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 No. 201880036745.X (PCT / IB2018 / 054140), with the filing date of June 8, 2018 and the invention title of "Method for manufacturing bispecific antibodies, bispecific antibodies and therapeutic uses of such antibodies".

[0002] Sequence Listing

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

[0004] The present invention relates to bivalent bispecific monoclonal antibodies (bbmAbs) or variants thereof, and methods for manufacturing 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 two different epitopes, are well known in the art. One method for generating bispecific antibodies is the so-called knob-into-hole (KiH) method, as described, for example, by Merchant et al., Nat. Biotechnol. [Nature Biotechnology], 16:677-681 (1998), where the first heavy chain IgG is modified by introducing point mutations such as Y349C, T366S, L368A, Y407V to display a hole-like structure; and the second heavy chain IgG is modified by introducing point mutations S354C, T366W (Merchant et al., Nat. Biotechnol. [Nature Biotechnology], 16:677-681 (1998), page 678, Table 1) to display a knob-like structure. Then, the two different IgG structures interact to form a bivalent bispecific antibody (bbmAb), i.e., a heterotetrameric protein composed of four different light chains and two different heavy chains.

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

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

[0008] Another method for generating bbmAbs is disclosed in WO 12023053A2 or WO 04009618A2, which utilizes shared heavy or light chains combined with different variable chains. However, keeping either heavy chain constant significantly reduces the diversity of the antibody library of screenable conjugates.

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

[0010] Thus, existing methods for generating bbmAbs may limit the diversity of the antibody library available for screening conjugates or may not provide sufficient overall yield, purity, and product quality in a cost-effective enough manner for large-scale production for clinical development and commercialization. Additionally, any modification of the protein chain inherently increases the risk of inducing anti-drug antibodies. Therefore, a method that requires 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 present invention particularly provides a method for producing bbmAbs that has one or more of the following advantages: it can use a large antibody library to identify conjugates since it does not require shared light or heavy chains, it does not require any extensive protein engineering except for mutations that drive H-chain dimerization, thus limiting the risk of anti-drug antibodies, it is cost-effective since it is completed 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 materials suitable for class use since product-related impurities can be effectively removed.

[0013] The present invention can be used to identify κ- and λ-type antibodies, where the light chains do not show strong promiscuous binding to the heavy chains of their counterparts. This makes the antibodies suitable for use in the methods of the present invention. The advantage of the method is that antibody combinations in which both light chains exchange their original heavy chain binding partners (which results in product-related H1L2-H2L1 type impurities) can be eliminated. This is advantageous because it is not easy to deplete such product-related impurities using prior art purification methods.

[0014] As shown below, the examples of the present invention enable the production of bbmAbs by using CHO co-expression, with a yield and quality suitable for the clinical development and commercialization of biopharmaceuticals.

[0015] In a first aspect of the present invention, there is provided a bispecific antibody suitable for co-expression in a common host cell, 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) (which 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) (which specifically binds to a second target different from the first target) and a second constant heavy chain (CH2) with a heterodimerization modification 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 antibody suitable for co-expression in a common host cell produces a bispecific antibody that is at least 60% (by mass), 70% (by mass), 80% (by mass), 85% (by mass) pure, for example at least 90% (by mass) pure, 95% (by mass), 96% (by mass), 97% (by mass), 98% (by mass) or 99% (by mass) pure after purifying the bispecific antibody by removing mismatched fragments from the correctly matched bispecific antibody.

[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 of the λ type and the second variable light chain is of the κ 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 can be IgG1, where the first constant heavy chain has a point mutation that produces a pestle structure and the second constant heavy chain has a point mutation that produces a mortar structure, or the first constant heavy chain has a point mutation that produces a mortar structure and the second constant heavy chain has a point mutation that produces a pestle structure. Optionally, the first constant heavy chain and the second constant heavy chain can 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 sequence): hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:76, CDR2 has the amino acid sequence SEQ ID NO:77, and CDR3 has the amino acid sequence SEQ ID NO:78; or hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:79, CDR2 has the amino acid sequence SEQ ID NO:80, and CDR3 has the amino acid sequence SEQ ID NO:81; and the first immunoglobulin VL1 domain comprises (e.g., in sequence): hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:92, CDR2 has the amino acid sequence SEQ ID NO:93, and CDR3 has the amino acid sequence SEQ ID NO:94 or hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:95, CDR2 has the amino acid sequence SEQ ID NO:96, and CDR3 has the amino acid sequence SEQ ID NO:97; the second immunoglobulin VH2 domain comprises (e.g., in sequence): hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:44, CDR2 has the amino acid sequence SEQ ID NO:45, and CDR3 has the amino acid sequence SEQ ID NO:46; or hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:47, CDR2 has the amino acid sequence SEQ ID NO:48, and CDR3 has the amino acid sequence SEQ ID NO:49; and the second immunoglobulin VL2 domain comprises (e.g., in sequence): hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:60, CDR2 has the amino acid sequence SEQ ID NO:61, and CDR3 has the amino acid sequence SEQ ID NO:62 or hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:63, CDR2 has the amino acid sequence SEQ ID NO:64, and CDR3 has the amino acid sequence 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 SEQ ID NO:85, the first immunoglobulin VL1 domain comprises the amino acid sequence SEQ ID NO:101, the second immunoglobulin VH2 domain comprises the amino acid sequence SEQ ID NO:53, and the second immunoglobulin VL2 domain comprises the amino acid sequence 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 SEQ ID NO:87, the first immunoglobulin light chain comprises the amino acid sequence SEQ ID NO:103, the second immunoglobulin heavy chain comprises the amino acid sequence SEQ ID NO:55, and the second immunoglobulin light chain comprises the amino acid sequence SEQ ID NO:71.

[0023] According to a second aspect, there is provided a method of selecting a bispecific antibody according to the first aspect, 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; and a third step: purifying the bispecific antibody by removing mismatched fragments from correctly matched bispecific antibodies. In one embodiment, the third step purifies to produce a bispecific antibody that is at least 60% (by mass), 70% (by mass), 80% (by mass), 85% (by mass) pure, such as at least 90% (by mass) pure, 95% (by mass), 96% (by mass), 97% (by mass), 98% (by mass) or 99% (by mass) pure.

[0024] According to a third aspect, there is provided a method of making a bispecific antibody according to the first aspect by co-expression in a common host cell, the method comprising: a first step: generating 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 bispecific antibody is expressed by the cells; and a fifth step: purifying the bispecific antibody, which is at least 60% (by mass), 70% (by mass), 80% (by mass), 85% (by mass) pure, such as at least 90% (by mass) pure, 95% (by mass), 96% (by mass), 97% (by mass), 98% (by mass) or 99% (by mass) pure.

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

[0026] According to a fourth aspect, an expression system comprising at least one vector (which comprises a polynucleotide encoding a first or a second part of a 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), which 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 mutant folate receptor, wherein the mutant folate receptor has a 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, which comprises at least a polynucleotide encoding the first selection marker (sm I) and at least a polynucleotide encoding the first part; a second vector, which comprises at least a polynucleotide encoding the second selection marker (sm II) and at least a polynucleotide encoding the 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 a fifth aspect, there is provided a method for selecting a common host cell for use in the method according to the foregoing aspects, 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 foregoing aspects; and culturing the plurality of host cells under conditions selective for the selection marker, thereby obtaining a host cell expressing the desired product.

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

[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, and wherein the fusion polypeptide is displayed on the surface of the host cell, and further comprises the steps of: 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 a 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, the 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 of capture is carried out using a principle selected from the group consisting of: Fc-binding affinity chromatography, such as Protein A or Protein G; λ light chain-specific affinity chromatography, which is well known in the art and commercially available, for example, LambdaFabSelect TM ; κ light chain-specific affinity chromatography, which is well known in the art and commercially available, for example, KappaSelect TM ; anti-idiotype affinity chromatography, such as the first or second portion; target-based affinity chromatography, such as affinity chromatography using the first or second target; ion exchange chromatography, which is well known in the art and commercially available, for example, Capto TM adhesion or Fractogel TM EMD SO 3; and hydrophobic interaction chromatography.

[0039] In one embodiment, the second step of purification is carried out 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 Adhere or Fractogel TM EMD SO 3 ; hydrophobic interaction chromatography; and virus inactivation.

[0040] In one embodiment, the third step of purification is carried out 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 Adhere or Fractogel TM EMD SO 3 ; hydrophobic interaction chromatography; and virus inactivation.

[0041] In one embodiment, the method includes a first step of Protein A capture, such as MabSelect TM SuRe TM ; a second step of lambda light chain affinity chromatography, such as LambdaFabSelect TM ; and a third step of kappa light chain affinity chromatography, such as KappaSelect TM ; or a first step of Protein A, such as MabSelect TM SuRe TM ; a second step of kappa light chain affinity chromatography, such as KappaSelect TM ; and a third step of lambda light chain affinity chromatography, such as LambdaFabSelect TM ; or a first step of kappa light chain affinity chromatography, such as KappaSelect TM ; and a second step of lambda light chain affinity chromatography, such as LambdaFabSelect TM; or a first step of lambda light chain affinity chromatography, such as LambdaFabSelect TM , and a second step of 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 (such as Sp2 / 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 (E. coli) or Pseudomonas fluorescence), plant-derived cells, algae, and ciliates.

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

[0044] 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 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 the treatment of inflammasome-related diseases.

[0046] According to an eighth aspect, there is provided an antibody according to the first aspect or a pharmaceutical composition according to the sixth aspect, for the treatment of inflammasome-related diseases, wherein the inflammasome-related diseases are selected from the group consisting of: sickle cell disease, angiopathy, ischemia-reperfusion injury, cardiovascular disease, peripheral artery 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-related disorder, the method comprising administering to a subject suffering from an inflammasome-related disorder an effective amount of an antibody according to the first aspect or a pharmaceutical composition according to the sixth aspect.

[0048] The inflammasome-related disorder can be sickle cell disease, angiopathy, ischemia-reperfusion injury, cardiovascular disease, peripheral artery 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 the carrier arrangement according to the embodiment;

[0050] Figures 2A - 2EShows a chromatogram according to an embodiment. Figure 2A Is the RP-UV chromatogram of deglycosylated intact bbmAb according to an embodiment. Figure 2B Is the deconvoluted mass spectrum of intact deglycosylated bbmAb1 according to an embodiment. Figure 2C Is the RP-UV chromatogram showing papain digested bbmAb fragments according to an embodiment. Figure 2D Is the RP-UV chromatogram showing IdeS digested fragments of bbmAb according to an embodiment. Figure 2E Is the RP-UV chromatogram showing deglycosylated and DTT-reduced bbmAb fragments according to an embodiment.

[0051] Figures 3A - 3D Shows the RP-UV chromatogram according to an embodiment. Figure 3A Is the chromatogram showing the expression purity profile of bbmAb after cultivation according to an embodiment. Figure 3B Is the bbmAb according to an embodiment after being captured by LambdaFabSelect TM The chromatogram after capture. Figure 3C Is the bbmAb according to an embodiment after being captured by MabSelect TM SuRe TM The chromatogram after capture by TM. Figure 3D Is the bbmAb according to an embodiment after being captured by LambdaFabSelect TM TM, purified by Fractogel TM EMD SO 3 The chromatogram after purification and ultrafiltration.

[0052] Figures 4A - 4M Is a schematic diagram of different options for bispecific mismatches. Figure 4A Is a schematic diagram of mAb1 pestle (λ) monomer, where the number 1 represents the variable heavy chain domain, the number 2 represents the first constant heavy chain domain, the number 3 represents the second constant heavy chain domain, the number 4 represents the third constant heavy chain domain. The number 5 represents the variable light chain domain, and the number 6 represents the variable heavy chain domain. Figure 4B Is a schematic diagram of mAb1 pestle (λ) homodimer. Figure 4C Is a schematic diagram of mAb2 mortar (κ) monomer, where the number 7 represents the variable heavy chain domain, the number 8 represents the first constant heavy chain domain, the number 9 represents the second constant heavy chain domain, the number 10 represents the third constant heavy chain domain. The number 11 represents the variable light chain domain, and the number 12 represents the constant heavy chain domain. Figure 4D Is a schematic diagram of mAb2 mortar (κ) homodimer. Figure 4E Is a schematic diagram of mAb1 pestle 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. Figure 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] Figures 6A - 6B Two diagrams according to examples are shown.

[0055] Figures 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] Figure 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 lambda (λ) light chains, which can be co-expressed with certain antibodies with kappa (κ) light chains to form the desired bbmAb, also referred to hereinafter as monospecific conjugates, can be generated by using techniques that provide the opportunity to obtain both types of antibodies, κ or λ, 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 antibodies (OMT), Kymouse TM (Kymab), Trianni Mouse TM (Trianni), or AlivaMab Mouse (Ablexis) (reference) can generate κ or λ antibodies. Methods for generating such monospecific conjugates are well known in the art and are widely used to generate diverse sets of κ or λ monospecific conjugates against a target of interest. Each monospecific conjugate is characterized in terms of relevant biological parameters such as affinity or potency and screened, for example, for physicochemical characteristics relevant to judging said developability characteristics (which are also well known in the art) (e.g., Lorenz et al., American Pharmaceutical Review, August 2014). The monospecific conjugate showing the best characteristics is ultimately co-expressed, for example, in CHO cells, as described in more detail below. Only test combinations of co-expression are made, in which a κ antibody binding to a first target is combined with a λ antibody binding to a second target and vice versa. A detailed characterization of the final co-expression product and the associated product-related impurities is aimed at selecting a combination that gives the best profile, especially those that only show a small amount of promiscuous binding of one light chain (e.g., L1, light chain 1, e.g., λ) to the wrong heavy chain (e.g., H2, heavy chain 2). The advantage of this method is that antibody combinations in which both light chains exchange their original heavy chain binding partners (which results in product-related H1L2-H2L1 type impurities) can be eliminated. This is advantageous because it is not easy to deplete such product-related impurities using purification methods of the prior art. The procedures for co-expressing individual antibodies and for analyzing the co-expression product are outlined in more detail below.

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

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

[0065] As shown in the following specific examples, the preferred embodiment bbmAb1 is expressed using a single common cell line, thus ensuring sufficient total yield, purity, and product quality required for biology or diagnostics for clinical development and commercialization.

[0066] 1. Definitions

[0067] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular form also include the plural form, and vice versa. Additional definitions are stated 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 specified, 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 Corporation Corporation) under product number #B001-5. Throughout the specification, the term IL-18 may interchangeably cover 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 a synonym for IL-1β polypeptide and interleukin 1β polypeptide. Unless otherwise specified, 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 typically comprise a tetramer, which generally consists 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 (usually consisting of three domains (CH1, CH2, and CH3)). The heavy chain can belong to any isotype, including IgG (IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM, and IgE. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Light chains include kappa (κ) chains and lambda (λ) chains. The heavy and light chain variable regions are generally responsible for antigen recognition, while the heavy and light chain constant regions can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). 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 is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: 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 "antigen-binding portion" (or simply "antigen portion") of an antibody refers to a full-length antibody or one or more fragments of an antibody that retain the ability to specifically bind to an IL-18 or IL-1β antigen. It has been shown that fragments of full-length antibodies can perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of VH domains (Ward et al., 1989, Nature 341:544-546); and isolated complementarity-determining regions (CDRs).

[0072] In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, the two domains can be joined using recombinant methods by a flexible linker that enables them to form a single protein chain, where the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc Natl Acad Sci USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and these 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 in a physical environment different from that in which it occurs in nature.

[0074] Throughout the specification, Complementary Determining Regions (“CDRs”) are defined according to the Kabat definition, unless it is specified that the CDRs are defined according to another definition. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest”, 5th ed., U.S. National Institutes of Health, Public Health Service, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), and the 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 Chothia, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL. According to IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3) (according to the “Kabat” numbering). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0075] By convention, the CDR regions in the heavy chain are usually designated as H-CDR1, H-CDR2, and H-CDR3, and the CDR regions in the light chain are usually designated as L-CDR1, L-CDR2, 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 a single molecular composition. Monoclonal antibody compositions exhibit 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 with sequences derived from human sources for both the framework regions and the CDR regions. In addition, if the antibody contains constant regions, the constant regions are 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 the analysis of human framework sequences, e.g., as described by Knappik et al., (2000), J Mol Biol; 296:57-86.

[0078] The human antibodies of the present invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random mutagenesis or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which the CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted onto human framework sequences.

[0079] The term "human monoclonal antibody" refers to an antibody having variable regions that exhibit a single binding specificity, wherein both the framework regions and the CDR regions are derived from human sequences.

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

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

[0082] As used herein, a binding molecule that "specifically binds IL-18" is intended to mean a binding molecule having a K of 100 nM or less, 10 nM or less, 1 nM or less D A binding molecule that binds human IL-18.

[0083] As used herein, a binding molecule that "specifically binds IL-1β" is intended to mean a binding molecule having a K of 100 nM or less, 10 nM or less, 1 nM or less D A binding molecule that binds human IL-1β.

[0084] A binding molecule that cross-reacts with an antigen other than IL-18 is a binding molecule having a K of 100 nM or less, 10 nM or less, 1 nM or less D A binding molecule that binds that antigen. A binding molecule that cross-reacts with an antigen other than IL-1β is a binding molecule having a K of 100 nM or less, 10 nM or less, 1 nM or less D A binding molecule that binds that antigen.

[0085] A binding molecule that "does not cross-react with a particular antigen" is intended to mean a binding molecule that exhibits substantially undetectable binding to those proteins in a standard binding assay.

[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 in the presence of IL-18 in a human cell assay such as an IL-18-dependent interferon-gamma (IFN-gamma) production assay. Examples of IL-18-dependent IFN-gamma production assays in human blood cells are described in more detail in the examples below.

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

[0088] A bispecific antibody is a "heterodimer," meaning that one part is from a first antibody specific for a first target and the other part is from a second antibody specific for a second target. "Heterodimer modification" is a modification of one or both parts of the antibodies that are intended to form a heterodimeric bispecific antibody, which is intended to facilitate such formation. An example of a heterodimer modification of the Fc domains of two IgG1 parts that are intended to form a bispecific antibody is the "stalk" with large amino acid (aa) side chains (S354C, T366W) in the first heavy chain and the "socket" with small amino acid side chains (Y349C, T366S, L368A, Y407V) introduced in the second heavy chain and an additional disulfide bridge connecting the two heavy chains in the CH3 region (Merchant et al., Nat. Biotechnol. [Nature Biotechnology], 16:677-681 (1998), page 678, Table 1).

[0089] As used herein, an antibody that is "agonist-inactive" is intended to mean 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-gamma production assay in the absence and / or presence of IL-18. Assays of this type are described in more detail in the examples below.

[0090] The term "K assoc " or "K a " as used herein is intended to refer to the association rate of a particular binding molecule-antigen interaction, while the term "K dis " or "K d " as used herein 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 the ratio of K d to K a (i.e., K d / K a) and expressed as molar concentration (M). The K of the antibody can be determined using methods well established in the art. D value. Methods for determining the K of an antibody D are by using 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" with respect to an antibody refers to an antibody having a KD of 1 nM or less for a target antigen.

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

[0094] The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles, etc.

[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 the production of cells or organisms (usually eukaryotic cells such as cells of Pichia pastoris, Chinese hamster ovary cells (CHO), or human cells). The optimized nucleotide sequence is engineered to exactly preserve 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. This identity can be expressed as a percentage of identity and can be determined by standard alignment algorithms (such as the Basic Local Alignment Search Tool (BLAST) (Altshul et al., (1990) J Mol Biol; 215:403 - 410)); the algorithm of Needleman et al., (1970) J Mol Biol; 48:444 - 453; or the algorithm of Meyers et al., (1988) Comput Appl Biosci; 4:11 - 17). A set of parameters can be the Blosum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. 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), can also be used to determine the percentage identity between two amino acid or nucleotide sequences using the PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percentage of identity is typically calculated by comparing sequences of similar length.

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

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

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

[0100] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, and polymers thereof. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequences. 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 a mixture of bases 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 "polynucleotide" or "nucleic acid" can include modifications, including base modifications such as bromouridine and inosine derivatives; ribose modifications such as phosphorothioate, phosphorodithioate, phosphororoselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, and phosphoroamidate.

[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 contains a nucleic acid sequence that directs and / or controls (in association with the 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 the polypeptides. Co-expression of a bispecific antibody refers to the expression of the 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 expressing 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 different parts of an expected protein complex (e.g., a bispecific antibody) not complexing as expected, meaning that the appearance or behavior of the protein complex does not conform to expectations. An example of a mismatch in the case of a bispecific antibody is shown in Figure 4.

[0105] A "conservative variant" of a sequence encoding a binding molecule, antibody or fragment thereof refers to a sequence that contains conservative amino acid modifications. "Conservative amino acid modifications" are intended to mean amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), 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 disclosure by standard techniques known in the art, which are 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, peptidomimetics (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 the present specification, the term "epitope" can be used interchangeably for conformational epitopes and linear epitopes. A conformational epitope is composed of discontinuous parts of an antigen amino acid sequence, while a linear epitope is formed by a continuous sequence of amino acids of an antigen.

[0107] The terms "treat", "treating", "treatment", "prevent", "preventing", "prevention" include therapeutic treatment, prophylactic treatment and applications which reduce the risk that a subject will develop a disorder or other risk factors. Treatment need not completely cure the disorder and includes alleviating symptoms or potential risk factors. As used herein, a human antibody or fragment thereof comprises a heavy or light chain variable region or 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 using human germline immunoglobulin genes. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with an antigen of interest or screening a human immunoglobulin gene library displayed on phage with an antigen of interest. A human antibody or fragment thereof that is a "product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is most closely related (i.e., highest % identity) to the sequence of the human antibody. A human antibody that is a "product of" or "derived from" a particular human germline immunoglobulin sequence can contain amino acid differences compared to the germline sequence due to, for example, naturally occurring somatic mutations or deliberately introduced site-directed mutations. However, the amino acid sequence of the selected human antibody typically is 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 a human antibody when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In some cases, the amino acid sequence of a human antibody can be at least 60%, 70%, 80%, 90% or at least 95% or even at least 96%, 97%, 98% or 99% identical to the amino acid sequence encoded by a germline immunoglobulin gene. Typically, a human antibody derived from a particular germline sequence will exhibit no more than 10 amino acid differences from the amino acid sequence encoded by a human germline immunoglobulin gene. In some cases, a human antibody can exhibit no more than 5 or even no more than 4, 3, 2 or 1 amino acid difference from the amino acid sequence encoded by a germline immunoglobulin gene.

[0108] Human antibodies can be produced by a number of methods known to those of skill in the art. Human antibodies can be prepared by hybridoma methods using human myeloma or mouse-human heteromyeloma cell lines (Kozbor, J Immunol; (1984) 133:3001; Brodeur, 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 12:433-455, Green LL, (1999) J Immunol Methods 231:11-23).

[0109] Several transgenic mouse lines are now available in which their murine 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). After antigen challenge, such mice are capable of generating a human antibody repertoire from which antibodies 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, Babcock et al, Proc Natl Acad Sci (1996) 93:7843-7848), in which human (or other species) lymphocytes are effectively passed through a large pooled in vitro antibody generation procedure followed by deconvolution, limiting dilution and selection procedures and Xenomouse TM (Abgenix). Another method is available from Morphotek using Morphodoma TM technology.

[0110] Phage display technology can be used to generate human antibodies and their fragments (McCafferty; (1990) Nature, 348:552-553 and Griffiths AD et al. (1994) EMBO 13:3245-3260). According to this technology, the isolated antibody variable domain genes are cloned in-frame into the major or minor coat of the protein gene of a filamentous phage (such as M13 or fd) and are displayed as functional isolated antibody fragments (usually with the help of a helper phage) on the surface of the phage particle. Selection based on the functional properties of the isolated antibody 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 taken from an individual suffering from a disease or disorder or alternatively from non-immunized human donors (Marks; J Mol Bio (1991) 222:581-591). When full-length human isolated antibodies containing the Fc domain are required, the phage-displayed derived fragments must be recloned into a mammalian expression vector containing the required constant regions and a stable expression cell line established.

[0111] Techniques for affinity maturation (Marks; Biotechnol (1992) 10:779-783) can be used to provide binding affinity, where the affinity of a human naïve isolated antibody is improved by sequentially replacing the variable regions of the H and L chains with naturally occurring variants and selecting on the basis of improved binding affinity. Variants of this technique are also now available, such as "epitope imprinting" (WO 93 / 06213; Waterhouse; Nucl Acids Res (1993) 21:2265-2266).

[0112] When used in the context of a purified bispecific antibody, the term "pure" relates to the purity and identity of different bispecific antibody combinations and constructs after co-expression in a selected cell under conditions where the cell expresses the bispecific antibody and after protein A purification using a complete UPLC-MS mass screening method. Pure or purity refers to the relative quantification of the heterodimers and homodimers of the bbmAb formed. Using the methods of the present invention, correctly formed heterodimers 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 used in the disclosed methods are human antibodies.

[0115] For ease of reference, Table 1 below provides the amino acid sequences of the hypervariable regions of a specific IL-18 antibody (referred to as mAb1) based on Kabat definition and Chothia definition, as well as the V L and V H domains and 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 VL of mAb1 is listed in SEQ ID NO:18. The DNA encoding VH of mAb1 is listed in SEQ ID NO:8.

[0117]

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

[0119] In one embodiment, the IL-18 antibody or its antigen-binding fragment comprises at least one immunoglobulin light chain variable domain (V L ) containing hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:11, CDR2 has the amino acid sequence SEQ ID NO:12, and CDR3 has the amino acid sequence SEQ ID NO:13. In one embodiment, the IL-18 antibody or its antigen-binding fragment comprises at least one immunoglobulin light chain variable domain (V L ) containing hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:14, CDR2 has the amino acid sequence SEQ ID NO:15, and CDR3 has the amino acid sequence 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 domain comprises (e.g., in sequence): i) hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:1, CDR2 has the amino acid sequence SEQ ID NO:2, and CDR3 has the amino acid sequence SEQ ID NO:3; or ii) hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:4, CDR2 has the amino acid sequence SEQ ID NO:5, and CDR3 has the amino acid sequence SEQ ID NO:6; and b) the immunoglobulin V L domain comprises (e.g., in sequence): i) hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:11, CDR2 has the amino acid sequence SEQ ID NO:12, and CDR3 has the amino acid sequence SEQ ID NO:13 or ii) hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:14, CDR2 has the amino acid sequence SEQ IDNO:15, and CDR3 has the amino acid sequence 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 H ) comprising the amino acid sequence set forth in SEQ ID NO:7; b) an immunoglobulin light chain variable domain (V L ) comprising the amino acid sequence set forth in SEQ ID NO:17; c) an immunoglobulin V H domain comprising the amino acid sequence set forth in SEQ ID NO:7 and an immunoglobulin V L domain comprising the amino acid sequence set forth in SEQ ID NO:17; d) an immunoglobulin V H domain comprising the hypervariable regions set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; e) an immunoglobulin V LDomain; f) an immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 H Domain; g) an immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16 L Domain; h) an immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 H Domain and an immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13 L Domain; i) an immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 H Domain and an immunoglobulin V domain comprising the hypervariable regions 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 the three CDRs of SEQ ID NO:7. In other embodiments, the IL-18 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:17. In other embodiments, the IL-18 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:7 and the three CDRs of SEQ ID NO:17. In some embodiments, the IL-18 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:9. In other embodiments, the IL-18 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:19. In other embodiments, the IL-18 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO:9 and the 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 human IL-18 antibodies, the human IL-18 antibody comprising at least: a) an immunoglobulin heavy chain or fragment thereof, the immunoglobulin heavy chain or fragment thereof comprising a variable domain and a constant portion of a human heavy chain or fragment thereof, the variable domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3; the CDR1 having the amino acid sequence SEQ ID NO:1, the CDR2 having the amino acid sequence SEQ ID NO:2, and the CDR3 having the amino acid sequence SEQ ID NO:3; and b) an immunoglobulin light chain or fragment thereof, the immunoglobulin light chain or fragment thereof comprising a variable domain and a constant portion of a human light chain or fragment thereof, the variable domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3, the CDR1 having the amino acid sequence SEQ ID NO:11, the CDR2 having the amino acid sequence SEQ ID NO:12, and the CDR3 having the amino acid sequence SEQ ID NO:13.

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

[0125] In one embodiment, the IL-18 antibody or antigen-binding fragment thereof is selected from single-chain antibodies or antigen-binding fragments thereof comprising an antigen-binding site, the antigen-binding site comprising: a) a first domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:1, CDR2 has the amino acid sequence SEQ ID NO:2, and CDR3 has the amino acid sequence SEQ ID NO:3; and b) a second domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:11, CDR2 has the amino acid sequence SEQ ID NO:12, and CDR3 has the amino acid sequence SEQ ID NO:13; and c) a peptide linker that binds 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 single-chain antibodies or antigen-binding fragments thereof comprising an antigen-binding site, the antigen-binding site comprising: a) a first domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:4, CDR2 has the amino acid sequence SEQ ID NO:5, and CDR3 has the amino acid sequence SEQ ID NO:6; and b) a second domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:14, CDR2 has the amino acid sequence SEQ ID NO:15, and CDR3 has the amino acid sequence SEQ ID NO:16; and c) a peptide linker that binds 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] The V H or V L domains of the IL-18 antibody or antigen-binding fragment thereof used in the disclosed method may have V H or V L domains that are substantially the same as the V H and / or V LDomain. The human IL-18 antibodies 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 antibodies disclosed herein may comprise: a heavy chain containing SEQ ID NO:9 and a light chain containing SEQ ID NO:19. The human IL-18 antibodies disclosed herein may comprise: a) a heavy chain comprising a variable domain having an amino acid sequence substantially identical to the amino acid sequence 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 (such as antibodies) for the disclosed methods, kits, and protocols are those listed below: U.S. Patent No. 9,376,489, which is incorporated herein by reference in its entirety.

[0129] 3. IL-1β Antibody

[0130] A particularly preferred IL-1β antibody or antigen-binding fragment thereof used in the disclosed methods is a human antibody.

[0131] For ease of reference, Table 2 below provides the amino acid sequences of the hypervariable regions of a specific IL-1β antibody (designated mAb2) based on Kabat definitions and Chothia definitions, as well as the V L and V H domains and the complete heavy and light chains.

[0132] Table 2. Amino acid sequences of the hypervariable regions (CDRs), variable domains (VH and VL), and full chains of mAb2. The DNA encoding VL of mAb2 is listed in SEQ ID NO:38. The DNA encoding 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), wherein the CDR1 has the amino acid sequence SEQ ID NO: 21, the CDR2 has the amino acid sequence SEQ ID NO: 22, and the CDR3 has the amino acid sequence SEQ ID NO: 23. In one embodiment, the IL-1β antibody or antigen-binding fragment thereof comprises at least one immunoglobulin heavy chain variable domain (V H ), wherein the CDR1 has the amino acid sequence SEQ ID NO: 24, the CDR2 has the amino acid sequence SEQ ID NO: 25, and the CDR3 has the amino acid sequence SEQ ID NO: 26.

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

[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 domain comprises (e.g., in sequence): i) hypervariable regions CDR1, CDR2, and CDR3, wherein 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; or ii) hypervariable regions CDR1, CDR2, and CDR3, wherein the CDR1 has the amino acid sequence SEQ ID NO: 24, the CDR2 has the amino acid sequence SEQ ID NO: 25, and the CDR3 has the amino acid sequence SEQ ID NO: 26; and b) the immunoglobulin V LThe domain contains (e.g., in sequence): i) hypervariable regions CDR1, CDR2, and CDR3, where CDR1 has the amino acid sequence SEQ ID NO:31, CDR2 has the amino acid sequence SEQ ID NO:32, and CDR3 has the amino acid sequence SEQ ID NO:33 or ii) hypervariable regions CDR1, CDR2, and CDR3, where CDR1 has the amino acid sequence SEQ ID NO:34, CDR2 has the amino acid sequence SEQ ID NO:35, and CDR3 has the amino acid sequence SEQ ID NO:36.

[0137] In one embodiment, the IL-1β antibody or its antigen-binding fragment comprises: a) an immunoglobulin heavy chain variable domain (V H ) containing the amino acid sequence set forth in SEQ ID NO:27; b) an immunoglobulin light chain variable domain (V L ) containing the amino acid sequence set forth in SEQ ID NO:37; c) an immunoglobulin V H domain containing the amino acid sequence set forth in SEQ ID NO:27 and an immunoglobulin V L domain containing the amino acid sequence set forth in SEQ ID NO:37; d) an immunoglobulin V H domain containing the hypervariable regions set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; e) an immunoglobulin V L domain containing the hypervariable regions set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; f) an immunoglobulin V H domain containing the hypervariable regions set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26; g) an immunoglobulin V L domain containing the hypervariable regions set forth in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36; h) an immunoglobulin V H domain containing the hypervariable regions set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 and an immunoglobulin V L domain containing the hypervariable regions set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; i) an immunoglobulin V Ha domain and an immunoglobulin V domain comprising hypervariable regions set forth in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36 L 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-1β antibody or an antigen-binding fragment thereof (e.g., mAb2) comprises three CDRs of SEQ ID NO:37. In other embodiments, the IL-1β antibody or an antigen-binding fragment thereof comprises three CDRs of SEQ ID NO:27. In other embodiments, the IL-1β antibody or an antigen-binding fragment thereof comprises three CDRs of SEQ ID NO:37 and three CDRs of SEQ ID NO:27. In some embodiments, the IL-1β antibody or an antigen-binding fragment thereof comprises three CDRs of SEQ ID NO:39. In other embodiments, the IL-1β antibody or an antigen-binding fragment thereof comprises three CDRs of SEQ ID NO:29. In other embodiments, the IL-1β antibody or an 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 an antigen-binding fragment thereof (e.g., mAb2) is selected from human IL-1β antibodies, the human IL-1β antibody comprising at least: a) an immunoglobulin heavy chain or a fragment thereof, the immunoglobulin heavy chain or a fragment thereof comprising a variable domain and a constant portion of a human heavy chain or a fragment thereof, the variable domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3; CDR1 having the amino acid sequence SEQ ID NO:21, CDR2 having the amino acid sequence SEQ ID NO:22, and CDR3 having the amino acid sequence SEQ ID NO:23; and b) an immunoglobulin light chain or a fragment thereof, the immunoglobulin light chain or a fragment thereof comprising a variable domain and a constant portion of a human light chain or a fragment thereof, the variable domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3, CDR1 having the amino acid sequence SEQ ID NO:31, CDR2 having the amino acid sequence SEQ ID NO:32, and CDR3 having the amino acid sequence SEQ ID NO:33.

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

[0141] In one embodiment, the IL-1β antibody or its antigen-binding fragment is selected from single-chain antibodies or their antigen-binding fragments comprising an antigen-binding site, and the antigen-binding site comprises: a) a first domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3, the CDR1 has the amino acid sequence SEQ ID NO:21, the CDR2 has the amino acid sequence SEQ ID NO:22, and the CDR3 has the amino acid sequence SEQ ID NO:23; and b) a second domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3, 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; and c) a peptide linker that binds 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 single-chain antibodies or antigen-binding fragments thereof comprising an antigen-binding site, the antigen-binding site comprising: a) a first domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:24, CDR2 has the amino acid sequence SEQ ID NO:25, and CDR3 has the amino acid sequence SEQ ID NO:26; and b) a second domain sequentially comprising hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:34, CDR2 has the amino acid sequence SEQ ID NO:35, and CDR3 has the amino acid sequence SEQ ID NO:36; and c) a peptide linker that binds 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] The V H or V L domains of the IL-1β antibody or antigen-binding fragment thereof used in the disclosed methods can have V H or V L domains that are substantially the same as the V H and / or V L domains listed in SEQ ID NO:27 and 37. The human IL-1β antibodies disclosed herein can comprise a heavy chain that is substantially the same as the heavy chain shown in SEQ ID NO:29 and / or a light chain that is substantially the same as the light chain shown in SEQ ID NO:39. The human IL-1β antibodies disclosed herein can comprise: a heavy chain containing SEQ ID NO:29 and a light chain containing SEQ ID NO:39. The human IL-1β antibodies disclosed herein can comprise: a) a heavy chain comprising a variable domain having an amino acid sequence substantially the same as 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 the same as the amino acid sequence shown in SEQ ID NO:37 and a constant portion of a human light chain.

[0144] Other preferred IL-1β antagonists (e.g., antibodies) for the disclosed methods, kits, and regimens are those listed below: U.S. Patent 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 or as an alternative to the modifications made in the framework or CDR regions, the antibodies of the present invention can be engineered to contain modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cell cytotoxicity. 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 alter their glycosylation, thereby again altering 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 according to 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 such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. The method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. Altering the number of cysteine residues in the CH1 hinge region is for example to facilitate the assembly of the 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 in vivo half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment such that the antibody has impaired Staphylococcus protein A (SpA) binding relative to native Fc hinge domain SpA binding. The method is further described in detail in U.S. Patent No. 6,165,745 to Ward et al.

[0149] In another embodiment, the antibody is modified to increase its in vivo half-life. Various methods can be employed. 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 in vivo half-life, the antibody can be altered in the CH1 or CL region to contain the salvage receptor binding epitope of two loops of the CH2 domain of the Fc region taken from IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al.

[0150] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues such that the antibody has an altered affinity for an effector ligand, while retaining the antigen-binding ability of the parental antibody. The effector ligand whose affinity is altered 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 to Winter et al.

[0151] In another embodiment, one or more amino acids selected from amino acid residues can be replaced with different amino acid residues such that the antibody has altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is further described in 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 alter the ability of the antibody to fix complement. This method is further described in PCT Publication WO 94 / 29351 to Bodmer et al.

[0153] In another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cell 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 FcγRl, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped and variants with improved binding have been described (see Shields, R.L. et al., 2001 J. Biol. Chen. 276:6591-6604).

[0154] In certain embodiments, the 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 cell cytotoxicity (ADCC) and / or bind to Fcγ receptors. Examples of IgG1 isotype silent mutants in which, as described by Hezareh et al., J. Virol (2001); 75(24):12161-8, the leucine residues at amino acid positions 234 and 235 are replaced with alanine residues.

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

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

[0157] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be prepared (i.e., the antibody lacks glycosylation). The glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished, for example, by altering 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 that site. This aglycosylation can increase the affinity of the antibody for the 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] Alternatively or additionally, antibodies with altered glycosylation patterns can be prepared, such as hypofucosylated 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 the antibodies. This carbohydrate modification can be achieved, for example, by expressing the antibody in host cells with an altered glycosylation machinery. Cells with an altered glycosylation machinery have been described in the art and can be used as host cells for expressing the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a disrupted FUT8 gene encoding fucosyltransferase, such that antibodies expressed in this cell line exhibit hypofucosylation. Thus, in one embodiment, the antibodies of the present invention are produced by recombinant expression in a cell line that exhibits a fucosylation pattern (e.g., a mammalian cell line defective in the expression of the FUT8 gene encoding fucosyltransferase). Presta describes in PCT publication WO03 / 035835 the variant CHO cell line Lecl3 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of the antibodies expressed in the host cells (see also Shields, R.L. et al., 2002 J. Biol. Chem. 277:26733-26740). PCT publication WO99 / 54342 by Umana et al. describes a cell line that is 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 result in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180). Alternatively, the antibodies of the present invention can be produced in yeast or filamentous fungi that are engineered for mammalian-like glycosylation patterns and are capable of producing antibodies lacking fucose as a glycosylation pattern (see, for example, EP1297172B1).

[0159] Another modification of the antibodies contemplated by the present invention is pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. Pegylation can be carried out using reactive PEG molecules (or similar reactive water-soluble polymers) by acylation or alkylation reactions. 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 a non-glycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, EP 0154 316 to Nishimura et al. and EP 0 401 384 to Ishikawa et al.

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

[0161] Another modification of the antibodies 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 the 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 by reference in their entirety.

[0162] For example, the use of a "knobs-into-holes" structure to generate bispecific antibodies is disclosed in PCT Publication No. WO 1996 / 027011, Ridgway et al., (1996) and Merchant et al. (1998).

[0163] (1) "Knobs-into-holes" structure (KIH)

[0164] The multispecific molecules (e.g., multispecific antibodies or antibody-like molecules) of the present invention may comprise one or more (e.g., multiple) mutations to one or more constant domains (e.g., to the CH3 domain). In one example, the multispecific molecule of the present invention comprises two polypeptides, each polypeptide comprising a heavy chain constant domain of an antibody, e.g., the CH2 or CH3 domain. In an example, the two heavy chain constant domains, e.g., the CH2 or CH3 domain of the multispecific molecule, comprise one or more mutations that permit heterodimeric association between the two chains. In one aspect, one or more mutations are disposed on the CH2 domains of the two heavy chains of a multispecific, e.g., bispecific antibody or antibody-like molecule. In one aspect, one or more mutations are disposed on the CH3 domains of at least two polypeptides of the multispecific molecule. In one aspect, one or more mutations to the first polypeptide of the multispecific molecule comprising a heavy chain constant domain create a "stalk" and one or more mutations to the second polypeptide of the multispecific molecule comprising a heavy chain constant domain create a "socket" such that heterodimerization of the polypeptides of the multispecific molecule comprising a heavy chain constant domain results in the "stalk" engaging the "socket" interface (e.g., interacting, 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 used herein, the term "stalk" 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 is thus locatable in a complementary "socket" in the interface with the second polypeptide of the multispecific molecule comprising a heavy chain constant domain to stabilize the heteropolymer and thus facilitate heteropolymer formation (e.g., relative to the homopolymer). The stalk may be present in the original interface or may be synthetically introduced (e.g., by altering the nucleic acid encoding the interface). Preferred input residues for forming the stalk are typically naturally occurring amino acid residues and may preferably be selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Most preferably are tryptophan and tyrosine. In a preferred embodiment, the original residue for forming the protrusion has a small side chain volume, e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0165] "Mortar" refers to at least one amino acid side chain that is recessed into the interface of the second polypeptide of a multispecific molecule comprising a heavy chain constant domain and thus accommodates a corresponding pestle on the adjacent junction surface of the first polypeptide of the multispecific molecule comprising the heavy chain constant domain. The mortar 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 mortar 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 original residue for forming the mortar has a large side chain volume, 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 produce a "pestle" or "mortar" (as described above), and the second CH3 domain that heterodimerizes with the first CH3 domain is mutated at the following positions: 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), according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85, to produce a "mortar" or "pestle" complementary to the "pestle" or "mortar" 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 produce a "pestle" or "mortar" (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 produce a "mortar" or "pestle" complementary to the "pestle" or "mortar" of the first CH3 domain. In one embodiment, the mutation of the first CH3 domain introduces a tyrosine (Y) residue at position 366. In an embodiment, the mutation of the first CH3 is T366Y. In one embodiment, the mutation of the first CH3 domain introduces a tryptophan (W) residue at position 366. In an embodiment, the mutation of 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., containing the mutation T366Y or T366W)) includes a mutation at position 366, a mutation at position 368, and a mutation at position 407, where the positions are 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 mutations include T366S, L368A, and Y407V. In one embodiment, the first CH3 domain of the multispecific molecule contains the mutation T366Y, and the second CH3 domain that heterodimerizes with the first CH3 domain contains the mutations T366S, L368A, and Y407V, and vice versa. In one embodiment, the first CH3 domain of the multispecific molecule contains the mutation T366W, and the second CH3 domain that heterodimerizes with the first CH3 domain contains the mutations T366S, L368A, and Y407V, and vice versa.

[0168] Additional space or “offset” (e.g., mortise and tenon structure) 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 by reference in their entirety. Examples of KIH variants include a first constant chain containing L368D and K370S mutations, which pairs with a second constant chain containing S364K and E357Q mutations.

[0169] Additional mortise and tenon structure mutations applicable to any multispecific molecule of the invention are further described, for example, in WO 1996 / 027011 and Merchant et al., Nat. Biotechnol. [Nature Biotechnology], 16:677-681 (1998), the contents of which are incorporated herein by reference in their entirety.

[0170] In any of the embodiments 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 introducing a pair of cysteine residues capable of forming a disulfide bond provides stability to the heterodimerizing multispecific molecule. In an embodiment, according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85, the first CH3 domain contains a cysteine at position 354, and the second CH3 domain that heterodimerizes with the first CH3 domain contains a cysteine at position 349 according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85. In an embodiment, the first CH3 domain of the multispecific molecule contains a cysteine at position 354 (e.g., contains the mutation S354C) and a tyrosine (Y) at position 366 (e.g., contains the mutation T366Y), and the second CH3 domain that heterodimerizes with the first CH3 domain contains a cysteine at position 349 (e.g., contains the mutation Y349C), a serine at position 366 (e.g., contains the mutation T366S), an alanine at position 368 (e.g., contains the mutation L368A), and a valine at position 407 (e.g., contains the mutation Y407V). In an embodiment, the first CH3 domain of the multispecific molecule contains a cysteine at position 354 (e.g., contains the mutation S354C) and a tryptophan (W) at position 366 (e.g., contains the mutation T366W), and the second CH3 domain that heterodimerizes with the first CH3 domain contains a cysteine at position 349 (e.g., contains the mutation Y349C), a serine at position 366 (e.g., contains the mutation T366S), an alanine at position 368 (e.g., contains the mutation L368A), and a valine at position 407 (e.g., contains the mutation Y407V).

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

[0172] On the one hand, heterodimerization of the polypeptide chains of the multispecific molecule (e.g., half-antibodies) is increased by introducing one or more mutations in the CH3 domain derived from the IgG1 antibody class. In one embodiment, according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85, the mutations include pairing a K409R mutation in one CH3 domain with an F405L mutation in the second CH3 domain. According to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85, additional mutations can also or alternatively be at positions 366, 368, 370, 399, 405, 407, and 409. Preferably, heterodimerization of the polypeptide containing such mutations is achieved under reducing conditions, e.g., at 25°C - 37°C, e.g., 25°C or 37°C, with 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. Generally, the DNA encoding one or more heavy chains is genetically engineered using the techniques described in Mutagenesis: a Practical Approach [Mutagenesis: A Practical Approach]. Oligonucleotide-mediated mutagenesis is the preferred method for preparing substitution variants of the DNA encoding two heterologous heavy chains. As described by Adelman et al., (1983) DNA, 2:183, this technique is well known in the art.

[0174] IgG heterodimerization strategies are described in, for example, 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 of the embodiments 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 introducing a pair of cysteine residues capable of forming a disulfide bond provides stability to the heterodimerizing multispecific molecule. In an embodiment, according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85, the first CH3 domain contains a cysteine at position 354, and the second CH3 domain heterodimerizing with the first CH3 domain contains a cysteine at position 349 according to the EU numbering scheme of Edelman et al., PNAS, May 1969, 63(1):78-85.

[0176] (3) Polar bridge

[0177] On the one hand, the heterodimerization of the polypeptide chains of the multispecific molecule (such as diabodies) is increased by introducing mutations based on the "polar bridging" principle, the basic principle being to create 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 such that in heterodimer formation, polar residues interact with polar residues, and hydrophobic residues interact with hydrophobic residues. In contrast, in homodimer formation, the mutant residues are such that polar residues interact with hydrophobic residues. The favorable interactions in the heterodimer configuration and the unfavorable interactions in the homodimer configuration together act to make the CH3 domain more likely to form a heterodimer than a homodimer.

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

[0179] On the one hand, 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 is replaced by leucine; Thr366Val: the original residue of threonine at position 366 is replaced by valine; Leu368Gln: the original residue of leucine at position 368 is replaced by glutamine; Asp399Lys: the original residue aspartic acid at position 399 is replaced by lysine; Phe405Ser: the original residue phenylalanine at position 405 is replaced by serine; Lys409Phe: the original residue lysine at position 409 is replaced by phenylalanine; Thr411Lys: the original residue of threonine at position 411 is replaced by lysine.)

[0180] On the other hand, one or more mutations selected from the group consisting of: Tyr407Phe, Lys409Gln, and Thr411Asp can be introduced into the other CH3: (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 of threonine at position 411 is replaced by aspartic acid).

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

[0182] In one exemplary embodiment, the original residue of threonine at position 366 of one CH3 domain is replaced with valine, while 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 original residue of phenylalanine at position 405 of one CH3 domain is replaced with serine and the original residue of lysine at position 409 of this CH3 domain is replaced with phenylalanine, while the original residue of lysine at position 409 of the other CH3 domain is replaced with glutamine.

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

[0186] The amino acid substitutions described herein are introduced into the CH3 domain using techniques well known in the art. Generally, the DNA encoding one or more heavy chains is engineered using the techniques described in Mutagenesis: a Practical Approach. Oligonucleotide-mediated mutagenesis is the preferred method for preparing substitution variants of the DNA encoding two heterologous 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, for example, in 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, for example, in 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 invariant chains containing the N208D, Q295E, N384D, Q418E, and N421D mutations.

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

[0190] Other strategies for enhancing heterodimerization are described, for example, in WO 2016 / 105450, WO 2016 / 086186, WO2016 / 086189, WO 2016 / 086196, WO 2016 / 141378, and WO 2014 / 145806 and WO 2014 / 110601, the entire contents of each of which are incorporated herein by reference in their entirety. Any of the said strategies can be used for the multispecific molecules described herein.

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

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

[0193] By way of example, the generation of the specific bbmAb is described below so that those skilled in the art can implement the present invention.

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

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

[0196] Table 3. Amino acid sequences of the 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 ) containing hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:76, CDR2 has the amino acid sequence SEQ ID NO:77, and CDR3 has the amino acid sequence SEQ ID NO:78. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin heavy chain variable domain (V H1), wherein the CDR1 has the amino acid sequence SEQ ID NO:79, the CDR2 has the amino acid sequence SEQ ID NO:80, and the CDR3 has the amino acid sequence SEQ ID NO:81. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin heavy chain variable domain (V H1 ), wherein the CDR1 has the amino acid sequence SEQ ID NO:82, the CDR2 has the amino acid sequence SEQ ID NO:83, and the CDR3 has the amino acid sequence SEQ ID NO:84.

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

[0201] In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin light chain variable domain (V L1 ), wherein the CDR1 has the amino acid sequence SEQ ID NO:92, the CDR2 has the amino acid sequence SEQ ID NO:93, and the CDR3 has the amino acid sequence SEQ ID NO:94. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin light chain variable domain (VL1 ), wherein the CDR1 has the amino acid sequence SEQ ID NO:95, the CDR2 has the amino acid sequence SEQ ID NO:96, and the CDR3 has the amino acid sequence SEQ ID NO:97. In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a first immunoglobulin light chain variable domain (V L1 ), wherein the CDR1 has the amino acid sequence SEQ ID NO:98, the CDR2 has the amino acid sequence SEQ ID NO:99, and the CDR3 has the amino acid sequence SEQ ID NO:100.

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

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

[0204] In one embodiment, the IL-18 / IL-1β bispecific antibody comprises a second immunoglobulin V H2 domain and a second immunoglobulin V L2 domain, wherein: a) said second immunoglobulin V H2The domain comprises (for example, in sequence): i) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:44, CDR2 has the amino acid sequence SEQ ID NO:45, and CDR3 has the amino acid sequence SEQ ID NO:46; or ii) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:47, CDR2 has the amino acid sequence SEQ ID NO:48, and CDR3 has the amino acid sequence SEQ ID NO:49; or iii) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:50, CDR2 has the amino acid sequence SEQ ID NO:51, and CDR3 has the amino acid sequence SEQ ID NO:52 and b) said second immunoglobulin V L2 The domain comprises (for example, in sequence): i) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:60, CDR2 has the amino acid sequence SEQ ID NO:61, and CDR3 has the amino acid sequence SEQ ID NO:62 or ii) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:63, CDR2 has the amino acid sequence SEQ ID NO:64, and CDR3 has the amino acid sequence SEQ ID NO:65 or iii) hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:66, CDR2 has the amino acid sequence SEQ ID NO:67, and CDR3 has the amino acid sequence 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 H1 ) comprising the amino acid sequence set forth in SEQ ID NO:85; b) a first immunoglobulin light chain variable domain (V L1 ) comprising the amino acid sequence set forth in SEQ ID NO:101; c) a first immunoglobulin V H1 domain comprising the amino acid sequence set forth in SEQ ID NO:85 and a first immunoglobulin V L1 domain comprising the amino acid sequence set forth in SEQ ID NO:101; d) a first immunoglobulin V H1 domain comprising the hypervariable regions set forth in SEQ ID NO:76, SEQ ID NO:77 and SEQ ID NO:78domain; e) 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 domain; f) a first immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81 H1 domain; g) 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; h) a first immunoglobulin V domain comprising the hypervariable regions set forth 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 domain. i) a first immunoglobulin V domain comprising the hypervariable regions set forth 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 H2 ) comprising the amino acid sequence set forth in SEQ ID NO:53; b) a second immunoglobulin light chain variable domain (V L2 ) comprising the amino acid sequence set forth in SEQ ID NO:69; c) a second immunoglobulin V H2 domain comprising the amino acid sequence set forth in SEQ ID NO:53 and a second immunoglobulin V L2 domain comprising the amino acid sequence set forth in SEQ ID NO:69; d) a second immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46 H2domain; e) 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 domain; f) a second immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49 H2 domain; g) 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; h) a second immunoglobulin V domain comprising the hypervariable regions set forth 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 domain. i) a second immunoglobulin V domain comprising the hypervariable regions set forth in SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49 H2 domain and a second immunoglobulin V 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 human IL-18 antibodies, which at least comprise: a) an immunoglobulin heavy chain or a fragment thereof, the immunoglobulin heavy chain or the fragment thereof comprising a variable domain and a constant part of the human heavy chain or a fragment thereof, the variable domain sequentially comprising hypervariable regions CDR1, CDR2 and CDR3; the CDR1 has the amino acid sequence SEQ ID NO:76, the CDR2 has the amino acid sequence SEQ ID NO:77, and the CDR3 has the amino acid sequence SEQ ID NO:78; and b) an immunoglobulin light chain or a fragment thereof, the immunoglobulin light chain or the fragment thereof comprising a variable domain and a constant part of the human light chain or a fragment thereof, the variable domain sequentially comprising hypervariable regions CDR1, CDR2 and CDR3, the CDR1 has the amino acid sequence SEQ ID NO:92, the CDR2 has the amino acid sequence SEQ ID NO:93, and the CDR3 has the amino acid sequence SEQ ID NO:94. In addition, the second part of the IL-18 / IL-1β bispecific antibody is selected from human IL-1β antibodies, which at least comprise: a) an immunoglobulin heavy chain or a fragment thereof, the immunoglobulin heavy chain or the fragment thereof comprising a variable domain and a constant part of the human heavy chain or a fragment thereof, the variable domain sequentially comprising hypervariable regions CDR1, CDR2 and CDR3; the CDR1 has the amino acid sequence SEQ ID NO:44, the CDR2 has the amino acid sequence SEQ ID NO:45, and the CDR3 has the amino acid sequence SEQ ID NO:46; and b) an immunoglobulin light chain or a fragment thereof, the immunoglobulin light chain or the fragment thereof comprising a variable domain and a constant part of the human light chain or a fragment thereof, the variable domain sequentially comprising hypervariable regions CDR1, CDR2 and CDR3, the CDR1 has the amino acid sequence SEQ ID NO:60, the CDR2 has the amino acid sequence SEQ ID NO:61, and the CDR3 has the amino acid sequence SEQ ID NO:62.

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

[0211] The first V of the IL-18 / IL-1β bispecific antibody used in the disclosed method H1 or V L1 domain may have a first V substantially the same as the V listed in SEQ ID NO:85 and 101 H or V L domain and / or a first V H1 and / or first V L1Domain. The IL-18 / IL-1β bispecific antibody disclosed herein may comprise a first heavy chain that is substantially identical to the heavy chain shown in SEQ ID NO:87 and / or a first light chain that is 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 containing SEQ ID NO:87 and a first light chain containing 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 with 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 effector mutations. In one embodiment, the human heavy chain IgG1 comprises a combination of silent mutations N297A, D265A, or L234A and L235A. In a particular embodiment, according to SEQ ID NO:87, the human heavy chain IgG1 comprises a silent mutation that is 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 domain may have a second V that is substantially identical to the V listed in SEQ ID NO:53 and 69 H or V L domain and / or a first V H2 and / or first V L2Domains. The IL-18 / IL-1β bispecific antibodies disclosed herein may comprise a second heavy chain that is substantially the same as the heavy chain shown in SEQ ID NO:55 and / or a second light chain that is substantially the same as the light chain shown in SEQ ID NO:71. The IL-18 / IL-1β bispecific antibodies disclosed herein may comprise: a second heavy chain containing SEQ ID NO:53 and a second light chain containing SEQ ID NO:69. The IL-18 / IL-1β bispecific antibodies disclosed herein may comprise: a) a second heavy chain comprising a variable domain having an amino acid sequence substantially the same as 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 the same as 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 human IgG1 without effector mutations. In one embodiment, the human heavy chain IgG1 comprises a combination of the silent mutations N297A, D265A, or L234A and L235A. In a particular embodiment, according to SEQ ID NO:55, the human heavy chain IgG1 comprises a silent mutation that is a combination of L234A and L235A.

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

[0214] Other preferred IL-1β antagonists (such as antibodies) for use as the second part of the bispecific antibody in the disclosed methods, kits, and protocols are those listed below: U.S. Patent No. 7,446,175 or 7,993,878 or 8,273,350, which are incorporated herein by reference in their entirety.

[0215] (1) Vector design

[0216] Two vectors, Vector A and Vector B, were generated according to the following settings. Vector A was designed for the antibody portion mAb1 (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 pestle structure and achieving Cys bridging. Additionally, 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 as seen at position 235 of SEQ ID NO:87 (the so-called LALA), for partial silencing of the FC effector function. The antibody has a light chain variable region of the λ1 type, 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 mortar structure and achieving additional Cys bridging. The mortar structure interacts with the pestle structure to facilitate the production of bispecific antibodies. Additionally, 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 the FC effector function. The antibody has a light chain variable region of the κ6 type, Vκ6.

[0218] Vectors A and B carry a combination of the DHFR and neomycin selection markers and a combination of the FOLR and hygromycin selection markers, respectively. Folic acid is a vitamin essential for purine and methionine synthesis and needs to be taken up by mammalian cells from the culture medium. The "folate receptor" (FolR) present on expression plasmid A is a mutant FolR with an altered affinity for folic acid, which promotes 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 (50 nM). 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 analogue of folic acid. It competes with the free binding sites on DHFR, thus blocking the enzyme. Cells overexpressing exogenous DHFR can handle high concentrations of MTX, giving cells growing in MTX-supplemented medium a distinct selective advantage. The 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 content of which is 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 content of which is 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 content of which is incorporated herein by reference.

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

[0221] (2) Host cell line and transfection

[0222] The parental CHO cell line is used as the host cell line for generating the cell line expressing bbmAb1. The host cell line is derived from the CHO-K1 cell line well known to those 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 the CHO line is used to prepare the bbmAb1 recombinant cell line. The CHO line is prepared in a chemically defined medium.

[0224] Cells are grown in a chemically defined medium.

[0225] One μg of SwaI-linearized plasmid DNA, expression vectors A&B encoding bbmAb, is added per transfection. The transfection reaction is carried out in a chemically defined medium.

[0226] Transfection is carried out by electroporation using an AMAXA Gene Pulser according to the manufacturer's instructions. The parental CHO cells used for transfection are in the exponential growth phase with a cell viability higher than 95%. In total, three transfections are carried out with 5x10 6 cells / transfection.

[0227] Cells are immediately transferred to a shake flask containing a medium-sized chemically defined medium after transfection.

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

[0229] (3) Cell selection and sorting

[0230] As described above, the selection process was carried out 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 analogue MTX into the cell, and the DHFR converts it into an important precursor for purine and methionine synthesis. Combining them as a selection principle, a specific strong selection scheme can be adopted to enrich recombinant cells expressing both recombinant proteins.

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

[0232] Protein A HPLC method was used to determine the complete species of all products with Fc part and related impurities, while reverse phase chromatography (RPC) was used to obtain the fingerprint of the distribution of each fraction - each peak was determined by MS method.

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

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

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

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

[0237] In the "single cell" mode, the "purity mask" is set to maximum, so that only droplets without particles or other cells are sorted.

[0238] The "phase mask" is set to half of the maximum value, so that only the particles at the center within the sorted droplets are deflected. The trajectory and counting accuracy of the droplets are optimized at the expense of yield to increase the likelihood that each droplet contains no more than 1 single cell.

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

[0240] The images of day 0 involving the production clones of bbmAb1 were visually inspected in duplicate to confirm that only one single cell could be identified in the images of each well taken by the imaging system.

[0241] This highlights the single cell origin of the bbmAb1 production clones.

[0242] (4) Cell expansion

[0243] After FACS cloning, the clones were processed through a robotic system in the initial few weeks and then manually, gradually scaling up from 96-well, 24-well to shaker flasks and finally to bioreactor culture to evaluate the effects (productivity and quality of bbmAb expression), which is well known to those skilled in the art.

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

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

[0246] The productivity was evaluated by Protein A HPLC and the initial product mass spectrum was determined by reverse phase chromatography (RPC).

[0247] All frozen stocks were generated 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 judge the product characteristics and quality parameters to ensure the selection of the most suitable production clones.

[0250] In addition, the production stability of the production clones was over-analyzed to ensure the selection of the most suitable production clones.

[0251] Use different analytical methods at the prior art level to evaluate clonal stability: affinity liquid chromatography, reverse-phase chromatography, FACS, and MS.

[0252] (6) Production

[0253] (a) Upstream processing

[0254] The bbmAb material is produced in shake flasks or wave fed-batch cultures. In a chemically defined medium, the frozen stock of the pool or clone (e.g., PSL) is thawed and amplified over the desired time to obtain the desired number of cells to inoculate the production culture, with a typical seeding cell density of 4.0x10 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 mass spectrum of the supernatant. At the end of the culture process, the cells are separated from the culture supernatant by centrifugation (e.g., shaker) or depth filtration, followed by sterile filtration and then further DSP processing.

[0255] (b) Downstream processing

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

[0257] Method I: Purification is carried out by capture on MabSelect TM SuRe TM , followed by polishing on LambdaFabSelect TM and KappaSelect TM

[0258] The bbmAb1 and bbmAb1 variants with Fc parts are captured from the cell-free supernatant by the first-step affinity liquid chromatography (ALC) step on MabSelect TM SuRe TM . The bbmAb1 variants containing only the κ light chain (mAb2 kappa, TM and Figure 4C and 4D ) and HCP are removed by the first-step polishing on LambdaFabSelect, and by KappaSelectTM perform the second step of purification on it to remove the bbmAb1 variant containing only the λ light chain (mAb1 pestle, Figure 4A and 4B ) and HCP.

[0259] Throughout the method, chromatography is performed at room temperature with a 4-minute residence time (RT). Before loading, all columns are equilibrated with 4 column volumes (CV) of 20 mM Na 2 HPO 4 / NaH 2 PO4 (pH 7.0). To remove non-specifically bound impurities from the product, such as host cell proteins (HCP), media components, and DNA, after loading the cell-free bbmAb1 supernatant from the shake flask onto the ALC column, the chromatography column is washed with 4 CV of 250 mM arginine-HCl, 1 M NaCl, 88 mM NaOH (pH 9.0), and 3 CV of equilibration buffer. bbmAb1 and potential bbmAb1 variants are eluted from KappaSelect TM and LambdaFabSelect TM using 50 mM acetic acid (pH 3.0) and 50 mM acetic acid / HCl (pH 2.0), respectively. The product peak collection starts and ends at 0.5 AU / cm or 0.25 Au / cm (280 nm). The pH of the bbmAb1 eluate is 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: Purification is performed by capturing on LambdaFabSelect TM and performing refinement on Capto TM adhere (Capto TM adhere) and Fractogel TM EMD SO 3

[0261] In the second method, intact bbmAb1 and bbmAb1 variants containing only the λ light chain (mAb1 pestle monomers and homodimers, TM are captured from the cell-free supernatant by affinity liquid chromatography on LambdaFabSelect Figure 4A and 4B ). To inactivate any possible enveloped viruses, the ALC eluate is subjected to low pH treatment, and then refined on Capto adhere and Fractogel TM EMD SO 3 ​Two chromatographic purification steps were carried out to remove product-related impurities, DNA, and HCP. Subsequently, any possible viruses were removed by nanofiltration, and then a final concentration and buffer exchange step was carried out using tangential flow filtration.

[0262] a) LambdaFabSelect TM Affinity liquid chromatography (ALC) on

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

[0264] b) Virus inactivation

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

[0266] c) Capto TM Adhered multimodal anion exchange chromatography (MAC)

[0267] Throughout the method, MAC is carried out at 18 °C - 28 °C with a residence time of 4 - 6 minutes in flow - through mode. First, the MAC column is equilibrated with 7 - 9 CV of 20 mM Tris / Tris - HCl (pH 7.5). Then the low - pH treated ALC eluate is loaded onto the Capto TM adhesion column at a loading density of 175 - 350 g / L. Product peak collection starts at 0.5 - 2.0 AU / cm (280 nm) rise. Then the MAC column is washed with 5 - 7 CV of equilibration buffer, and product peak collection ends at 0.5 - 2.0 AU / cm (280 nm) fall. Subsequently, the Capto TM adhesion column is stripped with 6 - 8 CV of 100 mM acetic acid, then an in - place cleaning step is performed with 3 - 5 CV of 0.5 M NaOH and stored in 3 - 5 CV of 0.1 M NaOH.

[0268] d) Cation - exchange chromatography on TM Fractogel 3 EMD SO

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

[0270] e) Nanofiltration

[0271] Any possible viruses are removed by using Planova TMNanofiltration was carried out using a 20N nanofilter and a 0.5 / 0.1μm Millipore SHR-P prefilter. Prefiltration and nanofiltration were carried out by applying a pressure difference of 0.7 - 0.9 bar.

[0272] f) Tangential flow filtration and formulation

[0273] To concentrate and diafilter bbmAb1, tangential flow filtration steps were carried out on a Millipore TM Pellicon TM 3RC 30 kDa membrane at 18°C - 28°C. First, the nanofiltered bbmAb1 protein solution was concentrated to a maximum loading density of 1000 g / m 2 to 60 - 80 g / L using a feed flow pressure of 0.5 to 1.2 bar and a transmembrane pressure (TMP) of 0.3 - 0.6 bar. Then, bbmAb1 was 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 carried out 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. Finally, the ultrafiltered bbmAb1 protein solution was 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 0.2μm 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] A 100 μg protein A-purified bbmAb sample was lyophilized in a 96-well plate and deglycosylated with PNGase F (New England Biolabs) in 100 μl of 50 mM Tris-HCl pH 7.5 buffer at 37 °C for 18 h. The sample was measured by LC-ESI-MS on an H-Class UPLC (Waters) connected to a Synapt G2 Q-TOF mass spectrometer (Waters). A MassPREP microdesalting column 2.1 x 5 mm (Waters) was used at a column temperature of 80 °C. The following linear gradient was applied at a flow rate of 0.3 ml / min: mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile: 0 - 2 min 5% B, 2 - 12 min 5% - 90% B, followed by a wash step at 0.5 ml / min. MS parameters: ESI+ resolution mode, capillary voltage 3 kV, sampling cone 40 V, source temperature 150 °C, desolvation temperature 400 °C. The system was calibrated with a NaCl calibration solution and the lock mass was leucine enkephalin. Data were processed using UNIFI 1.6 software (Waters) by automated MaxEnt1 deconvolution (mass range 60 kDa - 150 kDa, harmonic suppression). Identification and relative quantification of bbmAb species and mismatch variants were based on matching to the theoretical expected mass and relative mass signal intensity of the deconvoluted mass spectra.

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

[0278] Intact deglycosylated bbmAb: The purified bbmAb1 antibody was diluted to 1 mg / ml in 20 mM Tris-HCl pH 7.5 and deglycosylated with 2 μl of PNGase F enzyme (New England Biolabs) at 37 °C for 4 h. The digestion was terminated 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 of PNGase F was added and incubated at 37 °C for 4 h, then 80 μl of denaturing 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 h, the sample was acidified with 1 μl of TFA.

[0280] Papain digested 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 h with shaking. After incubation, the solution was terminated by adding iodoacetamide solution to a final concentration of 1.2 mM.

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

[0282] Reductive LysC digestion - peptide mapping. (According to Rombach-Riegraf et al., PlosOne, 2014) 200 μg of protein was denatured using 150 μl of denaturing solution (6 M guanidine hydrochloride, 50 mM Tris-HCl, 5 mM Na 2 EDTA, pH 8.0), and reduced by adding 1.5 μl of 1 M DTT and incubated at 37 °C for 1 h. 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. Then the sample was digested by adding 4 μl of 1 μg / μl of intracellular protease LysC solution (Wako, Osaka, Japan) twice and incubated at 37 °C for 1 h and 3 h respectively. 5 μl of TFA was added to quench the digestion.

[0283] LC-MS measurement. Using an instrument equipped with a BEH C4 RP column (1.7 μm, 2.1 x 100 mm, Waters UPLC H-Class of Waters Corporation (Waters Corporation) and Xevo G2 TOF mass spectrometer (Waters Corporation, Milford) were used for LC-MS system analysis of protein samples. 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, resolution 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 a NaCsI solution.

[0285] Peptide digests were analyzed by RP-LC-MS on an H-Class UPLC (Waters Corporation) coupled with a Synapt G2Q-TOF mass spectrometer (Waters Corporation) using a CSH130 C18 2.1 mm x 150, 1.7 μm (Waters Corporation, Milford). Mobile phase A: 0.1% TFA in water and mobile phase B: 0.09% TFA in acetonitrile. Peptides were eluted from the column with the following gradient: at a column temperature of 40 °C, 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%. The UV chromatogram was recorded at 214 nm, and MS was performed with low energy (4 eV) and high energy fragmentation (30 V - 55 V). E MS data acquisition was performed in positive ES(+) resolution mode. The lock mass was leucine enkephalin (Waters Corporation).

[0286] Data processing and evaluation were carried out using 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) Reverse-phase chromatography

[0288] The bbmAb sample was 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. The UV signal was recorded at 210 nm. Using Chromeleon TM 6.8 software (Thermo Scientific) to control and perform data acquisition and evaluation.

[0289] (d) Size exclusion chromatography

[0290] Using an Agilent 1260 system, the bbmAb1 sample was passed through a TSK gel G3000SWXL (Tosoh #808541, 5 μm, 7.8 mm x 300 mm) SEC column with a pore size of 250 Å. 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. The UV was recorded at 210 nm. Using Chromeleon TM 6.8 (Thermo Fisher Scientific) for data acquisition and peak integration.

[0291] (e) Capillary electrophoresis CE-SDS

[0292] For non-reducing CE-SDS, the bbmAb sample was mixed with sample buffer (0.1 sodium phosphate / 1.0% SDS, pH 6.6), and then mixed with iodoacetamide solution. For reducing CE-SDS, the protein was mixed with 0.1 M Tris / 1% SDS sample buffer (pH 8.0) and reduced with 5% (v / v) mercaptoethanol. Both samples were subjected to a thermal 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 sieving gel buffer. Separation was completed from the negative to the positive polarity at 15 kV and a capillary temperature of 25 °C, and detected by UV at 214 nm. Using Chromeleon TM6.8 Software processes and integrates the electrophoresis map.

[0294] (f) Capillary zone electrophoresis CZE

[0295] Separation was carried out 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 at 25 °C and a capillary voltage of 20 kV with positive polarity. Running buffer: 400 mM 6 - aminocaproic acid / acetic acid pH 5.7, containing 2 mM TETA and 0.03% Tween 20. Chromeleon TM 6.8 Software was used to perform peak integration.

[0296] (8) Analysis results

[0297] After protein A purification, the purity and identity of different bispecific antibody combinations and constructs after co - expression were analyzed using a complete UPLC - MS mass screening method. This method was used to confirm and relatively quantify heterodimers and homodimers from cell supernatants. Correctly formed heterodimers bbmAb1 and bbmAb2 could be observed, with a relative purity exceeding 85% based on the intact mass signal intensity. The main impurities observed in the screening were mismatched antibodies with two κ light chains, two λ light chains, and HC homodimer molecules.

[0298] Table 4. Summary of the analysis results of bbmAb1 - bbmAb11.

[0299]

[0300]

[0301] l is the λ chain and k is the κ 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 of the antibody types VH1_46 and Vk6.

[0310] mAb11 is of the antibody types VH3 and Vk3.

[0311] mAb12 is of the antibody types VH3 and Vk2.

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

[0313] To examine chain mispairing and other low-level impurities, the purified sample was digested with papain to analyze the 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 knobs-into-holes Fc fragment. Mispaired Fab fragments (Fab4, knob-κ) at levels <1% could be detected. Another method for generating Fab fragments by limited LysC digestion was tested, which allows for faster sample preparation during the pooling and clone selection process.

[0314] Another digestion strategy using the IdeS (Fabricator) enzyme was tested to generate Fc and F(ab’)2 fragments. In this experiment, the mass of the Fc heterodimer and the correctly formed heterodimeric F(ab’)2 were observed. The presence of the Fc heterodimer also demonstrated the correct formation of additional disulfide bonds in the Fc portion of bbmAb1, as otherwise only Fc / 2 fragments of smaller mass would be produced.

[0315] LysC peptide mapping by LC-MS could confirm the identity of the molecule, with a total peptide sequence coverage of 99%.

[0316] The specific results of the purified sample are shown in Figure 2. In particular, the RP-UV chromatogram of the deglycosylated full-length bispecific mAb is as Figure 2A shown. The papain-digested bbmAb1 fragments are as Figure 2C shown. The IdeS-digested bbmAb1 fragments are as Figure 2D shown. The deglycosylated and DTT-reduced bbmAb1 fragments are as Figure 2EAs shown. The deconvoluted mass spectrum of the fully deglycosylated bispecific mAb bbmAb1 is as Figure 2B shown.

[0317] The results are shown in Table 5.

[0318] Table 5. Mass of bbmAb1 Measured by RP-LC-MS Assignment

[0319]

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

[0321] The final two-step purified (λ / CEC) bispecific bbmAb1 was further analyzed by the methods listed in Table 6. Overall, the material showed high purity and low levels of aggregates or degradation products, which were detected by several separation methods (e.g., 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 different cell assays.

[0327] (1) Materials and Methods

[0328] (a) For Solution Equilibrium Titration (SET) Assay

[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 plate (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] bbmAb1 as described in Example 1.

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

[0343] Recombinant squirrel monkey IL-1β (Novartis)

[0344] Recombinant squirrel monkey IL-18 (Novartis)

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

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

[0347] Skin fibroblasts of cynomolgus monkeys (#42637F(510))

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

[0349] PBMC was isolated from buffy coat (obtained from Blustspendezentrum Bern)

[0350] Cynomolgus monkey blood was obtained from SILABE, Niederhausbergen

[0351] IL-6 ELISA: human (Biolegend, #430503); cynomolgus monkey (U-CyTech biosciences, CT974-5)

[0352] IFNγ ELISA: human (BD555142) and cynomolgus monkey (U-CyTech biosciences #CT340A)

[0353] QUANTI-Blue for detecting SEAP TM 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 LifeSciences #17-1440-02) PBS 1X, without calcium and magnesium (Gibco #14190094)

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

[0359] Falcon 50 ml polypropylene conical tube (BD #352070)

[0360] (c) Affinity measurement by SET

[0361] SET single target binding assay

[0362] Twenty-two consecutive 1.6-fold dilutions of the antigen (highest concentration: huIL-18, 5 nM; marIL-18, 10 nM; huIL-1β, 0.5 nM; marIL-1β, 0.5 nM) were prepared in sample buffer (PBS containing 0.5% bovine serum albumin (BSA) and 0.02% Tween-20), and a constant concentration of antibody was added (10 pM for IL-18 readings, 1 pM for IL-1β readings). Each antigen-antibody mixture with a volume of 60 μl / well was dispensed in duplicate into a 384-well polypropylene microtiter plate (MTP). Sample buffer served as the negative control, and the sample containing only the antibody served as the positive control (maximum electrochemiluminescence signal without antigen, B max ). The plate was sealed and incubated overnight (o / n, at least 16 hours) on a shaker at room temperature (RT).

[0363] IL-18 readings: A streptavidin-coated 384-well MSD array MTP was coated with 30 μl / well of biotinylated huIL-18 (0.1 μg / ml, PBS) and incubated for 1 hour at room temperature on an oscillator.

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

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

[0366] SET simultaneous target binding assay

[0367] Except for Assay A, the SET assay was performed as described above: the equilibration process (antibody / antigen mixture) was carried out in the presence of an excess of one target (500 pM of IL18 or IL-1β), while evaluating the K of the other target. D 。

[0368] Assay B: The equilibration process (antibody / antigen mixture) was carried out simultaneously in one mixture with two serially diluted targets (constant antibody concentration of 10 pM, highest antigen concentration as above). The free antibody concentration in the same mixture was then analyzed on IL18- and IL-1β-coated plates as described above.

[0369] SET data were exported to the MS Excel add-in software Xlfit. Mean ECL signals were calculated from 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. The K D value was determined by fitting the graph with the following:

[0370] 1:2 binding model for monospecific Ab

[0371]

[0372] 1:1 binding model for the pestle and mortar structure bispecific Ab

[0373]

[0374] where

[0375] y) minus the ECL signal of the blank

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

[0377] [IgG]: Antibody concentration applied

[0378] [Fab]: Total Fab concentration applied

[0379] K D : Dissociation equilibrium constant

[0380] x: Antigen concentration applied

[0381] (d) Cell culture

[0382] KG-1 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin / streptomycin at a density of 2x10 5 to 1x10 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 the starvation medium.

[0384] HEK-Blue TM IL-18 / IL-1β cells were grown 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 , 2 mM L-glutamine, supplemented with 30 μg / ml of blasticidin, 200 μg / ml of HygroGold TM and 100 μg / ml of Zeocin TM ).

[0385] Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from buffy coats using LeucoSep tubes according to the manufacturer's instructions. Briefly, 13 ml of Ficoll-Paque was preloaded into a 14 ml LeucoSep tube by centrifuging 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 continuously at 800×g for 15 min at room temperature. The cell suspension layer was collected, and the cells were washed twice in PBS (10 minutes each at 640×g and 470×g, two consecutive washes) and resuspended in medium, then counted.

[0386] Collect marmoset blood in heparinized tubes and filter 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 (Gram 2000) with minor modifications. Briefly, fibroblasts were seeded at a density of 5 x 103 cells / well (in 100 μl) in 96-well flat-bottom tissue culture plates. The next day, the cells were starved in starvation medium for 5 h before adding the recombinant IL-1β / compound solution mixture (IL-1β concentration as shown in the table). The IL-1β / compound solution mixture was pre-prepared by incubating recombinant IL-1β with a range of concentrations of the compound at 37 °C for 30 min. After incubation o / n at 37 °C, the cell supernatants were collected 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 3 x 10 5 cells / well (in 100 μl) in 96-well tissue culture plates and incubated with the recombinant IL-1β / compound solution mixture at 37 °C for 24 h (IL-1β concentration as shown in the table). The IL-1β / compound solution mixture was pre-prepared by incubating recombinant IL-1β with a range of concentrations of the compound at 37 °C for 30 min. After 24 h of stimulation, the cell supernatants were collected and the amount of released IL-6 was determined by ELISA.

[0389] (f) IL-18 neutralization assay

[0390] The assay was performed essentially as described below. KG-1 cells (pre-starved for 1 h in PBS + 1% FCS) or PBMCs at a density of 3 x 10 5 / well were seeded into round-bottom 96-well cell culture plates and incubated with a solution mixture of recombinant IL-18 / IL-12 and a range of concentrations of the compound (IL-18 / IL-12 concentrations as shown in the table). After incubation at 37 °C for 24 h, the supernatants were collected and the amount of released IFNγ was determined by ELISA. For the assay using 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 handling procedure. Briefly, HEK-Blue TM cells were seeded at 4 x 10 4Inoculated at a density of / holes into a 96-well cell culture plate and incubated with a solution mixture of recombinant IL-1β and IL-18 (to produce a 1:1 SEAP signal) and compounds in a concentration range. After incubation at 37 °C for 24 hours, the supernatant was collected and the amount of released SEAP was determined using QUANTI-Blue according to the manufacturer's instructions. TM The amount of released SEAP was determined by the method.

[0393] All data were exported to EXCEL software and the IC50 value was calculated by plotting the dose-response curve against the 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] The binding affinity of bbmAb1 for human and marmoset recombinant IL-1β and IL-18 proteins was measured by solution equilibrium titration (SET) titration ( Figure 5 ) and the resulting K D values were compared with the K D values of mAb2 for IL-1β and mAb1 for IL-18 binding. Figure 5 The titration curves for the ECL-based affinity assay in solution are shown, with a constant concentration of antibody: 10 pM for the IL-18 reading and 1 pM for the 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] In a single target binding assay comparing the binding affinities, bbmAb1 showed similar mean KD values for both human and marmoset IL-18 compared to mAb1 (Table 7). For human IL-1β binding, the mean KD value of bbmAb1 (2.6 pM) was slightly higher compared to mAb2 (0.6 pM), but still in the same low pM range. Subsequent measurements in a simultaneous dual target binding assay (Table 8) confirmed that the binding KD value of bbmAb1 for IL-1β was similar to that of mAb2 in both preclinical and clinical grade material cases. Thus, bbmAb1 has binding affinity for the targets in both human and marmoset, which are similar to mAb2 and mAb1 respectively.

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

[0399]

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

[0401] Table 8. Affinities for recombinant human (hu) and marmoset (mar) IL-1β and IL-18 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 bbmAb1 against the two cytokines (IL1β and IL-18) (mAb2mAb1) was evaluated. Additionally, the potency of bbmAb1 to neutralize marmoset IL-1β and IL-18 using the marmoset cell assay system was evaluated (see part d).

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

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

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

[0409]

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

[0411]

[0412] bbmAb1 was able to simultaneously neutralize the biological activities of IL-1β and IL-18, as shown by HEK Blue TM reporter cells (which respond to 1+1 stimulation with recombinant IL-1β and IL-18 by producing SEAP) (Table 11). Similar inhibition of SEAP in this assay system could only be achieved by the combination of mAb2 and mAb1, and not by using a single antibody.

[0413] Table 11. According to HEK Blue TMThe mean IC50 values for neutralizing both IL-1β and IL-18 while measuring SEAP reporter activity in cells. Shown are the means ± SEM of n = 5 experiments.

[0414]

[0415] (d) Neutralizing activity of bbmAb1 against cynomolgus IL-1β and cynomolgus IL-18 in cynomolgus cell assays

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

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

[0418]

[0419] The single-digit to double-digit nM IC50 values of bbmAb1 confirmed its neutralizing activity against cynomolgus IL-18 tested in the IFNγ production assay using cynomolgus blood cells (Tables 3-7). When measuring human IFNγ production, testing bbmAb1 on human PBMCs stimulated with cynomolgus IL-18 produced a similar inhibition profile.

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

[0421]

[0422] It has been demonstrated that, compared to the parental mAbs - mAb2 and mAb1, the KiH - type IL - 1β / IL - 18 bispecific mAb retains high - affinity binding to the two individual targets IL - 1β and IL - 18 and cytokine neutralizing potency in a variety of different cell assays. The dual IL - 1β and IL - 18 neutralizing properties of bbmAb1 have been demonstrated not only for human cytokines / cells but also for the corresponding cynomolgus monkey cytokines / cells, thus facilitating appropriate toxicology studies. Up to 2 - to 4 - fold higher IC50 values generated in some cell assays for IL - 1β and IL - 18 neutralization may be the result of monovalent binding of bbmAb1 rather than bivalent binding of mAb2 and mAb, respectively. However, the dual neutralization of cytokines by bbmAb1 may lead to 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 PBMC

[0424] Inflammasome - activation - dependent cleavage of the effector cytokines IL - 1β and IL - 18 leads to the induction of secondary pro - inflammatory mediators, promoting immune cell recruitment / activation not only systemically but also at the site of inflammation. In two different lethal systemic inflammation mouse models (a) LPS - injection model and (b) FCAS mice (a missense mutation in NLRP3 activation), the simultaneous absence / inhibition of IL - 1β and IL - 18 is more protective against lethality compared to the absence / inhibition of either single IL - 1β or single IL - 18, demonstrating an additive or synergistic mechanism of immune activation (Brydges 2013, van den Berghe 2014). bbmAb1 is a human / cynomolgus monkey IL - 1β / IL - 18 - reactive bispecific mAb with no rodent cross - reactivity and thus cannot be tested in mouse models. Therefore, we used LPS / IL - 12 in vitro to mimic inflammasome - dependent pathway activation to stimulate human PBMCs to reveal the additive or synergistic inhibitory effects of IL - 1β / IL - 18 neutralized by bbmAb1 and performed unbiased gene expression analysis using microarrays. As a complementary activity, we also compared the gene expression profiles of PBMCs from different donors stimulated with a combination of recombinant IL - 1β and recombinant IL - 18 or single cytokines 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 15 ml polypropylene conical tubes (BD#352096), Falcon 50 ml polypropylene conical tubes (BD#352070)

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

[0442] Cell strainer 70 μM, BD Biosciences #352350

[0443] Trypan blue, Sigma #T8154

[0444] RNA isolation, quantification and quality measurement, and qPCR:

[0445] Nuclease-free water, Ambion #AM9938

[0446] Rnase Zap, Ambion #AM9780

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

[0448] RLT buffer, Qiagen #1015762

[0449] Rneasy Mini Kit, Qiagen #74104

[0450] Set of DNase without RNase, Qiagen #79254

[0451] Agilent RNA 6000 Nano 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] 0.2 ml PCR tubes with nuclease-free thin walls and caps, Ambion #AM12225

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

[0459] TaqMan GenEx Premix, 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 buffy coats by Ficoll-Paque gradient centrifugation in Leucosep tubes according to the manufacturer's instructions. Briefly, 15 mL of Histopaque was placed into a 50 mL LeucosepTM tube and centrifuged at 1300 rpm for 30 seconds at room temperature. 30 mL of diluted suspension of buffy coat was added to the top of the Histopaque solution using a pipette and centrifuged continuously at 1000 g for 15 min at room temperature. Plasma (approx. 20 ml) was discarded, 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 of PBS containing 2% FCS and 2 mM EDTA. The cell suspension was filtered through a 70 μm cell strainer 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: Preparation for cytokine production in supernatant was carried out according to the following method. 250,000 cells / well (final volume of 100 ul) were dispensed into a 96-well round bottom plate. LPS at a concentration between 0.3 μg / ml and 3000 μg / ml was used, together with 10 ng / ml of recombinant IL-12. The supernatant was harvested after 24 hours at 37 °C and 10% CO 2 2.

[0464] RNA extraction from cell pellet was carried out according to the following. 3x10 6 cells / well were dispensed into a flat-bottom 24-well plate with a final volume of 1000 ul. 3 μg / ml of LPS was used together with 10 ng / ml of recombinant IL-12. The cells were harvested after 24 hours at 37 °C and 10% CO 2 2.

[0465] Stimulation of PBMCs with recombinant cytokines: 7x10 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, and 1 ng / ml recombinant IL-12. Supernatants and cells were collected after 4 hours and 24 hours at 37 °C and 10% CO 2 2. Supernatants and cells were collected after 4 hours and 24 hours at 37 °C and 10% CO

[0466] RNA isolation, quantification, and quality assessment: Cells were pelleted, and the pellet was 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, which were 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 column membrane. A DNase I incubation mix solution was prepared according to the manufacturer's instructions and added to the RNeasy column, and the column was incubated at room temperature for 15 min. After washing with 350 μl and 500 μl of buffer RW1, the RNeasy column was placed in a new 2 ml collection tube and centrifuged at full speed for 1 min. RNA was finally collected by directly adding 35 μl of RNase-free water to the column membrane and centrifuging at 8000 g for 1 min. The amount of RNA was measured using a Nanodrop ND-1000, and the RNA was stored at -20 °C. RIN measurements were performed according to the manufacturer's instructions to assess RNA quality. Briefly, 1 μl of RNA or a dilution series was pipetted into an Agilent RNA 6000 Nano chip, and measurements were performed using an Agilent 2100 Bioanalyzer.

[0467] Cytokine gene expression analysis 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 then 1 μl of cDNA was transferred to a 384-well reaction plate and mixed with 1 μl of 20X Gene Expression Assay Target FAM gene and 10 μl of 2x Gene Expression Premix and 10 μl of RNA / DNA-free water. The plate was loaded onto an Applied Biosystems ViiA TM 7 Real-Time PCR System, and the following instrument settings were used:

[0469]

[0470] The housekeeping genes used for this study were HPRT1 and RLP27. The relative expression levels of the 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] Microarrays were performed according to the following method. Samples were processed by CiToxLAB France on an Affymetrix HG_U133_Plus2 microarray. They were RMA-normalized and analyzed in GeneSpring 11.5.1 (Agilent Technologies, Santa Clara, CA). Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) and Nextbio (Illumina). The two data sets were processed independently.

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

[0478] Differentially expressed genes (DEGs) were identified using the "filter on volcano plot" function in GeneSpring. Filtered genes (expression between the 20.0th percentile - 100.0th percentile) with an unpaired T-test, a corrected p-value below 0.05 and a fold change above 2.0 were considered differentially expressed. Where possible, i.e., in the study of LPS (NUID-0000-0202-4150), Benjamini-Hochberg multiple testing correction was used.

[0479] For the cytokine stimulation experiment, 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, and the value represents the statistical significance of observing the overlap between the features and the "disease gene list" (lesion compared to non-lesion) of the public dataset. To this end, the lists are uploaded to the Illumina BaseSpace Correlation Engine (formerly Nextbio), and compared using the Meta-Analysis function and keyword search for the disease.

[0481] All data are exported to EXCEL software, and the IC is calculated by plotting the dose-response curve for the logistic curve fitting function using EXCEL / XLfit4 or GraphPad Prism software. 50 value. The differences between treatment groups are analyzed using GraphPad Prism software by one-way ANOVA followed by Dunnett's multiple comparison, and the results are 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] Exposing human whole blood to LPS supplemented with 10 ng / ml IL-12 results in an IFNγ response that depends largely (but not completely) on "natural" IL-18 produced by blood cells. The addition of IL-12 may enhance the LPS-induced IFNγ response by upregulating the IL-18 receptor on responding cells.

[0485] Under the experimental conditions used, IL-18 neutralization using mAb1 only results in incomplete inhibition of IFNγ production, while IL-1β blockade (using mAb2) has little effect on the IFNγ response. Interestingly, compared to single cytokine neutralization (Figure 6), the combined inhibition of IL-1β and IL-18 by bbmAb1 or the combination of mAb2 and mAb1 more profoundly and completely inhibits IFNγ production. 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 as Figure 6A shown). The percentage of IFNγ inhibition (mean and SEM) for n = 4 individual donors in whole blood using bbmAb1, mAb1, or mAb2 at 100 nM is as Figure 6B shown.

[0486] In our cell assays, none of the other tested cytokines (IL-2, -4, -6, -8, -10, -13, and TNFα), except IFNγ, were additively inhibited by the combined neutralization of IL-1β and IL-18 (data not shown). Considering the monovalent form of the bispecific molecule, the potency of bbmAb1 was in the same range as the combination (combo) 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 single IL-1β or IL-18 inhibition

[0488] Unbiased transcriptomic assessments are needed to reveal additional additive effects (except for IFNγ) by combined IL-1β / IL-18 inhibition using bbmAb1. Since whole blood is not the optimal material for transcriptomic analysis, we adapted the LPS / IL-12 stimulation assay conditions (as described in the Materials and Methods section above) to human PBMC samples. By using PBMCs from a total of 9 donors, we could confirm that bbmAb1 additively inhibited IFNγ protein secretion into the PBMC supernatant (Figure 7). Compared to the whole blood experiment, IFNγ production was inhibited 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), which confirmed the suitability of the samples for unbiased microarray-based gene expression analysis. Figure 7 shows the inhibition of LPS (0.3 μg / ml) / IL-12-induced IFNγ protein production ( Figure 7A ) and IFNγ gene expression ( Figure 7B ) by bbmAb1, mAb2, and mAb1 (each at a concentration of 10 nM) in human PBMCs. Shown are the percentages of inhibition in n = 9 donors ± SEM. ***p < 0.05 (one-way ANOVA)

[0489] The Affymetrix microarrays were performed with n = 5 individual donors from PBMCs, sampled from the LPS / IL-12 stimulation experiment described in the Materials and Methods section above. Unfortunately, the overall assessment 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, comparing the LPS / IL-12-stimulated samples with the samples stimulated with LPS plus bbmAb1 for differentially expressed genes revealed a list of genes downregulated by the combined IL-1β / IL-18 blockade using bbmAb1 (Table 14). In addition to the strong downregulation of the IFNγ gene, which reconfirmed our microarray data, the IL-26 gene was another cytokine gene that was cumulatively inhibited 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 the microarray data, as well as the inhibitory effects of bbmAb1, mAb2, and mAb1 (each 10 nM) at 24 hours in LPS (0.3 μg / ml) / IL-12-stimulated PBMCs. Figure 8A (IFNγ) and Figure 8B (IL-26) show values for individual donors, Figure 9A (IFNγ) and Figure 9B (IL-26) show the percentage inhibition (mean ± SEM) for n = 5 donors.

[0490] Table 14. Differentially expressed genes (genes downregulated only between bbmAb1 and the control group in LPS / IL-12-stimulated samples). FC = fold change.

[0491]

[0492]

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

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

[0495] (d) IL1β / IL18 signaling signature associated with disease

[0496] The previously established PBMC culture conditions (where recombinant IL-1β stimulation led to IL-6 production or recombinant IL-18 / IL-12 stimulation led to 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) for Affymetrix microarray assessment for unbiased assessment of gene expression profiles. Genes that were synergistically upregulated at 6 hours and 24 hours upon stimulation with the combination of IL-1β and IL-18 were revealed (data not shown). Adding IL-12 to the IL-1β / IL-18 combination greatly enhanced the synergy of a series of upregulated genes. The signaling signatures generated by single or combined IL-1β / IL-18 pathway stimulation (only upregulated genes) were used to interrogate datasets across patients with autoimmune diseases. For example, Figure 11Shows the correlation with the public sarcoidosis dataset. The P-values (calculated by Fisher's exact test) show significant correlations with several public studies comparing healthy tissues and diseased tissues from sarcoidosis patients. Tissues include skin as well as lung, lacrimal gland, and orbit. In all datasets, 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 to 5 sarcoidosis tissue "diseased vs healthy" DEGs. The P-values (y-axis) represent the statistical significance of the overlap observed between the feature and the "disease gene list". The black bars are skin from sarcoidosis lesions compared to skin from healthy patients. The light gray bars are skin from sarcoidosis lesions compared to non-lesion skin. The white bars are lacrimal glands from sarcoidosis patients compared to lacrimal glands from normal individuals. The dark gray bars are pre-orbital tissues from sarcoidosis patients compared to pre-orbital tissues from normal individuals. The striped bars are lung samples from progressive fibrotic, pulmonary sarcoidosis compared to lung samples from nodular self-limiting 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 hours of in vitro culture. It was demonstrated that the combined inhibition of IL-1β and IL-18 acted additively to reduce / suppress IFNγ production in LPS / IL-12-stimulated PBMCs by using bbmAb1. IL-12 has been previously described to act in synergy with IL-18 to induce IFNγ production in T, B, NK cells, macrophages, and dendritic cells (as described by Nakanishi in 2001), but it could now be demonstrated under the experimental conditions used that IL-1β had an additive stimulatory effect on IFNγ production. Thus, co-incubation of PBMCs with LPS / IL-12 effectively drove the production of "native" IL-1β and IL-18, both of which contributed to a strong IFNγ response. By 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. These included IL-26, which is a member of the IL-20 cytokine subfamily (IL-19, IL-20, IL-22, IL-24, and IL-26), conserved in most vertebrate species but absent in most rodent strains (including mice and rats) (Donnelly in 2010). It signals through a heterodimeric receptor complex composed of the IL-20R1 and IL-10R2 chains. The IL-26 receptor is mainly expressed on non-hematopoietic cell types, particularly epithelial cells. Elevated levels of IL-26 have been reported in serum and especially in the synovial fluid of RA patients (Corvaisier in 2012), where IL-26 may act as a factor promoting Th17 cell growth and differentiation. Unfortunately, the strong effect of LPS / IL-12 on stimulating PBMC samples hindered the discovery of additional genes / pathways induced by combined blockade of IL-1β and IL-18. However, IFNγ and IL-26, as well as IL-22 to some extent, 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. Thus, the IL-20 subfamily of cytokines (including IL-26 and IL-22) appears to be strongly dependent on simultaneous signals from IL-1β and IL-18. With due attention to the selectivity of individual signaling signatures and the potential efficacy of blockade, these comparisons helped to show that each pathway is active in diseases such as sarcoidosis.

[0499] 8. Example 4: Therapeutic uses

[0500] In inflammasome-driven inflammatory disorders 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 and endothelial cells, and pro-inflammatory cytokines (e.g., IL-6, IFNγ, and IL-17). Data obtained in a preclinical murine model of familial cold autoinflammatory syndrome (FCAS), which is driven by constitutive Nlrp3 inflammasome activation and overproduction of IL-1β and IL-18 (Brydges, 2013), support the advantage of blocking both IL-1β and IL-18. In this model, partial recovery of the FCAS condition was achieved in mice when either 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 diseased compared to 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 murine 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 the two cytokines by neutralizing antibodies completely prevented LPS lethality, while single cytokine deficiency / neutralization only partially protected.

[0501] The overall clinical strategy of bispecific antibodies that simultaneously target both IL-1β and IL-18 may represent a more effective therapeutic approach than currently available options. To identify candidate diseases, preclinical and translational studies were used to demonstrate that both IL-1β and IL-18 downstream pathways are actively involved in the underlying pathophysiology of the candidate disease. There is emerging evidence that chronic sarcoidosis is an inflammasome-driven disease that involves both innate and adaptive immunity. In addition, preliminary findings indicate that both IL-1β and IL-18 effector cytokines play important roles in this disease. Thus, sarcoidosis represents an ideal opportunity to demonstrate that bbmAb1 has dual specificity in a disease with established, chronic tissue inflammation. Efficacy of bbmAb1 in sarcoidosis may lead to the development of other interstitial lung diseases, such as allergic (occupational) lung diseases due to silica or beryllium. Other candidate diseases are granulomatous inflammation involving other organ tissues, such as Crohn's disease.

[0502] Vascular inflammation with tissue injury and endothelial dysfunction also represents a potential target for bbmAb1. Dysfunctional endothelial cells can respond to effective anti-inflammatory therapy, and this response can lead to improved blood flow even in the presence of fixed intravascular defects. Recent literature evidence has identified that sickle cell disease (SCD) has a strong inflammasome-driven component through a high proportion of constitutive intravascular hemolysis. Release of danger signals (uric acid, heme / Fe3+, other intracellular components) from chronic RBC lysis triggers inflammasome activation, which activates 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 injury. Preliminary in-house evidence supports the involvement of IL-18 and IL-1β in the underlying disease process of SCD. Thus, reducing the basal inflammation in SCD patients by treatment with bbmAb1 can mitigate chronic background inflammation and prevent acute crises associated with related end-organ damage, prevent acute sickle cell crises and related end-organ injury, and improve the quality of life of patients by reducing associated chronic pain and fatigue. Demonstrating the therapeutic efficacy of bbmAb1 in SCD patients may lead to the chronic or acute development of other chronic inflammatory conditions involving a high proportion of hemolysis, such as malaria and hemodialysis-dependent chronic kidney disease. Other indications that may benefit from the modulation of both IL-1β and IL-18 are those associated with ischemia-reperfusion tissue injury, such as cardiovascular disease or improved healing of all types of wounds, but especially the most severe soft tissue injuries of burns.

[0503] Accordingly, in one embodiment of the present invention, a method of treating an inflammasome-related disorder comprises administering to a subject having an inflammasome-related disorder an effective amount of a bbmAb disclosed herein, such as bbmAb1. Potential inflammasome-related disorders include cryopyrin-associated periodic syndromes (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 antibody (ANCA)-associated vasculitis, age-related macular degeneration (AMD), graft-versus-host disease, type 2 diabetes, acne, sickle cell disease, vascular lesions, ischemia-reperfusion injury, cardiovascular diseases, 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 the following diseases is provided by administering to a subject an effective amount of a bbmAb disclosed herein (such as bbmAb1), the method comprising: sickle cell disease, vascular lesions, ischemia-reperfusion injury, cardiovascular diseases, peripheral artery disease, atherosclerosis, vascular dysfunction, skeletal muscle ischemia, pulmonary sarcoidosis, fibrosis, malaria, hemodialysis-dependent chronic kidney disease or Crohn's disease.

[0505] 9. Example 5: Pharmaceutical Composition

[0506] The present invention provides a pharmaceutical composition 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. The pharmaceutically acceptable carrier enhances or stabilizes the composition, or may 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 a variety of methods known in the art. The route and / or mode of administration vary according to the desired result. Preferably, the administration can be intravitreal, intravenous, intramuscular, intraperitoneal, subcutaneous, or 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) may be encapsulated in a material to protect the compound from acids and other natural conditions that may inactivate the compound.

[0508] The composition should be sterile and fluid. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. In many cases, it is preferred to include isotonic agents such as sugars, polyols (e.g., mannitol or sorbitol), and sodium chloride in the composition. Prolonged absorption of an injectable composition can be achieved by including agents that delay absorption (e.g., aluminum monostearate or gelatin) in the composition.

[0509] The pharmaceutical compositions described herein can be prepared by methods well known and conventional in the art. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical compositions are preferably manufactured under GMP conditions. Generally, a therapeutically effective 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 optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, the term dosage unit form refers to physically discrete units suitable as unit doses for the subject to be treated; each unit containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0510] The actual dosage level of the active ingredient in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the 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 depends upon a variety of pharmacokinetic factors including the activity of the particular composition employed or its ester, salt or amide thereof used herein, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated.

[0511] A doctor or veterinarian can start with a dose of the antibodies described herein used in a pharmaceutical composition that is lower than the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Generally, the effective dose of the compositions described herein for treating the defecation disorders described herein varies according to many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. It is necessary to titrate the therapeutic dose to optimize safety and efficacy. For systemic administration with an antibody, the dose range is from about 0.0001 to 100 mg / kg of host body weight, and more typically from 0.01 to 15 mg / kg of host body weight. For intravitreal administration with an antibody, the dose can be in the range of 0.1 mg / eye to 5 mg / eye. Exemplary treatment regimens require systemic administration every two weeks or monthly or every three to six months. Exemplary treatment regimens require systemic administration every two weeks or monthly or every three to six months or as needed (PRN).

[0512] Biotherapies (such as bbmAb1) are typically administered in a variety of situations. The interval between single doses can be weekly, monthly, or annually. As shown by measuring the blood level of bbmAb1 in a patient, the interval can also be irregular. Additionally, alternative dosing intervals can be determined by a doctor and administered monthly or as needed for effectiveness. In some methods of systemic administration, the dose is adjusted to achieve a plasma antibody concentration of 1 - 1000 μg / ml, and in some methods to 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 exhibit longer half-lives than chimeric and non-human antibodies. The dose and frequency of administration can vary according to whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer relatively high doses at relatively short intervals until disease progression is reduced or terminated, and preferably until the patient shows partial or complete improvement of the disease symptoms. Thereafter, a prophylactic regimen can be administered to the patient.

[0513] Sequence Listing

[0514] Useful amino acid and nucleotide sequences for practicing the present invention are disclosed in Table 15.

[0515] Table 15. Sequences according to embodiments of the present invention

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526] Throughout the present application, if there are differences between the specification text (e.g., Table 15) and the sequence listing, the specification text shall prevail.

Claims

1. A bispecific antibody suitable for co-expression in a common host cell, wherein the antibody comprises: 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 a heterodimerization modification, wherein the VH1 specifically binds to a first target, and b. A second part, which is an immunoglobulin having a second variable light chain (VL2) of wild-type kappa and a second variable heavy chain (VH2) of wild-type, 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, wherein when the first part and the second part are co-expressed in a common host cell, a bispecific antibody is formed.

2. The bispecific antibody according to claim 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.

3. The bispecific antibody according to claim 2, wherein the first variable light chain is of the lambda1 type and the second variable light chain is of the kappa6 type.

4. The bispecific antibody according to claim 3, wherein the first and second constant heavy chains are IgG1, and wherein a. The first constant heavy chain has a point mutation that generates a stud structure, and the second constant heavy chain has a point mutation that generates a socket structure, or b. The first constant heavy chain has a point mutation that generates a socket structure, and the second constant heavy chain has a point mutation that generates a stud structure, and optionally c. The first and second constant heavy chains have mutations that result in disulfide bonds.

5. The bispecific antibody according to any one of the preceding claims, which comprises a first immunoglobulin VH1 domain, a first immunoglobulin VL1 domain, a second immunoglobulin VH2 domain and a second immunoglobulin VL2 domain, wherein: a. The first immunoglobulin VH1 domain comprises (for example, in sequence): i. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:76, CDR2 has the amino acid sequence SEQ ID NO:77, and CDR3 has the amino acid sequence SEQ ID NO:78; or ii. Hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:79, CDR2 has the amino acid sequence SEQ ID NO:80, and CDR3 has the amino acid sequence SEQ ID NO:81; and b. The first immunoglobulin VL1 domain comprises (for example, in sequence): i. Hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:92, CDR2 has the amino acid sequence SEQ ID NO:93, and CDR3 has the amino acid sequence SEQ ID NO:94 or ii. Hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:95, CDR2 has the amino acid sequence SEQ ID NO:96, and CDR3 has the amino acid sequence SEQ ID NO:97; and c. The second immunoglobulin VH2 domain comprises (e.g., in sequence): i. Hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:44, CDR2 has the amino acid sequence SEQ ID NO:45, and CDR3 has the amino acid sequence SEQ ID NO:46; or ii. Hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:47, CDR2 has the amino acid sequence SEQ ID NO:48, and CDR3 has the amino acid sequence SEQ ID NO:49; and d. The second immunoglobulin VL2 domain comprises (e.g., in sequence): i. Hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:60, CDR2 has the amino acid sequence SEQ ID NO:61, and CDR3 has the amino acid sequence SEQ ID NO:62 or ii. Hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:63, CDR2 has the amino acid sequence SEQ ID NO:64, and CDR3 has the amino acid sequence SEQ ID NO:

65.

6. The bispecific antibody according to any one of the preceding claims, which comprises a first immunoglobulin VH1 domain, a first immunoglobulin VL1 domain, a second immunoglobulin VH2 domain, and a second immunoglobulin VL2 domain, wherein: a. The first immunoglobulin VH1 domain comprises the amino acid sequence SEQ ID NO:85, b. The first immunoglobulin VL1 domain comprises the amino acid sequence SEQ ID NO:101, c. The second immunoglobulin VH2 domain comprises the amino acid sequence SEQ ID NO:53, and d. The second immunoglobulin VL2 domain comprises the amino acid sequence SEQ ID NO:

69.

7. The bispecific antibody according to any one of the preceding claims, which comprises a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain, wherein: a. The first immunoglobulin heavy chain comprises the amino acid sequence SEQ ID NO:87, b. The first immunoglobulin light chain comprises the amino acid sequence SEQ ID NO: 103, c. The second immunoglobulin heavy chain comprises the amino acid sequence SEQ ID NO: 55, and d. The second immunoglobulin light chain comprises the amino acid sequence SEQ ID NO:

71.

8. A method for selecting a bispecific antibody according to any one of claims 1-7, the method comprising: a. A first step: selecting the first part and the second part; b. 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; c. A third step: purifying the bispecific antibody by removing mismatched fragments from correctly matched bispecific antibodies.

9. The method according to claim 8, wherein the third step of purification yields a bispecific antibody that is at least 60% (by mass), 70% (by mass), 80% (by mass), 85% (by mass) pure, such as at least 90% (by mass) pure, 95% (by mass), 96% (by mass), 97% (by mass), 98% (by mass) or 99% (by mass) pure.

10. A method for manufacturing a bispecific antibody according to any one of claims 1-7 by co-expression in a common host cell, the method comprising a. A first step: generating at least one vector encoding the first part and the second part; b. A second step: introducing the at least one vector into the common host cell; c. A third step: selecting cells that specifically express the bispecific antibody; d. A fourth step: culturing the selected cells under conditions in which the bispecific antibody is expressed by the cells; and e. A fifth step: purifying the bispecific antibody, the bispecific antibody being at least 60% (by mass), 70% (by mass), 80% (by mass), or 85% (by mass) pure, such as at least 90% (by mass) pure, 95% (by mass), 96% (by mass), 97% (by mass), 98% (by mass) or 99% (by mass) pure.

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

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

13. The expression system according to claim 12, the expression system comprising: a. A polynucleotide encoding a first selection marker (sm I); b. A polynucleotide encoding a second selection marker (smII), the smII being different from the first selection marker (smI).

14. The expression system according to claim 12 or 13, wherein the first selection marker (smI) is a folate transporter or a polynucleotide encoding a mutant folate receptor, wherein the mutant folate receptor has a reduced folate binding affinity compared to the wild-type folate receptor, and the second selection marker (sm II) is DHFR.

15. The expression system according to any one of claims 12-14, wherein the first selection marker (sm I) is hygromycin, and the second selection marker (sm II) is Neo / G418.

16. The expression system according to any one of claims 12-15, the expression system comprising two expression vectors, wherein: a. A first vector, which comprises a polynucleotide encoding at least the first selection marker (smI) and a polynucleotide encoding at least the first part; and b. A second vector, which comprises a polynucleotide encoding at least the second selection marker (smII) and a polynucleotide encoding at least the second part.

17. The expression system according to any one of claims 12-16, the expression system 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.

18. A method of selecting a co-host cell for the method according to any one of claims 8-11, the method comprising a. A first step: providing a plurality of host cells, the plurality of host cells comprising the expression system according to any one of claims 12-17; and b. Culturing the plurality of host cells under conditions selective for the selection marker, thereby obtaining host cells expressing the desired product.

19. The method according to claim 18, wherein a selective medium is used: a. Comprising a limited concentration of folic acid; and / or b. Comprising a concentration of folic acid of 500 nM or lower; and / or c. Comprising a folic acid concentration selected from: i. 1000 nM - 100 pM; ii. 100 nM - 1 nM; iii. 15 nM - 1 nM; iv. 10 nM - 1 nM; and v. 10 nM - 2.5 nM; and / or d. Comprising a DHFR inhibitor; and / or e. Comprising an antifolate; and / or f. Comprising an antifolate at a concentration of 500 nM or lower; and / or g. Comprising an MTX concentration selected from: i. 500 nM - 3 nM; ii. 100 nM - 10 nM; iii. 50 nM - 10 nM; and iv. 50 nM and / or h. Comprising an antifolate at a concentration up to 20 times the folic acid concentration; and / or i. Comprising an antifolate at a concentration 10 - 20 times the folic acid concentration; and / or j. Comprising folic acid at a concentration up to 15 nM and an equimolar concentration of MTX up to 20 times.

20. The method according to claim 18 or 19, wherein the host cell comprises the expression system according to claim 17, wherein at least a portion of the first or second portion 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 comprises the following steps: a. contacting the plurality of host cells with a detection compound that binds to the fusion polypeptide; b. selecting at least one host cell based on the presence or amount of the detection compound bound to the cell surface.

21. The method according to claim 20, wherein the detection compound comprises the first or second target or a derivative thereof and at least one detection label.

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

23. The method according to claim 22, wherein the chromatography comprises a. a first step of capture; b. a second step of polishing; and optionally c. a third step of further polishing.

24. The method according to claim 23, wherein the first step is carried out by capturing using principles selected from the group consisting of: Fc-binding affinity chromatography, such as Protein A or Protein G; λ light chain-specific affinity chromatography, such as LambdaFabSelect TM ; κ 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.

25. The method according to claim 22 or 23, wherein the second step of purification is carried out by a principle selected from the group consisting of: Fc-binding affinity chromatography, such as Protein A or Protein G; λ light chain-specific affinity chromatography, such as LambdaFabSelect TM ; κ 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 virus inactivation.

26. The method according to claim 25, wherein said third step is further refined by a principle selected from the group consisting of: alone or in combination, Fc-binding affinity chromatography, such as Protein A or Protein G; λ light chain-specific affinity chromatography, such as LambdaFabSelect TM ; κ light chain-specific affinity chromatography, such as KappaSelect TM ; anti-idiotypic affinity chromatography, such as said first portion or said second portion; target-based affinity chromatography, such as affinity chromatography using said first target or said second target; ion exchange chromatography, such as Capto TM adhesion or Fractogel TM EMD SO 3 ; hydrophobic interaction chromatography; and virus inactivation.

27. The method according to any one of claim 23, the method selected from: a. First-step Protein A capture, e.g., MabSelect TM SuRe TM ; Second-step lambda light chain affinity chromatography, e.g., LambdaFabSelect TM ; And third-step kappa light chain affinity chromatography, e.g., KappaSelect TM ; or b. First-step protein A, e.g., MabSelect TM SuRe TM , second-step kappa light chain affinity chromatography, e.g., KappaSelect TM , and third-step lambda light chain affinity chromatography, e.g., LambdaFabSelect TM ; or c. First step of kappa light chain affinity chromatography, such as KappaSelect TM , and second step of lambda light chain affinity chromatography, such as LambdaFabSelect TM ; or d. The first step is lambda light chain affinity chromatography, such as LambdaFabSelect TM , and the second step is kappa light chain affinity chromatography, such as KappaSelect TM .

28. The method according to any one of claims 8-11 or 18-27, wherein the cell line is selected from the group consisting of: CHO cells; non-productive 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.

29. A pharmaceutical composition comprising the antibody according to any one of claims 1-7 and a pharmaceutically acceptable carrier.

30. The bispecific antibody according to any one of claims 1-7 or the pharmaceutical composition according to claim 29, for use as a medicament.

31. The bispecific antibody according to any one of claims 1-7 or the pharmaceutical composition according to claim 29, for the treatment of inflammasome-related diseases.

32. The bispecific antibody according to any one of claims 1-7 or the pharmaceutical composition according to claim 29, for the treatment of the inflammasome-related disease according to claim 31, wherein the inflammasome-related disease is selected from the group consisting of: sickle cell disease, angiopathy, 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.

33. A method for treating an inflammasome-related disorder, the method comprising administering to a subject suffering from an inflammasome-related disorder an effective amount of the bispecific antibody according to claims 1-7 or the pharmaceutical composition according to claim 29.

34. The method according to claim 33, wherein the inflammasome-related disorder is sickle cell disease, angiopathy, 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.

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