Methods of making bispecific antibodies, bispecific antibodies and therapeutic uses of such antibodies

Abstract

The present invention relates to divalent bispecific monoclonal antibodies (bbmAbs) or variants thereof, and methods of making such antibodies by co-expression of modified Fc-5 mutated derivatives of two different monoclonal antibodies in mammalian cell lines.

Description

[0001] This application is a divisional application of Chinese patent application 201880036745.X (PCT / IB2018 / 054140), whose application date is June 8, 2018 and whose invention name is “Methods for producing bispecific antibodies, bispecific antibodies and therapeutic uses of such antibodies”.

[0002] Sequence Listing

[0003] This application contains a sequence listing filed electronically in ASCII format and hereby incorporated by reference in its entirety. The ASCII copy was created on May 30, 2018, is named PAT057716-WO-PCT_SL.txt and is 68,498 bytes in size. Technical Field

[0004] The present invention relates to bivalent bispecific monoclonal antibodies (bbmAbs) or variants thereof, and methods for making such antibodies by co-expressing said knob-into-hole modified, FC mutated derivatives of two different monoclonal antibodies in a mammalian cell line. Background Art

[0005] Bispecific antibodies, i.e. antibodies that bind to two different epitopes, are well known in the art. One method of producing bispecific antibodies is the so-called knob-in-hole (KiH) method, e.g., as described by Merchant et al., Nat. Biotechnol., 16:677-681 (1998), wherein the first heavy chain IgG is modified to exhibit a hole-like structure by introducing point mutations such as Y349C, T366S, L368A, Y407V; and the second heavy chain IgG is modified to exhibit a knob-like structure by introducing point mutations S354C, T366W (Merchant et al., Nat. Biotechnol., 16:677-681 (1998), p. 678, Table 1). The two different IgG structures then interact to form a bivalent bispecific antibody (bbmAb), i.e., a heterotetrameric protein consisting of four different light chains and two different heavy chains.

[0006] When two KiH-modified mAbs were expressed in the same host cell line, the desired bbmAb statistically accounted for only 25% of the expressed protein, but 75% were so-called product-related impurities (Klein, Ch. et al., 2012).

[0007] Some approaches to address this problem are known in the art, such as promoting the correct formation of bbmAbs by applying further sequence modifications to promote correct HL binding (for an overview, see Klein, Ch. et al., 2012; Kontermann, R. and Brinkmann, U., 2015). However, such additional modifications may increase the risk of anti-drug antibodies.

[0008] Another approach to generate bbmAbs using a shared heavy or light chain in combination with different variable chains is disclosed in WO 12023053A2 or WO 04009618A2. However, keeping either heavy chain constant significantly reduces the diversity of the antibody library that can be screened for binders.

[0009] Another method for producing bbmAbs is disclosed in US9212230, which requires the separate expression and purification of mAbs carrying different modifications. Finally, the resulting mAbs are reorganized in vitro to form the desired bbmAbs. This in vitro reorganization is a complex additional process step that requires careful validation and analytical evaluation and may significantly increase costs.

[0010] Therefore, existing methods for generating bbmAbs may limit the diversity of antibody libraries that can be used to screen binders, or may not provide sufficient overall yield, purity, and product quality in a sufficiently cost-effective manner to allow large-scale production for clinical development and commercialization. In addition, any modification of the protein chain inherently increases the risk of inducing anti-drug antibodies. Therefore, methods that require only minimal protein engineering may be clinically advantageous. Summary of the invention

[0011] There is a need to provide an improved method for producing bivalent bispecific antibodies. In particular, there is a need for a method for manufacturing bivalent bispecific monoclonal antibodies (bbmAbs) that ensures sufficient overall yield, purity and product quality suitable for clinical development and commercial production at a reasonable cost.

[0012] The invention provides, inter alia, a method for producing bbmAbs having one or more of the following advantages: it can use large antibody libraries to identify binders as no light or heavy chains need to be shared, it does not require any extensive protein engineering other than mutations driving H chain dimerization, thus limiting the risk of anti-drug antibodies, it is cost-effective as expression is accomplished in a common cell line, so bbmAbs can be produced in one cell culture process without the need for specific in vitro shuffling, and it can produce high-quality material suitable for class use as product-related impurities can be effectively removed.

[0013] The present invention can be used to identify kappa and lambda type antibodies, where the light chain does not show strong promiscuous binding with the heavy chain of the counterpart. This makes the antibody suitable for use in the method of the present invention. The advantage of the method is that antibody combinations in which both light chains exchange the original heavy chain binding partner (which leads to product-related H1L2-H2L1 type impurities) can be eliminated. This is advantageous because such product-related impurities are not easily depleted using prior art purification methods.

[0014] As shown below, embodiments of the present invention enable the production of bbmAbs using CHO co-expression at yields and qualities suitable for clinical development and commercialization of biologics.

[0015] In a first aspect of the present invention, a bispecific antibody suitable for co-expression in a common host cell is provided, wherein the antibody comprises: a) a first part, which is an immunoglobulin having a λ wild-type variable light chain (VL1) and a wild-type variable heavy chain (VH1) that specifically binds to a first target and a first constant heavy chain (CH1) with a heterodimerization modification, and b) a second part, which is an immunoglobulin having a κ wild-type variable light chain (L2) and a wild-type variable heavy chain (H2) that specifically binds to a second target different from the first target and a second constant heavy chain (CH2) with a heterodimerization modification that is complementary to the heterodimerization modification of the first constant heavy chain, wherein when the first part and the second part are co-expressed in a common host cell, a bispecific antibody is formed.

[0016] In another embodiment of the first aspect, the bispecific antibodies suitable for co-expression in a common host cell produce at least 60% (mass), 70% (mass), 80% (mass), 85% (mass) pure, such as at least 90% (mass) pure, 95% (mass), 96% (mass), 97% (mass), 98% (mass) or 99% (mass) pure bispecific antibodies after purification of the bispecific antibodies by removing mismatched fragments from the correctly matched bispecific antibodies.

[0017] The first and second constant heavy chains of the bispecific antibody can be human IgA, IgD, IgE, IgG or IgM, preferably IgD, IgE or IgG. In a preferred embodiment, the first and second constant heavy chains are human IgG1, IgG2, IgG3 or IgG4, most preferably IgG1. In one embodiment, the first variable light chain is a lambda type, and the second variable light chain is a kappa type.

[0018] In a particularly preferred embodiment, the first variable light chain is of the λ1 type and the second variable light chain is of the κ6 type.

[0019] The first constant heavy chain and the second constant heavy chain may be IgG1, wherein the first constant heavy chain has a point mutation that generates a knob structure and the second constant heavy chain has a point mutation that generates a hole structure, or the first constant heavy chain has a point mutation that generates a hole structure and the second constant heavy chain has a point mutation that generates a knob structure. Optionally, the first constant heavy chain and the second constant heavy chain may additionally have mutations that result in disulfide bonds.

[0020] In one embodiment, the bispecific antibody comprises a first immunoglobulin VH1 domain, a first immunoglobulin VL1 domain, a second immunoglobulin VH2 domain, and a second immunoglobulin VL2 domain, wherein the first immunoglobulin VH1 domain comprises (e.g., in order): hypervariable regions CDR1, CDR2, and CDR3, wherein the CDR1 has an amino acid sequence of SEQ ID NO: 76, the CDR2 has an amino acid sequence of SEQ ID NO: 77, and the CDR3 has an amino acid sequence of SEQ ID NO: 78; or hypervariable regions CDR1, CDR2, and CDR3, wherein the CDR1 has an amino acid sequence of SEQ ID NO: 79, the CDR2 has an amino acid sequence of SEQ ID NO: 80, and the CDR3 has an amino acid sequence of SEQ ID NO: 81; and the first immunoglobulin VL1 domain comprises (e.g., in order): hypervariable regions CDR1, CDR2, and CDR3, wherein the CDR1 has an amino acid sequence of SEQ ID NO: 92, the CDR2 has an amino acid sequence of SEQ ID NO: 93, and the CDR3 has an amino acid sequence of SEQ ID NO: ID NO:94 or hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having an amino acid sequence of SEQ ID NO:95, said CDR2 having an amino acid sequence of SEQ ID NO:96, and said CDR3 having an amino acid sequence of SEQ ID NO:97; the second immunoglobulin VH2 domain comprises (e.g., in sequence): hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having an amino acid sequence of SEQ ID NO:44, said CDR2 having an amino acid sequence of SEQ ID NO:45, and said CDR3 having an amino acid sequence of SEQ ID NO:46; or hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having an amino acid sequence of SEQ ID NO:47, said CDR2 having an amino acid sequence of SEQ ID NO:48, and said CDR3 having an amino acid sequence of SEQ ID NO:49; and the second immunoglobulin VL2 domain comprises (e.g., in sequence): hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having an amino acid sequence of SEQ ID NO:4 NO:60, the CDR2 has the amino acid sequence of SEQ ID NO:61, and the CDR3 has the amino acid sequence of SEQ ID NO:62 or the hypervariable regions CDR1, CDR2 and CDR3, the CDR1 has the amino acid sequence of SEQ ID NO:63, the CDR2 has the amino acid sequence of SEQ ID NO:64, and the CDR3 has the amino acid sequence of SEQ ID NO:65.

[0021] In one embodiment, the bispecific antibody comprises a first immunoglobulin VH1 domain, a first immunoglobulin VL1 domain, a second immunoglobulin VH2 domain, and a second immunoglobulin VL2 domain, wherein: the first immunoglobulin VH1 domain comprises the amino acid sequence of SEQ ID NO:85, the first immunoglobulin VL1 domain comprises the amino acid sequence of SEQ ID NO:101, the second immunoglobulin VH2 domain comprises the amino acid sequence of SEQ ID NO:53, and the second immunoglobulin VL2 domain comprises the amino acid sequence of SEQ ID NO:69.

[0022] In one embodiment, the bispecific antibody comprises a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain, wherein: the first immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO:87, the first immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO:103, the second immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO:55, and the second immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO:71.

[0023] According to the second aspect, a method for selecting a bispecific antibody according to the first aspect is provided, the method comprising: a first step: selecting a first part and a second part; a second step: co-expressing the first part and the second part in a common host cell to produce a bispecific antibody comprising the first part and the second part; a third step: purifying the bispecific antibody by removing mismatched fragments from the correctly matched bispecific antibody. In one embodiment, the third step purifies and produces a bispecific antibody that is at least 60% (mass), 70% (mass), 80% (mass), 85% (mass) pure, for example, at least 90% (mass) pure, 95% (mass), 96% (mass), 97% (mass), 98% (mass) or 99% (mass) pure.

[0024] According to a third aspect, a method for producing a bispecific antibody by co-expression in a common host cell according to the first aspect is provided, the method comprising: a first step: producing at least one vector encoding a first part and a second part; a second step: introducing the at least one vector into a common host cell; a third step: selecting cells that specifically express the bispecific antibody; a fourth step: culturing the selected cells under conditions in which the cells express the bispecific antibody; and a fifth step: purifying the bispecific antibody, which is at least 60% (mass), 70% (mass), 80% (mass), 85% (mass) pure, for example, at least 90% (mass) pure, 95% (mass), 96% (mass), 97% (mass), 98% (mass) or 99% (mass) pure.

[0025] In one embodiment, the first step comprises generating a first vector encoding the first part and a second vector encoding the second part.

[0026] According to a fourth aspect, an expression system comprises at least one vector comprising a polynucleotide encoding the first part or the second part of the bispecific antibody according to the first aspect and a selection marker.

[0027] In one embodiment, the expression system comprises a polynucleotide encoding a first selection marker (sm I); and a polynucleotide encoding a second selection marker (sm II) that is different from the first selection marker (sm I).

[0028] In one embodiment, the first selection marker (sm I) is a folate transporter or a polynucleotide encoding a mutated folate receptor, wherein the mutated folate receptor has reduced folate binding affinity compared to the wild-type folate receptor, and the second selection marker (sm II) is DHFR.

[0029] In one embodiment, the first selection marker (sm I) is hygromycin and the second selection marker (sm II) is Neo / G418.

[0030] In one embodiment, the expression system comprises two expression vectors, wherein: a first vector comprising a polynucleotide encoding at least a first selection marker (sm I) and a polynucleotide encoding at least a first part; a second vector comprising a polynucleotide encoding at least a second selection marker (sm II) and a polynucleotide encoding at least a second part.

[0031] The expression system may comprise a stop codon downstream of the polynucleotide encoding the heavy chain and a polynucleotide encoding an immunoglobulin membrane anchor located downstream of the stop codon.

[0032] According to the fifth aspect, a method for selecting a common host cell used in a method according to the preceding aspects is provided, the method comprising a first step of providing a plurality of host cells, the plurality of host cells comprising an expression system according to the preceding aspects; and culturing the plurality of host cells under conditions selective for a selection marker, thereby obtaining host cells expressing a target product.

[0033] In one embodiment, the selective culture medium is selected from the group of culture media, the culture medium comprises a limited concentration of folic acid; and / or comprises a concentration of 500nM or less of folic acid; and / or comprises a concentration of folic acid selected from the following concentrations: 1000nM-100pM; 100nM-1nM; 15nM-1nM; 10nM-1nM; and 10nM-2.5nM; and / or comprises a DHFR inhibitor; and / or comprises an antifolate; and / or comprises an antifolate at a concentration of 500nM or less; and / or comprises MTX selected from the following concentrations: 500nM-3nM; 100nM-10nM; 50nM-10nM; and 50nM; and / or comprises an antifolate at a concentration up to 20 times the concentration of folic acid; and / or comprises an antifolate at a concentration 10-20 times the concentration of folic acid; and / or comprises a concentration up to 15nM and an equimolar concentration up to MTX 20 times the folic acid.

[0034] In one embodiment, the host cell comprises an expression system, wherein at least a portion of the first or second portion is expressed as a fusion polypeptide comprising an immunoglobulin transmembrane anchor or a fragment thereof, wherein the fusion polypeptide is displayed on the surface of the host cell, and further comprises the following steps: contacting a plurality of host cells with a detection compound that binds to the fusion polypeptide; and selecting at least one host cell based on the presence or amount of the detection compound bound to the cell surface.

[0035] In one embodiment, the detection compound comprises the first or second target or a derivative thereof and a detection label.

[0036] In one embodiment, the fifth step of purifying the bispecific antibody comprises affinity chromatography and / or ion exchange chromatography.

[0037] In one embodiment, chromatography comprises a first step of capture; a second step of polishing; and an optional third step of polishing.

[0038] In one embodiment, the first step capture is performed using a principle selected from the group consisting of: Fc binding affinity chromatography, such as protein A or protein G; lambda light chain specific affinity chromatography, which is well known in the art and commercially available, such as LambdaFabSelect TM κ light chain specific affinity chromatography, which is well known in the art and commercially available, such as KappaSelect TM ; anti-idiotypic affinity chromatography, such as the first part or the second part; target-based affinity chromatography, such as affinity chromatography using the first target or the second target; ion exchange chromatography, which is well known in the art and commercially available, such as Capto TM Adhesion or Fractogel TM EMD SO 3; and hydrophobic interaction chromatography.

[0039] In one embodiment, the second step polishing is performed using a principle selected from the group consisting of: Fc binding affinity chromatography, such as protein A or protein G; lambda light chain specific affinity chromatography, such as LambdaFabSelect TM ; kappa light chain specific affinity chromatography, such as KappaSelect TM ; anti-idiotypic affinity chromatography, such as the first part or the second part; target-based affinity chromatography, such as affinity chromatography using the first target or the second target; ion exchange chromatography, such as Capto TM Adhesion or Fractogel TM EMD SO 3 ; hydrophobic interaction chromatography; and viral inactivation.

[0040] In one embodiment, the third step of polishing is performed using a principle selected from the group consisting of: Fc binding affinity chromatography, such as protein A or protein G; lambda light chain specific affinity chromatography, such as LambdaFabSelect TM ; kappa light chain specific affinity chromatography, such as KappaSelect TM ; anti-idiotypic affinity chromatography, such as the first part or the second part; target-based affinity chromatography, such as affinity chromatography using the first target or the second target; ion exchange chromatography, such as Capto TM Adhesion or Fractogel TM EMD SO 3 ; hydrophobic interaction chromatography; and viral inactivation.

[0041] In one embodiment, the method comprises a first step of protein A capture, such as MabSelect TM SuRe TM ; Second step lambda light chain affinity chromatography, such as LambdaFabSelect TM ; and a third step kappa light chain affinity chromatography, such as KappaSelect TM ; or first step protein A, such as MabSelect TM SuRe TM , second step kappa light chain affinity chromatography, such as KappaSelect TM , and a third step lambda light chain affinity chromatography, such as LambdaFabSelect TM ; or a first step kappa light chain affinity chromatography, such as KappaSelect TM , and a second step lambda light chain affinity chromatography, such as LambdaFabSelect TM; or a first step lambda light chain affinity chromatography, such as LambdaFabSelect TM and a second step kappa light chain affinity chromatography, such as KappaSelect TM .

[0042] In one embodiment, the cell line is selected from the group consisting of: CHO cells, non-producing hybridomas (e.g., Sp2 / 0 or NS0), human-derived cell lines (e.g., HEK or PER.C6), baby hamster kidney (BHK)-derived cells, yeast or filamentous fungi, prokaryotic bacteria (e.g., E. coli or Pseudomonas fluorescence), plant-derived cells, algae, and ciliates.

[0043] According to a sixth aspect, there is provided a pharmaceutical composition comprising the antibody according to the first aspect and a pharmaceutically acceptable carrier.

[0044] According to a seventh aspect, there is provided the antibody according to the first aspect or the pharmaceutical composition according to the sixth aspect for use as a medicament.

[0045] According to a seventh aspect, there is provided an antibody according to the first aspect or a pharmaceutical composition according to the sixth aspect for use in treating an inflammasome-associated disease.

[0046] According to an eighth aspect, an antibody according to the first aspect or a pharmaceutical composition according to the sixth aspect is provided for use in treating an inflammasome-associated disease, wherein the inflammasome-associated disease is selected from the group consisting of sickle cell disease, vasculopathy, ischemia-reperfusion injury, cardiovascular disease, peripheral arterial disease, atherosclerosis, vascular dysfunction, skeletal muscle ischemia, pulmonary sarcoidosis, fibrosis, malaria, hemodialysis-dependent chronic kidney disease and Crohn's disease.

[0047] According to a ninth aspect, there is provided a method for treating an inflammasome-associated disorder, the method comprising administering an effective amount of the antibody according to the first aspect or the pharmaceutical composition according to the sixth aspect to a subject suffering from the inflammasome-associated disorder.

[0048] The inflammasome-associated disorder may be sickle cell disease, vasculopathy, ischemia-reperfusion injury, cardiovascular disease, peripheral arterial disease, atherosclerosis, vascular dysfunction, skeletal muscle ischemia, pulmonary sarcoidosis, fibrosis, malaria, hemodialysis-dependent chronic kidney disease, or Crohn's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is a schematic overview of a carrier arrangement according to an embodiment;

[0050] Figure 2A-2EA chromatogram according to an embodiment is shown. Figure 2A is a RP-UV chromatogram of a deglycosylated intact bbmAb according to the Examples. Figure 2B is a deconvoluted mass spectrum of intact deglycosylated bbmAbl according to the example. Figure 2C is a RP-UV chromatogram showing papain-digested bbmAb fragments according to the Examples. Figure 2D is a RP-UV chromatogram showing IdeS digestion fragments of bbmAb according to the examples. Figure 2E is a RP-UV chromatogram showing the deglycosylated and DTT-reduced bbmAb fragment according to the Examples.

[0051] Figures 3A-3D An RP-UV chromatogram according to an embodiment is shown. Figure 3A is a chromatogram showing the expression purity profile of bbmAb according to the example after culture. Figure 3B The bbmAb according to the embodiment is used in LambdaFabSelect TM Chromatogram after capture. Figure 3C The bbmAb according to the example is used in MabSelect TM SuRe TM Chromatogram after TM capture. Figure 3D The bbmAb according to the embodiment is used in LambdaFabSelect TM TM capture, using Fractogel TM EMD SO 3 Chromatogram after polishing and ultrafiltration.

[0052] Figures 4A-4M is a schematic diagram of different options for bispecific mismatching. Figure 4A Schematic diagram of mAb1 knob (λ) monomer, wherein number 1 represents the variable heavy chain domain, number 2 represents the first constant heavy chain domain, number 3 represents the second constant heavy chain domain, number 4 represents the third constant heavy chain domain, number 5 represents the variable light chain domain, and number 6 represents the variable heavy chain domain. Figure 4B Schematic representation of the mAb1 knob (λ) homodimer. Figure 4C Schematic diagram of mAb2 κ monomer, wherein number 7 represents the variable heavy chain domain, number 8 represents the first constant heavy chain domain, number 9 represents the second constant heavy chain domain, number 10 represents the third constant heavy chain domain, number 11 represents the variable light chain domain, and number 12 represents the constant heavy chain domain. Figure 4D Schematic representation of the mAb2 κ homodimer. Figure 4E Schematic diagram of the mAb1 knob homodimer with one CH / LC mismatch. Figure 4FSchematic diagram of the mAbl knob homodimer with two CH / LC mismatches. Figure 4G Schematic diagram of the mAb1 knob homodimer with one CH / LC mismatch. Figure 4H Schematic diagram of the mAb2 homodimer with one CH / LC mismatch. Fig. 4I Schematic diagram of the mAb2 homodimer with two CH / LC mismatches. Figure 4J Schematic diagram of the mAb2 homodimer with one CH / LC mismatch. Figure 4K Schematic diagram of bbmAb1 with one κ(CH / LC) mismatch. Figure 4L Schematic diagram of bbmAb1 with one λ(CH / LC) mismatch. Figure 4M Schematic diagram of bbmAb1 with two CH / LC mismatches.

[0053] Figure 5 Titration curves of ECL-based affinity assays according to examples are shown.

[0054] Figure 6A-6B Two diagrams according to examples are shown.

[0055] Figure 7A-7B Two diagrams according to examples are shown.

[0056] Figures 8A-8B The mRNA expression levels according to the examples are shown.

[0057] Figures 9A-9B The mRNA expression levels according to the examples are shown.

[0058] Figures 10A-10B Two diagrams according to examples are shown.

[0059] Fig.11 is a graph showing statistical correlation according to an example. DETAILED DESCRIPTION

[0060] The present disclosure is based, inter alia, on the unexpected discovery that certain antibodies having λ (lambda) type light chains can be co-expressed with certain antibodies having κ (kappa) type light chains to form desired bbmAbs.

[0061] Without wishing to be bound by theory, the CDRs of each light chain and / or heavy chain may significantly influence which lambda type light chains may be co-expressed with certain antibodies having kappa light chains to successfully obtain the resulting bbmAb.

[0062] Antibodies with λ (lambda) type light chains (which can be co-expressed with certain antibodies with κ (kappa) type light chains to form the desired bbmAb, also referred to below as monospecific binders) can be produced by using technologies that provide the opportunity to obtain both types of antibodies of κ or λ type, such as phage display libraries, such as HuUCAL or HuCAL (MorphoSys), or transgenic mice in which the relevant human immunoglobulin sequences have been introduced into the animal genome by genetic engineering, such as OmniAb (OMT), Kymouse TM (Kymab), Trianni Mouse TM (Trianni company) or AlivaMab Mouse (Ablexis company) (reference) can produce κ or λ type antibodies. The method of producing such monospecific binders is well known in the professional field and is widely used to produce a diverse group of κ or λ monospecific binders for the target of interest. Each monospecific binder is characterized with respect to relevant biological parameters such as affinity or efficacy, and is screened for physicochemical characteristics related to judging the developability characteristics (which are also well known in the field) (e.g., Lorenz et al., American Pharmaceutical Review [American Pharmaceutical Review], August 2014). The monospecific binders showing the best characteristics are ultimately co-expressed in, for example, CHO cells, as described in more detail below. Only by co-expression testing combinations, in which a κ type antibody that binds to a first target is combined with a λ type antibody that binds to a second target, and vice versa. Detailed characterization of the final co-expression products and associated product-related impurities is intended to select a combination that produces the best spectrum, especially those that only show a small amount of promiscuous binding of one light chain (e.g., L1, light chain 1, such as λ) to the wrong heavy chain (e.g., H2, heavy chain 2). The advantage of the method is that antibody combinations in which both light chains exchange the original heavy chain binding partner (which leads to product-related H1L2-H2L1 type impurities) can be eliminated. This is advantageous because such product-related impurities are not easily depleted using prior art purification methods. The procedures for how to co-express individual antibodies and how to analyze co-expression products are outlined in more detail below.

[0063] Taking specific antibodies as an example, mAb2 mainly binds to IL-1β with light chain Vκ6, while mAb1 binds to IL-18 with light chain Vλ1.

[0064] In a preferred embodiment, KiH modification of the Fc portion of two antibodies according to Ridgway et al., (1996) was used. Other antibodies were also tested.

[0065] As shown in the specific examples below, preferred example bbmAbl are expressed using a single common cell line, thereby ensuring sufficient overall yield, purity, and product quality for biological or diagnostic purposes for clinical development and commercialization.

[0066] 1. Definition

[0067] For purposes of interpreting this specification, the following definitions will apply and, where appropriate, terms used in the singular also include the plural, and vice versa. Additional definitions are set forth throughout the detailed description.

[0068] The term "IL-18" is a synonym for IL-18 polypeptide, interleukin-18 polypeptide, IFN-γ inducing factor or interferon-γ inducing factor or INF-γ inducing factor. Unless otherwise indicated, the term "IL-18" refers to human IL-18. IL-18 is well known to those skilled in the art and can be obtained, for example, from International Company( International Corporation) under product number # B001-5. Throughout the specification, the term IL-18 interchangeably encompasses pro-IL-18 (the precursor of mature IL-18 before protease cleavage) and mature IL-18 (after protease cleavage), unless specifically stated to mean the pro- or mature form.

[0069] The term "IL-1β" or "IL-1b" is synonymous with IL-1β polypeptide and interleukin 1β polypeptide. Unless otherwise indicated, the term "IL-1β" refers to human IL-1β. IL-1β is well known to those skilled in the art and can be obtained, for example, from Sino Biological under product number #10139-HNAE-5.

[0070] The term "antibody" refers to a complete immunoglobulin or a functional fragment thereof. Naturally occurring antibodies generally comprise a tetramer, which is generally composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (generally composed of three domains (CH1, CH2, and CH3)). The heavy chain may belong to any isotype, including IgG (IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM, and IgE. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Light chains include κ (kappa) chains and λ (lambda) chains. The heavy and light chain variable regions are generally responsible for antigen recognition, while the heavy and light chain constant regions can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0071] As used herein, the term "antigen-binding portion" of an antibody (or simply "antigen portion") refers to a full-length antibody or one or more fragments of an antibody that retain the ability to specifically bind to the IL-18 or IL-1β antigen. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an 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 bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment consisting of a VH domain (Ward et al., 1989, Nature 341:544-546); and isolated complementary determining regions (CDRs).

[0072] In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, recombinant methods can be used to connect the two domains through a flexible linker that enables them to form a single protein chain, in which the VL region and the VH region pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc Natl Acad Sc. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0073] Throughout the specification, the term "isolated" means that the immunoglobulin, antibody or polynucleotide (as the case may be) is present in a physical environment different from that of its natural environment.

[0074] Throughout the specification, complementarity determining regions ("CDRs") are defined according to the Kabat definition, unless it is indicated that a CDR is 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., National Institutes of Health, Public Health Division, Bethesda, Maryland ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Josiah" 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 the classical form, according to 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). According to Josiah, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acids in VL are numbered The amino acid residues are numbered as 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Josiah, 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. According to IMGT, the CDR amino acid residues in VH are numbered as approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered as approximately 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3) (according to "Kabat" numbering). Under IMGT, the program IMGT / DomainGap can be used. Align determines the CDR regions of antibodies.

[0075] By convention, the CDR regions in the heavy chain are usually referred to as H-CDR1, H-CDR2 and H-CDR3, and the CDR regions in the light chain are usually referred to as L-CDR1, LCDR2 and L-CDR3. They are numbered sequentially in the direction from the amino terminus to the carboxyl terminus.

[0076] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers 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.

[0077] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences, or mutant forms of human germline sequences or antibodies containing consensus framework sequences derived from human framework sequence analysis, such as, for example, as described in Knappik et al., (2000), J Mol Biol [Journal of Molecular Biology]; 296: 57-86.

[0078] 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, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences.

[0079] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity that have variable regions in which both the framework and CDR regions are derived from human sequences.

[0080] The term "recombinant human antibody" as used herein includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) (the animals are transgenic or transchromosomal for human immunoglobulin genes) or hybridomas prepared therefrom; antibodies isolated from host cells transformed to express human antibodies (e.g., from transfectomas); antibodies isolated from recombinant combinatorial human antibody libraries; and antibodies prepared, expressed, produced or isolated by any other means (which involves splicing of all or part of human immunoglobulin genes). Such recombinant human antibodies have variable regions in which framework regions and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when using animals of transgenic human Ig sequences, in vivo somatic cell mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from human germline VH and VL sequences and sequences related thereto, which may not naturally exist in the human antibody germline library in vivo.

[0081] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0082] As used herein, a binding molecule that "specifically binds to IL-18" is intended to refer to a binding molecule that binds to IL-18 with a K of 100 nM or less, 10 nM or less, 1 nM or less. D Binding molecules that bind to human IL-18.

[0083] As used herein, a binding molecule that "specifically binds to IL-1β" is intended to refer to a binding molecule that binds to IL-1β with a K of 100 nM or less, 10 nM or less, 1 nM or less. D Binding molecules that bind to human IL-1β.

[0084] Binding molecules that cross-react with antigens other than IL-18 are those with a K of 100 nM or less, 10 nM or less, or 1 nM or less. D A binding molecule that cross-reacts with an antigen other than IL-1β refers to a binding molecule that cross-reacts with an antigen other than IL-1β with a K of 100 nM or less, 10 nM or less, or 1 nM or less. D A binding molecule that binds to the antigen.

[0085] By binding molecules that "do not cross-react with specific antigens" is intended binding molecules that exhibit substantially undetectable binding to these proteins in standard binding assays.

[0086] As used herein, the term "antagonist" refers to a binding molecule that inhibits signaling activity in the presence of an activating compound. For example, in the case of IL-18, an IL-18 antagonist would be a binding molecule that inhibits signaling activity in human blood cells when IL-18 is present in a human cell assay such as an IL-18-dependent interferon-γ (IFN-γ) production assay. Examples of IL-18-dependent IFN-γ production assays in human blood cells are described in more detail in the following examples.

[0087] The term bivalent, bispecific antibody or bivalent, bispecific antibodies refers to an antibody that binds to two different targets (eg, IL-18 and IL-1β).

[0088] Bispecific antibodies are "heterodimers", which means that a portion comes from a first antibody specific to a first target, and another portion comes from a second antibody specific to a second target. "Heterodimerization modification" is a modification of one or two parts of an antibody forming a heterodimeric bispecific antibody, intended to promote this formation. An example of a heterodimerization modification of the Fc domain of the two IgG1 parts intended to form a bispecific antibody is a "knob" with a large amino acid (aa) side chain (S354C, T366W) in the first heavy chain and a "hole" with a small amino acid side chain (Y349C, T366S, L368A, Y407V) introduced in the second heavy chain, and an additional disulfide bridge (Merchant et al., Nat. Biotechnol. [Natural Biotechnology], 16: 677-681 (1998), page 678, Table 1) connecting two heavy chains in the CH3 region.

[0089] As used herein, an antibody with "no agonistic activity" is intended to refer to a binding molecule that does not significantly increase target-dependent signaling activity in the absence and / or presence of the target in a cell-based assay, e.g., in the case of IL-18, does not significantly increase IL-18-dependent signaling activity in a human blood cell IFN-γ production assay in the absence and / or presence of IL-18. Such assays are described in more detail in the Examples below.

[0090] As used herein, the term " assoc ” or “K a " is intended to refer to the association rate of a specific binding molecule-antigen interaction, while the term "K" as used herein dis ” or “K d " is intended to refer to the dissociation rate of a particular binding molecule-antigen interaction. As used herein, the term "K D ” is intended to refer to the dissociation constant, which is obtained from K d With K a The ratio (K d / K a) and expressed as a molar concentration (M). The K of an antibody can be determined using methods well established in the art. D Used to determine the K value of the antibody D The method is to use surface plasmon resonance, such as system.

[0091] As used herein, the term "affinity" refers to the strength of the interaction between a binding molecule and an antigen at a single antigenic site.

[0092] As used herein, the term "high affinity" for an antibody refers to an antibody that has a KD of 1 nM or less for the target antigen.

[0093] As used herein, the term "subject" includes human and non-human animals.

[0094] The term "non-human animals" includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like.

[0095] As used herein, the term "optimized nucleotide sequence" means that the nucleotide sequence has been altered to encode an amino acid sequence using codons preferred in a producing cell or organism (typically a eukaryotic cell, such as a cell of Pichia pastoris, a Chinese hamster ovary cell (CHO), or a human cell). The optimized nucleotide sequence is engineered to completely retain the amino acid sequence originally encoded by the starting nucleotide sequence, which is also referred to as the "parent" sequence. The sequences optimized herein have been engineered to have codons preferred in CHO mammalian cells; however, optimized expression of these sequences in other eukaryotic cells is also contemplated herein.

[0096] The term "identity" refers to the similarity between at least two different sequences. The identity can be expressed as a percent identity and can be determined by a standard alignment algorithm (e.g., Basic Local Alignment Tool (BLAST) (Altshul et al., (1990) J Mol Biol [Journal of Molecular Biology]; 215: 403-410); Needleman et al., (1970) J Mol Biol [Journal of Molecular Biology]; 48: 444-453 algorithm; or Meyers et al., (1988) Comput Appl Biosci [Computational Applications of Bioscience]; 4: 11-17 algorithm). One set of parameters can be a Blosum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1989) CABIOS; 4(1): 1-17, which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Percent identity is typically calculated by comparing sequences of similar length.

[0097] The term "immune response" refers to the action of lymphocytes, antigen presenting cells, phagocytes, granulocytes and soluble macromolecules (including antibodies, cytokines and complement) produced, for example, by the above cells or by the liver, which results in the selective damage, destruction or elimination from the human body of invading pathogens, cells or tissues infected by pathogens, cancer cells (or in the case of autoimmunity or pathological inflammation, normal human cells or tissues).

[0098] "Signal transduction pathway" or "signaling activity" refers to a biochemical cause-effect relationship that results in the transmission of a signal from one part of a cell to another part of the cell, usually through protein-protein interactions (such as the binding of a growth factor to a receptor). Typically, the transmission involves specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in a series of reactions that result in signal transduction. The penultimate process typically includes nuclear events that result in changes in gene expression.

[0099] Throughout the specification, the term "neutralize" and grammatical variations thereof refer to the total or partial reduction of a biological activity of a target in the presence of a binding protein or antibody, as the case may be.

[0100] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known natural nucleotide analogs that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as sequences explicitly specified. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0101] The nucleotides in a "polynucleotide" or "nucleic acid" may contain modifications, including base modifications, such as bromouridine and inosine derivatives; ribose modifications, such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, and phosphoramidate.

[0102] The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, phage or virus) that is suitable for transforming or transfecting a host cell and that contains a nucleic acid sequence that directs and / or controls (combines with a host cell) the expression of one or more heterologous coding regions operably linked thereto.

[0103] The term "co-expression" refers to the expression of different polypeptides together in a single host cell common to all polypeptides. Co-expression of bispecific antibodies refers to the expression of different parts that form a functional bispecific antibody in a single common host cell. Co-expression can be achieved by incorporating several expression vectors into the expression host cell, such as one vector for each half of the bispecific antibody, or by incorporating one expression vector encoding all parts of the bispecific antibody.

[0104] The term "mismatch" refers to the fact that different parts of an expected protein complex (e.g., a bispecific antibody) are not complexed as expected, meaning that the protein complex does not look or behave as expected. An example of mismatch in the context of a bispecific antibody is shown in FIG4 .

[0105] "Conservative variants" of sequences encoding binding molecules, antibodies or fragments thereof refer to sequences comprising conservative amino acid modifications. "Conservative amino acid modifications" are intended to refer to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Modifications can be introduced into the binding proteins of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions can also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. Non-naturally occurring amino acids include, but are not limited to, peptide mimetics (reverse or inverted forms of amino acid moieties).

[0106] The term "epitope" is a part of an antigen recognized by the immune system, such as an antibody or a fragment thereof. In this specification, the term "epitope" is used interchangeably for conformational epitopes and linear epitopes. Conformational epitopes consist of discontinuous parts of the amino acid sequence of the antigen, while linear epitopes are formed by a continuous sequence of amino acids of the antigen.

[0107] The terms "treat", "treating", "treatment", "prevent", "preventing", and "prevention" include therapeutic treatments, prophylactic treatments, and uses in which the risk of a subject developing a disorder or other risk factors is reduced. Treatment does not require a complete cure of the disorder, and includes alleviation of symptoms or potential risk factors. As used herein, a human antibody or fragment thereof comprises a heavy or light chain variable region or a full-length heavy or light chain that is a "product of" or "derived from" a particular germline sequence, if the variable region or full-length chain of the antibody is obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with an antigen of interest or screening human immunoglobulin gene libraries displayed on phages with an antigen of interest. Human antibodies or fragments thereof that are "the product of" or "derived from" human germline immunoglobulin sequences 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 to the sequence of the human antibody (i.e., highest % identity). Human antibodies that are "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence due to, for example, naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, the amino acid sequence of the selected human antibody is typically at least 90% identical to the amino acid sequence encoded by a human germline immunoglobulin gene, and contains amino acid residues that identify the human antibody as being a human antibody when compared to germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In some cases, the amino acid sequence of a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a specific 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 some cases, a human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0108] Human antibodies can be produced by many methods known to those skilled in the art. Human antibodies can be prepared by the hybridoma method using human myeloma or mouse-human heteromyeloma cell lines (Kozbor, J Immunol [Immunology]; (1984) 133: 3001; Brodeur, Monoclonal Isolated Antibody Production Techniques and Applications [Monoclonal Isolated Antibody Production Techniques and Applications], pp. 51-63, Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both of which utilize human variable region repertoires (Winter G; (1994) Annu Rev Immunol [Annual Review of Immunology] 12: 433-455, Green LL, (1999) J Immunol Methods [Immunology Magazine] 231: 11-23).

[0109] Several transgenic mouse strains are now available in which the mouse immunoglobulin loci have been replaced by human immunoglobulin gene segments (Tomizuka K, (2000) Proc Natl Acad Sci, 97:722-727; Fishwild DM (1996) Nature Biotechnol 14:845-851; Mendez MJ, (1997) Nature Genetics 15:146-156). Upon antigen challenge, such mice are capable of producing a human antibody repertoire from which the antibody of interest can be selected. Of particular note is the Trimera TM system (Eren R et al, (1988) Immunology 93:154-161) (in which human lymphocytes are transplanted into irradiated mice) the Selective Lymphocyte Isolation Antibody System (SLAM, Babcook et al, Proc Natl Acad Sci (1996) 93:7843-7848), in which human (or other species) lymphocytes are efficiently isolated in vitro through a large number of pools of antibody-generating procedures, followed by deconvolution, limiting dilution and selection procedures, and Xenomouse TM (Abgenix). Morphodoma is available from Morphotek TM Technology gets another way.

[0110] Phage display technology can be used to produce human antibodies and fragments thereof (McCafferty; (1990) Nature, 348:552-553 and Griffiths AD et al. (1994) EMBO 13:3245-3260). According to this technology, isolated antibody variable domain genes are cloned in-frame into the major or minor coat of a protein gene of a filamentous phage (e.g., M13 or fd) and displayed (usually with the aid of a helper phage) on the surface of the phage particle as functionally isolated antibody fragments. Selection based on the functional properties of the isolated antibodies results in the selection of genes encoding isolated antibodies that exhibit these properties. Phage display technology can be used to select antigen-specific antibodies from libraries prepared from human B cells, which are taken from individuals with a disease or disorder, or alternatively from unimmunized human donors (Marks; J Mol Bio (1991) 222:581-591). When fully human isolated antibodies containing the Fc domain are desired, the phage-displayed derived fragments must be recloned into mammalian expression vectors containing the desired constant regions and stable expressing cell lines established.

[0111] The technique of affinity maturation (Marks; Biotechnol [Biotechnology] (1992) 10: 779-783) can be used to improve binding affinity, wherein the affinity of human primary isolated antibodies is improved by sequentially replacing the H and L chain variable regions with naturally occurring variants and selecting on the basis of improved binding affinity. Variants of this technique are also currently available, such as "epitope imprinting" (WO 93 / 06213; Waterhouse; Nucl Acids Res [Nucleic Acids Research] (1993) 21: 2265-2266).

[0112] When used in the context of purified bispecific antibodies, the term "pure" relates to the purity and identity of different bispecific antibody combinations and constructs after co-expression in selected cells under conditions in which the cells express the bispecific antibodies and after protein A purification using an intact UPLC-MS mass screening method. Pure or purity refers to the relative quantification of the heterodimeric and homodimeric bbmAbs formed. Using the method of the present invention, correctly formed heterodimeric bbmAb1 and bbmAb2 can be observed with a relative purity of more than 85% based on the intact mass signal intensity.

[0113] 2. IL-18 Antibody

[0114] Particularly preferred IL-18 antibodies or antigen-binding fragments thereof for use in the disclosed methods are human antibodies.

[0115] For ease of reference, the hypervariable regions of specific IL-18 antibodies (referred to as mAb1) based on the Kabat definition and the Chothia definition, as well as the V L and V H domains and the amino acid sequences of the complete heavy and light chains.

[0116] 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 listed in SEQ ID NO: 18. The DNA encoding the VH of mAb1 is listed in SEQ ID NO:8.

[0117]

[0118] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain (V H ), the CDR1 has the amino acid sequence SEQ ID NO: 1, the CDR2 has the amino acid sequence SEQ ID NO: 2, and the CDR3 has the amino acid sequence SEQ ID NO: 3. In one embodiment, the IL-18 antibody or antigen-binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain (V HLD) comprising hypervariable regions CDR1, CDR2 and CDR3. H ), the CDR1 has the amino acid sequence of SEQ ID NO:4, the CDR2 has the amino acid sequence of SEQ ID NO:5, and the CDR3 has the amino acid sequence of SEQ ID NO:6.

[0119] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof comprises at least one immunoglobulin light chain variable domain (V LLD) comprising hypervariable regions CDR1, CDR2 and CDR3. L ), the CDR1 has the amino acid sequence of SEQ ID NO: 11, the CDR2 has the amino acid sequence of SEQ ID NO: 12 and the CDR3 has the amino acid sequence of SEQ ID NO: 13. In one embodiment, the IL-18 antibody or antigen-binding fragment thereof comprises at least one immunoglobulin light chain variable domain (V LLD) comprising hypervariable regions CDR1, CDR2 and CDR3. L ), the CDR1 has the amino acid sequence of SEQ ID NO:14, the CDR2 has the amino acid sequence of SEQ ID NO:15 and the CDR3 has the amino acid sequence of SEQ ID NO:16.

[0120] 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 The structural domain comprises (for example, in order): i) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence of SEQ ID NO: 1, CDR2 has an amino acid sequence of SEQ ID NO: 2, and CDR3 has an amino acid sequence of SEQ ID NO: 3; or ii) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence of SEQ ID NO: 4, CDR2 has an amino acid sequence of SEQ ID NO: 5, and CDR3 has an amino acid sequence of SEQ ID NO: 6; and b) the immunoglobulin V L The domain comprises (for example in order): i) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 11, the CDR2 has the amino acid sequence of SEQ ID NO: 12, and the CDR3 has the amino acid sequence of SEQ ID NO: 13 or ii) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 14, the CDR2 has the amino acid sequence of SEQ ID NO: 15, and the CDR3 has the amino acid sequence of SEQ ID NO: 16.

[0121] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable domain (V) comprising the amino acid sequence listed in SEQ ID NO: 7; H ); b) an immunoglobulin light chain variable domain (V L ); c) an immunoglobulin V comprising the amino acid sequence listed in SEQ ID NO: 7 H domain and an immunoglobulin V comprising the amino acid sequence set forth in SEQ ID NO: 17 L domain; d) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 H e) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 Lf) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 H g) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 L h) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 H domain and an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 L i) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 H domain and an immunoglobulin V comprising the hypervariable region set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16 L domain; j) a light chain comprising SEQ ID NO: 19; k) a heavy chain comprising SEQ ID NO: 9; or l) a light chain comprising SEQ ID NO: 19 and a heavy chain comprising SEQ ID NO: 9.

[0122] In some embodiments, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb1) comprises three CDRs of SEQ ID NO: 7. In other embodiments, the IL-18 antibody or antigen-binding fragment thereof comprises three CDRs of SEQ ID NO: 17. In other embodiments, the IL-18 antibody or antigen-binding fragment thereof comprises three CDRs of SEQ ID NO: 7 and three CDRs of SEQ ID NO: 17. In some embodiments, the IL-18 antibody or antigen-binding fragment thereof comprises three CDRs of SEQ ID NO: 9. In other embodiments, the IL-18 antibody or antigen-binding fragment thereof comprises three CDRs of SEQ ID NO: 19. In other embodiments, the IL-18 antibody or antigen-binding fragment thereof comprises three CDRs of SEQ ID NO: 9 and three CDRs of SEQ ID NO: 19.

[0123] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb1) is selected from a human IL-18 antibody, which comprises at least: a) an immunoglobulin heavy chain or a fragment thereof, comprising a variable domain and a constant part of a human heavy chain or a fragment thereof, wherein the variable domain sequentially comprises hypervariable regions CDR1, CDR2 and CDR3; wherein the CDR1 has an amino acid sequence of SEQ ID NO: 1, the CDR2 has an amino acid sequence of SEQ ID NO: 2, and the CDR3 has an amino acid sequence of SEQ ID NO: 3; and b) an immunoglobulin light chain or a fragment thereof, wherein the immunoglobulin light chain or a fragment thereof comprises a variable domain and a constant part of a human light chain or a fragment thereof, wherein the variable domain sequentially comprises hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has an amino acid sequence of SEQ ID NO: 11, the CDR2 has an amino acid sequence of SEQ ID NO: 12, and the CDR3 has an amino acid sequence of SEQ ID NO: 13.

[0124] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof (e.g., mAb1) is selected from a human IL-18 antibody, which comprises at least: a) an immunoglobulin heavy chain or a fragment thereof, comprising a variable domain and a constant part of a human heavy chain or a fragment thereof, wherein the variable domain comprises, in sequence, hypervariable regions CDR1, CDR2, and CDR3; wherein CDR1 has an amino acid sequence of SEQ ID NO: 4, wherein CDR2 has an amino acid sequence of SEQ ID NO: 5, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 6; and b) an immunoglobulin light chain or a fragment thereof, wherein the immunoglobulin light chain or a fragment thereof comprises a variable domain and a constant part of a human light chain or a fragment thereof, wherein the variable domain comprises, in sequence, hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has an amino acid sequence of SEQ ID NO: 14, wherein CDR2 has an amino acid sequence of SEQ ID NO: 15, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 16.

[0125] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof is selected from a single-chain antibody or antigen-binding fragment thereof comprising an antigen-binding site, wherein the antigen-binding site comprises: a) a first domain comprising hypervariable regions CDR1, CDR2 and CDR3 in sequence, wherein CDR1 has an amino acid sequence of SEQ ID NO: 1, wherein CDR2 has an amino acid sequence of SEQ ID NO: 2, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 3; and b) a second domain comprising hypervariable regions CDR1, CDR2 and CDR3 in sequence, wherein CDR1 has an amino acid sequence of SEQ ID NO: 11, wherein CDR2 has an amino acid sequence of SEQ ID NO: 12, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 13; and c) a peptide linker, which is bound to the N-terminus of the first domain and the C-terminus of the second domain or to the C-terminus of the first domain and the N-terminus of the second domain.

[0126] 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 comprising an antigen-binding site, wherein the antigen-binding site comprises: a) a first domain comprising hypervariable regions CDR1, CDR2 and CDR3 in sequence, wherein CDR1 has an amino acid sequence of SEQ ID NO: 4, wherein CDR2 has an amino acid sequence of SEQ ID NO: 5, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 6; and b) a second domain comprising hypervariable regions CDR1, CDR2 and CDR3 in sequence, wherein CDR1 has an amino acid sequence of SEQ ID NO: 14, wherein CDR2 has an amino acid sequence of SEQ ID NO: 15, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 16; and c) a peptide linker, which is bound to the N-terminus of the first domain and the C-terminus of the second domain or to the C-terminus of the first domain and the N-terminus of the second domain.

[0127] V of the IL-18 antibody or antigen-binding fragment thereof used in the disclosed method H or V L The domain may have the same V as listed in SEQ ID NO: 7 and 17. H or V L The domains are essentially the same as V H and / or V LDomain. The human IL-18 antibody disclosed herein may comprise a heavy chain substantially identical to the heavy chain shown in SEQ ID NO: 9 and / or a light chain substantially identical to the light chain shown in SEQ ID NO: 19. The human IL-18 antibody disclosed herein may comprise: a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 19. The human IL-18 antibody disclosed herein may comprise: a) a heavy chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 7 and a constant portion of a human heavy chain; and b) a light chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 17 and a constant portion of a human light chain.

[0128] Other preferred IL-18 antagonists (eg, antibodies) for use in the disclosed methods, kits, and regimens are those listed in U.S. Pat. No. 9,376,489, which is incorporated herein by reference in its entirety.

[0129] 3. IL-1β Antibody

[0130] Particularly preferred IL-1 β antibodies or antigen-binding fragments thereof for use in the disclosed methods are human antibodies.

[0131] For ease of reference, the hypervariable regions of specific IL-1β antibodies (referred to as mAb2) based on the Kabat definition and the Chothia definition, as well as the V L and V H domains and the amino acid sequences of the complete heavy and light chains.

[0132] Table 2. Amino acid sequences of hypervariable regions (CDRs), variable domains (VH and VL), and full chains of mAb2. The DNA encoding the VL of mAb2 is listed in SEQ ID NO: 38. The DNA encoding the VH of mAb2 is listed in SEQ ID NO: 27.

[0133]

[0134] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain (V H), the CDR1 has the amino acid sequence SEQ ID NO: 21, the CDR2 has the amino acid sequence SEQ ID NO: 22, and the CDR3 has the amino acid sequence SEQ ID NO: 23. In one embodiment, the IL-1β antibody or antigen-binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain (V HLD) comprising hypervariable regions CDR1, CDR2 and CDR3. H ), the CDR1 has the amino acid sequence of SEQ ID NO:24, the CDR2 has the amino acid sequence of SEQ ID NO:25, and the CDR3 has the amino acid sequence of SEQ ID NO:26.

[0135] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof comprises at least one immunoglobulin light chain variable domain (V L ), the CDR1 has the amino acid sequence SEQ ID NO: 31, the CDR2 has the amino acid sequence SEQ ID NO: 32 and the CDR3 has the amino acid sequence SEQ ID NO: 33. In one embodiment, the IL-1β antibody or antigen-binding fragment thereof comprises at least one immunoglobulin light chain variable domain (V LLD) comprising hypervariable regions CDR1, CDR2 and CDR3. L ), the CDR1 has the amino acid sequence of SEQ ID NO:34, the CDR2 has the amino acid sequence of SEQ ID NO:35 and the CDR3 has the amino acid sequence of SEQ ID NO:36.

[0136] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof comprises at least one immunoglobulin V H domain and at least one immunoglobulin V L domain, wherein: a) the immunoglobulin V H The structural domain comprises (for example, in order): i) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence of SEQ ID NO: 21, CDR2 has an amino acid sequence of SEQ ID NO: 22, and CDR3 has an amino acid sequence of SEQ ID NO: 23; or ii) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence of SEQ ID NO: 24, CDR2 has an amino acid sequence of SEQ ID NO: 25, and CDR3 has an amino acid sequence of SEQ ID NO: 26; and b) the immunoglobulin V LThe domain comprises (for example, in order): i) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO:31, CDR2 has the amino acid sequence of SEQ ID NO:32, and CDR3 has the amino acid sequence of SEQ ID NO:33 or ii) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO:34, CDR2 has the amino acid sequence of SEQ ID NO:35, and CDR3 has the amino acid sequence of SEQ ID NO:36.

[0137] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable domain (V) comprising the amino acid sequence listed in SEQ ID NO: 27; H ); b) an immunoglobulin light chain variable domain (V L ); c) an immunoglobulin V comprising the amino acid sequence listed in SEQ ID NO: 27 H domain and an immunoglobulin V comprising the amino acid sequence set forth in SEQ ID NO:37 L domain; d) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 H e) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33 L f) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 H g) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 L h) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 H domain and an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33 L i) an immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 Hdomain and an immunoglobulin V comprising the hypervariable region set forth in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36 L domain; j) a light chain comprising SEQ ID NO: 37; k) a heavy chain comprising SEQ ID NO: 29; or l) a light chain comprising SEQ ID NO: 39 and a heavy chain comprising SEQ ID NO: 29.

[0138] In some embodiments, the IL-1 β antibody or antigen-binding fragment thereof (e.g., mAb2) comprises three CDRs of SEQ ID NO: 37. In other embodiments, the IL-1 β antibody or antigen-binding fragment thereof comprises three CDRs of SEQ ID NO: 27. In other embodiments, the IL-1 β antibody or antigen-binding fragment thereof comprises three CDRs of SEQ ID NO: 37 and three CDRs of SEQ ID NO: 27. In some embodiments, the IL-1 β antibody or antigen-binding fragment thereof comprises three CDRs of SEQ ID NO: 39. In other embodiments, the IL-1 β antibody or antigen-binding fragment thereof comprises three CDRs of SEQ ID NO: 29. In other embodiments, the IL-1 β antibody or antigen-binding fragment thereof comprises three CDRs of SEQ ID NO: 39 and three CDRs of SEQ ID NO: 29.

[0139] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof (e.g., mAb2) is selected from a human IL-1β antibody, which comprises at least: a) an immunoglobulin heavy chain or a fragment thereof, comprising a variable domain and a constant part of a human heavy chain or a fragment thereof, wherein the variable domain sequentially comprises hypervariable regions CDR1, CDR2 and CDR3; the CDR1 has an amino acid sequence of SEQ ID NO: 21, the CDR2 has an amino acid sequence of SEQ ID NO: 22, and the CDR3 has an amino acid sequence of SEQ ID NO: 23; and b) an immunoglobulin light chain or a fragment thereof, wherein the immunoglobulin light chain or a fragment thereof comprises a variable domain and a constant part of a human light chain or a fragment thereof, wherein the variable domain sequentially comprises hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has an amino acid sequence of SEQ ID NO: 31, the CDR2 has an amino acid sequence of SEQ ID NO: 32, and the CDR3 has an amino acid sequence of SEQ ID NO: 33.

[0140] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof (e.g., mAb2) is selected from a human IL-1β antibody, which comprises at least: a) an immunoglobulin heavy chain or a fragment thereof, comprising a variable domain and a constant part of a human heavy chain or a fragment thereof, wherein the variable domain sequentially comprises hypervariable regions CDR1, CDR2 and CDR3; the CDR1 has an amino acid sequence of SEQ ID NO: 24, the CDR2 has an amino acid sequence of SEQ ID NO: 25, and the CDR3 has an amino acid sequence of SEQ ID NO: 26; and b) an immunoglobulin light chain or a fragment thereof, wherein the immunoglobulin light chain or a fragment thereof comprises a variable domain and a constant part of a human light chain or a fragment thereof, wherein the variable domain sequentially comprises hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has an amino acid sequence of SEQ ID NO: 34, the CDR2 has an amino acid sequence of SEQ ID NO: 35, and the CDR3 has an amino acid sequence of SEQ ID NO: 36.

[0141] In one embodiment, the IL-1β antibody or its antigen-binding fragment is selected from a single-chain antibody or its antigen-binding fragment comprising an antigen-binding site, wherein the antigen-binding site comprises: a) a first domain comprising hypervariable regions CDR1, CDR2 and CDR3 in sequence, wherein CDR1 has an amino acid sequence of SEQ ID NO: 21, wherein CDR2 has an amino acid sequence of SEQ ID NO: 22, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 23; and b) a second domain comprising hypervariable regions CDR1, CDR2 and CDR3 in sequence, wherein CDR1 has an amino acid sequence of SEQ ID NO: 31, wherein CDR2 has an amino acid sequence of SEQ ID NO: 32, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 33; and c) a peptide linker, which is bound to the N-terminus of the first domain and the C-terminus of the second domain or to the C-terminus of the first domain and the N-terminus of the second domain.

[0142] In one embodiment, the IL-1β antibody or antigen-binding fragment thereof (e.g., mAb2) is selected from a single-chain antibody or antigen-binding fragment thereof comprising an antigen-binding site, wherein the antigen-binding site comprises: a) a first domain comprising hypervariable regions CDR1, CDR2 and CDR3 in sequence, wherein CDR1 has an amino acid sequence of SEQ ID NO: 24, wherein CDR2 has an amino acid sequence of SEQ ID NO: 25, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 26; and b) a second domain comprising hypervariable regions CDR1, CDR2 and CDR3 in sequence, wherein CDR1 has an amino acid sequence of SEQ ID NO: 34, wherein CDR2 has an amino acid sequence of SEQ ID NO: 35, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 36; and c) a peptide linker, which is bound to the N-terminus of the first domain and the C-terminus of the second domain or to the C-terminus of the first domain and the N-terminus of the second domain.

[0143] V of the IL-1β antibody or antigen-binding fragment thereof used in the disclosed method H or V L The domain may have the same V as set forth in SEQ ID NOs: 27 and 37. H or V L The domains are essentially the same as V H and / or V L Domain. The human IL-1β antibody disclosed herein may comprise a heavy chain substantially identical to the heavy chain shown in SEQ ID NO: 29 and / or a light chain substantially identical to the light chain shown in SEQ ID NO: 39. The human IL-1β antibody disclosed herein may comprise: a heavy chain comprising SEQ ID NO: 29 and a light chain comprising SEQ ID NO: 39. The human IL-1β antibody disclosed herein may comprise: a) a heavy chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 27 and a constant portion of a human heavy chain; and b) a light chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 37 and a constant portion of a human light chain.

[0144] Other preferred IL-1 β antagonists (eg, antibodies) for use in the disclosed methods, kits, and regimens are those listed in: US Pat. Nos.: 7,446,175 or 7,993,878 or 8,273,350, which are incorporated herein by reference in their entirety.

[0145] 4. Fc modification

[0146] In addition to modifications made in the framework or CDR regions or as an alternative to modifications made in the framework or CDR regions, the antibodies of the present invention can be engineered to include modifications in the Fc region, typically to change one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular toxicity. In addition, the antibodies of the present invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to change its glycosylation, thereby again changing one or more functional properties of the antibody. Each of these embodiments is described in more detail below. The residue numbering in the Fc region is the numbering of the EU numbering scheme of Edelman et al., PNAS, May, 1969, 63 (1): 78-85.

[0147] In one embodiment, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is changed, for example, increased or decreased. The method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is changed, for example, to facilitate the assembly of light and heavy chains or to increase or decrease the stability of the antibody.

[0148] In another embodiment, the Fc hinge region of the antibody is mutated to shorten 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 so that the antibody has impaired staphylococcal protein A (SpA) binding relative to native Fc hinge domain SpA binding. This method is further described in detail in U.S. Pat. No. 6,165,745 to Ward et al.

[0149] In another embodiment, the antibody is modified to increase its biological half-life. Various methods can be used. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F as described by Ward in U.S. Patent No. 6,277,375. Alternatively, to increase the biological half-life, the antibody can be changed in the CH1 or CL region to contain a salvage receptor binding epitope of two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al.

[0150] In yet other embodiments, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues so that the antibody has a changed affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand that changes affinity can be, for example, an Fc receptor or the C1 component of complement. This method is further described in detail in U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al.

[0151] In another embodiment, one or more amino acids selected from the amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 to Idusogie et al.

[0152] 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 further described in PCT Publication WO 94 / 29351 by Bodmer et al.

[0153] In another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors by modifying one or more amino acids. This method is further described by Presta in PCT Publication WO 00 / 42072. In addition, the binding sites for FcγR1, FcγRII, FcγRIII and FcRn on human IgG1 have been located and variants with improved binding have been described (see Shields, RL et al., 2001 J. Biol. Chen. [Journal of Biological Chemistry] 276: 6591-6604).

[0154] In certain embodiments, an Fc domain of the IgG1 isotype is used. In some specific embodiments, a mutant variant of the IgG1 Fc fragment is used, such as a silent IgG1 Fc, which can reduce or eliminate the ability of the fusion polypeptide to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or bind to Fcγ receptors. An example of an IgG1 isotype silent mutant in which the leucine residues at amino acid positions 234 and 235 are replaced by alanine residues as described in Hezareh et al., J. Virol (2001); 75(24): 12161-8.

[0155] In certain embodiments, the Fc domain is a mutant that prevents glycosylation at position 297 of the Fc domain. For example, the Fc domain contains an amino acid substitution of an asparagine residue at position 297. An example of such an amino acid substitution is replacement of N297 with glycine or alanine.

[0156] Silent effector function can be obtained by mutations in the antibody Fc region and have been described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. [Current Biotechnology Opinion] vol. 20(6): 685-691); and D265A (Baudino et al., 2008, J. Immunol. [Journal of Immunology] 181: 6664-69; Strohl, W., supra); and DAPA (D265A and P329A) (Shields RL., J Biol Chem. [Journal of Biological Chemistry] 2001; 276(9): 6591-604; U.S. Patent Publication No. US2015 / 0320880). Examples of silent Fc IgG1 antibodies include the LALA mutant, which includes L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of silent IgG1 antibody comprises D265A mutation. Another example of silent IgG1 antibody is said DAPA mutant, which comprises D265A and P329A mutations in IgG1Fc amino acid sequence. Another silent IgG1 antibody comprises N297A mutation, which results in aglycosylated / non-glycosylated antibody. Other Fc mutations for providing silent effector functions are described in PCT Publication No. WO2014 / 145806 (e.g., in Fig. 7 of WO 2014 / 145806), which are incorporated herein by reference in their entirety. An example of silent IgG1 antibody from WO 2014 / 145806 comprises E233P, L234V, L235A and S267K mutations and the disappearance of G236 (G236del). Another example of a silent IgG1 antibody from WO 2014 / 145806 comprises E233P, L234V and L235A mutations, and a deletion of G236 (G236del).Another example of a silent IgG1 antibody from WO 2014 / 145806 comprises a S267K mutation.

[0157] In yet another embodiment, the glycosylation of the modified antibody. For example, an antibody without glycosylation can be prepared (i.e., the antibody lacks glycosylation). Glycosylation can be changed, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be accomplished in the following ways: for example, by changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, which results in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the site. This aglycosylation can increase the affinity of the antibody for an antigen. This method is described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

[0158] Additionally or alternatively, antibodies with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells in which recombinant antibodies of the present invention are expressed, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 of Hang et al. describes a cell line with a functionally disrupted FUT8 gene that encodes a fucosyltransferase, such that antibodies expressed in such a cell line exhibit low fucosylation. Therefore, in one embodiment, the antibodies of the present invention are produced by recombinant expression in a cell line exhibiting a fucosylation pattern (e.g., a mammalian cell line defective in the expression of a FUT8 gene encoding a fucosyltransferase). Presta describes in PCT Publication WO03 / 035835 a variant CHO cell line Lec13 cells that have reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields, RL et al., 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO99 / 54342 by Umana et al. describes a cell line engineered to express a glycoprotein modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac 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 may be produced in yeast or filamentous fungi engineered for a mammalian-like glycosylation pattern and capable of producing antibodies lacking fucose as a glycosylation pattern (see, e.g., EP1297172B1).

[0159] Another modification of the present invention's contemplated antibodies herein is pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. In order to pegylate an antibody, the antibody or its fragment is usually reacted with polyethylene glycol (PEG) (e.g., reactive esters or aldehyde derivatives of PEG) under conditions where one or more PEG groups are attached to the antibody or antibody fragment. Pegylation can be carried out using reactive PEG molecules (or similar reactive water-soluble polymers) by acylation or alkylation. As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to antibodies of the present invention. See, for example, EP 0 154 316 to Nishimura et al. and EP 0 401 384 to Ishikawa et al.

[0160] Another modification of the antibody contemplated by the present invention is a conjugate or protein fusion of at least the antigen binding region of the antibody of the present invention with a serum protein (e.g., human serum albumin or a fragment thereof) to increase the half-life of the resulting molecule. Such a method is described, for example, in Ballance et al. EP0322094.

[0161] Another modification of the antibody contemplated by the present invention is one or more modifications to increase the formation of heterodimeric bispecific antibodies. A variety of methods available in the art can be used to enhance the dimerization of two heavy chain domains of bispecific antibodies, such as bbmAbs, as disclosed in: EP 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1, and PCT Publication No. WO 2009 / 089004A1, the contents of which are incorporated herein in their entirety.

[0162] For example, the use of knob-in-hole structures to generate bispecific antibodies is disclosed in PCT Publication No. WO 1996 / 027011, Ridgway et al., (1996), and Merchant et al. (1998).

[0163] (1) Knob-and-Mortar (KIH)

[0164] The multispecific molecules of the present invention (e.g., multispecific antibodies or antibody-like molecules) may comprise one or more (e.g., multiple) mutations to one or more constant domains (e.g., to a CH3 domain). In one example, the multispecific molecules of the present invention comprise two polypeptides, each polypeptide comprising a heavy chain constant domain of an antibody, e.g., a CH2 or CH3 domain. In an example, two heavy chain constant domains, e.g., a CH2 or CH3 domain of a multispecific molecule, comprise one or more mutations that allow heterodimer association between the two chains. In one aspect, one or more mutations are arranged on the CH2 domains of two heavy chains of a multispecific, e.g., bispecific antibody or antibody-like molecule. In one aspect, one or more mutations are arranged on the CH3 domains of at least two polypeptides of a multispecific molecule. In one aspect, one or more mutations to a first polypeptide of a multispecific molecule comprising a heavy chain constant domain generate a "knob" and one or more mutations to a second polypeptide of a multispecific molecule comprising a heavy chain constant domain generate a "hole" such that heterodimerization of the polypeptides of the multispecific molecule comprising a heavy chain constant domain generates a "knob" to engage with the "hole" interface (e.g., interact, e.g., the CH2 domain of the first polypeptide interacts with the CH2 domain of the second polypeptide, or the CH3 domain of the first polypeptide interacts with the CH3 domain of the second polypeptide). As the term is used herein, a "knob" refers to at least one amino acid side chain that protrudes from the interface of the first polypeptide of the multispecific molecule comprising a heavy chain constant domain and can thus be positioned in a complementary "hole" in the interface with the second polypeptide of the multispecific molecule comprising a heavy chain constant domain to stabilize the heteromultimer and thereby favor heteromultimer formation (e.g., relative to homomultimer formation). The knob can be present in the original interface or can be introduced synthetically (e.g., by altering a nucleic acid encoding the interface). Preferred input residues for forming the knob are generally naturally occurring amino acid residues and may preferably be selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Most preferred are tryptophan and tyrosine. In a preferred embodiment, the initial residue for forming the protrusion has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.

[0165] "Hole" refers to at least one amino acid side chain that is recessed into the interface of a second polypeptide of a multispecific molecule comprising a heavy chain constant domain and thereby accommodates a corresponding knob on an adjacent interfacing surface of a first polypeptide of a multispecific molecule comprising a heavy chain constant domain. The hole may be present in the original interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). Preferred input residues for forming the hole are typically naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T) and valine (V). Most preferred are serine, alanine or threonine. In a preferred embodiment, the initial residue for forming the hole has a large side chain bulk, such as tyrosine, arginine, phenylalanine or tryptophan.

[0166] In a preferred embodiment, the first CH3 domain is mutated at residue 366, 405 or 407 (according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85) to generate a "knob" or "hole" (as described above), and the second CH3 domain that heterodimerizes with the first CH3 domain is mutated at the following positions: according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85, residue 407 (if residue 366 is mutated in the first CH3 domain), residue 349 (if residue 405 is mutated in the first CH3 domain), or residue 366 (if residue 407 is mutated in the first CH3 domain) to generate a "hole" or "knob" that is complementary to the "knob" or "hole" of the first CH3 domain.

[0167] In another preferred embodiment, the first CH3 domain is mutated at residue 366 (according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85) to generate a "knob" or "hole" (as described above), and the second CH3 domain that heterodimerizes with the first CH3 domain is mutated at residues 366, 368 and / or 407 (according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85) to generate a "hole" or "knob" that is complementary to the "knob" or "hole" of the first CH3 domain. In one embodiment, the mutation to the first CH3 domain introduces a tyrosine (Y) residue at position 366. In an embodiment, the mutation to the first CH3 is T366Y. In one embodiment, the mutation to the first CH3 domain introduces a tryptophan (W) residue at position 366. In an embodiment, the mutation to the first CH3 is T366W. In an embodiment, the mutation of the second CH3 domain that heterodimerizes with the first CH3 domain (mutated at position 366 (e.g., having a tyrosine (Y) or tryptophan (W) introduced at position 366, e.g., comprising the mutation T366Y or T366W)) comprises a mutation at position 366, a mutation at position 368, and a mutation at position 407, the positions being according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85. In an embodiment, the mutation at position 366 introduces a serine (S) residue, the mutation at position 368 introduces an alanine (A), and the mutation at position 407 introduces a valine (V). In an embodiment, the mutation comprises T366S, L368A, and Y407V. In one embodiment, the first CH3 domain of the multispecific molecule comprises the mutation T366Y, and the second CH3 domain that heterodimerizes with the first CH3 domain comprises the mutations T366S, L368A, and Y407V, or vice versa. In one embodiment, the first CH3 domain of the multispecific molecule comprises the mutation T366W, and the second CH3 domain that heterodimerizes with the first CH3 domain comprises the mutations T366S, L368A, and Y407V, or vice versa.

[0168] Additional spatial or "offset" (e.g., knob-in-hole) mutations are described in PCT Publication No. WO 2014 / 145806 (e.g., Figures 3, 4, and 12 of WO 2014 / 145806), PCT Publication No. WO 2014 / 110601, and PCT Publication Nos. WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, and WO 2016 / 182751 (the contents of which are incorporated herein in their entirety). An example of a KIH variant comprises a first constant chain comprising L368D and K370S mutations, paired with a second constant chain comprising S364K and E357Q mutations.

[0169] Additional knob-and-hole mutation pairs suitable for use in any of the multispecific molecules of the invention are further described, for example, in WO 1996 / 027011 and Merchant et al., Nat. Biotechnol., 16:677-681 (1998), the contents of which are incorporated herein by reference in their entireties.

[0170] In any embodiment described herein, the CH3 domain can be additionally mutated to introduce a pair of cysteine ​​residues. Without being bound by theory, it is believed that the introduction of a pair of cysteine ​​residues capable of forming a disulfide bond provides stability to the heterodimerized multispecific molecule. In an embodiment, according to Edelman et al., PNAS, May 1969, 63 (1): EU numbering scheme of 78-85, the first CH3 domain comprises cysteine ​​at position 354, and according to Edelman et al., PNAS, May 1969, 63 (1): EU numbering scheme of 78-85, the second CH3 domain heterodimerized with the first CH3 domain comprises cysteine ​​at position 349. In embodiments, a first CH3 domain of the multispecific molecule comprises a cysteine ​​at position 354 (e.g., comprising a mutation S354C) and a tyrosine (Y) at position 366 (e.g., comprising a mutation T366Y), and the second CH3 domain that heterodimerizes with the first CH3 domain comprises a cysteine ​​at position 349 (e.g., comprising a mutation Y349C), a serine at position 366 (e.g., comprising a mutation T366S), an alanine at position 368 (e.g., comprising a mutation L368A), and a valine at position 407 (e.g., comprising a mutation Y407V). In embodiments, a first CH3 domain of the multispecific molecule comprises a cysteine ​​at position 354 (e.g., comprising a mutation S354C) and a tryptophan (W) at position 366 (e.g., comprising a mutation T366W), and the second CH3 domain that heterodimerizes with the first CH3 domain comprises a cysteine ​​at position 349 (e.g., comprising a mutation Y349C), a serine at position 366 (e.g., comprising a mutation T366S), an alanine at position 368 (e.g., comprising a mutation L368A), and a valine at position 407 (e.g., comprising a mutation Y407V).

[0171] (2) Alternative Knob and Hole: IgG Heterodimerization

[0172] On the one hand, heterodimerization of the polypeptide chains (e.g., half antibodies) of the multispecific molecule is increased by introducing one or more mutations in a CH3 domain derived from an IgG1 antibody class. In one embodiment, according to Edelman et al., PNAS, May 1969, 63(1): EU numbering scheme of 78-85, mutations include a K409R mutation to one CH3 domain paired with a F405L mutation in a second CH3 domain. According to Edelman et al., PNAS, May 1969, 63(1): EU numbering scheme of 78-85, additional mutations may also or alternatively be at positions 366, 368, 370, 399, 405, 407, and 409. Preferably, heterodimerization of polypeptides comprising such mutations is achieved under reducing conditions, e.g., at 25°C-37°C, e.g., 25°C or 37°C, in 10-100 mM 2-MEA (e.g., 25, 50, or 100 mM 2-MEA) for 1-10, e.g., 1.5-5, e.g., 5 hours.

[0173] The amino acid substitutions described herein are introduced into the CH3 domain using techniques well known in the art. Typically, the DNA encoding one or more heavy chains is genetically engineered using the techniques described in Mutagenesis: a Practical Approach. Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution variants of the DNA encoding the two hybrid heavy chains. This technique is well known in the art as described by Adelman et al., (1983) DNA, 2:183.

[0174] IgG heterodimerization strategies are described, for example, in WO 2008 / 119353, WO 2011 / 131746 and WO 2013 / 060867, the contents of which are incorporated herein by reference in their entirety.

[0175] In any embodiment described herein, the CH3 domain can be additionally mutated to introduce a pair of cysteine ​​residues. Without being bound by theory, it is believed that the introduction of a pair of cysteine ​​residues capable of forming a disulfide bond provides stability to the heterodimerized multispecific molecule. In an embodiment, according to Edelman et al., PNAS, May 1969, 63 (1): EU numbering scheme of 78-85, the first CH3 domain comprises cysteine ​​at position 354, and according to Edelman et al., PNAS, May 1969, 63 (1): EU numbering scheme of 78-85, the second CH3 domain heterodimerized with the first CH3 domain comprises cysteine ​​at position 349.

[0176] (3) Polarity bridge

[0177] On the one hand, the heterodimerization of the polypeptide chains (e.g., half antibodies) of the multispecific molecule is increased by introducing mutations based on the "polar bridging" principle, the basic principle of which is to manufacture residues at the binding interface of the two polypeptide chains to interact with residues having similar (or complementary) physical properties in the heterodimer configuration, while interacting with residues having different physical properties in the homodimer configuration. In particular, these mutations are designed so that in heterodimer formation, polar residues interact with polar residues, and hydrophobic residues interact with hydrophobic residues. In contrast, in homodimer formation, the mutation residues so that polar residues interact with hydrophobic residues. The favorable interactions in the heterodimer configuration and the unfavorable interactions in the homodimer configuration act together to make the CH3 domains form heterodimers more likely than homodimers.

[0178] In an exemplary embodiment, the above mutations are made at one or more of residues 364, 368, 399, 405, 409 and 411 of the CH3 domain, amino acid numbering according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85.

[0179] In one aspect, one or more mutations selected from the group consisting of Ser364Leu, Thr366Val, Leu368Gln, Asp399Lys, Phe405Ser, Lys409Phe and Thr411Lys are introduced into one of the two CH3 domains. (Ser364Leu: the original residue of serine at position 364 was replaced by leucine; Thr366Val: the original residue of threonine at position 366 was replaced by valine; Leu368Gln: the original residue of leucine at position 368 was replaced by glutamine; Asp399Lys: the original residue aspartic acid at position 399 was replaced by lysine; Phe405Ser: the original residue phenylalanine at position 405 was replaced by serine; Lys409Phe: the original residue lysine at position 409 was replaced by phenylalanine; Thr411Lys: the original residue of threonine at position 411 was replaced by lysine.)

[0180] On the other hand, one or more mutations selected from the group consisting of: Tyr407Phe, Lys409Gln and Thr411Asp (Tyr407Phe: the original residue tyrosine at position 407 is replaced by phenylalanine; Lys409Glu: the original residue lysine at position 409 is replaced by glutamic acid; Thr411Asp: the original residue threonine at position 411 is replaced by aspartic acid) can be introduced into another CH3.

[0181] In another aspect, one CH3 domain has one or more mutations selected from the group consisting of Ser364Leu, Thr366Val, Leu368Gln, Asp399Lys, Phe405Ser, Lys409Phe and Thr411Lys, and the other CH3 domain has one or more mutations selected from the group consisting of Tyr407Phe, Lys409Gln and Thr411Asp.

[0182] In an exemplary embodiment, the original residue of threonine at position 366 of one CH3 domain is replaced with valine, and the original residue of tyrosine at position 407 of the other CH3 domain is replaced with phenylalanine.

[0183] In another exemplary embodiment, the original residue of serine at position 364 of one CH3 domain is replaced with leucine, and the original residue of leucine at position 368 of the same CH3 domain is replaced with glutamine.

[0184] In yet another exemplary embodiment, the initial residue of phenylalanine at position 405 of one CH3 domain is replaced with serine and the initial residue of lysine at position 409 of this CH3 domain is replaced with phenylalanine, while the initial residue of lysine at position 409 of the other CH3 domain is replaced with glutamine.

[0185] In yet another exemplary embodiment, the initial residue of aspartic acid at position 399 of one CH3 domain is substituted with lysine, and the initial residue of threonine at position 411 of the same CH3 domain is substituted with lysine, while the initial residue of threonine at position 411 of the other CH3 domain is substituted with aspartic acid.

[0186] The amino acid substitutions described herein are introduced into the CH3 domain using techniques well known in the art. Typically, the DNA encoding one or more heavy chains is genetically engineered using the techniques described in Mutagenesis: a Practical Approach. Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution variants of the DNA encoding the two hybrid heavy chains. This technique is well known in the art as described by Adelman et al., (1983) DNA, 2:183.

[0187] The polar bridge strategy is described in, for example, WO 2006 / 106905, WO 2009 / 089004 and K. Gunasekaran et al. (2010) The Journal of Biological Chemistry, 285: 19637-19646 (the contents of which are incorporated herein by reference in their entirety).

[0188] Additional polar bridge mutations are described in, e.g., PCT Publication No. WO 2014 / 145806 (e.g., FIG. 6 of WO 2014 / 145806), PCT Publication No. WO 2014 / 110601, and PCT Publication Nos. WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, and WO 2016 / 182751 (the contents of which are incorporated herein in their entirety). Examples of polar bridge variants include constant chains containing N208D, Q295E, N384D, Q418E, and N421D mutations.

[0189] In any embodiment described herein, the CH3 domain can be additionally mutated to introduce a pair of cysteine ​​residues. Without being bound by theory, it is believed that the introduction of a pair of cysteine ​​residues capable of forming a disulfide bond provides stability to the heterodimerized multispecific molecule. In an embodiment, according to Edelman et al., PNAS, May 1969, 63 (1): EU numbering scheme of 78-85, the first CH3 domain comprises cysteine ​​at position 354, and according to Edelman et al., PNAS, May 1969, 63 (1): EU numbering scheme of 78-85, the second CH3 domain heterodimerized with the first CH3 domain comprises cysteine ​​at position 349.

[0190] Other strategies for enhancing heterodimerization are described in, for example, WO 2016 / 105450, WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, WO 2016 / 141378 and WO 2014 / 145806 and WO 2014 / 110601 (each of which is incorporated herein by reference in its entirety in its entirety). Any of these strategies may be used in the multispecific molecules described herein.

[0191] In an embodiment, two or more modifications discussed herein are combined in a single bispecific antibody, eg, a bbmAb.

[0192] 5. Example 1: Generation of bbmAb bbmAb1

[0193] By way of example, the generation of specific bbmAbs is described below to enable one skilled in the art to practice the present invention.

[0194] The resulting bbmAb, bbmAb1, is a bispecific IgG1 with a LALA silent mutation that binds to two different targets, IL-1β and IL-18, simultaneously. The antibody binds to two different antigen-binding arms (Fab fragments), while the Fab for IL-1β is based on mAb2 and contains a kappa light chain (Vk6). The Fab for IL-18 is based on mAb1 and consists of a lambda light chain (Vλ1). In order to drive heterodimerization of the Fc domain during expression, a "knob" with large amino acid (aa) side chains (S354C and T366W) and a "hole" with small aa side chains (Y349C, T366S, L368A, Y407V) in the mAb1 heavy chain were introduced into the mAb2 heavy chain.

[0195] For ease of reference, the following Table 3 provides the CJM112 hypervariable regions based on the Kabat definition and the Chothia definition, as well as V L and V H domains and the amino acid sequences of the complete heavy and light chains.

[0196] Table 3. Amino acid sequences of hypervariable regions (CDRs), variable domains (VH and VL), and full chains of bbmAb1. The DNA encoding the first VL is listed in SEQ ID NO: 102, and the DNA encoding the second VL is listed in SEQ ID NO: 70. The DNA encoding the first VH is listed in SEQ ID NO: 86, and the DNA encoding the second VH is listed in SEQ ID NO: 54.

[0197]

[0198]

[0199] In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin heavy chain variable domain (V H1 ), the CDR1 has the amino acid sequence of SEQ ID NO: 76, the CDR2 has the amino acid sequence of SEQ ID NO: 77, and the CDR3 has the amino acid sequence of SEQ ID NO: 78. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin heavy chain variable domain (V HLD) comprising hypervariable regions CDR1, CDR2, and CDR3. H1), the CDR1 has the amino acid sequence of SEQ ID NO: 79, the CDR2 has the amino acid sequence of SEQ ID NO: 80, and the CDR3 has the amino acid sequence of SEQ ID NO: 81. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin heavy chain variable domain (V HLD) comprising hypervariable regions CDR1, CDR2, and CDR3. H1 ), the CDR1 has the amino acid sequence of SEQ ID NO:82, the CDR2 has the amino acid sequence of SEQ ID NO:83, and the CDR3 has the amino acid sequence of SEQ ID NO:84.

[0200] In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a second immunoglobulin heavy chain variable domain (V H2 ), the CDR1 has the amino acid sequence of SEQ ID NO: 44, the CDR2 has the amino acid sequence of SEQ ID NO: 45, and the CDR3 has the amino acid sequence of SEQ ID NO: 46. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a second immunoglobulin heavy chain variable domain (V HLD) comprising hypervariable regions CDR1, CDR2, and CDR3. H2 ), the CDR1 has the amino acid sequence of SEQ ID NO: 47, the CDR2 has the amino acid sequence of SEQ ID NO: 48, and the CDR3 has the amino acid sequence of SEQ ID NO: 49. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a second immunoglobulin heavy chain variable domain (V HLD) comprising hypervariable regions CDR1, CDR2, and CDR3. H2 ), the CDR1 has the amino acid sequence of SEQ ID NO:50, the CDR2 has the amino acid sequence of SEQ ID NO:51, and the CDR3 has the amino acid sequence of SEQ ID NO:52.

[0201] In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin light chain variable domain (V LCD) comprising hypervariable regions CDR1, CDR2 and CDR3. L1 ), the CDR1 has the amino acid sequence of SEQ ID NO: 92, the CDR2 has the amino acid sequence of SEQ ID NO: 93, and the CDR3 has the amino acid sequence of SEQ ID NO: 94. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin light chain variable domain (V LHD) comprising hypervariable regions CDR1, CDR2, and CDR3.L1 ), the CDR1 has the amino acid sequence of SEQ ID NO: 95, the CDR2 has the amino acid sequence of SEQ ID NO: 96, and the CDR3 has the amino acid sequence of SEQ ID NO: 97. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin light chain variable domain (V LHD) comprising hypervariable regions CDR1, CDR2, and CDR3. L1 ), the CDR1 has the amino acid sequence of SEQ ID NO:98, the CDR2 has the amino acid sequence of SEQ ID NO:99, and the CDR3 has the amino acid sequence of SEQ ID NO:100.

[0202] In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a second immunoglobulin light chain variable domain (V L2 ), the CDR1 has the amino acid sequence of SEQ ID NO: 60, the CDR2 has the amino acid sequence of SEQ ID NO: 61, and the CDR3 has the amino acid sequence of SEQ ID NO: 62. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a second immunoglobulin light chain variable domain (V LHD) comprising hypervariable regions CDR1, CDR2, and CDR3. L2 ), the CDR1 has the amino acid sequence of SEQ ID NO: 63, the CDR2 has the amino acid sequence of SEQ ID NO: 64, and the CDR3 has the amino acid sequence of SEQ ID NO: 65. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a second immunoglobulin light chain variable domain (V LHD) comprising hypervariable regions CDR1, CDR2, and CDR3. L2 ), the CDR1 has the amino acid sequence of SEQ ID NO:66, the CDR2 has the amino acid sequence of SEQ ID NO:67, and the CDR3 has the amino acid sequence of SEQ ID NO:68.

[0203] In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin V H1 domain and the first immunoglobulin V L1 domain, wherein: a) the first immunoglobulin V H1The structural domain comprises (for example, in order): i) hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence of SEQ ID NO: 76, the CDR2 has the amino acid sequence of SEQ ID NO: 77, and the CDR3 has the amino acid sequence of SEQ ID NO: 78; or ii) hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence of SEQ ID NO: 79, the CDR2 has the amino acid sequence of SEQ ID NO: 80, and the CDR3 has the amino acid sequence of SEQ ID NO: 81; or iii) hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence of SEQ ID NO: 82, the CDR2 has the amino acid sequence of SEQ ID NO: 83, and the CDR3 has the amino acid sequence of SEQ ID NO: 84 and b) the first immunoglobulin V L1 The domain comprises (e.g., in order): i) hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence of SEQ ID NO: 92, the CDR2 has the amino acid sequence of SEQ ID NO: 93, and the CDR3 has the amino acid sequence of SEQ ID NO: 94; or ii) hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence of SEQ ID NO: 95, the CDR2 has the amino acid sequence of SEQ ID NO: 96, and the CDR3 has the amino acid sequence of SEQ ID NO: 97; or iii) hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence of SEQ ID NO: 98, the CDR2 has the amino acid sequence of SEQ ID NO: 99, and the CDR3 has the amino acid sequence of SEQ ID NO: 100.

[0204] In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a second immunoglobulin V H2 domain and second immunoglobulin V L2 domain, wherein: a) the second immunoglobulin V H2The structural domain comprises (for example, in order): i) hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence of SEQ ID NO: 44, the CDR2 has the amino acid sequence of SEQ ID NO: 45, and the CDR3 has the amino acid sequence of SEQ ID NO: 46; or ii) hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence of SEQ ID NO: 47, the CDR2 has the amino acid sequence of SEQ ID NO: 48, and the CDR3 has the amino acid sequence of SEQ ID NO: 49; or iii) hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence of SEQ ID NO: 50, the CDR2 has the amino acid sequence of SEQ ID NO: 51, and the CDR3 has the amino acid sequence of SEQ ID NO: 52 and b) the second immunoglobulin V L2 The domain comprises (e.g., in order): i) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 60, CDR2 has the amino acid sequence of SEQ ID NO: 61, and CDR3 has the amino acid sequence of SEQ ID NO: 62; or ii) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 63, CDR2 has the amino acid sequence of SEQ ID NO: 64, and CDR3 has the amino acid sequence of SEQ ID NO: 65; or iii) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 66, CDR2 has the amino acid sequence of SEQ ID NO: 67, and CDR3 has the amino acid sequence of SEQ ID NO: 68.

[0205] In one embodiment, the IL-18 / IL-1β bispecific antibody comprises: a) a first immunoglobulin heavy chain variable domain (V HLD) comprising the amino acid sequence set forth in SEQ ID NO: 85; H1 ); b) a first immunoglobulin light chain variable domain (V L1 ); c) a first immunoglobulin V comprising the amino acid sequence listed in SEQ ID NO: 85 H1 domain and a first immunoglobulin V comprising the amino acid sequence set forth in SEQ ID NO: 101 L1 domain; d) a first immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78 H1e) a first immunoglobulin V domain comprising the hypervariable regions listed in SEQ ID NO: 92, SEQ ID NO: 93, and SEQ ID NO: 94 L1 f) a first immunoglobulin V domain comprising the hypervariable regions listed in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81 H1 g) a first immunoglobulin V domain comprising the hypervariable regions listed in SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97 L1 h) a first immunoglobulin V domain comprising the hypervariable regions listed in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78 H1 domain and a first immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO: 92, SEQ ID NO: 93, and SEQ ID NO: 94 L1 i) a first immunoglobulin V domain comprising the hypervariable regions listed in SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81. H1 domain and a first immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97 L1 domain; j) a first light chain comprising SEQ ID NO: 103; k) a first heavy chain comprising SEQ ID NO: 87; or l) a first light chain comprising SEQ ID NO: 103 and a first heavy chain comprising SEQ ID NO: 87.

[0206] In one embodiment, the IL-18 / IL-1β bispecific antibody comprises: a) a second immunoglobulin heavy chain variable domain (V HLD) comprising the amino acid sequence set forth in SEQ ID NO: 53; H2 ); b) a second immunoglobulin light chain variable domain (V L2 ); c) a second immunoglobulin V comprising the amino acid sequence listed in SEQ ID NO: 53 H2 domain and a second immunoglobulin V domain comprising the amino acid sequence set forth in SEQ ID NO: 69 L2 domain; d) a second immunoglobulin V comprising the hypervariable regions listed in SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46 H2e) a second immunoglobulin V domain comprising the hypervariable regions listed in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62 L2 f) a second immunoglobulin V domain comprising the hypervariable regions listed in SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49 H2 g) a second immunoglobulin V domain comprising the hypervariable regions listed in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65 L2 h) a second immunoglobulin V domain comprising the hypervariable regions listed in SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46 H2 domain and a second immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62 L2 i) a second immunoglobulin V domain comprising the hypervariable regions listed in SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49. H2 domain and a second immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65 L2 domain; j) a second light chain comprising SEQ ID NO:81; k) a second heavy chain comprising SEQ ID NO:55; or l) a second light chain comprising SEQ ID NO:81 and a second heavy chain comprising SEQ ID NO:55.

[0207] In some embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 53. In other embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 69. In other embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 53 and three CDRs of SEQ ID NO: 69. In some embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 85. In other embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 101. In other embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 85 and three CDRs of SEQ ID NO: 101.

[0208] In some embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 85. In other embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 101. In other embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 85 and three CDRs of SEQ ID NO: 101. In some embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 53. In other embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 69. In other embodiments, the IL-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO: 53 and three CDRs of SEQ ID NO: 69. In one embodiment, the L-18 / IL-1β bispecific antibody comprises three CDRs of SEQ ID NO:85, three CDRs of SEQ ID NO:101, three CDRs of SEQ ID NO:53, and three CDRs of SEQ ID NO:69.

[0209] In one embodiment, the first part of the IL-18 / IL-1β bispecific antibody is selected from a human IL-18 antibody, which comprises at least: a) an immunoglobulin heavy chain or a fragment thereof, comprising a variable domain and a constant part of a human heavy chain or a fragment thereof, wherein the variable domain comprises, in sequence, hypervariable regions CDR1, CDR2 and CDR3; wherein CDR1 has an amino acid sequence of SEQ ID NO: 76, wherein CDR2 has an amino acid sequence of SEQ ID NO: 77, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 78; and b) an immunoglobulin light chain or a fragment thereof, comprising a variable domain and a constant part of a human light chain or a fragment thereof, wherein the variable domain comprises, in sequence, hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence of SEQ ID NO: 92, wherein CDR2 has an amino acid sequence of SEQ ID NO: 93, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 94. Furthermore, the second part of the IL-18 / IL-1β bispecific antibody is selected from a human IL-1β antibody, which comprises at least: a) an immunoglobulin heavy chain or a fragment thereof, comprising a variable domain and a constant part of a human heavy chain or a fragment thereof, wherein the variable domain comprises, in sequence, hypervariable regions CDR1, CDR2 and CDR3; wherein CDR1 has an amino acid sequence of SEQ ID NO: 44, wherein CDR2 has an amino acid sequence of SEQ ID NO: 45, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 46; and b) an immunoglobulin light chain or a fragment thereof, comprising a variable domain and a constant part of a human light chain or a fragment thereof, wherein the variable domain comprises, in sequence, hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence of SEQ ID NO: 60, wherein CDR2 has an amino acid sequence of SEQ ID NO: 61, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 62.

[0210] In one embodiment, the first part of the IL-18 / IL-1β bispecific antibody is selected from a human IL-18 antibody, which comprises at least: a) an immunoglobulin heavy chain or a fragment thereof, comprising a variable domain and a constant part of a human heavy chain or a fragment thereof, wherein the variable domain comprises, in sequence, hypervariable regions CDR1, CDR2 and CDR3; wherein CDR1 has an amino acid sequence of SEQ ID NO: 76, wherein CDR2 has an amino acid sequence of SEQ ID NO: 77, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 78; and b) an immunoglobulin light chain or a fragment thereof, comprising a variable domain and a constant part of a human light chain or a fragment thereof, wherein the variable domain comprises, in sequence, hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence of SEQ ID NO: 92, wherein CDR2 has an amino acid sequence of SEQ ID NO: 93, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 94. Furthermore, the second part of the IL-18 / IL-1β bispecific antibody is selected from a human IL-1β antibody, which comprises at least: a) an immunoglobulin heavy chain or a fragment thereof, comprising a variable domain and a constant part of a human heavy chain or a fragment thereof, wherein the variable domain comprises, in sequence, hypervariable regions CDR1, CDR2 and CDR3; wherein CDR1 has an amino acid sequence of SEQ ID NO: 44, wherein CDR2 has an amino acid sequence of SEQ ID NO: 45, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 46; and b) an immunoglobulin light chain or a fragment thereof, comprising a variable domain and a constant part of a human light chain or a fragment thereof, wherein the variable domain comprises, in sequence, hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence of SEQ ID NO: 60, wherein CDR2 has an amino acid sequence of SEQ ID NO: 61, and wherein CDR3 has an amino acid sequence of SEQ ID NO: 62.

[0211] The first V of the IL-18 / IL-1β bispecific antibody used in the disclosed method H1 or V L1 The domain may have the same V as set forth in SEQ ID NOs: 85 and 101. H or V L The first V domain is substantially identical H1 and / or the first V L1Domain. The IL-18 / IL-1β bispecific antibody disclosed herein may comprise a first heavy chain substantially identical to the heavy chain shown in SEQ ID NO: 87 and / or a first light chain substantially identical to the light chain shown in SEQ ID NO: 103. The IL-18 / IL-1β bispecific antibody disclosed herein may comprise: a first heavy chain comprising SEQ ID NO: 87 and a first light chain comprising SEQ ID NO: 103. The IL-18 / IL-1β bispecific antibody disclosed herein may comprise: A) a first heavy chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 85 and a constant portion of a human heavy chain having a heterodimerization modification; and b) a first light chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 101 and a constant portion of a human light chain. The constant portion of the human heavy chain may be IgG1. In one embodiment, IgG1 is a human IgG1 without an effector mutation. In one embodiment, the human heavy chain IgG1 comprises the silent mutations N297A, D265A or a combination of L234A and L235A. In a specific embodiment, according to SEQ ID NO: 87, the human heavy chain IgG1 comprises the silent mutations which are a combination of L234A and L235A.

[0212] The second V of the IL-18 / IL-1β bispecific antibody used in the disclosed method H2 or V L2 The domain may have the same V as set forth in SEQ ID NOs: 53 and 69. H or V L The second V domain is substantially identical to H2 and / or the first V L2Domain. The IL-18 / IL-1β bispecific antibody disclosed herein may comprise a second heavy chain substantially identical to the heavy chain shown in SEQ ID NO: 55 and / or a second light chain substantially identical to the light chain shown in SEQ ID NO: 71. The IL-18 / IL-1β bispecific antibody disclosed herein may comprise: a second heavy chain comprising SEQ ID NO: 53 and a second light chain comprising SEQ ID NO: 69. The IL-18 / IL-1β bispecific antibody disclosed herein may comprise: a) a second heavy chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 53 and a constant portion of a human heavy chain having a heterodimerization modification (which is complementary to the heterodimerization of the first heavy chain); and b) a second light chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 69 and a constant portion of a human light chain. The constant portion of the human heavy chain may be IgG1. In one embodiment, IgG1 is a human IgG1 without an effector mutation. In one embodiment, the human heavy chain IgG1 comprises the silent mutations N297A, D265A or a combination of L234A and L235A. In a specific embodiment, according to SEQ ID NO: 55, the human heavy chain IgG1 comprises the silent mutations which are a combination of L234A and L235A.

[0213] Other preferred IL-18 antagonists (eg, antibodies) for use as the first part of the bispecific antibody in the disclosed methods, kits, and regimens are those listed in U.S. Pat. No.: 9,376,489, which is incorporated herein by reference in its entirety.

[0214] Other preferred IL-1 β antagonists (eg, antibodies) for use as the second part of the bispecific antibody in the disclosed methods, kits, and regimens are those listed in U.S. Pat. Nos.: 7,446,175 or 7,993,878 or 8,273,350, which are incorporated herein by reference in their entirety.

[0215] (1) Vector design

[0216] Two vectors, vector A and vector B, were generated according to the following setup. Vector A was designed for the antibody part mAbl (anti-IL18 IgG1). The constant region of heavy chain 1 was modified by two point mutations, T to W as seen at position 366 of SEQ ID NO:87, and S to C as seen at position 354 of SEQ ID NO:87, for generating a knob structure and achieving Cys bridging. In addition, the constant region of heavy chain 1 was modified by point mutations, L to A as seen at position 234 of SEQ ID NO:87, and L to A (said LALA) as seen at position 235 of SEQ ID NO:87, for partial silencing of FC effector function. The antibody has a light chain variable region of type λ1, Vλ1.

[0217] Vector B was designed for the antibody portion mAb2 (anti-IL-1β IgG1). The constant region of heavy chain 2 was modified by four point mutations, T to S as seen at position 366 of SEQ ID NO:55, L to A as seen at position 368 of SEQ ID NO:55, Y to V as seen at position 407 of SEQ ID NO:55, and Y to C as seen at position 349 of SEQ ID NO:55, for generating a hole structure and achieving additional Cys bridging. The hole structure interacts with the knob structure to facilitate the generation of bispecific antibodies. In addition, the constant region of the heavy chain 2 was modified with two LALA mutations, L to A as seen at position 234 of SEQ ID NO:55, and L to A as seen at position 235 of SEQ ID NO:55, for silencing of FC effector function. The antibody has a κ6-type light chain variable region, Vκ6.

[0218] Vectors A and B carry a combination of DHFR and neomycin selection markers and a combination of FOLR and hygromycin selection markers, respectively. Folic acid is a vitamin necessary for the synthesis of purine and methionine and needs to be absorbed by mammalian cells from the culture medium. The "folate receptor" (FolR) present on expression plasmid A is a mutated FolR whose affinity for folic acid is altered, promoting the transport of folic acid from the culture medium to mammalian cells. Given that the high-affinity folate receptor is only weakly expressed in cultured CHO cells, cells expressing recombinant FolR have a significant growth advantage under low folic acid conditions (50nM). The FolR selection marker is encoded at vector B.

[0219] In addition to FolR, "dihydrofolate reductase" (DHFR) is present on vector A as a selection marker. DHFR converts folic acid into an important precursor for purine and methionine synthesis. MTX is a chemical analog of folic acid. It competes with the free binding sites on DHFR, thereby blocking the enzyme. Cells that overexpress exogenous DHFR can handle high concentrations of MTX, so that cells grown in a culture medium supplemented with MTX have a significant selective advantage. Combined FolR and DHFR selection is well known to those skilled in the art, and is disclosed, for example, in patent document WO 2010 / 097240A1, the entire contents of which are incorporated herein by reference. MTX is well known to those skilled in the art, and is disclosed, for example, in patent document WO 2010 / 097239A1, the entire contents of which are incorporated herein by reference. Expression vectors are well known to those skilled in the art, and are disclosed, for example, in patent document WO 2009 / 080720A1, the entire contents of which are incorporated herein by reference.

[0220] Figure 1 Schematic diagrams of the two vectors are given.

[0221] (2) Host cell lines and transfection

[0222] The parental CHO cell line was used as host cell line for the production of a cell line expressing bbmAb 1. The host cell line was derived from the CHO-K1 cell line well known to the person skilled in the art in a manner described, for example, in patent applications WO 2015092737 and WO 2015092735, both of which are incorporated herein by reference in their entirety.

[0223] A single vial from a CHO line was used to prepare the bbmAb1 recombinant cell line. The CHO line was prepared in chemically defined medium.

[0224] Cells were grown in chemically defined medium.

[0225] One μg of SwaI linearized plasmid DNA, expression vectors encoding bbmAbs A & B was added per transfection. Transfection reactions were performed in chemically defined medium.

[0226] Transfections were performed by electroporation using an AMAXA Gene Pulser according to the manufacturer's instructions. Parental CHO cells used for transfection were in exponential growth phase with greater than 95% cell viability. In total, 5 x 10 6 The transfections were performed three times per cell / transfection.

[0227] Immediately after transfection, transfer the cells to a shake flask containing chemically defined medium with medium.

[0228] Before starting the selection process, the cell bank was maintained at 36.5 °C and 10% CO 2 Incubate for 48 hours.

[0229] (3) Cell selection and sorting

[0230] As described above, the selection process is performed using selection markers encoded by individual expression vectors A and B. Both proteins (FolR and DHFR) are involved in the same molecular pathway; the FolR transports folic acid and the folic acid analog MTX into the cell, and the DHFR converts it into important precursors for purine and methionine synthesis. Combining them as selection principles, specific strong selection schemes can be used to enrich for recombinant cells expressing both recombinant proteins.

[0231] After transfection and growth for 48 h under low folate conditions, additional selection pressure was applied by adding 10 nM MTX to chemically defined medium. Twenty-two days after the start of MTX selection, a pool population consisting mainly of MTX-resistant cells emerged. After pool recovery, cells were frozen and cell pellets were prepared. Standard batches were established in chemically defined medium to determine the concentration of bbmAb.

[0232] Protein A HPLC method was used to identify the complete identity of all products with Fc moiety and related impurities, whereas reverse phase chromatography (RPC) was used to obtain fingerprints regarding the distribution of each fraction - individual peaks were identified by MS method.

[0233] The CHO cell pool producing bbmAb1 has been used in a FACS cloning procedure to obtain individualized clonal cell lines as starting material for all further evaluations. Cell selection using FACS analysis is described, for example, in patent application US20110281751, the entire contents of which are incorporated herein by reference.

[0234] A single clonal CHO cell line expressing bbmAb1 was generated by fluorescence activated cell sorting (FACS). To enable FACS sorting, cells were incubated with FITC-labeled anti-IgG1 Fab for 30 minutes and washed twice in PBS before being used for FACS-assisted single cell sorting (a method well known to those skilled in the art).

[0235] FACS cell sorting was performed with a FACS Aria (Becton Dickinson) equipped with an automated cell deposition unit (ACDU) using FACS Diva software.

[0236] To ensure that only single cells were sorted by the FACS instrument, the settings were adjusted to single cell precision mode using a 130 μm nozzle and an appropriate flow rate to ensure good sorting quality.

[0237] In the Single Cell mode, the Purity Mask was set to maximum, so that only droplets that did not contain particles or other cells were sorted.

[0238] Set the "Phase Mask" to half of its maximum value, so that only particles in the center of the sorted droplet are deflected. Optimize droplet trajectories and counting accuracy at the expense of yield to increase the probability that each droplet contains no more than 1 single cell.

[0239] To verify and document the monoclonal origin and confirm the single-cell status on day 0 after FACS cloning, images of all wells of the 96-well plate were taken using an imaging system.

[0240] Day 0 images involving bbmAbl producing clones were visually inspected in duplicate to confirm that only a single cell could be identified in the image of each well captured by the imaging system.

[0241] This highlights the single-cell origin of the bbmAb1 producing clone.

[0242] (4) Cell expansion

[0243] After FACS cloning, clones are handled by a robotic system in the first few weeks, then manually, and gradually expanded from 96-well, 24-well, to shaker, and finally to bioreactor culture to evaluate the effect (productivity and quality of bbmAb expression), which is well known to those skilled in the art.

[0244] During expansion / cultivation, recombinant CHO cells were cultured in chemically defined medium supplemented with methotrexate (MTX) at a final concentration of 10 nM.

[0245] Cells were passaged 2-3 times per week into fresh medium and maintained in logarithmic growth phase throughout the study.

[0246] Productivity was assessed by Protein A HPLC and initial product mass spectra were determined by reverse phase chromatography (RPC).

[0247] All frozen stocks were produced in medium supplemented with 7.5% DMSO.

[0248] (5) Clone stability

[0249] The bbmAbs isolated from the pools and clones were carefully evaluated by different analytical methods to determine product characteristics and quality parameters to ensure the selection of the most appropriate production clones.

[0250] Additionally, production clones were over-analyzed for production stability to ensure the selection of the most appropriate production clones.

[0251] The stability of clones was assessed using different state of the art analytical methods: affinity liquid chromatography, reverse phase chromatography, FACS and MS.

[0252] (6) Production

[0253] (a) Upstream processing

[0254] bbmAb materials are produced in shake flasks or wave fed-batch cultures. Frozen stocks of pools or clones (e.g., PSL) are thawed and expanded for the desired time in chemically defined media to obtain the required number of cells to inoculate production cultures, with a typical seed cell density of 4.0 x 10 5 Cells / ml. The culture time for a single culture is 13-14 days. During the culture process, in-process controls are performed to monitor the concentration of bbmAb and the quality profile of the supernatant. At the end of the culture process, the cells are separated from the culture supernatant by centrifugation (e.g., shaking table) or depth filtration, followed by sterile filtration before further DSP processing.

[0255] (b) Downstream processing

[0256] Based on the format design and co-expression approach, not only intact product bbmAb1 and common impurities (e.g., aggregates, DNA, and host cell proteins), but also mAb1- and mAb2-derived monomers, homodimers, and mispaired light / heavy chain bbmAb1 variants (as shown in Figure 4) are expected in the supernatant after cell culture and cell debris removal. Figures 4E to 4M ) is suspected to have the same biophysical properties as intact bbmAb1, which is not easily removed at a manufacturing scale.

[0257] Method I: by using MabSelect TM SuRe TM Capture and purify on LambdaFabSelect TM and KappaSelect TM Refined

[0258] bbmAb1 and bbmAb1 variants with Fc moieties were identified by MabSelect TM SuRe TM The first affinity liquid chromatography (ALC) step is captured from the cell-free supernatant. TM The first step of purification was performed on the 500 μg / ml slurry to remove the bbmAb1 variant containing only the kappa light chain (mAb2, Figure 4C and 4D ) and HCP, and byTM A second polishing step was performed on the α-light chain to remove the bbmAb1 variant (mAb1 knob, Figure 4A and 4B ) and HCP.

[0259] Throughout the method, chromatography was performed at room temperature using a 4-minute residence time (RT). Prior to loading, 4 column volumes (CV) of 20 mM Na 2 HPO 4 / NaH 2 All columns were equilibrated with 4% PO4 (pH 7.0). To remove non-specifically bound impurities such as host cell proteins (HCPs), culture medium components, and DNA from the product, the cell-free bbmAb1 supernatant from the shake flask was loaded onto the ALC column and the column was washed with 4CV 250mM arginine-HCl, 1M NaCl, 88mM NaOH (pH 9.0), and 3CV equilibration buffer. bbmAb1 and potential bbmAb1 variants were isolated from KappaSelect 5000 by using 50mM acetic acid (pH 3.0) and 50mM acetic acid / HCl (pH 2.0), respectively. TM and LambdaFabSelect TM The product peak collection started and ended at 0.5 AU / cm or 0.25 Au / cm (280 nm). The pH of the bbmAb1 eluate was adjusted to ~ pH 5.0 with 0.1 M or 1 M Tris and then stored at 2°C-8°C for analytical evaluation.

[0260] Method II: By using LambdaFabSelect TM Captured on Capto TM Adhesion (Capto TM adhere) and Fractogel TM EMD SO 3 Refined

[0261] In the second method, by TM Affinity liquid chromatography was performed on the PBS to capture intact bbmAb1 and bbmAb1 variants containing only the λ light chain (mAb1 knob monomer and homodimer, Figure 4A and 4B To inactivate possible enveloped viruses, the ALC eluate was subjected to a low pH treatment and then to the Capto-adhesion and Fractogel TM EMD SO 3Two chromatographic polishing steps were performed on the HPLC column to remove product-related impurities, DNA and HCPs. Subsequently, any viruses present were removed by nanofiltration, followed by a final concentration and buffer exchange step using tangential flow filtration.

[0262] a)LambdaFabSelect TM Affinity Liquid Chromatography (ALC)

[0263] Throughout the method, ALC was performed at 18°C-28°C using a residence time (RT) of 3.6-4.4 minutes. First, the ALC column was heated with 4-6 CV of 20 mM Na 2 HPO 4 / NaH 2 PO4 (pH 7.0) equilibrated. The clarified cell-free bbmAb1 supernatant from the wave or bioreactor was then loaded onto LambdaFabSelect at a loading density of 7-23 g / L TM Column. Wash the column with 4-6CV 250mM arginine-HCl, 1M NaCl, 88mM NaOH (pH 9.0) and a second wash with 3-5CV equilibration buffer before eluting the product with 4-6CV 50mM acetic acid. Collect product peaks from 0.5-2.0Au / cm (280nm) rising and 0.5-2.0Au / cm (280nm) falling. Wash LambdaFabSelect with 3-5CV 120mM phosphoric acid, 167mM acetic acid (pH 1.5) TM The column was then washed with 3-5 CV 20 mM Na 2 HPO 4 / NaH 2 PO4 (pH 7.0) and stored in 4-6 CV 20% ethanol.

[0264] b) Virus inactivation

[0265] The pH of the ALC eluate was adjusted to pH 3.4-3.6 using 0.3M phosphoric acid. The protein solution was then incubated at this low pH for 60-90 minutes before the pH was adjusted to 7.3-7.7 using 1M Tris. A depth filtration step was performed using a Millipore B1HC Pod filter at a flow rate of 100-300LMH, followed by a 0.45 / 0.2μm Sartopore TM Sterile filter for sterile filtration.

[0266] c) Capto TM Multimodal Anion Exchange Chromatography (MAC) on Adhesion

[0267] Throughout the method, MAC was performed at 18°C-28°C in flow-through mode using a residence time of 4-6 minutes. First, the MAC column was equilibrated with 7-9 CV 20 mM Tris / Tris-HCl (pH 7.5). The low pH treated ALC eluate was then loaded onto Capto TM Adhesion column. Product peak collection starts at 0.5-2.0 AU / cm (280 nm) rise. Then wash the MAC column with 5-7 CV equilibration buffer, and end product peak collection at 0.5-2.0 AU / cm (280 nm) fall. Subsequently, Capto TM The column was adhered and then subjected to a cleaning in place step with 3-5CV 0.5M NaOH and stored in 3-5CV 0.1M NaOH.

[0268] d) Fractogel TM EMD SO 3 Cation Exchange Chromatography

[0269] Fractogel TM EMD SO 3 CEC on 100 μL was performed in bind-elute mode at 18°C-28°C. A residence time of 6-8 minutes was used during equilibration, stripping, CIP and storage, and a residence time of 8-10 minutes was used during loading, washing and elution. The CEC column was equilibrated with 6-8CV 20mM succinic acid, 35.1mM NaOH (pH 6.0). The MAC permeate was then loaded onto the column at a loading density of 35-70g / L. Subsequently, the CEC column was washed with 5-7CV equilibration buffer. Elution was performed using a linear salt gradient of 20mM succinic acid, 500mM NaCl, 37.4mM NaOH (pH 6.0) from 10% to 90% over 15CV. Collection of bbmAb1 product peaks started at 0.1-0.4AU / cm rise to 20%-40% maximum peak height at 300nm. Fractogel was stripped with 3-5CV 1M NaCl TM EMD SO 3 The column was then subjected to a cleaning in place step with 3-5 CV 0.5 M NaOH and stored in 3-5 CV 0.1 M NaOH.

[0270] e) Nanofiltration

[0271] Possible presence of viruses by using Planova TMNanofiltration removal was performed using a 20N nanofilter and a 0.5 / 0.1 μm Millipore SHR-P prefilter. Prefiltration and nanofiltration were performed by applying a pressure difference of 0.7-0.9 bar.

[0272] f) Tangential flow filtration and formulation

[0273] To concentrate and diafilter bbmAb1, perform centrifugation at 18°C-28°C in a Millipore TM Pellicon TM The tangential flow filtration step was performed on a 3RC 30 kDa membrane. First, a feed pressure of 0.5 to 1.2 bar and a transmembrane pressure (TMP) of 0.3-0.6 bar were used at 1000 g / m 2 The nanofiltered bbmAb1 protein solution was concentrated to a maximum loading density of 60-80 g / L. The bbmAb1 was then diafiltered with 7-9 diafiltration volumes of 20 mM histidine / histidine-HCl (pH 6.0) at a feed flow pressure of 0.8 to 1.8 bar and a TMP of 0.4-0.9 bar. A second concentration step was performed to 134 ± 10 g / L at a feed flow pressure of 1.4-3.0 bar and a TMP of 0.7-1.5 bar. The ultrafiltered bbmAb1 protein solution was finally formulated to a concentration of 100 ± 10 g / L and 0.04% (w / v) polysorbate 20. The final drug substance (DS) was filtered through a 0.2 μm filter and stored frozen at ≤-60°C.

[0274] (7) Analytical characterization and purity assessment

[0275] (a) LC-MS screening of intact bbmAb and its variants

[0276] 100 μg protein A purified bbmAb samples were lyophilized in 96-well plates and deglycosylated by PNGaseF (New England Biolabs) in 100 μl 50 mM Tris-HCl pH 7.5) buffer at 37°C for 18 hours. The samples were measured by LC-ESI-MS on an H-Class UPLC (Waters) connected to a Synapt G2 Q-TOF mass spectrometer (Waters). A MassPREP micro desalting column 2.1 x 5 mm (Waters) was used at 80°C column temperature. The following linear gradient was applied at a speed of 0.3 ml / min: mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% Fa in acetonitrile: 0-2 min 5% B, 2-12 min 5%-90% B, followed by a wash step at a speed of 0.5 ml / min. MS parameters: ESI+deconvolution mode, capillary voltage 3 kV, sampling cone 40 V, source temperature 150 ° C, desolvation temperature 400 ° C. The system was calibrated with NaCl calibration solution and the lock mass was Leucin Enkephalin. Data were processed using UNIFI 1.6 software (Waters) by automatic MaxEnt1 deconvolution (mass range 60 kDa-150 kDa, harmonic suppression). Identification and relative quantification of bbmAb species and mismatch variants were based on matching with theoretical expected masses and relative mass signal intensities of deconvoluted mass spectra.

[0277] (b) LC-MS characterization of bbmAb1

[0278] Intact deglycosylated bbmAb: Purified bbmAb1 antibody was diluted to 1 mg / ml in 20 mM Tris-HCl pH 7.5 and deglycosylated using 2 μl PNGaseF enzyme (New England Biolabs) for 4 hours at 37. Digestion was stopped by adding trifluoroacetic acid (TFA) to 2%.

[0279] Reduced deglycosylated bbmAb: bbmAb1 was diluted to 5 mg / ml in 20 μl of 0.1 M Tris-HCl pH 7.5. 2 μl PNGase F was added and incubated at 37°C for 4 hours, then 80 μl denaturation buffer (50 mM Tris-HCl pH 8.0, 6 M guanidine hydrochloride) and 1 μl of 1 M DTT were added to the mixture. After incubation at 37°C for 1 hour, the sample was acidified with 1 μl TFA.

[0280] Papain digests bbmAb into Fab and Fc. bbmAb1 was mixed with digestion buffer (20 mM succinic acid, 35.1 mM NaOH, pH 6.0, 1 mM Cys-HCl, 1 mM EDTA) to 5 mg / ml, then papain (Roche, Germany) was added to a final concentration of 5 μg / ml (protease / protein ratio of 1:1000) and incubated at 37°C for 2 hours with shaking. After incubation, the solution was stopped by adding iodoacetamide solution to a final concentration of 1.2 mM.

[0281] IdeS digests bbmAb into F(ab') 2 100 μg of bbmAb1 was mixed with cleavage buffer (50 mM sodium phosphate, 150 mM NaCl, pH 6.6) and digested with 100 U IdeS protease (Fabricator, Genovis) at 37° C. overnight. After incubation, the solution was terminated by adding TFA to a final concentration of 2%.

[0282] Reduced LysC digestion - peptide mapping. (According to Rombach-Riegraf et al., PlosOne, 2014) 150 μl of denaturing solution (6 M guanidine-HCl, 50 mM Tris-HCl, 5 mM Na 2 200 μg protein was denatured with 1 M EDTA, pH 8.0, and reduced by adding 1.5 μl of 1 M DTT and incubated at 37°C for 1 hour. Alkylation was performed by adding 3 μl of 1 M iodoacetamide and then incubating at 37°C in the dark. The reaction was quenched with 1 μl of 1 M DTT. After reduction / alkylation, 750 μl of digestion buffer (50 mM Tris-HCl, pH 8) was added to the sample. The sample was then digested by adding 4 μl of 1 μg / μl intracellular protease LysC solution (Wako Co., Ltd. (Osaka, Japan)) twice, and incubated at 37°C for 1 hour and 3 hours, respectively. 5 μl of TFA was added to quench the digestion.

[0283] LC-MS measurement was performed using a BEH C4 RP column (1.7 μm, 2.1 x 100 mm, Protein samples were analyzed by LC-MS system using a Waters UPLC H-Class with a HPLC-HPLC system (Waters Corporation) and a Xevo G2 TOF mass spectrometer (Waters Corporation, Milford). The eluents were A: 0.1% TFA in water and B: 0.09% TFA in acetonitrile. The column was set at 80°C. The flow rate was 0.2 ml / min. The protein was eluted with the following gradient over 40 min: 0-5 min 10% B, 5-10 min 10%-30% B, 10-25 min 30%-40% B, 25-26 min 40%-95% B, 26-28 min 95%, 28-40 min 10% B.

[0284] MS settings: ESI (+) TOF mode, analytical mode, mass range 400 Da-4000 Da, scan time 1 s, capillary voltage 3 kV, sampling cone 25 V-40 V. The system was calibrated using NaCsI solution.

[0285] The peptide digests were analyzed by RP-LC-MS on an H-Class UPLC (Waters) coupled to a Synapt G2Q-TOF mass spectrometer (Waters) using a CSH130 C18 2.1 mm x 150, 1.7 μm (Waters, Milford). Mobile phase A: 0.1% TFA in water and mobile phase B: 0.09% TFA in acetonitrile. The peptides were eluted from the column with the following gradient: 0-5 min 0% B, 5-10 min 0-2% B, 10-40 min 2%-20% B, 40-120 min 20%-40% B, 120-135 min 40%-70% at 40°C column temperature. UV chromatograms were recorded at 214 nm and MS with low energy (4 eV) and high energy fragmentation (30 V-55 V) was used. E The MS data were collected in positive ES (+) desorption mode. The lock mass was leucine enkephalin (Waters Corporation).

[0286] Data processing and evaluation were performed by MassLynx 4.2 or UNIFI 1.6 software (Waters Corporation). The MaxEnt1 algorithm was used for deconvolution of protein mass spectra. Theoretical mass calculations were performed using GPMAW 9.2 software (Lighthouse data).

[0287] (c) Reversed Phase Chromatography

[0288] The bbmAb samples were analyzed on an Agilent 1260 HPLC using a Poroshell 300SB-C8 RP column (2.1 mm x 75 mm, 5 μm (Agilent)). The column temperature was set at 80 °C, the flow rate was 2 ml / min, mobile phase A: 90% water, 10% acetonitrile, 0.1% TFA, 0.3% PEG-300, mobile phase B: 10% water, 90% acetonitrile, 0.1% TFA, 0.3% PEG-300. The gradient used was: 0-5 min 22%-37% B, 5.0-5.1 min 100% B, 5.1-6 min 100% B, 6.1.-8.5 min 22% B. UV signal was recorded at 210 nm. The chromatographic analysis was performed using a Chromeleon TM Data acquisition and evaluation were controlled and performed using the 6.8 software (Thermo Scientific).

[0289] (d) Size Exclusion Chromatography

[0290] Using Agilent 1260 system, bbmAb1 sample was passed through TSK gel G3000SWXL (Tosoh #808541, 5 μm, 7.8 mm x 300 mm) SEC column with pore size of 250A. The mobile phase was 150 mM potassium phosphate solution, pH 6.5, the flow rate was 0.4 ml / min, and the column temperature was 30 °C. UV was recorded at 210 nm. Chromeleon TM Data acquisition and peak integration were performed using ELISA kit 6.8 (Thermo Fisher Scientific).

[0291] (e) Capillary electrophoresis CE-SDS

[0292] For non-reducing CE-SDS, bbmAb samples were mixed with sample buffer (0.1 sodium phosphate / 1.0% SDS, pH 6.6) and then mixed with iodoacetamide solution. For reducing CE-SDS, proteins were mixed with 0.1M Tris / 1% SDS sample buffer (pH 8.0) and reduced with 5% (v / v) mercaptoethanol. Both samples were subjected to a heat denaturation step at 70°C for 10 min.

[0293] The samples were analyzed on a Beckman PA 800 system equipped with a bare fused silica capillary (50 μm, 375 OD, 67 cm, Beckman) with a total capillary length of 30 cm and filled with Beckman SDS MW Sizing Gel Buffer. Separation was done from negative to positive polarity at 15 kV and 25°C capillary temperature with UV detection at 214 nm. Chromeleon TM6.8 Software processing and integration of electropherograms.

[0294] (f) Capillary zone electrophoresis (CZE)

[0295] Separation was performed on a Beckman Coulter PA 800 Pharmaceutical Analysis System equipped with a 214 nm UV detector. Proteins were separated on a fused silica capillary (50 μm ID) with a total length of 40 cm using a capillary voltage of 20 kV at 25°C and positive polarity. Running buffer: 400 mM 6-aminohexanoic acid / acetic acid pH 5.7 with 2 mM TETA and 0.03% Tween 20. A Chromeleon TM 6.8 software performs peak integration.

[0296] (8) Analysis results

[0297] The purity and identity of different bispecific antibody combinations and constructs after co-expression were analyzed using an intact UPLC-MS mass screening approach after protein A purification. This approach was used to confirm and relatively quantify heterodimers and homodimers from cell supernatants. Correctly formed heterodimers, bbmAb1 and bbmAb2, were observed with relative purities exceeding 85% based on intact mass signal intensity. The major impurities observed in the screen were mispaired antibodies with two kappa light chains, two lambda light chains, and HC dimer molecules.

[0298] Table 4. Summary of analytical results for bbmAb1-bbmAb11.

[0299]

[0300]

[0301] The designation l is for lambda chain and k is for kappa chain.

[0302] mAb3 is antibody type VH3, Vk1.

[0303] mAb4 is antibody type VH3, Vk1.

[0304] mAb5 is a grafted version of mAb1 (VH1, Vl1).

[0305] mAb6 is a grafted version of mAb2 (VH1_46, Vk3).

[0306] mAb7 is antibody type VH3, Vk1.

[0307] mAb8 is antibody type VH1_2, Vk2.

[0308] mAb9 is antibody type VH5, Vk6.

[0309] mAb10 is antibody type VH1_46, Vk6.

[0310] mAb11 is an antibody type VH3, Vk3.

[0311] mAb12 is antibody type VH3, Vk2.

[0312] bbmAb1 was characterized in more detail to evaluate all product variants and impurities formed after the different purification steps by LC-MS using several sample preparation methods as well as other separation techniques. The quality of the intact 2-step purification (λ / CEC) product was determined after deglycosylation with PNGaseF enzyme and subsequent injection into the RP-LC-MS device. The deconvoluted mass spectrum of the intact bbmAb1 confirmed the correct formation of the knob-in-hole heterodimer after co-expression and λ-selection purification. No major impurities were detected after λ-selection purification as homodimers or partial antibodies. After sample reduction and deglycosylation, the identity of the four different antibody chains could be confirmed.

[0313] To check for chain mispairing and other low-level impurities, purified samples were digested with papain to analyze individual Fab and Fc fragments. The measured fragment masses again confirmed the correct formation of the different Fab arms (knob-λ, hole-κ) and the correct formation of the knob-hole structure Fc fragment. Mispaired Fab fragments (Fab4, knob-κ) with a level <1% can be found. Another method for generating Fab fragments by limited LysC digestion was tested, which can allow faster sample preparation during merging and clonal selection.

[0314] Another digestion strategy using IdeS (Fabricator) enzyme was tested to generate Fc and F(ab')2 fragments. In this experiment, the mass of Fc heterodimers and correctly formed heterodimeric F(ab')2 were observed. The presence of Fc heterodimers also proves that the additional disulfide bonds in the Fc part of bbmAb1 are correctly formed, because otherwise only Fc / 2 fragments of smaller mass would be generated.

[0315] The identity of the molecule was confirmed by LysC peptide mapping using LC-MS, with a total sequence coverage of 99% for the peptides.

[0316] Detailed results for the purified samples are shown in Figure 2. In particular, the RP-UV chromatogram of the deglycosylated intact bispecific mAb is shown in Figure 2. Figure 2A The papain-digested bbmAb1 fragments are shown in Figure 2C The bbmAb1 fragment digested with IdeS is shown in Figure 2D The deglycosylated and DTT-reduced bbmAb1 fragments are shown in Figure 2EThe deconvoluted mass spectrum of the intact deglycosylated bispecific mAb bbmAb1 is shown in Figure 2B shown.

[0317] The results are shown in Table 5.

[0318] Table 5. bbmAb1 masses determined by RP-LC-MS partitioning

[0319]

[0320] The improved purity obtained after applying the different steps described above is shown in Figure 3. Figure 3A is a chromatogram showing the expression profile after culture, Figure 3B Use LambdaFabSelect TM The chromatogram after capture, Figure 3C MabSelect TM SuRe TM The chromatogram after capture, Figure 3D Use LambdaFabSelect TM Captured by Fractogel TM EMD SO 3 Chromatogram after purification and ultrafiltration.

[0321] The final 2-step purified (λ / CEC) bispecific bbmAb1 was further analyzed by the methods listed in Table 6. Overall, the material showed high purity with low levels of aggregates or degradation products as detected by several separation methods such as size exclusion chromatography (SEC), CE-SDS, and capillary zone electrophoresis (CZE).

[0322] Table 6. Purity analysis of purified bbmAb1

[0323]

[0324] Additional combinations of other antibodies have also been tested.Table 4 shows a summary of the analytical results obtained.

[0325] 6. Example 2: In vitro activity of bbmAb1

[0326] The binding activity of bbmAb1 was tested in various cellular assays.

[0327] (1) Materials and methods

[0328] (a) For solution equilibrium titration (SET) determination

[0329] The following materials were used:

[0330] Biotinylated recombinant human IL-18 (BTP25828)

[0331] Recombinant cynomolgus monkey IL-1β (Novartis)

[0332] SULFO-TAG labeled anti-human IgG antibody (Meso Scalediscovery (MSD) #R32AJ-5)

[0333] Goat anti-human Fab specific antibody conjugated with MSD SULFO-TAG NHS ester (Jackson Immuno Research #109-005-097, MSD #R91AN-1), BSA (Sigma #A-9647)

[0334] MSD Read Buffer T with Surfactant (MSD#R92TC-1)

[0335] Phosphate buffered saline (PBS) 10x (Teknova #P0195) Tris buffered saline pH 7.5 (TBS) 10x (Teknova #T1680) Tween-20 (Fluka #93773)

[0336] Polypropylene microtiter plates (MTP) (Greiner #781280)

[0337] 384-well plate, standard (MSD#L21XA)

[0338] (b) For cell assays and SET assays

[0339] mAb2 as described in the IL-1β antibody section.

[0340] mAb1 as described in the IL-18 antibody section.

[0341] As described in Example 1 bbmAb1.

[0342] Recombinant human IL-18 (BTP 25829) purchased from MBL Int. Corp. (#B001-5)

[0343] Recombinant marmoset IL-1β (Novartis)

[0344] Recombinant marmoset IL-18 (Novartis)

[0345] Recombinant human IL-12 (#573008) was purchased from Biolegend KG-1 cell line (ATCC#CCL-246)

[0346] Normal human skin fibroblasts (#CC-2509) were purchased from Lonza

[0347] Marmoset skin fibroblasts (#42637F(510))

[0348] HEK-Blue TM IL-18 / IL-1β cells (#hkb-il18) were purchased from InvivoGen

[0349] PBMCs were isolated from buffy coat (obtained from Blustspendezentrum Bern)

[0350] Marmoset blood was obtained from SILABE, Niederhausbergen

[0351] IL-6 ELISA: Human (Biotech, #430503); Marmoset (U-CyTech Biosciences, CT974-5)

[0352] IFNγ ELISA: Human (BD555142) and Marmoset (U-CyTech Biosciences #CT340A)

[0353] QUANTI-Blue for SEAP detection TM The assay (#rep-qb1) was purchased from InvivoGen

[0354] Cell culture medium: RPMI 1640 (Invitrogen #31870) supplemented with 10% fetal bovine serum (Invitrogen #10108-157), 1% L-glutamine (Invitrogen #25030-03), 1% penicillin / streptomycin (Invitrogen #15140-148), 10 μM 2-mercaptoethanol (Gibco #31350-010), 5 mM Hepes (Gibco #15630-080)

[0355] Round bottom tissue culture treated 96-well plates (Costar #3799)

[0356] Flat-bottom tissue culture treated 96-well plates (Costar #3596)

[0357] Ficoll-Pacque TMPlus (GE Healthcare Life Sciences #17-1440-02) PBS 1X without calcium and magnesium (Gibco #14190094)

[0358] Leucosep tubes with porous barrier, 50 ml, polypropylene (Greiner bio-one #227290) Falcon 15 ml polypropylene conical tubes (BD #352096)

[0359] Falcon 50ml Polypropylene Conical Tube (BD#352070)

[0360] (c) Affinity measurement by SET

[0361] SET single target binding assay

[0362] 22 serial 1.6n dilutions of the antigen (highest concentration: huIL-18, 5nM; marIL-18, 10nM; huIL-1β, 0.5nM; marIL-1β, 0.5nM) 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 (10pM for IL-18 reading and 1pM for IL-1β reading). Each antigen-antibody mixture was dispensed in duplicate into a 384-well polypropylene microtiter plate (MTP) at a volume of 60μl / well. Sample buffer served as a negative control and samples containing only antibody served as positive controls (maximum electrochemiluminescent signal without antigen, B max The plate was sealed and incubated overnight (o / n, at least 16 h) on a shaker at room temperature (RT).

[0363] IL-18 Readout: Streptavidin-coated 384-well MSD array MTPs were coated with 30 μl / well biotinylated huIL-18 (0.1 μg / ml in PBS) and incubated for 1 hour at room temperature on a shaker.

[0364] IL-1β readout: A standard 384-well MSD array MTP was coated with 30 μl / well of huIL-1 (3 μg / ml, PBS) diluted in PBS (as capture agent) and incubated overnight at 4°C.

[0365] The plates were blocked for 1 hour (h) at room temperature (RT) with 50 μl / well of blocking buffer (PBS containing 5% BSA). After washing (TBST, TBS containing 0.05% Tween 20), a volume of 30 μl / well of the equilibrium antigen-antibody mixture was transferred from the polypropylene MTP to the coated MSD plate and incubated for 20 min at room temperature. After an additional washing step, 30 μl of sulfo-tagged anti-IgG detection antibody (0.5 μg / ml) diluted in sample buffer was added to each well and incubated on a shaker at room temperature for 30 min. The MSD plate was washed and 35 μl / well of MSD reading buffer was added and incubated for 5 min at room temperature. Electrochemiluminescence (ECL) signals were generated and measured by MSD Sector Imager 6000.

[0366] SET simultaneous target binding assay

[0367] SET assays were performed as described above, except for assay A: the equilibrium process (antibody / antigen mixture) was performed in the presence of an excess of one target (500 pM of IL18 or IL-1β), while the K of the other target was assessed. D .

[0368] Assay B: Equilibrium process (antibody / antigen mixture) was performed simultaneously with serial dilutions of both targets in one mixture (constant concentration of antibody 10 pM, highest antigen concentration see above). The same mixture was then analyzed for free antibody concentration on IL18 and IL-1β coated plates as described above.

[0369] SET data were exported to the MS Excel add-in software Xlfit. The average ECL signal was calculated from the replicate measurements in each assay. The data were baseline adjusted by subtracting the lowest value from all data points and plotted against the corresponding antigen concentration to generate a titration curve. D The value is determined by fitting the graph with:

[0370] 1:2 binding model of monospecific Ab

[0371]

[0372] 1:1 binding model of a knob-and-hole bispecific Ab

[0373]

[0374] in

[0375] y) Subtract the blank ECL signal

[0376] B max : Maximum ECL signal when the antigen concentration is zero

[0377] [IgG]: Antibody concentration used

[0378] [Fab]: Total Fab concentration applied

[0379] K D : Dissociation equilibrium constant

[0380] x: applied antigen concentration

[0381] (d) Cell culture

[0382] KG-1 cells were grown at a density of 2 x 10 in RPMI1640 supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin / streptomycin. 5 Up to 1x 10 6 viable cells / mL.

[0383] Normal human fibroblasts and marmoset fibroblasts were grown in FBM (Clonetics, CC-3131) containing bFGF (1 ng / ml, CC-4065), insulin (5 μg / ml, CC-4021) and 2% FCS (CC-4101). Fibroblast basal medium (LONZA #CC-3131) was used as starvation medium.

[0384] HEK-Blue TM IL-18 / IL-1β cells were cultured in growth medium (DMEM, 4.5 g / l glucose, 10% (v / v) fetal bovine serum, 50 U / ml penicillin, 50 mg / ml streptomycin, 100 mg / ml normocin TM , 2mM L-glutamine, supplemented with 30μg / ml blasticidin, 200μg / ml HygroGold TM and 100 μg / ml Zeocin TM ) grows in.

[0385] Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from the buffy coat using LeucoSep tubes according to the manufacturer's instructions. Briefly, 13 ml of Ficoll-Paque was pre-loaded in a 14 ml LeucoSep tube by centrifugation at 1,000 × g for 30 seconds. The heparinized whole blood sample was diluted with an equal volume of PBS, and 25 ml of the diluted blood was added to the LeucoSep tube. The cell separation tube was centrifuged at 800 × g for 15 min at room temperature without interruption. The cell suspension layer was collected, and the cells were washed twice in PBS (640 × g and 470 × g for 10 minutes, respectively, two consecutive washes) and resuspended in culture medium before counting.

[0386] Marmoset blood was collected in heparinized tubes and filtered using a 70 μm cell strainer (BD Biosciences #352350).

[0387] (e) IL-1β neutralization assay

[0388] The IL-1β-induced IL-6 production assay in fibroblasts was performed essentially as described in (Gram 2000) with only minor modifications. Briefly, fibroblasts were seeded in 96-well flat-bottom tissue culture plates at a density of 5 x 103 cells / well (in 100 μl). The next day, the cells were starved in starvation medium for 5 hours before adding the recombinant IL-1β / compound solution mixture (IL-1β concentrations shown in the table). The IL-1β / compound solution mixture was prepared in advance by incubating recombinant IL-1β with a concentration range of compounds at 37°C for 30 min. Cell supernatants were collected after o / n incubation at 37°C, and the amount of released IL-6 was determined by ELISA. The IL-1β-induced IL-6 production assay in PBMCs was performed as follows. PBMCs were seeded at 3x10 5 Cells / well (in 100 μl) were seeded in 96-well tissue culture plates and incubated with recombinant IL-1β / compound solution mixtures at 37°C for 24 hours (IL-1β concentrations shown in the table). IL-1β / compound solution mixtures were prepared in advance by incubating recombinant IL-1β with a concentration range of compounds at 37°C for 30 min. Cell supernatants were collected after 24 hours of stimulation, and the amount of released IL-6 was determined by ELISA.

[0389] (f) IL-18 neutralization assay

[0390] The determination was performed essentially as follows. 5 KG-1 cells (previously starved for 1 hour in PBS + 1% FCS) or PBMCs were seeded / well into a round-bottom 96-well cell culture plate and incubated with a solution mixture of recombinant IL-18 / IL-12 and a concentration range of compounds (IL-18 / IL-12 concentrations shown in the table). After incubation for 24 hours at 37°C, the supernatant was collected and the amount of released IFNγ was determined by ELISA. For assays with marmoset blood, 85 μl of blood / well was used.

[0391] (g)HEK-Blue TM Dual IL1β / IL-18 neutralization in cells

[0392] The assay was performed essentially as described in the manufacturer's protocol. TM Cells were cultured at 4 x 104 Cells were seeded at a density of 100 μg / well in 96-well cell culture plates and incubated with a solution mixture of recombinant IL-1β and IL-18 (to generate a 1:1 SEAP signal) and a concentration range of compounds. After incubation at 37°C for 24 h, supernatants were collected and analyzed using QUANTI-Blue according to the manufacturer's instructions. TM The method determines the amount of SEAP released.

[0393] All data were exported to EXCEL software and IC50 values ​​were calculated by plotting dose response curves against a logistic curve fitting function using EXCEL / XLfit4 or GraphPad Prism software.

[0394] (2) Results

[0395] (a) Affinity for recombinant human and marmoset IL1β and IL-18

[0396] Titration by solution equilibrium titration (SET) Figure 5 ) measured the binding affinity of bbmAb1 to human and marmoset recombinant IL-1β and IL-18 proteins, and the resulting K D The K values ​​of mAb2 for IL-1β and mAb1 for IL-18 were compared with those of mAb1 for IL-18. D The values ​​are compared. Figure 5 Titration curves for ECL-based affinity assays in solution are shown, constant concentration of antibody: 10 pM for IL-18 reading, 1 pM for IL-1β reading; antigen dilution: highest concentration: huIL-18, 5 nM; marIL-18, 10 nM; huIL-1β, 0.5 nM; marIL-1β, 0.5 nM. The solid line represents the fit of the data using the above model. The dashed line indicates the 95% confidence interval, n=3.

[0397] Comparing binding affinities in a single target binding assay, bbmAb1 showed similar average KDs for both human and marmoset IL-18 compared to mAb1 (Table 7). For human IL-1β binding, the average KD values ​​for bbmAb1 (2.6 pM) were slightly higher than those for mAb2 (0.6 pM), but were still in the same low pM range. Subsequent measurements in a simultaneous dual target binding assay (Table 8) confirmed that the KD values ​​for bbmAb1 binding to IL-1β were similar to those for mAb2 in both preclinical and clinical grade material. Thus, bbmAb1 has binding affinities for targets in both humans and marmosets that are similar to mAb2 and mAb1, respectively.

[0398] Table 7. Affinity for recombinant human (hu) and marmoset (mar) IL-1β and IL-18 as determined by SET (single target binding assay)

[0399]

[0400] In addition to single target binding results, the binding K D The simultaneous dual target binding affinity of bbmAb1 was investigated by either using an excess of one target relative to the other during the quantification process (Assay A) or by applying a mixture of both targets in serial dilutions (Assay B) (Table 8). Simultaneous IL-1β / IL-18 affinity assays showed no significant differences between Assay A (one antigen in excess) and Assay B (a mixture of both antigens in serial dilutions), demonstrating that both targets bind simultaneously without affecting the binding of the other. In addition, the K values ​​obtained using the simultaneous dual binding assays were D The values ​​were comparable to those obtained using the standard assay (Table 7; in the absence of the second antigen). D The values ​​were similar, demonstrating that bbmAb1 can bind both antigens independently. Thus, bbmAb1 binds both human IL-1β and IL-18 simultaneously and independently, and fully cross-reacts with the corresponding marmoset proteins.

[0401] Table 8. Affinity for recombinant human (hu) and marmoset (mar) IL-1β and IL-18 as determined by SET (simultaneous target binding assay)

[0402]

[0403]

[0404] (b) Neutralizing activity of bbmAb1 in human and marmoset cell assays

[0405] The neutralizing activity of bbmAbl on two cytokines (IL1β and IL-18) was evaluated (mAb2mAb1).In addition, the potency of bbmAbl in neutralizing marmoset IL-1β and IL-18 was evaluated using the marmoset cell assay system (see section d).

[0406] (c) Individual and simultaneous IL-1β and IL-18 neutralization in human cells

[0407] The neutralizing activity of bbmAb1 against IL-1β was evaluated by inhibiting IL-6 production induced by recombinant IL-1β in human skin fibroblasts (IL-1β was used at 6pM) and human PBMCs (IL-1β was used at 60pM). The neutralizing activity of bbmAb1 against IL-18 was measured by inhibiting IFN-γ production induced by recombinant IL-18 in KG-1 cells and human PBMCs (both cells were activated by 3nM recombinant human IL-18 and 1ng / ml recombinant human IL-12). The inhibitory potency of bbmAb1 against IL-1β and IL-18 was always compared with mAb2 or mAb1, respectively. Depending on the assay, the average IC50 values ​​of bbmAb1 were in the sub-nM or single-digit nM range, while directly up to 2-4 times higher than mAb2 (for IL-1β) and mAb1 (for IL-18), respectively (Tables 9 and 10). The monovalent format of bbmAb1, but also possibly the KiH mutation, is responsible for the slight difference in potency of bbmAb1 compared to the bivalent format of mAb2 / mAb1.

[0408] Table 9. Mean IC50 values ​​for neutralization of IL-1β by bbmAb1 compared to mAb2 in human skin fibroblasts and human PBMCs. *Inhibition of IL-6 production in human skin fibroblasts or PBMCs stimulated with recombinant human IL-1β (6 pM for skin fibroblasts and 60 pM for PBMCs). Shown are mean ± SEM (n = 3 PBMCs and n = 6 human skin fibroblasts)

[0409]

[0410] Table 10. Mean IC50 values ​​for neutralization of IL-18 by bbmAb1 compared to mAb1 in KG-1 cells and human PBMCs. **Inhibition of IFNγ production in KG-1 cells or PBMCs stimulated with recombinant human IL-18 (3 nM) and human IL-12 (1 ng / ml). Mean ± SEM (n = 3 KG-1 and n = 4 PBMCs) are shown

[0411]

[0412] bbmAb1 can simultaneously neutralize the biological activities of IL-1β and IL-18, such as HEK Blue TM The results are shown in Table 11 for reporter cells that produce SEAP in response to 1+1 stimulation with recombinant IL-1β and IL-18. Similar inhibition of SEAP could be achieved in this assay system only by the combination of mAb2 and mAb1, but not by using the individual antibodies.

[0413] Table 11. Based on HEK Blue TMMean IC50 values ​​for SEAP reporter activity in cells and simultaneous neutralization of IL-1β and IL-18. Shown are means ± SEM of n = 5 experiments.

[0414]

[0415] (d) Neutralization activity of bbmAb1 against marmoset IL-1β and marmoset IL-18 in marmoset cell assay

[0416] To demonstrate the inhibitory activity of bbmAb1 in marmosets, similar in vitro assays were performed with marmoset cells and human cells, but using recombinant marmoset IL-1β and IL-18 for stimulation. When evaluating the inhibition of IL-6 production induced by recombinant marmoset IL-1β in marmoset skin fibroblasts, bbmAb1 exhibited sub-nM potency with IC50 values ​​2- to 3-fold higher than mAb2 (Table 12). Testing bbmAb1 with human skin fibroblasts stimulated with marmoset IL-1β produced an inhibition profile similar to that with human IL-6.

[0417] Table 12. bbmAb1 inhibits recombinant marmoset IL-1β-induced IL-6 production in marmoset and human fibroblasts. * Inhibition of IL-6 production in marmoset or human skin fibroblasts stimulated by recombinant marmoset IL-1β (18 pM). Results of 3 separate experiments (A, B and C) are shown.

[0418]

[0419] Single to double digit nM IC50 values ​​for bbmAbl demonstrated neutralizing activity of bbmAbl against marmoset IL-18 tested in an IFNγ production assay with marmoset blood cells (Tables 3-7). Testing bbmAbl with human PBMCs stimulated with marmoset IL-18 yielded a similar inhibition profile when measuring human IFNγ production.

[0420] Thus, in a functional assay using marmoset responder cells, bbmAb1 showed complete cross-reactivity with marmoset IL-1β and marmoset IL-18. Table 13. Mean IC50 values ​​for inhibition of IFNγ production induced by recombinant marmoset IL-18 in marmoset whole blood or human PBMCs. **Inhibition of IFNγ production in marmoset whole blood (n=3 per compound / condition) or human PBMCs (n=6) stimulated with recombinant marmoset IL-18 (indicated concentrations) and human IL-12 (10 ng / ml). Shown are mean ± SEM

[0421]

[0422] It has been demonstrated that the KiH-type IL-1β / IL-18 bispecific mAbs retain high affinity binding to two separate targets, IL-1β and IL-18, as well as cytokine neutralization potency in multiple different cell-based assays compared to the original mAbs, mAb2 and mAb1. The dual IL-1β and IL-18 neutralization properties of bbmAb1 were demonstrated not only against human cytokines / cells but also against the corresponding marmoset cytokines / cells, facilitating appropriate toxicology studies. The up to 2- to 4-fold higher IC50 values ​​generated in some of the cell-based assays for IL-1β and IL-18 neutralization may be a result of monovalent binding of bbmAb1 rather than bivalent binding of mAb2 and mAb1, respectively. However, dual neutralization of cytokines by bbmAb1 may result in additive or synergistic inhibitory activity in vivo, which may not be fully reflected in our in vitro cell system.

[0423] 7. Example 3: Effects of combined stimulation and blockade of IL-1β and IL-18 in PBMCs

[0424] Inflammasome activation-dependent cleavage of the effector cytokines IL-1β and IL-18 leads to the induction of secondary proinflammatory mediators that promote immune cell recruitment / activation not only systemically but also at the site of inflammation. In two different mouse models of lethal systemic inflammation (a) the LPS injection model and (b) FCAS mice (activating missense mutations in NLRP3), the simultaneous absence / inhibition of IL-1β and IL-18 was more protective against lethality than the absence / inhibition of either IL-1β or IL-18 alone, demonstrating an additive or synergistic mechanism of immune activation (Brydges 2013, van den Berghe 2014). bbmAb1 is a human / marmoset IL-1β / IL-18 reactive bispecific mAb with no rodent cross-reactivity and therefore could not be tested in the mouse model. Therefore, we stimulated human PBMCs with LPS / IL-12 in vitro to mimic inflammasome-dependent pathway activation to reveal the additive or synergistic inhibitory effects of bbmAb1 neutralization and combined IL-1β / IL-18 and performed unbiased gene expression analysis using microarrays. As a complementary activity, we also compared gene expression profiles of PBMCs from different donors stimulated with a combination of recombinant IL-1β and recombinant IL-18 or with either single cytokine alone.

[0425] (1) Materials and methods

[0426] (a) Cell culture and ELISA

[0427] RPMI 1640 (Invitrogen #31870 or Gibco #61870-010) supplemented with 10% fetal bovine serum (Invitrogen #10108-157), 1% L-glutamine (Invitrogen #25030-03), 1% penicillin / streptomycin (Invitrogen #15140-148), 10 μM 2-mercaptoethanol (Gibco #31350-010), 5 mM Hepes (Gibco #15630-080)

[0428] Recombinant human IL-1β was purchased from Sino Biological (#10139-HNAE-5)

[0429] Recombinant human IL-18 was purchased from MBL (#B001-5)

[0430] Recombinant human IL-12 was purchased from Biochi (#573008)

[0431] IFNγ ELISA: MAX Standard Kit, Biochi, #430103 or BD OptEIA Human IFNγ ELISA Kit, BD #555142

[0432] IL-6 ELISA: MAX Standard Kit, Biochi, #430503

[0433] IL-26 ELISA: Cloud Clone Corp #SEB695Hu

[0434] mAb2 as described in the IL-1β antibody section.

[0435] mAb1 as described in the IL-18 antibody section.

[0436] As described in Example 1 bbmAb1.

[0437] LPS from Salmonella Enteritidis serotype Enteritidis, Sigma #L7770

[0438] PBMCs were isolated from buffy coat (obtained from Blustspendezentrum Bern)

[0439] Round-bottom tissue culture treated 96-well plates (Costar #3799) Flat-bottom tissue culture treated 96-well plates (Costar #3596) Ficoll-Pacque TM Plus (GE Healthcare Life Sciences #17-1440-02) PBS 1X without calcium and magnesium (Gibco #14190094)

[0440] Falcon 15ml polypropylene conical tube (BD#352096) Falcon 50ml polypropylene conical tube (BD#352070)

[0441] LeucosepTM tube with porous barrier, 50 ml, Greiner bio-one #227290

[0442] CellScreen 70 μM, BD Biosciences #352350

[0443] Trypan blue, Sigma #T8154

[0444] RNA isolation, quantity and quality measurements, and qPCR:

[0445] Nuclease-free water, Ambion #AM9938

[0446] RNase Zap, Ambion #AM9780

[0447] 1.5 ml Eppendorf tube, sterile, RNase and DNase free

[0448] RLT buffer, Qiagen #1015762

[0449] RNeasy Mini Kit, Qiagen #74104

[0450] RNase-free DNase kit, Qiagen #79254

[0451] Agilent RNA 6000Nano Kit, Agilent #5067-1511

[0452] Chip Priming Station, Agilent #5065-4401

[0453] IKA Vortex Mixer

[0454] Ambion #9780

[0455] Agilent 2100 Bioanalyzer

[0456] High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, #PN4374966

[0457] Nase-free thin-walled 0.2 ml PCR tube with cap, Ambion #AM12225

[0458] MicroAmp Optical 384-well reaction plate, Applied Biosystems #4309849

[0459] TaqMan GenEx Master Mix, Applied Biosystems #4369514

[0460] PCR primers (Applied Biosystems)

[0461] Target Assay ID Taqman Color / Quencher IFNγ Hs00989291_m1 FAM-MGB IL-26 Hs00218189_m1 FAM-MGB RPL27 Hs03044961_g1 FAM-MGB HPRT1 Hs02800695_m1 FAM-MGB

[0462] PBMC preparation: PBMCs were isolated from the buffy coat by Ficoll-Paque gradient centrifugation in Leucosep tubes according to the manufacturer's instructions. Briefly, 15 mL of Histopaque was placed in a 50 mL Leucosep™ tube and centrifuged at 1300 rpm for 30 seconds at room temperature. 30 mL of the diluted suspension of the buffy coat was added to the top of the Histopaque solution with a pipette and centrifuged at 1000 g for 15 min without interruption at room temperature. Plasma (approximately 20 ml) was discarded, and the interface ring (= human PBMC) was collected and transferred to a 50 ml falcon tube. The tube was filled with 50 mL of sterile PBS and centrifuged once at 1200 rpm for 5 min at room temperature. This centrifugation was repeated 2 times. The supernatant was gently discarded and the cells were resuspended in 50 mL PBS containing 2% FCS and 2 mM EDTA. The cell suspension was filtered using a 70 μm cell sieve and the cells were counted using trypan blue staining (500 μL trypan blue + 200 μL cells + 300 μL PBS).

[0463] LPS / IL-12 stimulation of PBMCs: Prepare cytokine production in supernatant according to the following method. Dispense 250,000 cells / well (final volume 100ul) into 96-well round bottom plates. Use LPS at concentrations between 0.3ug / ml and 3000ug / ml, along with 10ng / ml of recombinant IL-12. Incubate at 37°C and 10% CO 2 The supernatant was harvested after 24 h.

[0464] RNA was extracted from the cell pellet as follows. 3 x 10 6 1000 μl of LPS was used together with 10 ng / ml of recombinant IL-12. The cells were cultured at 37°C and 10% CO. 2 After 24 hours, cells were harvested.

[0465] Stimulate PBMCs with recombinant cytokines: 7 x 10 6PBMCs were used in 1.5 ml of final complete RPMI medium. Recombinant cytokines were added at the following final concentrations: 10 ng / ml recombinant IL-1β, 3 nM recombinant IL-18, 1 ng / ml recombinant IL-12. Incubate at 37°C and 10% CO 2 Supernatants and cells were collected after 4 and 24 hours.

[0466] RNA isolation, quantity and quality assessment: Cells were pelleted, the pellets were lysed in 350 μl of Qiagen RTL buffer containing 2% β-mercaptoethanol, and frozen at -20°C or -80°C until all study samples were collected. RNA isolation was performed using the Qiagen standard protocol. Briefly, 350 μl of 70% ethanol was added to all samples, then transferred to an RNeasy spin column and centrifuged at 8000 g for 15 seconds. After discarding the flow-through, 350 μl of buffer RW1 was added and the column was centrifuged at 8000 g for 15 seconds 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 at room temperature for 15 min. After washing with 350 μl and 500 μl of buffer RW1, the RNeasy spin column was placed in a new 2 ml collection tube and centrifuged at full speed for 1 min. RNA was finally collected by adding 35 μl of RNase-free water directly to the spin column membrane and centrifuging at 8000 g for 1 min 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 to assess RNA quality according to the manufacturer's instructions. Briefly, 1 μl of RNA or gradient was pipetted into an Agilent RNA 6000 Nano chip and measured using an Agilent 2100 Bioanalyzer.

[0467] Analysis of cytokine gene expression by qPCR:

[0468] The method was performed according to the manufacturer's instructions. Briefly, 400 ng of RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit according to the instructions. The cDNA solution was diluted 1 / 10 in RNA / DNA-free water, and 1 μl of cDNA was transferred to a 384-well reaction plate and then mixed with 1 μl of 20X Gene expression assay target FAM gene and 10 μl 2x Gene Expression Master Mix and 10 μl RNA / DNA-free water. Load the plate into the Applied Biosystems ViiA TM 7 Real-time PCR system and use the following instrument settings:

[0469]

[0470] The housekeeping genes used for this study were HPRT1 and RLP27. The relative expression levels of target genes were calculated using the following formula:

[0471] 1) Ct[reference] = (Ct[HPRT1] + Ct[RLP27]) / 2

[0472] 2) dCt[reference] = 40 - Ct[reference]

[0473] 3) dCt[target] = Ct[target] - Ct[reference]

[0474] 4) ddCt = dCt [reference] - dCt [target]

[0475] 5) Relative target gene expression = 2^ddCt

[0476] Microarray was performed according to the following method. Samples were processed by CiToxLAB France on Affymetrix HG_U133_Plus2 microarray. In GeneSpring 11.5.1 (Agilent Technologies, Santa Clara, California), they were subjected to RMA normalization and analysis. Path analysis was performed using Ingenuity Pathway Analysis (IPA) and Nextbio (Illumina). These two data sets were processed independently.

[0477] Initially, the data were subjected to standard quality control (QC) by CiToxLAB, internal QC using R scripts (MA_AffyQC.R) in the Rstudio suite and GeneSpring (PCA, hybridization controls). Subsequently, they were filtered to eliminate unreliable expression levels: entities (probe sets) were retained for which at least 100% of the samples in any 1 experimental condition had a value above the 20th percentile.

[0478] Differentially expressed genes (DEGs) were identified using the "filter on volcano plot" function in GeneSpring. Filtered genes (expression between 20.0 percentile-100.0 percentile) with unpaired T-test were used, and probe sets with corrected p-values ​​lower than 0.05 and fold changes higher than 2.0 were considered to be differentially expressed. Where possible, i.e., in the study of LPS (NUID-0000-0202-4150), Benjamini-Hochberg multiple testing correction was used.

[0479] For cytokine stimulation experiments, synergy was calculated using the following formula: Signal A + B / (Signal A + Signal B - Control) ≥ 1.5

[0480] The respective features (or DEG lists) are used to calculate the p-value in the case of Fisher's exact test, which represents the statistical significance of the overlap between the "disease gene list" (lesion compared to non-lesion) of the observed features and the public data set. For this reason, the lists are uploaded into the Innomina Basic Spatial Association Engine (formerly Nextbio) and compared using the Meta-Analysis function and keyword searches for diseases.

[0481] Export all data to EXCEL software and calculate IC by plotting dose-response curves against a logistic curve fitting function using EXCEL / XLfit4 or GraphPad Prism software. 50 The differences between treatment groups were analyzed by one-way ANOVA followed by Dunnett's multiple comparisons using GraphPad Prism software, and the results were considered statistically significant at p < 0.05.

[0482] (2) Results

[0483] (a) bbmAb1 is highly effective in inhibiting LPS / IL-12-induced IFNγ production in whole blood

[0484] Exposure of human whole blood to LPS supplemented with 10 ng / ml IL-12 results in an IFNγ response that is largely (but not entirely) dependent on "natural" IL-18 produced by blood cells. The addition of IL-12 may enhance the LPS-induced IFNγ response by upregulating IL-18 receptors on responding cells.

[0485] Under the experimental conditions used, IL-18 neutralization using mAb1 resulted in only incomplete inhibition of IFNγ production, while IL-1β blockade (using mAb2) had little effect on the IFNγ response. Interestingly, combined inhibition of IL-1β and IL-18 by bbmAb1 or the combination of mAb2 and mAb1 resulted in a more profound and complete inhibition of IFNγ production compared to single cytokine neutralization (Figure 6). Figure 6 shows the inhibition of LPS (0.3 μg / ml) / IL-12-induced IFNγ in whole blood by bbmAb1, mAb2, mAb1 or the combined mAb2 & mAb1 (Combo) (typical inhibition curves are shown in Figure 6). Fig. 6A Percent inhibition of IFNγ in whole blood from n=4 individual donors using bbmAb1, mAb1 or mAb2 at 100 nM (mean and SEM as shown). Figure 6B shown).

[0486] In our cell assay, except for IFNγ, none of the other tested cytokines (IL-2, -4, -6, -8, -10, -13 and TNFα) were additively inhibited by the combined neutralization of IL-1β and IL-18 (data not shown). Considering the monovalent format of the bispecific molecule, the potency of bbmAb1 is in the same range as the combination of mAb2 and mAb1.

[0487] (b) In LPS / IL-12 activated human PBMCs, IFNγ was additively inhibited by bbmAb1 (i.e., combined IL-1β / IL-18 inhibition) compared to inhibition by either IL-1β or IL-18 alone.

[0488] Unbiased transcriptomic assessments were needed to reveal additional additive effects (except for IFNγ) by combined IL-1β / IL-18 inhibition using bbmAb1. Since whole blood is not the best material for transcriptomic analysis, we adapted the LPS / IL-12 stimulation assay conditions (as described in the Materials and Methods section above) to human PBMC samples. By using PBMCs from a total of 9 donors, we could confirm that bbmAb1 additively inhibited IFNγ protein secretion into the PBMC supernatant (Figure 7). IFNγ production was inhibited compared to the whole blood experiments at approximately 10-fold lower concentrations than the individual mAbs used. Importantly, a similar pattern of inhibition was shown at the mRNA level of IFNγ (Figure 7), confirming that the samples are suitable for unbiased microarray-based gene expression analysis. Figure 7 shows the effects of bbmAb1, mAb2 and mAb1 (each at a concentration of 10 nM) on LPS (0.3 μg / ml) / IL-12-induced IFNγ protein production in human PBMCs ( Fig. 7A ) and IFNγ gene expression ( Figure 7B ). Shown are percentage inhibition in n=9 donors ± SEM. ***p<0.05 (one-way ANOVA)

[0489] Affymetrix microarrays were performed with n=5 single donors from PBMCs sampled from the LPS / IL-12 stimulation experiments described in the Materials and Methods section above. Unfortunately, overall evaluation of the gene expression profiles confirmed a strong LPS / IL-12 stimulation effect, with PCA showing clustering of each donor, rather than compounds within the stimulated or unstimulated groups. However, comparison of LPS / IL-12 stimulated samples with stimulated plus bbmAb1 for differentially expressed genes revealed a list of genes downregulated by combined IL-1β / IL-18 blockade using bbmAb1 (Table 14). In addition to the strong downregulation of the IFNγ gene, which reconfirms our microarray data, the IL-26 gene is also another cytokine gene that is additively suppressed by bbmAb1 compared to single IL-1β inhibition (by mAb2) or IL-18 inhibition (by mAb1) (see Figure 8). Figures 8 and 9 show the gene expression levels of IFNγ and IL-26 derived from microarray data, and the inhibitory effects of bbmAb1, mAb2 and mAb1 (10 nM each) in LPS (0.3 ug / ml) / IL-12 stimulated PBMCs at 24 hours. Fig. 8A (IFNγ) and Figure 8B (IL-26) shows the values ​​for a single donor, Fig.9A (IFNγ) and Fig. 9B Percent inhibition (mean ± SEM) of n = 5 donors is shown in (IL-26).

[0490] Table 14. Differentially expressed genes (genes down-regulated between bbmAbl and control groups only in LPS / IL-12 stimulated samples). FC = fold change.

[0491]

[0492]

[0493] (c) IL-26 is another proinflammatory cytokine that is additively inhibited by bbmAb1 in LPS / IL-12-stimulated PBMCs

[0494] To further confirm that LPS / IL-12 driven IL-26 gene expression and protein production were most effectively inhibited using bbmAb1 by combined IL-1β / IL-18 blockade, the study was extended to a total of n=9 PBMC donors and IL-26 gene expression was investigated by qPCR and IL-26 protein production by ELISA. As shown in Figure 10, it largely confirmed the inhibition of IL-26 gene expression obtained by the microarray approach ( Fig. 10AInterestingly, the level of IL-26 protein in the supernatant was only partially reduced at 24 h by the addition of mAb ( Fig. 10B ). The reason for this difference is unclear, but may be related to the kinetic differences between IL-26 gene expression and protein production and the difference in IL-26 consumption compared to IFNγ. However, bbmAb1 showed an advantage in reducing IL-26 protein levels in PBMC supernatants compared to mAb2 and mAb1. Figure 10 shows the effects of bbmAb1, mAb2 and mAb1 (each 10 nM) on LPS (0.3 ug / ml) / IL-12-induced IL-26 gene expression (by qPCR) in human PBMCs ( Fig. 10A ) and IL-26 protein levels ( Fig. 10B ). n=9 individual PBMC donors. Percent inhibition (mean and SEM). ***p<0.05 (one-way ANOVA).

[0495] (d) Disease-related IL1β / IL18 signaling features

[0496] Previously established PBMC culture conditions, in which recombinant IL-1β stimulation resulted in IL-6 production or recombinant IL-18 / IL-12 stimulation resulted in IFNγ production, were combined to reveal additive or synergistic downstream target genes or signatures (data not shown). PBMCs from n=4 donors were sampled at two different time points (6 hours and 24 hours) and evaluated on Affymetrix microarrays for unbiased assessment of gene expression profiles. Genes that were synergistically upregulated at 6 and 24 hours upon stimulation with a combination of IL-1β and IL-18 were revealed (data not shown). The addition of IL-12 to the IL-1β / IL-18 combination greatly enhanced the synergistic effects of a range of upregulated genes. Signal signatures generated by single or combined IL-1β / IL-18 pathway stimulation (upregulated genes only) were used to interrogate a dataset across patients with autoimmune diseases. For example, Fig.11Correlation with public sarcoidosis data sets is shown. P values ​​(calculated by Fisher's exact test) show significant correlation with several public studies comparing healthy tissues and diseased tissues of sarcoidosis patients. Tissues include skin as well as lungs, lacrimal glands and orbits. In all data sets, the combination of IL1β / IL18 signaling showed the best correlation with the disease, followed by IL-1β and IL-18. IL-1β / IL-18 differentially upregulated genes (DEGs) (x-axis) in PBMC compared with 5 sarcoidosis tissue "sick relative to healthy" DEGs. P values ​​(y-axis) represent the statistical significance of the overlap between the observed features and the "disease gene list". The black bars are skin from skin sarcoidosis lesions relative to skin from healthy patients. The light gray bars are skin from skin sarcoidosis lesions relative to non-lesioned skin. The white bars are lacrimal glands from sarcoidosis patients relative to lacrimal glands from normal people. The dark gray bars are pre-orbital tissues from sarcoidosis patients for pre-orbital tissues from normal people. The striped bars are lung samples with progressive fibrosis, pulmonary sarcoidosis versus lung samples with nodular, self-limited pulmonary sarcoidosis.

[0497] (e) Conclusion

[0498] LPS and recombinant IL-12 were used to mimic pathogen-associated molecular pattern (PAMP)-dependent NLRP3 inflammasome activation within the first 24 h of in vitro culture. It was demonstrated that the combined inhibition of IL-1β and IL-18, using bbmAb1, acted additively to reduce / inhibit IFNγ production in LPS / IL-12 stimulated PBMCs. IL-12 was previously described to act synergistically with IL-18 to induce IFNγ production in T, B, NK cells, macrophages and dendritic cells (as described by Nakanishi, 2001), but it was now possible to demonstrate that IL-1β exerted an additive stimulatory effect on IFNγ under the experimental conditions used. Thus, co-incubation of PBMCs with LPS / IL-12 efficiently drove the production of “natural” IL-1β and IL-18, both of which contribute to a strong IFNγ response. Using unbiased microarray transcriptomics, additional genes were identified that were additively downregulated by combined IL-1β / IL-18 neutralization relative to single IL-1β or IL-18 blockade. Among these was IL-26, a member of the IL-20 cytokine subfamily (IL-19, IL-20, IL-22, IL-24, and IL-26) that is conserved in most vertebrate species but absent from most rodent strains, including mice and rats (Donnelly 2010). It signals through a heterodimeric receptor complex composed of IL-20R1 and IL-10R2 chains. The IL-26 receptor is primarily expressed on non-hematopoietic cell types, particularly on epithelial cells. IL-26 levels have been reported to be elevated in serum and, in particular, in the synovial fluid of RA patients (Corvaisier 2012), where IL-26 may function as a factor that promotes the growth and differentiation of Th17 cells. Unfortunately, the strong effects of LPS / IL-12 stimulation of PBMC samples hampered the discovery of additional genes / pathways induced by the combined blockade of IL-1β and IL-18. However, IFNγ and IL-26 and to some extent IL-22 were also among the genes synergistically upregulated by combined stimulation of recombinant IL-1β and IL-18 in PBMCs, confirming that these two factors are downstream effectors of this activation pathway. Therefore, the IL-20 subfamily of cytokines (including IL-26 and IL-22) appears to be strongly dependent on simultaneous signals from IL-1β and IL-18. With due attention to the selectivity of individual signal features and the potential efficacy of blockade, these comparisons help to show that each pathway is active in diseases such as sarcoidosis.

[0499] 8. Example 4: Therapeutic Use

[0500] In inflammasome-driven inflammatory conditions involving both innate and adaptive immune components, combined targeting of IL-1β and IL-18 may represent a more effective therapeutic strategy than single cytokine blockade. Simultaneous neutralization of IL-1β and IL-18 targets both innate and adaptive immune components, including neutrophils, Th1 / Tc1 and NK cells, adhesion molecules on immune cells and endothelial cells, and proinflammatory cytokines (e.g., IL-6, IFNγ, and IL-17). Data obtained in a preclinical mouse model of familial cold autoimmune syndrome (FCAS), which is driven by constitutive Nlrp3 inflammasome activation and overproduction of IL-1β and IL-18, support the advantages of blocking both IL-1β and IL-18 (Brydges, 2013). In the model, partial recovery of FCAS disease was achieved in mice when IL-1β or IL-18 signaling was genetically ablated, demonstrating that both cytokines are involved in the pathogenesis of the disease. Importantly, FCAS mice lacking both IL-18 and IL-1β signaling were less sick than mice in which only one of the two cytokines was inactivated, demonstrating the additive effect of dual IL-1β / IL-18 neutralization. The additive effect of IL-1β and IL-18 neutralization was also demonstrated in another mouse model in which mice were injected with high doses of LPS to induce septic shock (van den Berghe, 2014). In this model, genetic deficiency of both IL-1β and IL-18 or combined neutralization of both cytokines by neutralizing antibodies completely protected against LPS lethality, whereas single cytokine deficiency / neutralization only partially conferred protection.

[0501] The overall clinical strategy of bispecific antibodies targeting both IL-1β and IL-18 simultaneously may represent a more effective treatment than currently available options. In order to identify candidate diseases, preclinical and translational studies are used to prove that both IL-1β and IL-18 downstream pathways are actively involved in the potential pathophysiology of candidate diseases. There is new evidence that chronic pulmonary sarcoidosis is a disease driven by inflammasomes, which involves both innate and adaptive immunity. In addition, preliminary findings indicate that IL-1β and IL-18 effector cytokines play an important role in this disease. Therefore, sarcoidosis represents an ideal opportunity to prove that bbmAb1 has dual specificity in diseases with determined, chronic tissue inflammation. The efficacy of bbmAb1 in sarcoidosis may lead to the development of other interstitial lung diseases, such as allergic (occupational) lung diseases caused by silica or beryllium. Other candidate diseases are granulomatous inflammation involving other organ tissues, such as Crohn's disease.

[0502] Vascular inflammation with tissue damage and endothelial dysfunction also represents a potential target for bbmAb1. Dysfunctional endothelial cells can respond to effective anti-inflammatory treatments, which can lead to improved vascular flow even in the presence of fixed intravascular defects. Recent literature evidence identifies sickle cell disease (SCD) as having a strong inflammasome-driven component through a high rate of constitutive intravascular hemolysis. Inflammasome activation caused by the release of danger signals (uric acid, heme / Fe3+, other intracellular components) from chronic RBC lysis triggers inflammasome receptors, triggers the activation of the inflammasome receptors and leads to an intravascular inflammatory cascade, resulting in upregulation of endothelial cell adhesion molecules, activation of neutrophils and platelets, which leads to chronic activation of endothelial cells. Such persistent vascular inflammation in SCD patients leads to recurrent, painful vaso-occlusive crises and acute episodes of chronic tissue damage. Preliminary in-house evidence supports the involvement of IL-18 and IL-1β in the underlying disease process of SCD. Thus, reduction of basal inflammation in SCD patients by bbmAb1 treatment could reduce chronic background inflammation and prevent acute crises associated with end-organ damage, prevent acute sickle cell crises and associated end-organ damage, and improve patients' quality of life by reducing associated chronic pain and fatigue. Demonstration of therapeutic efficacy of bbmAb1 in SCD patients could lead to the chronic or acute development of other chronic inflammatory conditions involving high rates of hemolysis, such as malaria and hemodialysis-dependent chronic kidney disease. Other indications that could benefit from modulation of both IL-1β and IL-18 are those associated with ischemia-reperfusion tissue damage, such as cardiovascular disease or improved healing of all types of wounds, but especially the most severe soft tissue injuries of burns.

[0503] Therefore, in one embodiment of the invention, a method of treating an inflammasome-associated disorder comprises administering an effective amount of a bbmAb disclosed herein, such as bbmAb1, to a subject suffering from an inflammasome-associated disorder. Potential inflammasome-related disorders include cryopyrin-associated autoinflammatory syndrome (CAPS), familial Mediterranean fever (FMF), systemic juvenile idiopathic arthritis (SJIA), lupus nephritis, diabetic nephropathy, acute kidney injury, renal hypertension, IgA nephropathy, glomerulonephritis (GN), frontotemporal dementia (FTD), Alzheimer's disease (AD), epilepsy, stroke, Parkinson's disease (PD), depression, sarcoidosis such as pulmonary sarcoidosis, pancreatitis, idiopathic pulmonary fibrosis (IPF), non-alcoholic steatohepatitis (NASH), atherosclerosis, giant cell arteritis, anti-neutrophil cytoplasmic antibodies (ANSA), and inflammatory bowel disease (ADA). Anti-cancer antibody (ANCA)-associated vasculitis, age-related macular degeneration (AMD), graft-versus-host disease, type 2 diabetes, acne, sickle cell disease, vasculopathy, ischemia-reperfusion injury, cardiovascular disease, peripheral artery disease (PAD), atherosclerosis, vascular dysfunction, skeletal muscle ischemia, fibrosis, malaria, hemodialysis-dependent chronic kidney disease, or Crohn's disease.

[0504] In one embodiment, a method of treating sickle cell disease, vasculopathy, ischemia-reperfusion injury, cardiovascular disease, peripheral arterial disease, atherosclerosis, vascular dysfunction, skeletal muscle ischemia, pulmonary sarcoidosis, fibrosis, malaria, hemodialysis-dependent chronic kidney disease, or Crohn's disease is provided by administering to a subject an effective amount of a bbmAb disclosed herein (e.g., bbmAbl).

[0505] 9. Example 5: Pharmaceutical composition

[0506] Provided herein are pharmaceutical compositions comprising a bbmAb antibody, such as bbmAb1, formulated with a pharmaceutically acceptable carrier. The composition may additionally contain one or more other therapeutic agents suitable for treating a medical condition. A pharmaceutically acceptable carrier enhances or stabilizes the composition, or can be used to facilitate the preparation of the composition. Pharmaceutically acceptable carriers include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, among others.

[0507] The pharmaceutical compositions described herein can be administered by various methods known in the art. The route and / or mode of administration vary depending on the desired result. Preferably, administration can be intravitreal, intravenous, intramuscular, intraperitoneal or subcutaneous or administered near the target site. The pharmaceutically acceptable carrier should be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (i.e., bbmAb) can be coated in a material to protect the compound from the effects of acid and other natural conditions that may inactivate the compound.

[0508] The composition should be sterile and mobile. Suitable fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the required particle size in the case of a dispersion, and by using a surfactant. In many cases, it is preferred to include isotonic agents such as sugars, polyols (such as mannitol or sorbitol) and sodium chloride in the composition. Long-term absorption of injectable compositions can be achieved by including an agent (such as aluminum monostearate or gelatin) that delays absorption in the composition.

[0509] The pharmaceutical compositions described herein can be prepared according to methods well known and routinely practiced in the art. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th edition, 2000; and Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective (effective or efficacious) dose of bbmAb is used in the pharmaceutical compositions described herein. The bbmAb is formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, as indicated by the urgency of the treatment situation, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased. It is particularly advantageous to formulate parenteral compositions in dosage unit form for uniform administration and dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0510] Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein can be varied so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition used as described herein, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and prior medical history of the patient being treated.

[0511] Doctors or veterinarians can start the dosage of the antibody described herein used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Generally, the effective dose of the composition described herein for treating defecation disorders described herein varies according to many different factors, including mode of administration, target site, patient's physiological state, whether the patient is a human or human animal, other drugs administered, and whether the treatment is preventive or therapeutic. The therapeutic dose needs to be titrated to optimize safety and efficacy. For systemic administration with antibodies, the dosage range is about 0.0001 to 100 mg / kg host body weight, and more generally 0.01 to 15 mg / kg host body weight. For intravitreal administration with antibodies, the dosage can be in the range of 0.1 mg / eye to 5 mg / eye. Exemplary treatment regimens require systemic administration every two weeks or once a month or once every 3 to 6 months. Exemplary treatment regimens require systemic administration every two weeks or once a month or once every 3 to 6 months or as needed (PRN).

[0512] Biological therapies (such as bbmAbl) are usually administered in a variety of situations. The interval between single doses can be weekly, monthly or annual. As shown by measuring the blood level of bbmAbl in the patient, the interval can also be irregular. In addition, alternative dosing intervals can be determined by a doctor and administered monthly or as needed for effectiveness. In some systemic administration methods, the dose is adjusted to reach a plasma antibody concentration of 1-1000 μg / ml, and in some methods to reach 25-500 μg / ml. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency vary according to the half-life of the antibody in the patient. Generally, human antibodies show a longer half-life than chimeric antibodies and non-human antibodies. The dose and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer a relatively high dose at relatively short intervals until disease progression is reduced or terminated, and preferably until the patient shows partial or complete improvement in disease symptoms. Thereafter, a preventive regimen may be administered to the patient.

[0513] Some embodiments of the present invention:

[0514] 1. A bispecific antibody suitable for co-expression in a common host cell, wherein the antibody comprises:

[0515] a. a first part, which is an immunoglobulin having a first variable light chain (VL1) of wild type lambda and a first variable heavy chain (VH1) of wild type and a first constant heavy chain (CH1) with heterodimerization modification, wherein the VH1 specifically binds to a first target, and

[0516] b. a second part, which is an immunoglobulin having a kappa wild-type second variable light chain (VL2) and a wild-type second variable heavy chain (VH2) and a second constant heavy chain (CH2) with a heterodimerization modification complementary to the heterodimerization modification of the first constant heavy chain, wherein the VH2 specifically binds to a second target different from the first target,

[0517] Wherein when the first part and the second part are co-expressed in a common host cell, a bispecific antibody is formed.

[0518] 2. The bispecific antibody according to embodiment 1, wherein the first and second constant heavy chains are human IgA, IgD, IgE, IgG or IgM, preferably IgD, IgE or IgG, such as human IgG1, IgG2, IgG3 or IgG4, preferably IgG1.

[0519] 3. The bispecific antibody according to embodiment 2, wherein the first variable light chain is of λ1 type and the second variable light chain is of κ6 type.

[0520] 4. The bispecific antibody according to embodiment 3, wherein the first and second constant heavy chains are IgG1, and wherein

[0521] a. the first constant heavy chain has a point mutation that generates a knob structure, and the second constant heavy chain has a point mutation that generates a hole structure, or

[0522] b. the first constant heavy chain has a point mutation that generates a hole structure, and the second constant heavy chain has a point mutation that generates a knob structure, and optionally

[0523] c. The first and second constant heavy chains have mutations that result in disulfide bonds.

[0524] 5. The bispecific antibody according to any one of the preceding embodiments, comprising a first immunoglobulin VH1 domain, a first immunoglobulin VL1 domain, a second immunoglobulin VH2 domain, and a second immunoglobulin VL2 domain, wherein:

[0525] a. The first immunoglobulin VH1 domain comprises (for example in sequence):

[0526] i. hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 76, CDR2 has the amino acid sequence of SEQ ID NO: 77, and CDR3 has the amino acid sequence of SEQ ID NO: 78; or

[0527] ii. hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 79, CDR2 has the amino acid sequence of SEQ ID NO: 80, and CDR3 has the amino acid sequence of SEQ ID NO: 81; and

[0528] b. The first immunoglobulin VL1 domain comprises (for example in sequence):

[0529] i. hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 92, CDR2 has the amino acid sequence of SEQ ID NO: 93, and CDR3 has the amino acid sequence of SEQ ID NO: 94 or

[0530] ii. hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 95, CDR2 has the amino acid sequence of SEQ ID NO: 96, and CDR3 has the amino acid sequence of SEQ ID NO: 97; and

[0531] c. The second immunoglobulin VH2 domain comprises (for example in sequence):

[0532] i. hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 44, CDR2 has the amino acid sequence of SEQ ID NO: 45, and CDR3 has the amino acid sequence of SEQ ID NO: 46; or

[0533] ii. hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 47, CDR2 has the amino acid sequence of SEQ ID NO: 48, and CDR3 has the amino acid sequence of SEQ ID NO: 49; and

[0534] d. the second immunoglobulin VL2 domain comprises (for example in sequence):

[0535] i. hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO: 60, CDR2 has the amino acid sequence of SEQ ID NO: 61, and CDR3 has the amino acid sequence of SEQ ID NO: 62 or

[0536] ii. hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence of SEQ ID NO:63, CDR2 has the amino acid sequence of SEQ ID NO:64, and CDR3 has the amino acid sequence of SEQ ID NO:65.

[0537] 6. The bispecific antibody according to any one of the preceding embodiments, comprising a first immunoglobulin VH1 domain, a first immunoglobulin VL1 domain, a second immunoglobulin VH2 domain, and a second immunoglobulin VL2 domain, wherein:

[0538] a. the first immunoglobulin VH1 domain comprises the amino acid sequence SEQ ID NO: 85,

[0539] b. the first immunoglobulin VL1 domain comprises the amino acid sequence SEQ ID NO: 101,

[0540] c. the second immunoglobulin VH2 domain comprises the amino acid sequence SEQ ID NO: 53, and

[0541] d. The second immunoglobulin VL2 domain comprises the amino acid sequence of SEQ ID NO:69.

[0542] 7. The bispecific antibody according to any one of the preceding embodiments, comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain, wherein:

[0543] a. the first immunoglobulin heavy chain comprises the amino acid sequence SEQ ID NO: 87,

[0544] b. the first immunoglobulin light chain comprises the amino acid sequence SEQ ID NO: 103,

[0545] c. the second immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO: 55, and

[0546] d. The second immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO:71.

[0547] 8. A method for selecting a bispecific antibody according to any one of embodiments 1-7, the method comprising:

[0548] a. The first step: selecting the first part and the second part;

[0549] b. the second step: co-expressing the first part and the second part in a common host cell to produce a bispecific antibody comprising the first part and the second part;

[0550] c. Third step: purifying the bispecific antibody by removing mismatched fragments from the correctly matched bispecific antibody.

[0551] 9. A method according to embodiment 8, wherein the third step of purification produces a bispecific antibody that is at least 60% (mass), 70% (mass), 80% (mass), 85% (mass) pure, for example at least 90% (mass) pure, 95% (mass), 96% (mass), 97% (mass), 98% (mass) or 99% (mass) pure.

[0552] 10. A method for producing a bispecific antibody according to any one of embodiments 1-7 by co-expression in a common host cell, the method comprising

[0553] a. The first step: generating at least one vector encoding the first part and the second part;

[0554] b. The second step: introducing the at least one vector into the common host cell;

[0555] c. The third step: selecting cells that specifically express the bispecific antibody;

[0556] d. Step 4: culturing the selected cells under conditions where the cells express the bispecific antibody; and

[0557] e. Fifth step: purifying the bispecific antibody, wherein the bispecific antibody is at least 60% (mass), 70% (mass), 80% (mass), or 85% (mass) pure, for example, at least 90% (mass) pure, 95% (mass), 96% (mass), 97% (mass), 98% (mass) or 99% (mass) pure.

[0558] 11. The method according to embodiment 10, wherein the first step comprises generating a first vector encoding the first part and a second vector encoding the second part.

[0559] 12. An expression system comprising at least one vector and a selection marker, wherein the at least one vector comprises a polynucleotide encoding the first part or the second part of the bispecific antibody according to any one of embodiments 1-7.

[0560] 13. The expression system according to embodiment 12, comprising:

[0561] a. a polynucleotide encoding a first selection marker (sm I);

[0562] b. a polynucleotide encoding a second selection marker (sm II), said sm II being different from said first selection marker (sm I).

[0563] 14. An expression system according to embodiment 12 or 13, wherein the first selection marker (sm I) is a folate transporter or a polynucleotide encoding a mutated folate receptor, wherein the mutated folate receptor has a reduced folate binding affinity compared to the wild-type folate receptor, and the second selection marker (sm II) is DHFR.

[0564] 15. The expression system of any one of embodiments 12-14, wherein the first selection marker (smI) is hygromycin and the second selection marker (sm II) is Neo / G418.

[0565] 16. The expression system according to any one of embodiments 12-15, comprising two expression vectors, wherein:

[0566] a. a first vector comprising a polynucleotide encoding at least a first selection marker (sm I) and a polynucleotide encoding at least said first portion; and

[0567] b. A second vector comprising a polynucleotide encoding at least a second selection marker (sm II) and a polynucleotide encoding at least said second part.

[0568] 17. An expression system according to any one of embodiments 12-16, comprising a stop codon downstream of the polynucleotide encoding the heavy chain and a polynucleotide encoding an immunoglobulin membrane anchor located downstream of the stop codon.

[0569] 18. A method for selecting a common host cell for use in the method according to any one of embodiments 8-11, the method comprising

[0570] a. The first step: providing a plurality of host cells, wherein the plurality of host cells comprises an expression system according to any one of embodiments 12-17; and

[0571] b. Cultivating the plurality of host cells under conditions that are selective for the selection marker, thereby obtaining host cells that express a target product.

[0572] 19. The method according to embodiment 18, wherein a selective medium is used:

[0573] a. contain limited concentrations of folic acid; and / or

[0574] b. Containing folic acid at a concentration of 500 nM or less; and / or

[0575] c. Contains folic acid in a concentration selected from:

[0576] i. 1000nM-100pM;

[0577] ii.100nM-1nM;

[0578] iii.15nM-1nM;

[0579] iv. 10nM-1nM; and

[0580] v. 10nM-2.5nM; and / or

[0581] d. comprising a DHFR inhibitor; and / or

[0582] e. Contains antifolate; and / or

[0583] f. comprising an antifolate at a concentration of 500 nM or less; and / or

[0584] g. comprising MTX at a concentration selected from:

[0585] i.500nM-3nM;

[0586] ii.100nM-10nM;

[0587] iii. 50nM-10nM; and

[0588] iv.50nM and / or

[0589] h. an antifolate comprising a concentration up to 20 times the folic acid concentration; and / or

[0590] i. an antifolate comprising 10-20 times the concentration of folic acid; and / or

[0591] j. Contains folic acid at concentrations up to 15 nM and equimolar concentrations up to 20-fold that of MTX.

[0592] 20. The method according to embodiment 18 or 19, wherein the host cell comprises the expression system according to embodiment 17, wherein at least a portion of the first or second part is expressed as a fusion polypeptide comprising the immunoglobulin transmembrane anchor or a fragment thereof, and wherein the fusion polypeptide is displayed on the surface of the host cell, the method further comprising the steps of:

[0593] a. contacting the plurality of host cells with a detection compound that binds to the fusion polypeptide;

[0594] b. selecting at least one host cell based on the presence or amount of the test compound bound to the cell surface.

[0595] 21. A method according to embodiment 20, wherein the detection compound comprises the first or second target or its derivative and at least one detection label.

[0596] 22. The method according to embodiment 10 or 11, wherein the fifth step of purifying the bispecific antibody comprises affinity chromatography and / or ion exchange chromatography.

[0597] 23. The method according to embodiment 22, wherein the chromatogram comprises

[0598] a. The first step is capture;

[0599] b. a second step of refining; and optionally

[0600] c. The third step is further refinement.

[0601] 24. The method according to embodiment 23, wherein the first step of capturing is performed using a principle selected from the group consisting of: Fc binding affinity chromatography, such as protein A or protein G; lambda light chain specific affinity chromatography, such as LambdaFabSelect TM ; kappa light chain specific affinity chromatography, such as KappaSelect TM ; anti-idiotypic affinity chromatography, such as the first part or the second part; target-based affinity chromatography, such as affinity chromatography using the first target or the second target; ion exchange chromatography, such as Capto TM Adhesion or Fractogel TM EMD SO 3 ; and hydrophobic interaction chromatography.

[0602] 25. The method according to embodiment 22 or 23, wherein the second step of refining is performed using a principle selected from the group consisting of: Fc binding affinity chromatography, such as protein A or protein G; lambda light chain specific affinity chromatography, such as LambdaFabSelect TM ; kappa light chain specific affinity chromatography, such as KappaSelect TM ; anti-idiotypic affinity chromatography, such as the first part or the second part; target-based affinity chromatography, such as affinity chromatography using the first target or the second target; ion exchange chromatography, such as Capto TM Adhesion or Fractogel TM EMD SO 3 ; hydrophobic interaction chromatography; and viral inactivation.

[0603] 26. The method according to embodiment 25, wherein the third step of further refining is performed using a principle selected from the group consisting of: Fc binding affinity chromatography, such as protein A or protein G, alone or in combination; lambda light chain specific affinity chromatography, such as LambdaFabSelect TM ; kappa light chain specific affinity chromatography, such as KappaSelect TM ; anti-idiotypic affinity chromatography, such as the first part or the second part; target-based affinity chromatography, such as affinity chromatography using the first target or the second target; ion exchange chromatography, such as Capto TM Adhesion or Fractogel TM EMD SO 3 ; hydrophobic interaction chromatography; and viral inactivation.

[0604] 27. The method according to any one of Embodiment 23, wherein the method is selected from:

[0605] a. First step protein A capture, such as MabSelect TM SuRe TM ; Second step lambda light chain affinity chromatography, such as LambdaFabSelect TM ; and a third step kappa light chain affinity chromatography, such as KappaSelect TM ;or

[0606] b. First step protein A, such as MabSelect TM SuRe TM , second step kappa light chain affinity chromatography, such as KappaSelect TM , and a third step lambda light chain affinity chromatography, such as LambdaFabSelect TM ;or

[0607] c. First step kappa light chain affinity chromatography, such as KappaSelect TM , and a second step lambda light chain affinity chromatography, such as LambdaFabSelect TM ;or

[0608] d. First step lambda light chain affinity chromatography, such as LambdaFabSelect TM and a second step kappa light chain affinity chromatography, such as KappaSelect TM .

[0609] 28. A method according to any one of embodiments 8-11 or 18-27, wherein the cell line is selected from the group consisting of: CHO cells; non-producing hybridomas, such as Sp 2 / 0 or NS0; human cell lines, such as HEK or PER.C6; baby hamster kidney (BHK)-derived cells; yeast or filamentous fungi; prokaryotic bacteria, such as Escherichia coli or Pseudomonas fluorescens; plant-derived cells; algae; and ciliates.

[0610] 29. A pharmaceutical composition comprising the antibody according to any one of embodiments 1-7, and a pharmaceutically acceptable carrier.

[0611] 30. The bispecific antibody according to any one of embodiments 1-7 or the pharmaceutical composition according to embodiment 29, for use as a medicament.

[0612] 31. The bispecific antibody according to any one of embodiments 1-7 or the pharmaceutical composition according to embodiment 29, for use in treating an inflammasome-associated disease.

[0613] 32. The bispecific antibody according to any one of embodiments 1-7 or the pharmaceutical composition according to embodiment 29, for treating the inflammasome-associated disease according to embodiment 31, wherein the inflammasome-associated disease is selected from the group consisting of sickle cell disease, vasculopathy, ischemia-reperfusion injury, cardiovascular disease, peripheral arterial disease, atherosclerosis, vascular dysfunction, skeletal muscle ischemia, pulmonary sarcoidosis, fibrosis, malaria, hemodialysis-dependent chronic kidney disease, and Crohn's disease.

[0614] 33. A method for treating an inflammasome-associated disorder, the method comprising administering an effective amount of the bispecific antibody according to embodiments 1-7 or the pharmaceutical composition according to embodiment 29 to a subject suffering from the inflammasome-associated disorder.

[0615] 34. The method of embodiment 33, wherein the inflammasome-associated disorder is sickle cell disease, vasculopathy, ischemia-reperfusion injury, cardiovascular disease, peripheral arterial disease, atherosclerosis, vascular dysfunction, skeletal muscle ischemia, pulmonary sarcoidosis, fibrosis, malaria, hemodialysis-dependent chronic kidney disease, or Crohn's disease.

[0616] Sequence Listing

[0617] Useful amino acid and nucleotide sequences for practicing the present invention are disclosed in Table 15.

[0618] Table 15. Sequences according to embodiments of the present invention

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629] Throughout this application, if there is a discrepancy between the specification text (eg, Table 15) and the sequence listing, the specification text shall prevail.

Claims

1. A bispecific antibody comprising: a. a first part, wherein the first part is an immunoglobulin having a first light chain (LC1) and a first heavy chain (HC1) that specifically bind to IL-18, and b. a second part, wherein the second part is an immunoglobulin having a second light chain (LC2) and a second heavy chain (HC2) that specifically binds to IL-1β, in: (a) the first immunoglobulin heavy chain (HC1) comprises the amino acid sequence SEQ ID NO: 87, (b) the first immunoglobulin light chain (LC1) comprises the amino acid sequence of SEQ ID NO: 103, (c) the second immunoglobulin heavy chain (HC2) comprises the amino acid sequence of SEQ ID NO: 55, and (d) the second immunoglobulin light chain (LC2) comprises the amino acid sequence of SEQ ID NO:

71.

2. Use of the bispecific antibody according to claim 1 in the preparation of a medicament for treating a patient suffering from an inflammasome-associated disease.

3. Use of the bispecific antibody according to claim 2 in the preparation of a medicament for treating a patient suffering from an inflammasome-associated disease, wherein the inflammasome-associated disease is selected from the group consisting of sickle cell disease, vasculopathy, ischemia-reperfusion injury, cardiovascular disease, peripheral arterial disease, atherosclerosis, vascular dysfunction, skeletal muscle ischemia, pulmonary sarcoidosis, fibrosis, malaria, hemodialysis-dependent chronic kidney disease and Crohn's disease.

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