 and IL-18, bbmAb has potential for superior clinical efficacy in autoinflammatory conditions where the inflammasome is overactivated and where both I L-1 p and IL-18 directly contribute to the disease pathophysiology, such as in hypogammaglobulinemia, cytopaenias, inflammatory bowel disease, abdominal pain and diarrhea, recurrent fevers, splenomegaly, haemophagocytic lymphohistiocytosis, neonatal-onset cytopenia, syndromic form of thrombocytopenia, Macrothrombocytopenia, dyshematopoiesis, autoinflammation, syndromic immunedeficiency, or recurrent haemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome or AIFEC.

Non-clinical data

Non-clinical Pharmacology bbmAb binds simultaneously to both I L-1 p and IL-18 with single to double digit pM affinities resulting in sub-nM inhibition of the cytokine signals in most cellular assays. Although bbmAb has monovalent binding to I L-1 and IL-18, the in vitro potencies for human I L-1 p and IL-18 neutralization by bbmAb are in the same ranges as the bivalent mAb1 and mAb2, with similar inhibitory activity on primary marmoset cells.

Clinical data

Clinical Human Pharmacokinetics

Preliminary pharmacokinetic data from the ongoing FIH trial in healthy volunteers (HV’s) were in line with predictions based on marmoset monkey data and modelling (i.e., typical of a human I gG 1 immunoglobulin). bbmAb peak serum concentrations were observed shortly after the end of the i.v. infusion. The median Tmax was approximately 3 hours from the start of the infusion (120 minutes in duration). The Cmax and AUCinf increased with increasing doses in a slightly over-proportional manner. The mean terminal elimination half-life (T 1/2) ranged from ~ 21 days at 0.1 mg/kg to ~ 28 days at 30 mg/kg. Volume of distribution was low with the mean Vz between ~ 5.2 L and ~ 6.4 L. Additionally, bbmAb was administered subcutaneously at a dose of 100 mg, 300 mg and 600 mg. The mean Cmax of bbmAb ranged from 7.1 to 54.7 ug/mL at about 7 days post dose. Exposure in Japanese subjects at 10 mg/kg i.v. is approximately 1.16-fold higher than in non-Japanese subjects while after 600 mg s.c. the exposure was 1.25-fold higher compared to the non-Japanese subjects based on AUCinf. For the Japanese cohorts 600 mg s.c. dose compared to the 10 mg/kg i.v. dose, bioavailability was approximately 65%. For the non-Japanese cohorts 100 mg s.c., 300 mg s.c. and 600 mg s.c. dose compared to the 10 mg/kg i.v. dose, bioavailability was approximately 45%, 69% and 65%, respectively.

2. XIAP deficiency, or CDC42 mutations related Objectives and endpoints

Table 5 Objectives and related endpoints

Objective(s) Endpoint(s)

Primary objective(s) Endpoint(s) for primary objective(s)

• To determine the efficacy of bbmAb in • Cohort 1 : Occurrence of disease flare prevention of flares in NLRC4-GOF patients in BBMAB1 treated patients compared with placebo during the 24-week Objective(s) Endpoint(s)

Randomized Withdrawal Period assessed by PGA and inflammatory markers

Secondary objective(s) Endpoint(s) for secondary objective(s)

• To evaluate the safety and tolerability of All Cohorts: All safety endpoints bbmAb in patients with XIAP deficiency, or (including physical examination CDC42 mutations findings, growth, vital signs, ECG parameters, safety laboratory, and adverse events)

• To evaluate the immunogenicity (IG) of All Cohorts: Confirmation and titer of bbmAb in patients with XIAP deficiency, or anti- bbmAb antibodies CDC42 mutations

• To evaluate the efficacy of bbmAb to improve • Cohort 1 : Response by Day 29, end of the clinical status of patients with XIAP Period 1 and Period 2 defined by PGA deficiency, or CDC42 mutations and inflammatory markers

• To evaluate the efficacy of bbmAb to achieve • Cohort 1 : Serological remission at serological remission Day 29, end of Period 1 and Period 2 defined by inflammatory markers

• To evaluate the effects of bbmAb on • Cohort 1 : Glucocorticoid therapy to < concomitant glucocorticoid administration 0.2 mg/kg/day by end of Period 1

• To evaluate the effect of bbmAb on the time to • Cohort 1 : The time to first first flare in patients with XIAP deficiency, or flare observed during the 24-week CDC42 mutations Randomized Withdrawal Period

• To evaluate the efficacy of bbmAb to improve • All Cohorts: Physician Severity the signs and symptoms of XIAP deficiencies Assessment of Disease Signs and or CDC42 mutation based autoinflammation Symptoms scale or disease

• To evaluate the effect of bbmAb on patient • All cohorts: Patient’s I Parent’s global reported outcomes in patients with XIAP assessment of disease activity deficiencies or CDC42 mutation based (PPGA) scale autoinflammation or disease over time

Primary estimands

The primary clinical question regarding XIAP deficiencies or CDC42 mutation based autoinflammation or disease of interest is: What is the effect of continuation of bbmAb treatment in patients with XIAP deficiencies or CDC42 mutation based autoinflammation or disease who have achieved a complete clinical response after approximately 28 weeks of bbmAb treatment despite discontinuation of glucocorticoid on disease flares over 24 weeks?

The primary estimand includes the following components: 1. Population: Patients with a XIAP deficiencies or CDC42 mutation based autoinflammation or disease who have achieved complete response after approximately 28 weeks of bbmAb treatment and have discontinued cyclosporin and glucocorticoids or are on a maintenance/replacement dose (<0.2 mg/kg/day) of glucocorticoids. 2. Endpoint: Occurrence of disease flare within 24 weeks. 3. Treatment of interest: The randomized study treatment (the investigational treatment of bbmAb or placebo).

4. Handling of intercurrent events: The treatment policy strategy will be adopted for primary analysis thus treatment discontinuation due to any reason other than occurrence of a disease flare will be ignored. Patients who discontinue treatment prematurely (not due to disease flare) in Period 2 will be analyzed in the same manner as those that continue the treatment as planned.

5. Summary: Difference in proportions of patients with disease flare between treatment groups.

3. Study design

As depicted in Fig. 1 , this is a three-period study, with an open-label, single-arm active treatment in Period 1 followed by a randomized-withdrawal, placebo-controlled, double-blinded design in Period 2 and an open label, long-term safety follow up in Period 3. Patients who are receiving bbmAb with a diagnosis of XIAP-deficiency or CDC42 mutation who meet criteria will be eligible to directly enter into Period 3 for open-label long-term safety follow-up.

Patients will be assigned to the relevant cohort:

• Cohort 1: patients with a confirmed diagnosis of XIAP deficiency, or CDC42 mutation will be included in to Period 1 of the study in order to randomize patients into Period 2 of the study.

• Cohort 2: additional patients with a confirmed diagnosis of XIAP- deficiency or CDC42 mutation with bbmAb may be enrolled directly into Period 3 of the study after completing Screening/Baseline. The total study duration from Screening until end of study (EoS) is expected to be between 3-4 years.

The overall study design is outlined in Figure 1.

This three-period study includes:

Screening:

Period of approximately 30 days to confirm that the study inclusion and exclusion criteria are met. The Screening Period also allows for safe discontinuation or stabilization of doses of permitted medications in Period 1 for Cohort 1. For Cohort 2, patients may enroll directly into Period 3 after Screening and Baseline are completed. The required assessments may be conducted over several days if it is in the best interest of the patient, or for logistical reasons. Laboratory tests that have been completed as part of the patient’s routine care in the days preceding Screening may be used to avoid taking additional blood samples from the patient, providing the results are available. The Screening window may be extended in the following situations (providing Informed Consent has been obtained):

• in order to allow enough time to prove presence of active disease following discontinuation of current treatment as outlined in the inclusion and exclusion criteria for Cohort 1.

• to allow results from the molecular diagnosis to be available for patients who have no documented molecular diagnosis of NLRC4, XIAP-deficiency or CDC42 mutations (Cohort 1). All other Screening assessments (apart from Informed Consent) should be performed after the molecular diagnosis is available and within the Screening window.

Baseline:

Patients who meet the eligibility criteria will be admitted, if not already hospitalized, and assessed at a Baseline visit; this may be conducted on Day -1 or may be combined with Day 1 prior to dosing.

To mitigate potential SARS-CoV-2 infections among patients, guidance and requirements provided by the local regulatory authorities or local site-specific SOPs will be followed (e.g., patients may be screened for SARS-CoV-2 by PCR or comparable approved methodology prior to admission at the study/hospital site for any overnight stays following local site-specific SOPs).

All Baseline safety evaluation results must be available prior to dosing. Laboratory tests that have been completed as part of the patient’s routine care in the days preceding Baseline may be used to avoid taking additional blood samples from the patient, providing the results are available.

Period 1, Open Label Treatment Period:

Applicable for Cohort 1 only.

Period 1 is an Open-Label Treatment Period to identify responders to bbmAb treatment and to allow for these patients to taper their glucocorticoid dose and/or discontinue cyclosporin treatment. Period 1 is separated into 3 sub-parts (Period 1a, 1b and 1c).

Patients that meet the eligibility criteria will enter Period 1 and receive their first dose of bbmAb (10 mg/kg) as an i.v. infusion on Day 1 in Period 1a. Due to the nature of the disease, it is anticipated that patients may remain hospitalized for the duration of Period 1, however, this is not mandatory and investigators should use their judgement on when a patient can be discharged based on the patient’s condition. During Period 1, patients will undergo the efficacy, PK and PD assessments as outlined in the assessment schedule (Table 14). Period 1a

The duration of Period 1a is 4 weeks with bbmAb dosing every 2 weeks.

For patients currently on stable doses of glucocorticoids and/or cyclosporin, these doses will remain stable throughout Period 1a. No tapering of glucocorticoids or cyclosporin is allowed.

At Day 29 (Week 4) an assessment of response will be completed using the PGA, CRP and ferritin and those patients with at least a partial response will continue into Period 1b of the study. Patients who have not achieved a partial response during Period 1a will be withdrawn from the study.

If a patient discontinues during Period 1a, the patient should return within approximately 1 month of discontinuation to complete the End of Period 1 assessments (i.e. Period 1c, Week 28 in the assessment schedule) as the End of Treatment visit.

Period 1 b (Glucocorticoid tapering and Cyclosporin discontinuation)

The duration of Period 1b is a maximum of 20 weeks with bbmAb dosing every 2 weeks. Patients will enter Period 1 b after they have successfully completed Period 1a.

Patients on a stable dose of a glucocorticoid will be gradually tapered down to the lowest dose possible for 4 weeks prior to randomization at the start of the Randomized Withdrawal Period (Period 2). Similarly, any patients on a stable dose of cyclosporin will have the dose reduced with the aim of achieving discontinuation of cyclosporin for 4 weeks prior to randomization at the start of the Randomized Withdrawal Period (Period 2). Guidelines for tapering glucocorticoids, discontinuing cyclosporin and eligibility for patients to move to Period 1c and Period 2 are given in Section 6.2.1.1.

Weekly site phone calls to patients/parents/caregivers will be performed for patients who are not hospitalized during Period 1b to monitor the response during the glucocorticoid tapering.

Patients who meet the eligibility criteria to move to Period 1c earlier than Week 24 may move to Period 1c earlier but must complete the assessments listed for Week 24 as detailed in the assessment schedule before moving to Period 1c.

For patients who are glucocorticoid and cyclosporin free at study entry (Period 1a) i.e. require no glucocorticoid tapering or cyclosporin discontinuation in Period 1 b, on completion of Period 1a patients will enter Period 1 b and only complete the assessments and treatment as listed for Week 22 and Week 24 of Period 1 b before moving to Period 1c. This will ensure that all patients receive a minimum of 12 weeks treatment with bbmAb in Period 1 of the study.

Patients who are unable to reduce their glucocorticoid dose or stop treatment with cyclosporin by Week 24 may be discontinued from the study. If a patient discontinues during Period 1b, the patient should return within approximately 1 month of discontinuation to complete the End of Period 1 assessments (i.e. Period 1c, Week 28 in the assessment schedule) as the End of Treatment visit. Patients who have achieved a partial response (with or without the glucocorticoid tapering, but having discontinued cyclosporin) as assessed at the End of Period 1 visit, may enroll straight into Period 3 open label treatment at the investigator’s and family’s discretion.

Period 1c

The duration of Period 1c is 4 weeks with bbmAb dosing every 2 weeks.

Patients will enter Period 1c at the time of their next scheduled bbmAb dose after they have successfully completed Period 1b.

Any patients continuing glucocorticoid treatment must maintain a stable dose for the full duration of Period 1c. No tapering of glucocorticoids is allowed in Period 1c, no treatment with cyclosporin is allowed in Period 1c.

The purpose of Period 1c is to ensure that all patients who have stopped cyclosporin treatment and/or tapered glucocorticoids and are maintained on a low dose of glucocorticoids are clinically stable for at least 4 weeks before entering Period 2.

At the end of Period 1c (Day 197, Week 28) an assessment of response will be completed using the PGA, CRP and ferritin and those patients with a complete response will be randomized into Period 2 of the study. Patients who have not met the complete response criteria but have achieved a partial response (with or without the glucocorticoid tapering, but having discontinued cyclosporin) may enroll straight into Period 3 open label treatment at the investigator’s and family’s discretion.

If a patient discontinues during Period 1c, the patient should return within approximately 1 month of discontinuation to complete the End of Period 1 assessments (i.e. Period 1c, Week 28 in the assessment schedule) as the End of Treatment visit.

Period 2, Randomized Withdrawal Period:

Applicable for Cohort 1

Period 2 consists of a 24-week placebo-controlled, double-blind, randomized withdrawal period, primarily to assess the efficacy of bbmAb compared to placebo. At the beginning of Period 2 bbmAb responders (complete response to treatment at the end of the open label treatment in Period 1) will be randomized in a ratio of 1:1 to bbmAb treatment (i.e. continue with 10 mg/kg) or placebo.

The first scheduled blinded dosing following randomization in Period 2 will be 2 weeks after the last dose in Period 1c and will continue with dosing every 2 weeks until either disease flare occurs or 24 weeks has elapsed in Period 2. It is anticipated that patients may remain hospitalized for duration of Period 2, however, this is not mandatory and investigators should use their judgement on when a patient can be discharged based on the patient’s condition. If a patient meets the flare criteria in Period 2, an unscheduled visit should be performed as detailed in the assessment schedule, blinded treatment will be stopped and patients transferred to open label bbmAb treatment in order to continue the remainder of Period 2.

If a patient discontinues during Period 2, the patient should return within approximately 1 month of discontinuation to complete the End of Period 2 assessments (i.e. Period 2, Week 24 in the assessment schedule) as the End of Treatment visit.

Period 3, Long Term Safety, Open Label Treatment:

Period 3 consists of a 3-year long-term safety phase, with open-label bbmAb treatment (10 mg/kg).

The first scheduled dosing in Period 3 will be 2 weeks after the last dose in Period 1 or Period 2 and will continue with dosing separated by a minimum of 2 weeks. For Cohort 2, dosing will be scheduled following completion of the Screening and Baseline visits and confirmation that all cohort-specific eligibility criteria have been met.

Dosing in Period 3 will continue every approximately every 2 weeks, protocol assessments have a reduced visit frequency as outlined in the assessment schedule. Any of the visits in Period 3 may be performed at an alternative Sponsor-approved site (e.g., satellite site that may be more convenient to the patient) if agreed by the Principal Investigator who will maintain the investigator responsibilities, and only as permitted by local regulations.

At the discretion of the investigator, and provided the patient remains clinically stable without evidence of elevated disease activity, dosing in Period 3 may be administered approximately every 14 days, with a window specified in Table 14 to allow flexibility based on individual patient response.

For patients on a maintenance dose of glucocorticoids it is recommended to consider further tapering of glucocorticoids to complete discontinuation if possible. All patients who do not maintain a minimum partial response may be discontinued unless their loss of response is considered a consequence of glucocorticoid tapering. Patients in Cohort 2 who have transitioned into the study would continue in Period 3 as long as there is clinical benefit as judged by the treating physician.

If a patient discontinues during Period 3, the patient should return within approximately 1 month of discontinuation to complete the End of Period 3 assessments (i.e. Period 3, Week 152 in the assessment schedule) as the End of Treatment visit.

The Screening and Baseline visits will be used to confirm that the study inclusion and exclusion criteria are met and for performing Baseline clinical observations and biological sampling. The patients who are enrolled in this study may have been treated with anakinra, canakinumab, emapalumab and/or investigational I L-18/IL-1/IFN- y binding or blocking therapy and may be screened. Entry into the study will take place within approximately 30 days of Screening with bbmAb treatment administered as soon as the criteria for active disease are fulfilled. This run-in phase for previously treated patients holds a reduced time for wash-out compared to classical designs, but is considered justified due to the fact subjects may require emergent enrollment to bbmAb after other treatment options have failed.

Period 1 is an open-label, active treatment period to identify patients with monogenic IL-18 driven autoinflammatory diseases, including XIAP-deficiency and CDC42 mutations who respond to bbmAb treatment and then allow patients on glucocorticoids and/or cyclosporin to taper/discontinue these therapies. Period 1a patients will initially be treated with two doses of bbmAb to ensure they respond to treatment at Day 29 and specifically, achieve simultaneous control of MAS and resolution of gut clinical manifestations (enterocolitis) over several weeks. In Period 1b, apparent bbmAb responders on cyclosporin and glucocorticoids will discontinue cyclosporin and gradually taper off or reduce glucocorticoids to a maintenance/replacement dose over a maximum duration of 20 weeks to avoid the long-term morbidity associated with both treatments in pediatric populations. Period 1c ensures patients are clinically stable for at least 4 weeks off glucocorticoids or on a maintenance/replacement dose prior to assessment of response to bbmAb treatment at the end of Period 1.

At the start of the Randomized Withdrawal Period (Period 2) patients with a complete response to bbmAb and have discontinued glucocorticoids or on a maintenance/replacement dose will be randomized 1 :1 ratio in a double-blind manner to receive either treatment with bbmAb or matching placebo. This design allows patients who have bbmAb treatment withdrawn (placebo patients) to immediately re-start bbmAb treatment after they have reached the study endpoint (occurrence of disease flare), thereby addressing both clinical concerns and patient preferences about placebo assignment by minimizing the time that patients are on potentially non effective therapy. All patients who flare at any time during Period 2, blinded treatment will be stopped and patients transferred to open label bbmAb treatment in order to continue the remainder of Period 2 at both investigator’s and family’s discretion. The 1 :1 randomization ratio was chosen to maximize the statistical power for the primary analysis whilst minimizing the overall sample size given the rarity of the condition. Blinding is justified to prevent conscious or unconscious bias in the study design and how it is conducted. The duration of the Randomized Withdrawal Period of the study, is based on clinical trial experience of flares in similar pediatric populations with autoinflammatory conditions treated with canakinumab (e.g., CAPS and SJIA) and from modeling of free IL-18 where levels are anticipated to increase to result in a flare in patients with monogenic IL-18 driven autoinflammatory diseases, including XIAP deficiency, and CDC42 mutations in the absence of effective therapy.

The Long-Term Safety, Open Label Treatment (Period 3) in patients who have responded to bbmAb, will allow them to continue with bbmAb treatment and provide long term safety data.

Rationale for dose/regimen

Intravenous administration of bbmAb has been evaluated in a FIH single dose ascending study up 30 mg/kg i.v. in healthy volunteers and 10 mg/kg i.v. in COVID-19 patients without any drug related SAEs; the PK of bbmAb in humans is as expected for a typical lgG1 antibody binding to a soluble ligand cytokine target. In a preplanned PK analysis of the FIH study to enable subcutaneous dosing, bbmAb showed a dose proportional increase in exposure matching the predicted human PK. bbmAb peak serum concentration was observed shortly following its i.v. infusion. The median Tmax was approximately 0.146 day, or approximately 3.5 hours, from the start of the infusion. The Cmax and AUCO-inf increased with increasing doses in a dose-proportional manner. bbmAb concentrations decreased exponentially with a mean terminal elimination half-life (T1/2) ranging from 21.1 to 286.3 days. Volume of distribution was low with the mean Vz between 0.06662 and 0.083 mL/kg. Additionally bbmAb was administered subcutaneously at a dose of 100 mg, Cmax of approximately 7 pg/mL at about 9 days post dose was observed. Bioavailability was estimated to be 70% by comparing AUCinf divided by dose of the 100 mg s.c. to AUCinf divided by dose of the 1 mg/kg i.v. in duration. The Cmax and AUCinf increased with increasing doses in a slightly over-proportional manner. The mean terminal elimination half-life (T1/2) is ranging from ~ 21 days at 0.1 mg/kg to ~ 28 days at 30 mg/kg. Volume of distribution was low with the mean Vz between ~ 5.2 L and ~ 6.4 L. Additionally, bbmAb was administered subcutaneously at a dose of 100 mg, 300 mg and 600 mg. The mean Cmax of bbmAb ranged from 7.1 to 54.7 ug/mL at about 7 days post dose. Exposure in Japanese subjects at 10 mg/kg i.v. was approximately 1.16- fold higher than in non-Japanese subjects while after 600 mg s.c. the exposure was 1.25- fold higher compared to non-Japanese subjects based on AUCinf. For the Japanese cohorts 600 mg s.c. dose compared to the 10 mg/kg i.v. dose, bioavailability was approximately 65%. For the non-Japanese cohorts 100 mg s.c., 300 mg s.c. and 600 mg s.c. dose compared to the 10 mg/kg i.v. dose, bioavailability was approximately 45%, 69% and 65%, respectively. (Figure 5)

All subjects in all the cohorts (including bbmAb treated and placebo subjects) tested negative (i.e. , treatment with anti-bbmAb antibodies), except one subject from cohort A4 (3 mg/kg i.v.) 29 days post dose, one subject from cohort A5 (10 mg/kg i.v.) 29 days post dose, one subject from cohort A6 (30 mg/kg i.v.) 197 days post dose, and one subject from cohort D1 (10 mg/kg, i.v. Japanese), 197 days post dose, which were therefore all considered as transient ADA responses. On day 15, one subject from cohort A5 (10 mg/kg i.v.) tested positive. All the subjects from cohort A6 (bbmAb treated and placebo subjects) tested negative on Day 253.

4. Rationale

Rationale for dose/regimen

Patients with monogenic IL-18 driven autoinflammatory diseases, including XIAP deficiency, or CDC42 mutations have grossly and chronically elevated free IL-18 in serum. The dynamics of free IL-18 potentially limit the efficacy of the bispecific antibody and therefore guide the dosing principle in these patients. Under normal physiological conditions almost all circulating IL-18 is biologically inactive bound to its binding protein (IL-18BP), however, in severe inflammatory conditions the levels of IL-18 exceed the available IL 18BP, resulting in a higher fraction of free/bioactive IL-18 to drive pathology. Modeling utilizing measurements of total IL-18, IL-18BP and free IL-18 from pediatric patients with NLRC4-GOF mutations (Weiss et al 2018) confirms a bbmAb dose of 10mg mg/kg i.v. is expected to achieve rapid and sustained reduction of free IL-18 in those with NLRC4-GOF, XIAP deficiency, or CDC42 mutations in both pediatric and adult populations. bbmAb

The model used to predict the dynamics of the anti-IL-18/IL-1 p bispecific antibody and its targets in serum consists of a general competitive binding model (Yan et al 2012) describing the free and total IL-18 dynamics for the IL-18 arm and the previously published model of canakinumab (Chakraborty et al 2012) with parameters adapted to bbmAb for the IL-1 arm. Parameters for baseline values of free IL-18, total IL-18 and IL-18BP in serum from patients across several autoimmune diseases including NLRC4-GOF (Weiss et al 2018) and in-house measurements were used for model adjustment and a body weight of 3 kg for a newborn patient was assumed. Based on the simulation of the effect of bbmAb on free IL-18 when given intravenously, full control (neutralization) of free IL-18 at a dose of 10 mg/kg is anticipated for approximately 14 days while also neutralizing IL- 1 B to fully control the inflammatory syndrome, to allow for reversal of clinical features such as gastrointestinal pathology and MAS or HLH in these patients during treatment.

The simulations of the effects of bbmAb 10 mg/kg on patients with elevated free IL- 18 and IL-1 p suggest immediate and durable responses. In comparison to the 2 mg/kg q2d dose of rhlL-18BP (tadekinig alfa) (Tak et al 2006), therapeutic range shown in grey shading (Figure 5), that has been reported to be clinically efficacious in NLRC4-GOF infants (Canna et al 2017, Moghaddas et al 2018) and is currently undergoing evaluation in phase 3 study (NCT03113760), a 10 mg/kg q2W dose of bbmAb is predicted to neutralize free IL-18 equivalently at 2 weeks and then to fully suppress free/bioactive IL- 18 to an undetectable levels similar to healthy individuals in subsequent weeks.

The bbmAb dose of 10 mg/kg i.v. q2w for this study is further justified by;

• The dose is predicted to lead to rapid and simultaneous neutralization of free IL-18 and IL-1 p to rapidly induce a clinical response in patients where hyper-elevated levels of IL-1 and IL-18 have been measured and are expected in pediatric patients enrolled in this study.

• The 10 mg/kg i.v single dose was administered to healthy volunteers in the FIH to establish the safety of this dose to enable the treatment of patients with gain of function mutations leading to overexpression of IL-ip and IL-18 (e.g., NLRC4 mutations (Romberg et al 2014). This dose in healthy volunteers has provided several months of combined IL-18 and IL-1 p inhibition and is currently administered to COVID- 19 patients with no identified safety concerns.

• Modeling of IL-18BP treatment of NLRC4-GOF pediatric patients to estimate clinically efficacious bbmAb dose suggests that patients with IL-18 driven disease, including XIAP-deficiency and CDC42 mutations should achieve a sustained clinical response at 10 mg/kg i.v. q2w with neutralization of free IL-18 for approximately 14 days - lower doses with greater dose intervals may not achieve a complete response and risk patients flaring due to inadequate treatment. In Open-Label Period 3, patients with stable disease may go up to 28 days at maximum between doses, with close monitoring of the clinical status of the patient by the Investigator.

• The clinical experience of canakinumab treatment in pediatric patients with severe CAPS, where these patients, in particular pediatric patients less than age 2 may require higher than usual doses (greater than canakinumab 8 mg/kg) to attain full clinical response and may require more frequent dose titration compared to adults.

• Allometric scaling of pop-PK parameters of bbmAb from 70-kg human to 3-kg newborn predicts a higher clearance per weight in neonates and young infants. Therefore expected lower exposure per dose provides a rational for a high dose in pediatric population to maximize clinical responses. No significant accumulation is anticipated at the proposed dose of 10 mg/kg i.v. q2w after multiple dosing in this population. Similarly, body weight based clearance (L/day/kg) was found increased and exposure of canakinumab decreased in younger pediatric patients (Zhung et al 2019).

• The systemic exposure in the study is expected to be considerably lower than the exposures reached in the 26-week marmoset study, with a predicted exposure ratio to non-clinical NOEL exposure of 14.4-fold for AUG and 14.4-fold for Cmax for a 3 kg newborn. • In the NHP 26-week toxicology study, 100 mg/kg, twice a week had no adverse events with no observed effect level.

In summary, depending on the disease status, the doses of 10 mg/kg q2w to q4w haves been defined as likely efficacious, while having minimal risk in the pediatric and adult population based on available data.

5. Study Population

The study population includes a total of approximately 18 patients:

• Cohort 1 : patients with monogenic IL-18 driven autoinflammatory diseases, including XIAP deficiency, or CDC42 mutation

• Cohort 2: patients with a confirmed diagnosis of XIAP deficiency or CDC42 mutation entering directly into Period 3

Inclusion criteria

For all patients: Patients eligible for inclusion in this study must meet all of the following criteria:

1. Male and female patients weighing at least 3 kg, at time of Screening.

2. Written informed consent by parent(s)/legal guardian(s) for the pediatric patients and assent by the pediatric patient (depending on local requirements) must be obtained before any study-specific assessment is performed. For adult patients, written informed consent by patients capable of giving consent, or, when the patient is not capable of giving consent, by his or her legal/authorized representative (if allowed according to local requirements).

Cohort 1 specific inclusion criteria:

3. Patients with monogenic IL-18 driven autoinflammatory disease, with a genetic diagnosis of either XIAP deficiency, or CDC42 mutation (this analysis may be performed as part of Screening procedures if not already available).

• 3a. Patients with XIAP deficiency or CDC42 mutation and a previous bone marrow transplantation may be permitted, if they show evidence of primary or secondary graft failure, or failure to achieve phenotypic correction with evidence of XIAP/CDC42- related disease recurrence or clinically significant mixed chimerism.

4. Clinical history and investigations consistent with autoinflammation and infantile enterocolitis, XIAP or CDC42, including elevated IL-18 levels (this analysis may be performed as part of Screening procedures if not already available).

• 4a. XIAP patients only: Patients must have persistent disease or be resistant to escalating therapy. 5. At first treatment (Day 1 of Period 1), evidence of active disease as assessed by: a. PGA of disease activity > minimal and b. Ferritin > 600 ng/ml or c. elevation of CRP > 20mg/l.

Cohort 2 specific inclusion criteria:

Patients with a genetic diagnosis of XIAP deficiency, or CDC42 mutations, with elevated IL-18 and/or IL-1 p, who are being treated with bbmAb in a Novartis Managed Access Program (MAP), and who have demonstrated treatment failure on other immunomodulatory therapies and have no alternative treatment options available according to their treating physician.

Exclusion criteria

Patients meeting any of the following criteria are not eligible for inclusion in this study:

1. History of hypersensitivity to any of the study drugs or to drugs of similar chemical classes or to any of the excipients.

2. Signs and symptoms, in the judgment of the investigator, of clinically significant active bacterial, fungal, or viral infections. Infections are considered controlled if appropriate therapy has been instituted and, at the time of Screening, no signs of infection progression are present. Progression of infection is defined as hemodynamic instability attributable to sepsis, new symptoms, worsening physical signs or radiographic findings attributable to infection. Persisting fever without other signs or symptoms will not be interpreted as progressing infection.

3. COVID-19 specific: If in line with health and governmental authority guidance, it is highly recommended that PCR or comparable approved methodology for COVID- 19 be completed within 1 week prior to first dosing. If testing is performed, negative test results are required prior to enrolment into the study. Additional testing may occur at the discretion of the investigating physician. COVID-19 testing should be completed via nasal or throat swabs or other approved route for pediatric patients. If testing is not performed, the investigator must document their discussion with the patient/parent/caregiver regarding testing, and the rationale for not testing, in the source documentation. This requirement may be ignored if the pandemic is declared ended by the country where the site is located and resumed if the pandemic recurs. Any conditions or significant medical problems, which in the opinion of the investigator places the patient at unacceptable risk for bbmAb therapy (this can be discussed with Novartis on a case by case basis in case of uncertainty). Previous treatment with anti-rejection and/or immunomodulatory drugs within the past 28 days or 5 half-lives (whichever is the longer) for immunomodulatory therapeutic antibodies (or as listed in Section 6.2.2 prohibited treatments) prior to bbmAb treatment.

The exceptions are:

• Drugs listed in the Prohibited medication Section 6.2.2, for which the washout periods should be followed as outlined there.

• Stable dose of glucocorticoids < 2.0 mg/kg/day (maximum 60 mg/day for patients over 30 kg) of oral prednisone (or equivalent, regardless of route of administration or dosing schedule) for at least 24 hours prior to treatment with bbmAb

• Stable dose of cyclosporin < 5mg/kg/day for at least 3 days prior to treatment with bbmAb

• Anakinra, canakinumab, emapalumab and/or investigational I L-18/IL-1/IFN- y binding or blocking therapy, must be discontinued (See Section 6.2.2) prior to treatment with bbmAb. As soon as the criteria for evidence of active disease are met, patients can receive bbmAb treatment (per inclusion 5).

5. Participation in any other investigational trials within 4 weeks prior to dosing or longer if required by local regulation with the exception of treatment with anakinra, canakinumab, emapalumab and/or investigational IL-18/IL-18BP/IL-1/IFN-D binding or blocking therapy. Participation in a Novartis MAP with bbmAb is not exclusionary.

6. A positive HIV test result (ELISA and Western blot) at Screening. Evidence of prior testing within 3 months is sufficient.

7. A positive Hepatitis B surface antigen (HBsAg) or Hepatitis C test result at Screening. Evidence of prior testing within 3 months is sufficient.

8. Presence of tuberculosis infection as defined by a positive TB test at Screening. Evidence of prior testing within 3 months is sufficient.

9. Live vaccinations within 1 month prior to bbmAb treatment, during the trial, and up to 3 months following the last dose.

10. History of malignancy of any organ system, including post-transplant lymphoproliferative disorder (other than adequately treated localized basal cell carcinoma of the skin or in-situ cervical cancer), treated or untreated, within the past 5 years, regardless of whether there is evidence of local recurrence or metastases. 11 . Pregnant or nursing (lactating) females, where pregnancy is defined as the state of a female after conception and until the termination of gestation, confirmed by a positive hCG laboratory test.

12. Female patients of child-bearing potential (or Tanner stage 2 or above) who are or might become sexually active, must be informed of the potential teratogenic risk with bbmAb and the need, and agree to use highly effective contraceptive methods to prevent pregnancy while on bbmAb therapy;

• Highly effective contraception (abstinence, oral, injected or implanted hormonal methods of contraception or placement of an intrauterine device (IUD) or intrauterine system (I US) or other forms of hormonal contraception that have comparable efficacy (failure rate <1%), for example hormone vaginal ring or transdermal hormone contraception) must be used during the study and for 5 months after stopping treatment with bbmAb, when it is predicted that IL-18 and I L-113 will not be neutralized by bbmAb. The decision on the contraceptive method should be reviewed at least every 3 months to evaluate the individual need and compatibility of the method chosen.

Note that only highly effective contraceptive methods that are approved as per local regulations must be used.

• Women are considered post-menopausal if they have had 12 months of natural (spontaneous) amenorrhea with an appropriate clinical profile (e.g., age appropriate history of vasomotor symptoms). Women are considered not of childbearing potential if they are post-menopausal or have had surgical bilateral oophorectomy (with or without hysterectomy), total hysterectomy or bilateral tubal ligation at least six weeks ago. In the case of oophorectomy alone, only when the reproductive status of the woman has been confirmed by follow up hormone level assessment is she considered not of childbearing potential.

13. Patients weighing >160 kg at Screening.

14. For CDC42 mutation patients: Takenouchi-Kosaki syndrome - CDC42 mutations associated with a diverse syndrome characterized by variable development delays, cardiac, brain and hematological abnormalities.

6. Treatment

6.1. Study treatment

Details on the requirements for storage and management of study treatment, and instructions to be followed for patient numbering, prescribing/dispensing, and taking study treatment are outlined in the Pharmacy Manual. 6.1.1. Investigational and control drugs bbmAb is still in development, the investigational and control drug will be same as administered in the Phase 1 study and Phase 2 study. The investigational drug, bbmAb and the matching placebo will be prepared by Novartis and supplied as open labeled bulk medication to the unblinded site pharmacist. An unblinded pharmacist or authorized designee is required to dispense the study drug. Drug will be administered over approximately 120 minutes as i.v. infusion at the clinical site by the study personnel in accordance with the specified study procedures.

During an epidemic or pandemic (e.g., COVID-19 pandemic) that limits or prevents on-site study visits, visits by site staff to a patient’s home may be arranged in order to continue study treatment per protocol, as permitted by local regulations.

Table 13 Investigational drug

Investigational Pharmaceutical Route of Supply Type Sponsor

Drug(Name and Dosage Form Administration (global or local)

Strength) bbmAb 100 Concentrate for Intravenous use Open label Sponsor (global) mg/mL solution for patient specific infusion supply ; vials

Matching Concentrate for Intravenous use Open label Sponsor (global)

Placebo solution for patient specific infusion supply; vials

6.1.2. Additional study treatments

No other treatment beyond they investigational drug are included in this trial. Administered supportive treatment in addition to study treatment will be supplied by the investigational site.

6.1.3. Treatment arms/group

Period 1 - Open Label Treatment Period (Cohort 1)

Patients will be assigned at Day 1 to a dose of bbmAb 10mg/kg q2w i.v.

Period 2 - Randomized Withdrawal Period (Cohort 1)

Responsive patients will be assigned at randomization to one of the following treatment arms/groups in a ratio of 1 :1 at the end of Period 1 :

• bbmAb 10 mg/kg i.v. q2w

• Matching placebo i.v. q2w Period 3 - Open Label Long Term Safety (All cohorts)

Patients will receive a dose of bbmAb 10mg/kg q2w i.v. infusion starting with Period 3 Day 1 (Week 0). The frequency of administrations may be adjusted within the permitted windows listed in Table 14, and per the judgement of Investigator.

6.1.4. Post- Trial Access

Novartis will offer to provide the bbmAb to a patient who completes participation in the study for as long as there is evidence of clinical benefit for the patient, as required or permitted by local legislation, or until:

• the Investigator discontinues treatment,

• the product or an alternative treatment becomes available commercially.

6.2. Other treatment(s)

6.2.1. Concomitant therapy

All key medications, procedures, and significant non-drug therapies (including physical therapy and blood transfusions) administered for the treatment of the diseases being investigated in the six months (if available) before the patient was enrolled into the study should be recorded on the appropriate Case Report Forms.

All medications, procedures, and significant non-drug therapies (including physical therapy and blood transfusions) administered after the patient was enrolled into the study must be recorded on the appropriate Case Report Forms.

Each concomitant drug must be individually assessed against all exclusion criteria/prohi bited medication. If in doubt, the investigator should contact the Novartis medical monitor before enrolling a patient or allowing a new medication to be started. If the patient is already enrolled, contact Novartis to determine if the patient should continue participation in the study.

During the course of the study and also prior to Screening, patients may receive gastric protection, folic acid, paracetamol, NSAIDs, analgesics, antibiotics, vasopressors and nutrition supplementation (e.g., vitamins, liquid supplements, enteral nutrition, total parenteral nutrition) and other agents/treatments where these form part of supportive treatment of the diseases being investigated at their participating site (per medical judgement).

6.2.1.1. Permitted concomitant therapy requiring caution and/or action

Period 1 - Glucocorticoids taper

This is applicable for Cohort 1 only. During the 4-week treatment in Period 1a to stabilize patients on bbmAb, patients on stable doses of glucocorticoids are permitted entry into the study and receive the first dose of i.v. dose of bbmAb (10 mg/kg). The glucocorticoids dose should be maintained for the full duration of Period 1a until at least Day 29.

Following Day 29, investigators should introduce a glucocorticoid taper to gradually decrease the dose with the aim of achieving discontinuation of glucocorticoids (per medical judgment) or a stable maintenance dose of < 0.2 mg/kg/day of glucocorticoids (prednisone or equivalent) for 4 weeks prior to randomization at the start of the Randomized Withdrawal Period (Period 2).

• Steroid tapering after Day 29 may be initiated if a patient has achieved at least a partial response (Section 8.3.5).

Table 16 Glucocorticoids tapering guideline

Prednisone (or equivalent) dose Amount of reduction

> 0.2 mg/kg/day Taper at 0.1 mg/kg per week until at dose of 0.1 mg/kg/day

0.1 mg/kg/day Taper to dose of 0.05 mg/kg/day and take for every 24 hours for 1 week

< 0.05 mg/kg/day Alternate dosing days (i.e. take dose every 48 hours) for 2 weeks and then discontinue

• Tapering can be further continued if:

1. Between study visits during the weekly site phone calls patient/parents do not report any loss of response based on response to phone scripted questionnaire.

2. During site visits the patient maintains at least a partial response to bbmAb.

• Glucocorticoid tapering will continue until one of the following occurs first:

1. Patient achieved discontinuation of glucocorticoids and is steroid free.

2. Patient has reached maximal duration of 20 weeks for the glucocorticoid taper.

3. Patient has failed three glucocorticoid tapering attempts.

• All patients who fail to maintain at least a partial response to bbmAb may be discontinued from the study unless the loss of response is considered a consequence of glucocorticoid tapering.

1. If the patient loses response to bbmAb while tapering glucocorticoids, the glucocorticoid dose will be increased to the previous level and the patient will be permitted to remain in Period 1b; the increased steroid dose will be maintained for at least 2 weeks. If the patients fails to respond for more than 2 weeks after the initial event after increasing to the previous steroid dose, the patient may be discontinued from the study.

2. For patients who lose response during tapering, a subsequent attempt of steroid tapering may occur only if the patient has been on stable steroid dose for at least 2 weeks and patient at least has a partial response.

Patients will be eligible for direct entry into Period 2 of the study if one of the following is met:

1. Any patient who achieved discontinuation of glucocorticoids and is steroid free for 4 weeks of Period 1c.

2. Patient who achieved a stable maintenance dose of < 0.2 mg/kg/day of glucocorticoids (for patients over 30 kg) or < 0.4 mg/kg/day (for pediatric patients where higher replacement doses maybe required) of glucocorticoids (prednisone or equivalent) for the 4 weeks of Period 1c.

If a patient discontinues during Period 1, the End of Period 1 assessments (i.e. Period 1c, Week 28 in the assessment schedule) should be completed as the End of Treatment visit. Patients who have achieved a partial response (with or without the glucocorticoid tapering but having discontinued cyclosporin) as assessed at the End of Period 1 visit, may enroll straight into Period 3 open label treatment at the investigator’s and family’s discretion.

Period 2 - Glucocorticoids (Randomized Withdrawal Period)

During Period 2, patients who achieved a stable maintenance dose of < 0.2 mg/kg/day (for patients over 30 kg) or < 0.4 mg/kg/day (for pediatric patients) of glucocorticoids (prednisone or equivalent) in Period 1c should be maintained on this dose and no tapering of glucocorticoids is allowed in Period 2. Rescue medication - glucocorticoids

Patients who flare (see Section 8.3.5) are permitted to receive increases of glucocorticoid maintenance doses or intermittent glucocorticoid treatment as rescue medication for a limited period as per medical judgement and local guidance.

Period 1 - Cyclosporin discontinuation

During the 4-week treatment in Period 1a to stabilize patients on bbmAb, patients on stable doses of cyclosporin are permitted to receive bbmAb treatment. The cyclosporin dose should be maintained stable by investigators for the full duration of Period 1a until at least Day 29.

Following Day 29, investigators should gradually decrease the cyclosporin dose (per medical judgment) with the aim of achieving discontinuation of cyclosporin for 4 weeks prior to randomization at the start of the Randomized Withdrawal Period (Period 2).

• Reduction in cyclosporin may be initiated if a patient has achieved at least a partial response (Section 8.3.5).

• Cyclosporin dose reduction will continue until one of the following occurs first:

• Patient achieved discontinuation of cyclosporin.

• Patient has reached maximal duration of 20 weeks for cyclosporin discontinuation.

• Patient has failed three attempts at cyclosporin discontinuation.

Patients will be eligible for direct entry into Period 2 of the study if they have discontinued cyclosporin for 4 weeks.

All patients who fail to maintain at least a partial response to bbmAb and discontinue cyclosporin may be discontinued from the study. Patients who have achieved a partial response (with or without the glucocorticoid tapering, but having discontinued cyclosporin) as assessed at the End of Period 1 visit, may enroll straight into Period 3 open label treatment at the investigator’s and family’s discretion.

If a patient discontinues during Period 1 , the End of Period 1 assessments (i.e. Period 1c, Week 28 in the assessment schedule) should be completed as the End of Treatment visit. Patients who have achieved a partial response (with or without the glucocorticoid tapering but having discontinued cyclosporin) as assessed at the End of Period 1 visit, may enroll straight into Period 3 open label treatment at the investigator’s and family’s discretion.

Contraception

Use of oral, injected or implanted hormonal methods of contraception are allowed while on bbmAb.

6.2.2. Prohibited medication

The following treatments are NOT allowed prior to Period 1 Day 1 (time intervals prior to Day 1 are detailed below) AND during the entire study:

Etanercept in 1 week prior to Day 1

Adalimumab in the 4 weeks prior to Day 1

Infliximab in the 2 weeks prior to Day 1

Tocilizumab in the 3 weeks prior to Day 1

Vedolizumab in the 4 weeks prior to Day 1 i.v. immunoglobulin (i.v. Ig) in the 4 weeks prior to Day 1, except for replacement therapy for patients with a diagnosis of hypogammaglobulinemia. Refer to Section 6.2.1.1 for Immunoglobulin therapy discontinuation guidance.

Any other investigational or non-investigational immunomodulatory therapeutic antibodies in past 30 days or 5 half-lives (whichever is the longer) prior to Day 1 Leflunomide in the 4 weeks prior to Day 1

Thalidomide in the 4 weeks prior to Day 1

6-Mercaptopurine, azathioprine, cyclophosphamide, or chlorambucil in the 12 weeks prior to Day 1

Tacrolimus in the 4 weeks prior to Day 1

Colchicine, dapsone, mycophenolate mofetil in the 4 weeks prior to Day 1 Ruxolitinib and other JAK inhibitors 4 weeks prior to Day 1

Any other investigational or non-investigational anti-rejection and immunomodulatory drugs within the past 28 days prior to Day 1

Anti-thymocyte globulin and Campath in the 12 weeks prior to Day 1

Patients on treatment with glucocorticoids may continue as needed depending on the clinical condition of the patient. The dose of glucocorticoids should be stable for at least 24 hours before treatment with bbmAb (see Section 6.2.1.1 for glucocorticoid taper during Period 1).

Patients on treatment with cyclosporin may continue as needed depending on the clinical condition of the patient. The dose of cyclosporin should be stable for at least 3 days before treatment with bbmAb (see Section 6.2.1.1 for cyclosporin discontinuation during Period 1).

Patients on treatment with Anakinra, canakinumab, emapalumab and/or investigational I L-18/I L-1/IFN-y binding or blocking therapy, need to discontinue this treatment. As soon as the criteria for evidence of active disease (Section 5.1 Inclusion Criteria), patients can receive bbmAb treatment. This run-in phase reduces the classical wash out phase to a medically meaningful time and avoids unnecessary suffering for the patient in case a predefined wash-out period per protocol is too long for an individual patient.

No live vaccinations within 4 weeks prior to Period 1 , Day 1 , during the trial, and up to 3 months following the last dose. Approved (including HA conditional marketing authorization) killed, inactivated, peptide, DNA and RNA vaccines may be permitted according to the investigator’s discretion and per local guidance.

Patients who have entered the study from a MAP with bbmAb will be required to maintain the requirements from the MAP in regards to prohibited and concomitant medications which were permitted while in the MAP.

Novartis qualified medical personnel will be readily available to advise investigators on trial related medical questions about concomitant therapy and prohibited medications. 6.2.3. Rescue medication

Increase of glucocorticoids maintenance dose or intermittent steroid treatment may be used as rescue therapy. Information regarding the administration of glucocorticoids to study patients is found in Section 6.2.1.1, which describes use and tapering of glucocorticoids during this study.

Use of rescue medication must be recorded on the concomitant medications page in the CRF.

Patients who do not improve with treatment, do not meet the partial response criteria at Day 29 in Period 1, or flare, not due to glucocorticoids tapering during Period 1c, may discontinue to study and be treated as per medical judgement and local practice. Patients who have achieved at least a partial response and discontinued cyclosporin may be eligible to enter Part 3 where they will receive bbmAb.

Instruction for prescribing and taking study treatment

Table 6 Dose and treatment schedule

(a) Table 7 Dose and treatment schedule

Investigational Drug Dose Frequency and/or Regimen

(Name and Strength) bbmAb 100 mg/mL 10 mg/kg Periods 1 , 2, and 3: q2w by i.v. infusion over approx. 60 to 120 minutes

Period 3 only: Frequency / dosing window may be altered per Table 8- 1.

Placebo 0 mg/mL 0 mg/kg q2w by i.v. infusion over approx. 60 to 120 minutes (Period 2 only)

Note: Infusion time with i.v. bag or pump syringe is approximately 60 to 120 minutes in total.

8. Visit schedule and assessments

The Assessment Schedule (Table 14) lists all of the assessments when they are performed. All data obtained from these assessments must be supported in the patient’s source documentation.

Patients should be seen for all visits/assessments as outlined in the assessment schedule (Table 14) or as close to the designated day/time as possible.

Missed or rescheduled visits should not lead to automatic discontinuation.

Patients who prematurely discontinue the study for any reason should be scheduled for a visit as soon as possible, at which time all of the assessments listed for the final visit will be performed. At this final visit, all dispensed investigational product should be reconciled, and the adverse event and concomitant medications recorded on the CRF.

In Period 3, in order to maintain q2w dosing in-between scheduled study visits as outlined in Table 14, home dosing visits by a mobile nurse may be possible, depending on local regulations and capabilities. At each dosing, weight will be measured and any AEs will be evaluated (AE assessment will be performed by the investigator or appropriate delegated study team member based on information obtained from the mobile nurse). The weight from the previous dosing day may be used for the dose calculation for a home dosing visit.

If an epidemic or pandemic (e.g., COVID-19 pandemic) limits or prevents on-site study visits, alternative methods of providing continuing care may be implemented. Phone calls, virtual contacts (e.g., teleconsult) to the patient or visits by site staff to a patient’s home depending on local regulations and capabilities, can replace on-site study visits, for the duration of the pandemic until it is safe for the patient to visit the site again.

able 14 Assessment Schedule

Period 1, Open Label Treatment Period

Period 2, Randomized Withdrawal Period

Period 3, Long Term Safety, Open Label Treatment

^x Assessment to be recorded in the clinical database or received electronically from a vendor. s Assessment to be recorded in the source documentation only.

¹ Evidence of active disease must be present before the first dosing as assessed by PGA of disease activity > minimal and Ferritin > 600g/ml and/or elevation of CRP > 20mg/l. Screening window may be extended in order to allow enough time to prove presence of active disease.

² Baseline assessments may be performed pre-dose on Day 1.

³ Visits may be skipped if glucocorticoid tapering I cyclosporin discontinuation criteria has been achieved earlier as described in Section.2.1.1 , but assessments from Period 1 b Week 24 must be completed. For patients free from glucocorticoids/cyclosporin on study entry, a inimum of 4 weeks in Period 1b must be completed (i.e. on completion of Period 1a, move to Period 1 b Week 22 and Week 24 in thessessment schedule) before moving to Period 1c.

⁴ End of T reatment visit: Week 28 is the End of Period 1 visit that should be performed for all patients including those who discontinuearly from Period 1 . Week 24 is the End of Period 2 visit that should be performed for all patients including those who discontinue early fromeriod 2. Week 152 is the End of Period 3 visit that should be performed for all patients including those who discontinue early from Period 3.

⁵ If a patient flares in Period 2 blinded treatment may be stopped and open label bbmAb treatment may be given for the remainder oferiod 2.

⁶ In case of a flare in-between scheduled visits, an unscheduled visit assessment should be performed.

⁷ If a patient discontinues early from the study, the assessments in the Follow-up visit should be performed 16 weeks after the last dosef study treatment.

⁸ Based on the Investigator’s judgement, patients can be hospitalized for dosing. Hospitalizations for this reason will not be recorded as aAE.

⁹ Optional consent.

¹⁰ NLRC4-GOF treatment, surgical/medical procedures information should be collected from the previous 6 months where possible.

¹¹ Detailed medical history of the patient’s condition should be documented to demonstrate how the patient’s condition has been anaged and diagnosed (Section 8.2). Molecular diagnosis of NLRC4 mutation and IL-18 must occur at Screening if not already available in theatient history. The Screening window may be extended in order to allow results from Molecular diagnosis of NLRC4 mutation to be available, allther Screening assessments (apart from Informed Consent) should be performed after the Molecular diagnosis is available and within the creening window.

¹² Test result from the previous 3 months can be accepted if already available in the patient history. TB testing includes QuantiFERON® r Chest X-ray.

¹³ A short (abbreviated) physical examination can be conducted except at Screening, Period 1 D29 and Week 28, Period 2 Week 24, andt the Follow up visit.

¹⁴ Length for small infants less than 2 years old.

¹⁵Head (Occipito-frontal) circumference should be measured for all patients aged 3 years and under (age at the time of Screening and ntil the patient turns 3 years old).

¹⁶ At Screening, an ECG performed within the previous 3 months may be used if already available in the patient history. An ECG may beonducted at any point throughout the study if deemed necessary in the judgement of the Investigator.

¹⁷ for children >8 years of age until reach stage 5.

¹⁸ Females with Tanner stage 2 and above: should be reviewed at least every 3 months to evaluate the need and compatibility of the ethod of contraception and need for pregnancy testing.

¹⁹ Safety laboratory assessments will be performed at the local laboratory. Safety blood samples have priority over the PK, IG and iomarker sample collection where TBV may be limited. Additional safety laboratory assessments can be done based on the investigatorsudgement according to standard of care.

²⁰ Where possible, CRP and ferritin should be included as part of SoC chemistry sampling, rather than collecting a separate sample.

²¹ Weekly site phone calls to patients/parents/caregivers will be performed for patients not hospitalized during Period 1b to monitoresponse during the glucocorticoid tapering.

²² Only for hospitalized patients.

²³ Sample collection at home 1-2 days prior to clinic visit is possible. Keep sample ambient or as per local laboratory guidance.

²⁴ Study drug administration will continue every 2 weeks. Home nursing visits by a mobile nurse may be possible, depending on localegulations and capabilities. Weight and AEs will be assessed at each dosing. AE assessment will be performed by the investigator orppropriate delegated study team member based on information obtained from the mobile nurse. Weight from the previous dosing day may besed for the dose calculation for a home dosing visit.

²⁵ PK sampling immediately after infusion on Day indicated (see Section 9.1.1).

²⁶ Pre-dose.

²⁷ In case of suspected allergic hypersensitivity, the patient should return to the site and a sample to assess immunogenicity will beollected.

²⁸ Only collected if optional consent given. Can be collected any time during the study.

²⁹ Optional sample (only if patient weight is above 5 kg).

³⁰ Priority list for the analytics (in case limited amount of blood is available): minimum PK > IG> target capture biomarkers > immuneiomarkers> protein profiling > DNA, for Baseline PK, IG and biomarker must be analyzed.

8.1. Screening

Screening

It is permissible to re-screen a patient if s/he fails the initial Screening; however, each case must be discussed and agreed with the Sponsor on a case-by-case basis. Patients who are re-screened must be re-consented and a re-screening CRF must be completed.

In the case where a safety laboratory assessment at Screening is outside of the range specified in the exclusion criteria, the assessment may be repeated once prior to entering treatment. If the repeat value remains outside of the specified ranges, the patient must be excluded from the study.

8.1.1. Eligibility Screening

8.1.1.1. Hepatitis screen, HIV screen

Where appropriate, patients may be screened for Hepatitis B surface antigen (HBsAg) and, if standard local practice, Hepatitis B core antigen (HBcAg). Screening for Hepatitis C will be based in HCV antibodies and if positive, HCV RNA levels should be determined. Negative test results from the previous 3 months may be used if available.

Evaluation for HIV seropositivity will be performed, and, if positive, confirmation by a second technique available at the laboratory site e.g., Western blot. Appropriate counseling will be made available by the Investigator in the event of a positive confirmatory test. Notification of state and federal authorities, as required by law, will be the responsibility of the Investigator. Negative test results from the previous 3 months may be used if available.

8.1.2. Tuberculosis (TB) testing

In order to evaluate the TB status of the patient, a TB test may be performed at Screening as per local regulations/guidelines using one of the following methods:

• QuantiFERON®-TB assay

• Chest x-ray

Negative test results from the previous 3 months may be used if available.

Any significant findings will be recorded in the Relevant medical history/Current medical conditions section of the eCRF as necessary.

8.1.3. Information to be collected on screening failures

Patients (or parent/legal guardian) who sign an informed consent/assent form and where the patient subsequently found to be ineligible will be considered a screen failure. The reason for screen failure should be entered on the applicable Case Report Form (Disposition form). The visit information, demographic information, informed consent, Inclusion/Exclusion, disease specific (e.g., XIAP deficiency, or CDC42 mutation) medical history and Disposition pages must also be completed for screen failure patient. No other data will be entered into the clinical database for patients who are screen failures, unless the patient experienced a serious adverse event during the Screening phase. Adverse events that are not SAEs will be followed by the investigator and only recorded in the patient’s source data.

Patients (or parent/legal guardian) who sign an informed consent/assent form and where the patient is considered eligible but fail to be started on treatment for any reason will be considered an early terminator. The reason for early termination should be captured on the appropriate disposition Case Report Form (Disposition page). If a patient (or parent/legal guardian) voluntarily withdraws participation from the study during the Screening phase then visit information, demographic information, informed consent, NLRC4-GOF medical history, Inclusion/Exclusion pages, Withdrawal of Informed Consent and Disposition must be completed.

8.2. Patient demographics/other baseline characteristics

Demographic information

Country-specific regulations should be considered for the collection of demographic and baseline characteristics in alignment with CRF. Patient race and ethnicity are collected and analyzed to identify variations in safety or efficacy due to these factors as well as to assess the diversity of the study population as required by Health Authorities.

Patient demographics: year of birth (age), sex, race, predominant ethnicity (if permitted) and relevant medical history/current medical conditions (until date of signature of informed consent) will be recorded in the eCRF. Where possible, the diagnosis and not symptoms should be recorded. Vaccination status should be recorded as part of the collection of medical history/current medical conditions.

Disease specific (e.g., NLRC4-GOF, XIAP deficiency, or CDC42 mutation) medical history and diagnosis

A detailed medical history of the patient’s condition should be documented in the eCRF to demonstrate how the patient’s condition has been managed and diagnosed. Details (including dates of assessments) should include: presenting symptoms

Molecular diagnosis of XIAP deficiency or CDC42 mutation (can be performed by site per local approved procedures for diagnosis at Screening if not already available) IL-18 assay level (can be performed by site per local approved procedures for diagnosis at Screening if not already available) treatment interventions and outcomes/response time to and number of flares laboratory values of clinical significance e.g., cytokines, CRP, ferritin nutritional support hospitalization family history any other investigations any other clinically relevant information considered clinically relevant to support the wider understanding of the disease or diagnosis.

8.3. Efficacy

Efficacy assessments will be performed at the timepoints defined in the Assessment Schedule (Table 14).

If an epidemic or pandemic (e.g., COVID-19 pandemic) limits or prevents on-site study visits, alternative methods of providing continuing care and collecting efficacy assessments may be implemented.

8.3.1. Physician Global Assessment of Disease Activity (PGA)

Physician Global Assessment (PGA) of disease (Appendix 4) will be evaluated at the timepoints outlined in the Assessment Schedule (Table 14).

PGA will be performed prior to the CRP results being available from the local laboratory in order to prevent bias in the evaluation. It is encouraged that one investigator assess the same patient throughout the study to ensure consistency between assessments.

The physician’s global assessment will be based on a 5-point scale:

• 0 = Absent (no) disease associated clinical signs and symptoms

• 1 = Minimal disease associated signs and symptoms

• 2 = Mild disease associated signs and symptoms

• 3 = Moderate disease associated signs and symptoms

• 4 = Severe disease associated signs and symptoms

8.3.2. Physician's Severity Assessment of Disease Signs and Symptoms

Physician’s severity assessment of key disease-specific signs and symptoms (Appendix 5) will be assessed at the timepoints outlined in the Assessment Schedule (Table 14). A different assessment is used for each disease diagnosis, and the appropriate questionnaire should be completed per diagnosis. It is encouraged that one investigator assess the same patient throughout the study to ensure consistency between assessments. The following signs and symptoms will be assessed:

The following signs and symptoms will be assessed for patients with XIAP deficiency:

Gut disease

Fever

Cytopenia

Skin disease

Infection

Splenomegaly

CNS involvement

The following signs and symptoms will be assessed for patients with CDC42 mutations:

Gut disease

Fever

Skin disease

Splenomegaly

CNS involvement

Physician severity assessment of key disease-specific signs and symptoms will be based on a 5-point scale:

0 = Absent

1 = Minimal

2 = Mild

3 = Moderate

4 = Severe

8.3.3. Inflammatory markers

CRP and ferritin will be measured as shown in Table 14 by the local laboratory. Where possible, analysis should be included as part of the routine safety laboratory monitoring to avoid additional sample collection.

8.3.4. Patient's/Parent's Global Assessment of Disease Activity (PPGA)

Patient’s assessment of disease activity (PPGA) will be collected on a paper CRF to be transcribed into the electronic CRF (Appendix 6).

The PPGA should be completed prior to any clinical assessments at any given visit. This instrument needs to be filled out at the timepoints outlined in Table 14 by the patient or parent/caregiver depending on the age of the patient, as per local guidance. Where possible, a parent/caregiver can assist. However, the same evaluator (same patient or parent/caregiver) should perform the assessment throughout the study for consistency.

Patients or parents/caregivers will be instructed to complete the PPGA.

The investigator or site staff should not give verbal or non-verbal cues to influence the answers to the PPGA. The investigator or site staff will only be allowed to review the instrument for completeness.

The PPGA is based on a 5-point scale:

• 0 = Absent (no) disease associated clinical signs and symptoms

• 1 = Minimal disease associated signs and symptoms

• 2 = Mild disease associated signs and symptoms

• 3 = Moderate disease associated signs and symptoms

• 4 = Severe disease associated signs and symptoms

8.3.5. Response to treatment criteria

Response to treatment will be collected through the PGA (Section 8.3.1) and inflammatory markers (Section 8.3.3).

Period 1

Complete response: A patient is considered to have a complete response if (to be assessed on the same day):

• The physician global assessment of disease activity is minimal or better and

• There is either a 60% or greater reduction from Baseline or a normalization of either ferritin (< 400 ng/mL) and/or CRP (< 10 mg/L).

Partial response criteria: A patient is considered to have an incomplete (partial) response if (to be assessed on the same day):

• The physician global assessment of disease activity improvement over Baseline by one step and

• There is either a 30% or greater reduction from Baseline of either ferritin or CRP.

Period 2

Flare criteria: A patient is considered to have a flare if (to be assessed on the same day):

• Physician global assessment of disease activity > minimal and

• There is either a 60% or greater increase from entry levels in Period 2 or in patients who have normalized their levels an increase in ferritin > 2500 ng/mL and/or elevation of CRP > 20mg/L.

Investigators when accessing the inflammatory markers for the response to treatment criteria, should as per medical judgment exclude alternative common causes of changes in CRP or ferritin (e.g., childhood infections, iron supplementation, blood transfusions) in this pediatric study population.

Table 8 Clinical Laboratory Safety Assessments (local) 9. Efficacy and/or Pharmacodynamic endpoint(s)

The FAS of respective study periods will be used for analyses in this section.

The following secondary efficacy endpoints will be analyzed:

Response at Day 29, end of Periods 1 and 2 (for cohort 1)

Serological remission at Day 29, end of Periods 1 and 2 (for cohort 1) Glucocorticoid therapy in Period 1 (for cohort 1)

Time to first flare in Period 2 (for cohort 1)

Physician’s severity assessment of disease signs and symptoms (all cohorts) Patient's/ parent's global assessment of disease activity (all cohorts) Physician’s global assessment of disease activity (all cohorts)

The proportion of patients who respond to bbmAb treatment on Day 29 and thereafter until the end of Period 1 , will be calculated. The definition of an bbmAb treatment complete responder is given in Section 8.3.6 of the protocol. The proportion of patients who respond to bbmAb and placebo will also be evaluated at the end of Period

2.

The inflammatory markers (CRP and ferritin) will be summarized by treatment and visit.

The proportion of patients who achieve serological remission will be evaluated at Day 29, end of Period 1 and 2.

The proportion of patients who achieve glucocorticoid taper and cyclosporin reduction after Day 29 until end of Period 1 will be calculated. The definition of the glucocorticoid taper and cyclosporin reduction is given in Section 6.2.1.1 of the protocol.

Flare

Flare will be assessed by Physician's global assessment of disease activity, ferritin and/or CRP as given in flare criteria in Section 8.3.6 of the protocol.

The time to first flare will be summarized by treatment for Period 2. Kaplan Meier graphs with separate lines by treatment group will be provided for graphical presentation.

Other efficacy endpoints

Summary statistics for the absolute values and change from Baseline in the Physician’s severity assessment of disease sign and symptoms, Patient’ s/parent’s global assessment of disease activity and Physician’s global assessment of disease activity will be provided by treatment and visit. Frequency tables for each symptom assessed by the physician and patient/parent will be presented by visit. The frequency distribution of the severity scores (absent, minimal, mild, moderate, severe) will be calculated by treatment and visit.

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Appendix 1 : Clinically notable laboratory values and vital signs

The following defined notable laboratory or vital sign abnormalities will be communicated at the same time as they are available to investigators to Novartis. Novartis will determine if further consultations with Investigator(s) are appropriate.

Newly occurring selected notable laboratory abnormalities in pediatric patients (< 16 years of age):

Albumin: < LLN

>3x-, 5x-, 10x-, and 20xllLN elevations of AST, ALT, and either ALT or AST*

Any elevations of bilirubin; elevated bilirubin to >1.5xULN, and to >2xULN*

Any elevations of ALP >1.5xULN*

Elevation of ALT and/or AST (>3xULN) accompanied by elevated bilirubin

(>1.5xULN, >2xULN)*

Gamma-Glutamyltransferase (GGT): > 3 x ULN

Creatinine (serum): > 1.5 x ULN

Potassium: > 5.5 mmol/L, or < 3.5 mmol/L

Magnesium: > 1.2 mmol/L, or < 0.7 mmol/L

Sodium: >150 mmol/L, or < 130 mmol/L

Hemoglobin: > 2 g/dL decrease from Baseline, or < 8.5 g/dL

Platelet count: < Lower Limit of Normal (LLN)

White blood cell count: < 0.8 x LLN or > 1.2 x ULN

Neutrophils: < 0.9 x LLN or > 1.2 x ULN

Eosinophils: > 1.1 x ULN

Lymphocytes: < LLN or > 1.1 x ULN

Protein urine dipstick: Positive (Trace, > +)

Notable vital signs abnormalities in pediatric patients (< 16 years of age):

Systolic/diastolic blood pressure¹:

High: > 95th percentile of the age and height group

Low: < 5th percentile of the age and height group

Oral body temperature (°C)

High: > 38.4 °C

Low: < 35.0 °C

Pulse (bpm): refer to Table 17-1

Table 9 Abnormal pulse rate (bpm) in pediatric population² Age High Low

1-6 months >160 <120

6-12 months >150 <110

12-18 months >140 <103

18-24 months >135 <98

2-3 years >128 <92

3-4 years >123 <86

4-6 years >117 <81

6-8 years >111 <74

8-12 years >103 <67

12-15 years >96 <62

>15 years >92 <58

• Weight:

• High: increase from Baseline³ of >2 BMI-for-age percentile categories⁴

• Low: decrease from Baseline³ of >2 BMI-for-age percentile categories⁴

• Respiratory rate: refer to Table 17-2

Table 10 Abnormal respiratory rate (breaths per minute) in pediatric population^{2 5}

Age High Low

1-6 months >55 <33

6-12 months >50 <30

12-18 months >46 <28

18-24 months >40 <25

2-3 years >34 <22

3-4 years >29 <21

4-6 years >27 <20

6-8 years >24 <18

8-12 years >22 <16

12-15 years >21 <15

>15 years >20 <13

¹ Blood pressure percentiles are calculated for each blood BP record using the method described in Appendix B of The Fourth Report on the Diagnosis, Evaluation and Treatment of High Blood Pressure in Children and Adolescents (Anon 2004). 2 Fleming S et al 2011.

³ Baseline BMI-for-age weight status categories are underweight (less than the

5th percentile), healthy weight (5th percentile to less than the 85th percentile), overweight (85th to less than the 95th percentile) and obese (equal to or greater than the 95th percentile);

⁴ BMI-for-age percentiles categories (P3, P5, P10, P25, P50, P75, P85, P90, P95, P97) are obtained from the WHO Growth Charts (www.who.int/childgrowth/en/) (WHO 2020);

Note: For patients less than 2 years old, growth charts are based on recumbent length instead of height;

⁵ Kou R and Shuei L 2009.

Note: Only post-Baseline values will be flagged as notable abnormalities

Newly occurring selected notable laboratory abnormalities in adult patients (>16 years of age):

Albumin: < LLN

3x-, 5x-, 10x-, and 20xllLN elevations of AST, ALT, and either ALT or AST*

Any elevations of bilirubin; elevated bilirubin to >1.5xULN, and to >2xULN*

Any elevations of ALP >1.5xULN*

Elevation of AST and/or ALT (>3xULN) accompanied by elevated bilirubin (>1.5xULN, >2xULN)*

Gamma-Glutamyltransferase (GGT): > 3 x ULN

Creatinine (serum): > 1 .5 x ULN

Creatinine clearance: (Cockroft-Gault formula)§: > 25% decrease from Baseline

Potassium: > 5.5 mmol/L, or < 3.0 mmol/L

Magnesium: > 1.5 mmol/L, or < 0.5 mmol/L

Sodium: > 150 mmol/L, or < 130 mmol/L

Calcium: > 1 .2 x ULN or < Lower Limit of Normal (LLN)

Hemoglobin: >2 g/dL decrease from Baseline, or < 10.0 g/dL

Platelet count: < LLN

White blood cell count: < 0.8 x LLN or > 1.2 x ULN

Neutrophils: <0.9 x LLN or > 1 .2 x ULN

Eosinophils: > 1.1 x ULN

Lymphocytes: < LLN or > 1.1 x ULN

Protein urine dipstick: > ++

Newly occurring selected notable vital signs abnormalities in adult patients (> 16 years of age):

Systolic/Diastolic blood pressure: >25% decrease or >25 % increase from Baseline or to >140/90 • Pulse: >110 bpm with >15% change from Baseline, or < 50 bpm with >15% change from Baseline

* Source: Draft October 2007 FDA Guidance for Industry Drug-Induced Liver

Injury: Premarketing Clinical Evaluation (FDA 2007)

Cockroft-Gault formula (Men): Creatinine clearance (mL/min) = [((140-age

(years)) x weight (kg)) I (serum creatinine (pmol/L) 188.4) (mg/dL) x 72]

Cockroft-Gault formula (Women): Creatinine clearance (mL/min) = [((140-age

(years)) x weight (kg)) I (serum creatinine (pmol/L) 188.4) (mg/dL) x 72] x 0.85

Note: Only post-Baseline values will be flagged as notable abnormalities

Table 11 Recommended Dimensions for Blood Pressure Cuff Bladders

Source: Feld and Corey Pediatrics in review (2007)

Appendix 2: Anaphylaxis

Anaphylaxis is highly likely when any one of the following 3 criteria are fulfilled:

1. Acute onset of an illness (minutes to several hours) with involvement of the skin, mucosal tissue, or both (e.g., generalized hives, pruritus or flushing, swollen lips- tongue- uvula)

AND AT LEAST ONE OF THE FOLLOWING: a. Respiratory compromise (e.g., dyspnea, wheeze-bronchospasm, stridor, reduced PEF, hypoxemia) b. Reduced BP or associated symptoms of end-organ dysfunction (e.g., hypotonia [collapse], syncope, incontinence)

2. Two or more of the following that occur rapidly after exposure to a likely allergen for that patient (minutes to several hours): a. Involvement of the skin mucosal tissue (e.g., generalized hives, pruritus or flushing, swollen lips-tongue-uvula) b. Respiratory compromise (e.g., dyspnea, wheeze-bronchospasm, stridor, reduced PEF, hypoxemia) c. Reduced BP or associated symptoms (e.g., hypotonia [collapse], syncope, incontinence) d. Persistent gastrointestinal symptoms (e.g., crampy abdominal pain, vomiting)

3. Reduced BP after exposure to known allergen for that patient (minutes to several hours) a. Adults: systolic BP of less than 90 mm Hg or greater than 30% decrease from Baseline.

Appendix 3: The Toronto Hospital for Sick Children Research Ethics Board (REB) Blood Sampling Guidelines

The following recommendations by the Toronto Hospital for Sick Children Research Ethics Board will be used to guide the investigator on the maximum blood volume collected during the study. Investigators should closely monitor total blood collection to ensure that the limits outlined in the guidance or per local restrictions from the IRB/EC are adhered to.

For research of infants, children and adolescents, the guidance allows total blooddrawing of up to 5% of the patient’s total blood volume over an eight week period, on a single occasion or in divided portions.

Table 12 Blood volume changes with age, thus amount available per kg will be:

Calculation of 5% Blood Volume by weight

Neonates (*4% of TBV) *3.0 mL/kg 1 month to 10 years 4.0 mL/kg 10-15 years 3.7 mL/kg

Greater than 15 years 3.6 mL/kg

Adapted from The Hospital for Sick Children Research Ethics Board Blood Sampling Guide (Anon 2017). SEQUENCE TABLE

Useful amino acid and nucleotide sequences for practicing the invention are disclosed in Table 13. Table 13. Sequences according to embodiments of the invention

Throughout the text of this application, should there be a discrepancy between the text of the specification (e.g., Table 13) and the sequence listing, the text of the specification shall prevail.

Claims

WHAT IS CLAIMED IS:

1. A method for the treatment or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a bispecific antibody, wherein the antibody comprises a. a first part which is an immunoglobulin with a first variable light chain of (VL1) and a first variable heavy chain (VH1), that binds specifically to a IL1 p, and a first constant heavy chain (CH1) with a hetero-dimerization modification, and b. a second part which is an immunoglobulin with a second variable light chain (VL2) and a second variable heavy chain (VH2), that binds specifically to IL-18 and a second constant heavy chain (CH2) with a hetero-dimerization modification which is complementary to the hetero-dimerization modification of the first constant heavy chain.

2. The method according to claim 1 , wherein the first and second constant heavy chains of the bispecific antibody are lgG1 , and wherein a. the first constant heavy chain has point mutations generating a knob structure and the second constant heavy has point mutations generating a hole structure, or b. the first constant heavy chain has point mutations generating a hole structure and the second constant heavy has point mutations generating a knob structure, and optionally c. the first and second constant heavy chains have mutations resulting in a disulfide bridge.

3. The method according to claims 1-2, wherein: a. 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 ii. 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 NQ:80, and said CDR3 having the amino acid sequence SEQ ID NO:81 ; and d. the first immunoglobulin VL1 domain of the bispecific antibody comprises 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; and e. the second immunoglobulin VH2 domain of the bispecific antibody comprises 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. 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 f. the second immunoglobulin VL2 domain of the bispecific antibody comprises i. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NQ: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.

4. The method according to any of the preceding claims, wherein: 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, and d. the second immunoglobulin VL2 domain of the bispecific antibody comprises the amino acid sequence SEQ ID NO: 69.

5. The method according to 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.

6. The method according to any one of the preceding claims, wherein said subject has a loss-of-function mutation in the XIAP/BIRC4 gene encoding the XIAP protein.

7. The method according to any one of the preceding claims, wherein said subject has macrophage activation syndrome (MAS).

8. The method according to any one of the preceding claims, wherein said subject has autoinflammation with infantile enterocolitis (AIFEC).

9. The method according to any one of the preceding claims, wherein said subject has hyper elevated levels of serum IL-18 and serum IL-1 p as compared to a control population of subjects that do not have XIAP deficiency based autoinflammation or disease.

10. The method according to any one of the preceding claims, wherein the XIAP deficiency based autoinflammation or disease patient is suffering from an inflammatory bowel disease, abdominal pain and diarrhea, recurrent fevers, splenomegaly or haemophagocytic lymphohistiocytosis.

11. The method according to any one of the preceding claims, wherein said subject has a serum ferritin level of > 600 ng/mL or a serum C-reactive protein (CRP) of > 20 mg/L.

12. The method according to any one of the preceding claims, wherein said XIAP deficiency based autoinflammation or disease is resistant to treatment with cyclosporin, anti-TNFa treatments, systemic glucocorticoids and anti-IL-1 therapies as a monotherapy, or as a combination thereof.

13. A bispecific antibody comprising a. a first part which is an immunoglobulin with a first variable light chain of (VL1) and a first variable heavy chain (VH1), that binds specifically to a IL1 p, and a first constant heavy chain (CH1) with a hetero-dimerization modification, and b. a second part which is an immunoglobulin with a second variable light chain (VL2) and a second variable heavy chain (VH2), that binds specifically to IL-18 and a second constant heavy chain (CH2) with a hetero-dimerization modification which is complementary to the hetero-dimerization modification of the first constant heavy chain, for use in the treatment or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof.

14. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to claim 13, wherein the first and second constant heavy chains of the bispecific antibody are lgG1 , and wherein a. the first constant heavy chain has point mutations generating a knob structure and the second constant heavy has point mutations generating a hole structure, or b. the first constant heavy chain has point mutations generating a hole structure and the second constant heavy has point mutations generating a knob structure, and optionally c. the first and second constant heavy chains have mutations resulting in a disulfide bridge.

15. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to any one of claims 13-14, wherein: a. 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 ii. 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 NQ:80, and said CDR3 having the amino acid sequence SEQ ID NO:81 ; and b. the first immunoglobulin VL1 domain of the bispecific antibody comprises iii. 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 iv. 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 c. 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 vi. 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 vii. hypervariable regions CDR1 , CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NQ: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 viii. 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.

16. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to any one of claims 13-15, wherein: 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, and d. the second immunoglobulin VL2 domain of the bispecific antibody comprises the amino acid sequence SEQ ID NO: 69.

17. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to any one of claims 13-16, 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.

18. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to any one of claims 13-17, wherein said subject has a loss-of-function mutation in the XIAP gene.

19. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to any one of claims 13-18, wherein said XIAP deficiency based autoinflammation or disease patient is suffering from an inflammatory bowel disease, abdominal pain and diarrhea, recurrent fevers, splenomegaly or haemophagocytic lymphohistiocytosis (HLH).

20. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to any one of claims 13-19, wherein said subject has macrophage activation syndrome (MAS).

21. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to any one of claims 13-20, wherein said subject has autoinflammation with infantile enterocolitis (AIFEC).

22. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to any one of claims 13-21 , wherein said subject has hyper elevated levels of serum IL-18 and serum I L-1 as compared to a control population of subjects that does not have an XIAP deficiency based autoinflammation or disease.

23. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to any one of claims 13-22, wherein said subject has a serum ferritin level of > 600 ng/mL or a serum C-reactive protein (CRP) of > 20 mg/L.

24. The method according to any one of claims 1-12, comprising administering about 1 mg/kg to about 35 mg/kg of the bispecific antibody to the subject.

25. The method according to claim 24, comprising administering about 10 mg/kg of the bispecific antibody to said subject.

26. The method according to claim 25, wherein the bispecific antibody is administered intravenously or subcutaneously.

27. The method according to claims 25 or 26, wherein the dose of the administered bispecific antibody is about 10 mg/kg administered intravenously, optionally wherein the bispecific antibody is administered every other week.

28. The method according to claim 26, wherein the dose of the administered bispecific antibody is about 50 mg to about 900 mg administered subcutaneously.

29. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to any one of the claims 13-23, comprising administering 1 mg/kg to 35 mg/kg of the bispecific antibody to the subject.

30. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to claim 29, comprising administering 10 mg/kg intravenously of the bispecific antibody to said subject.

31. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to any one of claims 13-23, wherein the bispecific antibody is administered at a dose of about 50 mg to about 900 mg subcutaneously.

32. A method for the treatment or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a bispecific antibody, wherein the antibody comprises a. a first part which is an immunoglobulin with a first variable light chain of (VL1) and a first variable heavy chain (VH1), that binds specifically to a IL1 p, and a first constant heavy chain (CH1) with a hetero-dimerization modification, and b. a second part which is an immunoglobulin with a second variable light chain (VL2) and a second variable heavy chain (VH2), that binds specifically to IL-18 and a second constant heavy chain (CH2) with a hetero-dimerization modification which is complementary to the hetero-dimerization modification of the first constant heavy chain.

33. The method according to claim 32, wherein the first and second constant heavy chains of the bispecific antibody are lgG1 , and wherein a. the first constant heavy chain has point mutations generating a knob structure and the second constant heavy has point mutations generating a hole structure, or b. the first constant heavy chain has point mutations generating a hole structure and the second constant heavy has point mutations generating a knob structure, and optionally c. the first and second constant heavy chains have mutations resulting in a disulfide bridge.

34. The method according to claims 32-33, wherein: a. 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 ii. 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 NQ:80, and said CDR3 having the amino acid sequence SEQ ID NO:81 ; and b. the first immunoglobulin VL1 domain of the bispecific antibody comprises iii. 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 iv. 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 c. 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 vi. 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 vii. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NQ: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 viii. 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.

35. The method according to claims 32-34, wherein: 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, and d. the second immunoglobulin VL2 domain of the bispecific antibody comprises the amino acid sequence SEQ ID NO: 69.

36. The method according to claims 32-35, 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.

37. The method according claims 32-36, wherein said subject has a mutation in the CDC42 gene resulting in aberrant palmitoylation of CDC42 protein or missense mutation at the C-terminus of the CDC42 protein affecting localization of the CDC42 protein.

38. The method according to claims 32-37, wherein said subject has macrophage activation syndrome (MAS).

39. The method according to claims 32-37, wherein said subject has autoinflammation with infantile enterocolitis (AIFEC).

40. The method according to claims 32-39, wherein said subject has hyper elevated levels of serum IL-18 and serum I L-113 as compared to a control population of subjects that does not have CDC42 mutation based autoinflammation or disease.

41. The method according to claims 32-40, wherein the CDC42 mutation based autoinflammation or disease patient is suffering from neonatal-onset cytopenia, autoinflammation or recurrent haemophagocytic lymphohistiocytosis (HLH).

42. The method according to claims 32-41 , wherein said subject has a serum ferritin level of > 600 ng/mL or a serum C-reactive protein (CRP) of > 20 mg/L.

43. The method according to any one of the claims 32-42, wherein said CDC42 mutation based autoinflammation or disease patient is resistant to treatment with cyclosporin, anti-TNFa treatments, systemic glucocorticoids and anti-l L-1 p therapies as a monotherapy, or as a combination thereof.

44. A bispecific antibody comprising a. a first part which is an immunoglobulin with a first variable light chain of (VL1) and a first variable heavy chain (VH1), that binds specifically to a IL1 p, and a first constant heavy chain (CH1) with a hetero-dimerization modification, and b. a second part which is an immunoglobulin with a second variable light chain (VL2) and a second variable heavy chain (VH2), that binds specifically to IL-18 and a second constant heavy chain (CH2) with a hetero-dimerization modification which is complementary to the hetero-dimerization modification of the first constant heavy chain, for use in the treatment or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof.

45. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to claim 44, wherein the first and second constant heavy chains of the bispecific antibody are lgG1 , and wherein d. the first constant heavy chain has point mutations generating a knob structure and the second constant heavy has point mutations generating a hole structure, or e. the first constant heavy chain has point mutations generating a hole structure and the second constant heavy has point mutations generating a knob structure, and optionally f. the first and second constant heavy chains have mutations resulting in a disulfide bridge.

46. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to any one of claims 44-45, wherein: e. 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 ii. 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 NQ:80, and said CDR3 having the amino acid sequence SEQ ID NO:81 ; and f. the first immunoglobulin VL1 domain of the bispecific antibody comprises iii. 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 iv. 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 g. 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 vi. 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 h. the second immunoglobulin VL2 domain of the bispecific antibody comprises vii. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NQ: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 viii. 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.

47. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to any one of claims 44--46, wherein: e. the first immunoglobulin VH1 domain of the bispecific antibody comprises the amino acid sequence SEQ ID NO: 85, f. the first immunoglobulin VL1 domain of the bispecific antibody comprises the amino acid sequence SEQ ID NO: 101, g. the second immunoglobulin VH2 domain of the bispecific antibody comprises the amino acid sequence SEQ ID NO: 53, and h. the second immunoglobulin VL2 domain of the bispecific antibody comprises the amino acid sequence SEQ ID NO: 69.

48. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to any one of claims 44-47, wherein: e. the first immunoglobulin heavy chain of the bispecific antibody comprises the amino acid sequence SEQ ID NO: 87, f. the first immunoglobulin light chain of the bispecific antibody comprises the amino acid sequence SEQ ID NO: 103, g. the second immunoglobulin heavy chain of the bispecific antibody comprises the amino acid sequence SEQ ID NO: 55, and h. the second immunoglobulin light chain of the bispecific antibody comprises the amino acid sequence SEQ ID NO: 71.

49. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to any one of claims 44-48, wherein said subject has a mutation in the CDC42 gene resulting in aberrant palmitoylation of CDC42 protein or missense mutation at the C-terminus of the CDC42 protein affecting localization of the CDC42 protein.

50. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to any one of claims 44-49, wherein said patient is suffering from neonatal-onset cytopenia, autoinflammation or recurrent haemophagocytic lymphohistiocytosis (HLH).

51. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to any one of claims 44-50, wherein said subject has macrophage activation syndrome (MAS).

52. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to claim 50, wherein said subject has autoinflammation with infantile enterocolitis (AIFEC).

53. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to any one of claims 44-52, wherein said subject has hyper elevated levels of serum IL-18 and serum IL-1 as compared to a control population of subjects that does not have an CDC42 mutation based autoinflammation or disease.

54. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to any one of claims 44-53, wherein said subject has a serum ferritin level of > 600 ng/mL or a serum C-reactive protein (CRP) of > 20 mg/L.

55. The method according to any one of claims 33-43, comprising administering about 1 mg/kg to about 35 mg/kg of the bispecific antibody to the subject.

56. The method according to claim 55, comprising administering about 10 mg/kg of the bispecific antibody to said subject.

57. The method according to claim 56, wherein the bispecific antibody is administered intravenously or subcutaneously.

58. The method according to claims 56-57, wherein the dose of the administered bispecific antibody is about 10 mg/kg administered intravenously, optionally wherein the bispecific antibody is administered every other week.

59. The method according to claim 57, wherein the dose of the administered bispecific antibody is about 50 mg to about 900 mg administered subcutaneously.

60. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to any one of the claims 44-54, comprising administering 1 mg/kg to 35 mg/kg of the bispecific antibody to the subject.

61. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to claim 60, comprising administering 10 mg/kg intravenously of the bispecific antibody to said subject.

62. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to claims 60-61 , wherein the bispecific antibody is administered at a dose of about 50 mg to about 900 mg subcutaneously.

63. The method of treatment according to claims 1-12, wherein said subject has a loss-of- function mutation in the XIAP gene.

64. The method of treatment according to claims 32-43, wherein said subject has a loss- of-function mutation in the CDC42 gene.

65. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to any one of claims 44-54 and 60-62, wherein said subject has a loss-of-function mutation in the CDC42 gene.

66. The bispecific antibody for the use in the treatment of or prevention of an XIAP deficiency based autoinflammation or disease in a subject in need thereof according to claims 15-23 and 29-31 , wherein the subject is resistant to treatment with cyclosporin, anti- TNFa treatments, systemic glucocorticoids and anti-l L-1 p therapies as a monotherapy, or as a combination thereof.

67. The bispecific antibody for the use in the treatment of or prevention of an CDC42 mutation based autoinflammation or disease in a subject in need thereof according to claims 44-54 and 60-62, wherein the subject is resistant to treatment with cyclosporin, anti- TNFa treatments, systemic glucocorticoids and anti-l L-1 therapies as a monotherapy, or as a combination thereof.