Patent Grant
10457742
The present disclosure generally provides polypeptide heterodimers, compositions thereof, and methods for making and using such polypeptide heterodimers. More specifically, the present invention relates to thermo-stable multispecific, including bispecific, antibodies comprising a heterodimeric Fc domain.
Bispecific therapeutics are antibody-based molecules that can simultaneously bind two separate and distinct targets or different epitopes of the same antigen. Bispecific antibodies are comprised of the immunoglobulin domain based entities and try to structurally and functionally mimic components of the antibody molecule. One use of bispecific antibodies has been to redirect cytotoxic immune effector cells for enhanced killing of tumor cells, such as by antibody dependent cellular cytotoxicity (ADCC). In this context, one arm of the bispecific antibody binds an antigen on the tumor cell, and the other binds a determinant expressed on effector cells. By cross-linking tumor and effector cells, the bispecific antibody not only brings the effector cells within the proximity of the tumor cells but also simultaneously triggers their activation, leading to effective tumor cell-killing. Bispecific antibodies have also been used to enrich chemo- or radiotherapeutic agents in tumor tissues to minimize detrimental effects to normal tissue. In this setting, one arm of the bispecific antibody binds an antigen expressed on the cell targeted for destruction, and the other arm delivers a chemotherapeutic drug, radioisotope, or toxin. Going beyond bispecifics, there is a need for protein therapeutics to achieve their efficacies by targeting multiple modalities concurrently. Such complex and novel biological effects can be obtained with protein therapeutics by designing multi-target binding and multi-functional aspects into the protein.
A robust scaffold that provides a framework to fuse other functional war-heads or target protein binding domains in order to design these multifunctional and multi-target binding therapeutics is required. Ideally, the scaffold should not only provide the framework but also make available a number of other therapeutically relevant and valuable features to the designed therapeutic. A major obstacle in the general development of antibody-based bispecific and multifunctional therapeutics has been the difficulty of producing materials of sufficient quality and quantity for both preclinical and clinical studies. There remains a need in the art for polypeptide constructs that comprise single variable domains as the protein binding domains that are linked to a variant Fc region, said variant Fc comprising CH3 domains, which have been modified to select for heterodimers with an increased stability and purity.
There is provided according to one aspect of the invention an isolated heteromultimer Fc construct comprising a modified heterodimeric CH3 domain, said modified CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide; wherein at least one of said first and second CH3 domain polypeptides comprises an amino acid modification of K392J wherein J is selected from L, I or an amino acid with a side chain volume not substantially larger than the side chain volume of K; wherein said first and second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain with a melting temperature (Tm) of at least about 74° C. and a purity of at least 95%; and wherein at least one amino acid modification is not of an amino acid which is at the interface between said first and said second CH3 domain polypeptides. In certain embodiments is a heteromultimer Fc construct described herein, comprising at least one T350X modification, wherein X is a natural or non-natural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments is an isolated heteromultimer Fc construct described herein, comprising at least one T350V modification. In an embodiment is an isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In an embodiment is the isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a Tm of about 70° C. or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80° C. or greater. Provided in certain embodiments is an isolated heteromultimer Fc construct described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of at least one of L351, F405, and Y407. In some embodiments is an isolated heteromultimer Fc construct, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide further comprising an amino acid modification of T366. In certain embodiments is an isolated heteromultimer Fc construct described herein, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising amino acid modifications at positions L351, F405, and Y407, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising amino acid modifications at positions T366, K392, and T394. In an embodiment is the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A, and Y407V, and said second CH3 domain polypeptide comprising amino acid modifications T366L, K392M, and T394W. In some embodiments is the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A, and Y407V, and said second CH3 domain polypeptide comprising amino acid modifications T366L, K392L, and T394W. In a further embodiment is the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A, and Y407V, and said second CH3 domain polypeptide comprising amino acid modifications T366I, K392M, and T394W. In some embodiments is the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A, and Y407V, and said second CH3 domain polypeptide comprising amino acid modifications T366I, K392L, and T394W. In certain embodiments is the isolated heteromultimer Fc construct described herein, wherein at least one of said first and second CH3 domain polypeptides is a modified CH3 domain polypeptide comprising an amino acid modification at position S400. In a further embodiment is the isolated heteromultimer Fc construct described herein, comprising the modification S400Z, wherein Z is selected from a positively charged amino acid and a negatively charged amino acid. In some embodiments, the positively charged amino acid is lysine or arginine and the negatively charged amino acid is aspartic acid or glutamic acid. In certain embodiments is the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide comprising an amino acid modification selected from S400E and S400R. In some embodiments is provided the isolated heteromultimer Fc construct described herein, wherein at least one of said first and second CH3 domain polypeptides is a modified CH3 domain polypeptide comprising an amino acid modification at position N390. In some embodiments, the modification of N 390 is N390Z, wherein Z is selected from a positively charged amino acid and a negatively charged amino acid. In an embodiment, N390Z is N390R. In certain embodiments of the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising the amino acid modification S400E and said second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising the amino acid modification N390R. In some embodiments of the isolated heteromultimer Fc construct described herein, each of the first and second CH3 domain polypeptide is a modified CH3 domain polypeptide, one said modified CH3 domain polypeptide comprising the amino acid modification Q347R and the other modified CH3 domain polypeptide comprising the amino acid modification K360E.
Provided in one aspect is an isolated heteromultimer Fc construct comprising a modified heterodimeric CH3 domain, said modified CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide; wherein at least one of said first and second CH3 domain polypeptides comprises an amino acid modification of K392J wherein J is selected from L, I or an amino acid with a side chain volume not substantially larger than the side chain volume of K; wherein said first and second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain with a melting temperature (Tm) of at least about 74° C. and a purity of at least 95%; and wherein at least one amino acid modification is not of an amino acid which is at the interface between said first and said second CH3 domain polypeptides. In certain embodiments is a heteromultimer Fc construct described herein, comprising at least one T350X modification, wherein X is a natural or non-natural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments is an isolated heteromultimer Fc construct described herein, comprising at least one T350V modification. In an embodiment is an isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In an embodiment is the isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a Tm of about 77° C. or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80° C. or greater. In an embodiment is the isolated heteromultimer Fc construct described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of at least one of K409 and T411. In certain embodiments is the isolated heteromultimer Fc construct described herein, comprising at least one of K409F, T411E and T411 D. In some embodiments is the isolated heteromultimer Fc construct described herein wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of D399. In some embodiments, the amino acid modification of D399 is at least one of D399R and D399K.
Provided in one aspect is an isolated heteromultimer Fc construct comprising a modified heterodimeric CH3 domain, said modified CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide; wherein at least one of said first and second CH3 domain polypeptides comprises an amino acid modification of K392J wherein J is selected from L, I or an amino acid with a side chain volume not substantially larger than the side chain volume of K; wherein said first and second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain with a melting temperature (Tm) of at least about 74° C. and a purity of at least 95%; and wherein at least one amino acid modification is not of an amino acid which is at the interface between said first and said second CH3 domain polypeptides. In certain embodiments is a heteromultimer Fc construct described herein, comprising at least one T350X modification, wherein X is a natural or non-natural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments is an isolated heteromultimer Fc construct described herein, comprising at least one T350V modification. In an embodiment is an isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In an embodiment is the isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a Tm of about 77° C. or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80° C. or greater. In certain embodiments of the isolated heteromultimer Fc construct described herein, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising at least one amino acid modification selected from K409F, T411E and T411D, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising at least one amino acid modification selected from Y407A, Y407I, Y407V, D399R and D399K. In some embodiments is any one of the isolated heteromultimer Fc constructs described herein, further comprising a first modified CH3 domain comprising one of amino acid modifications T366V, T366I, T366A, T366M, and T366L; and a second modified CH3 domain comprising the amino acid modification L351Y. In some embodiments is any one of the isolated heteromultimer Fc constructs described herein, comprising a first modified CH3 domain comprising one of amino acid modifications K392L or K392E; and a second modified CH3 domain comprising one of the amino acid modifications S400R or S400V.
Provided herein is an isolated heteromultimer Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, each modified CH3 domain polypeptide comprising at least four amino acid mutations, wherein at least one of said first and said second modified CH3 domain polypeptide comprises a mutation selected from N390Z and S400Z, wherein Z is selected from a positively charged amino acid and a negatively charged amino acid, and wherein said first and second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain with a melting temperature (Tm) of at least about 70° C. and a purity of at least 90%. In an embodiment is provided the isolated heteromultimer Fc construct, wherein said first modified CH3 domain polypeptide comprising amino acid modifications at positions F405 and Y407 and said second modified CH3 domain polypeptide comprises amino acid modification at position T394. In an embodiment is provided the isolated heteromultimer Fc construct, the first modified CH3 domain polypeptide comprising an amino acid modification at position L351. In certain embodiments, is the isolated heteromultimer described herein, said second modified CH3 domain polypeptide comprising a modification of at least one of positions T366 and K392. In some embodiments, is the isolated heteromultimer described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. and is formed with a purity of at least about 95%. In certain embodiments, is the isolated heteromultimer described herein, at least one modified CH3 domain polypeptide comprising amino acid modifications of at least one of N390R, S400E and S400R. In some embodiments is an isolated heteromultimer described herein, one of said first and second modified CH3 domain polypeptide comprising amino acid modifications of position 347 and the other modified CH3 domain polypeptide comprising amino acid modification at position 360. In certain embodiments is the isolated heteromultimer described herein, at least one of said first and second modified CH3 domain polypeptides comprising amino acid modification of T350V. In specific embodiments is an isolated heteromultimer described herein, said first modified CH3 domain polypeptide comprising at least one amino acid modification selected from L351Y, F405A and Y407V; and said second modified CH3 domain polypeptide comprising at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In certain embodiments described herein is an isolated heteromultimer, the first modified CH3 domain polypeptide comprising amino acid modifications at positions D399 and Y407, and a second modified CH3 domain polypeptide comprising amino acid modification at positions K409 and T411. In some embodiments is an isolated heteromultimer described herein, the first CH3 domain polypeptide comprising amino acid modification at position L351, and the second modified CH3 domain polypeptide comprising amino acid modifications at position T366 and K392. In specific embodiments are isolated heteromultimers described herein, at least one of said first and second CH3 domain polypeptide comprising amino acid modification of T350V. In certain embodiments are isolated heteromultimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater and is formed with a purity of at least about 95%. Provided in certain embodiments are isolated heteromultimer Fc constructs described herein, said first modified CH3 domain polypeptide comprising amino acid modifications selected from L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A, and Y407V; and said second modified CH3 domain polypeptide comprising amino acid modifications selected from T366V, T366I, T366L, T366M, N390D, N390E, K392L, K392I, K392D, K392E, K409F, K409W, T411D and T411E.
Provided herein is an isolated heteromultimer Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, each modified CH3 domain polypeptide comprising at least three amino acid mutations, wherein one of said first and said second modified CH3 domain polypeptide comprises a mutation selected from T411E and T411D, and wherein said first and second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain with a melting temperature (Tm) of at least about 70° C. and a purity of at least 90%. In an embodiment is provided the isolated heteromultimer Fc construct wherein said first modified CH3 domain polypeptide comprising amino acid modifications at positions F405 and Y407 and said second modified CH3 domain polypeptide comprises amino acid modification at position T394. In an embodiment is provided the isolated heteromultimer Fc construct, the first modified CH3 domain polypeptide comprising an amino acid modification at position L351. In certain embodiments, is the isolated heteromultimer described herein, said second modified CH3 domain polypeptide comprising a modification of at least one of positions T366 and K392. In some embodiments, is the isolated heteromultimer described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. and is formed with a purity of at least about 95%. In certain embodiments, is the isolated heteromultimer described herein, at least one modified CH3 domain polypeptide comprising amino acid modifications of at least one of N390R, S400E and S400R. In some embodiments is an isolated heteromultimer described herein, one of said first and second modified CH3 domain polypeptide comprising amino acid modifications of position 347 and the other modified CH3 domain polypeptide comprising amino acid modification at position 360. In certain embodiments is the isolated heteromultimer described herein, at least one of said first and second modified CH3 domain polypeptides comprising amino acid modification of T350V. In specific embodiments is an isolated heteromultimer described herein, said first modified CH3 domain polypeptide comprising at least one amino acid modification selected from L351Y, F405A and Y407V; and said second modified CH3 domain polypeptide comprising at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In certain embodiments described herein is an isolated heteromultimer, the first modified CH3 domain polypeptide comprising amino acid modifications at positions D399 and Y407, and a second modified CH3 domain polypeptide comprising amino acid modification at positions K409 and T411. In some embodiments is an isolated heteromultimer described herein, the first CH3 domain polypeptide comprising amino acid modification at position L351, and the second modified CH3 domain polypeptide comprising amino acid modifications at position T366 and K392. In specific embodiments are isolated heteromultimers described herein, at least one of said first and second CH3 domain polypeptide comprising amino acid modification of T350V. In certain embodiments are isolated heteromultimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater and is formed with a purity of at least about 95%. Provided in certain embodiments are isolated heteromultimer Fc constructs described herein, said first modified CH3 domain polypeptide comprising amino acid modifications selected from L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A, and Y407V; and said second modified CH3 domain polypeptide comprising amino acid modifications selected from T366V, T366I, T366L, T366M, N390D, N390E, K392L, K392I, K392D, K392E, K409F, K409W, T411O and T411E.
Provided herein is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366I, K392M and T394W.
Provided in an aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366I, K392L and T394W.
Provided in a certain aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366L, K392M and T394W.
Provided in some aspects is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366L, K392L and T394W.
Provided in an aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T350V, L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, K392L and T394W.
Provided in an aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T350V, L351Y, S400R, F405A, Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, K392M and T394W.
Provided in an aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T350V, L351Y, S400E, F405A, Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, N390R, K392M and T394W.
Provided in an aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T350V, L351Y, F405A, Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, K392L and T394W.
Provided in one aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T366V, K392L, K409F and T411E; and a second modified CH3 domain polypeptide comprising amino acid modifications L351Y, D399R, and Y407A.
Provided in one aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T366V, K392LE K409F and T411E; and a second modified CH3 domain polypeptide comprising amino acid modifications L351Y, D399R, S400R and Y407A.
There is provided according to one aspect of the invention an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation, wherein the heterodimer Fc region further comprises a variant CH2 domain comprising at least one asymmetric amino acid modification to promote selective binding of a Fcgamma receptor. In one embodiment the variant CH2 domain selectively binds Fcgamma IiIa receptor as compared to wild-type CH2 domain. In one embodiment, the modified CH3 domain has a melting temperature (Tm) of about 70° C. or greater. In certain embodiments, the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. In some embodiments, the modified CH3 domain has a melting temperature (Tm) of at least about 80° C.
There is provided in another aspect an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations, wherein the modified CH3 domain has a melting temperature (Tm) of about 70° C. or greater, and wherein said modified CH3 domain results in the formation of heterodimer Fc region with increased stability as compared to a CH3 domain that does not comprise amino acid mutations. In one embodiment, heterodimer Fc region does not comprise an additional disulfide bond in the CH3 domain relative to a wild type Fc region. In an alternative embodiment, the heterodimer Fc region comprises at least one additional disulfide bond in the modified CH3 domain relative to a wild type Fc region, with the proviso that the melting temperature (Tm) of about 70° C. or greater is in the absence of the additional disulfide bond. In another embodiment, the heterodimer Fc region comprises at least one additional disulfide bond in the modified CH3 domain relative to a wild type Fc region, and wherein the modified CH3 domain has a melting temperature (Tm) of about 77.55° C. or greater.
Provided in one embodiment, an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations, wherein the modified CH3 domain has a melting temperature (Tm) of about 70° C. or greater and the heterodimer Fc region is formed with a purity greater than about 90%, or the heterodimer Fc region is formed with a purity of about 95% or greater or the heterodimer Fc region is formed with a purity of about 98% or greater.
Also provided in certain embodiments is an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising one or more amino acid mutations that result in the formation of heterodimer Fc region with increased stability as compared to a CH3 domain that does not comprise said one or more amino acid mutations, wherein the modified CH3 domain has a melting temperature (Tm) of about 70° C. or greater or the Tm is about 71° C. or greater or the Tm is about 74° C. or greater. In another embodiment, the heterodimer Fc region is formed in solution with a purity of about 98% or greater, and Tm about 73° C. or wherein the heterodimer Fc region is formed with a purity of about 90% or greater, and Tm about 75° C.
Provided in certain embodiments is an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first and a second CH3 domain polypeptides, wherein at least one of said first and second CH3 domain polypeptides comprises amino acid modification T350V. Provided in certain embodiments is an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising amino acid modification T350V and a second CH3 domain polypeptide also comprising amino acid modification T350V. Provided in certain embodiments is an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising amino acid modification at positions F405 and Y407 and a second CH3 domain polypeptide comprising amino acid modification at position T394. a first CH3 domain polypeptide comprises amino acid modifications at positions D399 and Y407 and a second CH3 domain polypeptide comprises amino acid modification at positions K409 and T411. Provided in certain embodiments is an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising amino acid modifications L351Y and Y407A and a second CH3 domain polypeptide comprising amino acid modifications T366A and K409F. In one aspect, the first CH3 domain polypeptide or the second CH3 domain polypeptide comprises a further amino acid modification at position T411, D399, S400, F405, N390, or K392. The amino acid modification at position T411 is selected from T411N, T411R, T411Q, T411K, T411D, T411E or T411W. The amino acid modification at position D399 is selected from D399R, D399W, D399Y or D399K. The amino acid modification at position S400 is selected from S400E, S400D, S400R, or S400K. The amino acid modification at position F405 is selected from F405I, F405M, F405T, F4058, F405V or F405W. The amino acid modification at position N390 is selected from N390R, N390K or N390D. The amino acid modification at position K392 is selected from K392V, K392M, K392R, K392L, K392F or K392E.
In certain embodiments is provided an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising amino acid modifications T350V and L351Y and a second CH3 domain polypeptide also comprising amino acid modifications T350V and L351Y.
In another embodiment is provided an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising amino acid modification Y407A and a second CH3 domain polypeptide comprising amino acid modifications T366A and K409F. In one aspect the first CH3 domain polypeptide or the second CH3 domain polypeptide comprises further amino acid modifications K392LE, T411E, D399R and S400R. In another aspect, the first CH3 domain polypeptide comprises amino acid modification D399R, S400R and Y407A and the second CH3 domain polypeptide comprises amino acid modification T366A, K409F, K392E and T411E. In a further embodiment the modified CH3 domain has a melting temperature (Tm) of about 74° C. or greater and the heterodimer has a purity of about 95% or greater.
Provided in another embodiment is an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising an amino acid modification at positions L351 and amino acid modification Y407A and a second CH3 domain polypeptide comprises an amino acid modification at position T366 and amino acid modification K409F. In one aspect the amino acid modification at position L351 is selected from L351Y, L3511, L3510, L351R or L351F. In another aspect, the amino acid modification at position Y407 is selected from Y407A, Y407V or Y4078. In yet another aspect the amino acid modification at position T366 is selected from T366A, T366I, T366L, T366M, T366Y, T3668, T366C, T366V or T366W. In one embodiment the modified CH3 domain has a melting temperature (Tm) of about 75° C. or greater and the heterodimer has a purity of about 90% or greater.
Provided in another embodiment is an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising an amino acid modification at position F405 and amino acid modifications L351Y and Y407V and a second CH3 domain polypeptide comprises amino acid modification T394W. In one aspect the first CH3 domain polypeptide or the second CH3 domain polypeptide comprise an amino acid modification at positions K392, T411, T366, L368 or S400. The amino acid modification at position F405 is F405A, F405I, F405M, F405T, F4058, F405V or F405W. The amino acid modification at position K392 is K392V, K392M, K392R, K392L, K392F or K392E. The amino acid modification at position T411 is T411N, T411R, T411Q, T411K, T411D, T411E or T411W. The amino acid modification at position S400 is S400E, S4000, S400R or S400K. The amino acid modification at position T366 is T366A, T366I, T366L, T366M, T366Y, T3668, T366C, T366V or T366W. The amino acid modification at position L368 is L368D, L368R, L368T, L368M, L368V, L368F, L3688 and L368A.
In another embodiment is provided an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising an amino acid modifications L351Y, F405A and Y407V and a second CH3 domain polypeptide comprises amino acid modification T394W. In one aspect, the second CH3 domain polypeptide comprises amino acid modification T366L or T366I.
In yet another embodiment is provided an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising at least one of amino acid modifications Y349C, F405A and Y407V and a second CH3 domain polypeptide comprises amino acid modifications T366I, K392M and T394W.
In certain embodiments are provided an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V and a second CH3 domain polypeptide comprises amino acid modifications K392M and T394W, and one of T366L and T366I.
In another embodiment is provided an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising amino acid modifications F405A and Y407V and a second CH3 domain polypeptide comprises amino acid modifications T366L and T394W.
In another embodiment is provided an isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first CH3 domain polypeptide comprising amino acid modifications F405A and Y407V and a second CH3 domain polypeptide comprises amino acid modifications T366I and T394W.
In certain embodiments of the heteromultimer is provided bispecific antibody or a multispecific antibody.
In another embodiment is provided a composition comprising a heteromultimer of the invention and a pharmaceutically acceptable carrier.
In another embodiment is provided a host cell comprising nucleic acid encoding the heteromultimer of the invention.
In certain embodiments is provided heteromultimer, wherein the heteromultimer comprises at least one therapeutic antibody. In one aspect the therapeutic antibody is selected from the group consisting of abagovomab, adalimumab, alemtuzumab, aurograb, bapineuzumab, basiliximab, belimumab, bevacizumab, briakinumab, canakinumab, catumaxomab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, galiximab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lumiliximab, mepolizumab, motavizumab, muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab, ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab, reslizumab, rituximab, teplizumab, tocilizumab/atlizumab, tositumomab, trastuzumab, Proxinium™, Rencarex™, ustekinumab, and zalutumumab.
In another embodiment of the heteromultimer of the invention is provided a method of treating cancer in a patient having a cancer characterized by a cancer antigen, said method comprising administering to said patient a therapeutically effective amount of a heteromultimer.
In another embodiment of the heteromultimer of the invention is provided a method of treating immune disorders in a patient having an immune disorder characterized by an immune antigen, said method comprising administering to said patient a therapeutically effective amount of a heteromultimer.
In certain embodiments, the modified Fc region utilized in the heteromultimer constructs described herein comprises type G immunoglobulins for instance immunoglobulins which are defined as immunogloblilins of class 2 (IgG2) or immunoglobulins of class 3 (IgG3). In some embodiments, the modified Fc region utilized in the heteromultimer constructs described herein comprises Immunoglobulin M, or IgM. In some embodiments, the modified Fc region utilized in the heteromultimer constructs described herein comprises Immunoglobulin A, or IgA. In some embodiments, the modified Fc region utilized in the heteromultimer constructs described herein comprises Immunoglobulin D, or IgD. In some embodiments, the modified Fc region utilized in the heteromultimer constructs described herein comprises Immunoglobulin E, or IgE. In certain embodiments, the modified Fc region utilized in the heteromultimer constructs described herein comprises all cases of immunoglobulins G isotypes for instance immunoglobulins which are defined as immunogloblilins of class 1 (IgG1), class 2 (IgG2), class 3 (IgG3) or class 4 (IgG4).
a(M-1) of homodimeric IgG1 Fc, the heterodimeric variants het1(Control1): A:Y349C_T366S_L368A_Y407V/B:S354C_T366W and het2(Control4): A:K409D_K392D/B:D399K_D356K binding to the six Fcgamma receptors. The heterodimeric Fc variants tend to show slightly altered binding to the Fcgamma receptors compared to the wild type IgG1 Fc. See, Example 7
a(M−1)) of various Fc's and its variants with a number of FcgRIIa, FcgRIIb and FcgRIIIa allotypes. The Ka of wild type IgG1 Fc to various Fcg receptors is represented as columns with horizontal shade. The bars with vertical shades (homodimer base2) represent the Ka of homodimeric Fc with the mutations S239D/D265S/I332E/S298A. The columns with the slanted shade represent the Ka of heterodimeric Fc with asymmetric mutations A:S239D/D265S/I332E/E269K and B:S239D/D265S/S298A in the CH2 domain. The introduction of asymmetric mutations is able to achieve increased selectivity between the IiIa and IIa/IIb receptors. The Heterodimeric Fc here is comprised of CH3 mutations as indicated for the variant het2 (Control 4) in
a(M−1)) for wild type IgG1 and three other variants involving homodimeric or asymmetric mutations in the CH2 domain of the Fc region. The Ka of wild type Fc is represented in the column shaded with grids. The Ka of Fc variant with the base mutation S239D/K326E/A330L/I332E/S298A introduced in a homodimeric manner (homodimer base1) on both the chains of Fc is shown with the slanted patterned column. Introduction of related mutations in an asymmetric manner in chains A and B of a heterodimeric Fc (hetero base1) is shown with the horizontal lines. The column with vertical shaded lines represents the asymmetric variant including the E269K mutation (hetero base 1+PO). The Heterodimeric Fc here is comprised of CH3 mutations as indicated for the variant het2 (Control 4) in
Provided herein are modified CH3 domains comprising specific amino acid modifications to promote heteromultimer formation. In one embodiment, the modified CH3 domains comprise specific amino acid modifications to promote heterodimer formation (See, for example Tables 1.1-1.3). In another embodiment the modified CH3 domains comprise specific amino acid modifications to promote heterodimer formation with increased stability (See, for example Table 4, Table 6 and Table 7). Stability is measured as the melting temperature (Tm) of the CH3 domain and an increased stability refers to a Tm of about 70° C. or greater. The CH3 domains form part of the Fc region of a heteromultimeric, multispecific antibody. Provided herein in one embodiment are heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation wherein the modified CH3 domains are selected from the variants listed in Table 1. In a second embodiment, provided are heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) of about 70° C. or greater.
Amino acid modifications utilized to generate a modified CH3 domain include, but are not limited to, amino acid insertions, deletions, substitutions, and rearrangements. The modifications of the CH3 domain and the modified CH3 domains are referred to herein collectively as “CH3 modifications”, “modified CH3 domains”, “modified CH3 domains” or “CH3 variants”. In certain embodiments, the modified CH3 domains are incorporated into a molecule of choice. Accordingly, in one embodiment are provided molecules, for instance polypeptides, such as immunoglobulins (e.g., antibodies) and other binding proteins, comprising an Fc region (as used herein “Fc region” and similar terms encompass any heavy chain constant region domain comprising at least a portion of the CH3 domain) incorporating a modified CH3 domain. Molecules comprising Fc regions comprising a modified CH3 domain (e.g., a CH3 domain comprising one or more amino acid insertions, deletions, substitutions, or rearrangements) are referred to herein as “Fc variants”, “heterodimers” or “heteromultimers”. The present Fc variants comprise a CH3 domain that has been asymmetrically modified to generate heterodimer Fc variants or regions. The Fc region is comprised of two heavy chain constant domain polypeptides—Chain A and Chain B, which can be used interchangeably provided that each Fc region comprises one Chain A and one Chain B polypeptide. The amino acid modifications are introduced into the CH3 in an asymmetric fashion resulting in a heterodimer when two modified CH3 domains form an Fc variant (See, e.g., Table 1). As used herein, asymmetric amino acid modifications are any modification wherein an amino acid at a specific position on one polypeptide (e.g., “Chain A”) is different from the amino acid on the second polypeptide (e.g., “Chain B”) at the same position of the heterodimer or Fc variant. This can be a result of modification of only one of the two amino acids or modification of both amino acids to two different amino acids from Chain A and Chain B of the Fc variant. It is understood that the modified CH3 domains comprise one or more asymmetric amino acid modifications.
An amino acid which is at the interface between the first and said second CH3 domain polypeptides is any amino acid on the first or the second CH3 domain polypeptide which interacts with an amino acid on the other CH3 domain polypeptide resulting in the formation of the dimeric CH3 domain. An amino acid that is not at the interface between the first and said second CH3 domain polypeptides is any amino acid on the first or the second CH3 domain polypeptide which does not interact with an amino acid on the other CH3 domain polypeptide. In embodiments described herein, a modified amino acid that is not at the interface between the first and said second CH3 domain polypeptides is any amino acid on the first or the second CH3 domain polypeptide which after it is modified as described herein, does not interact with an amino acid on the other CH3 domain polypeptide. For instance, in certain embodiments described herein, are provided modifications of the amino acid position T350. As demonstrated by the crystal structure provided in Example 12 and shown in
In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. As used herein, “about” means ±10% of the indicated range, value, sequence, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated or dictated by its context. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms “include” and “comprise” are used synonymously. In addition, it should be understood that the individual single chain polypeptides or heterodimers derived from various combinations of the structures and substituents (e.g., modified CH3 domains) described herein are disclosed by the present application to the same extent as if each single chain polypeptide or heterodimer were set forth individually. Thus, selection of particular components to form individual single chain polypeptides or heterodimers is within the scope of the present disclosure.
The “first polypeptide” is any polypeptide that is to be associated with a second polypeptide, also referred to herein as “Chain A”. The first and second polypeptide meet at an “interface”. The “second polypeptide” is any polypeptide that is to be associated with the first polypeptide via an “interface”, also referred to herein as “Chain B”. The “interface” comprises those “contact” amino acid residues in the first polypeptide that interact with one or more “contact” amino acid residues in the interface of the second polypeptide. As used herein, the interface comprises the CH3 domain of an Fc region that preferably is derived from an IgG antibody and most preferably a human IgG1 antibody.
As used herein, “isolated” heteromultimer means a heteromultimer that has been identified and separated and/or recovered from a component of its natural cell culture environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the heteromultimer, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
An amino acid with a side chain volume “not substantially larger” than a first amino acid is any amino acid that has a side chain volume not more than 20 A3 larger than the first amino acid based on side chain volume values from A. A. Zamyatnin, Prog. Biophys. Mol. Bioi. 24: 107-123, 1972. In certain embodiments, the volume is not more than 10 A3 larger than the first amino acid. In some embodiments, the volume is not more than 5 A3 larger than the first amino acid. For instance in certain embodiments described herein, are mutations of lysine (K) such as K392J wherein J is selected from L, I or an amino acid with a side chain volume not substantially larger than the side chain volume of K.
The variant Fc heterodimers are generally purified to substantial homogeneity. The phrases “substantially homogeneous”, “substantially homogeneous form” and “substantial homogeneity” are used to indicate that the product is substantially devoid of by-products originated from undesired polypeptide combinations (e.g. homodimers). Expressed in terms of purity, substantial homogeneity means that the amount of by-products does not exceed 10%, and preferably is below 5%, more preferably below 1%, most preferably below 0.5%, wherein the percentages are by weight.
Terms understood by those in the art of antibody technology are each given the meaning acquired in the art, unless expressly defined differently herein. Antibodies are known to have variable regions, a hinge region, and constant domains. Immunoglobulin structure and function are reviewed, for example, in Harlow et al, Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).
The design of variant Fc heterodimers from wildtype homodimers is illustrated by the concept of positive and negative design in the context of protein engineering by balancing stability vs. specificity, wherein mutations are introduced with the goal of driving heterodimer formation over homodimer formation when the polypeptides are expressed in cell culture conditions. Negative design strategies maximize unfavorable interactions for the formation of homodimers, by either introducing bulky sidechains on one chain and small sidechains on the opposite, for example the knobs-into-holes strategy developed by Genentech (Ridgway J B, Presta L G, Carter P. ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996 July; 9(7):617-21; Atwell S, Ridgway J B, Wells J A, Carter P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J Mol Bioi. 270(1):26-35 (1997))), or by electrostatic engineering that leads to repulsion of homodimer formation, for example the electrostatic steering strategy developed by Amgen (Gunaskekaran K, et al. Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. JBC 285 (25): 19637-19646 (2010)). In these two examples, negative design asymmetric point mutations were introduced into the wild-type CH3 domain to drive heterodimer formation. To date, only negative design strategies have been used to develop Fc heterodimers. Published results show that heterodimers designed using only a negative design approach leads to high specificity with >95% heterodimers, but destabilizes the complex considerably (Supra). These negative design heterodimers posses a melting temperature, of the modified CH3 domain, of 69° C. or less, absent additional disulfide bonds as compared to the wild type. See, Table A below.
The melting temperature for wild-type IgG1 is shown as a range from 81-83 as the values in the literature vary depending on the assay system used, we report a value of 81.5° C. in our assay system.
In contrast to negative design, a general concept used to engineer proteins is positive design. In this instance amino acid modifications are introduced into polypeptides to maximize favorable interactions within or between proteins. This strategy assumes that when introducing multiple mutations that specifically stabilize the desired heterodimer while neglecting the effect on the homodimers, the net effect will be better specificity for the desired heterodimer interactions over the homodimers and hence a greater heterodimer specificity. It is understood in the context of protein engineering that positive design strategies optimize the stability of the desired protein interactions, but rarely achieve >90% specificity (Havranek J J & Harbury P B. Automated design of specificity in molecular recognition. Nat Struct Bioi. 10(1):45-52 (2003); Bolon O N, Grant R A, Baker T A, Sauer R T. Specificity versus stability in computational protein design. Proc Natl Acad Sci USA. 6; 102(36):12724-9 (2005); Huang P S, Love J J, Mayo S L. A de novo designed protein interface Protein Sci. 16(12):2770-4 (2007)). Prior to this disclosure positive design strategies have not been used to design Fc heterodimers as more attention was devoted to specificity as compared to stability for therapeutic antibody manufacturing and development. In addition, beneficial positive design mutations can be hard to predict. Other methodologies for improving stability, such as additional disulfide bonds, have been tried to improve stability in Fc heterodimers with limited success on improvements to the molecule. (See, Table A) This may be because all engineered Fc CH3 domain disulfide bonds are solvent exposed, which results in a short lifetime of the disulfide bond and therefore a significant impact on the long-term stability of the heterodimer—especially when the engineered CH3 domain has a Tm of less than 70° C. without the additional disulfide bond (as in Control 4 which has a Tm of 69° C. without the disulfide (see Control 2). It is contemplated that other methodologies to improve stability, such as disulfide bonds, can also be used with the present Fc variants, provided the intrinsic stability (measured as melting temperature) of the CH3 domain is 70° C. or greater without the disulfide bond, in particular when the intrinsic stability (measured as melting temperature) of the CH3 domain is 72° C. or greater without the disulfide bond.
Therefore, we herein disclose a novel method for designing Fc heterodimers that results in both stable and highly specific heterodimer formation. This design method combines both negative and positive design strategies along with structural and computational modeling guided protein engineering techniques. This powerful method has allowed us to design novel combinations of mutations in the IgG1 CH3 domain wherein using only standard cell culture conditions heterodimers were formed with more than 90% purity compared to homodimers and the resulting heterodimers had a melting temperature of 70° C. or greater. In exemplary embodiments, the Fc variant heterodimers have a melting temperature of 73° C. or greater and a purity of greater than 98%. In other exemplary embodiments, the Fc variant heterodimers have a melting temperature of 75° C. or greater and a purity of greater than 90%. In certain embodiments of the heterodimer Fc variants described herein, the Fc variant heterodimers have a melting temperature of 77° C. or greater and a purity of greater than 98%. In some embodiments of the heterodimer Fc variants described herein, the Fc variant heterodimers have a melting temperature of 78° C. or greater and a purity of greater than 98%. In certain embodiments of the heterodimer Fc variants described herein, the Fc variant heterodimers have a melting temperature of 79° C. or greater and a purity of greater than 98%. In certain embodiments of the heterodimer Fc variants described herein, the Fc variant heterodimers have a melting temperature of 80° C. or greater and a purity of greater than 98%. In certain embodiments of the heterodimer Fc variants described herein, the Fc variant heterodimers have a melting temperature of 81° C. or greater and a purity of greater than 98%.
In certain embodiments, an isolated heteromultimer comprising a heterodimer Fc region is provided wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) of 70° C. or greater. As used herein “increased stability” or “stable heterodimer”, refers to a modified CH3 domain, in heterodimer formation, with a melting temperature of about 70° C. or greater. In certain embodiments, “increased stability” or “stable heterodimer”, refers to a modified CH3 domain, in heterodimer formation, with a melting temperature of about 72° C. or greater. In certain embodiments, “increased stability” or “stable heterodimer”, refers to a modified CH3 domain, in heterodimer formation, with a melting temperature of about 74° C. or greater. In certain embodiments, “increased stability” or “stable heterodimer”, refers to a modified CH3 domain, in heterodimer formation, with a melting temperature of about 75° C. or greater. In certain embodiments, “increased stability” or “stable heterodimer”, refers to a modified CH3 domain, in heterodimer formation, with a melting temperature of about 76° C. or greater. In certain embodiments, “increased stability” or “stable heterodimer”, refers to a modified CH3 domain, in heterodimer formation, with a melting temperature of about 78° C. or greater. In certain embodiments, “increased stability” or “stable heterodimer”, refers to a modified CH3 domain, in heterodimer formation, with a melting temperature of about 79° C. or greater. In certain embodiments, “increased stability” or “stable heterodimer”, refers to a modified CH3 domain, in heterodimer formation, with a melting temperature of about 80° C. or greater. In certain embodiments, “increased stability” or “stable heterodimer”, refers to a modified CH3 domain, in heterodimer formation, with a melting temperature of about 81° C. or greater. In addition, it is understood that the term “to promote heterodimer formation” refers herein to the amino acid mutations in the CH3 domain that result in greater than 90% heterodimer formation compared to homodimer formation.
In a further embodiment, this increased stability is in the absence of an additional disulfide bond. Specifically, the increased stability is in the absence of an additional disulfide bond in the CH3 domain. In one embodiment, the modified CH3 domain does not comprise an additional disulfide bond as compared to wild-type CH3 domain. In an alternative embodiment, the modified CH3 comprises at least one disulfide bond as compared to wild-type CH3 domain, provided that the modified CH3 has a melting temperature of 70° C. or greater in the absence of the disulfide bond. In one embodiments, the modified CH3 domain comprises at least one disulfide bond as compared to wild-type CH3 domain, and the modified CH3 domain has a melting temperature (Tm) of about 77.5° C. or greater. In an embodiment, the modified CH3 domain comprises at least one disulfide bond as compared to wild-type CH3 domain, and the modified CH3 domain has a melting temperature (Tm) of about 78° C. or greater. In another embodiment, the modified CH3 domain comprises at least one disulfide bond as compared to wild-type CH3 domain, and the modified CH3 domain has a melting temperature (Tm) of greater than about 78° C., or greater than about 78.5° C., or greater than about 79° C., or greater than about 79.5° C., or greater than about 80° C., or greater than about 80.5° C., or greater than about 81° C., or greater than about 81.5° C., or greater than about 82° C., or greater than about 82.5° C., or greater than about 83° C.
In one embodiment, the modified CH3 domain has a melting temperature of greater than about 70° C., or greater than about 70.5° C., or greater than about 71° C., or greater than about 71.5° C., or greater than about 72° C., or greater than about 72.5° C., or greater than about 73° C., or greater than about 73.5° C., or greater than about 74° C., or greater than about 74.5° C., or greater than about 75° C., or greater than about 75.5° C., or greater than about 76° C., or greater than about 76.5° C., or greater than about 77° C., or greater than about 77.5° C., or greater than about 78° C., or greater than about 78.5° C., or greater than about 79° C., or greater than about 79.5° C., or greater than about 80° C., or greater than about 80.5° C., or greater than about 81° C., or greater than about 81.5° C., or greater than about 82° C., or greater than about 82.5° C., or greater than about 83° C. In another embodiment, the modified CH3 domain has a melting temperature of about 70° C., or about 70.5° C., or about 71° C., or about 71.5° C., or about 72° C., or about 72.5° C., or about 73° C., or about 73.5° C., or about 74° C., or about 74.5° C., or about 75° C., or about 75.5° C., or about 76° C., or about 76.5° C., or about 77° C., or about 77.5° C., or about 78° C., or about 78.5° C., or about 79° C., or about 79.5° C., or about 80° C., or about 80.5° C., or about 81° C. In yet another embodiment, the modified CH3 domain has a melting temperature of about 70° C. to about 81° C., or about 70.5° C. to about 81° C., or about 71° C. to about 81° C., or about 71.5° C. to about 81° C., or about 72° C. to about 81° C., or about 72.5° C. to about 81° C., or about 73° C. to about 81° C., or about 73.5° C. to about 81° C., or about 74° C. to about 81° C., or about 74.5° C. to about 81° C., or about 75° C. to about 81° C., or about 75.5° C. to about 81° C., or 76° C. to about 81° C., or about 76.5° C. to about 81° C., or about 77° C. to about 81° C., or about 77.5° C. to about 81° C., or about 78° C. to about 81° C., or about 78.5° C. to about 82° C., or about 79° C. to about 81° C. In yet another embodiment, the modified CH3 domain has a melting temperature of about 71° C. to about 76° C., or about 72° C. to about 76° C., or about 73° C. to about 76° C., or about 74° C. to about 76° C.
In addition to improved stability, the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation. It is understood that these amino acid mutations to promote heterodimer formation are as compared to homodimer formation. This heterodimer formation as compared to homodimer formation is referred jointly herein as “purity” or “specificity” or “heterodimer purity” or “heterodimer specificity”. It is understood that the heterodimer purity refers to the percentage of desired heterodimer formed as compared to homodimer species formed in solution under standard cell culture conditions prior to selective purification of the heterodimer species. For instance, a heterodimer purity of 90% indicates that 90% of the dimer species in solution is the desired heterodimer. In one embodiment, the Fc variant heterodimers have a purity of greater than about 90%, or greater than about 91%, or greater than about 92%, or greater than about 93%, or greater than about 94%, or greater than about 95%, or greater than about 96%, or greater than about 97%, or greater than about 98%, or greater than about 99%. In another embodiment, the Fc variant heterodimers have a purity of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100%.
In a specific embodiment, the isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) of 70° C. or greater and the resulting heterodimer has a purity greater than 90%. In one aspect, the resulting Fc variant heterodimer has a purity greater than 98% and the modified CH3 domain has a melting temperature of greater than about 70° C., or greater than about 71° C., or greater than about 72° C., or greater than about 73° C., or greater than about 74° C., or greater than about 75° C., or greater than about 76° C., or greater than about 77° C., or greater than about 78° C., or greater than about 79° C., or greater than about 80° C. or greater than about 81° C. In a further aspect, the modified CH3 domain has a melting temperature of 70° C. or greater and the resulting Fc variant heterodimer has a purity greater than about 90%, or greater than about 91%, or greater than about 92%, or greater than about 93%, or greater than about 94%, or greater than about 95%, or greater than about 96%, or greater than about 97%, or greater than about 98%, or greater than about 99%.
In order to design these Fc variants with improved stability and purity we employed an iterative process of computational design and experimental screening to select the most successful combinations of positive and negative design strategies (See,
Specifically, in the initial design phase different negative design Fc variant heterodimers were made and tested for expression and stability as described in Examples 1-3. The initial design phase included Fc variant heterodimers AZ1-AZ16 (See, Table 1). From this initial set of negative design Fc variant heterodimers, which were expected to have low stability (e.g., a Tm of less than 71° C.), the Fc variant heterodimers with greater than 90% purity and a melting temperature of about 68° C. or greater were selected for further development. This included Fc variant heterodimers AZ6, AZ8 and AZ15. In the second design phase, those selected Fc variant heterodimers were further modified to drive both stability and purity using positive design strategies following a detailed computational and structural analysis. The selected Fc variant heterodimers (AZ6, AZ8, and AZ15) were each analyzed with computational methods and comprehensive structure function analysis to identify the structural reasons these Fc variants had a lower stability than the wild-type Fc homodimer, which is 81° C. for IgG1. See, Table 4 for the list of Fc variant heterodimers and the Tm values.
In certain embodiments, the modified CH3 domain is selected from AZ1, or AZ2, or AZ3, or AZ4, or AZ5, or AZ6, or AZ7, or AZ8, or AZ9, or AZ10, or AZ11, or AZ12, or AZ13, or AZ14, or AZ15, or AZ16. In selected embodiments, the modified CH3 domain is AZ6, or AZ8 or AZ15.
The computational tools and structure-function analysis included, but were not limited to molecular dynamic analysis (MD), sidechain/backbone re-packing, Knowledge Base Potential (KBP), cavity and (hydrophobic) packing analysis (LJ, CCSD, SASA, dSASA(carbon/all-atom)), electrostatic-GB calculations, and coupling analysis. (See,
An aspect of the protein engineering approach relied on combining structural information of the Fc IgG protein derived from X-ray crystallography with computational modeling and simulation of the wild type and variant forms of the CH3 domain. This allowed us to gain novel structural and physico-chemical insights about the potential role of individual amino acids and their cooperative action. These structural and physico-chemical insights, obtained from multiple modified CH3 domains, along with the resulting empirical data pertaining to their stability and purity helped us develop an understanding for the relationship between purity and stability of the Fc heterodimer as compared to the Fc homodimers and the simulated structural models. In order to execute our simulations we started by building complete and realistic models and refining the quality of the wild type Fc structure of an IgG1 antibody. Protein structures derived from X-ray crystallography are lacking in detail regarding certain features of the protein in aqueous medium under physiological condition and our refinement procedures addressed these limitations. These include building missing regions of the protein structure, often flexible portions of the protein such as loops and some residue side chains, evaluating and defining the protonation states of the neutral and charged residues and placement of potential functionally relevant water molecules associated with the protein.
Molecular dynamics (MD) algorithms are one tool we used, by simulating the protein structure, to evaluate the intrinsic dynamic nature of the Fc homodimer and the modified CH3 domains in an aqueous environment. Molecular dynamics simulations track the dynamic trajectory of a molecule resulting from motions arising out of interactions and forces acting between all the atomic entities in the protein and its local environment, in this case the atoms constituting the Fc and its surrounding water molecules. Following molecular dynamics simulations, various aspects of the trajectories were analyzed to gain insight into the structural and dynamic characteristics of the Fc homodimer and variant Fc heterodimer, which we used to identify specific amino acid mutations to improve both purity and stability of the molecule.
Therefore, the generated MD trajectories were studied using methods such as the principal component analysis to reveal the intrinsic low frequency modes of motion in the Fc structure. This provides insight into the potential conformational sub-states of the protein (See,
The generated MD trajectories were also studied to determine the mutability of specific amino acid residue positions in the Fc structure based on profiling their flexibility and analysis of their environment. This algorithm allowed us to identify residues that could affect protein structure and function, providing unique insight into residue characteristics and mutability for subsequent design phases of the modified CH3 domains. This analysis also enabled us to compare multiple simulations, and assess mutability based on outliers following profiling.
The generated MD trajectories were also studied to determine correlated residue motions in the protein and the formation of networks of residues as a result of coupling between them. Finding dynamic correlations and networks of residues within the Fc structure is a critical step in understanding the protein as a dynamic entity and for developing insight into the effects of mutations at distal sites. See, e.g. Example 6.
Thus, we studied in detail the impact of mutations on the local environment of the site of mutation. The formation of a well packed core at the CH3 interface between chain A and B is critical for the spontaneous pairing of the two chains in a stable Fc structure. Good packing is the result of strong structural complementarity between interacting molecular partners coupled with favorable interactions between the contacting groups. The favorable interactions result from either buried hydrophobic contacts well removed from solvent exposure and/or from the formation of complementary electrostatic contacts between hydrophilic polar groups. These hydrophobic and hydrophilic contacts have entropic and enthalpic contributions to the free energy of dimer formation at the CH3 interface. We employ a variety of algorithms to accurately model the packing at the CH3 interface between chain A and chain B and subsequently evaluate the thermodynamic properties of the interface by scoring a number of relevant physicochemical properties.
We employed a number of protein packing methods including flexible backbones to optimize and prepare model structures for the large number of variants we computationally screened. Following packing we evaluated a number of terms including contact density, clash score, hydrogen bonds, hydrophobicity and electrostatics. The use of the solvation models allowed us to more accurately address the effect of solvent environment and contrast the free energy differences following mutation of specific positions in the protein to alternate residue types. Contact density and clash score provide a measure of complementarity, a critical aspect of effective protein packing. These screening procedures are based on the application of knowledge-based potentials or coupling analysis schemes relying on pair-wise residue interaction energy and entropy computations.
This comprehensive in-silico analysis provided a detailed understanding of the differences of each Fc variant compared to wild-type with respect to interface hotspots, sites of asymmetry, cavities and poorly packed regions, structural dynamics of individual sites and sites of local unfolding. These combined results of the described computational analysis identified specific residues, sequence/structural motifs and cavities that were not optimized and in combination responsible for the lower stability (e.g., Tm of 68° C.) and/or lower specificity of <90% purity. In the second design phase we used targeted positive design to specifically address these hypothesis by additional point-mutations and tested these by in-silico engineering using the above described methodology and analysis (See,
In certain embodiments, provided herein are isolated heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain is AZ17, or AZ18, or AZ19, or AZ20, or AZ21, or AZ22, or AZ23, or AZ24, or AZ25, or AZ26, or AZ27, or AZ28, or AZ29, or AZ30, or AZ21, or AZ32, or AZ33, or AZ34, or AZ35, or AZ36, or AZ37, or AZ38, or AZ39, or AZ40, or AZ41, or AZ42, or AZ43, or AZ44, or AZ45, or AZ46, or AZ47, or AZ48, or AZ49, or AZ50, or AZ51, or AZ52, or AZ53, or AZ54, or AZ55, or AZ56 or AZ57, or AZ58, or AZ59, or AZ60, or AZ61, or AZ62, or AZ63, or AZ64, or AZ65, or AZ66, or AZ67, or AZ68, or AZ69, or AZ70, or AZ71, or AZ72, or AZ73, or AZ74, or AZ75, or AZ76, or AZ77, or AZ78, or AZ79, or AZ80, or AZ81, or AZ82, or AZ83, or AZ84, or AZ85, or AZ86, or AZ87, or AZ88, or AZ89, or AZ90, or AZ91, or AZ92, or AZ93, or AZ94, or AZ95, or AZ96, or AZ97, or AZ98, or AZ99, or AZ100 or AZ101. In an exemplary embodiment, the modified CH3 domain is AZ17, or AZ18, or AZ19, or AZ20, or AZ21, or AZ22, or AZ23, or AZ24, or AZ25, or AZ26, or AZ27, or AZ28, or AZ29, or AZ30, or AZ21, or AZ32, or AZ33, or AZ34, or AZ38, or AZ42, or AZ43, or AZ 44, or AZ45, or AZ46, or AZ47, or AZ48, or AZ49, or AZ50, or AZ52, or AZ53, or AZ54, or AZ58, or AZ59, or AZ60, or AZ61, or AZ62, or AZ63, or AZ64, or AZ65, or AZ66, or AZ67, or AZ68, or AZ69, or AZ70, or AZ71, or AZ72, or AZ73, or AZ74, or AZ75, or AZ76, or AZ77, or AZ78, or AZ79, or AZ81, or AZ82, or AZ83, or AZ84, or AZ85, or AZ86, or AZ87, or AZ88, or AZ89, or AZ91, or AZ92, or AZ93, or AZ94, or AZ95, or AZ98, or AZ99, or AZ100 or AZ101. In a specific embodiment, the modified CH3 domain is AZ33 or AZ34. In another embodiment, the modified CH3 domain is AZ70 or AZ90.
In an exemplary embodiment, the CH3 domain comprises a first and second polypeptide (also referred to herein as Chain A and Chain B) wherein the first polypeptide comprises amino acid modifications L351Y, F405A, and Y407V and wherein the second polypeptide comprises amino acid modifications T366I, K392M and T394W. In another embodiment, a first polypeptide comprises amino acid modifications L351Y, S400E, F405A and Y407V and the second polypeptide comprises amino acid modifications T366I, N390R, K392M and T394W.
This iterative process of computational structure-function analysis, targeted engineering and experimental validation was used to design the remaining Fc variants listed in Table 1 in subsequent design phases and resulting in Fc variant heterodimers with a purity greater than 90% and an increased stability with a CH3 domain melting temperature greater than 70° C. In certain embodiments, the Fc variants comprise amino acid mutations selected from AZ1 to AZ 136. In further embodiments, the Fc variants comprise amino acid mutations selected from the Fc variants listed in Table 4.
From the first and second design phases two core scaffolds were identified, Scaffold 1 and Scaffold 2, wherein additional amino acid modifications were introduced into these scaffolds to fine tune the purity and stability of the Fc variant heterodimers. See Example 5 for a detailed description of the development of Scaffold 1 including AZ8, AZ17-62 and the variants listed in Table 6. See Example 6 for a detailed description of the development of Scaffold 2 including AZ15 and AZ63-101 and the variants listed in Table 7.
The core mutations of Scaffold 1 comprise L351Y_F405A_Y407V/T394W. Scaffold 1a comprises the amino acid mutations T366I_K392M_T394W/F405A_Y407V and Scaffold 1 b comprises the amino acid mutations T366L_K392M_T394W/F405A_Y407V. See, Example 5.
In certain embodiments, the modified CH3 domain comprises a first and second polypeptide (also referred to herein as Chain A and Chain B) wherein the first polypeptide comprises amino acid modifications I351Y, F405A and Y407V and the second polypeptide comprises amino acid modification T394W. In one aspect the modified CH3 domain further comprises point mutations at positions F405 and/or K392. These mutations at position K392 include, but are not limited to, K392V, K392M, K392R, K392L, K392F or K392E. These mutations at position F405 include, but are not limited to, F405I, F405M, F405S, F405S, F405V or F405W. In another aspect, the modified CH3 domain further comprises point mutations at positions T411 and/or S400. These mutations at position T411 include, but are not limited to, T411N, T411R, T411Q, T411K, T411D, T411E or T411W. These mutations at position S400 include, but are not limited to, S400E, S400D, S400R or S400K. In yet another embodiment, the modified CH3 domain comprises a first and second polypeptide wherein the first polypeptide comprises amino acid modifications L351Y, F405A and Y407V and the second polypeptide comprises amino acid modification T394W, wherein the first and/or second polypeptide comprises further amino acid modifications at positions T366 and/or L368. These mutations at position T366 include, but are not limited to, T366A, T366I, T366L, T366M, T366Y, T366S, T366C, T366V or T366W. In an exemplary embodiment, the amino acid mutation at position T366 is T366I. In another exemplary embodiment, the amino acid mutation at position T366 is T366L. The mutations at position L368 include, but are not limited to, L368D, L368R, L368T, L368M, L368V, L368F, L368S and L368A.
In certain embodiments, the modified CH3 domain comprises a first and second polypeptide (also referred to herein as Chain A and Chain B) wherein the first polypeptide comprises amino acid modifications L351Y, F405A and Y407V and the second polypeptide comprises amino acid modifications T366L and T394W. In another embodiment, the modified CH3 domain comprises a first and second polypeptide wherein the first polypeptide comprises amino acid modifications L351Y, F405A and Y407V and the second polypeptide comprises amino acid modifications T366I and T394W.
In certain other embodiments, the modified CH3 domain comprises a first and second polypeptide (also referred to herein as Chain A and Chain B) wherein the first polypeptide comprises amino acid modifications L351Y, F405A and Y407V and the second polypeptide comprises amino acid modifications T366L, K392M and T394W. In another embodiment, the modified CH3 domain comprises a first and second polypeptide wherein the first polypeptide comprises amino acid modifications L351Y, F405A and Y407V and the second polypeptide comprises amino acid modifications T366I, K392M and T394W.
In yet another embodiment, the modified CH3 domain comprises a first and second polypeptide (also referred to herein as Chain A and Chain B) wherein the first polypeptide comprises amino acid modifications F405A and Y407V and the second polypeptide comprises amino acid modifications T366L, K392M and T394W. In another embodiment, the modified CH3 domain comprises a first and second polypeptide wherein the first polypeptide comprises amino acid modifications F405A and Y407V and the second polypeptide comprises amino acid modifications T366I, K392M and T394W.
In certain embodiments, the modified CH3 domain comprises a first and second polypeptide (also referred to herein as Chain A and Chain B) wherein the first polypeptide comprises amino acid modifications F405A and Y407V and the second polypeptide comprises amino acid modifications T366L and T394W. In another embodiment, the modified CH3 domain comprises a first and second polypeptide wherein the first polypeptide comprises amino acid modifications F405A and Y407V and the second polypeptide comprises amino acid modifications T366I and T394W.
In an exemplary embodiment, provided herein are isolated heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) of about 74° C. or greater. In another embodiment, provided herein are isolated heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) of about 74° C. or greater and the heterodimer has a purity of about 98% or greater.
In certain embodiments, the isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) greater than 70° C. and the modified CH3 domains are selected from Table 6.
The core mutations of Scaffold 2 comprise L351Y_Y407A/T366A_K409F. Scaffold 2a comprises the amino acid mutations L351Y_Y407A/T366V_K409F and Scaffold 2b comprises the amino acid mutations Y407A/T366A_K409F. See, Example 6.
In certain embodiments, the modified CH3 domain comprises a first and second polypeptide (also referred to herein as Chain A and Chain B) wherein the first polypeptide comprises amino acid modifications L351Y and Y407A and the second polypeptide comprises amino acid modifications T366A and K409F. In one aspect the modified CH3 domain further comprises point mutations at positions T366, L351, and Y407. These mutations at position T366 include, but are not limited to, T366I, T366L, T366M, T366Y, T366S, T366C, T366V or T366W. In a specific embodiment, the mutation at position T366 is T366V. The mutations at position L351 include, but are not limited to, L3511, L351 D, L351R or L351F. The mutations at position Y407 include, but are not limited to, Y407V or Y407S. See, CH3 variants AZ63-AZ70 in Table 1 and Table 4 and Example 6.
In an exemplary embodiment, the modified CH3 domain comprises a first and second polypeptide (also referred to herein as Chain A and Chain B) wherein the first polypeptide comprises amino acid modifications L351Y and Y407A and the second polypeptide comprises amino acid modification T366V and K409F.
In an exemplary embodiment, provided herein are isolated heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) of about 75.5° C. or greater. In another embodiment, provided herein are isolated heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) of about 75° C. or greater and the heterodimer has a purity of about 90% or greater.
In other certain embodiments, the modified CH3 domain comprises a first and second polypeptide (also referred to herein as Chain A and Chain B) wherein the first polypeptide comprises amino acid modifications L351Y and Y407A and the second polypeptide comprises amino acid modification T366A and K409F, wherein the modified CH3 domain comprises one or more amino acid modifications at positions T411, D399, S400, F405, N390, and/or K392. These mutations at position D399 include, but are not limited to, D399R, D399W, D399Y or D399K. The mutations at position T411 includes, but are not limited to, T411N, T411R, T411Q, T411K, T411O, T411E or T411W. The mutations at position S400 includes, but are not limited to, S400E, S400D, S400R, or S400K. The mutations at position F405 includes, but are not limited to, F405I, F405M, F405S, F405S, F405V or F405W. The mutations at position N390 include, but are not limited to, N390R, N390K or N390D. The mutations at position K392 include, but are not limited to, K392V, K392M, K392R, K392L, K392F or K392E. See, CH3 variants AZ71-101 in Table 1 and Table 4 and Example 6.
In an exemplary embodiment, the modified CH3 domain comprises a first and second polypeptide (also referred to herein as Chain A and Chain B) wherein the first polypeptide comprises amino acid modification Y407A and the second polypeptide comprises amino acid modification T366A and K409F. In one aspect, this modified CH3 domain further comprises the amino acid modifications K392E, T411E, D399R and S400R. In a further embodiment, the modified CH3 domain comprises a first and second polypeptide wherein the first polypeptide comprises amino acid modification D399R, S400R and Y407A and the second polypeptide comprises amino acid modification T366A, K409F, K392E and T411E.
In an exemplary embodiment, provided herein are isolated heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) of about 74° C. or greater. In another embodiment, provided herein are isolated heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) of about 74° C. or greater and the heterodimer has a purity of about 95% or greater.
In certain embodiments, provided herein are isolated heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) greater than 70° C. and the modified CH3 domains are selected from Table 7.
Furthermore, this new method of designing Fc variant heterodimers with improved stability and purity can be applied to other classes and isotypes of Fc regions. In certain embodiments, the Fc region is a human IgG Fc region. In further embodiments, the human IgG Fc region is a human IgGI, IgG2, IgG3, or IgG4 Fc region. In some embodiments the Fc regions is from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE and IgM. In some embodiments, the IgG is of subtype selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3 and IgG4.
The Fc region as defined herein comprises a CH3 domain or fragment thereof, and may additionally comprise one or more addition constant region domains, or fragments thereof, including hinge, CH1, or CH2. It will be understood that the numbering of the Fc amino acid residues is that of the EU index as in Kabat et al., 1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va. The “EU index as set forth in Kabat” refers to the EU index numbering of the human IgG1 Kabat antibody. For convenience, Table B provides the amino acids numbered according to the EU index as set forth in Kabat of the CH2 and CH3 domain from human IgG1.
There is provided according to one aspect of the invention an isolated heteromultimer Fc construct comprising a modified heterodimeric CH3 domain, said modified CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide; wherein at least one of said first and second CH3 domain polypeptides comprises an amino acid modification of K392J wherein J is selected from L, I or an amino acid with a side chain volume not substantially larger than the side chain volume of K; wherein said first and second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain with a melting temperature (Tm) of at least about 74DC and a purity of at least 95%; and wherein at least one amino acid modification is not of an amino acid which is at the interface between said first and said second CH3 domain polypeptides. In certain embodiments is a heteromultimer Fc construct described herein, comprising at least one T350X modification, wherein X is a natural or non-natural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments is an isolated heteromultimer Fc construct described herein, comprising at least one T350V modification. In an embodiment is an isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In an embodiment is the isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a Tm of about 70° C. or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80° C. or greater. Provided in certain embodiments is an isolated heteromultimer Fc construct described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of at least one of L351, F405, and Y407. In some embodiments is an isolated heteromultimer Fc construct, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide further comprising an amino acid modification of T366. In certain embodiments is an isolated heteromultimer Fc construct described herein, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising amino acid modifications at positions L351, F405, and Y407, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising amino acid modifications at positions T366, K392, and T394. In an embodiment is the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A, and Y407V, and said second CH3 domain polypeptide comprising amino acid modifications T366L, K392M, and T394W. In some embodiments is the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A, and Y407V, and said second CH3 domain polypeptide comprising amino acid modifications T366L, K392L, and T394W. In a further embodiment is the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A, and Y407V, and said second CH3 domain polypeptide comprising amino acid modifications T366I, K392M, and T394W. In some embodiments is the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A, and Y407V, and said second CH3 domain polypeptide comprising amino acid modifications T366I, K392L, and T394W. In certain embodiments is the isolated heteromultimer Fc construct described herein, wherein at least one of said first and second CH3 domain polypeptides is a modified CH3 domain polypeptide comprising an amino acid modification at position S400. In a further embodiment is the isolated heteromultimer Fc construct described herein, comprising the modification S400Z, wherein Z is selected from a positively charged amino acid and a negatively charged amino acid. In some embodiments, the positively charged amino acid is lysine or arginine and the negatively charged amino acid is aspartic acid or glutamic acid. In certain embodiments is the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide comprising an amino acid modification selected from S400E and S400R. In some embodiments is provided the isolated heteromultimer Fc construct described herein, wherein at least one of said first and second CH3 domain polypeptides is a modified CH3 domain polypeptide comprising an amino acid modification at position N390. In some embodiments, the modification of N 390 is N390Z, wherein Z is selected from a positively charged amino acid and a negatively charged amino acid. In an embodiment, N390Z is N390R. In certain embodiments of the isolated heteromultimer Fc construct described herein, said first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising the amino acid modification S400E and said second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising the amino acid modification N390R. In some embodiments of the isolated heteromultimer Fc construct described herein, each of the first and second CH3 domain polypeptide is a modified CH3 domain polypeptide, one said modified CH3 domain polypeptide comprising the amino acid modification Q347R and the other modified CH3 domain polypeptide comprising the amino acid modification K360E.
Provided in one aspect is an isolated heteromultimer Fc construct comprising a modified heterodimeric CH3 domain, said modified CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide; wherein at least one of said first and second CH3 domain polypeptides comprises an amino acid modification of K392J wherein J is selected from L, I or an amino acid with a side chain volume not substantially larger than the side chain volume of K; wherein said first and second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain with a melting temperature (Tm) of at least about 74° C. and a purity of at least 95%; and wherein at least one amino acid modification is not of an amino acid which is at the interface between said first and said second CH3 domain polypeptides. In certain embodiments is a heteromultimer Fc construct described herein, comprising at least one T350X modification, wherein X is a natural or non-natural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments is an isolated heteromultimer Fc construct described herein, comprising at least one T350V modification. In an embodiment is an isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In an embodiment is the isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a Tm of about 70° C. or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80° C. or greater. In an embodiment is the isolated heteromultimer Fc construct described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of at least one of K409 and T411. In certain embodiments is the isolated heteromultimer Fc construct described herein, comprising at least one of K409F, T411E and T411 D. In some embodiments is the isolated heteromultimer Fc construct described herein wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of D399. In some embodiments, the amino acid modification of D399 is at least one of D399R and D399K.
Provided in one aspect is an isolated heteromultimer Fc construct comprising a modified heterodimeric CH3 domain, said modified CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications as compared to a wild-type CH3 domain polypeptide; wherein at least one of said first and second CH3 domain polypeptides comprises an amino acid modification of K392J wherein J is selected from L, I or an amino acid with a side chain volume not substantially larger than the side chain volume of K; wherein said first and second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain with a melting temperature (Tm) of at least about 74° C. and a purity of at least 95%; and wherein at least one amino acid modification is not of an amino acid which is at the interface between said first and said second CH3 domain polypeptides. In certain embodiments is a heteromultimer Fc construct described herein, comprising at least one T350X modification, wherein X is a natural or non-natural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments is an isolated heteromultimer Fc construct described herein, comprising at least one T350V modification. In an embodiment is an isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In an embodiment is the isolated heteromultimer Fc construct described herein, wherein the modified CH3 domain has a Tm of about 70° C. or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80° C. or greater. In certain embodiments of the isolated heteromultimer Fc construct described herein, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising at least one amino acid modification selected from K409F, T411E and T4110, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising at least one amino acid modification selected from Y407A, Y407I, Y407V, D399R and D399K. In some embodiments is any one of the isolated heteromultimer Fc constructs described herein, further comprising a first modified CH3 domain comprising one of amino acid modifications T366V, T366I, T366A, T366M, and T366L; and a second modified CH3 domain comprising the amino acid modification L351Y. In some embodiments is any one of the isolated heteromultimer Fc constructs described herein, comprising a first modified CH3 domain comprising one of amino acid modifications K392L or K392E; and a second modified CH3 domain comprising one of the amino acid modifications S400R or S400V.
Provided herein is an isolated heteromultimer Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, each modified CH3 domain polypeptide comprising at least four amino acid mutations, wherein at least one of said first and said second modified CH3 domain polypeptide comprises a mutation selected from N390Z and S400Z, wherein Z is selected from a positively charged amino acid and a negatively charged amino acid, and wherein said first and second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain with a melting temperature (Tm) of at least about 70° C. and a purity of at least 90%. In an embodiment is provided the isolated heteromultimer Fc construct, wherein said first modified CH3 domain polypeptide comprising amino acid modifications at positions F405 and Y407 and said second modified CH3 domain polypeptide comprises amino acid modification at position T394. In an embodiment is provided the isolated heteromultimer Fc construct, the first modified CH3 domain polypeptide comprising an amino acid modification at position L351. In certain embodiments, is the isolated heteromultimer described herein, said second modified CH3 domain polypeptide comprising a modification of at least one of positions T366 and K392. In some embodiments, is the isolated heteromultimer described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. and is formed with a purity of at least about 95%. In certain embodiments, is the isolated heteromultimer described herein, at least one modified CH3 domain polypeptide comprising amino acid modifications of at least one of N390R, S400E and S400R. In some embodiments is an isolated heteromultimer described herein, one of said first and second modified CH3 domain polypeptide comprising amino acid modifications of position 347 and the other modified CH3 domain polypeptide comprising amino acid modification at position 360. In certain embodiments is the isolated heteromultimer described herein, at least one of said first and second modified CH3 domain polypeptides comprising amino acid modification of T350V. In specific embodiments is an isolated heteromultimer described herein, said first modified CH3 domain polypeptide comprising at least one amino acid modification selected from L351Y, F405A and Y407V; and said second modified CH3 domain polypeptide comprising at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In certain embodiments described herein is an isolated heteromultimer, the first modified CH3 domain polypeptide comprising amino acid modifications at positions D399 and Y407, and a second modified CH3 domain polypeptide comprising amino acid modification at positions K409 and T411. In some embodiments is an isolated heteromultimer described herein, the first CH3 domain polypeptide comprising amino acid modification at position L351, and the second modified CH3 domain polypeptide comprising amino acid modifications at position T366 and K392. In specific embodiments are isolated heteromultimers described herein, at least one of said first and second CH3 domain polypeptide comprising amino acid modification of T350V. In certain embodiments are isolated heteromultimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater and is formed with a purity of at least about 95%. Provided in certain embodiments are isolated heteromultimer Fc constructs described herein, said first modified CH3 domain polypeptide comprising amino acid modifications selected from L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A, and Y407V; and said second modified CH3 domain polypeptide comprising amino acid modifications selected from T366V, T366I, T366L, T366M, N390D, N390E, K392L, K392I, K392D, K392E, K409F, K409W, T4110 and T411E.
Provided herein is an isolated heteromultimer Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, each modified CH3 domain polypeptide comprising at least three amino acid mutations, wherein one of said first and said second modified CH3 domain polypeptide comprises a mutation selected from T411E and T4110, and wherein said first and second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain with a melting temperature (Tm) of at least about 70° C. and a purity of at least 90%. In an embodiment is provided the isolated heteromultimer Fc construct wherein said first modified CH3 domain polypeptide comprising amino acid modifications at positions F405 and Y407 and said second modified CH3 domain polypeptide comprises amino acid modification at position T394. In an embodiment is provided the isolated heteromultimer Fc construct, the first modified CH3 domain polypeptide comprising an amino acid modification at position L351. In certain embodiments, is the isolated heteromultimer described herein, said second modified CH3 domain polypeptide comprising a modification of at least one of positions T366 and K392. In some embodiments, is the isolated heteromultimer described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. and is formed with a purity of at least about 95%. In certain embodiments, is the isolated heteromultimer described herein, at least one modified CH3 domain polypeptide comprising amino acid modifications of at least one of N390R, S400E and S400R. In some embodiments is an isolated heteromultimer described herein, one of said first and second modified CH3 domain polypeptide comprising amino acid modifications of position 347 and the other modified CH3 domain polypeptide comprising amino acid modification at position 360. In certain embodiments is the isolated heteromultimer described herein, at least one of said first and second modified CH3 domain polypeptides comprising amino acid modification of T350V. In specific embodiments is an isolated heteromultimer described herein, said first modified CH3 domain polypeptide comprising at least one amino acid modification selected from L351Y, F405A and Y407V; and said second modified CH3 domain polypeptide comprising at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In certain embodiments described herein is an isolated heteromultimer, the first modified CH3 domain polypeptide comprising amino acid modifications at positions D399 and Y407, and a second modified CH3 domain polypeptide comprising amino acid modification at positions K409 and T411. In some embodiments is an isolated heteromultimer described herein, the first CH3 domain polypeptide comprising amino acid modification at position L351, and the second modified CH3 domain polypeptide comprising amino acid modifications at position T366 and K392. In specific embodiments are isolated heteromultimers described herein, at least one of said first and second CH3 domain polypeptide comprising amino acid modification of T350V. In certain embodiments are isolated heteromultimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 750C or greater and is formed with a purity of at least about 95%. Provided in certain embodiments are isolated heteromultimer Fc constructs described herein, said first modified CH3 domain polypeptide comprising amino acid modifications selected from L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A, and Y407V; and said second modified CH3 domain polypeptide comprising amino acid modifications selected from T366V, T366I, T366L, T366M, N390D, N390E, K392L, K392I, K392D, K392E, K409F, K409W, T411D and T411E.
Provided herein is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366I, K392M and T394W.
Provided in an aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366I, K392L and T394W.
Provided in a certain aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366L, K392M and T394W.
Provided in some aspects is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366L, K392L and T394W.
Provided in an aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T350V, L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, K392L and T394W.
Provided in an aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T350V, L351Y, S400R, F405A, Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, K392M and T394W.
Provided in an aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T350V, L351Y, S400E, F405A, Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, N390R, K392M and T394W.
Provided in an aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T350V, L351Y, F405A, Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, K392L and T394W.
Provided in one aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T366V, K392L, K409F and T411E; and a second modified CH3 domain polypeptide comprising amino acid modifications L351Y, D399R, and Y407A.
Provided in one aspect is an isolated heteromultimer Fc construct, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T366V, K392LE K409F and T411E; and a second modified CH3 domain polypeptide comprising amino acid modifications L351Y, D399R, S400R and Y407A.
In certain embodiments, the Fc variant comprises a CH2 domain. In some embodiments, the CH2 domain is a variant CH2 domain. In some embodiments, the variant CH2 domains comprise asymmetric amino acid substitutions in the first and/or second polypeptide chain. In some embodiments, the heteromultimer comprises asymmetric amino acid substitutions in the CH2 domain such that one chain of said heteromultimer selectively binds an Fc receptor.
In certain embodiments, the heteromultimer selectively binds an Fc receptor. In some embodiments, Fc receptor is a member of Fcγ receptor family. In some embodiments, the receptor is selected from FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIb. In one embodiment, the CH2 domain comprises asymmetric amino acid modifications that promote selective binding to Fcgamma receptors.
In some embodiments, the heteromultimer binds selectively to FcγRIIIa. In some embodiments, the heteromultimer comprises asymmetric amino acid substitutions selected from S267D, K392D and K4090. In some embodiments, the heteromultimer binds selectively to FcγRIIa. In some embodiments, the heteromultimer comprises asymmetric amino acid substitutions selected from S239D, K326E, A330L and I332E. In some embodiments, the heteromultimer binds selectively to FcγRIIb. In some embodiments, the heteromultimer comprises asymmetric amino acid substitutions selected from S239D, D265S, E269K and I332E. In some embodiments, the heteromultimer binds selectively to FcγRIIIa and FcγRIIa. In some embodiments, the heteromultimer comprises asymmetric amino acid substitutions selected from S239D, D265S, and S298A. In some embodiments, the heteromultimer binds selectively to FcγRIIIa and FcγRIIb. In some embodiments, the heteromultimer comprises asymmetric amino acid substitutions selected from S239D, S298A, K326E, A330L and I332E. In some embodiments, the heteromultimer binds selectively to FcγRIIa and FcγRIIb. In some embodiments, the heteromultimer comprises asymmetric amino acid substitutions selected from S239D, D265S, S298A and I332E.
In certain embodiments is provided a method of designing multi-functional therapeutics comprising heteromultimer described herein. In some embodiments is provided method of designing bi-functional therapeutics comprising a variant Fc heterodimer. In some embodiments is provided a method for the design of asymmetric mutations in the CH2 domain of a variant Fc heterodimer derived with mutations in the CH3 domain. In some embodiments is provided a method to design selectivity for the different Fc gamma receptors based on the mutations in the asymmetric Fc. In certain embodiments is provided a method to design mutations that bias binding of the Fc gamma receptors to one face of the Fc molecule. In certain embodiments is provided a method to design polarity drivers that bias the Fcγ receptors to interact with only one face of the asymmetric Fc scaffold of the heteromultimer described herein.
In some embodiments, is provided a polypeptide comprising mutations in the CH2 domain of the asymmetric Fc that lead to preferential Fc gamma receptor selectivity profiles. In some embodiments mutations in the CH3 domain lead to preferential formation of heterodimeric Fc. In certain embodiments is a method for designing bispecific therapeutic entities based on the asymmetric Fc described herein. In certain embodiments is a method to design multi-specific therapeutic entities based on the asymmetric Fc described herein.
Monoclonal antibodies such as IgG are symmetric molecules composed of two equivalent heavy and two light polypeptide chains (
The symmetric nature of the monoclonal antibodies structure results in both Fab arms having their antigen binding capability affinity matured to recognize the same epitope. At the other end, the Fc portion of the antibody molecule is involved in interactions with various receptor molecules on the immune or “effector” cells, and some of these interactions are responsible for mediating effector functions such as antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and complement activation. Generally, the effector function involves immune responses leading to pathogen or toxin neutralization and elimination, complement activation, and phagocytic response from the humoral immune system. The Fcγ receptor (FcγR) molecules on the effector cells contact the Fc of the activated IgG antibody involved in integral antibody-antigen immune complex to mediate and regulate the effector response. Optimizing the interaction of monoclonal antibody based protein therapeutic agents to these Fcγ receptors can lead to improvements in the efficacy of these drug candidates.
In humans there are three known classes of FcγR's with further polymorphic types within each class. The Fc in the IgG1 molecule is known to bind FcγRI (CD64) with dissociation constants in the nanomolar range while FcγRII (CD32) and FcγRIII (CD16) binding occurs at the micromolar range [Bruhns P.; Iannascoli B.; England P.; Mancardi D. A.; Fernandez N.; Jorieux S. and Daeron M. (2009) Blood 113: 3716-25]. The high affinity FcγRI receptors can bind IgG in monomeric forms while the low affinity FcγRII and FcγRIII receptors can only bind antigen-antibody immune complexes or IgG aggregates as a result of avidity effects. The different IgG forms have varying affinities for the different FcγR's; in particular, the IgG1 and IgG3 exhibit stronger activity. The Fcγ receptors are the extracellular domains of trans-membrane proteins and possess cytoplasmic domains that are involved in regulating signaling pathways within the cell. When clustered on the immune cell surface on association with the antibody mediated immune complexes, depending on the nature of signaling units linked to the FcγR's on the cytoplasmic end of these cell surface receptors, these molecules regulate the effector response [Nimmerjahn F. and Ravetch J. V. (2008) Nature Immu Rev 8(1):34-47].
At the human chromosomal level, three genes encode the FcγRI (FcγRIA, FcγRIB, FcγRIC) and FcγRII (FcγRIIA, FcγRIIB, FcγRIIC) and two genes encode the FcγRIII (FcγRIIIA, FcγRIIIB). Among the IgG binding human Fcγ receptors, the FcγRIA, FcγRIC and FcγRIIIA types have been shown to be membrane associated with a common γ-chain signal adaptor protein which contains a cytoplasmic immunoreceptor tyrosine based activation motif (ITAM) that leads to the activation of effector function. The FcγRIIA and FcγRIIC also comprise a cytoplasmic ITAM, but without the common γ-chain signal adaptor protein. At the same time, the FcγRIIB is linked to an immunoreceptor tyrosine-based inhibitory motif (ITIM). Activation of FcγRIIB resulting in ITIM phosphorylation results in inhibition of the activating signaling cascade. The FcγRIIIB, while lacking either of the tyrosine based immune-modulatory cytoplasmic tails, has a GPI (glycosyl-phosphatidyl-inositol) anchor and has been shown to contribute to activation of some granulocytes in the presence of FcγRIIA.
While the functional role of ITAM and ITIM motifs and the associated receptor molecules are known, the nature and mechanisms of the modulation of signaling in combination is not completely understood, especially when combined with the activity of a host of other immune cell surface receptors and adaptor molecules (e.g. BCR's, CD22, CD45 etc) involved in signal transduction. In this context, the design of Fc-like molecules that can interact with these Fcγ receptors with exquisite selectivity profiles is a valuable scaffold in any attempt to de-convolute and modulate the effect of such receptor molecules with subtle regulatory activities.
In the context of designing antibody molecules that can differentiate the FcγR's, the effort is complicated by the fact that the extracellular Fc binding sections of the FcγRII and FcγRII) receptor types exhibit high sequence similarity (
Complicating matters further, each of the receptor class presents multiple single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). The resulting receptor diversity differentially impact their affinity to IgG's and its mechanism of action. These genetic variations could affect the affinity of particular IgG subclasses for the Fcγ receptors, alter the downstream effector events or impact mechanisms that alter the levels of receptor expression resulting in functionally relevant phenotypes, non-functional or functionally unknown receptor variants (Bournazos S.; Woof J. M.; Hart S. P. and Dransfield I. (2009) Clinical and Experimental Immunology 157(2):244-54). They potentially lead to complex effects, altering the balance between activating and inhibitory receptor signaling, resulting in the creation of disease susceptible phenotypes.
Some of these allelic variations are listed in Table C. Notably, the R131 variant in FcγRIIa is a high responder with IgG1 while the alternate H131 variants show more efficient interactions with IgG2 and IgG3. In the case of FcγRIIIa, donors homozygous for V at position 158 exhibit increased NK cell activity in comparison to homozygous F/F158 individuals due to higher affinity of the former allotype for human IgG1, IgG3 and IgG4. The allelic variants NA1 and NA2 of FcγRIIIb is the result of a four amino acid substitution which in turn leads to differences in the glycosylation of the receptor. The NA1 allele presents enhanced binding and phagocytosis of the immune complex by neutrophils. The FcγRIIB has two known allelic variants, 2321 and 232T. The 232T variant is known to be strongly impaired in its negative regulatory activity. The frequencies of FcγR polymorphisms and its associations to differential responsiveness to infections or predisposition to disease conditions such as systemic lupus erthematosus (SLE), rheumatoid arthritis (RA), vasculitis, immune-mediated thrombocytic purpura (ITP), myasthenia gravis, multiple sclerosis (MS), and immuno neuropathies (Guillian-Barre syndrome (GBS)) have been reported.
Copy number variation in the locus of FcγR genes, in particular for FcγRIIIB, FcγRIIc and FcγRIIIA has been demonstrated, and further correlation of these differences to cell surface expression of these receptors have been noted. In contrast FcγRIIa and FcγRIIb do not show gene copy number variation. Low copy number of FcγRIIIb has in fact been associated with glomerulonephritis in the autoimmune disease systemic lupus erythematosus (SLE) [Aitman T J et al. (2006) Nature 16; 439(7078):851-5]. This is particularly interesting given the fact that a non-signaling GPI module anchors the FcγRIIIb receptor. It can be hypothesized that the presence of these FcγRIIIb receptors could potentially act as competitive inhibitors of Fc interactions with other signaling FcγR's. The effect of copy number variation in FcγRIIc is also especially interesting. A CIT SNP at position 202 in FcγRIIc converts a glutamine residue to a stop codon preventing the generation of a functional protein. The functional open reading frame of FcγRIIc is expressed in 9% of healthy individuals (white population) and there is a significant overrepresentation (19%) of the allele in the ITP population implying a predisposition of these phenotypes for ITP [Breunis W B et al. (2008) Blood 111(3):1029-38]. It has been demonstrated that in individuals expressing functional FcγRIIc on NK cells, the ADCC achieved is mediated by these receptors to a greater extent than the FcγRIIIa. Such complexities associated with these polymorphisms and genetic variations highlights the need for personalized treatment strategies requiring high tailored therapeutics.
The various effector cells differ in the presentation of these Fcγ receptors as well as in their humoral and tissue distribution, thus contributing to variations in their mechanism of activation and action [Table D]. Tuning the selectivity of therapeutic antibodies towards the recognition of specific FcγR types and modulating the impact of certain classes of effector cells, leads to optimization of the effector mechanism for particular disease conditions. This is meant to selectively activate or inhibit specific effector modalities, depending on the disease condition being treated.
In addition, FcγR's are also expressed by follicular dendritic cells, endothelial cells, microglial cells, osteoclasts and mesangial cells. Currently, the functional significance of FcγR expression on these other cells is not known.
The high affinity FcγRI is composed of three C-type immunoglobulin superfamily (IgSF) domains while the low affinity FcγRII and FcγRIII are constituted of two C-type IgSF domains each. The structure of FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb receptor proteins has been solved by crystallography. The two IgSF domains in these structures are positioned 50-55 degrees relative to each other and are connected by a hinge.
The publicly available structure of an Fc-FcγR co-complex is that of the Fc-FcγRIIIb system and the FcγR geometry in the complex is maintained very close to that observed in the apo state of the protein [Sondermann P.; Huber R.; Oosthuizen V. and Jacob U. (2000) Nature 406, 267-273.; Radaev S.; Motyaka S.; Fridman W.; Sautes-Fridman C. and Sun P. O. (2001) J Bioi Chem 276, 16469-16477; Sondermann P. et al. Biochem Soc Trans. 2002 August; 30(4):481-6; Sondermann P, Oosthuizen V. Immunol Lett. 2002 Jun. 3; 82(1-2):51-6; Radaev S, Sun P. MolImmunol. 2002 May; 38(14):1073-83.][
Prior to the structural evaluation of Fc-FcγR complex based on crystallography, there were questions if the 2-fold axis of symmetry in the Fc molecule means two potential binding sites and an effective 2:1 stoichiometry for the Fc-FcγR association. Nuclear magnetic resonance (NMR) based structural studies of Fc-FcγR interactions indicate that binding an Fc to one FcγR on one face of the molecule induces a conformational change that precludes the binding of a second FcγR molecule to the Fc of the same antibody molecule [Kato K. et al (2000) J Mol Bioi. 295(2):213-24]. The geometry of the available co-crystal complex of the Fc-FcγRIIIb confirms the association of the FcγR to Fc in an asymmetric orientation with a 1:1 stoichiometry. As shown in
Alanine scanning mutagenesis [Shields R L et al. (2001) JBC 276(9): 6591-604] provides insight on the residues of the Fc interfacing with the diverse receptor types and hence involved in the Fc-FcγR interaction and recognition. Traditionally, optimization of the therapeutic antibodies has been focused around mutations that exhibit increased binding to the activating receptors FcγRIII [U.S. Pat. No. 6,737,056] or decreased affinity to FcγRIIb [US2009/0010920A1]. In all these alternate variants, mutations are introduced concurrently in both the chains.
Monoclonal antibodies often exhibit their therapeutic activity by inducing spatial localization of the target and effector immune cells. A natural antibody mediates this by interacting with the target using its Fab domains and the effector cell using Fc domain. They are able to juxtaposition the immune complex vis-a-vis the effector cell such that the cell mediated response can be induced. Avidity effects required for FcγR signaling, originating in the formation of immune complexes involving the targeting of a single target by multiple antibody molecules, is another example of significance of spatia-temporal organization in immune action.
There is also a spatia-temporal aspect to the cell signaling that is induced as part of the effector activity of mAb molecules. Cell signaling such as those based on FcγR molecule activation involves localization of the relevant receptor molecules within a region of membrane domain referred to as lipid rafts. Lipid rafts are enriched with glycosphingolipid and cholesterol and several classes of upstream signal transducers including the Src family kinases. Upon cell stimulation various signaling molecules, adaptor proteins and the signaling kinases as well as phosphatases are recruited. Molecular assembly at lipid rafts is important for signal transduction.
A non-natural design strategy, combining different antigen specificities and increased avidity to provide better binding properties is the basis of bispecific therapeutic design. Bispecific antibodies or other forms of bispecific or multifunctional protein therapeutics are designed to mediate interactions between the target and a variety of effector cells [Muller & Kontermann (2010) BioDrugs 24(2):89-98]. Multispecific therapeutic molecules are engineered to redirect the Helper T-cells or other immune effector cells against specific target cells.
In another embodiment, the invention relates to a method for identifying Fc variant polypeptides in silico based on calculated binding affinities to FcγRIIa, FcγRIIb and/or FcγRIIIa. In another embodiment, the method further comprises calculating in silico electrostatics, solvation, packing, packing density, hydrogen binding, and entropic effects of said Fc variant polypeptides. In yet another embodiment, the method of the current invention further includes constructing the Fc variant polypeptides and expressing said polypeptides in the context of a therapeutic antibody and further expressing said antibody in mammalian cells. In still another embodiment the method of the current invention comprises constructing the Fc variant polypeptides identified in silico by site directed mutagenesis, PCR based mutagenesis, cassette mutagenesis or de novo synthesis.
Factors taken into account in the design of the synthetic Fc scaffold include in silica calculations for steric repulsion, change in buried interface area, relative contact density, relative solvation and electrostatic effect. All these matrices were used to arrive at an affinity score.
In one aspect, this application describes a molecular design for achieving exquisite FcγR selectivity profiles via the design of an asymmetric scaffold built on a heterodimeric Fc. This scaffold allows for asymmetric mutations in the CH2 domain to achieve a variety of novel selectivity profiles. Further, the scaffold has inherent features for the engineering of multifunctional (bi, tri, tetra or penta functional) therapeutic molecules.
In certain embodiments, the asymmetric scaffold is optimized for pH dependent binding properties to the neonatal Fc receptor (FcRn) to enable better recycling of the molecule and enhance its half life and related pharmacokinetic properties.
The asymmetric scaffold can be optimized for binding to the functionally relevant FcγRI receptor allotypes. FcγRI is a prominent marker on macrophages that are involved in chronic inflammatory disorders such as Rheumatoid Arthritis, Atopic Dermatitis, Psoriasis and a number of pulmonary diseases.
The asymmetric scaffold can be optimized for protein A binding. Protein A binding is often employed for separation and purification of antibody molecules. Mutations can be introduced in the asymmetric scaffold to avoid aggregation of the therapeutic during storage.
Therefore, it is specifically contemplated that the Fc variants of the invention may contain inter alia one or more additional amino acid residue substitutions, mutations and/or modifications which result in an antibody with preferred characteristics including but not limited to: increased serum half life, increase binding affinity, reduced immunogenicity, increased production, enhanced or reduced ADCC or CDC activity, altered glycosylation and/or disulfide bonds and modified binding specificity.
It is contemplated that the Fc variants of the invention may have other altered characteristics including increased in vivo half-lives (e.g., serum half-lives) in a mammal; in particular a human, increased stability in vivo (e.g., serum half-lives) and/or in vitro (e.g., shelf-life) and/or increased melting temperature (Tm), relative to a comparable molecule. In one embodiment, an Fc variant of the invention has an in vivo half-life of greater then 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. In another embodiment, an Fc variant of the invention has an in vitro half-live (e.g, liquid or powder formulation) of greater then 15 days, greater than 30 days, greater than 2 months, greater than 3 months, greater than 6 months, or greater than 12 months, or greater than 24 months, or greater than 36 months, or greater than 60 months.
It will also be appreciated by one skilled in the art that the Fc variants of the invention may have altered immunogenicity when administered to a subject. Accordingly, it is contemplated that the modified CH3 domain, which minimize the immunogenicity of the Fc variant are generally more desirable for therapeutic applications.
The Fc variants of the present invention may be combined with other Fc modifications, including but not limited to modifications that alter effector function. The invention encompasses combining an Fc variant of the invention with other Fc modifications to provide additive, synergistic, or novel properties in antibodies or Fc fusion proteins. Such modifications may be in the hinge, CH1, or CH2, (or CH3 provided it does not negatively alter the stability and purity properties of the present modified CH3 domains) domains or a combination thereof. It is contemplated that the Fc variants of the invention enhance the property of the modification with which they are combined. For example, if an Fc variant of the invention is combined with a mutant known to bind FcγRIIIA with a higher affinity than a comparable molecule comprising a wild type Fc region; the combination with a mutant of the invention results in a greater fold enhancement in FcγRIIIA affinity.
In one embodiment, the Fc variants of the present invention may be combined with other known Fc variants such as those disclosed in Duncan et al, 1988, Nature 332:563-564; Lund et al., 1991, J Immunol 147:2657-2662; Lund et al., 1992, Mol Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol Lett. 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett 54:101-104; Lund et al, 1996, Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol 164:4178-4184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol 166:2571-2575; Shields et al., 2001, J Bioi Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); U.S. Pat. Nos. 5,624,821; 5,885,573; 6,194,551; U.S. Patent Application Nos. 60/601,634 and 60/608,852; PCT Publication Nos. WO 00/42072 and WO 99/58572.
One skilled in the art will understand that the Fc variants of the invention may have altered Fc ligand (e.g., FcγR, C1q) binding properties (examples of binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (K0), dissociation and association rates (Kaff and Kan respectively), binding affinity and/or avidity) and that certain alterations are more or less desirable. It is well known in the art that the equilibrium dissociation constant (Ko) is defined as kofffkon. It is generally understood that a binding molecule (e.g., and antibody) with a low K0 is preferable to a binding molecule (e.g., and antibody) with a high K0. However, in some instances the value of the kon or koff may be more relevant than the value of the K0. One skilled in the art can determine which kinetic parameter is most important for a given antibody application. For example a modified CH3 and/or CH2 that enhances Fc binding to one or more positive regulators (e.g., FcγRIIIA) while leaving unchanged or even reducing Fc binding to the negative regulator FcγRIIB would be more advantageous for enhancing ADCC activity. Alternatively, a modified CH3 and/or CH2 that reduced binding to one or more positive regulator and/or enhanced binding to FcγRIIB would be advantageous for reducing ADCC activity. Accordingly, the ratio of binding affinities (e.g., equilibrium dissociation constants (K0)) can indicate if the ADCC activity of an Fc variant is enhanced or decreased. For example a decrease in the ratio of FcγRIIIA/FcγRIIB equilibrium dissociation constants (K0), will correlate with improved ADCC activity, while an increase in the ratio will correlate with a decrease in ADCC activity.
As part of the characterization of the Fc variants they were tested for their binding affinity to FcγRIIIA (CD16a) and FcγRIIB(CD32b) reported as a ratio in comparison to wild-type IgG1. (See, Example 4 and Table 5)1n this instance it was possible to evaluate the impact of the CH3 domain mutations on binding to these activating and inhibitory Fc receptors. In one embodiment, provided herein are isolated heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) greater than 70° C., wherein the heterodimer binding to CD16a is about the same as compared to wild-type homodimer. In certain embodiments the heterodimer binding to CD16a is increased as compared to wild-type homodimer. In an alternative embodiment, the heterodimer binding to CD16a is reduced as compared to wild-type homodimer.
In certain embodiments, provided herein are isolated heteromultimers comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) greater than 70° C., wherein the heterodimer binding to CD32b is about the same as compared to wild-type homodimer. In certain embodiments the heterodimer binding to CD32b is increased as compared to wild-type homodimer. In an alternative embodiment, the heterodimer binding to CD32b is reduced as compared to wild-type homodimer.
One of skill in the art will understand that instead of reporting the K0 of binding CD16a and CD32b as a ratio Fc variant to wild-type homodimer, the K0 could be reported as a ratio of Fc variant binding to CD16a to Fc variant binding to CD32b (data not shown). This ratio would provide an indication of the modified CH3 domain mutation on ADCC, either unchanged, increased to decreased compared to wild-type, described below in more detail.
The affinities and binding properties of the Fc variants of the invention for an FcγR are initially determined using in vitro assays (biochemical or immunological based assays) known in the art for determining Fc-FcγR interactions, i.e., specific binding of an Fc region to an FcγR including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays (See section entitled “Characterization and Functional Assays” infra) and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.
It is contemplated that the binding properties of the molecules of the invention are also characterized by in vitro functional assays for determining one or more FcγR mediator effector cell functions (See section entitled “Characterization and Functional Assays” infra). In certain embodiments, the molecules of the invention have similar binding properties in in vivo models (such as those described and disclosed herein) as those in in vitro based assays. However, the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.
The invention encompasses Fc variants that bind FcγRIIIA (CD16a) with increased affinity, relative to a comparable molecule. In a specific embodiment, the Fc variants of the invention bind FcγRIIIA with increased affinity and bind FcγRIIB (CD32b) with a binding affinity that is either unchanged or reduced, relative to a comparable molecule. In yet another embodiment, the Fc variants of the invention have a ratio of FcγRIIIA/FcγRIIB equilibrium dissociation constants (Ko) that is decreased relative to a comparable molecule.
Also encompassed by the present invention are Fc variants that bind FcγRIIIA (CD16a) with decreased affinity, relative to a comparable molecule. In a specific embodiment, the Fc variants of the invention bind FcγRIIIA with decreased affinity, relative to a comparable molecule and bind FcγRIIB with a binding affinity that is unchanged or increased, relative to a comparable molecule.
In one embodiment, the Fc variants bind with increased affinity to FcγRIIIA. In a specific embodiment, said Fc variants have affinity for FcγRIIIA that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold greater than that of a comparable molecule. In other embodiments, the Fc variants have an affinity for FcγRIIIA that is increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least SO %, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule.
In another embodiment, the Fc variant has an equilibrium dissociation constant (Ko) for an Fc ligand (e.g., FcγR, C1q) that is decreased between about 2 fold and 10 fold, or between about 5 fold and 50 fold, or between about 25 fold and 250 fold, or between about 100 fold and 500 fold, or between about 250 fold and 1000 fold relative to a comparable molecule.
In a another embodiment, said Fc variants have an equilibrium dissociation constant (Ko) for FcγRIIIA that is reduced by at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold, or at least 400 fold, or at least 600 fold, relative to a comparable molecule. In another embodiment, the Fc variants have an equilibrium dissociation constant (Ko) for FcγRIIIA that is reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule.
In one embodiment, the Fc variant binds to FcγRIIB with an affinity that is unchanged or reduced. In a specific embodiment, said Fc variants have affinity for FcγRIIB that is unchanged or reduced by at least 1 fold, or by at least 3 fold, or by at least 5 fold, or by at least 10 fold, or by at least 20 fold, or by at least 50 fold, or by at least 100 fold, relative to a comparable molecule. In other embodiments, the Fc variants have an affinity for FcγRIIB that is unchanged or reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule.
In another embodiment, the Fc variants have an equilibrium dissociation constant (Ko) for FcγRIIB that is unchanged or increased by at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least SO fold, or at least 90 fold, or at least 100 fold, or at least 200 fold relative to a comparable molecule. In another specific embodiment, the Fc variants have an equilibrium dissociation constant (Ko) for FcγRIIB that is unchanged or increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule.
In still another embodiment, the Fc variants bind FcγRIIIA with increased affinity, relative to a comparable molecule and bind FcγRIIB with an affinity that is unchanged or reduced, relative to a comparable molecule. In a specific embodiment, the Fc variants have affinity for FcγRIIIA that is increased by at least 1 fold, or by at least 3 fold, or by at least 5 fold, or by at least 10 fold, or by at least 20 fold, or by at least 50 fold, or by at least 100 fold, relative to a comparable molecule. In another specific embodiment, the Fc variants have affinity for FcγRIIB that is either unchanged or is reduced by at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 50 fold, or at least 100 fold, relative to a comparable molecule. In other embodiments, the Fc variants have an affinity for FcγRIIIA that is increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule and the Fc variants have an affinity for FcγRIIB that is either unchanged or is increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule.
In yet another embodiment, the Fc variants have a ratio of FcγRIIIA/FcγRIIB equilibrium dissociation constants (Ko) that is decreased relative to a comparable molecule. In a specific embodiment, the Fc variants have a ratio of FcγRIIIA/FcγRIIB equilibrium dissociation constants (Ko) that is decreased by at least 1 fold, or by at least 3 fold, or by at least 5 fold, or by at least 10 fold, or by at least 20 fold, or by at least 50 fold, or by at least 100 fold, relative to a comparable molecule. In another specific embodiment, the Fc variants have a ratio of FcγRIIIA/FcγRIIB equilibrium dissociation constants (Ko) that is decreased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule.
In another embodiment, the Fc variants bind FcγRIIIA with a decreased affinity, relative to a comparable molecule. In a specific embodiment, said Fc variants have affinity for FcγRIIIA that is reduced by at least 1 fold, or by at least 3 fold, or by at least 5 fold, or by at least 10 fold, or by at least 20 fold, or by at least 50 fold, or by at least 100 fold, relative to a comparable molecule. In other embodiments, the Fc variants have an affinity for FcγRIIIA that is decreased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule.
In still another embodiment, the Fc variants bind FcγRIIIA with decreased affinity and bind FcγRIIB with an affinity that is either unchanged or increased, relative to a comparable molecule. In a specific embodiment, the Fc variants have affinity for FcγRIIIA that is reduced by at least 1 fold, or by at least 3 fold, or by at least 5 fold, or by at least 10 fold, or by at least 20 fold, or by at least 50 fold, or by at least 100 fold relative to a comparable molecule. In another specific embodiment, the Fc variants have affinity for FcγRIIB that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 50 fold, or at least 100 fold, greater than that of a comparable molecule. In other embodiments, the Fc variants have an affinity for FcγRIIIA that is decreased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule and the Fc variants have an affinity for FcγRIIB that is increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule.
In still another embodiment, the Fc variants have an equilibrium dissociation constant (Ko) for FcγRIIIA that are increased by at least 1 fold, or by at least 3 fold, or by at least 5 fold or by at least 10 or by at least 20 fold, or by at least 50 fold when compared to that of a comparable molecule. In a specific embodiment, said Fc variants have equilibrium dissociation constant (Ko) for FcγRIIB that are decreased at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 50 fold or at least 100 fold, relative to a comparable molecule.
CH2 Variations for FcγR Selectivity
The Fc-FcγR protein-protein interaction in this complex indicates that the two chains in the Fc molecule interact with two distinct sites on the FcγR molecule. Although there is symmetry in the two heavy chains in the natural Fc molecules, the local FcγR environment around residues on one chain is different from the FcγR residues surrounding the same residue position on the opposite Fc chain. The two symmetry related positions interact with different selection of FcγR residues.
Given the asymmetry in the association of Fc to FcγR, concurrent mutations in chain A and B of the Fc molecule do not impact the interactions with FcγR in a symmetric manner. When introducing mutations to optimize interactions on one chain of the Fc with its local FcγR environment, in a homodimeric Fc structure, the corresponding mutation in the second chain may be favorable, unfavorable or non-contributing to the required FcγR binding and selectivity profile.
Using a structure and computation guided approach, asymmetric mutations are engineered in the two chains of the Fc to overcome these limitations of traditional Fc engineering strategies, which introduce the same mutations on both the chains of Fc. One can achieve better binding selectivity between the receptors if the two chains of Fc are optimized independently for enhanced binding to their corresponding face of the receptor molecule.
For instance, mutations at a particular position on one chain of the Fc can be designed to enhance selectivity to a particular residue, a positive design effort, while the same residue position can be mutated to unfavorably interact with its local environment in an alternate Fcγ receptor type, a negative design effort, hence achieving better selectivity between the two receptors. In certain embodiments, is provided a method for designing asymmetric amino acid modifications in the CH2 domain that selectively bind one Fc gamma receptor as compared to a different Fc gamma receptor (e.g., selectively bind FcgRIIIa instead of FcgRIIb). In other certain embodiments, is provided a method for the design of asymmetric amino acid modifications in the CH2 domain of a variant Fc heterodimer comprising amino acid modifications in the CH3 domain to promote heterodimer formation. In another embodiment, is provided a method to design selectivity for the different Fc gamma receptors based on a variant Fc heterodimer comprising asymmetric amino acid modifications in the CH2 domain. In yet another embodiment, is provided a method for designing asymmetric amino acid modifications that bias binding of the Fc gamma receptors to one face of the Fc molecule. In other certain embodiments, is provided a method for designing polarity drivers that bias the Fcgamma receptors to interact with only one face of the variant Fc heterodimer comprising asymmetric amino acid modifications in the CH2 domain.
The asymmetric design of mutations in the CH2 domain can be tailored to recognize the FcγR on one face of the Fc molecule. This constitutes the productive face of the asymmetric Fc scaffold while the opposite face presents wild type like interaction propensity without the designed selectivity profile and can be considered a non-productive face. A negative design strategy can be employed to introduce mutations on the non-productive face to block FcγR interactions to this side of the asymmetric Fc scaffold, there by forcing the desired interaction tendencies to the Fcγ receptors.
The present invention also relates to fusion polypeptides comprising a binding domain fused to an Fc region, wherein the Fc region comprising a modified CH3 domain, comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) greater than 70° C. It is specifically contemplated that molecules comprising a heterodimer comprising a modified CH3 domain may be generated by methods well known to one skilled in the art. Briefly, such methods include but are not limited to, combining a variable region or binding domain with the desired specificity (e.g., a variable region isolated from a phage display or expression library or derived from a human or non-human antibody or a binding domain of a receptor) with a variant Fc heterodimers. Alternatively, one skilled in the art may generate a variant Fc heterodimer by modifying the CH3 domain in the Fc region of a molecule comprising an Fc region (e.g., an antibody).
In one embodiment, the Fc variants are antibodies or Fc fusion proteins. In a specific embodiment, the invention provides antibodies comprising an Fc region comprising a modified CH3 domain, comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) greater than 70° C. Such antibodies include IgG molecules that naturally comprise an Fc region containing a CH3 domain that can be modified to generate an Fc variant, or antibodies derivatives that have been engineered to contain an Fc region comprising a modified CH3 domain. Fc variants of the invention includes any antibody molecule that binds, preferably, specifically (i.e., competes off non-specific binding as determined by immunoassays well known in the art for assaying specific antigen-antibody binding) an antigen which comprises an Fc region incorporating a modified CH3 domain. Such antibodies include, but are not limited to, polyclonal, monoclonal, mono-specific, bi-specific, multi-specific, human, humanized, chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)2 fragments, disulfide-linked Fvs, and fragments containing either a VL or VH domain or even a complementary determining region (CDR) that specifically binds an antigen, in certain cases, engineered to contain or fused to a variant Fc heterodimer.
“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted antibody bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to bind specifically to an antigen-healing target cell and subsequently kill the target cell with cytotoxins. Specific high-affinity IgG antibodies directed to the surface of target cells “arm” the cytotoxic cells and are absolutely required for such killing. Lysis of the target cell is extracellular, requires direct cell-to-cell contact, and does not involve complement.
The ability of any particular antibody to mediate lysis of the target cell by ADCC can be assayed. To assess ADCC activity an antibody of interest is added to target cells in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cytolysis is generally detected by the release of label (e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Specific examples of in vitro ADCC assays are described in Wisecarver et al., 1985, 79:277; Bruggemann et al., 1987, J Exp Med 166:1351; Wilkinson et al., 2001, J Immunol Methods 258:183; Patel et al., 1995 J Immunol Methods 184:29 and herein (see section entitled “Characterization and Functional Assays” infra). Alternatively, or additionally, ADCC activity of the antibody of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, PNAS USA95:652.
It is contemplated that the Fc variants of the invention are characterized by in vitro functional assays for determining one or more FcγR mediator effector cell functions. In specific embodiments, the molecules of the invention have similar binding properties and effector cell functions in in vivo models (such as those described and disclosed herein) as those in in vitro based assays However, the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.
The present invention further provides Fc variants with enhanced CDC function. In one embodiment, the Fc variants have increased CDC activity. In one embodiment, the Fc variants have CDC activity that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 10 fold, or at least 50 fold, or at least 100 fold greater than that of a comparable molecule. In another embodiment, the Fc variants bind C1q with an affinity that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 50 fold, or at least 100 fold, greater than that of a comparable molecule. In yet another embodiment, the Fc variants have CDC activity that is increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule. In a specific embodiment, the Fc variants of the invention bind C1q with increased affinity; have enhanced CDC activity and specifically bind to at least one antigen.
The present invention also provides Fc variants with reduced CDC function. In one embodiment, the Fc variants have reduced CDC activity. In one embodiment, the Fc variants have CDC activity that is at least 2 fold, or at least 3 fold, or at least 5 fold or at least 10 fold or at least 50 fold or at least 100 fold less than that of a comparable molecule. In another embodiment, an Fc variant binds C1q with an affinity that is reduced by at least 1 fold, or by at least 3 fold, or by at least 5 fold, or by at least 10 fold, or by at least 20 fold, or by at least 50 fold, or by at least 100 fold, relative to a comparable molecule. In another embodiment, the Fc variants have CDC activity that is decreased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%, relative to a comparable molecule. In a specific embodiment, Fc variants bind to C1q with decreased affinity have reduced CDC activity and specifically bind to at least one antigen.
In some embodiments, the Fc variants comprise one or more engineered glycoforms, i.e., a carbohydrate composition that is covalently attached to a molecule comprising an Fc region. Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example 13(1,4)-N-acetylglucosaminyltransferase III (GnTI11), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Bioi Chem 277:26733-26740; Shinkawa et al., 2003, J Bioi Chem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S. Ser. Nos. 10/277,370; 10/113,929; PCTWO 00/61739A1; PCTWO 01/292246A1; PCTWO 02/311140A1; PCTWO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland). See, e.g., WO 00061739; EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.
It is contemplated that Fc variants include antibodies comprising a variable region and a heterodimer Fc region, wherein the heterodimer Fc region comprises a modified CH3 domain comprising amino acid mutations to promote heterodimer formation with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) greater than 70° C. The Fc variants which are antibodies may be produced “de novo” by combing a variable domain, of fragment thereof, that specifically binds at least one antigen with a heterodimer Fc region comprising a modified CH3 domain. Alternatively, heterodimer Fc variants may be produced by modifying the CH3 domain of an Fc region containing antibody that binds an antigen.
Antibodies of the invention may include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, monospecific antibodies, multispecific antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain FvFcs (scFvFc), single-chain Fvs (scFv), and anti-idiotypic (anti-Id) antibodies. In particular, antibodies used in the methods of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
Antibodies of the invention may be from any animal origin including birds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken). In a specific embodiment, the antibodies are human or humanized monoclonal antibodies, in particular bi-specific monoclonal antibodies. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from mice that express antibodies from human genes.
Antibodies like all polypeptides have an Isoelectric Point (pi), which is generally defined as the pH at which a polypeptide carries no net charge. It is known in the art that protein solubility is typically lowest when the pH of the solution is equal to the isoelectric point (pi) of the protein. It is possible to optimize solubility by altering the number and location of ionizable residues in the antibody to adjust the pI. For example the pi of a polypeptide can be manipulated by making the appropriate amino acid substitutions (e.g., by substituting a charged amino acid such as a lysine, for an uncharged residue such as alanine). Without wishing to be bound by any particular theory, amino acid substitutions of an antibody that result in changes of the pi of said antibody may improve solubility and/or the stability of the antibody. One skilled in the art would understand which amino acid substitutions would be most appropriate for a particular antibody to achieve a desired pI. The pi of a protein may be determined by a variety of methods including but not limited to, isoelectric focusing and various computer algorithms (see for example Bjellqvist et al., 1993, Electrophoresis 14:1023). In one embodiment, the pi of the Fc variants of the invention is between pH 6.2 and pH 8.0. In another embodiment, the pi of the antibodies of the invention is between pH 6.8 and pH 7.4. In one embodiment, substitutions resulting in alterations in the pi of the Fc variant of the invention will not significantly diminish its binding affinity for an antigen. It is contemplated that the modified CH3 domain with an increased stability may also result in a change in the pI. In one embodiment, variant Fc heterodimers are specifically chosen to effect both the increased stability and purity and, any desired change in pI.
Antibodies of the invention may be monospecific, bispecific, trispecific or have greater multispecificity. Multispecific antibodies may specifically bind to different epitopes of desired target molecule or may specifically bind to both the target molecule as well as a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., International Publication Nos. WO 94/04690; WO 93/17715; WO 92/08802; WO 91/00360; and WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920 and 5,601,819 and Kostelny et al., 1992, J. Immunol. 148:1547).
Various embodiments of multifunctional targeting molecules can be designed on the basis of this asymmetric scaffold as shown in
Multispecific antibodies have binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by the instant invention. Examples of BsAbs include without limitation those with one arm directed against a tumor cell antigen and the other arm directed against a cytotoxic molecule, or both arms are directed again two different tumor cell antigens, or both arms are directed against two different soluable ligands, or one arm is directed against a soluable ligand and the other arm is directed against a cell surface receptor, or both arms are directed against two different cell surface receptors. Methods for making bispecific antibodies are known in the art.
According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion may be with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is contemplated that the first heavy-chain constant region (CH1) containing the site necessary for light chain binding is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. See, Example 1 and Table 2. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when, the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
Antibodies with more than two valencies incorporating modified CH3 domains and resulting Fc heterodimers of the invention are contemplated. For example, trispecific antibodies can be prepared. See, e.g., Tutt et al. J. Immunol. 147: 60 (1991).
Antibodies of the present invention also encompass those that have half-lives (e.g., serum half-lives) in a mammal, (e.g., a human), of greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-lives of the antibodies of the present invention in a mammal, (e.g., a human), results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus, reduces the frequency of the administration of said antibodies or antibody fragments and/or reduces the concentration of said antibodies or antibody fragments to be administered. Antibodies having increased in vitro half-lives can be generated by techniques known to those of skill in the art. For example, antibodies with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., International Publication Nos. WO 97/34631; WO 04/029207; U.S. Pat. No. 6,737,056 and U.S. Patent Publication No. 2003/0190311).
In a specific embodiment the variant Fc heterodimer comprising the modified CH3 domain is a multi-specific antibody (referred to herein as an antibody of the invention), the antibody of the invention specifically binds an antigen of interest. In particular the antibody of the invention is a bi-specific antibody. In one embodiment, an antibody of the invention specifically binds a polypeptide antigen. In another embodiment, an antibody of the invention specifically binds a nonpolypeptide antigen. In yet another embodiment, administration of an antibody of the invention to a mammal suffering from a disease or disorder can result in a therapeutic benefit in that mammal.
Virtually any molecule may be targeted by and/or incorporated into a variant Fc heterodimer construct provided herein (e.g., antibodies, Fc fusion proteins) including, but not limited to, the following list of proteins, as well as subunits, domains, motifs and epitopes belonging to the following list of proteins: renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VII, factor VIIIC, factor IX, tissue factor (TF), and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-alpha); a serum albumin such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors such as, for example, EGFR, VEGFR; interferons such as alpha interferon (a-IFN), beta interferon (13-IFN) and gamma interferon (y-IFN); protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor; platelet-derived growth factor (PDGF); fibroblast growth factor such as AFGF and PFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-1, TGF-2, TGF-3, TGF-4, or TGF-5; insulin-like growth factor-1 and -II (IGF-1 and IGF-11); des (1-3)-IGF-1 (brain IGF-1), insulin-like growth factor binding proteins; CD proteins such as C02, CD3, CD4, CDS, CD11a, CD14, CD1S, CD19, CD20, CD22, CD23, CD25, CD33, CD34, CD40, CD40L, CD52, CD63, CD64, COSO and CD147; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), such as M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-13; TNFa, superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS envelope, e.g., gp120; transport proteins; homing receptors; addressins; regulatory proteins; cell adhesion molecules such as LFA-1, Mac 1, p150.95, VLA-4, ICAM-1, ICAM-3 and VCAM, a4/p7 integrin, and (Xv/p3 integrin including either a or subunits thereof, integrin alpha subunits such as CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, alpha?, alphaS, alpha9, alphaD, CD11a, CD11b, CD51, CD11c, CD41, alphaIib, alphaiELb; integrin beta subunits such as, CD29, CD 1S, CD61, CD104, beta5, beta6, beta7 and betaS; Integrin subunit combinations including but not limited to, aVI33, aVI35 and a4137; a member of an apoptosis pathway; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mp1 receptor; CTLA-4; protein C; an Eph receptor such as EphA2, EphA4, EphB2, etc.; a Human Leukocyte Antigen (HLA) such as HLA-DR; complement proteins such as complement receptor CR1, C1Rq and other complement factors such as C3, and C5; a glycoprotein receptor such as GpIba, GPIIb/IIIa and CD200; and fragments of any of the above-listed polypeptides.
Also provided are antibodies of the invention that specifically bind cancer antigens including, but not limited to, ALK receptor (pleiotrophin receptor), pleiotrophin, KS 1/4 pan-carcinoma antigen; ovarian carcinoma antigen (CA125); prostatic acid phosphate; prostate specific antigen (PSA); melanoma-associated antigen p97; melanoma antigen gp75; high molecular weight melanoma antigen (HMW-MAA); prostate specific membrane antigen; carcinoembryonic antigen (CEA); polymorphic epithelial mucin antigen; human milk fat globule antigen; colorectal tumor-associated antigens such as: CEA, TAG-72, CO17-1A, GICA 19-9, CTA-1 and LEA; Burkitt's lymphoma antigen-3S.13; CD19; human B-lymphoma antigen-CD20; CD33; melanoma specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3; tumor-specific transplantation type cell-surface antigen (TSTA); virally-induced tumor antigens including T-antigen, DNA tumor viruses and Envelope antigens of RNA tumor viruses; oncofetal antigen-alpha-fetoprotein such as CEA of colon, 5T4 oncofetal trophoblast glycoprotein and bladder tumor oncofetal antigen; differentiation antigen such as human lung carcinoma antigens L6 and L20; antigens of fibrosarcoma; human leukemia T cell antigen-Gp37; neoglycoprotein; sphingolipids; breast cancer antigens such as EGFR (Epidermal growth factor receptor); NY-BR-16; NY-BR-16 and HER2 antigen (p185HER2); polymorphic epithelial mucin (PEM); malignant human lymphocyte antigen-APO-1; differentiation antigen such as I antigen found in fetal erythrocytes; primary endoderm I antigen found in adult erythrocytes; preimplantation embryos; l(Ma) found in gastric adenocarcinomas; M18, M39 found in breast epithelium; SSEA-1 found in myeloid cells; VEP8; VEP9; Myl; Va4-D5; D156-22 found in colorectal cancer; TRA-1-85 (blood group H); SCP-1 found in testis and ovarian cancer; C14 found in colonic adenocarcinoma; F3 found in lung adenocarcinoma; AH6 found in gastric cancer; Y hapten; Ley found in embryonal carcinoma cells; TL5 (blood group A); EGF receptor found in A431 cells; E1 series (blood group B) found in pancreatic cancer; FC10.2 found in embryonal carcinoma cells; gastric adenocarcinoma antigen; C0-514 (blood group Lea) found in Adenocarcinoma; NS-10 found in adenocarcinomas; C0-43 (blood group Leb); G49 found in EGF receptor of A431 cells; MH2 (blood group ALeb/Ley) found in colonic adenocarcinoma; 19.9 found in colon cancer; gastric cancer mucins; Ty found in myeloid cells; R24 found in melanoma; 4.2, Go3, D1.1, OFA-1, GM2.OFA-2, G02, and M1:22:25:8 found in embryonal carcinoma cells and SSEA-3 and SSEA-4 found in 4 to 8-cell stage embryos; Cutaneous Tcell Lymphoma antigen; MART-1 antigen; Sialy Tn (STn) antigen; Colon cancer antigen NY-C0-45; Lung cancer antigen NY-LU-12 valiant A; Adenocarcinoma antigen ART1; Paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen); Neuro-oncological ventral antigen 2 (NOVA2); Hepatocellular carcinoma antigen gene 520; TUMOR-ASSOCIATED ANTIGEN C0-029; Tumor-associated antigens MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4-a, MAGE-4-b and MAGE-X2; Cancer-Testis Antigen (NY-EOS-1) and fragments of any of the above-listed polypeptides.
In certain embodiments, the heteromultimer described herein, comprises at least one therapeutic antibody. In some embodiments, the therapeutic antibody binds a cancer target antigen. In an embodiment, the therapeutic antibody may be one of is selected from the group consisting of abagovomab, adalimumab, alemtuzumab, aurograb, bapineuzumab, basiliximab, belimumab, bevacizumab, briakinumab, canakinumab, catumaxomab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, galiximab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lumiliximab, mepolizumab, motavizumab, muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab, ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab, reslizumab, rituximab, teplizumab, tocilizumab/atlizumab, tositumomab, trastuzumab, Proxinium™, Rencarex™, ustekinumab, zalutumumab, and any other antibodies.
Antibodies of the invention include derivatives that are modified (i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment). For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
Antibodies or fragments thereof with increased in vivo half-lives can be generated by attaching polymer molecules such as high molecular weight polyethyleneglycol (PEG) to the antibodies or antibody fragments. PEG can be attached to the antibodies or antibody fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of said antibodies or antibody fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography.
Further, antibodies can be conjugated to albumin in order to make the antibody or antibody fragment more stable in vivo or have a longer half life in vivo. The techniques are well known in the art, see e.g., International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP 413,622. The present invention encompasses the use of antibodies or fragments thereof conjugated or fused to one or more moieties, including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.
The present invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, for example, to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. For example, antibodies may be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating the antibodies to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., International publication No. WO 93/21232; European Patent No. EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452.
The present invention further includes compositions comprising heterologous proteins, peptides or polypeptides fused or conjugated to antibody fragments. For example, the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)2fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragment thereof. Methods for fusing or conjugating polypeptides to antibody portions are well known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851 and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vii et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341.
Additional fusion proteins, e.g. of antibodies that specifically bind an antigen (e.g., supra), may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252 and 5,837,458, and Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2): 76-82; Hansson, et al., 1999, J. Mol. Bioi. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2): 308-313. Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. One or more portions of a polynucleotide encoding an antibody or antibody fragment, which portions specifically bind to an antigen may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
The present invention further encompasses uses of variant Fc heterodimers or fragments thereof conjugated to a therapeutic agent.
An antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include ribonuclease, monomethylauristatin E and F, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). A more extensive list of therapeutic moieties can be found in PCT publications WO 03/075957.
Further, an antibody or fragment thereof may be conjugated to a therapeutic agent or drug moiety that modifies a given biological response. Therapeutic agents or drug moieties are not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, Onconase (or another cytotoxic RNase), pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, 13-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-a, TNF-13, AIM I (see, International Publication No. WO 97/33899), AIM II (see, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., 1994, J. Immunol., 6:1567), and VEGI (see, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, a biological response modifier such as, for example, a lymphokine (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”)), or a growth factor (e.g., growth hormone (“GH”)).
Moreover, an antibody can be conjugated to therapeutic moieties such as a radioactive materials or macrocyclic chelators useful for conjugating radiometal ions (see above for examples of radioactive materials). In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule. Such linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4:2483; Peterson et al., 1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Bioi. 26:943.
Methods for fusing or conjugating antibodies to polypeptide moieties are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851 and 5,112,946; EP 307,434; EP 367,166; PCT Publications WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, PNAS USA 88:10535; Zheng et al., 1995, J Immunol 154:5590; and Vii et al., 1992, PNAS USA 89:11337. The fusion of an antibody to a moiety does not necessarily need to be direct, but may occur through linker sequences. Such linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res 4:2483; Peterson et al., 1999, Bioconjug Chem 10:553; Zimmerman et al., 1999, Nucl Med Biol 26:943; Garnett, 2002, Adv Drug Deliv Rev 53:171.
Recombinant expression of an Fc variant, derivative, analog or fragment thereof, (e.g., an antibody or fusion protein of the invention), requires construction of an expression vector containing a polynucleotide that encodes the Fe variant (e.g., antibody, or fusion protein). Once a polynucleotide encoding an Fc variant (e.g., antibody, or fusion protein) has been obtained, the vector for the production of the Fc variant (e.g., antibody, or fusion protein) may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an Fc variant (e.g., antibody, or fusion protein) encoding nucleotide sequence are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing Fc variant (e.g., antibody, or fusion protein) coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an Fc variant of the invention, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication No. WO 86/05807; International Publication No. WO 89/01036; and U.S. Pat. No. 5,122,464 and the variable domain of the antibody, or a polypeptide for generating an Fc variant may be cloned into such a vector for expression of the full length antibody chain (e.g. heavy or light chain), or complete Fc variant comprising a fusion of a non-antibody derived polypeptide and an Fc region incorporating at least the modified CH3 domain.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an Fc variant of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an Fc variant of the invention, operably linked to a heterologous promoter. In specific embodiments for the expression of Fc variants comprising double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the Fc variants of the invention (e.g., antibody or fusion protein molecules) (see, e.g., U.S. Pat. No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an Fc variant of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing Fc variant coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing Fc variant coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing Fc variant coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing Fc variant coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In certain embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells, are used for the expression of an Fc variant, which is a recombinant antibody or fusion protein molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio/Technology 8:2). In a specific embodiment, the expression of nucleotide sequences encoding an Fc variant of the invention (e.g., antibody or fusion protein) is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the Fc variant (e.g., antibody or fusion protein) being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an Fc variant, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the Fc variant coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a lac Z-fusion protein is produced; piN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Bioi. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The Fc variant (e.g., antibody or fusion protein) coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the Fc variant (e.g., antibody or fusion protein) coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the Fc variant (e.g., antibody or fusion protein) in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:516-544).
The expression of an Fc variant (e.g., antibody or fusion protein) may be controlled by any promoter or enhancer element known in the art. Promoters which may be used to control the expression of the gene encoding an Fc variant (e.g., antibody or fusion protein) include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), the tetracycline (Tet) promoter (Gossen et al., 1995, Proc. Nat. Acad. Sci. USA 89:5547-5551); prokaryotic expression vectors such as the 13-lactamase promoter (Villa-Kamaroff et al, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25; see also “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94); plant expression vectors comprising the nopaline synthetase promoter region (Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Bioi. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Bioi. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Bioi. 5:1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286); neuronal-specific enolase (NSE) which is active in neuronal cells (Morelli et al., 1999, Gen. Virol. 80:571-83); brain-derived neurotrophic factor (BDNF) gene control region which is active in neuronal cells (Tabuchi et al., 1998, Biochem. Biophysic. Res. Com. 253:818-823); glial fibrillary acidic protein (GFAP) promoter which is active in astrocytes (Gomes et al., 1999, Braz J Med Bioi Res 32(5): 619-631; Morelli et al., 1999, Gen. Virol. 80:571-83) and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).
Expression vectors containing inserts of a gene encoding an Fc variant of the invention (e.g., antibody or fusion protein) can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of “marker” gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a gene encoding a peptide, polypeptide, protein or a fusion protein in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted gene encoding the peptide, polypeptide, protein or the fusion protein, respectively. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain “marker” gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a nucleotide sequence encoding an antibody or fusion protein in the vector. For example, if the nucleotide sequence encoding the Fc variant (e.g., antibody or fusion protein) is inserted within the marker gene sequence of the vector, recombinants containing the gene encoding the antibody or fusion protein insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the gene product (e.g., antibody or fusion protein) expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the fusion protein in in vitro assay systems, e.g., binding with anti-bioactive molecule antibody.
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered fusion protein may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins). Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system will produce an unglycosylated product and expression in yeast will produce a glycosylated product. Eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript (e.g., glycosylation, and phosphorylation) of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MOCK, 293, 3T3, WI38, NSO, and in particular, neuronal cell lines such as, for example, SK-N-AS, SK-N-FI, SK-N-DZ human neuroblastomas (Sugimoto et al., 1984, J. Natl. Cancer Inst. 73: 51-57), SK-N-SH human neuroblastoma (Biochim. Biophys. Acta, 1982, 704: 450-460), Daoy human cerebellar medulloblastoma (He et al., 1992, Cancer Res. 52: 1144-1148) DBTRG-05MG glioblastoma cells (Kruse et al., 1992, In Vitro Cell. Dev. Bioi. 28A: 609-614), IMR-32 human neuroblastoma (Cancer Res., 1970, 30: 2110-2118), 1321N1 human astrocytoma (Proc. Natl. Acad. Sci. USA, 1977, 74: 4816), MOG-G-CCM human astrocytoma (Br. J. Cancer, 1984, 49: 269), U87MG human glioblastoma-astrocytoma (Acta Pathol. Microbial. Scand., 1968, 74: 465-486), A172 human glioblastoma (Oiopade et al., 1992, Cancer Res. 52: 2523-2529), C6 rat glioma cells (Benda et al., 1968, Science 161: 370-371), Neuro-2a mouse neuroblastoma (Proc. Natl. Acad. Sci. USA, 1970, 65: 129-136), NB41A3 mouse neuroblastoma (Proc. Natl. Acad. Sci. USA, 1962, 48: 1184-1190), SCP sheep choroid plexus (Bolin et al., 1994, J. Virol. Methods 48: 211-221), G355-5, PG-4 Cat normal astrocyte (Haapala et al., 1985, J. Virol. 53: 827-833), Mpf ferret brain (Trowbridge et al., 1982, In Vitro 18: 952-960), and normal cell lines such as, for example, CTX TNA2 rat normal cortex brain (Radany et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6467-6471) such as, for example, CRL7030 and Hs578Bst. Furthermore, different vector/host expression systems may effect processing reactions to different extents.
For long-term, high-yield production of recombinant proteins, stable expression is often preferred. For example, cell lines that stably express an Fc variant of the invention (e.g., antibody or fusion protein) may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched medium, and then are switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines that express an Fc variant that specifically binds to an Antigen. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the activity of an Fc variant (e.g., a polypeptide or a fusion protein) that specifically binds to an antigen.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Bioi. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147) genes.
Once an Fc variant (e.g., antibody, or a fusion protein) of the invention has been produced by recombinant expression, it may be purified by any method known in the art for purification of a protein, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
The Fc variant is generally recovered from the culture medium as a secreted polypeptide, although it also may be recovered from host cell lysate when directly produced without a secretory signal. If the Fc variant is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100).
When the Fc variant is produced in a recombinant cell other than one of human origin, it is completely free of proteins or polypeptides of human origin. However, it is necessary to purify the Fc variant from recombinant cell proteins or polypeptides to obtain preparations that are substantially homogeneous as to the Fc variant. As a first step, the culture medium or lysate is normally centrifuged to remove particulate cell debris.
Fc heterodimers having antibody constant domains can be conveniently purified by hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, with affinity chromatography being the preferred purification technique. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin Sepharose, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the polypeptide to be recovered. The suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain that is used. Protein A can be used to purify immunoglobulin Fc regions that are based on human y1, y2, or y4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human y3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. The conditions for binding an immuno adhesin to the protein A or G affinity column are dictated entirely by the characteristics of the Fc domain; that is, its species and isotype. Generally, when the proper ligand is chosen, efficient binding occurs directly from unconditioned culture fluid. Bound variant Fc heterodimers can be efficiently eluted either at acidic pH (at or above 3.0), or in a neutral pH buffer containing a mildly chaotropic salt. This affinity chromatography step can result in a variant Fc heterodimer preparation that is >95% pure.
The expression levels of an Fc variant (e.g., antibody or fusion protein) can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). For example, when a marker in the vector system expressing an antibody or fusion protein is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody or fusion protein will also increase (Crouse et al., 1983, Mol. Cell. Bioi. 3:257).
The host cell may be co-transfected with two expression vectors of the invention. For example, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers, which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, a fusion protein or both heavy and light chain polypeptides. The coding sequences for the fusion protein or heavy and light chains may comprise eDNA or genomic DNA.
Characterization and Functional Assays
Fc variants (e.g., antibodies or fusion proteins) of the present invention may be characterized in a variety of ways. In one embodiment, purity of the variant Fc heterodimers is assessed using techniques well known in the art including, but not limited to, SDS-PAGE gels, western blots, densitometry or mass spectrometry. Protein stability can be characterized using an array of techniques, not limited to, size exclusion chromatography, UV Visible and CD spectroscopy, mass spectroscopy, differential light scattering, bench top stability assay, freeze thawing coupled with other characterization techniques, differential scanning calorimetry, differential scanning fluorimetry, hydrophobic interaction chromatorgraphy, isoelectric focusing, receptor binding assays or relative protein expression levels. In en exemplary embodiment, stability of the variant Fc heterodimers is assessed by melting temperature of the modified CH3 domain, as compared to wild-type CH3 domain, using techniques well known in the art such as Differential Scanning calorimetryor differential scanning flourimetry.
Fc variants of the present invention may also be assayed for the ability to specifically bind to a ligand, (e.g., FcγRIIIA, FcγRIIB, C1q). Such an assay may be performed in solution (e.g., Houghten, Bio/Techniques, 13:412-421, 1992), on beads (Lam, Nature, 354:82-84, 1991, on chips (Fodor, Nature, 364:555-556, 1993), on bacteria (U.S. Pat. No. 5,223,409) on plasmids (Cull et al., Proc. Natl. Acad. Sci. USA, 89:1865-1869, 1992) or on phage (Scott and Smith, Science, 249:386-390, 1990; Devlin, Science, 249:404-406, 1990; Cwirla et al., Proc. Natl. Acad. Sci. USA, 87:6378-6382, 1990; and Felici, J. Mol. Bioi., 222:301-310, 1991). Molecules that have been identified to specifically bind to a ligand, (e.g., FcγRIIIA, FcγRIIB, C1q or to an antigen) can then be assayed for their affinity for the ligand.
Fc variants of the invention may be assayed for specific binding to a molecule such as an antigen (e.g., cancer antigen and cross-reactivity with other antigens) or a ligand (e.g., FcγR) by any method known in the art. Immunoassays which can be used to analyze specific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).
The binding affinity of the Fc variants of the present invention to a molecule such as an antigen or a ligand, (e.g., FcγR) and the off-rate of the interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled ligand, such as FcγR (e.g., 3H or 1251 with a molecule of interest (e.g., Fc variants of the present invention) in the presence of increasing amounts of unlabeled ligand, such as FcγR, and the detection of the molecule bound to the labeled ligand. The affinity of the molecule of the present invention for the ligand and the binding off-rates can be determined from the saturation data by scatchard analysis.
The kinetic parameters of an Fc variant may also be determined using any surface plasmon resonance (SPR) based assays known in the art (e.g., BIAcore kinetic analysis). For a review of SPR-based technology see Mullet et al., 2000, Methods 22: 77-91; Dong et al., 2002, Review in Mol. Biotech., 82: 303-23; Fivash et al., 1998, Current Opinion in Biotechnology 9: 97-101; Rich et al., 2000, Current Opinion in Biotechnology 11:54-61. Additionally, any of the SPR instruments and SPR based methods for measuring protein-protein interactions described in U.S. Pat. Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215; 6,268,125 are contemplated in the methods of the invention.
Fluorescence activated cell sorting (FACS), using any of the techniques known to those skilled in the art, can be used for characterizing the binding of Fc variants to a molecule expressed on the cell surface (e.g., FcγRIIIA, FcγRIIB). Flow sorters are capable of rapidly examining a large number of individual cells that contain library inserts (e.g., 10-100 million cells per hour) (Shapiro et al., Practical Flow, Cytometry, 1995). Flow cytometers for sorting and examining biological cells are well known in the art. Known flow cytometers are described, for example, in U.S. Pat. Nos. 4,347,935; 5,464,581; 5,483,469; 5,602,039; 5,643,796; and 6,211,477. Other known flow cytometers are the FACS Vantage™ system manufactured by Becton Dickinson and Company, and the COPAS™ system manufactured by Union Biometrica.
The Fc variants of the invention can be characterized by their ability to mediate FcγR-mediated effector cell function. Examples of effector cell functions that can be assayed include, but are not limited to, antibody-dependent cell mediated cytotoxicity (ADCC), phagocytosis, opsonization, opsonophagocytosis, C1q binding, and complement dependent cell mediated cytotoxicity (CDC). Any cell-based or cell free assay known to those skilled in the art for determining effector cell function activity can be used (For effector cell assays, see Perussia et al., 2000, Methods Mol. Bioi. 121: 179-92; Baggiolini et al., 1998 Experientia, 44(10): 841-8; Lehmann et al., 2000 J. Immunol. Methods, 243(1-2): 229-42; Brown E J. 1994, Methods Cell Bioi., 45: 147-64; Munn et al., 1990 J. Exp. Med., 172: 231-237, Abdul-Majid et al., 2002 Scand. J. Immunol. 55: 70-81; Ding et al., 1998, Immunity 8:403-411).
In particular, the Fc variants of the invention can be assayed for FcγR-mediated ADCC activity in effector cells, (e.g., natural killer cells) using any of the standard methods known to those skilled in the art (See e.g., Perussia et al., 2000, Methods Mol. Bioi. 121: 179-92). An exemplary assay for determining ADCC activity of the molecules of the invention is based on a 51Cr release assay comprising of: labeling target cells with [51Cr]Na2 CrO4 (this cell-membrane permeable molecule is commonly used for labeling since it binds cytoplasmic proteins and although spontaneously released from the cells with slow kinetics, it is released massively following target cell necrosis); osponizing the target cells with the Fc variants of the invention; combining the opsonized radiolabeled target cells with effector cells in a microtitre plate at an appropriate ratio of target cells to effector cells; incubating the mixture of cells for 16-18 hours at 3r C.; collecting supernatants; and analyzing radioactivity. The cytotoxicity of the molecules of the invention can then be determined, for example using the following formula: % lysis=(experimental cpm−target leak cpm)/(detergent lysis cpm−target leak cpm)×100%. Alternatively, % lysis=(ADCC−AICC)/(maximum release−spontaneous release). Specific lysis can be calculated using the formula: specific lysis=% lysis with the molecules of the invention−% lysis in the absence of the molecules of the invention. A graph can be generated by varying either the target:effector cell ratio or antibody concentration.
Method to characterize the ability of the Fc variants to bind C1q and mediate complement dependent cytotoxicity (CDC) are well known in the art. For example, to determine C1q binding, a C1q binding ELISA may be performed. An exemplary assay may comprise the following: assay plates may be coated overnight at 4 C with polypeptide variant or starting polypeptide (control) in coating buffer. The plates may then be washed and blocked. Following washing, an aliquot of human C1q may be added to each well and incubated for 2 hrs at room temperature. Following a further wash, 100 uL a sheep anti-complement C1q peroxidase conjugated antibody may be added to each well and incubated for 1 hour at room temperature. The plate may again be washed with wash buffer and 100 ul of substrate buffer containing OPD (0-phenylenediamine dihydrochloride (Sigma)) may be added to each well. The oxidation reaction, observed by the appearance of a yellow color, may be allowed to proceed for 30 minutes and stopped by the addition of 100 ul of 4.5 NH2 SO4. The absorbance may then read at (492-405) nm.
To assess complement activation, a complement dependent cytotoxicity (CDC) assay may be performed, (e.g. as described in Gazzano-Santoro et al., 1996, J. Immunol. Methods 202:163). Briefly, various concentrations of Fc variant and human complement may be diluted with buffer. Cells which express the antigen to which the Fc variant binds may be diluted to a density of about 1×106 cells/mi. Mixtures of the Fc variant, diluted human complement and cells expressing the antigen may be added to a flat bottom tissue culture 96 well plate and allowed to incubate for 2 hrs at 37 C. and 5% CO2 to facilitate complement mediated cell lysis. 50 uL alamar blue (Accumed International) may then be added to each well and incubated overnight at 37 C. The absorbance is measured using a 96-well fluorometer with excitation at 530 nm n and emission at 590 nm. The results may be expressed in relative fluorescence units (RFU). The sample concentrations may be computed from a standard curve and the percent activity, relative to a comparable molecule (i.e., a molecule comprising an Fc region with an unmodified or wild type CH3 domain) is reported for the Fc variant of interest.
Complement assays may be performed with guinea pig, rabbit or human serum. Complement lysis of target cells may be detected by monitoring the release of intracellular enzymes such as lactate dehydrogenase (LDH), as described in Korzeniewski et al., 1983, Immunol. Methods 64(3): 313-20; and Decker et al., 1988, J. Immunol Methods 115(1): 61-9; or the release of an intracellular label such as europium, chromium 51 or indium 111 in which target cells are labeled.
Methods
The present invention encompasses administering one or more Fc variant of the invention (e.g., antibodies) to an animal, in particular a mammal, specifically, a human, for preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection. The Fc variants of the invention are particularly useful for the treatment or prevention of a disease or disorder where an altered efficacy of effector cell function (e.g., ADCC, CDC) is desired. The Fc variants and compositions thereof are particularly useful for the treatment or prevention of primary or metastatic neoplastic disease (i.e., cancer), and infectious diseases. Molecules of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein. As detailed below, the molecules of the invention can be used in methods of treating or preventing cancer (particularly in passive immunotherapy), autoimmune disease, inflammatory disorders or infectious diseases.
The Fc variants of the invention may also be advantageously utilized in combination with other therapeutic agents known in the art for the treatment or prevention of a cancer, autoimmune disease, inflammatory disorders or infectious diseases. In a specific embodiment, Fc variants of the invention may be used in combination with monoclonal or chimeric antibodies, lymphokines, or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), which, for example, serve to increase the number or activity of effector cells which interact with the molecules and, increase immune response. The Fc variants of the invention may also be advantageously utilized in combination with one or more drugs used to treat a disease, disorder, or infection such as, for example anti-cancer agents, anti-inflammatory agents or anti-viral agents.
Accordingly, the present invention provides methods for preventing, treating, or ameliorating one or more symptoms associated with cancer and related conditions by administering one or more Fc variants of the invention. Although not intending to be bound by any mechanism of actions, an Fc variant of the invention that binds FcγRIIIA and/or FcγRIIA with a greater affinity than a comparable molecule, and further binds FcγRIIB with a lower affinity than a comparable molecule, and/or said Fc variant has an enhanced effector function, e.g., ADCC, CDC, phagocytosis, opsonization, etc. will result in the selective targeting and efficient destruction of cancer cells.
The invention further encompasses administering one or more Fc variants of the invention in combination with other therapies known to those skilled in the art for the treatment or prevention of cancer, including but not limited to, current standard and experimental chemotherapies, hormonal therapies, biological therapies, immunotherapies, radiation therapies, or surgery. In some embodiments, the molecules of the invention may be administered in combination with a therapeutically or prophylactically effective amount of one or more anti-cancer agents, therapeutic antibodies or other agents known to those skilled in the art for the treatment and/or prevention of cancer. Examples of dosing regimes and therapies which can be used in combination with the Fc variants of the invention are well known in the art and have been described in detail elsewhere (see for example, PCT publications WO 02/070007 and WO 03/075957).
Cancers and related disorders that can be treated or prevented by methods and compositions of the present invention include, but are not limited to, the following: Leukemias, lymphomas, multiple myelomas, bone and connective tissue sarcomas, brain tumors, breast cancer, adrenal cancer, thyroid cancer, pancreatic cancer, pituitary cancers, eye cancers, vaginal cancers, vulvar cancer, cervical cancers, uterine cancers, ovarian cancers, esophageal cancers, stomach cancers, colon cancers, rectal cancers, liver cancers, gallbladder cancers, cholangiocarcinomas, lung cancers, testicular cancers, prostate cancers, penal cancers; oral cancers, salivary gland cancers pharynx cancers, skin cancers, kidney cancers, bladder cancers (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America).
The invention further contemplates engineering any of the antibodies known in the art for the treatment and/or prevention of cancer and related disorders, so that the antibodies comprise an Fc region incorporating a modified CH3 domain of the invention.
In a specific embodiment, a molecule of the invention (e.g., an antibody comprising a variant Fc heterodimer inhibits or reduces the growth of primary tumor or metastasis of cancerous cells by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the growth of primary tumor or metastasis in the absence of said molecule of the invention.
The present invention encompasses the use of one or more Fc variants of the invention for preventing, treating, or managing one or more symptoms associated with an inflammatory disorder in a subject. Although not intending to be bound by any mechanism of actions, Fc variants with enhanced affinity for FcγRIIB will lead to a dampening of the activating receptors and thus a dampening of the immune response and have therapeutic efficacy for treating and/or preventing an autoimmune disorder. Furthermore, antibodies binding more than one target, such as bispecific antibodies comprising a variant Fc heterodimer, associated with an inflammatory disorder may provide synergist effects over monovalent therapy.
The invention further encompasses administering the Fc variants of the invention in combination with a therapeutically or prophylactically effective amount of one or more anti-inflammatory agents. The invention also provides methods for preventing, treating, or managing one or more symptoms associated with an autoimmune disease further comprising, administering to said subject an Fc variant of the invention in combination with a therapeutically or prophylactically effective amount of one or more immunomodulatory agents. Examples of autoimmune disorders that may be treated by administering the Fc variants of the invention include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis. Examples of inflammatory disorders include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacteria infections. Some autoimmune disorders are associated with an inflammatory condition, thus, there is overlap between what is considered an autoimmune disorder and an inflammatory disorder. Therefore, some autoimmune disorders may also be characterized as inflammatory disorders. Examples of inflammatory disorders which can be prevented, treated or managed in accordance with the methods of the invention include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacteria infections.
Fc variants of the invention can also be used to reduce the inflammation experienced by animals, particularly mammals, with inflammatory disorders. In a specific embodiment, an Fc of the invention reduces the inflammation in an animal by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the inflammation in an animal, which is not administered the said molecule.
The invention further contemplates engineering any of the antibodies known in the art for the treatment and/or prevention of autoimmune disease or inflammatory disease, so that the antibodies comprisea variant Fc heterodimer of the invention.
The invention also encompasses methods for treating or preventing an infectious disease in a subject comprising administering a therapeutically or prophylactically effective amount of one or more Fc variants of the invention. Infectious diseases that can be treated or prevented by the Fc variants of the invention are caused by infectious agents including but not limited to viruses, bacteria, fungi, protozae, and viruses.
Viral diseases that can be treated or prevented using the Fc variants of the invention in conjunction with the methods of the present invention include, but are not limited to, those caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-1), herpes simplex type II (HSV-11), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, small pox, Epstein Barr virus, human immunodeficiency virus type I (HIV-1), human immunodeficiency virus type II (HIV-11), and agents of viral diseases such as viral meningitis, encephalitis, dengue or small pox.
Bacterial diseases that can be treated or prevented using the Fc variants of the invention in conjunction with the methods of the present invention, that are caused by bacteria include, but are not limited to, mycobacteria rickettsia, mycoplasma, neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus, streptococcus, staphylococcus, mycobacterium, tetanus, pertissus, cholera, plague, diptheria, chlamydia, S. aureus and legionella. Protozoal diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention, that are caused by protozoa include, but are not limited to, leishmania, kokzidioa, trypanosoma or malaria. Parasitic diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention, that are caused by parasites include, but are not limited to, chlamydia and rickettsia.
In some embodiments, the Fc variants of the invention may be administered in combination with a therapeutically or prophylactically effective amount of one or additional therapeutic agents known to those skilled in the art for the treatment and/or prevention of an infectious disease. The invention contemplates the use of the molecules of the invention in combination with other molecules known to those skilled in the art for the treatment and or prevention of an infectious disease including, but not limited to, antibiotics, antifungal agents and anti-viral agents.
The invention provides methods and pharmaceutical compositions comprising Fc variants of the invention (e.g., antibodies, polypeptides). The invention also provides methods of treatment, prophylaxis, and amelioration of one or more symptoms associated with a disease, disorder or infection by administering to a subject an effective amount of at least one Fc variant of the invention, or a pharmaceutical composition comprising at least one Fc variant of the invention. In a one aspect, the Fc variant, is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side-effects this includes homodimers and other cellular material). In a specific embodiment, the subject is an animal, such as a mammal including non-primates (e.g., cows, pigs, horses, cats, dogs, rats etc.) and primates (e.g., monkey such as, a cynomolgous monkey and a human). In a specific embodiment, the subject is a human. In yet another specific embodiment, the antibody of the invention is from the same species as the subject.
The route of administration of the composition depends on the condition to be treated. For example, intravenous injection may be preferred for treatment of a systemic disorder such as a lymphatic cancer or a tumor that has metastasized. The dosage of the compositions to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. Depending on the condition, the composition can be administered orally, parenterally, intranasally, vaginally, rectally, lingually, sublingually, buccally, intrabuccally and/or transdermally to the patient.
Accordingly, compositions designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example, with an inert diluent or with an edible carrier. The composition may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the pharmaceutical compositions of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums, and the like.
Tablets, pills, capsules, troches and the like may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and/or flavoring agents. Some examples of binders include microcrystalline cellulose, gum tragacanth and gelatin. Examples of excipients include starch and lactose. Some examples of disintegrating agents include alginic acid, cornstarch, and the like. Examples of lubricants include magnesium stearate and potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweetening agents include sucrose, saccharin, and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavoring, and the like. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
The pharmaceutical compositions of the present invention can be administered parenterally, such as, for example, by intravenous, intramuscular, intrathecal and/or subcutaneous injection. Parenteral administration can be accomplished by incorporating the compositions of the present invention into a solution or suspension. Such solutions or suspensions may also include sterile diluents, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol and/or other synthetic solvents. Parenteral formulations may also include antibacterial agents, such as, for example, benzyl alcohol and/or methyl parabens, antioxidants, such as, for example, ascorbic acid and/or sodium bisulfite, and chelating agents, such as EDTA. Buffers, such as acetates, citrates and phosphates, and agents for the adjustment of tonicity, such as sodium chloride and dextrose, may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes and/or multiple dose vials made of glass or plastic. Rectal administration includes administering the composition into the rectum and/or large intestine. This can be accomplished using suppositories and/or enemas. Suppository formulations can be made by methods known in the art. Transdermal administration includes percutaneous absorption of the composition through the skin. Transdermal formulations include patches, ointments, creams, gels, salves, and the like. The compositions of the present invention can be administered nasally to a patient. As used herein, nasally administering or nasal administration includes administering the compositions to the mucous membranes of the nasal passage and/or nasal cavity of the patient.
The pharmaceutical compositions of the invention may be used in accordance with the methods of the invention for preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection. It is contemplated that the pharmaceutical compositions of the invention are sterile and in suitable form for administration to a subject.
In one embodiment the compositions of the invention are pyrogen-free formulations that are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside a microorganism and are released when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drug solutions. The Food & Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeia! Convention, Pharmacopeia! Forum 26 (1):223 (2000)). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram body weight, as can be the case with monoclonal antibodies, it is advantageous to remove even trace amounts of endotoxin. In a specific embodiment, endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
The invention provides methods for preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection, said method comprising: (a) administering to a subject in need thereof a dose of a prophylactically or therapeutically effective amount of a composition comprising one or more Fc variants and (b) administering one or more subsequent doses of said Fc variants, to maintain a plasma concentration of the Fc variant at a desirable level (e.g., about 0.1 to about 100 IJg/ml), which continuously binds to an antigen. In a specific embodiment, the plasma concentration of the Fc variant is maintained at 10 IJg/ml, 15 IJg/ml, 20 IJg/ml, 25 IJg/ml, 30 IJg/ml, 35 IJg/ml, 40 IJg/ml, 45 IJg/ml or 50 IJg/ml. In a specific embodiment, said effective amount of Fc variant to be administered is between at least 1 mg/kg and 8 mg/kg per dose. In another specific embodiment, said effective amount of Fc variant to be administered is between at least 4 mg/kg and 5 mg/kg per dose. In yet another specific embodiment, said effective amount of Fc variant to be administered is between 50 mg and 250 mg per dose. In still another specific embodiment, said effective amount of Fc valiant to be administered is between 100 mg and 200 mg per dose.
The present invention also encompasses protocols for preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection which an Fc variant is used in combination with a therapy (e.g., prophylactic or therapeutic agent) other than an Fc variant and/or variant fusion protein. The invention is based, in part, on the recognition that the Fc variants of the invention potentiate and synergize with, enhance the effectiveness of, improve the tolerance of, and/or reduce the side effects caused by, other cancer therapies, including current standard and experimental chemotherapies. The combination therapies of the invention have additive potency, an additive therapeutic effect or a synergistic effect. The combination therapies of the invention enable lower dosages of the therapy (e.g., prophylactic or therapeutic agents) utilized in conjunction with Fc variants for preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection and/or less frequent administration of such prophylactic or therapeutic agents to a subject with a disease disorder, or infection to improve the quality of life of said subject and/or to achieve a prophylactic or therapeutic effect. Further, the combination therapies of the invention reduce or avoid unwanted or adverse side effects associated with the administration of current single agent therapies and/or existing combination therapies, which in turn improves patient compliance with the treatment protocol. Numerous molecules which can be utilized in combination with the Fc variants of the invention are well known in the art. See for example, PCT publications WO 02/070007; WO 03/075957 and U.S. Patent Publication 2005/064514.
The present invention provides kits comprising one or more Fc variants with altered binding affinity to FcγRs and/or C1q and altered ADCC and/or CDC activity that specifically bind to an antigen conjugated or fused to a detectable agent, therapeutic agent or drug, in one or more containers, for use in monitoring, diagnosis, preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection.
The examples below are given so as to illustrate the practice of this invention. They are not intended to limit or define the entire scope of this invention.
The genes encoding the antibody heavy and light chains were constructed via gene synthesis using codons optimized for human/mammalian expression. The Fab sequences were generated from a known Her2/neu binding Ab (Carter P. et al. (1992) Humanization of an anti P185 Her2 antibody for human cancer therapy. Proc Nat/Acad Sci 89, 4285.) and the Fc was an IgG1 isotype (SEQ ID NO:1). The final gene products were sub-cloned into the mammalian expression vector pTT5 (NRC-BRI, Canada) (Durocher, Y., Perret, S. & Kamen, A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human HEK293-EBNA1 cells. Nucleic acids research 30, E9 (2002)). The mutations in the CH3 domain were introduced via site-directed mutagenesis of the pTT5 template vectors. See Table 1 and Table 6 and Table 7 for a list of the modified CH3 domain mutations made.
In order to estimate the formation of heterodimers and determine the ratio of homodimers vs. heterodimers the two heterodimer heavy chains were designed with C-terminal extensions of different size (specifically, chain A with C-terminal HisTag and chain B with C-terminal mRFP plus StrepTagII). This difference in molecular weight allows differentiation of homodimers vs. heterodimer in non-reducing SDS-PAGE as illustrated in
The HEK293 cells were transfected in exponential growth phase (1.5 to 2 million cells/ml) with aqueous 1 mg/m2l 5 kDa polyethylenimine (PEI, Polysciences) at a PEI:DNA ratio of 2.5:1. (Raymond C. et al. A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications. Methods. 55(1):44-51 (2011)). In order to determine the optimal concentration range for forming heterodimers, the DNA was transfected in three separate ratios of the two heavy chains. For example, this was done in 2 ml culture volume and transfection DNA, comprised of 5% GFP, 45% salmon sperm DNA, 25% light chain and 25% total heavy chains, where the heavy chain A plasmid (with C-terminal His-Tag) and the heavy chain B plasmid (with C-terminal StrepTagII plus RFP) at 65%/55%/35% or 10%/20%/40%) were sampled at 3 different relative ratios (chain_A(His)/chain_B(mRFP)) of 10%/65%; 20%/55%; 40%/35% (the apparent 1:1 expression ratio of a WT_His/WT_mRFP heterodimer was determined to be close to the DNA ratio 20%/55%). At 4 to 48 hours after transfection in F17 serum-free media (Gibco), TN1 peptone is added to a final concentration of 0.5%. Expressed antibody was analyzed by SDS-PAGE to determine the best ratio of heavy to light chain for optimal heterodimer formation (See
A selected DNA ratio, for example 50% light chain plasmid, 25% heavy chain A plasmid, 25% heavy chain B of AZ33 and AZ34, with 5% GFP, and 45% salmon sperm DNA was used to transfect 150 mol f cell culture as described above. Transfected cells were harvested after 5-6 days with the culture medium collected after centrifugation at 4000 rpm and clarified using a 0.451-Jm filter. See Table 2 below, for a list of the percentage of light and heavy chain A and B plasmids used in the scale up transfection assays for each of the antibodies with CH3 mutations generated for further analysis, including determination of purity and melting temperature.
The clarified culture medium was loaded onto a MabSelect SuRe (GE Healthcare) protein-A column and washed with 10 column volumes of PBS buffer at pH 7.2. The antibody was eluted with 10 column volumes of citrate buffer at pH 3.6 with the pooled fractions containing the antibody neutralized with TRIS at pH 11. The protein was finally desalted using an Econo-Pac 1ODG column (Bio-Rad). The C-terminal mRFP tag on the heavy chain B was removed by incubating the antibody with enterokinase (NEB) at a ratio of 1:10,000 overnight in PBS at 25° C. The antibody was purified from the mixture by gel filtration. For gel filtration, 3.5 mg of the antibody mixture was concentrated to 1.5 mL and loaded onto a Sephadex 200 Hiload 16/600 200 pg column (GE Healthcare) via an AKTA Express FPLC at a flow-rate of 1 ml/min. PBS buffer at pH 7.4 was used at a flow-rate of 1 ml/min. Fractions corresponding to the purified antibody were collected, concentrated to ˜1 mg/mL and stored at −80° C.
Formation of heterodimers, as compared to homodimers, was assayed using non-reducing SDS-PAGE and mass spectrometry. Protein A purified antibody was run on a 4-12% gradient SDS-PAGE, non-reducing gel to determine the percentage of heterodimers formed prior to enterokinase (EK) treatment (See,
All DSC experiments were carried out using a GE VP-Capillary instrument. The proteins were buffer-exchanged into PBS (pH 7.4) and diluted to 0.4 to 0.5 mg/mL 0.137 ml loaded into the sample cell and measured with a scan rate of 1° C./min from 20 to 100° C. Data was analyzed using the Origin software (GE Healthcare) with the PBS buffer background subtracted. (See,
All binding experiments were carried out using a BioRad ProteOn XPR36 instrument at 25° C. with 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.05% Tween 20 at pH 7.4. Recombinant HER-2/neu(p185, ErbB-2 (eBiosciences, Inc.)) was captured on the activated GLM sensorchip by injecting 4.01-Jg/min 1 OmM NaOAc (pH 4.5) at 251-JL/min until approx. 3000 resonance units (RUs) were immobilized with the remaining active groups quenched. 401 Jg/mol f purified anti-HER-2/neu antibodies comprising the modified CH3 domains were indirectly captured on the sensorchip by binding the Her-2/neu protein when injected at 251-JL/min for 240 s (resulting in approx. 500 RUs) following a buffer injection to establish a stable baseline. FcgammaR (CD16a(f allotype) and CD32b) concentrations (6000, 2000, 667, 222, and 74.0 nM) were injected at 601-JL/min for 120 s with a 180 s dissociation phase to obtain a set of binding sensograms. Resultant K0 values were determined from binding isotherms using the Equilibrium Fit model with reported values as the mean of three independent runs. Comparisons were made with the wild-type IgG1 Fc domain and binding is expressed as a ratio of the WT kD to the variant kD (See, Table 5).
To obtain AZ variants with high stability and purity, the structural and computational strategies described above were employed. (See,
Consequently, we identified the key residues/sequence motifs responsible for the low stability of AZ8 compared to WT. To improve the stability and heterodimer specificity of
AZ8 the subsequent positive design engineering efforts were therefore specifically focused on stabilizing the loop conformation of positions 399-401 in a more ‘closed’-WT like conformation (See,
To achieve this stabilization of the loop conformation of positions 399-401 the described computational approach was used to evaluate our different targeted design ideas. Specifically, three different independent options for Fc variant AZ8 were analyzed to optimize the identified key regions for improving stability. First, the binding pocket close to position K409 and F405A was evaluated for better hydrophobic packing to both protect the hydrophobic core and stabilize the loop conformation of 399-400 (See,
Fc variant AZ33 is an example of the development of an Fc variant wherein Scaffold 1 was modified resulting in Scaffold 1a mutations to improve stability and purity. This Fc variant was designed based on AZ8 with the goal improving the hydrophobic packing at positions 392-394-409 and 366 to both protect the hydrophobic core and stabilize the loop conformation of 399-400. This Fc variant AZ33 heterodimer has two additional point mutations different from the core mutations of AZ8, K392M and T366I. The mutations T366I_K392M_T394W/F405A_Y407V are referred to herein as “Scaffold 1a” mutations. The mutation K392M was designed to improve the packing at the cavity close to position K409 and F405A to protect the hydrophobic core and stabilize the loop conformation of 399-400 (See,
Development of Fc Variants Using Scaffold 1 Mutations in Phase Three Design of Fc Variant Heterodimers
Although AZ33 provides a significant stability and specificity (or purity) improvement over the initial starting variant AZ8, our analysis indicates that further improvements to the stability of the Fc variant heterodimer can be made with further amino acid modifications using the experimental data of AZ33 and the above described design methods. The different design ideas have been independently tested for expression and stability, but the independent design ideas are transferable and the most successful heterodimer will contain a combination of the different designs. Specifically, for the optimization of AZ8 packing mutations at the cavity close to K409-F405A-K392 have been evaluated independently from mutations that optimize the core packing at residues L366T-L368. These two regions 366-368 and 409-405-392 are distal from each other and are considered independent. Fc variant AZ33 for example has been optimized for packing at 409-405-392, but not at 366-368, because these optimization mutations were separately evaluated. The comparison of the 366-368 mutations suggests that T366L has an improved stability over T366 and also T366I, the point mutation used in the development of Fc variant AZ33. Consequently, the presented experimental data immediately suggest further optimization of AZ33 by introducing T366L instead of T366I, for example. Therefore, the amino acid mutations in the CH3 domain T366L-K392M-T394W/F405A-Y407V are herein referred to as “Scaffold 1b” mutations.
In a similar manner the complete experimental data has been analyzed to identify point mutations that can be used to further improve the current Fc variant heterodimer AZ33. These identified mutations were analyzed by the above described computational approach and ranked to yield the list of additional Fc variant heterodimers based on AZ33 as shown in Table 6.
To improve the initial negative design phase Fc variant AZ15 for stability and purity, the structural and computational strategies described above were employed (See,
Development of Fc Variants Using Scaffold 2 Mutations and the Further Development of Scaffold 2a Mutations
In-silico analysis indicated a non-optimal packing of previous Fc variant designs such as AZ15 mutations K409F_T366A_Y407A and an overall decreased packing of the hydrophobic core due to the loss of the WT-Y407-Y407 interactions. The heteromultimers described herein are designed with more optimal packing. Some of the positive design efforts described herein were focused on point mutations to compensate for packing deficits in the initial Fc variant AZ15. The targeted residues included positions T366, L351, and Y407. Different combinations of these were tested in-silica and the best-ranked Fc variants using the computational tools (AZ63-AZ70) were validated experimentally for expression and stability as described in Examples 1-4.
Fc variant AZ70 is an example of the development of an Fc variant wherein Scaffold 2 was modified resulting in Scaffold 2a mutations to improve stability and purity. This Fc variant was designed based on AZ15 with the goal of achieving better packing at the hydrophobic core as described above. Fc variant AZ70 has the same Scaffold 2 core mutations (L351Y_Y407A I T366A_K409F) as described above except that T366 was mutated to T366V instead of T366A (
Development of Fc Variants Using Scaffold 2 Mutations and the Further Development of Scaffold 2b Mutations
The Molecular Dynamics simulation (MD) and packing analysis showed a preferred more ‘open’ conformation of the loop 399-400, which was likely due to the loss of the WT salt bridge K409-D399. This also results in the unsatisfied D399, which in turn preferred a compensating interaction with K392 and induced a more ‘open’ conformation of the loop. This more ‘open’ loop conformation results in an overall decreased packing and higher solvent accessibility of the core CH3 domain interface residues, which in turn significantly destabilized the heterodimer complex. Therefore, one of the targeted positive design efforts was the tethering of this loop in a more ‘closed’, WT-like conformation by additional point mutations that compensate for the loss of the D399-K409 salt bridge and the packing interactions of K409. The targeted residues included positions T411, D399, S400, F405, N390, K392 and combinations thereof. Different packing, hydrophobic- and electrostatic positive engineering strategies were tested in silica with respect to the above positions and the best-ranked Fc variants determined using the computational tools (AZ71-AZ101) were validated experimentally for expression and stability as described in Examples 1-4.
Fc variant AZ94 is an example of the development of an Fc variant wherein Scaffold 2 is modified resulting in Scaffold 2b mutations along with additional point mutations to improve stability and purity. This Fc variant was designed based with the goal of tethering loop 399-400 in a more ‘closed’, WT-like conformation and compensating for the loss of the D399-K409 salt bridge as described above. Fc variant AZ94 has four additional point mutations to Scaffold 2 (L351Y_Y407A/T366A_K409F) and returns L351Y to wild-type L351 leaving (Y407A/T366A_K409F) as the core mutations for this Fc variant. The core mutations Y407A/T366A K409F are herein referred to as “Scaffold 2b” mutations. The four additional point mutations of AZ94 are K392E_T411E/D399R_S400R. The mutations T411E/D399R were engineered to form an additional salt bridge and compensate for the loss of the K409/D399 interaction (
Development of Fc Variants Using Scaffold 2 Mutations in Phase Three Design of Fc Variant Heterodimers
Fc variants AZ70 and AZ94 provide a significant improvement in stability and purity over the initial negative design Fc variants such as AZ15, but our analysis and the comparison of AZ70 and AZ94 directly indicate that unexpected improvements to the stability of the Fc variant heterodimer can be made with further amino acid modifications. For example, Fc variants AZ70 and AZ94 were designed to target two distinct non-optimized regions in the initial variant AZ15, which was accomplished by improving packing at the hydrophobic core and making mutations outside of the core interface residues resulting in additional salt bridges and hydrogen bonding to stabilize the loop conformation of positions 399-401. The additional point mutations of Fc variants AZ70 and AZ94 are distal from each other and are therefore independent and transferable to other Fc variants designed around the same Scaffold 2 core mutations, including 2a and 2b mutations. Specifically, AZ70 only carries the optimized core mutations L351Y_Y407A/T366A_K409F, but no additional salt bridges, whereas AZ94 comprises four additional electrostatic mutations (K392E_T411E/D399R_S400R), but has one less mutation in the hydrophobic core interface (Y407AI T366A_K409F). These Scaffold 2b mutations are less stable than AZ70 (See, for example AZ63, which has equivalent core mutations as AZ94 and Tm of 72° C.), but are compensated for by the addition of K392E_T411E/D399R_S400R mutations. The presented experimental stability and purity data indicates that combining the mutations of AZ70, which optimizes the hydrophobic core, and the electrostatic mutations of AZ94 should further improve stability and purity of the Fc variant heterodimers. In a similar manner the complete experimental data for Scaffold 2 Fc variants (AZ63-101) has been analyzed to identify point mutations that can be used to further improve the Fc variant heterodimers AZ70 and AZ94. These identified mutations were further analyzed by the above described computational approach and ranked to yield the list of additional Fc variant heterodimers based on AZ70 and AZ94 as shown in Table 7.
As a prototypical example of heterodimeric Fc activity with FcgR, two variant antibodies with heterodimeric Fc region were tested A:K409D_K392DIB:D399K_D356K (Control1 (het 1 in
Mutation of Serine at position 267 in the CH2 domain of the Fc region to an Aspartic acid (S267D) is known to enhance binding to Fcgamma IIbF, IIbY & IiaR receptors when introduced in a homodimeric manner in the two chains of CH2 domain. This mutation can be introduced on only one of the CH2 domains in an heterodimeric Fc molecule to gain roughly half the improvement in binding strength relative to when this mutation is introduced in a homodimeric CH2 Fc as the data presented in
In a subsequent experiment, we have altered the selectivity profile of the base Fc mutant S239D_D265S_I332E_S298A that shows increased binding strength to the Fcgamma IIIaF and IIIaV receptors while continuing to exhibit weaker binding to the Fcgamma IiaR, IIbF and IIbY receptors. This is shown in the binding profile shown in
In another example shown in
The reagents employed in the examples are commercially available or can be prepared using commercially available instrumentation, methods, or reagents known in the art. The foregoing examples illustrate various aspects of the invention and practice of the methods of the invention. The examples are not intended to provide an exhaustive description of the many different embodiments of the invention. Thus, although the forgoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will realize readily that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.
Binding to FcRn was determined by SPR in two different orientations.
Bispecific binding was demonstrated using an Fc heterodimer with the mutations Chain-A: L351Y_F405A_Y407V, Chain-B: T366L_K392M_T394W and anti-HER2 and anti-HER3 scFvs fused to the N-terminus of Chain-A and Chain-B of the Fc heterodimer. The resultant variants bispecific HER21HER3 variant and the two monovalent-monospecific HER2, HER3 variants are illustrated in
Heterodimeric variants AZ133 (A: L351Y/F405A/Y407V, B: T366L/K392M/T394W), AZ138 (A: F405A/Y407V, B: T366L/K392M/T394W), AZ3002 (A: T350V/L351Y/F405A/Y407V, B: T350V/T366L/K392M/T394W), AZ3003 (A: T350V/L351Y/F405A/Y407V, B: T350V/T366L/K392L/T394W), and other AZ constructs AZ3000-AZ3021 were generated and purified as described in Examples 1 and 2. In order to estimate the robustness of heterodimer formation and the effect of excess of one of the heterodimer chains on heterodimer purity, the selected heterodimers were transiently expressed using 3 different DNA ratios of the two heavy chain A and B (e.g. ratios A:B=1:1.5; 1:1; 1.5:1).
The genes encoding the heterodimer heavy and light chains were constructed via gene synthesis using codons optimized for human/mammalian expression, as described in detail in Example 1. The Fab sequences were generated from a known Her2/neu binding Ab (Carter P. et al. (1992) Humanization of an anti P185 Her2 antibody for human cancer therapy. Proc Natl Acad Sci 89, 4285) and the Fc was an IgG1 isotype (SEQ ID NO:1). The variant was expressed by transient co-expression as described in the Examples 1-2 using 3 different Heavy Chain-A to Heavy Chain-B ratios of 1:1.5, 1:1 and 1.5:1. The samples were purified by protein-A affinity chromatography and preparative gel filtration (see Example 2 for details). The purified samples were de-glycosylated with PNGaseF overnight at 37° C. Prior to MS analysis the samples were injected onto a Poros R2 column and eluted in a gradient with 20-90% ACN, 0.2% FA in 3 minutes. The peak of the LC column was analyzed with an LTQ-Orbitrap XL mass spectrometer (Cone Voltage: 50 V′ Tube lens: 215 V; FT Resolution: 7,500) and integrated with the software Promass to generate molecular weight profiles.
The relative peak heights for the heterodimer and homodimers were used to estimate the heterodimer purity (see
Heterodimeric Fc constructs of AZ3002 and AZ3003 were transiently expressed in CHO and purified to homogeneity by pA and SEC. The purified Fc heterodimers were crystallized at 18° C. after −24 hours of incubation via hanging drop vapor diffusion method at a ratio of 2:1 above a mother liquor solution composed of 5% (v/v) ethylene glycol, 18% (w/v) polyethylene glycol 3350, and 0.15 M ammonium iodide with aid of microseeding. Crystals were cryoprotected by increasing the concentration of ethylene glycol to 30% (v/v) and subsequently flash cooled in liquid nitrogen. Diffraction data from both crystals were collected at 100 K, using 0.5 degree oscillations for 200 degrees total, and processed with XDS.1 The structure of AZ3002 was solved via molecular replacement with Phaser using PDBID: 2J6E as a query protein.2 The structure of AZ3002 was then used to solve AZ3003 in similar fashion. In order to accommodate the perfect twin, reciprocal relationship of the Azymetric heterodimer present in the crystallographic asymmetric unit (eg. the occupancy of molecule A can be equally be described by molecule B and vice versa), two possible heterodimer pairs, each with 0.5 atomic occupancies, were modeled with Coot, refined with Refmac.3 4 Diffraction data processing and structure refinement statistics are presented in Table 9.
An superimposition of the crystal structures is shown in
The AZ3003 heterodimer was expressed and purified as described in Example 11. Glycans were analysed with GlykoPrep™ Rapid N-Giycan Preparation with InstantAB™ (Prozyme) using the standard manufacturer protocol.
The results are shown in
The stability of the AZ3003 heterodimer was assessed by incubation under forced degradation conditions. The stability of a mAb under forced degradation conditions can be good estimate for the long term and formulation stability.
Purified heterodimer sample (expression and purification as described in Example 11) was concentrated to 100 mg/ml without signs of aggregation. The sample was diluted into the appropriate buffer and evaluated under forced degradation conditions as described in Table 10 below. The treated samples were analysed by SDS-PAGE and HPLC-SEC.
SDS-PAGE was performed under reducing (R) and non-reducing (NR) conditions with precast gradient gels purchased from LONZA. Protein bands were visualized by staining with Coomassie Brilliant Blue G-250.
Analytical SEC-HPLC was performed using either a Phenomenex, BIOSEP-SEC-S400D or BioRad Bio-Sil TSK 4000 HPLC column at 0.8 ml/min flow rate with 10 mM sodium phosphate, 0.14 M NaCl, 10% isopropanol as a running buffer. This allowed the quantification of potential higher and lower molecular with species.
The results are shown in
Manufacturability assessment of AZ3003 was performed to evaluate the behavior of AZ3003 using the industry-standard antibody purification process scheme as shown in
During manufacturability assessment, chromatographic behavior, protein stability and product quality were evaluated using the industry-standard purification process shown in
Standard industrial purification processes were used to verify the stability, chromatographic behavior and product quality of AZ3003.
1.1 Protein A Capture
CM expressing AZ3003 was 0.22-IJm filtered using a bottle-top filter (PES) from Millipore and applied to Mab Select SuRe (1.6×25 em) column equilibrated with 5 CV of 20 mM Tris-HCl, 0.14 M NaCl, pH 7.5. After loading, the column was extensively washed with equilibration buffer until A280 absorbance reached a stable baseline. AZ3003 was eluted with 0.1 M acetate buffer, pH 3.6 and immediately titrated to pH 5.2 by the addition of 1/10 volume of 1 M tris base.
After the elution step, the column with washed with 0.1 M acetate, pH 3.0. SDS-PAGE analysis shows that all the Mab was bound to the column as no Mab was detected in the column FT. Highly purified Mab was detected in the pH 3.6 elution buffer. The initial capture and purification step using Protein A affinity chromatography yields a product with >90% purity.
1.2 Low pH Hold Study
The next step in the downstream process is the low pH hold, which is performed to inactivate viruses. After elution from the Protein A column, the Mab (−10 mg/ml, pH 4.0) was titrated to pH 3.6 with 10% acetic acid and incubated at RT with stirring for 90 min. The stability of AZ3003 against the low pH treatment was evaluated by SDS-PAGE, SEC-HPLC and by turbidity measurements at A410 nm. AZ3003 tolerated well the low pH hold step, showing no changes in SDS-PAGE or SEC-HPLC. In addition, no increase in turbidity was detected after 90-min incubation, indicating the absence of the formation of insoluble aggregates that can be problematic during the purification (i.e. clogging of in-process filters and columns, loss of product). These data shows that AZ30003 is stable to the low pH hold step.
1.3 Cation Exchange Chromatography (CEX)
CEX was investigated as the second step in the purification process. Two resins were evaluated: Fractogel EMD S03 (M) from Merk/Millipore and SP HP from GE Lifesciences. Fractogel EMD S03 (M), pH 5.2: The Mab Select SuRe pool (35 mg) was titrated to pH 5.2 by the addition of 10% (v/v) of 1M tris base and then, 2-fold diluted with equilibration buffer, 20 mM acetate, pH 5.2. This pool was applied to a Fractogel EMD S03 (M) column equilibrated with 5 CV of 20 mM acetate, pH 5.2. The column was washed with equilibration buffer until A280 absorbance reached a stable baseline. The Mab was eluted from the column with a linear salt gradient from 0 to 600 mM NaCl, pH 5.2 over 10 CV. The remaining contaminants were stripped off the column with 20 mM acetate, 1 M NaCl, pH 5.2 followed by treatment with 1 N NaOH. SDS-PAGE and SEC-HPLC analysis was performed to monitor HMW levels and their removal from the main Mab fraction on this column. The step yield (based upon A280 nm readings) for was 73%.
SP HP, pH 5.2: The Mab Select SuRe pool (50 mg) was titrated to pH 5.2 by the addition of 10% (v/v) of 1M tris base, and then equally diluted with equilibration buffer, 20 mM acetate, pH 5.2. This pool was applied to aSP HP column (1.6×2.5 em/5 ml) equilibrated with 5 CV of 20 mM acetate, pH 5.2 (
The step yield (based upon A280 nm readings) was 87%.
1.4 Anion Exchange Chromatography (AEX)
Anion-exchangers (e.g. quaternary amine, Q) have been widely used in monoclonal antibody purification. The AEX medium is operated in flow-through mode, with the Mab appearing in the FT while allowing retention of HCP, DNA, viruses and endotoxin.
The Fractogel S03 M pool pH 5.2 (25 mg) was titrated to pH 7.0 with 1 M tris base and applied to 1 ml HiTrap Q FF equilibrated with 5 CV of 10 mM phosphate, pH 7.0. The column was washed with equilibration buffer. However in this case, a stable baseline was not reached. Consequently the column was washed with PBS to elute any residually bound Mab. Then, the column was washed with 10 mM phosphate, 1 M NaCl, pH 7.0 to remove any bound contaminants. This step needs further optimization so that all the Mab fraction will be present in the flow through. The step yield (based upon A280 nm readings) was 82% with an estimated purity >98% by SEC-HPLC.
1.5 SDS-PAGE Analysis of Downstream Purification
SDS-PAGE (
1.7 Estimation of Purity by SEC-HPLC
Purified AZ3003 after the three-column chromatography steps Protein A; CEX; AEX flow through mode; was evaluated by SEC (size exclusion)-HPLC under native conditions (
AZ3003 shows a single peak eluting within the expected 150 kDa region for native IgG1. Purity was estimated at >98%.
1.8 Process Yields for the Purification of AZ3003
Process yields were calculated for the downstream purification process and listed in
AZ3003 was successfully purified from CM using the industry-standard purification process (Protein A affinity resin, CEX, followed by AEX).
These results indicated that AZ3003 is comparable to standard mAb's in overall recovery yield (see Kelley B. Biotechnol. Prog. 2007, 23, 995-1008) and minimally-observed aggregation. AZ3003 was also evaluated with respect to low pH hold & CHT (ceramic hydroxyapatite) and HIC (hydrophobic interaction chromatography) (Phenyl HP pH 5 and pH 7) with good aggregate-free recovery.
All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.
This application is a division of U.S. application Ser. No. 13/668,098, filed Nov. 2, 2012, now U.S. Pat. No. 9,574,010, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 61/556,090, filed Nov. 4, 2011; U.S. Provisional Patent Application No. 61/557,262 filed Nov. 8, 2011; and U.S. Provisional Patent Application No. 61/645,547 filed May 10, 2012, each of which is incorporated herein by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4816567 | Cabilly et al. | Mar 1989 | A |
| 5624821 | Winter et al. | Apr 1997 | A |
| 5731168 | Carter et al. | Mar 1998 | A |
| 5807706 | Carter et al. | Sep 1998 | A |
| 5821333 | Carter et al. | Oct 1998 | A |
| 5885573 | Bluestone et al. | Mar 1999 | A |
| 6194551 | Idusogie et al. | Feb 2001 | B1 |
| 6331415 | Cabilly et al. | Dec 2001 | B1 |
| 6737056 | Presta | May 2004 | B1 |
| 6809185 | Schoonjans et al. | Oct 2004 | B1 |
| 7183076 | Arathoon et al. | Feb 2007 | B2 |
| 7635472 | Kufer et al. | Dec 2009 | B2 |
| 7642228 | Carter et al. | Jan 2010 | B2 |
| 7695936 | Carter et al. | Apr 2010 | B2 |
| 7769573 | Fejes et al. | Aug 2010 | B2 |
| 7923221 | Cabilly et al. | Apr 2011 | B1 |
| 7951917 | Arathoon et al. | May 2011 | B1 |
| 8501185 | Heitner Hansen et al. | Aug 2013 | B2 |
| 8592562 | Kannan et al. | Nov 2013 | B2 |
| 8623361 | Beirnaert et al. | Jan 2014 | B2 |
| 8771988 | Goepfert et al. | Jul 2014 | B2 |
| 9079965 | Zhou et al. | Jul 2015 | B2 |
| 9499634 | Dixit et al. | Nov 2016 | B2 |
| 9527927 | Chowdhury et al. | Dec 2016 | B2 |
| 9562109 | Von Kreudenstein et al. | Feb 2017 | B2 |
| 9574010 | Spreter Von et al. | Feb 2017 | B2 |
| 9732155 | Spreter Von Kreudenstein et al. | Aug 2017 | B2 |
| 9914785 | Corper et al. | Mar 2018 | B2 |
| 9988460 | Spreter Von Kreudenstein et al. | Jun 2018 | B2 |
| 10000576 | Weisser et al. | Jun 2018 | B1 |
| 10077298 | Corper et al. | Sep 2018 | B2 |
| 20030190311 | Dall'Acqua et al. | Oct 2003 | A1 |
| 20040071696 | Adams et al. | Apr 2004 | A1 |
| 20050069549 | Herman | Mar 2005 | A1 |
| 20050227324 | Huang et al. | Oct 2005 | A1 |
| 20060074225 | Chamberlain et al. | Apr 2006 | A1 |
| 20060106905 | Chren, Jr. | May 2006 | A1 |
| 20060160184 | Mattheus Hoogenboom et al. | Jul 2006 | A1 |
| 20060263882 | Fazio et al. | Nov 2006 | A1 |
| 20070041987 | Carter et al. | Feb 2007 | A1 |
| 20070196363 | Arathoon et al. | Aug 2007 | A1 |
| 20070274985 | Dubel et al. | Nov 2007 | A1 |
| 20070276791 | Fejes et al. | Nov 2007 | A1 |
| 20070278170 | Wiebe | Dec 2007 | A1 |
| 20080050370 | Glaser et al. | Feb 2008 | A1 |
| 20080147360 | Fejes et al. | Jun 2008 | A1 |
| 20090162360 | Klein et al. | Jun 2009 | A1 |
| 20090182127 | Kjaergaard et al. | Jul 2009 | A1 |
| 20090226466 | Fong et al. | Sep 2009 | A1 |
| 20090232811 | Klein et al. | Sep 2009 | A1 |
| 20090263392 | Igawa et al. | Oct 2009 | A1 |
| 20100104564 | Hansen et al. | Apr 2010 | A1 |
| 20100105874 | Schuurman et al. | Apr 2010 | A1 |
| 20100149876 | Mokhlesi | Jun 2010 | A1 |
| 20100166749 | Presta | Jul 2010 | A1 |
| 20100196265 | Adams et al. | Aug 2010 | A1 |
| 20100256338 | Brinkmann et al. | Oct 2010 | A1 |
| 20100286374 | Kannan et al. | Nov 2010 | A1 |
| 20100322934 | Imhof-Jung et al. | Dec 2010 | A1 |
| 20100322935 | Croasdale et al. | Dec 2010 | A1 |
| 20100331527 | Davis et al. | Dec 2010 | A1 |
| 20110008345 | Ashman et al. | Jan 2011 | A1 |
| 20110053261 | Lario et al. | Mar 2011 | A1 |
| 20110059076 | McDonagh et al. | Mar 2011 | A1 |
| 20110200596 | Huang et al. | Aug 2011 | A1 |
| 20110274691 | Arvedson et al. | Nov 2011 | A1 |
| 20110275787 | Kufer et al. | Nov 2011 | A1 |
| 20110287009 | Scheer et al. | Nov 2011 | A1 |
| 20110293613 | Brinkmann et al. | Dec 2011 | A1 |
| 20120076728 | Wu et al. | Mar 2012 | A1 |
| 20120143580 | Constantine et al. | Jun 2012 | A1 |
| 20120149876 | Von Kreudenstein et al. | Jun 2012 | A1 |
| 20120244577 | Dixit et al. | Sep 2012 | A1 |
| 20120244578 | Kannan et al. | Sep 2012 | A1 |
| 20120270801 | Frejd et al. | Oct 2012 | A1 |
| 20130078249 | Ast et al. | Mar 2013 | A1 |
| 20130195849 | Spreter Von et al. | Aug 2013 | A1 |
| 20130238299 | Ohrn | Sep 2013 | A1 |
| 20130245963 | Ohrn et al. | Sep 2013 | A1 |
| 20130336973 | Spreter Von et al. | Dec 2013 | A1 |
| 20140051835 | Dixit et al. | Feb 2014 | A1 |
| 20140066378 | Dixit et al. | Mar 2014 | A1 |
| 20140072581 | Dixit et al. | Mar 2014 | A1 |
| 20140154253 | Ng et al. | Jun 2014 | A1 |
| 20140200331 | Corper et al. | Jul 2014 | A1 |
| 20150051889 | Ohrn et al. | Feb 2015 | A1 |
| 20150125449 | Ng et al. | May 2015 | A1 |
| 20150142326 | Lakatos et al. | May 2015 | A1 |
| 20150211001 | Ohrn et al. | Jul 2015 | A1 |
| 20150220681 | Dixit | Aug 2015 | A1 |
| 20150284470 | Von et al. | Oct 2015 | A1 |
| 20150307594 | Corper et al. | Oct 2015 | A1 |
| 20160083480 | Ng et al. | Mar 2016 | A1 |
| 20160257763 | Von Kreudenstein et al. | Sep 2016 | A1 |
| 20160289335 | Weisser et al. | Oct 2016 | A1 |
| 20160297891 | Ng et al. | Oct 2016 | A1 |
| 20170158779 | Dixit et al. | Jun 2017 | A1 |
| 20180030150 | Spreter Von Kreudenstein et al. | Feb 2018 | A1 |
| 20180179296 | Corper et al. | Jun 2018 | A1 |
| 20180194860 | Von Kreudenstein et al. | Jul 2018 | A1 |
| 20180280429 | Wang et al. | Oct 2018 | A1 |
| Number | Date | Country |
|---|---|---|
| 2548757 | Jul 2005 | CA |
| 1176659 | Mar 1998 | CN |
| 368684 | May 1990 | EP |
| 1870459 | Dec 2007 | EP |
| 2011508604 | Mar 2011 | JP |
| 2013536966 | Sep 2013 | JP |
| 2014504265 | Feb 2014 | JP |
| 9308829 | May 1993 | WO |
| 9404690 | Mar 1994 | WO |
| 9627011 | Sep 1996 | WO |
| 9734631 | Sep 1997 | WO |
| 9958572 | Nov 1999 | WO |
| 0042072 | Jul 2000 | WO |
| 03031464 | Apr 2003 | WO |
| 2004029207 | Apr 2004 | WO |
| 2005018629 | Mar 2005 | WO |
| 2006003388 | Jan 2006 | WO |
| 2006030220 | Mar 2006 | WO |
| 2007110205 | Oct 2007 | WO |
| 2008131242 | Oct 2008 | WO |
| 2008141449 | Nov 2008 | WO |
| 2009089004 | Jul 2009 | WO |
| 2010068722 | Jun 2010 | WO |
| 2010085682 | Jul 2010 | WO |
| 2010115553 | Oct 2010 | WO |
| 2011005621 | Jan 2011 | WO |
| 2011028952 | Mar 2011 | WO |
| 2011063348 | May 2011 | WO |
| 2011066655 | Jun 2011 | WO |
| 2011120134 | Oct 2011 | WO |
| 2011120135 | Oct 2011 | WO |
| 2011143545 | Nov 2011 | WO |
| 2011133886 | Dec 2011 | WO |
| 2011147982 | Dec 2011 | WO |
| 2012006635 | Jan 2012 | WO |
| 2012037659 | Mar 2012 | WO |
| 2012040833 | May 2012 | WO |
| 2012058768 | May 2012 | WO |
| 2012116453 | Sep 2012 | WO |
| 2012143523 | Oct 2012 | WO |
| 2013002362 | Jan 2013 | WO |
| 2013060867 | May 2013 | WO |
| 2013063702 | May 2013 | WO |
| 2013166594 | Nov 2013 | WO |
| 2013166604 | Nov 2013 | WO |
| 2014004586 | Jan 2014 | WO |
| 2014012082 | Jan 2014 | WO |
| 2014012085 | Jan 2014 | WO |
| 2014018572 | Jan 2014 | WO |
| 2014067011 | May 2014 | WO |
| 2014082179 | Jun 2014 | WO |
| 2014182970 | Nov 2014 | WO |
| 2014186905 | Nov 2014 | WO |
| 2015006749 | Jan 2015 | WO |
| 2015181805 | Dec 2015 | WO |
| Entry |
|---|
| NCI Dictionary of Cancer Terms, Proxinium, Sep. 15, 2016. |
| U.S. Appl. No. 13/289,934, Notice of Allowance dated Sep. 29, 2016. |
| U.S. Appl. No. 13/289,934, Notice of Allowance dated Apr. 25, 2016. |
| U.S. Appl. No. 13/289,934, Advisory Action dated Feb. 5, 2016, 5 pages. |
| U.S. Appl. No. 13/289,934, Final Office Action dated Nov. 16, 2015, 19 pages. |
| U.S. Appl. No. 13/289,934, Non-Final Office Action dated Feb. 27, 2015, 15 pages. |
| U.S. Appl. No. 13/289,934, Non-Final Office Action dated May 13, 2015, 17 pages. |
| U.S. Appl. No. 13/289,934, Restriction Requirement dated Sep. 16, 2014, 6 pages. |
| U.S. Appl. No. 13/668,098, Final Office Action dated Nov. 17, 2015, 16 pages. |
| U.S. Appl. No. 13/668,098, Non-Final Office Action dated Apr. 3, 2015, 18 pages. |
| U.S. Appl. No. 13/668,098, Notice of Allowance dated Sep. 23, 2016, 12 pages. |
| U.S. Appl. No. 13/668,098, Restriction Requirement dated Dec. 5, 2014, 10 pages. |
| U.S. Appl. No. 13/892,198, Non-Final Office Action dated Oct. 6, 2015, 23 pages. |
| U.S. Appl. No. 13/892,198, Restriction Requirement dated Jul. 10, 2015, 12 pages. |
| U.S. Appl. No. 13/927,065, Final Office Action dated Feb. 22, 2016, 6 pages. |
| U.S. Appl. No. 13/927,065, Non-Final Office Action dated Oct. 7, 2015, 10 pages. |
| U.S. Appl. No. 13/927,065, Notice of Allowance dated Aug. 26, 2016, 7 pages. |
| U.S. Appl. No. 13/927,065, Restriction Requirement dated Apr. 15, 2015, 9 pages. |
| U.S. Appl. No. 13/941,449, Final Office Action dated Mar. 30, 2017. |
| U.S. Appl. No. 13/941,449, Non-Final Office Action dated Dec. 21, 2017. |
| U.S. Appl. No. 13/941,449, Final Office Action dated Jul. 24, 2018. |
| U.S. Appl. No. 13/941,449, Non-Final Office Action dated Apr. 13, 2016, 41 pages. |
| U.S. Appl. No. 13/941,449, Restriction Requirement dated Dec. 3, 2015, 10 pages. |
| U.S. Appl. No. 14/092,804, Non-Final Office Action dated Sep. 10, 2015, 33 pages. |
| U.S. Appl. No. 14/092,804, Restriction Requirement dated Jun. 18, 2015, 5 pages. |
| U.S. Appl. No. 14/092,804, Restriction Requirement dated May 12, 2016, 5 pages. |
| U.S. Appl. No. 14/648,222, Restriction Requirement dated May 9, 2016, 14 pages. |
| U.S. Appl. No. 14/888,580, Restriction Requirement dated Jan. 30, 2017. |
| U.S. Appl. No. 14/888,580, Non-Final Office Action dated Apr. 12, 2017. |
| U.S. Appl. No. 14/888,580, Final Office Action dated Sep. 7, 2017. |
| U.S. Appl. No. 14/888,580, Non-Final Office Action dated Mar. 1, 2018. |
| U.S. Appl. No. 14/888,580, Final Office Action dated Aug. 7, 2018. |
| U.S. Appl. No. 14/893,706, Restriction Requirement dated Apr. 4, 2017. |
| U.S. Appl. No. 14/893,706, Non-Final Office Action dated Jun. 30, 2017. |
| U.S. Appl. No. 14/893,706, Final Office Action dated Dec. 1, 2017. |
| U.S. Appl. No. 14/893,706, Restriction Requirement dated Aug. 24, 2018. |
| U.S. Appl. No. 14/989,648, Restriction Requirement dated Jan. 9, 2018. |
| U.S. Appl. No. 14/989,648, Non-Final Office Action dated Aug. 24, 2018. |
| U.S. Appl. No. 14/439,532, Restriction Requirement dated Nov. 8, 2011. |
| U.S. Appl. No. 14/439,532, Notice of Allowance dated Feb. 9, 2017. |
| U.S. Appl. No. 14/439,532, Notice of Allowance dated May 19, 2017. |
| U.S. Appl. No. 15/675,248, Notice of Allowance dated Mar. 22, 2018. |
| U.S. Appl. No. 15/298,625, Restriction Requiremement dated Jan. 17, 2018. |
| U.S. Appl. No. 15/298,625, Non-Final Office Action dated Jun. 1, 2018. |
| U.S. Appl. No. 15/355,019, Non-Final Office Action dated Jul. 21, 2017. |
| U.S. Appl. No. 15/355,019, Notice of Allowance dated Nov. 17, 2017. |
| U.S. Appl. No. 15/355,019, Notice of Allowance dated May 22, 2018. |
| U.S. Appl. No. 15/036,176, Restriction Requirement dated Jul. 28, 2017. |
| U.S. Appl. No. 15/036,176, Non-Final Office Action dated Mar. 14, 2018. |
| U.S. Appl. No. 15/036,175, Restriction Requirement dated Jun. 28, 2018. |
| Alegre et al., A Non-activating Humanized Anti-CD3 Monoclonal Antibody Retains Immunosuppressive Properties in Vivo, Transplantation, vol. 57, No. 11, Jun. 15, 1994, pp. 1537-1543. |
| Altintas et al., Targeting Epidermal Growth Factor Receptor in Tumors: From Conventional Monoclonal Antibodies via Heavy Chain-only Antibodies to Nanobodies, Eur J Pharm Sci., vol. 45, No. 4, Mar. 12, 2012, pp. 399-407. |
| Aitman et al., Copy Number Polymorphism in Fcgr3 Predisposes to Glomerulonephritis in Rats and Humans, Nature, vol. 439, No. 7078, Feb. 16, 2006, pp. 851-855. |
| Arnold et al., The Impact of Glycosylation on the Biological Function and Structure of Human Immunoglobulins, Annu Rev Immunol., vol. 25, Apr. 23, 2007, pp. 21-50. |
| Atwell et al., Stable Heterodimers From Remodeling the Domain Interface of a Homodimer Using a Phage Display Library, Journal of Molecular Biology, vol. 270, No. 1, Jul. 4, 1997, pp. 26-35. |
| Barthelemy et al., Comprehensive Analysis of the Factors Contributing to the Stability and Solubility of Autonomous Human Vh Domains, J. Bioi. Chem., vol. 283, No. 6, Feb. 2008, pp. 3639-3654. |
| Beck et al., Strategies and Challenges for the Next Generation of Therapeutic Antibodies, Nature Reviews Immunology, vol. 10, No. 5, May 2010, pp. 345-352. |
| Beckman, R. A., et al., Antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors. Cancer. Jan. 15, 2007; 109(2):170-9. |
| Bell et al., Differential Tumor-targeting Abilities of Three Single-domain Antibody Formats, Cancer Letters, vol. 289, No. 1, Mar. 1, 2010, pp. 81-90. |
| Bolon et al., Specificity Versus Stability in Computational Protein Design, Proceedings of the National Academy of Sciences, vol. 102, No. 36, Sep. 6, 2005, pp. 12724-12749. |
| Carter et al., Humanization of an Anti-p185HER2 Antibody for Human Cancer Therapy, Proc. Natl. Acad. Sci. USA, vol. 89, No. 10, May 1992, pp. 4285-4289. |
| Carter, Introduction to Current and Future Protein Therapeutics: a Protein Engineering Perspective, Experimental Cell Research, vol. 317, No. 9, May 15, 2011, pp. 1261-1269. |
| Cespedes, M.V., et al., Mouse models in oncogenesis and cancer therapy. Clin Transl Oncol. May 2006; 8(5):318-29. |
| Chames et al., Therapeutic Antibodies: Successes, Limitations and Hopes for the Future, British Journal of Pharmacology, vol. 157, No. 2, May 2009, pp. 220-223. |
| Cochlovius et al., Treatment of Human B Cell Lymphoma Xenografts With a CD3 X CD19 Diabody and T Cells, Journal of Immunology, vol. 165, No. 2, 2000, pp. 888-895. |
| Colbere-Garapin et al., A New Dominant Hybrid Selective Marker for Higher Eukaryotic Cell, Journal of Molecular Biology, vol. 150, No. 1, Jul. 25, 1981, pp. 1-14. |
| Colman, Effects of Amino Acid Sequence Changes on Antibody-antigen Interactions, Research in Immunology, vol. 145, No. 1, Jan. 1994, pp. 33-36. |
| Coloma et al., Design and Production of Novel Tetravalent Bispecific Antibodies, Nature Biotechnology, vol. 15, No. 2, Feb. 1997, pp. 159-163. |
| Dall'Acqua et al., Contribution of Domain Interface Residues to the Stability of Antibody CH3 Domain Homodimers, Biochemistry, American Chemical Society, US, vol. 37, No. 26, Jun. 30, 1998, pp. 9266-9273. |
| Davis et al., SEEDbodies: Fusion Proteins Based on Strand-exchange Engineered Domain (SEED) CH3 Heterodimers in an Fc Analogue Platform for Asymmetric Binders or Immunofusions and Bispecific Antibodies, Protein Engineering, Design & Selection, vol. 23, No. 4, Feb. 4, 2010, pp. 195-202. |
| De Kruif et al., Leucine zipper dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library, Journal of Biological Chemistry, vol. 271, No. 13, Mar. 29, 1996, pp. 7630-7634. |
| Demarest et al., Antibody Therapeutics, Antibody Engineering, and the Merits of Protein Stability, Current Opinion in Drug Discovery and Development vol. 11, No. 5, Sep. 2008, pp. 675-687. |
| Demarest et al., Optimization of the Antibody CH3 Domain by Residue Frequency Analysis of IgG Sequences, Journal of Molecular Biology, vol. 335, No. 1, Jan. 2, 2004, pp. 41-48. |
| Dennis, C., Cancer: Off by a whisker. Nature. Aug. 16, 2006; 442; 739-741. |
| Dockal et al., Conformational Transitions of the Three Recombinant Domains of Human Serum Albumin Depending on pH, The Journal of Biological Chemistry, vol. 275, No. 5, Feb. 4, 2000, pp. 3042-3050. |
| Dockal et al., Five Recombinant Fragments of Human Serum Albumin-tools for the Characterization of the Warfarin Binding Site, Protein Science, vol. 9, No. 8, 2000, pp. 1455-1465. |
| Ducry et al., Antibody-drug conjugates: linking cytotoxic payloads to monoclonal antibodies, Bioconjugate Chem., vol. 21, No. 1, Jan. 2010, pp. 5-13. |
| Duncan et al., Localization of the Binding Site for the Human High-affinity FC Receptor on IgG, Nature, vol. 332, No. 7, Apr. 7, 1988, pp. 563-564. |
| Durocher et al., High-Level and High-Throughput Recombinant Protein Production by Transient Transfection of Suspension-Growing Human HEK293-EBNA1 Cells, Nucleic acids research, vol. 30, No. 2, Jan. 2002, p. e9. |
| Fujimori, J., et al., A modeling analysis of monoclonal antibody percolation through tumors: a binding-site barrier. J Nucl Med. Jul. 1990; 3(17):1191-8. |
| Grabulovski et al., A Novel, Non-Immunogenic Fyn SH3-derived Binding Protein With Tumor Vascular Targeting Properties, J Bioi Chem., vol. 282, No. 5, Feb. 2007, pp. 3196-3204. |
| Groot et al., Identification by Phage Display of Single-domain Antibody Fragments Specific for the Odd Domain in Hypoxia-inducible Factor 1 Alpha, Lab Invest, vol. 86, No. 4, Apr. 2006, pp. 345-356. |
| Gunasekaran et al., Enhancing Antibody Fc Heterodimer Formation Through Electrostatic Steering Effects: Applications to Bispecific Molecules and Monovalent IgG, J. Biol. Chem., vol. 285, No. 25, Jun. 18, 2010, pp. 19637-19646. |
| Guss et al., Structure of the IgG-binding Regions of Streptococcal Protein G, The EMBO Journal, vol. 5, No. 7, 1986, pp. 1567-1575. |
| Hardy et al., Valency of Antibody Binding to Enveloped Virus Particles as Determined by Surface Plasmon Resonance, J Viral., vol. 77, no. 2, 2003, pp. 1649-1652. |
| Havranek et al., Automated Design of Specificity in Molecular Recognition, Nature Structure Biology, vol. 10, No. 1, Jan. 2003, pp. 45-52. |
| Hennecke et al., Composition and Arrangement of Genes Define the Strength of IRES-driven Translation in Bicistronic mRNAs, Nucleic Acids Res., vol. 29, No. 16, Aug. 15, 2001, pp. 3327-3334. |
| Holt et al., Domain Antibodies: Proteins for Therapy, Trends in Biotechnology vol. 21, No. 11, Nov. 2003, pp. 484-490. |
| Huang et al., A De Novo Designed Protein Protein Interface, Protein Science, vol. 16, No. 12, 2007, pp. 2770-2774. |
| Huang, C., et al., Recombinant immuno-therapeutics: current state and perspectives regarding the feasibility and market. Appl Microbiol Biotechnol. Jun. 2010; 87(2):401-410. |
| Hust et al., Single chain Fab (scFab) Fragment, BMC Biotechnology, vol. 7, No. 14, Available online at: httn://www.biomedcentral.com/1472-6750/7/14, 2007, pp. 1-15. |
| Hutchins et al., Improved Biodistribution, Tumor Targeting, and Reduced Immunogenicity in Mice With a Gamma 4 Variant of Campath-1H, Proc. Natl. Acad. Sci., vol. 92, No. 26, Dec. 1995, pp. 11980-11984. |
| Idusogie et al., Engineered Antibodies With Increased Activity to Recruit Complement, J. Immunol., vol. 166, No. 4, Feb. 15, 2001, pp. 2571-2575. |
| Idusogie et al., Mapping of the C1q Binding Site on Rituxan, a Chimeric Antibody With a Human IgG1 Fc, J. Immunol., vol. 164, No. 8, Apr. 15, 2000, pp. 4178-4184. |
| Igawa et al., VH/VL Interface Engineering to Promote Selective Expression and Inhibit Conformational Isomerization of Thrombopoietin Receptor Against Single-chain Diabody, Protein Engineering, Design & Selection, vol. 23, No. 8, Aug. 2010, pp. 667-677. |
| Ito et al., Effects of Subclass Change on the Structural Stability of Chimeric, Humanized, and Human Antibodies under Thermal Stress, Protein Science: A Publication of the Protein Society, vol. 22, No. 11, 2013, pp. 1542-1551. |
| Jackman et al., Development of a Two-part Strategy to Identify a Therapeutic Human Bispecific Antibody That Inhibits IgE Receptor Signaling, J Biol Chem., vol. 285, No. 27, Jul. 2, 2010, pp. 20850-20859. |
| Jaeger, Clinical Immunology and Allergology, M.: Medicine, 2nd edition, vol. 2, 1990, pp. 484-485. |
| Janeway et al., Structure of the Antibody Molecule and Immunoglobulin Genes, Immunology Third Edition, Garland Publishing Inc. Chapter 3, 1997, pp. 3:1-3:11. |
| Jefferis et al., Interaction Sites on Human IgG-fc for Fcgammar: Current Models, Immunol. Lett., vol. 82, No. 1-2, 2002, pp. 57-65. |
| Jefferis et al., Modulation of Fc(Gamma)R and Human Complement Activation by IgG3-core Oligosaccharide Interactions, Immunol. Lett., vol. 54, No. 2-3, Dec. 1996, pp. 101-104. |
| Jefferis et al., Recognition Sites on Human IgG for Fc Gamma Receptors: the Role of Glycosylation, Immunol. Lett., vol. 44, No. 2-3, 1995, pp. 111-117. |
| Jin et al., MetMAb, the One-armed 5D5 Anti-c-met Antibody, Inhibits Orthotopic Pancreatic Tumor Growth and Improves Survival, Cancer Res., vol. 68, No. 11, Jun. 1, 2008, pp. 4360-4368. |
| Kabat et al., Sequences of Proteins of Immunological Interest, DIANE publishing, 5th ed., vol. 1, 1991, pp. 688-695. |
| Kang et al., Engineering Multivalent Antibodies to Target Heregulin-induced HER3 Signaling in Breast Cancer Cells, MAbs, vol. 6, Dec. 26, 2013, pp. 340-353. |
| Kelley, Very Large Scale Monoclonal Antibody Purification: the Case for Conventional Unit Operations, Biotechnology Progress, vol. 23, No. 5, Sep.-Oct. 2007, pp. 995-1008. |
| Klein et al., Progress in Overcoming the Chain Association No. in Bispecific Heterodimeric LgG Antibodies, Mabs, vol. 4, No. 6, Nov. 2012, pp. 653-663. |
| Kontermann, Dual Targeting Strategies with Bispecific Antibodies., mABs, Voume 4, No. 2, Mar.-Apr. 2012, pp. 182-197. |
| Lewis et al., Generation of Bispecific IgG Antibodies by Structure-Based Design of an Orthogonal Fab Interface, Nature Biotechnology, vol. 32, No. 2, Jan. 26, 2014, pp. 191-198. |
| Li et al., Bispecific Antibody to Erbb2 Overcomes Trastuzumab Resistance Through Comprehensive Blockade of Erbb2 Heterodimerization, Cancer Research, vol. 73, No. 21, Sep. 2013, pp. 6471-6483. |
| Lindhofer et al., Preferential Species-restricted Heavy/light Chain Pairing in Rat/mouse Quadromas. Implications for a Single-step Purification of Bispecific Antibodies, The Journal of Immunology, vol. 155, No. 1, Jul. 1, 1995, pp. 219-225. |
| Lorusso et al., Trastuzumab emtansine: a unique antibody-drug conjugate in development for human epidermal growth factor receptor 2-positive cancer, Clinical Cancer Research, vol. 17, No. 20, Oct. 15, 2011, pp. 6437-6447. |
| Lu et al., Fab-scFv Fusion Protein: an Efficient Approach to Production of Bispecific Antibody Fragments, Journal of Immunological Methods, vol. 267, No. 2, 2002, pp. 213-226. |
| Lund et al., Human Fc Gamma RI and Fe Gamma RII Interact With Distinct but Overlapping Sites on Human IgG, J. Immunol., vol. 147, No. 8, Oct. 15, 1991, pp. 2657-2662. |
| Lund et al., Multiple Binding Sites on the CH2 Domain of IgG for Mouse Fc Gamma R11, Mol. Immunol., vol. 29, No. 1, 1992, pp. 53-59. |
| Lund et al., Multiple Interactions of IgG With Its Core Oligosaccharide Can Modulate Recognition by Complement and Human Fc Gamma Receptor I and Influence the Synthesis of Its Oligosaccharide Chains, J. Immunol., vol. 157, No. 11, Dec. 1, 1996, pp. 4963-4969. |
| Lund et al., Oligosaccharide-protein Interactions in IgG Can Modulate Recognition by Fc Gamma Receptors, Faseb J., vol. 9, No. 1, Jan. 1995, pp. 115-119. |
| MacCallum et al., Antibody-antigen Interactions: Contact Analysis and Binding Site Topography, Journal of Molecular Biology, vol. 262, No. 5, Oct. 1996, pp. 732-745. |
| Marqusee et al., Helix Stabilization by Glu- . . . Lys+ Salt Sridges in Short Septides of De Novo Design, Proc Natl Acad Sci U S A., vol. 84, No. 24, Dec. 1987, pp. 8898-8902. |
| McDonagh et al., Antitumor Activity of a Novel Bispecific Antibody that Targets the Erbb2/erbb3 Oncogenic Unit and Inhibits Heregulin-induced Activation of Erbb3, Mol. Cancer Ther., vol. 11, No. 3, Jan. 2012, pp. 582-593. |
| Merchant et al., An Efficient Route to Human Bispecific IgG, Nature Biotechnology, vol. 16, No. 7, Jul. 16, 1998, pp. 677-681. |
| Milstein et al., Hybrid Hybridomas and Their Use in Immunohistochemistry, Nature, vol. 305, No. 6, Oct. 6, 1983, pp. 537-540. |
| Moore et al., A Novel Bispecific Antibody Format Enables Simultaneous Bivalent and Monovalent Co-engagement of Distinct Target Antigens, mAbs, vol. 3, No. 6, Nov. 1, 2011, pp. 546-557. |
| Moore et al., Engineered Fc Variant Antibodies With Enhanced Ability to Recruit Complement and Mediate Effector Functions, Mabs, Landes Biosciences , vol. 2, No. 2, Mar. 1, 2010, pp. 181-189. |
| Omidfar et al., Single Domain Antibodies: a New Concept for Epidermal Growth Factor Receptor and EGFRvIII Targeting, DNA Cell Biol., vol. 31, No. 6, Jun. 2012, pp. 1015-1026. |
| Omidfar et al., Studies of Thermostability in Camelus bactrianus (Bactrian Camel) Single-domain Antibody Specific for the Mutant Epidermal-growth-factor Receptor Expressed by Pichia, Biotechnol. Appl. Biochem., vol. 46, Jan. 2007, pp. 41-49. |
| Osborn et al., Pharmacokinetic and Pharmacodynamics Studies of a Human Serum Albumin-interferon-a Fusion Protein in Cynomolgus Monkeys, J. Pharamcology and Experiemental Therapeutics, vol. 330, Nov. 2002, pp. 540-548. |
| Paul, Protein and Polypeptide Antigenic Determinants, Fundamental Immunology, 3d ed, 1993, p. 242. |
| Pham et al., Large-scale Transfection of Mammalian Cells for the Fast Production of Recombinant Protein, Molecular Biotechnology, vol. 34, No. 2, 2006, pp. 225-237. |
| Pluckthun, Antibodies From Escherichia coli. In: The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore eds, Springer-Verlag, vol. 113, chapter 11, 1994, pp. 269-315. |
| Portolano et al., Lack of Promiscuity in Autoantigen-Specific H and L Chain Combinations as Revealed by Human H and L Chain Roulette, J Immunol., vol. 150, No. 3, Feb. 1, 1993, pp. 880-887. |
| Presta, et al., Engineering Therapeutic Antibodies for Improved Function, Biochem. Soc. Trans., vol. 30, No. 4, Aug. 2002, pp. 487-490. |
| Rakestraw et al., Secretion-and-Capture Cell-surface Display for Selection of Target-Binding Proteins, Protein Engineering, Design and Selection, vol. 24, No. 6, 2011, pp. 525-530. |
| Raymond et al., A Simplified Polyethylenimine-mediated Transfection Process for Large-scale and High-throughput Applications, Methods, vol. 55, No. 1, 2011, pp. 44-51. |
| Reddy et al., Elimination of Fc Receptor-Dependent Effector Functions of a Modified IgG4 Monoclonal Antibody to Human CD4, J. Immunol., vol. 164, No. 4, Feb. 15, 2000, pp. 1925-1933. |
| Ridgway et al., Knobs-into-Holes' Engineering of Antibody CH3 Domains for Heavy Chain Heterodimerization, Protein Engineering, vol. 9, No. 7, Jul. 1996, pp. 617-621. |
| Robinson et al., Targeting ErbB2 and ErbB3 With a Bispecific Single-chain Fv Enhances 1-5 Targeting Selectivity and Induces a Therapeutic Effect in Vitro, Br. J. Cancer, vol. 99, Oct. 7, 2008, pp. 1415-1425. |
| Rudikoff et al., Single Amino Acid Substitution altering Antigen-binding Specificity, Proc. Natl Acad Sci., vol. 79,No. 6, 1982, pp. 1979-1983. |
| Segal et al., Introduction: Bispecific Antibodies, Journal of Immunological Methods, vol. 248, No. 1-2, Feb. 1, 2001, pp. 1-6. |
| Shields et al., High Resolution Mapping of the Binding Site on Human IgG1 for Fc Gamma RI, Fc Gamma RII, Fc Gamma RIII, and FcRn and Design of IgG1 Variants With Improved Binding to the Fc Gamma R, Journal of Biological Chemistry, vol. 276, No. 9, Mar. 2, 2001, pp. 6591-6604. |
| Stancovski et al., Mechanistic Aspects of the Opposing Effects of Monoclonal Antibodies to the ERBB2 Receptor on Tumor Growth, Proceedings of the National Academy of Sciences, vol. 88, Nov. 1991, pp. 8691-8695. |
| Stanglmaier et al., Bi20 (fBTA05), a Novel Trifunctional Bispecific Antibody (Anti-CD20 X Anti-CD3), Mediates Efficient Killing of B-cell Lymphoma Cells Even With Very Low CD20 Expression Levels, International Journal of Cancer, vol. 123, Sep. 1, 2008, pp. 1181-1189. |
| Strohl et al., Cell Line Development, Therapeutic Antibody Engineering, Cambridge Woodhead Publishing, 2012, pp. 420-437. |
| Strop et al., Generating Bispecific Human IgG1 and IgG2 antibodies from any Antibody Pair., Journal of Molecular Biology, vol. 420, No. 3, Jul. 13, 2012, pp. 204-219. |
| Suresh et al., Bispecific Monoclonal Antibodies From Hybrid Hybridomas, Methods in Enzymology, vol. 121, 1986, pp. 210-228. |
| Talmadge, J.E., et al., Murine models to evaluate novel and conventional therapeutic strategies for cancer. Am J Pathol. Mar. 2007; 170(3):793-804. |
| Tamaskovic et al., Designed Ankyrin Repeat Proteins (DARPins): From Reserach to Therapy, Methods in Enzymology, vol. 503, 2012, pp. 101-134. |
| Thurber, G.M., et al., Antibody tumor penetration: transport opposed by systemic and antigen-mediated clearance. Adv Drug Deliv Rev. Sep. 2008; 60(12):1421-34. |
| Traunecker et al., Bispecific Single Chain Molecules (Janusins) Target Cytotoxic Lymphocytes on HIV Infected Cell, EMBO Journal, vol. 10, No. 12, Dec. 1991, pp. 3655-3699. |
| Troise et al., Differential Binding of Human Immunoagents and Herceptin to the ErbB2 Receptor, FEBS Journal, vol. 275, No. 20, 2008, pp. 4967-4979. |
| Verheesen et al., Selection by Phage Display of Single Domain Antibodies Specific to Antigens in Their Native Conformation, Methods Mo Bio., Chapter 6, vol. 911, Jul. 12, 2012, pp. 81-104. |
| Vie et al., Human Fusion Proteins Between Interleukin 2 and LgM Heavy Chain Are Cytotoxic for Cells Expressing the Interleukin 2 Receptor, Proc. Natl. Acad. Sci., vol. 89, Dec. 1992, pp. 11337-11341. |
| Vitetta et al., Considering Therapeutic Antibodies, Immunology, Science, vol. 313, No. 5785, Jul. 21, 2006, pp. 308-309. |
| Von Kreudenstein et al., Protein Engineering and the Use of Molecular Modeling and Simulation: The Case of Heterodimeric Fc Engineering, Methods, vol. 65, No. 1, Jan. 1, 2014, pp. 77-94. |
| Von Kreudenstein, T. S., et al., Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design. MAbs. Sep.-Oct. 2013; 5(5):646-654. |
| Voskoglou-Nomikos, T., et al., Clinical predictive value of the in vitro cell line, human xenograft, and mouse allograft preclinical cancer models. Clin Can Res. Sep. 15, 2006; 9(11):4227-39. |
| Wang , Protein Aggregation and Its Inhibition in Biopharmaceutics, International Journal of Pharmaceutics, vol. 289, No. 1-2, Jan. 31, 2005, pp. 1-30. |
| Ward et al., Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli, Nature, vol. 341, No. 6242, Oct. 12, 1989, pp. 544-546. |
| Wesolowski et al., Single Domain Antibodies: Promising Experimental and Therapeutic Tools in Infection and Immunity, Med Microbiol Immunol., vol. 198, No. 3, Aug. 2009, pp. 157-174. |
| Wiens et al., Mutation of a Single Conserved Residue in VH Complementarity-determining Region 2 Results in a Severe Ig Secretion Defect., J Immunology, vol. 167, No. 4, Aug. 2001, pp. 2179-2186. |
| Woods et al., LC-MS Characterization and Purity Assessment of a Prototype Bispecific Antibody, MABS, vol. 5, No. 5, Sep. 1, 2013, pp. 711-722. |
| Wu et al., Arming Antibodies: Prospects and Challenges for Immunoconjugates, Nature Biotech., vol. 23, No. 9, Sep. 2005, pp. 1137-1146. |
| Wu et al., Humanization of a Murine Monoclonal Antibody by Simultaneous Optimization of Framework and CDR Residues, Journal of Molecular Biology, vol. 294, Nov. 1999, pp. 151-162. |
| Xu et al., In Vitro Characterization of Five Humanized OKT3 Effector Function Variant Antibodies, Cell. Immunol., vol. 200, No. 1, Feb. 25, 2000, pp. 16-26. |
| Yarilin, Fundamentals of Immunology, M.: Medicine, 1999, pp. 169-179. |
| Zhu et al., Remodeling Domain Interfaces to Enhance Heterodimer Formation, Protein Science, vol. 6, No. 4, Apr. 1997, pp. 781-788. |
| U.S. Appl. No. 15/409,456, Restriction Requirement dated Jan. 10, 2019. |
| U.S. Appl. No. 15/355,019: Non-Final Office Action dated Jan. 8, 2019, 5 pages. |
| U.S. Appl. No. 14/989,648: Non-Final Office Action dated Mar. 29, 2019. |
| U.S. Appl. No. 15/409,456: Non-Final Office Action dated May 23, 2019. |
| Number | Date | Country | |
|---|---|---|---|
| 20180016347 A1 | Jan 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61645547 | May 2012 | US | |
| 61557262 | Nov 2011 | US | |
| 61556090 | Nov 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13668098 | Nov 2012 | US |
| Child | 15411799 | US |
