Mixtures of antibodies

Abstract

Described herein are antibodies and mixtures of antibodies optionally produced by a host cell line, nucleic acids encoding the antibodies and mixtures of antibodies, host cells containing such nucleic acids, and methods of treatment using the antibodies, mixtures of antibodies, or nucleic acids encoding the antibodies or mixtures of antibodies. Also described are methods of producing mixtures of antibodies in host cells.

1 - 81 . (canceled) 
     
     
         82 . A mixture of antibodies comprising two and not more than three different major antibody species, which comprises
 (a) a first antibody comprising two chains of a first heavy chain (HC1), each having the same first amino acid sequence, and two chains of a first light chain (LC1), each having the same second amino acid sequence, and   (b) a second antibody comprising two chains of a second heavy chain (HC2), each having the same third amino acid sequence, and two chains of a second light chain (LC2), each having the same fourth amino acid sequence,   wherein:
 (1) the first and second antibodies are full-length human and/or humanized IgG antibodies; 
 (2) the HC1 and the HC2 have different amino acid sequences, and the LC1 and the LC2 have different amino acid sequences; 
 (3) the first heavy chain constant (CH1) and the light chain constant (CL) domains of the first antibody and/or the second antibody comprise one or more partner-directing alterations that create one or more pairs of contacting cysteine residues, wherein one cysteine residue of the pair(s) is in the CH1 domain and the other is in the CL domain of the antibody or antibodies comprising the pair(s) of contacting cysteine residues, wherein (A) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG1 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG1 antibody or antibodies is (are) selected from the group consisting of: positions 126 and 124; positions 128 and 118; positions 133 and 117; positions 133 and 209; positions 134 and 116; positions 168 and 174; positions 170 and 162; positions 170 and 176; positions 173 and 160; and positions 183 and 176, (B) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG2 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG2 antibody or antibodies is (are) selected from the group consisting of: positions 170 and 162; and positions 173 and 160, and (C) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG4 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG4 antibody or antibodies is (are) selected from the group consisting of: positions 126 and 121; positions 126 and 124; positions 127 and 121; positions 128 and 118; positions 168 and 174; positions 170 and 162; and positions 173 and 162; and 
 (4) if both the first and second antibodies comprise a pair of contacting cysteine residues as recited in (3) above, then the first and second antibodies do not comprise contacting cysteine residues at the same pairs of positions. 
   
     
     
         83 . The mixture of  claim 82 , wherein the first antibody is an IgG1, IgG2, or IgG4 antibody, and the second antibody is an IgG1, IgG2, or IgG4 antibody. 
     
     
         84 . The mixture of  claim 83 , wherein the mixture comprises a third major antibody species comprising one chain of the HC1, one chain of the LC1, one chain of the HC2, and one chain of the LC2. 
     
     
         85 . The mixture of  claim 83 ,
 wherein the first antibody and/or the second antibody comprise(s) (a) charge pair(s),   wherein one amino acid of the charge pair(s) is in the CH1 and the other amino acid of the charge pair(s) is in the CL of the antibody or antibodies comprising the charge pair, and   wherein:
 (1) if the antibody or antibodies comprising the charge pair(s) is (are) (an) IgG1 antibody or antibodies, then the charge pair(s) is (are) selected from the group consisting of the following pairs of residues, which are specified by their the heavy and light chain positions, respectively, and the amino acids occupying those positions: 147D/E and 124K/R; 147K/R and 124D/E; 147D/E and 129K/R; 147K/R and 129D/E; 147D/E and 131K/R; 147K/R and 131D/E; 147D/E and 180K/R; 147K/R and 180D/E; 168D/E and 164K/R; 168K/R and 164D/E; 168D/E and 167K/R; 168K/R and 167D/E; 168D/E and 174K/R; and 168K/R and 174D/E; 
 (2) if the antibody or antibodies comprising the charge pair(s) is (are) (an) IgG2 antibody or antibodies, then the charge pair(s) is (are) selected from the group consisting of the following pairs of residues, which are specified by their the heavy and light chain positions, respectively, and the amino acids occupying those positions: 147D/E and 131K/R; and 147K/R and 131D/E; and 
 (3) if the antibody or antibodies comprising the charge pair(s) is (are) (an) IgG4 antibody or antibodies, then the charge pair(s) is (are) selected from the group consisting of the following pairs of residues, which are specified by their the heavy and light chain positions, respectively, and the amino acids occupying those positions: 133D/E and 117K/R; 133K/R and 117D/E; 137K/R and 114D/E; 137D/E and 114K/R; 137K/R and 116D/E; 137D/E and 116K/R; 147D/E and 124K/R; 147K/R and 124D/E; 147D/E and 129K/R; 147K/R and 129D/E; 147D/E and 131K/R; 147K/R and 131D/E; 147D/E and 178K/R; 147K/R and 178D/E; 147D/E and 180K/R; 147K/R and 180D/E; 168D/E and 164K/R; 168K/R and 164D/E; 168D/E and 167K/R; 168K/R and 167D/E; 168D/E and 173K/R; 168K/R and 173D/E; 168D/E and 174K/R; and 168K/R and 174D/E. 
   
     
     
         86 . The mixture of  claim 85 , wherein the pair of contacting cysteine residues in the first or second antibody is at positions 170 of HC1 and 162 of LC1 or positions 170 of HC2 and 162 of LC2, and wherein the amino acids comprising the charge pair in the first or second antibody are 147D/E in the heavy chain of the first or second antibody and 131R/K in the cognate light chain. 
     
     
         87 . The mixture of  claim 83 ,
 wherein the first antibody comprises one or more partner-directing alteration(s) and the second does not; and   wherein (1) if the first antibody is an IgG1 antibody, then the cysteine at position 220 in the HC1 is substituted with another amino acid, and the cysteine at position 214 in the LC1 is substituted with another amino acid, and (2) if the first antibody is an IgG2 or IgG4 antibody, then the cysteine at position 131 in the HC1 is substituted with another amino acid, and the cysteine at position 214 in the LC1 is substituted with another amino acid.   
     
     
         88 . The mixture of  claim 87 , wherein
 (a) the first antibody is an IgG1 antibody, and it comprises the alterations C220G/A/S in the HC1 and C214S/A/G in the LC1; or   (b) the first antibody is an IgG2 or IgG4 antibody, and it comprises the alterations C131SING in the HC1 and C214S/A/G in the LC1.   
     
     
         89 . The mixture of  claim 88 ,
 wherein the first antibody is an IgG1 antibody and comprises 147D, 170C, 173C, and 220G in the HC1 and 131K, 160C, 162C, and 214S in the LC1; or   wherein the first antibody is an IgG2 antibody and comprises 147D, 170C, 173C, and 131S/A/G in the HC1 and 131K, 160C, 162C, and 214S in the LC1.   
     
     
         90 . The mixture of  claim 88 , wherein the first antibody comprises 399R and 409E and the second antibody comprises 409R. 
     
     
         91 . The mixture of  claim 82 , wherein
 both the first and second antibodies comprise one or more partner-directing alteration(s),   the HC1 and the HC2 comprise a charged amino acid at an HC residue at the same position in both the HC1 and the HC2,   the charged amino acid at the HC residue in the HC1 is opposite in charge to the charged amino acid at the HC residue at the same position in the HC2,   the LC1 and the LC2 comprise a charged amino acid at an LC residue at the same position in both the LC1 and the LC2,   the LC residue contacts the HC residue,   the charged amino acid at the LC residue in the LC1 is opposite in charge to the charged amino acid at the LC residue in the LC2, and   the charged amino acid at the LC residue in the LC1 is opposite in charge to the amino acid at the HC residue in the HC1.   
     
     
         92 . The mixture of  claim 91 , wherein:
 both the HC1 and the HC2 comprise charged amino acids at positions 44, 105, 147, and 168; and   both the LC1 and the LC2 comprise charged amino acids at positions 43, 100, 131, and 174.   
     
     
         93 . The mixture of  claim 91 , wherein one of the first and second antibodies comprises 399R and 409E and the other comprises 409R. 
     
     
         94 . The mixture of  claim 83 , wherein if the HC1 and/or the HC2 is an IgG4 HC, then the IgG4 HC(s) comprise(s) 228P. 
     
     
         95 . The mixture of  claim 83 , wherein the in vivo half lives of the first and second antibodies differ by at least one week, and wherein the antibody with the shorter in vivo half life comprises at least one of the following alterations: M252A, M252L, M252S, M252R, R255K or H435R. 
     
     
         96 . The mixture of  claim 82 , wherein
 (a) one of the first and second antibodies binds to human CTLA4 and the other binds to human PD1,   (b) both the first and second antibodies bind to human HER2, but they do not compete for binding to HER2,   (c) one of the first and second antibodies binds to human LAG3 and the other binds to human PD1,   (d) one of the first and second antibodies binds to human GITR and the other binds to human PD1,   (d) one of the first and second antibodies binds to human VEGF and the other binds to human PD1,   (f) one of the first and second antibodies binds to human CSFR1a and the other binds to human PD1,   (g) one of the first and second antibodies binds to human OX40 and the other binds to human PD1,   (h) one of the first and second antibodies binds to human TIGIT and the other binds to human PD1,   (i) one of the first and second antibodies binds to human CTLA4 and the other binds to human PDL1,   (j) one of the first and second antibodies binds to human VEGF and the other binds to human PDL1,   (k) one of the first and second antibodies binds to human OX40 and the other binds to human PDL1,   (l) one of the first and second antibodies binds to human CSFR1a and the other binds to human PDL1,   (m) one of the first and second antibodies binds to human TIGIT and the other binds to human PDL1,   (n) one of the first and second antibodies binds to human Tim3 and the other binds to human PDL1,   (o) one of the first and second antibodies binds to human CTLA4 and the other binds to human VEGF,   (p) one of the first and second antibodies binds to human CTLA4 and the other binds to human 41BB,   (q) one of the first and second antibodies binds to human CD20 and the other binds to human CD37,   (r) one of the first and second antibodies binds to human ANG2 and the other binds to human VEGF,   (s) one of the first and second antibodies binds to human TNF and the other binds to human IL17a,   (t) one of the first and second antibodies binds to human CD38 and the other binds to human CD138,   (u) one of the first and second antibodies binds to human EGFR and the other binds to human HER2,   (v) one of the first and second antibodies binds to human EGFR and the other binds to human HER3,   (w) one of the first and second antibodies binds to human MET and the other binds to human VEGF   (x) one of the first and second antibodies binds to human MET and the other binds to human EGFR,   (y) one of the first and second antibodies binds to human TSLP and the other binds to human IL33,   (z) one of the first and second antibodies binds to human IL4 and the other binds to human IL13,   (aa) one of the first and second antibodies binds to human CSF1R protein and the other binds to a human SIRP-alpha protein,   (bb) one of the first and second antibodies binds to human PD1 protein and the other binds to a human SIRP-alpha protein, or   (cc) one of the first and second antibodies binds to human PD1 and the other binds to human CCR8.   
     
     
         97 . One or more nucleic acid(s) encoding a mixture of antibodies comprising two and not more than three different major antibody species, which include
 (a) a first antibody comprising two chains of a first heavy chain (HC1), each having the same first amino acid sequence, and two chains of a first light chain (LC1), each having the same second amino acid sequence, and   (b) a second antibody comprising two chains of a second heavy chain (HC2), each having the same third amino acid sequence, and two chains of a second light chain (LC2), each having the same fourth amino acid sequence,   wherein:
 (1) the first and second antibodies are full-length human and/or humanized IgG antibodies; 
 (2) the HC1 and the HC2 have different amino acid sequences, and the LC1 and the LC2 have different amino acid sequences; 
 (3) the first heavy chain constant (CH1) and the light chain constant (CL) domains of the first antibody and/or the second antibody comprise one or more partner-directing alterations that create one or more pairs of contacting cysteine residues, wherein one cysteine residue of the pair(s) is in the CH1 domain and the other is in the CL domain of the antibody or antibodies comprising the pair(s) of contacting cysteine residues, wherein (A) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG1 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG1 antibody or antibodies is (are) selected from the group consisting of: positions 126 and 124; positions 128 and 118; positions 133 and 117; positions 133 and 209; positions 134 and 116; positions 168 and 174; positions 170 and 162; positions 170 and 176; positions 173 and 160; and positions 183 and 176, (B) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG2 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG2 antibody or antibodies is (are) selected from the group consisting of: positions 170 and 162; and positions 173 and 160, and (C) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG4 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG4 antibody or antibodies is (are) selected from the group consisting of: positions 126 and 121; positions 126 and 124; positions 127 and 121; positions 128 and 118; positions 168 and 174; positions 170 and 162; and positions 173 and 162; and 
 (4) if both the first and second antibodies comprise a pair of contacting cysteine residues as recited in (3) above, then the first and second antibodies do not comprise contacting cysteine residues at the same pairs of positions. 
   
     
     
         98 . The nucleic acid(s) of  claim 97 ,
 wherein the first antibody is an IgG1, IgG2, or IgG4 antibody and the second antibody is an IgG1, IgG2, or IgG4 antibody.   
     
     
         99 . The nucleic acid(s) of  claim 97 , which include a mammalian expression vector or a viral vector, and wherein the viral vector is selected from the group consisting of: an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a vaccinia virus vector, a modified vaccinia virus Ankara (MVA) vector, a herpes virus vector, a lentivirus vector, and a poxvirus vector. 
     
     
         100 . A method of making a mixture of antibodies comprising the steps of:
 (a) introducing one or more nucleic acid(s) encoding the mixture of antibodies into a host cell line,   (b) culturing the host cell line expressing the mixture of antibodies in a culture medium, and   (c) recovering the mixture of antibodies from the cell mass or the culture medium,   wherein
 (1) the mixture comprises (A) a first antibody comprising two chains of a first heavy chain (HC1) each having the same first amino acid sequence and two chains of a first light chain (LC1) each having the same second amino acid sequence and (B) a second antibody comprising two chains of a second heavy chain (HC2) each having the same third amino acid sequence and two chains of a second light chain (LC2) each having the same fourth amino acid sequence, 
 (2) the first and second antibodies are full-length human and/or humanized IgG antibodies; 
 (3) the HC1 and the HC2 have different amino acid sequences, and the LC1 and the LC2 have different amino acid sequences; and 
 (4) the mixture comprises not more than three different major species of full-length IgG antibodies. 
   
     
     
         101 . The method of  claim 100 , wherein:
 (a) the nucleic acid(s) encoding the HC1, HC2, LC1, and LC2 was (were) introduced at the same time;   (b) the nucleic acid(s) encoding the HC1 and LC1 was (were) introduced before the nucleic acid(s) encoding the HC2 and LC2; or   (c) the nucleic acid(s) encoding the HC2 and LC2 was (were) introduced before the nucleic acid(s) encoding the HC1 and LC1.   
     
     
         102 . The method of a  claim 101 , wherein the host cell line is a mammalian cell line. 
     
     
         103 . A host cell line that contains nucleic acid(s) encoding a mixture of antibodies comprising two and not more than three different major antibody species, which includes
 (a) a first antibody comprising two chains of a first heavy chain (HC1), each having the same first amino acid sequence, and two chains of a first light chain (LC1), each having the same second amino acid sequence, and   (b) a second antibody comprising two chains of a second heavy chain (HC2), each having the same third amino acid sequence, and two chains of a second light chain (LC2), each having the same fourth amino acid sequence,   wherein:
 (1) the first and second antibodies are full-length human and/or humanized IgG antibodies; 
 (2) the HC1 and the HC2 have different amino acid sequences, and the LC1 and the LC2 have different amino acid sequences; 
 (3) the first heavy chain constant (CH1) and the light chain constant (CL) domains of the first antibody and/or the second antibody comprise one or more partner-directing alterations that create one or more pairs of contacting cysteine residues, wherein one cysteine residue of the pair(s) is in the CH1 domain and the other is in the CL domain of the antibody or antibodies comprising the pair(s) of contacting cysteine residues, wherein (A) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG1 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG1 antibody or antibodies is (are) selected from the group consisting of: positions 126 and 124; positions 128 and 118; positions 133 and 117; positions 133 and 209; positions 134 and 116; positions 168 and 174; positions 170 and 162; positions 170 and 176; positions 173 and 160; and positions 183 and 176, (B) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG2 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG2 antibody or antibodies is (are) selected from the group consisting of: positions 170 and 162; and positions 173 and 160, and (C) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG4 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG4 antibody or antibodies is (are) selected from the group consisting of: positions 126 and 121; positions 126 and 124; positions 127 and 121; positions 128 and 118; positions 168 and 174; positions 170 and 162; and positions 173 and 162; and 
 (4) if both the first and second antibodies comprise a pair of contacting cysteine residues as recited in (3) above, then the first and second antibodies do not comprise contacting cysteine residues at the same pairs of positions. 
   
     
     
         104 . The host cell line of  claim 103 , which is a CHO cell line. 
     
     
         105 . A method of treating a disease comprising administering to a patient having the disease a mixture of antibodies or (a) nucleic acid(s) encoding the mixture of antibodies,
 wherein:   (a) the disease is a cancer, a metabolic disease, an infectious disease, or an autoimmune or inflammatory disease;   (b) the mixture of antibodies comprises not more than three major species of antibodies including (1) a first antibody comprising two chains of a first heavy chain (HC1) each having the same first amino acid sequence and two chains of a first light chain (LC1) each having the same second amino acid sequence and (2) a second antibody comprising two chains of a second heavy chain (HC2) each having the same third amino acid sequence and two chains of a second light chain (LC2) each having the same fourth amino acid sequence;   (c) the first and second antibodies are full-length human and/or humanized IgG antibodies, the HC1 and the HC2 have different amino acid sequences, the LC1 and the LC2 have different amino acid sequences, and the mixture comprises not more than three different major species of full-length IgG antibodies;   (d) the CH1 and CL domains of the first antibody and/or the second antibody comprise one or more partner-directing alters creating a pair of contacting cysteine residues, wherein one cysteine residue of the pair is in the CH1 domain and the other is in the CL domain of the antibody or antibodies comprising the pair(s) of contacting cysteine residues;   (e) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG1 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG1 antibody or antibodies is (are) selected from the group consisting of: positions 126 and 124; positions 128 and 118; positions 133 and 117; positions 133 and 209; positions 134 and 116; positions 168 and 174; positions 170 and 162; positions 170 and 176; positions 173 and 160; and positions 183 and 176;   (f) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG2 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG2 antibody or antibodies is (are) selected from the group consisting of: positions 170 and 162; and positions 173 and 160,   (g) if the first and/or second antibody comprising the contacting cysteine residues is (are) (an) IgG4 antibody or antibodies, then the pair(s) of heavy chain and light chain positions, respectively, of the pair(s) of contacting cysteine residues in the IgG4 antibody or antibodies is (are) selected from the group consisting of: positions 126 and 121; positions 126 and 124; positions 127 and 121; positions 128 and 118; positions 168 and 174; positions 170 and 162; and positions 173 and 162; and   (h) if both the first and second antibodies comprise a pair of contacting cysteine residues as recited in (d)-(g), then the first and second antibodies do not comprise contacting cysteine residues at the same pairs of positions.   
     
     
         106 . The method of  claim 105 , wherein the disease is a cancer and wherein (1) the first antibody in the mixture of antibodies binds to human CTLA4 and the second antibody in the mixture of antibodies binds to human PD1, or vice versa, (2) both the first and second antibodies in the mixture of antibodies bind to human HER2, but they do not compete for binding to HER2, or (3) the first antibody in the mixture of antibodies binds to human CD20 and the second antibody in the mixture of antibodies binds to human CD37, or vice versa. 
     
     
         107 . The method of  claim 106 , wherein the disease is a cancer and the patient has a tumor, and wherein the method comprises injecting the mixture of antibodies or the nucleic acid(s) encoding the mixture of antibodies into the tumor.