Method of manufacturing bispecific antibodies, bispecific antibodies and therapeutic use of such antibodies

Abstract

The invention relates to bivalent bispecific monoclonal antibodies (bbmAb) or variants thereof, and methods of manufacturing such antibodies by co-expressing modified Fc-mutated derivatives of two different monoclonal antibodies in mammalian cell lines.

What is claimed is: 
     
         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 with a first variable light chain of lambda wild type (VL1) and a first variable heavy chain of wild type (VH1), that binds specifically to a first target, and a first constant heavy chain (CH1) with a hetero-dimerization modification, and   b. a second part which is an immunoglobulin with a second variable light chain of kappa wild type (VL2) and a second variable heavy chain of wild type (VH2), that binds specifically to a second target, different from the first target, and a second constant heavy chain (CH2) with a hetero-dimerization modification which is complementary to the hetero-dimerization modification of the first constant heavy chain,   
       wherein the first part and the second part, when co-expressed in a common host cell, form a bispecific antibody. 
     
     
         2 . The bispecific antibody according to  claim 1 , wherein the first and second constant heavy chain 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 wherein the first variable light chain is of lamba1 type, and the second variable light chain is of kappa 6 type. 
     
     
         4 . The bispecific antibody according to  claim 3 , wherein the first and second constant heavy chain are IgG1, and wherein
 a. the first constant heavy chain has point mutations generating a knob structure and the second constant heavy has point mutations generating a hole structure, or   b. the first constant heavy chain has point mutations generating a hole structure and the second constant heavy has point mutations generating a knob structure, and optionally   c. the first and second constant heavy chains have mutations resulting in a disulfide bridge.   
     
     
         5 . The bispecific antibody of  claim 1 , comprising 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 (e.g. in sequence):
 i. hypervariable regions complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3), said CDR1 having the amino acid sequence SEQ ID NO:76, said CDR2 having the amino acid sequence SEQ ID NO:77, and said CDR3 having the amino acid sequence SEQ ID NO:78; or 
 ii. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:79, said CDR2 having the amino acid sequence SEQ ID NO:80, and said CDR3 having the amino acid sequence SEQ ID NO:81; and 
   b. the first immunoglobulin VL1 domain comprises (e.g. in sequence):
 i. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:92, said CDR2 having the amino acid sequence SEQ ID NO:93, and said CDR3 having the amino acid sequence SEQ ID NO:94 or 
 ii. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:95, said CDR2 having the amino acid sequence SEQ ID NO:96, and said CDR3 having the amino acid sequence SEQ ID NO:97; and 
   c. the second immunoglobulin VH2 domain comprises (e.g. in sequence):
 i. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:44, said CDR2 having the amino acid sequence SEQ ID NO:45, and said CDR3 having the amino acid sequence SEQ ID NO:46; or 
 ii. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:47, said CDR2 having the amino acid sequence SEQ ID NO:48, and said CDR3 having the amino acid sequence SEQ ID NO:49; and 
   d. the second immunoglobulin VL2 domain comprises (e.g. in sequence):
 i. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:60, said CDR2 having the amino acid sequence SEQ ID NO:61, and said CDR3 having the amino acid sequence SEQ ID NO:62 or 
 ii. hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:63, said CDR2 having the amino acid sequence SEQ ID NO:64, and said CDR3 having the amino acid sequence SEQ ID NO:65. 
   
     
     
         6 . The bispecific antibody according to  claim 1 , comprising 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  claim 1 , comprising 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  claim 1 , said method comprising;
 a. a first step of selecting the first part, and the second part;   b. a second step of co-expressing the first part and the second part in a common host cell, resulting in a bispecific antibody comprising the first part and the second part;   c. a third step of purifying the bispecific antibody by removing mismatched fragments from the correctly matched bispecific antibody.   
     
     
         9 . The method according to  claim 8 , wherein the third step of purification results in a bispecific antibody which is at least 60% (mass), 70% (mass), 80% (mass), 85% (mass), 90% (mass), 95% (mass), 96% (mass), 97% (mass), 98% (mass), or 99% (mass) pure. 
     
     
         10 . A method for manufacturing a bispecific antibody according to  claim 1  by co-expression in a common host cell, said method comprising;
 a. a first step of generating at least one vector encoding the first part and the second part; 
 b. a second step of introducing the at least one vector into the common host cell; 
 c. a third step of selecting cells specifically expressing the bispecific antibody; 
 d. a forth step of culturing the selected cells under conditions wherein the cells express the bispecific antibody; and 
 e. a fifth step of purifying the bispecific antibody which is at least 60% (mass), 70% (mass), 80% (mass), or 85% (mass), 90% (mass), 95% (mass), 96% (mass), 97% (mass), 98% (mass), or 99% (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 comprising a polynucleotide encoding the first part or the second part of the bispecific antibody according to  claim 1 , and a selectable marker. 
     
     
         13 . The expression system according to  claim 12  comprising
 a polynucleotide encoding a first selectable marker (sm I); and 
 a polynucleotide encoding a second selectable marker (sm II), which differs from the first selectable marker (sm I). 
 
     
     
         14 . The expression system according to  claim 13 , wherein the first selectable marker (sm I) is a folate transporter or a polynucleotide encoding a mutated folate receptor, wherein the mutated folate receptor has a decreased folate binding affinity compared to the wildtype folate receptor and the second selectable marker (sm II) is dihydrofolate reductase (DHFR). 
     
     
         15 . The expression system according to  claim 13 , wherein the first selectable marker (sm I) is Hygromycine and the second selectable marker (sm II) is Neo/G418. 
     
     
         16 . The expression system according to  claim 12 , comprising two expression vectors wherein:
 a. a first vector comprising polynucleotide encoding at least a first selectable marker (sm I) and at least polynucleotides encoding the first part; and   b. a second vector comprising polynucleotide encoding at least a second selectable marker (sm II) and at least polynucleotides encoding the second part.   
     
     
         17 . The expression system according to  claim 12 , comprising a stop codon downstream of the polynucleotides encoding the heavy chain and a polynucleotide encoding an immunoglobulin membrane anchor located downstream of the stop codon. 
     
     
         18 . A method for selecting a common host cell for use in the methods according to  claim 8 , comprising:
 a. a first step of providing a plurality of host cells, comprising an expression system comprising at least one vector comprising a polynucleotide encoding the first part or the second part of the bispecific antibody, and a selectable marker; and   b. culturing said plurality of host cells under conditions selective for the selectable marker, thereby obtaining a host cell expressing the product of interest.   
     
     
         19 . The method according to  claim 18 , wherein a selective culture medium is used:
 a. comprising a limiting concentration of folate; and/or   b. comprising a folic acid in a concentration of 500 nM or less; and/or   c. comprising a folic acid in a 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 in a concentration of 500 nM or less; and/or   g. comprising MTX in a 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 a concentration of antifolate up to 20-fold of the folate concentration; and/or   i. comprising a concentration of antifolate 10-20 fold of the folate concentration; and/or   j. comprising a folic acid in a concentration up to 15 nM and an equimolar concentration up to 20-fold of MTX.   
     
     
         20 . The method according to  claim 18 , wherein the host cell comprises an expression system comprising a stop codon downstream of the polynucleotides encoding the heavy chain and a polynucleotide encoding an immunoglobulin membrane anchor located downstream of the stop codon, wherein at least a portion of the first or second part is expressed as a fusion polypeptide comprising the immunoglobulin membrane anchor or fragment thereof, and wherein said fusion polypeptide is being displayed on the surface of said host cell, further comprising a step of:
 a. contacting the plurality of host cells with a detection compound binding the fusion polypeptide;   b. selecting at least one host cell based upon 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 derivatives thereof, and at least one detection label. 
     
     
         22 . The method according to  claim 10 , 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 first step of capturing and a second step of polishing.   
     
     
         24 . The method according to  claim 23 , further comprising a third step of further polishing. 
     
     
         25 . The method according to  claim 23 , wherein the first step of capturing is performed with a principle selected from the group consisting of Fc-binding affinity chromatography, lambda light chain specific affinity chromatography, kappa light chain specific affinity chromatography, anti-idiotypic affinity chromatography, a target based affinity chromatography, ion exchange chromatography, and hydrophobic interaction chromatography. 
     
     
         26 . The method according to  claim 23 , wherein the second step of polishing is performed with a principle selected from the group consisting of Fc-binding affinity chromatography, lambda light chain specific affinity chromatography, kappa light chain specific affinity chromatography, anti-idiotypic affinity chromatography, a target based affinity chromatography, such as, ion exchange chromatography, hydrophobic interaction chromatography, and virus inactivation. 
     
     
         27 . The method according to  claim 24 , wherein the third step of further polishing is performed with a principle selected from the group consisting of Fc-binding affinity chromatography, lambda light chain specific affinity chromatography, kappa light chain specific affinity chromatography, anti-idiotypic affinity chromatography, a target based affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and virus inactivation, alone or in combination. 
     
     
         28 . The method according to  claim 23 , selected from:
 a. a first step of Protein A capturing, a second step of lambda light chain affinity chromatography, and comprising a third step of further polishing comprising kappa light chain affinity chromatography; or   b. a first step of Protein A, a second step of kappa light chain affinity chromatography, and comprising a third step of further polishing comprising lambda light chain affinity chromatography; or   c. a first step of kappa light chain affinity chromatography and a second step of lambda light chain affinity chromatography; or   d. a first step of lambda light chain affinity chromatography and a second step of kappa light chain affinity chromatography.   
     
     
         29 . The method according to  claim 8 , wherein the host cell is selected from the group consisting of a CHO cell, a non-producing hybridoma, a human derived cell line, a baby hamster kidney (BHK) derivative, a yeast or filamentous fungi, a prokaryotic bacteria, a plant derivative, an algae and a ciliate. 
     
     
         30 . A pharmaceutical composition comprising the antibody according to  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . A method of treating an inflammasome related disorder comprising administering to a subject afflicted with an inflammasome related disorder an effective amount of a bispecific antibody according to  claim 1 . 
     
     
         35 . The method according to  claim 34 , wherein the inflammasome related disorder is sickle cell disease, vasculopathy, 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.