Modified antibodies containing modified igg2 domains which elicit agonist or antagonistic properties and use thereof

Abstract

Through a combination of in vitro and in vivo approaches, the inventors show that human lgG2 (h2) delivers unique FcγR-independent agonistic activity to anti-CD40 antibodies and to antibodies specific to other immunostimulatory receptors, including 4-1BB and CD28. Investigation of an anti-human CD40 mAb, LOB7.4, revealed that the unique activity of h2 was dependent upon the precise arrangement of hinge and CH1 disulfide bonds. Chemical ‘shuffling’ or mutagenesis to ‘lock’ LOB7.4 into either a more flexible ‘h2A’ or more compact ‘h2B’ conformation endowed antagonistic and agonistic properties, respectively. Engineering of h2 in this way allows development of reagents with either immunostimulatory or immunosuppressive characteristics, with direct implication for the design of therapeutic mAb agents and fusion proteins.

1 - 88 . (canceled) 
     
     
         89 . A modified antibody that specifically binds to a receptor expressed on the surface of human cells, wherein said modified antibody agonizes one or more biological activities elicited by said receptor, or biological activities elicited by the interaction of a ligand and said receptor, wherein said antibody is other than a human IgG2 antibody, and further wherein the entire or substantially the heavy chain hinge and C H 1 domains and optionally the light chain constant region of said modified antibody, which is other than a human IgG2, are replaced with the corresponding entire or substantially the entire hinge and C H 1 domains and optionally the light chain constant regions of an hIgG2 antibody and wherein:
 (i) either or both of the heavy chain cysteine residue at position 127 and the light chain cysteine residue at position 214 (wherein numbering is according to Kabat) are deleted or changed to a different amino acid residue, resulting in an increase in the agonistic properties of the resultant modified antibody relative to an otherwise identical antibody wherein either or both of said cysteine residues are unchanged;   (ii) the only cysteine residue in the H2 regions of said modified antibody which is mutated or deleted is the cysteine at position 214 in the light chain and said modified antibody comprises either (1) a heavy chain wherein no cysteine residues are deleted or modified in the H2 regions or (2) one or more of the cysteine residues at positions 127, 232 or 233 of the heavy chain are deleted or mutated, resulting in a modified antibody wherein the agonistic properties of the resultant modified antibody are increased relative to an otherwise identical antibody lacking said deletions or modifications;   (iii) is an hIgG1, hIgG3, or hIgG4, wherein the entire or substantially the entire hinge and C H 1 domains of said antibody and optionally the entire or substantially the entire light chain constant region have been replaced with the corresponding entire or substantially the entire light chain, and the hinge and CH1 domains (“H2 regions” or “H2 domains”) of hIgG2;   (iv) is a modified hIgG1 or hIgG3;   (v) specifically binds to an antigen expressed on human immune cells;   (vi) it specifically binds to an antigen expressed on human dendritic cells, mast cells, monocytes, macrophages, NK cells, B lymphocytes, T lymphocytes or any combination of the foregoing;   (vii) absent said modifications the antibody lacks agonistic activity;   (viii) absent said modifications the antibody possesses no more than 10%, 20%, 30%, 40%, 50% or no more than 50-80%, of the agonistic activity of the modified antibody, wherein agonistic activity is detected in an accepted assay for quantifying agonism;   (ix) the only cysteine residue in the hIgG2 heavy chain H2 regions which is removed, modified or substituted with another amino acid residue comprises the heavy chain cysteine residue at position 127;   (x) it comprises additional modifications outside the H2 regions which do not affect or appreciably affect (no more than 10% change) the agonistic properties of the modified antibody;   (xi) it specifically binds to a TNF or B7 superfamily member expressed on the surface of human immune cells;   (xii) it comprises the entire human IgG2 C H 1 and hinge and light chain constant regions;   (xiii) the agonistic properties of said antibody are FcγR independent;   (xiv) the antibody specifically binds to a TNFR superfamily member selected from CD40, LTα, LTβ, CD30, CD27, OX40, 4-1BB, TNF-R, TRANCE-R, GITR or “glucocorticoid-induced TNF receptor”, TWEAK, and FN14 or to a B7/CD28 family member selected from B7.1 (CD80), B7.2 (CD86), B7-H1, B7-H2, B7-H3 (CD276), B7-H4 (VTCN1), B7-H5 (VISTA), B7-H6 (NCR3LG1), B7-H7 (HHLA2), PD-1 (CD279), CD28 , CTLA-4 (CD152), ICOS(CD278), BTLA, NCR3, CD28H, and NKp30;   (xv) the antibody specifically binds to a TNFR superfamily member selected from 4-1BB, CD40, or CD27 or the B7/CD28 family member is CTLA-4 or CD28;   or any combination of the foregoing.   
     
     
         90 . A modified human IgG2 antibody (hIgG2) that specifically binds to a receptor expressed on human cells, wherein said antibody agonizes one or more biological activities elicited by said receptor or ligand, or biological activities elicited by the interaction of said ligand and its corresponding receptor, and further wherein said modified antibody comprises at least the light chain and the C H 1 and hinge of heavy chain regions of hIgG2 (“H2 regions”), wherein either or both of the cysteine residues selected from the heavy chain cysteine residue at position 127 and the light chain cysteine residue at position 214 are removed or changed to a different amino acid residue, resulting in an increase in the agonistic properties of the resultant agonistic antibody relative to an otherwise identical antibody wherein both of said cysteine residues are unchanged (wherein amino acid numbering is according to Kabat). 
     
     
         91 . A modified antibody according to  claim 89 , wherein:
 (i) the cysteine residue of the light chain of said modified antibody at position 214 is deleted or substituted with another amino acid and the modified antibody comprises either (1) a heavy chain wherein no cysteine residues within the H2 regions are deleted or mutated or (2) said modified antibody comprises a heavy chain wherein at least one of the cysteine residues at positions 127, 232 or 233 are deleted or mutated;   (ii) no cysteine residues within the H2 regions of the heavy chain are deleted or mutated;   (iii) at least one of the cysteine residues at positions 127, 232 or 233 in the heavy chain are deleted or mutated;   (iv) any combination of one or two of the cysteine residues at positions 127, 232 or 233 in the heavy chain are deleted or mutated;   (v) the H2 regions of the modified antibody are in the h2B conformation(heavy chain C127 and light chain C214 form disulfide bridges with heavy chain cysteines 232 and 233);   (vi) the only cysteine residue in the hIgG2 heavy chain H2 regions which is removed, modified or substituted with another amino acid residue comprises the heavy chain cysteine residue at position 127;   (vii) no cysteine residues in the light chain H2 regions are removed, modified or substituted with another amino acid residue;   (viii) the only cysteine residue in the hIgG2 light chain regions which is removed, modified or substituted with another amino acid residue comprises the light chain cysteine residue at position 214;   (ix) the only cysteine residue in the hIgG2 H2 regions which is removed, modified or substituted with another amino acid residue is selected from the heavy chain cysteine residue at position 127 and the light chain cysteine residue at position 214;   (x) a serine is substituted for the cysteine at position 214 in the light chain and/or a serine residue substituted for the cysteine residue at position 127 in the heavy chain;   (xi) it comprises additional modifications outside the H2 regions which do not affect or appreciably affect (no more than 10% change) the agonistic properties of the modified antibody;   (xii) it specifically binds to a TNF or B7 superfamily member expressed on the surface of human immune cells;   (xiii) it comprises the entire human IgG2 C H 1 and hinge and light chain constant regions;   (xiv) the agonistic properties of said antibody are FcγR independent;   (xv) the antibody specifically binds to a TNFR superfamily member selected from CD40, LTα, LTβ, CD30, CD27, OX40, 4-1BB, TNF-R, TRANCE-R, GITR or “glucocorticoid-induced TNF receptor”, TWEAK, and FN14 or to a B7/CD28 family member selected from B7.1 (CD80), B7.2 (CD86), B7-H1, B7-H2, B7-H3 (CD276), B7-H4 (VTCN1), B7-H5 (VISTA), B7-H6 (NCR3LG1), B7-H7 (HHLA2), PD-1 (CD279), CD28 , CTLA-4 (CD152), ICOS(CD278), BTLA, NCR3, CD28H, and NKp30;   (xvi) the antibody specifically binds to a TNFR superfamily member selected from 4-1BB, CD40, or CD27 or the B7/CD28 family member is CTLA-4 or CD28;   or any combination of the foregoing.   
     
     
         92 . A modified hIgG1 or hIgG3 or hIgG4 antibody according to  claim 89 , wherein the entire hinge and C H 1 regions of said hIgG1, hIgG3 or hIgG4 and optionally the light chain constant region (H2 regions) are replaced with the corresponding hinge and light chain constant regions of C H 1 regions of hIgG2 and further comprising one or more modified cysteine residues within the H2 regions which provide for increased agonism compared to an otherwise identical antibody lacking said cysteine modifications. 
     
     
         93 . A pharmaceutical composition comprising an effective amount of a modified antibody according to  claim 89 , optionally comprising another immune agonist. 
     
     
         94 . A pharmaceutical composition comprising an effective amount of a modified antibody according to  claim 90 , optionally comprising another immune agonist. 
     
     
         95 . A therapeutic method which comprises the administration of an effective amount at least one modified antibody or pharmaceutical composition containing according to  claim 89 , to a subject in need thereof. 
     
     
         96 . A therapeutic method which comprises the administration of an effective amount at least one modified antibody or pharmaceutical composition containing according to  claim 90 , to a subject in need thereof. 
     
     
         97 . The method of  claim 95 , which is used to treat a condition selected from cancer, infection, allergy, autoimmunity or inflammation. 
     
     
         98 . The method of  claim 96 , which is used to treat a condition selected from cancer, infection, allergy, autoimmunity or inflammation. 
     
     
         99 . A modified antibody that specifically binds to a receptor or ligand expressed on the surface of human cells, wherein said modified antibody antagonizes one or more biological activities elicited by said receptor or ligand, or biological activities elicited by the interaction of said ligand and its corresponding receptor, wherein said antibody is other than a human IgG2 antibody, and further wherein the entire or substantially the entire hinge and C H 1 domains and optionally the light chain constant region of said modified antibody, which is other than a human IgG2, are replaced with the corresponding entire or substantially the entire hinge and C H 1 domains and light chain constant region (“H2 regions” or “H2 domains”) of an hIgG2 antibody, wherein any combination of at least one cysteine residue contained in said IgG2 H2 regions which are selected from the cysteine residues at positions 232, 233, 236 and 239 are removed or changed to a different amino acid residue, resulting in an increase in the antagonistic properties of the resultant modified antibody relative to an otherwise identical antibody wherein said one or more cysteine residues are unchanged. 
     
     
         100 . A modified antibody according to  claim 99 , which is produced by combining an optionally modified light chain containing an hIgG2 constant region wherein C214 is unchanged and a light chain variable region of an antibody that is specific to a desired human ligand or receptor expressed on human cells, and a modified heavy chain comprising the C H 1 and hinge regions of hIgG2 and a variable region of the same antibody specific to the same human ligand or receptor as the light chain variable region, said modified heavy chain comprising one or more modifications comprising the deletion or substitution of one or more (any combination of) the cysteine residues at positions 232, 233, 236 and 239, wherein such combination selectively results in a modified antibody in the h2A conformation which specifically binds and antagonizes biological activities elicited by particular human ligand or receptor bound thereby. 
     
     
         101 . The modified antibody of  claim 99 , which is selected from an hIgG1, hIgG3, or hIgG4, wherein the entire or substantially the entire hinge and C H 1 domains of said antibody have been replaced with the corresponding entire or substantially the entire hinge and C H 1 domains (“H2 regions” or “H2 domains”) of hIgG2. 
     
     
         102 . A modified antibody according to  claim 99 , wherein any combination of at least one cysteine residue contained in said IgG2 H2 regions which are selected from the cysteine residues at positions 232, 233, 236 and 239 are removed or changed to a different amino acid residue, resulting in an increase in the antagonistic properties of the resultant modified antibody relative to an otherwise identical antibody wherein said one or more cysteine residues are unchanged. 
     
     
         103 . A modified antibody according to  claim 99 , wherein
 (i) the cysteine at residue 214 in the light chain and the cysteine at residue 127 in the heavy chain of the modified antibody are not deleted, modified or substituted with another amino acid residue;   (ii) the cysteine modifications within the H2 regions of the antibody selectively favor or result in the association of the heavy and light chains of the modified antibody via the cysteine at position 214 in the light chain and the cysteine at position 127 in the heavy chain;   (iii) the H2 regions of the modified antibody are in the h2A conformation;   (iv) at least one of the cysteine residues in the H2 other than C214 and C127 are changed to a serine residue;   (v) it comprises a serine substituted for the cysteine at position 232 in the heavy chain;   (vi) it comprises a serine substituted for the cysteine at position 233 in the heavy chain;   (vii) it is a modified hIgG1 or hIgG3;   (viii) it specifically binds to a receptor expressed on human immune cells;   (viii) it specifically binds to a receptor expressed on human immune cells selected from dendritic cells, mast cells, monocytes, macrophages, NK cells, B lymphocytes, T lymphocytes or any combination of the foregoing;   (ix) absent said modifications the antibody lacks antagonistic activity;   (x) absent said modifications the antibody possesses no more than 10%, 20%, 30%, 40%, 50% of the antagonistic activity of the modified antibody, wherein antagonistic activity is detected in an accepted assay for quantifying antagonism; or   any combination of the foregoing.   
     
     
         104 . A modified antibody that specifically binds to a receptor expressed on human cells, wherein said antibody antagonizes one or more biological activities elicited by said receptor or ligand, or biological activities elicited by the interaction of said ligand and its corresponding receptor, and further wherein said antagonistic antibody comprises at least the C H 1 and hinge heavy and light chain constant regions of human IgG2 (“H2 regions”), wherein at least one cysteine residue of said IgG2 H2 regions selected from the cysteine residues at positions 232, 233, 236 and 239 are removed or changed to a different amino acid residue, resulting in an increase in the antagonistic properties of the resultant antagonistic antibody relative to an otherwise identical antibody wherein said one or more cysteine residues are unchanged. 
     
     
         105 . The modified antibody of  claim 104 , which
 (i) is produced by combining an optionally modified light chain containing an hIgG2 constant region wherein C214 is unchanged and a light chain variable region that is specific to a desired human ligand or receptor expressed on human cells, and a modified heavy chain comprising the C H 1 and hinge regions of hIgG2 and a variable region specific to the same human ligand or receptor as the light chain variable region, said modified heavy chain comprising one or more modifications comprising the deletion or substitution of one or more (any combination of) the cysteine residues at positions 232, 233, 236 and 239 , wherein such combination selectively results in a modified antibody in the h2A conformation which specifically binds and antagonizes biological activities elicited by particular human ligand or receptor bound thereby;   (ii) the cysteine residues at position 214 in the light chain and position 127 in the heavy chain are not deleted, modified or substituted with another amino acid;   (iii) one or more cysteine or other amino acid residues in the hIgG2 H2 regions are removed, modified or substituted with another amino acid residue resulting in the H2 regions being in the h2A conformation, wherein h2A conformation means that the heavy chain C127 in C H 1 is linked to C214 in the light chain and optionally none, one, two, or three inter-HC disulfide bonds are present between opposing hinge cysteines 232, 233, 236 and 239;   (iv) at least one, two, three or all of said the H2 region cysteine residues other than C127 and C214 in the hIgG2 H2 regions are changed to a serine residue, and C127 and C214 are unmodified;   (v) it comprises a serine substituted for the cysteine at position 232 in the heavy chain or a serine substituted for the cysteine at position 233, or a serine substituted for the cysteine at position 236 or a serine substituted for the cysteine at position 239, or any combination of two, three or all of said modifications;   (vi) it comprises a serine substituted for the cysteine at position 233 in the heavy chain;   (vii) it specifically binds to a TNF or TNFR superfamily member or B7 family member expressed on the surface of human cells;   (viii) it specifically binds to a receptor expressed on T, B, mast, macrophage, monocyte, NK, or dendritic cells;   (ix) the antagonistic properties of said antibody are FcγR independent;   (x) the antibody specifically binds to a TNFR superfamily member selected from CD40, CD40L (CD154), LTα, LTβ, FASL (CD178), CD30, CD30L (CD153), CD27, CD27L (CD70), OX40, OX40L, TRAIL/APO-2L, 4-1BB,4-1BBL, TNF, TNF-R, TRANCE, TRANCE-R, GITR or “glucocorticoid-induced TNF receptor”, TWEAK, and FN14;   (xi) the antibody specifically binds to a TNFR or B7 family member selected from 4-1BB, 4-1BBL, CD40, CD40L, CD27, CD28, B7.1, B7.2, or CD70;   or any combination of any of the foregoing.   
     
     
         106 . A pharmaceutical composition containing a purified modified antibody according to  claim 99 , which is substantially homogeneous, i.e., the composition is substantially devoid of any other “h2a” or “h2b” forms of said antibody. 
     
     
         107 . A pharmaceutical composition containing a purified modified antibody according to  claim 104 , which is substantially homogeneous, i.e., the composition is substantially devoid of any other “h2a” or “h2b” forms of said antibody. 
     
     
         108 . A therapeutic method which comprising the administration of at least one antagonistic antibody or composition containing to a subject in need thereof, wherein the method comprises the administration of a modified antibody according to  claim 99 , optionally a condition selected from cancer, allergy, infectious disease, autoimmunity or an inflammatory condition. 
     
     
         109 . A therapeutic method which comprising the administration of at least one antagonistic antibody or composition containing to a subject in need thereof, wherein the method comprises the administration of a modified antibody according to  claim 104 , optionally a condition selected from cancer, allergy, infectious disease, autoimmunity or an inflammatory condition.