RU2810540C2 - DEVELOPMENT OF STABLE HETERODIMERIC ANTIBODY WITH MUTATIONS IN Fc DOMAIN - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: heterodimeric Fc constructs have been proposed to impart thermostability to the antibody. Fc constructs are based on human IgG1 and contain modified CH3 domains including amino acid modifications compared to the wild-type CH3 domain polypeptide. Heat-stable antibodies containing these Fc constructs are also proposed.

EFFECT: invention provides increased stability and purity of the obtained Fc heterodimer variants.

18 cl, 52 dwg, 16 tbl, 15 ex

Description

LINK TO RELATED APPLICATIONS

This application claims priority under 35 USC § 119(e) to US Provisional Patent Application No. 61/556,090, filed November 4, 2011; US Provisional Patent Application Number 61/557262, filed November 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.

TECHNICAL FIELD

The present invention generally provides polypeptide heterodimers, compositions thereof, and methods for preparing and using such polypeptide heterodimers. In particular, the present invention relates to heat-stable multispecific, including bispecific, antibodies containing a heterodimeric Fc domain.

BACKGROUND OF THE ART

Bispecific drugs are antibody molecules that can simultaneously bind two separate and distinct targets or different epitopes of the same antigen. Bispecific antibodies are composed of immunoglobulin domain-based compounds and seek to structurally and functionally mimic components of the antibody molecule. One application of bispecific antibodies is to retarget cytotoxic effector cells of the immune system to enhance the killing of tumor cells, for example, through antibody-dependent cellular cytotoxicity (ADCC). In this case, one arm of the bispecific antibody binds an antigen on the surface of the tumor cell, and the other arm binds a determinant expressed on effector cells. By cross-linking tumor and effector cells, the bispecific antibody not only brings the effector cells closer to the tumor cells, but also simultaneously triggers the activation of the former, which leads to the effective destruction of tumor cells. Bispecific antibodies are also used to enrich tumor tissues with chemo- or radiotherapeutic agents to minimize harmful effects on normal tissue. In this context, one arm of the bispecific antibody binds an antigen expressed on the surface of the cell being targeted for destruction, and the other arm delivers a chemotherapeutic agent, radioactive isotope, or toxin. Moving beyond bispecificity, there is a need to achieve protein drug efficacy by targeting multiple mechanisms simultaneously. Such complex and novel biological effects of a protein drug can be achieved by engineering a protein that binds multiple targets and has multiple functions.

To develop such a multifunctional, multi-target drug, a robust scaffold is required that will provide a basis for fusion with other functional reactive groups or domains that bind the target protein. Ideally, the scaffold should not only provide a scaffold, but also make available to the developed therapeutic agent a variety of other therapeutically important and beneficial properties. A major barrier to the overall development of bispecific and multifunctional antibody therapeutics is the difficulty of obtaining materials of sufficient quality and quantity for both preclinical and clinical studies. There remains a need in the art for polypeptide constructs that contain distinct variable domains as protein binding domains that are linked to a variant Fc region, wherein the variant Fc region contains CH3 domains that are modified to select heterodimers with increased stability and purity.

BRIEF DESCRIPTION OF THE INVENTION

According to one aspect of the present invention, there is provided an isolated heteromultimeric Fc construct comprising a modified heterodimeric CH3 domain, wherein said modified CH3 domain comprises: a first modified CH3 domain polypeptide comprising at least three amino acid modifications relative to a wild-type CH3 domain polypeptide, and a second modified polypeptide a CH3 domain containing at least three amino acid modifications compared to a wild type CH3 domain polypeptide; wherein at least one of the first and second CH3 domain polypeptides contains the amino acid modification K392J, where J is selected from L, I, or an amino acid with a side chain volume not significantly greater than the volume of side chain K; wherein said first and second modified CH3 domain polypeptides preferably form a heterodimeric CH3 domain with a melting point (Tm) of at least about 74° C. and a purity of at least 95%; and wherein the at least one amino acid modification does not affect an amino acid that is located at the boundary between said first and said second CH3 domain polypeptides. In some embodiments, a heteromultimeric Fc construct described herein is provided that contains at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants of these amino acids. In some embodiments, an isolated Fc heteromultimeric construct described herein is provided that contains at least one T350V modification. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a Tm of approximately 77°C or greater. In some embodiments, the modified CH3 domain has a Tm of about 80°C or more. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein the at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising a modification of at least one of amino acids L351, F405, and Y407. In some embodiments, an isolated heteromultimeric Fc construct is provided, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide further comprising a T366 amino acid modification. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide containing amino acid modifications at positions L351, F405, and Y407, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide. , containing amino acid modifications at positions T366, K392 and T394. In one embodiment, there is provided an isolated heteromultimeric Fc construct described herein, wherein said first CH3 domain polypeptide contains amino acid modifications L351Y, F405A and Y407V, and said second CH3 domain polypeptide contains amino acid modifications T366L, K392M and T394W. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein said first CH3 domain polypeptide contains amino acid modifications L351Y, F405A, and Y407V, and said second CH3 domain polypeptide contains amino acid modifications T366L, K392L, and T394W. In a further embodiment, there is provided an isolated heteromultimeric Fc construct described herein, wherein said first CH3 domain polypeptide contains amino acid modifications L351Y, F405A and Y407V, and said second CH3 domain polypeptide contains amino acid modifications T366I, K392M and T394W. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein said first CH3 domain polypeptide comprises amino acid modifications L351Y, F405A and Y407V, and said second CH3 domain polypeptide contains amino acid modifications T366I, K392L and T394W. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein at least one of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide containing an amino acid modification at position S400. In an additional embodiment, an isolated heteromultimeric Fc construct is provided as described herein, comprising an S400Z modification in which 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 some embodiments, an isolated heteromultimeric Fc construct is provided as described herein, wherein said first CH3 domain polypeptide comprises an amino acid modification selected from S400E and S400R. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein at least one of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide containing an amino acid modification at position N390. In some embodiments, the N390 modification is N390Z, where Z is selected from a positively charged amino acid and a negatively charged amino acid. In one embodiment, N390Z is N390R. In some embodiments of the isolated heteromultimeric Fc construct described herein, said first CH3 domain polypeptide is a modified CH3 domain polypeptide containing the S400E amino acid modification, and said second CH3 domain polypeptide is a modified CH3 domain polypeptide containing the N390R amino acid modification. In some embodiments of the isolated heteromultimeric Fc construct described herein, each of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide, one modified CH3 domain polypeptide contains a Q347R amino acid modification, and the other modified CH3 domain polypeptide contains a K360E amino acid modification .

In one aspect, an isolated heteromultimeric Fc construct is provided comprising a modified heterodimeric CH3 domain, wherein said modified CH3 domain comprises: a first modified CH3 domain polypeptide containing at least three amino acid modifications relative to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide containing at least three amino acid modifications compared to the wild type CH3 domain polypeptide; wherein at least one of the first and second polypeptides of the CH3 domain contains the amino acid modification K392J, where J is selected from L, I or an amino acid with a side chain volume not significantly exceeding the volume of the side chain K; wherein said first and second modified CH3 domain polypeptides preferably form a heterodimeric CH3 domain with a melting point (Tm) of at least about 74° C. and a purity of at least 95%; and wherein the at least one amino acid modification does not affect an amino acid that is located at the boundary between said first and said second CH3 domain polypeptides. In some embodiments, a heteromultimeric Fc construct described herein is provided that contains at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants of these amino acids. In some embodiments, a heteromultimeric Fc construct is provided. embodiments of the proposed isolated heteromultimeric construct Fc, described in this application, which contains at least one modification T350V. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a Tm of approximately 77°C or greater. In some embodiments, the modified CH3 domain has a Tm of about 80°C or more. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising a modification of at least one of amino acids K409 and T411. In some embodiments, an isolated Fc heteromultimeric construct described herein is provided containing at least one of the K409F, T411E, and T411D modifications. In some embodiments, an isolated heteromultimeric Fc construct is provided as described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide containing a D399 amino acid modification. In some embodiments, the amino acid modification D399 is at least one of D399R and D399K.

In one aspect, an isolated heteromultimeric Fc construct is provided comprising a modified heterodimeric CH3 domain, wherein said modified CH3 domain comprises: a first modified CH3 domain polypeptide comprising at least three amino acid modifications relative to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide containing at least three amino acid modifications compared to the wild type CH3 domain polypeptide; wherein at least one of the first and second CH3 domain polypeptides includes the amino acid modification K392J, where J is selected from L, I, or an amino acid with a side chain volume not significantly greater than the volume of the side chain K; wherein said first and second modified CH3 domain polypeptides preferably form a heterodimeric CH3 domain with a melting point (Tm) of at least about 74° C. and a purity of at least 95%; and wherein the at least one amino acid modification does not affect an amino acid that is located at the boundary between said first and said second CH3 domain polypeptides. Some embodiments provide a heteromultimeric Fc construct described herein that includes at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants of these amino acids. In some embodiments, an isolated Fc heteromultimeric construct described herein is provided that includes at least one T350V modification. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a Tm of approximately 77°C or greater. In some embodiments, the modified CH3 domain has a Tm of about 80°C or more. In some embodiments, an isolated heteromultimeric Fc construct described herein, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide containing at least one amino acid modification selected from K409F, T411E, and T411D, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide containing at least one amino acid modification selected from Y407A, Y407I, Y407V, D399R and D399K. In some embodiments, any of the isolated heteromultimeric Fc constructs described herein are provided, further comprising a first modified CH3 domain containing one of the amino acid modifications T366V, T366I, T366A, T366M, and T366L; and a second modified CH3 domain containing the amino acid modification L351Y. In some embodiments, any of the isolated heteromultimeric Fc constructs described herein are provided, comprising a first modified CH3 domain containing one of amino acid modifications K392L or K392E; and a second modified CH3 domain containing one of the amino acid modifications S400R or S400V.

This application provides an isolated heteromultimeric Fc construct containing a modified CH3 domain, comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, wherein each modified CH3 domain polypeptide includes at least four amino acid mutations, wherein at least one of the above the first and said second modified CH3 domain polypeptides comprise 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 preferably form a heterodimeric CH3 domain with a melting point (Mn ), being at least about 70°C, and having a purity of at least 90%. In one embodiment, an isolated heteromultimeric Fc construct is provided wherein said first modified CH3 domain polypeptide includes amino acid modifications at positions F405 and Y407 and said second modified CH3 domain polypeptide includes an amino acid modification at position T394. In one embodiment, an isolated heteromultimeric Fc construct is provided in which the first modified CH3 domain polypeptide includes an amino acid modification at position L351. In some embodiments, an isolated heteromultimer is provided as described herein, wherein said second modified CH3 domain polypeptide includes a modification at at least one of positions T366 and K392. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the modified CH3 domain has a melting point (Tm) of at least about 75° C. and is obtained at a purity of at least about 95%. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the at least one modified CH3 domain polypeptide includes at least one of the amino acid modifications N390R, S400E, and S400R. In some embodiments, an isolated heteromultimer is provided as described herein, wherein one of the first and second modified CH3 domain polypeptides includes an amino acid modification at position 347 and the other modified CH3 domain polypeptide includes an amino acid modification at position 360. In some embodiments, an isolated heteromultimer is provided. a heteromultimer described herein, wherein at least one of said first and second modified CH3 domain polypeptides includes a T350V amino acid modification. In specific embodiments, an isolated heteromultimer is provided as described herein, wherein said first modified CH3 domain polypeptide includes at least one amino acid modification selected from L351Y, F405A, and Y407V; and said second modified CH3 domain polypeptide includes at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In some embodiments described herein, an isolated heteromultimer is provided in which the first modified CH3 domain polypeptide includes amino acid modifications at positions D399 and Y407 and the second modified CH3 domain polypeptide includes amino acid modifications at positions K409 and T411. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the first CH3 domain polypeptide includes an amino acid modification at position L351 and the second modified CH3 domain polypeptide includes amino acid modifications at positions T366 and K392. In specific embodiments, isolated heteromultimers are provided as described herein, wherein at least one of the first and second CH3 domain polypeptides includes the T350V amino acid modification. Some embodiments provide the isolated heteromultimers described herein, wherein the modified CH3 domain has a melting point (Tm) of at least about 75° C. or greater and is obtained at a purity of at least about 95%. Some embodiments provide the isolated heteromultimeric Fc constructs described herein, wherein said first modified CH3 domain polypeptide includes amino acid modifications selected from L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A, and Y407V; and said second modified CH3 domain polypeptide includes amino acid modifications selected from T366V, T366I, T366L, T366M, N390D, N390E, K392L, K392I, K392D, K392E, K409F, K409W, T411D and T411E.

This application provides an isolated heteromultimeric Fc construct containing a modified CH3 domain, comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, wherein each modified CH3 domain polypeptide includes mutations of at least three amino acids, wherein one of said first and said second modified CH3 domain polypeptides comprises a mutation selected from T411E and T411D, wherein said first and second modified CH3 domain polypeptides preferably form a heterodimeric CH3 domain with a melting point (Tm) of at least about 70° C. and a purity of at least 90%. In one embodiment, an isolated heteromultimeric Fc construct is provided wherein said first modified CH3 domain polypeptide includes amino acid modifications at positions F405 and Y407 and said second modified CH3 domain polypeptide includes an amino acid modification at position T394. In one embodiment, an isolated heteromultimeric Fc construct is provided in which the first modified CH3 domain polypeptide includes an amino acid modification at position L351. In some embodiments, an isolated heteromultimer is provided as described herein, wherein said second modified CH3 domain polypeptide includes a modification at at least one of positions T366 and K392. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the modified CH3 domain has a melting point (Tm) of at least about 75° C. and is obtained at a purity of at least about 95%. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the at least one modified CH3 domain polypeptide includes at least one of the amino acid modifications N390R, S400E, and S400R. In some embodiments, an isolated heteromultimer is provided as described herein, wherein one of the first and second modified CH3 domain polypeptides includes an amino acid modification at position 347 and the other modified CH3 domain polypeptide includes an amino acid modification at position 360. In some embodiments, an isolated heteromultimer is provided. a heteromultimer described herein, wherein at least one of said first and second modified CH3 domain polypeptides includes a T350V amino acid modification. In specific embodiments, an isolated heteromultimer is provided as described herein, wherein said first modified CH3 domain polypeptide includes at least one amino acid modification selected from L351Y, F405A, and Y407V; and said second modified CH3 domain polypeptide includes at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In some embodiments described herein, an isolated heteromultimer is provided in which the first modified CH3 domain polypeptide includes amino acid modifications at positions D399 and Y407 and the second modified CH3 domain polypeptide includes amino acid modifications at positions K409 and T411. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the first CH3 domain polypeptide includes an amino acid modification at position L351 and the second modified CH3 domain polypeptide includes amino acid modifications at positions T366 and K392. In specific embodiments, isolated heteromultimers are provided as described herein, wherein at least one of the first and second CH3 domain polypeptides includes the T350V amino acid modification. Some embodiments provide the isolated heteromultimers described herein, wherein the modified CH3 domain has a melting point (Tm) of at least about 75° C. or greater and is obtained at a purity of at least about 95%. Some embodiments provide the isolated heteromultimeric Fc constructs described herein, wherein said first modified CH3 domain polypeptide includes amino acid modifications selected from L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A, and Y407V; and said second modified CH3 domain polypeptide includes amino acid modifications selected from T366V, T366I, T366L, T366M, N390D, N390E, K392L, K392I, K392D, K392E, K409F, K409W, T411D and T411E.

This application provides an isolated heteromultimeric Fc construct containing a modified CH3 domain containing a first modified CH3 domain polypeptide containing amino acid modifications L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide containing amino acid modifications T366I, K392M and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T366I, K392L and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T366L, K392M and T394W.

In some aspects, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T366L, K392L and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising the amino acid modifications T350V, L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide containing amino acid modifications T350V, T366L, K392L and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T350V, T366L, K392M and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T350V, T366L, N390R, K392M and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T350V, T366L, K392L and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising the amino acid modifications T366V, K392L, K409F and T411E; and a second modified CH3 domain polypeptide containing amino acid modifications L351Y, D399R and Y407A.

In one aspect, an isolated heteromultimeric Fc construct is provided comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising the amino acid modifications T366V, K392L, K409F and T411E; and a second modified CH3 domain polypeptide containing amino acid modifications L351Y, D399R, S400R and Y407A.

According to one aspect of the present invention, there is provided an isolated heteromultimer comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain containing amino acid mutations that promote heterodimer formation, wherein the heterodimeric Fc region further comprises a variant CH2 domain comprising at least one asymmetric amino acid modification that promotes selective binding of the Fc-gamma receptor. In one embodiment, the variant CH2 domain selectively binds the Fc-gamma IIIa receptor over the wild-type CH2 domain. In one embodiment, the modified CH3 domain has a melting point (Tm) of about 70°C or more. In some embodiments, the modified CH3 domain has a melting point (Tm) of at least about 75°C. In some embodiments, the modified CH3 domain has a melting point (Tm) of at least about 80°C.

In another aspect, an isolated heteromultimer is provided comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain containing amino acid mutations, wherein the modified CH3 domain has a melting temperature (Tm) of about 70° C. or greater, and wherein this modified CH3 domain promotes the formation of a heterodimeric Fc region with increased stability compared to the CH3 domain, which does not contain amino acid mutations. In one embodiment, the heterodimeric Fc region does not contain an additional disulfide bond in the CH3 domain compared to the wild type Fc region. In an alternative embodiment, the heterodimeric Fc region contains at least one additional disulfide bond in the modified CH3 domain compared to the wild type Fc region, provided that the melting temperature (Tm) is approximately 70°C or more in the absence of an additional disulfide bond . In another embodiment, the heterodimeric Fc region contains at least one additional disulfide bond in the modified CH3 domain compared to the wild type Fc region, and wherein the modified CH3 domain has a melting temperature (Tm) of approximately 11.5°C or more.

In one embodiment, an isolated heteromultimer is provided comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain containing amino acid mutations, wherein the modified CH3 domain has a melting temperature (Tm) of about 70° C. or greater, and the heterodimeric Fc region is obtained with a purity of greater than about 90%, or the heterodimeric Fc region is obtained with a purity of approximately 95% or greater, or the heterodimeric Fc region is obtained with a purity of approximately 98% or greater.

Also in some embodiments, an isolated heteromultimer is provided containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing one or more amino acid mutations that result in the formation of a heterodimeric Fc region with increased stability compared to the CH3 domain which does not contain said one or more amino acid mutations, wherein the modified CH3 domain has a melting temperature (Tm) of about 70°C or more, or a Tm of about 71°C or more, or a Tm of about 74°C or more . In another embodiment, the heterodimeric Fc region is prepared in solution with a purity of about 98% or greater and a Tm of approximately 73° C., or wherein the heterodimeric Fc region is prepared with a purity of approximately 90% or greater and a Tm of approximately 75°C.

In some embodiments, an isolated heteromultimer is provided comprising a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first and a second CH3 domain polypeptide, wherein at least one of the first and second CH3 domain polypeptides includes a T350V amino acid modification. In some embodiments, an isolated heteromultimer is provided containing a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide containing the T350V amino acid modification and a second CH3 domain polypeptide also containing the T350V amino acid modification. In some embodiments, an isolated heteromultimer is provided containing a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide containing amino acid modifications at positions F405 and Y407, and a second CH3 domain polypeptide containing an amino acid modification at position T394. The first CH3 domain polypeptide includes amino acid modifications at positions D399 and Y407 and the second CH3 domain polypeptide includes amino acid modifications at positions K409 and T411. In some embodiments, an isolated heteromultimer is provided containing a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide containing the amino acid modifications L351Y and Y407A, and a second CH3 domain polypeptide containing the amino acid modifications T366A and K409F. In one aspect, the first CH3 domain polypeptide or the second CH3 domain polypeptide includes an additional 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, F405S, 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 some embodiments, an isolated heteromultimer is provided comprising a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide comprising the T350V and L351Y amino acid modifications and a second CH3 domain polypeptide also comprising the T350V and L351Y amino acid modifications.

In another embodiment, an isolated heteromultimer is provided containing a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide containing the amino acid modification Y407A and a second CH3 domain polypeptide containing the amino acid modifications T366A and K409F. In one aspect, the first CH3 domain polypeptide or the second CH3 domain polypeptide includes additional amino acid modifications K392E, T411E, D399R and S400R. In another aspect, the first CH3 domain polypeptide includes the amino acid modifications D399R, S400R and Y407A and the second CH3 domain polypeptide includes the amino acid modifications T366A, K409F, K392E and T411E. In a further embodiment, the modified CH3 domain has a melting point (Tm) of about 74° C. or greater, and the heterodimer has a purity of about 95% or greater.

In another embodiment, an isolated heteromultimer is provided containing a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide containing an amino acid modification at position L351 and an amino acid modification Y407A, and a second CH3 domain polypeptide containing 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, L351I, L351D, L351R, or L351F. In another aspect, the amino acid modification at position Y407 is selected from Y407A, Y407V or Y407S. In another further aspect, the amino acid modification at position T366 is selected from T366A, T366I, T366L, T366M, T366Y, T366S, T366C, T366V or T366W. In one embodiment, the modified CH3 domain has a melting point (Tm) of about 75° C. or more and the heterodimer has a purity of about 90% or more.

In another embodiment, an isolated heteromultimer is provided containing a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide containing an amino acid modification at position F405 and amino acid modifications L351Y and Y407V, and a second CH3 domain polypeptide containing the T394W amino acid modification. In one aspect, the first CH3 domain polypeptide or the second CH3 domain polypeptide includes amino acid modifications at positions K392, T411, T366, L368, or S400. The amino acid modification at position F405 is F405A, F405I, F405M, F405T, F405S, 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, S400D, S400R or S400K. The amino acid modification at position T366 is T366A, T366I, T366L, T366M, T366Y, T366S, T366C, T366V or T366W. The amino acid modification at position L368 is L368D, L368R, L368T, L368M, L368V, L368F, L368S and L368A.

In another embodiment, an isolated heteromultimer is provided containing a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide containing the amino acid modifications L351Y, F405A and Y407V, and a second CH3 domain polypeptide containing the amino acid modification T394W. In one aspect, the second CH3 domain polypeptide includes the amino acid modification T366L or T366I.

In yet another embodiment, an isolated heteromultimer is provided comprising a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide containing at least one of the amino acid modifications Y349C, F405A, and Y407V, and a second CH3 domain polypeptide containing the amino acid modifications T366I, K392M and T394W.

In some embodiments, an isolated heteromultimer is provided comprising a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V, and a second CH3 domain polypeptide comprising amino acid modifications K392M and T394W, and one from T366L and T366I.

In another embodiment, an isolated heteromultimer is provided containing a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide containing the amino acid modifications F405A and Y407V, and a second CH3 domain polypeptide containing the amino acid modifications T366L and T394W.

In another embodiment, an isolated heteromultimer is provided containing a heterodimeric Fc region, wherein the heterodimeric Fc region includes a first CH3 domain polypeptide containing the amino acid modifications F405A and Y407V, and a second CH3 domain polypeptide containing the amino acid modifications T366I and T394W.

In some embodiments of the heteromultimer, a bispecific antibody or a multispecific antibody is provided.

In another embodiment, a composition is provided comprising a heteromultimer of the present invention and a pharmaceutically acceptable carrier.

In another embodiment, a host cell is provided containing a nucleic acid encoding a heteromultimer of the present invention.

In some embodiments, a heteromultimer is provided, wherein said heteromultimer includes at least one therapeutic antibody. In one aspect, the therapeutic antibody is selected from the group consisting of abagovomab, adalimumab, alemtuzumab, aurograb, bapineizumab, basiliximab, belimumab, bevacizumab, briakinumab, canakinumab, catumaxomab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, g aliximab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lumiliximab, mepolizumab, motavizumab, muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab, ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab, reslizumab ba, rituximab, teplizumab, tocilizumab/atlizumab, tositumomab, trastuzumab , Proxinium™, Rencarex™, ustekinumab and zalutumumab.

In another embodiment of the heteromultimer, the present invention provides a method of treating cancer in a patient suffering from cancer characterized by the presence of a cancer antigen, comprising administering to said patient a therapeutically effective amount of the heteromultimer.

In another embodiment of the heteromultimer, the present invention provides a method of treating an immunological disorder in a patient suffering from an immunological disorder characterized by the presence of an immune antigen, comprising administering to the patient a therapeutically effective amount of the heteromultimer.

In some embodiments, the modified Fc region used in the heteromultimeric constructs described herein includes type G immunoglobulins, for example, immunoglobulins referred to as immunoglobulin class 2 (IgG2) or immunoglobulin class 3 (IgG3). In some embodiments, the modified Fc region used in the heteromultimeric constructs described herein includes immunoglobulin M, or IgM. In some embodiments, the modified Fc region used in the heteromultimeric constructs described herein includes immunoglobulin A, or IgA. In some embodiments, the modified Fc region used in the heteromultimeric constructs described herein includes immunoglobulin D, or IgD. In some embodiments, the modified Fc region used in the heteromultimeric constructs described herein includes immunoglobulin E, or IgE. In some embodiments, the modified Fc region used in the heteromultimeric constructs described herein includes all immunoglobulin G isotype variants, e.g., immunoglobulins referred to as immunoglobulin class 1 (IgG1), class 2 (IgG2), class 3 (IgG3) ) or class 4 (IgG4).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a three-dimensional graphical structure of a wild-type antibody, showing the CH3 (top), CH2 (middle), and receptor regions. The dashed box on the left is enlarged on the right and shows two sections, Section 1 and Section 2, of the CH3 target region.

Figure 2 shows a three-dimensional graphical representation of the wild-type residue at position 368.

Figure 3 shows a three-dimensional graphical representation of Site 1, which shows the mutation at position 368.

Figure 4 shows a three-dimensional graphical representation of additional mutations in Site 2.

Figure 5 is a table of computer calculations of the collision index, contact area differences, stacking differences, electrostatic interaction energy differences, and overall “affinity score” for the first three options AZ1, AZ2, and AZ3.

Figure 6 is a three-dimensional graphical representation showing variants AZ2 and AZ3, which "build on" variant AZ1.

Figure 7 shows three-dimensional graphical representations of variants AZ2 and AZ3.

Figure 8 shows the same table as Figure 5, but only for heterodimers AZ1, AZ2 and AZ3, and potential homodimers. The affinity index is not shown for homodimers because it is not applicable.

Figure 9 shows a three-dimensional graphical representation of wild-type AZ4 (left) and mutated AZ4 (right).

Figure 10 is the same table as Figure 5, showing computer calculations for the AZ4 heterodimer and potential homodimers.

Figure 11 shows a graphical representation of the CH3 variants AZ5 (left) and AZ6 (right).

Figure 12 is the same table as Figure 5, showing the computer calculation results for AZ4, AZ5 and AZ6.

Figure 13 shows a three-dimensional graphical representation of the antibody on the left, depicting variant binding features at the receptor site using a heterodimeric approach.

Figure 14 shows a schematic representation of an IgG molecule.

Figure 15 shows an alignment of multiple Fcγ receptor sequences. Genebank/Uniprot sequence identification numbers: FcγRIIA (sp P12318), FcγRIIB (sp P31994), FcγRIIC (gi 1261 16592), FcγRIIIA (sp P08637), FcγRIIIB (sp O75015).

Figure 16 shows a diagram of the crystal structure of the Fc-FcγRIIIb complex [PDB ID: 1T83, Radaev & Sun]. A 1:1 complex of Fc and Fcγ receptor is observed, with asymmetric contact between the two chains of Fc and FcγR.

Figure 17 shows a diagram of multifunctional molecules based on an asymmetric Fc backbone formed by the heterodimeric variants described herein: Asymmetric Fc backbone and Asymmetric Fc monomer arm of IgG.

Figure 18 shows a diagram of multifunctional molecules based on an asymmetric Fc framework formed by the heterodimeric variants described in this application: Asymmetric Fc-monospecific IgG arms and Asymmetric Fc-bispecific arms IgG (common light chain).

Figure 19 shows an image of multifunctional molecules based on the asymmetric Fc framework formed by the heterodimeric variants described in this application. Asymmetric Fc-bispecific arms of IgG and a functional molecule such as a toxin.

Figure 20 illustrates multifunctional molecules based on the asymmetric Fc scaffold formed by the heterodimeric variants described herein: Asymmetric Fc-individual scFv arm and Asymmetric Fc-bispecific scFv arms.

Figure 21 illustrates alternative multifunctional molecules based on the asymmetric Fc scaffold formed by the heterodimeric variants described herein: Asymmetric Fc trispecific scFv arms and Asymmetric Fc tetraspecific scFv arms.

Figure 22 shows an asymmetric pattern of mutations on one side of the Fc to improve selectivity for FcγR and shows the productive side for interactions with FcγR and the non-productive side with wild-type interactions. Mutations on the nonproductive side of the Fc can be introduced to block interactions with FcR and shift the polarity of the Fc so that the interaction occurs only on the productive side.

Figure 23 shows the amino acid sequence of wild-type human IgG1.

Figure 24 shows the iterative development process of an Fc heterodimer, combining positive and negative development strategies detailed below.

Figures 25A-25C show the in vitro assay used to determine heterodimer purity. This assay is based on a full-length monospecific antibody framework with two Fc heavy chains of different molecular weights; heavy chain A contains a C-terminal histidine tag (His) and heavy chain B contains a C-terminal cleavable tag - monomeric red fluorescent protein (RFP). The two heavy chains A (His) and B (RFP) are expressed in different relative ratios together with a fixed amount of light chain, allowing for 3 possible types of dimers with different molecular weights: a) homodimer Chain A (His/Chain A (His) (~ 150 kDa) b) Chain A (His)/Chain B (RFP) heterodimer (~175 kDa); c) Chain B (RFP)/Chain B (RFP) homodimer (~200 kDa). Following expression as described in Example 2, the ratio of heterodimer versus two homodimers is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, which allows separation of the 3 dimer species by molecular weight. The PAGE/SDS gels were stained with Coomassie brilliant blue. In Figure 25A: the variants examined were only wild-type Chain A (His); wild-type Chain B (RFP) only; Wild type A chain (His) plus B chain (RFP); Control 1 chain A (His) plus chain B (RFP), the heterodimer purity of which was known to be >95%. The composition of the dimer bands was checked by Western blotting with antibodies directed against the IgG-Fc (anti-Fc), against the monomeric RFP tag (anti-mRFP), and against the histidine tag (anti-His), as illustrated above. A single His/His homodimer band, a double His/RFP heterodimer band, and several RFP homodimer bands were visible on the SDS-PAGE gel. These few bands were an artifact of monomeric RFP labeling and were ensured that this did not affect the physical properties of the Fc heterodimer. Figure 25 B: confirmed the validity of the SDS-PAGE analysis using published Controls 1-4 Fc heterodimer variants as controls, see Table A. These variants were expressed with different relative ratios of chain A (His) and chain B (RFP): In particular , for LC,HC_His,HC_mRFP the ratio 1:3 is equivalent to the ratio 25%, 10%, 65%; a 1:1 ratio is equivalent to a ratio of 25%, 20%, 55% and a 3:1 ratio is equivalent to a ratio of 25%, 40%, 35%, respectively (the apparent 1:1 ratio of expression of chain A (His) to chain B (RFP) determined as close to 20%/55% (His/RFP) for wild type (WT) Fc). Figure 25C shows a non-reducing SDS-PAGE assay to determine the heterodimer purity of the Framework 1 variants. Fc variants were expressed with different relative ratios of chain A (His) to chain B (RFP) and analyzed by PAGE/SDS under non-reducing conditions described in Figure 2. In particular, for LC,HC_His,HC_mRFP, the ratio of 1:3 is equivalent to the ratio of 25%, 10%, 65%; a 1:1 ratio is equivalent to a ratio of 25%, 20%, 55% and a 3:1 ratio is equivalent to a ratio of 25%, 40%, 35%, respectively (the apparent 1:1 ratio of expression of chain A (His) to chain B (RFP) determined as close to 20%/55% (His/RFP) for Fc DT).

Figures 26A-26B show Fc heterodimer variants expressed at a specific ratio of A chain (His) to B chain (RFP) (see Table 2), purified by Protein A affinity chromatography and analyzed by SDS-PAGE under non-reducing conditions. described in Figures 25A-25C. Figure 26A illustrates the classification of heterodimers based on purity observed by visual inspection of the SDS-PAGE results. For comparison, an equivalent amount of protein A purified product was loaded onto the gel. This purity determination based on SDS-PAGE under non-reducing conditions was confirmed for selected variants using a combination of liquid chromatography-mass spectrometry (LC/MS) (see Figure 28). Figure 26B shows examples of SDS-PAGE results for selected heterodimer variants purified on Protein A (AZ94, AZ86, AZ70, AZ33 and AZ34).

Figures 27A-27B illustrate differential scanning calorimetry (DSC) analyzes to determine the melting point of the CH3-CH3 heterodimeric domain formed by variants of the heterodimer described herein. Two independent methods were used to determine melting points. Figure 27A shows thermograms fitted to 4 independent non-biphasic transitions and optimized to obtain values for the CH2 and Fab transitions close to the published values for Herceptin in the literature of ~72°C (CH2) and ~82°C (Fab). Figure 27B shows the normalized and background-corrected thermograms for the heterodimer variants that were subtracted from the DT to obtain a positive and negative difference peak for the CH3 transition only.

Figure 28 illustrates the LC/MS analysis of the AZ70 variant as described in Example 2. The expected (calculated average) masses of the glycosylated heterodimer and homodimers are indicated. The region corresponding to the heterodimer mass includes major peaks corresponding to the loss of glycine (−57 Da) and the insertion of 1 or 2 hexoses (+162 Da and +324 Da, respectively). Heterodimer purity is classified as >90% if there are no significant peaks corresponding to any of the homodimers.

Figures 29A-29D show the CH3:Fc DT interface in FIG. 29A, AZ6 in FIG. 29B, AZ33 in Fig. 29C, AZ19 in FIG. 29D. Detailed computer analysis described in the detailed description section and comparison of variants with DT showed that one of the reasons for the decrease in stability of the original AZ33 heterodimer compared to DT was the loss of interaction/folding of the Y407 and T366 core. The original AZ33 exhibits suboptimal folding of this hydrophobic core, illustrated in FIG. 29B, suggesting that optimization of this region, especially at position T366, will improve the stability of AZ33. This optimization is shown in Fig. 29C and Fig. 29D with replacements T366I and T366L. The results of this experiment correlate with the results of the structural analysis and show that the T366L substitution results in the most significant improvement in Tm. See Example 5.

Figure 30 illustrates the usefulness and importance of conformational dynamics analysis using the original AZ8 scaffold 1 as an example. The structure after computational mutagenesis (scaffold conformation close to that of DT) was superimposed on a typical structure obtained using molecular dynamics (MD) simulations for 50 ns . The figure highlights a large conformational difference in the D399-S400 loop region of the AZ8 variant compared to DT, which, in turn, exposes the hydrophobic core to the solvent, which leads to a decrease in the stability of the AZ8 heterodimer.

Figures 31A-31C illustrate how information obtained from detailed computer analysis and MD simulations is used to describe a positive development strategy. Figure 30 illustrates that one of the reasons for the reduced stability of AZ8 compared to DT is the weakened interaction of the 399-400 loop with 409, which is mainly due to the loss of F405 stacking interactions (see comparison of Figure 31A (DT) with Figure 31B (AZ8) ). One positive design strategy was to optimize the hydrophobic folding of the region to stabilize the 399–400 loop conformation. This was achieved by introducing the K392M mutation, which is shown in Figure 31 C. In FIG. 31C shows the AZ33 heterodimer, which has a Tm of 74°C, compared to 68°C for the original negative design AZ8.

Figures 32A-32B illustrate the dynamics of an Fc molecule observed using molecular dynamics principal component analysis. In FIG. 32A shows a frame trace of an Fc structure as a reference. In FIG. 32B and C show a superposition of the dynamics observed for the 2 most important modes of motion of the Fc structure. In the CH2 domains, chains A and B showed significant opening/closing motion relative to each other, whereas the CH3 domains were relatively stationary. Mutations at the CH3 boundary affected the relative flexibility and dynamics of this opening/closing movement in the CH2 domains.

Figures 33A-33C illustrate the stacking of the hydrophobic core of the two frame options 2 compared to the DT. In FIG. 33A represents Fc DT; in Fig. 33B is represented by AZ63; and Fig. 33C introduced AZ70. Detailed computer analysis of the original scaffold 2 variant suggested that the loss of the Y407-T366 interaction in the WT core is one of the reasons for the reduced stability of the original scaffold 2 variants relative to WT. The loss of Y407-T366 is partially compensated by the K409F mutations, but, as shown in Figure 33 B, in particular, the T366A mutation leaves a cavity in the hydrophobic core, which destabilizes this variant compared to WT. Introduction of additional mutations T366V_L351Y into this hydrophobic core, as shown for the Fc variant AZ70 in FIG. 33C, proved successful; AZ70 has an experimentally determined Tm of 75.5°C. See Table 4 and Example 6.

Figures 34A-34C illustrate the interactions of the loop 399-400 of the two frame options 2 compared to the DT: FIG. 34A represents Fc DT; in Fig. 34 V is represented by AZ63; and Fig. 34C is represented by AZ94. Detailed computer analysis of the original scaffold 2 suggested that the loss of the K409-D399 DT salt bridge (Fig. 34A) as a result of the K409F mutation and, consequently, the unsaturation of D399 (Fig. 34B) leads to a more “open” conformation of loop 399-400. Moreover, this leads to greater exposure of the hydrophobic core to the solvent and additional destabilization of this variant compared to DT. One strategy used to stabilize the 399-400 loop and compensate for the loss of the K409-D399 interaction is the creation of additional salt bridges D399R-T411E and S400R-K392E, shown in FIG. 34С for variant AZ94. The experimental results showed a purity of >95% and a Tm of 74°C. See Table 4 and Example 6. Additionally, although AZ94 had significantly greater purity and stability compared to the original scaffold 2 (purity < 90%, Tm is 71°C), mutations in the hydrophobic core in AZ94 are less favored than " best" mutations in the hydrophobic core found in the AZ70 variant (Figure 33). Because the mutations in the hydrophobic core in AZ70 (T366V_L351Y) are distant from the salt bridge mutations in loop 399-400 of AZ94, the combination of amino acid mutations in AZ70 and additional mutations in AZ94 is expected to result in a higher melting temperature than either AZ70 or AZ94. Such a combination can be tested as described in Examples 1-4.

Figure 35 illustrates the association constant (Ka(M ^-1 )) of homodimeric Fc IgG1, heterodimeric variants het1 (Control 1) A:Y349C_T366S_L368A_Y407V/B:S354C_T366W and het2 (Control 4) A:K409D_K392D/B:D399K_D35 6K to bind to six receptors Fc-gamma. There is a tendency for heterodimeric Fc variants to show a slight change in binding to Fc-gamma receptors compared to wild-type IgG1 Fc. See Example 7.

Figure 36A shows the relative binding strength of wild-type IgG1 Fc and various homodimeric and asymmetrically mutated forms thereof to receptors IIbF, IIbY and IIaR, compared with the binding strength of wild-type control. (HomoFc+S267D) refers to the binding strength of the homodimeric Fc to the S267D mutation on both strands. (HetFc + asymmS267D) refers to the binding strength of the heterodimeric Fc to the S267D mutation introduced into one of the two Fc chains. The average binding strength obtained for introducing a mutation into each of the two Fc chains is shown. Introducing a given mutation into one strand reduced the binding strength to approximately half the strength observed for the same mutation in a homodimer. (HetFc + asymS267D + asymE269K) refers to the binding strength of the heterodimeric Fc to both the S267D and E269K mutations introduced in an asymmetric manner into one of the two Fc chains. The E269K mutation blocks the interaction of FcγR with one of the Fc surfaces and is capable of reducing the binding strength by approximately half of that observed for the variant with asymmetric S267D (HetFc+S267D) alone. Here hetFc includes the CH3 mutations indicated for the het2 variant (Control 4) in Figure 35.

Figure 36 B shows the association constant (Ka(M ^-1 )) of various Fcs and their variants with a variety of FcγRIIa, FcγIIIb and FcγRIIIa allotypes. Wild-type IgG1 Ka Fc with different Fcγ receptors are shown as bars with horizontal shading. Vertical hatched bars (homodimeric backbone2) represent Ka of homodimeric Fc with mutations S239D/D265S/I332E/S298A. Columns with slanted shading 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 helps to achieve increased selectivity for receptors IIIa and IIa/IIb. Here, the heterodimeric Fc includes the CH3 mutations indicated for the het2 variant (Control 4) in Figure 35.

Figure 36C shows the association association constant (Ka(M ^-1 )) for wild-type IgG1 and three other variants containing homodimeric or asymmetric mutations in the CH2 domain of the Fc region. Wild-type Ka Fc is shown as bars with grid shading. The Ka variant of Fc with the major mutation in the backbone S239D/K326E/A330L/I332E/S298A introduced in a homodimeric manner (homodimeric backbone 1) into both Fc chains is presented as bars with slanted shading. Introduction of the corresponding mutations in an asymmetric manner into the A and B chains of the heterodimeric Fc (heterobase 1) is presented in the form of horizontally shaded bars. Vertical hatched bars represent an asymmetric variant containing the E269K mutation (heterobase1 + PD). Here, the heterodimeric Fc includes the CH3 mutations indicated for the het2 variant (Control 4) in Figure 35.

Figure 37 shows Table 6, which provides a list of CH3 domain options based on the third phase of development described in Example 5 for Framework 1.

Figure 38 shows Table 7, which provides a list of modified CH3 domains based on the third phase of development described in Example 6 for Framework 2.

Figure 39A-39B illustrates the determination of the purity of variants without any C-terminal tags using LC/MS. In FIG. 39A shows the LC/MS spectra of one typical variant (AZ162: L351Y_F405A_Y407V/ T366L_K392L_T394W). This variant was expressed by transient co-expression as described in the Examples using 3 different ratios of heavy chain A to heavy chain B: 1:1.5 (AZ133-1), 1:1 (AZ133-2) and 1.5:1 (AZ133-3). Samples were purified and deglycosylated using Endo S endoglycosadase for 1 h at 37°C. Before MS analysis, samples were applied to a Poros R2 column and eluted with a gradient of 20-90% acetonitrile, 0.2% formic acid (FA) over 3 minutes. The peak from the LC column was analyzed using an LTQ-Orbitrap XL mass spectrometer (cone voltage: 50 V, cylindrical lens: 215 V; Fourier transform (FT) resolution: 7500) and installed Promass software to obtain molecular weight profiles. In FIG. 39B shows LC/MS spectra for sample Control 2, which is a peak-to-valley design. This variant was expressed by transient co-expression as described in the Examples using 3 different ratios of heavy chain A to heavy chain B: 1:1.5 (Control 2-1), 1:1 (Control 2-2) and 1.5 :1 (Control 2-3). Samples were purified and deglycosylated using Endo S for 1 h at 37°C. Before MS analysis, samples were applied to a Poros R2 column and eluted with a gradient of 20-90% acetonitrile, 0.2% MK over 3 minutes. The peak from the LC column was analyzed using an LTQ-Orbitrap XL mass spectrometer (cone voltage: 50 V, cylindrical lens: 215 V; PF resolution: 7500) and installed Promass software to obtain molecular weight profiles.

Figures 40A-40B demonstrated bispecific binding using scFvs against HER2 (anti-HER2) and HER3 (anti-HER3) Fc heterodimer fused to the N-terminus of chain A and chain B of the Fc heterodimer. The resulting variants, a bispecific HER2/HER3 variant and two monovalent-monospecific variants HER2, HER3, are shown in Figure 40-A (chain A dark gray; chain B light gray). Figure 40-B demonstrates a bispecific binding test.

Figure 41 illustrates a computational model comparing wild-type IgG1 Fc and AZ3003. The computational model for AZ3002 is the same as for AZ3003 in position T350. The table summarizes the selected heterodimer variants and the stabilizing effect of the T350V mutation on the CH3 melting point. The figure shows heterodimer variants expressed and purified as described in Example 11. Differential scanning calorimetry (DSC) was performed as detailed in Example 3, and LC/MS quantitation was performed as detailed in Example 11.

Figure 42 illustrates a comparison of the crystal structure and the predicted model of the main heterodimer. The mutated surface residues (listed in the table) are highlighted in the graphical representation.

Figure 43 shows an analysis of the glycosylation pattern of the purified core heterodimer.

Figure 44 illustrates the results of an assessment of the accelerated degradation of the purified basic heterodimer.

Figure 45 depicts an industry-standardized antibody purification process flow chart.

Figure 46 summarizes the downstream purification evaluation of the AZ3003 heterodimer variant and shows the yields from each step and the overall yield (see Example 15 for more details). This heterodimer was prepared in 10 L of transiently transfected CHO cells as detailed in Example 11.

DETAILED DESCRIPTION OF THE INVENTION

This application provides modified CH3 domains containing certain amino acid modifications to promote heteromultimer formation. In one embodiment, the modified CH3 domains include certain amino acid modifications to promote heterodimer formation (see, for example, Tables 1.1-1.3). In another embodiment, the modified CH3 domains include certain amino acid modifications to promote heterodimer formation with increased stability (see, for example, Table 4, Table 6 and Table 7). Stability was measured as the melting temperature (Tm) of the CH3 domain, and increased stability refers to a Tm of approximately 70° C. or more. The CH3 domains form part of the Fc region of a heteromultimeric multispecific antibody. In one embodiment, this application provides heteromultimers containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote heterodimer formation, wherein the modified CH3 domains are selected from the options listed in Table 1. In the second An embodiment provides heteromultimers containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, wherein the modified CH3 domain has a melting temperature (Tm) of approximately 70°C or more.

Amino acid modifications used to produce a modified CH3 domain include, but are not limited to, insertions, deletions, substitutions and amino acid permutations. CH3 domain modifications and modified CH3 domains are collectively referred to herein as “CH3 modifications,” “modified CH3 domains,” or “CH3 variants.” In some embodiments, the modified CH3 domains are included in the selected molecule. Accordingly, in one embodiment, there are provided molecules, such as polypeptides, such as immunoglobulins (e.g., antibodies) and other binding proteins, containing an Fc region (as used herein, the term "Fc region" and related terms includes any heavy chain constant region domain). chain containing at least part of a CH3 domain) containing a modified CH3 domain. Molecules containing Fc regions containing a modified CH3 domain (eg, a CH3 domain containing one or more insertions, deletions, substitutions or rearrangements of amino acids) are referred to herein as "Fc variants", "heterodimers" or "heteromultimers". The present Fc variants include a CH3 domain that has been asymmetrically modified to produce Fc variants or heterodimer regions. The Fc region consists of two heavy chain constant domain polypeptides, chain A and chain B, and these terms can be used interchangeably when each Fc region contains one chain A polypeptide and one chain B polypeptide. Amino acid modifications are introduced into CH3 asymmetrically by producing a heterodimer in which two modified CH3 domains form an Fc variant (see, for example, Table 1). As used herein, asymmetric amino acid modifications are any modification in which the amino acid at a specific position on one polypeptide (eg, "chain A") is different from the amino acid on a second polypeptide (eg, "chain B") at the same position of the heterodimer or variant Fc. This can be achieved by modifying only one of the two amino acids or modifying both amino acids to two different amino acids in chain A and chain B of the Fc variant. It will be apparent that the modified CH3 domains include one or more asymmetric amino acid modifications.

An amino acid that lies at the boundary between the first and second CH3 domain polypeptides is any amino acid on the first or second CH3 domain polypeptide that reacts with an amino acid on another CH3 domain polypeptide, resulting in the formation of a dimeric CH3 domain. An amino acid that is not at the boundary between the first and second CH3 domain polypeptides is any amino acid on the first or second CH3 domain polypeptide that does not interact with an amino acid on another CH3 domain polypeptide. In embodiments described herein, a modified amino acid that is not at the boundary between the first and second CH3 domain polypeptides is any amino acid on the first or second CH3 domain polypeptide that, after it has been modified as described in this application does not interact with an amino acid on another CH3 domain polypeptide. For example, some embodiments described herein provide amino acid modifications at position T350. The crystal structure presented in Example 12 and shown in Figure 42 demonstrates that T350 is not involved in the interactions between the two CH3 domain polypeptides. Any modifications at the T350 position have been shown to have little effect on the formation of CH3 dimers, as described by Carter et al. Biochemistry 1998, 37, 9266. In the heteromultimeric Fc constructs described herein, modifications at the T350 position have been found to have an unexpected effect stabilizing effect on CH3 domain variants, despite the fact that they are not directly involved in the formation of the CH3 dimer itself. For example, variants containing at least one modification T350X, where X is a natural or non-natural amino acid selected from valine, isoleucine, leucine, methionine and derivatives or variants of these amino acids, form very stable CH3 domain variants. Some embodiments described herein provide an isolated Fc heteromultimeric construct described herein containing at least one T350V modification. In some embodiments, the first and second CH3 domain variant polypeptides include the T350V modification, which imparts unexpected stability to the CH3 domain variant compared to a corresponding CH3 domain not containing the modification.

As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the described range and, when appropriate, parts thereof (such as one-tenth and one-hundredth of an integer) if not otherwise stated. As used herein, “about” means ± 10% of the stated range, value, sequence or structure unless otherwise stated. It should be clear that the singular terms used in this application refer to “one or more” of the listed components unless otherwise indicated or the context requires. The use of an alternative (such as the conjunction "or") should be understood to mean either one, both, or any combination of alternatives. In this application, the terms “comprise” and “include” are used synonymously. In addition, it should be apparent that individual single chain polypeptides or heterodimers derived from the various combinations of structures and substituents (e.g., modified CH3 domains) described herein are described herein equally as if each single chain polypeptide or heterodimer has been described separately. Thus, the selection of specific components to obtain individual single chain polypeptides or heterodimers is within the scope of the present description.

A "first polypeptide" is any polypeptide that is to be linked to a second polypeptide and is also referred to herein as the "A chain". The first and second polypeptides "meet" at the "border". A “second polypeptide” is any polypeptide that must be linked to the first polypeptide across a “border” and is also referred to herein as the “B chain”. "Border" includes those "contacting" amino acid residues in the first polypeptide that interact with one or more "contacting" amino acid residues at the boundary of the second polypeptide. In this application, the boundary includes a CH3 domain of the Fc region, which is preferably derived from an IgG antibody and, most preferably, from a human IgG1 antibody.

As used herein, an “isolated” heteromultimer means a heteromultimer that has been detected and separated and/or recovered from components of its natural cell culture environment. Contaminating components of its natural environment are materials that will interfere with the heteromultimer's diagnostic or therapeutic applications, and may include enzymes, hormones, and other protein or non-protein solutes.

An amino acid with a side chain volume "not substantially greater" than that of the first amino acid is any amino acid whose side chain volume is no more than 20 Å ³ greater than that of the first amino acid, based on side chain volume values from AA Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107-123, 1972. In some embodiments, the volume is no more than 10 Å ³ greater than that of the first amino acid. In some embodiments, the volume is no more than 5 Å ³ greater than that of the first amino acid. For example, some embodiments described herein provide lysine (K) mutations, such as K392J, wherein J is selected from L, I, or an amino acid with a side chain volume not significantly greater than the side chain volume of K.

The Fc heterodimer variants are typically purified to a substantially homogeneous state. The phrases “substantially homogeneous,” “substantially homogeneous form,” and “substantially homogeneous state” are used to indicate that the product is substantially free of by-products resulting from the formation of undesirable combinations of polypeptides (eg, homodimers). In terms of purity, a substantially homogeneous state means that the amount of by-products does not exceed 10%, and is preferably below 5%, more preferably below 1%, most preferably below 0.5%, where percentages are by weight.

All terms as understood by those skilled in the art of antibody technology have the meanings acquired in the art unless otherwise clearly defined in this application. Antibodies are known to have variable regions, a hinge region, and constant domains. For a review of the structure and function of immunoglobulins, see, for example, Harlow et al., eds., Antibodies: A Laboratory Manual, chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

The development of Fc heterodimer variants from wild-type homodimers is illustrated by the principles of positive and negative engineering in the context of protein engineering by achieving a balance between stability and specificity, with mutations introduced to cause heterodimer but not homodimer formation when polypeptides are expressed under culture conditions cells. Negative engineering strategies maximize unfavorable interactions to form homodimers, either by introducing bulky side chains on one chain and small side chains on the opposite chain, such as the ridge-to-hole strategy developed by Genentech (Ridgway JB, Presta LG, Carter P. engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. July 1996; 9(7):617-21; Atwell S, Ridgway JB, Wells JA, Carter P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J Mol Biol. 270(1): 26-35 (1997)), or by electrostatic engineering that results in repulsion and prevents homodimer formation, such as the electrostatic engineering 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 negative engineering examples, asymmetric point mutations were introduced into the wild-type CH3 domain to cause heterodimer formation. To date, only negative design strategies have been used to develop Fc heterodimers. Published results show that heterodimer design using only a negative design approach results in high specificity with >95% heterodimers, but also significant destabilization of the complex (above).

Heterodimers produced using the negative design approach have a modified CH3 domain melting temperature of 69°C or less and lack additional disulfide bonds compared to the wild type. See Table A below.

* According to the observations of the present inventors, the purity was >90% for Control 1 in the assay system used by the present inventors, but not 100% as previously described in the literature.

** According to the observations of the authors of the present invention, Tm was more than 77°C for Control 4 in the analysis system used by the authors of the present invention; Tm for this variant has not been published in the literature.

BUT - Tm for Control 3 was not published and was not tested in our analysis systems.

The melting point of wild-type IgG1 is reported as a range from 81°C to 83°C, since values in the literature vary depending on the assay system used, for the assay system used by the present inventors, a value of 81.5°C has been published by the present inventors.

In contrast to negative engineering, the basic principle used for protein engineering is positive engineering. In this case, amino acid modifications are introduced into polypeptides to maximize favorable interactions within or between proteins. This strategy assumes that by introducing several mutations that specifically stabilize a desired heterodimer while neglecting the effect on homodimer formation, the net effect will be better specificity for the desired heterodimeric, but not homodimeric, interactions and therefore greater specificity for the desired heterodimeric, but not homodimeric, interactions. heterodimers. In the context of protein engineering, it is clear that positive design strategies optimize the stability of desired protein interactions, but rarely achieve specificity >90% (Havranek JJ & Harbury RW. Automated design of specificity in molecular recognition. Nat Struct Biol. 10(1):45-52 (2003); Bolon DN, Grant RA, Baker TA, Sauer RT. Specificity versus stability in computational protein design. Proc Natl Acad Sci USA. 6; 102(36): 12724-9 (2005); Huang PS, Love JJ, Mayo SL, A de novo designed protein-protein interface, Protein Sci 16(12):2770-4 (2007). Prior to the present description, positive design strategies have not been used to design Fc heterodimers for therapeutic antibody production and development, as more emphasis has been placed on specificity rather than stability. Additionally, beneficial mutations in a positive development strategy may be difficult to predict. Other stability-enhancing techniques, such as the introduction of additional disulfide bonds, have been tried to increase the stability of Fc heterodimers with limited success in improving the molecule (see Table A). This may be due to the fact that all artificial disulfide bonds of the Fc domains of CH3 domains are exposed to the solvent, which leads to rapid disintegration of the disulfide bond and, therefore, has a significant impact on the long-term stability of the heterodimer, especially if the artificial CH3 domain has a Tm less than 70°C , without an additional disulfide bond (as in Control 4, which has a Tm of 69°C, without a disulfide bond (see Control 2)). It is contemplated that other stability enhancing techniques other than disulfide bond formation may also be used for the present Fc variants, provided that the intrinsic stability (measured as melting point) of the CH3 domain is 70° C. or more without disulfide bonding, particularly if the internal stability (measured as melting point) of the CH3 domain is 72°C or more without a disulfide bond.

Therefore, in this application, the present inventors have proposed a new method for developing Fc heterodimers, which allows obtaining both a stable and highly specific heterodimer. This design method combines negative and positive design strategies, along with structural and computer modeling using protein engineering techniques. This efficient method allowed us to develop new combinations of mutations in the CH3 domain of IgG1, whereby using only standard cell culture conditions, heterodimers were formed with a purity of greater than 90% relative to homodimers, and the resulting heterodimers had a melting point of 70°C or more. In typical embodiments, the Fc heterodimer variants have a melting point of 73° C. or greater and a purity greater than 98%. In other exemplary embodiments, Fc heterodimer variants have a melting point of 75° C. or greater and a purity greater than 90%. In some embodiments of the Fc heterodimer variants described herein, the Fc heterodimer variants have a melting point of 77° C. or greater and a purity of greater than 98%. In some embodiments of the Fc heterodimer variants described herein, the Fc heterodimer variants have a melting point of 78° C. or greater and a purity of greater than 98%. In some embodiments of the Fc heterodimer variants described herein, the Fc heterodimer variants have a melting point of 79° C. or greater and a purity of greater than 98%. In some embodiments of the Fc heterodimer variants described herein, the Fc heterodimer variants have a melting point of 80° C. or greater and a purity of greater than 98%. In some embodiments of the Fc heterodimer variants described herein, the Fc heterodimer variants have a melting point of 81° C. or greater and a purity of greater than 98%.

In some embodiments, an isolated heteromultimer is provided containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, and wherein the modified CH3 domain has a melting temperature (Tm) of 70°C or more. As used herein, “enhanced stability” or “stable heterodimer” refers to a modified CH3 domain within the heterodimer having a melting point of approximately 70° C. or greater. In some embodiments, “enhanced stability” or “stable heterodimer” refers to a modified CH3 domain within the heterodimer having a melting point of about 72° C. or greater. In some embodiments, “enhanced stability” or “stable heterodimer” refers to a modified CH3 domain within the heterodimer having a melting point of about 74° C. or greater. In some embodiments, “enhanced stability” or “stable heterodimer” refers to a modified CH3 domain within the heterodimer having a melting point of about 75° C. or greater. In some embodiments, “enhanced stability” or “stable heterodimer” refers to a modified CH3 domain within the heterodimer having a melting point of about 76° C. or greater. In some embodiments, “enhanced stability” or “stable heterodimer” refers to a modified CH3 domain within the heterodimer having a melting point of about 78° C. or greater. In some embodiments, “enhanced stability” or “stable heterodimer” refers to a modified CH3 domain within the heterodimer having a melting point of about 79° C. or greater. In some embodiments, “enhanced stability” or “stable heterodimer” refers to a modified CH3 domain within the heterodimer having a melting point of about 80° C. or greater. In some embodiments, “enhanced stability” or “stable heterodimer” refers to a modified CH3 domain within the heterodimer having a melting point of about 81° C. or greater. In addition, it should be apparent that the term “heterodimer-promoting” as used herein refers to amino acid mutations in the CH3 domain that result in heterodimer formation more than 90% of the time compared to homodimer formation.

In a further embodiment, this increased stability is observed in the absence of an additional disulfide bond. In particular, increased stability is observed in the absence of an additional disulfide bond in the CH3 domain. In one embodiment, the modified CH3 domain does not contain an additional disulfide bond compared to the wild type CH3 domain. In an alternative embodiment, the modified CH3 domain contains at least one disulfide bond compared to the wild type CH3 domain if the modified CH3 domain has a melting point of 70° C. or more in the absence of a disulfide bond. In one embodiment, the modified CH3 domain contains at least one disulfide bond compared to the wild type CH3 domain, and the modified CH3 domain has a melting temperature (Tm) of approximately 77.5°C or greater. In one embodiment, the modified CH3 domain contains at least one disulfide bond compared to the wild type CH3 domain, and the modified CH3 domain has a melting temperature (Tm) of about 78°C or more. In another embodiment, the modified CH3 domain contains at least one disulfide bond compared to the wild type CH3 domain, and the modified CH3 domain has a melting temperature (Tm) 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 point 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 point 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 approximately 73°C, or approximately 73.5°C, or approximately 74°C, or approximately 74.5°C, or approximately 75°C, or approximately 75.5°C, or approximately 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 point of from about 70°C to about 81°C, or from about 70.5°C to about 81°C, or from about 71°C to about 81°C , or from about 71.5°C to about 81°C, or from about 72°C to about 81°C, or from about 72.5°C to about 81°C, or from about 73°C to about 81 °C, or from about 73.5°C to about 81°C, or from about 74°C to about 81°C, or from about 74.5°C to about 81°C, or from about 75°C to about 81°C, or about 75.5°C to about 81°C, or about 76°C to about 81°C, or about 76.5°C to about 81°C, or about 77° From to about 81°C, or from about 77.5°C to about 81°C, or from about 78°C to about 81°C, or from about 78.5°C to about 82°C, or from about 79°C to approximately 81°C. In yet another embodiment, the modified CH3 domain has a melting point of from about 71°C to about 76°C, or from about 72°C to about 76°C, or from about 73°C to about 76°C, or from about 74°C to about 76°C.

In addition to increased stability, the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote heterodimer formation. It should be obvious that these amino acid mutations favor heterodimer formation over homodimer formation. This heterodimer formation versus homodimer formation is collectively referred to herein as “purity” or “specificity” or “heterodimer purity” or “heterodimer specificity”. It should be apparent that heterodimer purity refers to the percentage of the desired heterodimer compared to the homodimer species formed in solution under standard cell culture conditions prior to selective purification of the heterodimer species. For example, a heterodimer purity of 90% indicates that 90% of the dimer species in solution are the desired heterodimer. In one embodiment, the Fc heterodimer variants have 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 another embodiment, the Fc heterodimer variants 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 includes a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote formation of a heterodimer with increased stability, and wherein the modified CH3 domain has a melting temperature (Tm) of 70 °C or more, and the resulting heterodimer has a purity greater than 90%. In one aspect, the resulting Fc heterodimer variant has a purity greater than 98%, and said modified CH3 domain has a melting point 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 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 point of 70° C. or greater, and the resulting Fc heterodimer variant 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 approximately 99%.

To develop these Fc variants with increased stability and purity, we used an iterative computer-aided design process and experimental screening to select the most successful combinations of positive and negative development strategies (see Figure 24).

Specifically, during the initial phase of development, various Fc heterodimer variants were generated through negative engineering and their expression and stability were examined as described in Examples 1-3. The initial phase of development included Fc heterodimer variants AZ1-AZ16 (see Table 1). From this initial set of negatively engineered Fc heterodimer variants that were expected to have low stability (e.g., Tm less than 71°C), Fc heterodimer variants with greater than 90% purity and a melting point of approximately 68°C were selected for further development. C or more. The variants selected included the Fc heterodimer variants AZ6, AZ8 and AZ15. In the second phase of development, these selected Fc heterodimer variants were further modified to improve both stability and purity using positive design strategies following detailed computational and structural analysis. Each of the selected Fc heterodimer variants (AZ6, AZ8, and AZ15) was analyzed using computational methods and detailed structure-function analysis to determine the structural reasons why these Fc variants have lower stability than the wild-type Fc homodimer, which has a melting temperature of is 81°C for IgG1. For a list of Fc heterodimer variants and Tm values, see Table 4.

In some 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.

Computational tools and structure-function analysis included, but were not limited to: molecular dynamics (MD), side chain/framework refolding, statistical potential (KBP), cavity and (hydrophobic) stacking analysis (LJ, CCSD, SASA, dSASA( carbon/all atoms)), electrostatic-GB calculations and binding analysis (Figure 24 provides an overview of the computational strategy).

The protein engineering aspect of the approach relied on combining structural information about the Fc of the IgG protein obtained from X-ray diffraction analysis with computer modeling and modeling of the wild-type CH3 domain and its variant forms. This approach allowed us to obtain new structural and physicochemical insights into the potential role of individual amino acids and their joint action. This structural and physicochemical insight obtained for several modified CH3 domains, along with the obtained empirical data regarding their stability and purity, helped us understand the relationship between the purity and stability of the Fc heterodimer, in comparison with Fc homodimers and simulated structural models. To implement the simulations, the present inventors began by constructing complete and realistic models and improving the quality of the Fc structure of the wild type IgG1 antibody. Protein structures obtained from X-ray diffraction analysis lacked detail regarding some properties of the protein in aqueous environments under physiological conditions, and our detailing procedures were aimed at addressing these limitations. These included constructing missing regions in the protein structure, often mobile regions of the protein such as loops and side chains of certain residues, assessing and determining the protonation levels of neutral and charged residues, and introducing potentially functionally significant water molecules associated with the protein.

Molecular dynamics (MD) algorithms are one tool that the present inventors have used in protein structure modeling to evaluate the intrinsic dynamic nature of the Fc homodimer and modified CH3 domains in an aqueous environment. Molecular dynamics simulation allows one to trace the dynamic trajectory of a molecule resulting from the movements generated by the interactions and forces acting between all the atomic units in the protein and the local environment surrounding it, in this case, between the atoms that make up Fc and the surrounding water molecules. Following molecular dynamics simulations, various aspects of the trajectories were analyzed to understand the structural and dynamic properties of the Fc homodimer and Fc heterodimer variant that the present inventors used to identify specific amino acid mutations to improve both the purity and stability of the molecule.

Therefore, the resulting MD trajectories were examined using techniques such as principal component analysis to detect internal low-frequency oscillations in the motion of the Fc structure. This gave us insight into potential conformational substates of the protein (see Figure 32). Although the critical protein-protein interactions between chains A and B in the Fc region occurred at the boundary of the CH3 domains, simulations performed by the present inventors showed that this boundary acts as a hinge in the movement, which includes "opening" and " closing" of the N-termini of CH2 domains relative to each other. The CH2 domain interacts at this end with FcγR, as can be seen in Figure 16. Thus, without being bound by any particular theory, it appears that the introduction of amino acid mutations at the CH3 boundary affects the amplitude and nature of the opening/closing movement of the N-terminus of Fc and , therefore, on how Fc interacts with FcγR. See Example 4 and Table 5.

The obtained MD trajectories were also examined to determine the possibility of introducing mutations at certain positions of amino acid residues in the Fc structure based on determining their flexibility profile and analysis of their environment. This algorithm allowed us to identify residues that may affect protein structure and function and provided unique insight into the properties of residues and the possibility of introducing mutations for subsequent phases of the development of modified CH3 domains. This analysis also allowed us to compare multiple models and evaluate the possibility of introducing mutations based on outliers after profiling.

The resulting MD trajectories were also examined to determine the coordinated movements of residues in the protein and the formation of networks of residues resulting from the formation of bonds between them. Discovering the dynamic relationships and networks of residues within the Fc structure is an important step for understanding the protein as a dynamic molecule and for gaining insight into the impact of mutations in peripheral regions. See, for example, Example 6.

Thus, the present inventors have examined in detail the contribution of mutations to the local environment of the mutation site. The formation of a close-packed core at the CH3 interface between chains A and B is necessary for the spontaneous joining of the two chains into a stable Fc structure. Tight folding is achieved as a result of strong structural complementarity between interacting molecular partners, coupled with favorable interactions between contacting groups. Favorable interactions are formed as a result of hidden hydrophobic contacts, well removed from the interaction with the solvent, and/or as a result of the formation of complementary electrostatic contacts between hydrophilic polar groups. These hydrophobic and hydrophilic contacts make an entropic and enthalpic contribution to the free energy of dimer formation at the CH3 interface. The present inventors use a variety of algorithms to create an accurate model of stacking at the CH3 interface between chain A and chain B, and then evaluate the thermodynamic properties of the interface by assessing a variety of important physicochemical properties.

The present inventors use a variety of protein folding techniques, including flexible scaffolds, to optimize and prepare model structures for a large number of variants, which the present inventors have subjected to computational screening. After installation, the present inventors evaluated a variety of parameters, including contact density, collision rate, hydrogen bonding, hydrophobicity, and electrostatics. The use of solvation models allowed us to more closely examine the influence of the solvent environment and compare the free energy differences following mutation at specific protein positions to alternative residue types. The contact density and collision index allowed the measurement of complementarity, a critical aspect for efficient protein folding. These screening procedures are based on the application of science-intensive capabilities or binding analysis schemes that rely on calculations of pairwise residue interaction energies and entropy.

This detailed computational analysis provided an in-depth understanding of the differences of each Fc variant compared to wild-type Fc with respect to mutagenesis hotspots at the interface, sites of asymmetry, cavities and poorly folded regions, structural dynamics of individual sites, and sites of local unfolding. These combined results from the described computational analysis identified specific residues, sequence/structure motifs, and cavities that were not optimized and, in combination, resulted in lower stability (e.g., Tm of 68°C) and/or lower specificity with purity <90%. In the second phase of development, the present inventors used directed positive engineering to specifically explore this hypothesis by introducing additional point mutations and investigated them through computational engineering using the methodology and analysis described above (see Figure 24). The expression and stability of Fc heterodimer variants designed to improve stability and purity for each phase two design target (Fc heterodimer variants AZ17-AZ101) were tested empirically as described in Examples 1-4.

In some embodiments, this application provides isolated heteromultimers comprising a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote formation of a heterodimer with increased stability, and 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 AZ31 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 AZ31, 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 a second polypeptide (also referred to herein as chain A and chain B), wherein the first polypeptide includes amino acid modifications L351Y, F405A and Y407V and wherein the second polypeptide includes amino acid modifications T366I, K392M and T394W . In another embodiment, the first polypeptide includes amino acid modifications L351Y, S400E, F405A and Y407V and the second polypeptide includes amino acid modifications T366I, N390R, K392M and T394W.

This iterative process of computer-aided structure-function analysis, targeted design, and experimental validation was used to develop the remaining Fc variants listed in Table 1 in subsequent development phases, resulting in Fc heterodimer variants with greater than 90% purity and improved temperature stability. melting of the CH3 domain above 70°C. In some embodiments, the Fc variants include amino acid mutations selected from AZ1-AZ136. In further embodiments, the Fc variants include amino acid mutations selected from the Fc variants listed in Table 4.

During the first and second phases of development, two basic scaffolds were discovered, Scaffold 1 and Scaffold 2, with additional amino acid modifications introduced into these scaffolds to fine-tune the purity and stability of the Fc heterodimer variants. Example 5 provides a detailed description of the development of Frame 1, including AZ8, AZ17-62, and the options listed in Table 6. Example 6 provides a detailed description of the development of Frame 2, including AZ15 and AZ63-101, and the options listed in Table 7.

Mutations in Core Framework 1 include L351 Y_F405A_Y407V/T394W. Framework 1a includes amino acid mutations T366I_K392M_T394W/F405A_Y407V and Framework 1b includes amino acid mutations T366L_K392M_T394W/F405A_Y407V. See Example 5.

In some embodiments, the modified CH3 domain comprises a first and a second polypeptide (also referred to herein as chain A and chain B), wherein the first polypeptide includes the amino acid modifications L351Y, F405A, and Y407V and the second polypeptide includes the amino acid modification T394W. In one aspect, the modified CH3 domain further includes 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 includes 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 includes a first and a second polypeptide, wherein the first polypeptide includes amino acid modifications L351Y, F405A, and Y407V and the second polypeptide includes the amino acid modification T394W, and wherein the first and/or second polypeptide includes additional 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. Mutations at position L368 include, but are not limited to, L368D, L368R, L368T, L368M, L368V, L368F, L368S, and L368A.

In some embodiments, the modified CH3 domain includes a first and a second polypeptide (also referred to herein as chain A and chain B), wherein the first polypeptide includes amino acid modifications L351Y, F405A, and Y407V and the second polypeptide includes amino acid modifications T366L and T394W. In another embodiment, the modified CH3 domain includes a first and a second polypeptide, wherein the first polypeptide includes amino acid modifications L351Y, F405A and Y407V and the second polypeptide includes amino acid modifications T366I and T394W.

In other specific embodiments, the modified CH3 domain includes a first and a second polypeptide (also referred to herein as chain A and chain B), wherein the first polypeptide includes amino acid modifications L351Y, F405A, and Y407V and the second polypeptide includes amino acid modifications T366L, K392M, and T394W . In another embodiment, the modified CH3 domain includes a first and a second polypeptide, wherein the first polypeptide includes amino acid modifications L351Y, F405A and Y407V and the second polypeptide includes amino acid modifications T366I, K392M and T394W.

In yet another embodiment, the modified CH3 domain includes a first and a second polypeptide (also referred to herein as chain A and chain B), wherein the first polypeptide includes amino acid modifications F405A and Y407V and the second polypeptide includes amino acid modifications T366L, K392M, and T394W. In another embodiment, the modified CH3 domain includes a first and a second polypeptide, wherein the first polypeptide includes the amino acid modifications F405A and Y407V and the second polypeptide includes the amino acid modifications T366I, K392M, and T394W.

In some embodiments, the modified CH3 domain includes a first and a second polypeptide (also referred to herein as chain A and chain B), wherein the first polypeptide includes amino acid modifications F405A and Y407V and the second polypeptide includes amino acid modifications T366L and T394W. In another embodiment, the modified CH3 domain includes a first and a second polypeptide, wherein the first polypeptide includes the amino acid modifications F405A and Y407V and the second polypeptide includes the amino acid modifications T366I and T394W.

In an exemplary embodiment, this application provides isolated heteromultimers containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, wherein the modified CH3 domain has a melting temperature (Tm ) of approximately 74°C or more. In another embodiment, this application provides isolated heteromultimers containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, wherein the modified CH3 domain has a melting temperature (Tm ) of about 74°C or more, and the heterodimer has a purity of about 98% or more.

In some embodiments, an isolated heteromultimer is provided comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain containing amino acid mutations that promote formation of a heterodimer with increased stability, and wherein the modified CH3 domain has a melting temperature (Tm) greater than than 70°C, and the specified modified CH3 domains are selected from Table 6.

Mutations in Core Framework 2 include L351Y_Y407A/T366A_K409F. Scaffold 2a includes amino acid mutations L351Y_Y407A/T366V_K409F and Scaffold 2b includes amino acid mutations Y407A/T366A_K409F. See Example 6.

In some embodiments, the modified CH3 domain includes a first and a second polypeptide (also referred to herein as chain A and chain B), wherein the first polypeptide includes amino acid modifications L351Y and Y407A and the second polypeptide includes amino acid modifications T366A and K409F. In one aspect, the modified CH3 domain further includes 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. Mutations at position L351 include, but are not limited to, L351I, L351D, L351R, or L351F. 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 a second polypeptide (also referred to herein as chain A and chain B), the first polypeptide comprising amino acid modifications L351Y and Y407A and the second polypeptide comprising amino acid modifications T366V and K409F.

In an exemplary embodiment, this application provides isolated heteromultimers containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, wherein the modified CH3 domain has a melting temperature (Tm ) of approximately 75.5°C or more. In another embodiment, this application provides isolated heteromultimers containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, wherein the modified CH3 domain has a melting point (Tm ) of about 75°C or more, and the heterodimer has a purity of about 90% or more.

In some other embodiments, the modified CH3 domain includes a first and a second polypeptide (also referred to herein as chain A and chain B), wherein the first polypeptide includes amino acid modifications L351Y and Y407A and the second polypeptide includes amino acid modifications T366A and K409F, and wherein the modified CH3 domain includes 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. Mutations at position T411 include, but are not limited to, T411N, T411R, T411Q, T411K, T411D, T411E, or T411W. Mutations at position S400 include, but are not limited to, S400E, S400D, S400R, or S400K. Mutations at position F405 include, but are not limited to, F405I, F405M, F405S, F405S, F405V, or F405W. Mutations at position N390 include, but are not limited to, N390R, N390K, or N390D. Mutations at position K392 include, but are not limited to, K392V, K392M, K392R, K392L, K392F, or K392E. See CH3 versions of AZ71-101 in Table 1 and Table 4 and Example 6.

In an exemplary embodiment, the modified CH3 domain comprises a first and a second polypeptide (also referred to herein as chain A and chain B), the first polypeptide comprising the amino acid modification Y407A and the second polypeptide comprising the amino acid modifications T366A and K409F. In one aspect, the modified CH3 domain further includes the amino acid modifications K392E, T411E, D399R and S400R. In a further embodiment, the modified CH3 domain includes a first and a second polypeptide, wherein the first polypeptide includes the amino acid modifications D399R, S400R, and Y407A and the second polypeptide includes the amino acid modifications T366A, K409F, K392E, and T411E.

In an exemplary embodiment, this application provides isolated heteromultimers containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, wherein the modified CH3 domain has a melting temperature (Tm ) of approximately 74°C or more. In another embodiment, this application provides isolated heteromultimers containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, wherein the modified CH3 domain has a melting temperature (Tm ) of about 74°C or more, and the heterodimer has a purity of about 95% or more.

In some embodiments, this application provides isolated heteromultimers comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain containing amino acid mutations that promote formation of a heterodimer with increased stability, and wherein the modified CH3 domain has a melting point of ( Tm) greater than 70°C, and the modified CH3 domains are selected from Table 7.

Moreover, this new method for developing Fc heterodimer variants with increased stability and purity can be applied to other classes and isotypes of Fc regions. In some embodiments, the Fc region is a human IgG Fc region. In further embodiments, the human IgG Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc regions are immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. In some embodiments, the IgG is of a subtype selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3, and IgG4.

The Fc region as defined herein includes a CH3 domain or a fragment thereof, and may further include one or more additional constant region domains, or fragments thereof, including a hinge region, CH1 or CH2. It is apparent that the numbering of the Fc amino acid residues corresponds to the EU index described in Kabat et al., 1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA. “Kabat EU index” refers to the Kabat EU index numbering of a human IgG1 antibody. For convenience, Table B shows the amino acids numbered according to the EU index described by Kabat, the CH2 and CH3 domains of human IgG1.

According to one aspect of the present invention, there is provided an isolated heteromultimeric Fc construct comprising a modified heterodimeric CH3 domain, wherein said modified CH3 domain comprises: a first modified CH3 domain polypeptide comprising at least three amino acid modifications relative to a wild-type CH3 domain polypeptide, and a second modified polypeptide a CH3 domain containing at least three amino acid modifications compared to a wild type CH3 domain polypeptide; wherein at least one of the first and second CH3 domain polypeptides includes the amino acid modification K392J, where J is selected from L, I, or an amino acid with a side chain volume not significantly greater than the volume of the side chain K; wherein said first and second modified CH3 domain polypeptides preferably form a heterodimeric CH3 domain with a melting point (Tm) of at least about 74° C. and a purity of at least 95%; and wherein the at least one amino acid modification does not affect an amino acid that is located at the boundary between said first and said second CH3 domain polypeptides. Some embodiments provide a heteromultimeric Fc construct described herein that includes at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants of these amino acids. In some embodiments, an isolated Fc heteromultimeric construct described herein is provided that includes at least one T350V modification. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a Tm of approximately 77°C or greater. In some embodiments, the modified CH3 domain has a Tm of about 80°C or more. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein the at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising a modification of at least one of amino acids L351, F405, and Y407. In some embodiments, an isolated heteromultimeric Fc construct is provided, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide further comprising a T366 amino acid modification. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide containing amino acid modifications at positions L351, F405, and Y407, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide. , containing amino acid modifications at positions T366, K392 and T394. In one embodiment, there is provided an isolated heteromultimeric Fc construct described herein, wherein said first CH3 domain polypeptide includes amino acid modifications L351Y, F405A and Y407V, and said second CH3 domain polypeptide includes amino acid modifications T366L, K392M and T394W. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein said first CH3 domain polypeptide includes amino acid modifications L351Y, F405A, and Y407V, and said second CH3 domain polypeptide includes amino acid modifications T366L, K392L, and T394W. In a further embodiment, there is provided an isolated heteromultimeric Fc construct described herein, wherein said first CH3 domain polypeptide includes amino acid modifications L351Y, F405A and Y407V, and said second CH3 domain polypeptide includes amino acid modifications T366I, K392M and T394W. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein said first CH3 domain polypeptide includes amino acid modifications L351Y, F405A, and Y407V, and said second CH3 domain polypeptide includes amino acid modifications T366I, K392L, and T394W. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein at least one of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide containing an amino acid modification at position S400. In an additional embodiment, an isolated heteromultimeric Fc construct is provided as described herein, comprising the S400Z modification, where 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 some embodiments, an isolated heteromultimeric Fc construct is provided as described herein, wherein said first CH3 domain polypeptide includes an amino acid modification selected from S400E and S400R. Some embodiments provide an isolated heteromultimeric Fc construct described herein, wherein at least one of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide containing an amino acid modification at position N390. In some embodiments, the N390 modification is N390Z, where Z is selected from a positively charged amino acid and a negatively charged amino acid. In one embodiment, N390Z is N390R. In some embodiments of the isolated heteromultimeric Fc construct described herein, said first CH3 domain polypeptide is a modified CH3 domain polypeptide containing the S400E amino acid modification, and said second CH3 domain polypeptide is a modified CH3 domain polypeptide containing the N390R amino acid modification. In some embodiments of the isolated heteromultimeric Fc construct described herein, each of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide, wherein one modified CH3 domain polypeptide includes a Q347R amino acid modification and the other modified CH3 domain polypeptide includes an amino acid modification K360E.

In one aspect, an isolated heteromultimeric Fc construct is provided comprising a modified heterodimeric CH3 domain, wherein said modified CH3 domain comprises: a first modified CH3 domain polypeptide comprising at least three amino acid modifications relative to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide containing at least three amino acid modifications compared to the wild type CH3 domain polypeptide; wherein at least one of the first and second CH3 domain polypeptides includes the amino acid modification K392J, where J is selected from L, I, or an amino acid with a side chain volume not significantly greater than the volume of side chain K; wherein said first and second modified CH3 domain polypeptides preferably form a heterodimeric CH3 domain with a melting point (Tm) of at least about 74° C. and a purity of at least 95%; and wherein the at least one amino acid modification does not affect an amino acid that is located at the boundary between said first and said second CH3 domain polypeptides. Some embodiments provide a heteromultimeric Fc construct described herein that includes at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants of these amino acids. In some embodiments, an isolated Fc heteromultimeric construct described herein is provided that includes at least one T350V modification. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a Tm of approximately 77°C or greater. In some embodiments, the modified CH3 domain has a Tm of about 80°C or more. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising a modification of at least one of amino acids K409 and T411. In some embodiments, an isolated Fc heteromultimeric construct described herein is provided containing at least one of the K409F, T411E, and T411D modifications. In some embodiments, an isolated heteromultimeric Fc construct is provided as described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide containing a D399 amino acid modification. In some embodiments, the amino acid modification D399 is at least one of D399R and D399K.

In one aspect, an isolated heteromultimeric Fc construct is provided comprising a modified heterodimeric CH3 domain, wherein said modified CH3 domain comprises: a first modified CH3 domain polypeptide comprising at least three amino acid modifications relative to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide containing at least three amino acid modifications compared to the wild type CH3 domain polypeptide; wherein at least one of the first and second CH3 domain polypeptides includes the amino acid modification K392J, where J is selected from L, I, or an amino acid with a side chain volume not significantly greater than the volume of side chain K; wherein said first and second modified CH3 domain polypeptides preferably form a heterodimeric CH3 domain with a melting point (Tm) of at least about 74° C. and a purity of at least 95%; and wherein the at least one amino acid modification does not affect an amino acid that is located at the boundary between said first and said second CH3 domain polypeptides. Some embodiments provide a heteromultimeric Fc construct described herein that includes at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants of these amino acids. In some embodiments, an isolated Fc heteromultimeric construct described herein is provided that includes at least one T350V modification. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75° C. or greater. In one embodiment, an isolated heteromultimeric Fc construct is provided as described herein, wherein the modified CH3 domain has a Tm of approximately 77°C or greater. In some embodiments, the modified CH3 domain has a Tm of about 80°C or more. In some embodiments of the isolated heteromultimeric Fc construct described herein, the first CH3 domain polypeptide is a modified CH3 domain polypeptide containing at least one amino acid modification selected from K409F, T411E, and T411D, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide. CH3 domain containing at least one amino acid modification selected from Y407A, Y407I, Y407V, D399R and D399K. In some embodiments, any of the isolated heteromultimeric Fc constructs described herein are provided, further comprising a first modified CH3 domain containing one of the amino acid modifications T366V, T366I, T366A, T366M, and T366L; and a second modified CH3 domain containing the amino acid modification L351Y. In some embodiments, any of the isolated heteromultimeric Fc constructs described herein are provided, comprising a first modified CH3 domain containing one of amino acid modifications K392L or K392E; and a second modified CH3 domain containing one of the amino acid modifications S400R or S400V.

This application provides an isolated heteromultimeric Fc construct containing a modified CH3 domain, comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, wherein each modified CH3 domain polypeptide includes at least four amino acid mutations, wherein at least one of the above the first and said second modified CH3 domain polypeptides comprise 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 preferably form a heterodimeric CH3 domain with a melting point (Tm) , being at least about 70°C, and a purity of at least 90%. In one embodiment, an isolated heteromultimeric Fc construct is provided wherein said first modified CH3 domain polypeptide includes amino acid modifications at positions F405 and Y407 and said second modified CH3 domain polypeptide includes an amino acid modification at position T394. In one embodiment, an isolated heteromultimeric Fc construct is provided in which the first modified CH3 domain polypeptide includes an amino acid modification at position L351. In some embodiments, an isolated heteromultimer is provided as described herein, wherein said second modified CH3 domain polypeptide includes a modification at at least one of positions T366 and K392. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the modified CH3 domain has a melting point (Tm) of at least about 75° C. and is obtained at a purity of at least about 95%. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the at least one modified CH3 domain polypeptide includes at least one of the amino acid modifications N390R, S400E, and S400R. In some embodiments, an isolated heteromultimer is provided as described herein, wherein one of the first and second modified CH3 domain polypeptides includes an amino acid modification at position 347 and the other modified CH3 domain polypeptide includes an amino acid modification at position 360. In some embodiments, an isolated heteromultimer is provided. a heteromultimer described herein, wherein at least one of said first and second modified CH3 domain polypeptides includes a T350V amino acid modification. In specific embodiments, an isolated heteromultimer is provided as described herein, wherein said first modified CH3 domain polypeptide includes at least one amino acid modification selected from L351Y, F405A, and Y407V; and said second modified CH3 domain polypeptide includes at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In some embodiments described herein, an isolated heteromultimer is provided in which the first modified CH3 domain polypeptide includes amino acid modifications at positions D399 and Y407 and the second modified CH3 domain polypeptide includes amino acid modifications at positions K409 and T411. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the first CH3 domain polypeptide includes an amino acid modification at position L351 and the second modified CH3 domain polypeptide includes amino acid modifications at positions T366 and K392. In specific embodiments, isolated heteromultimers are provided as described herein, wherein at least one of the first and second CH3 domain polypeptides includes the T350V amino acid modification. Some embodiments provide isolated heteromultimers as described herein, wherein the modified CH3 domain has a melting point (Tm) of at least about 750° C. or greater and is obtained at a purity of at least about 95%.

Some embodiments provide the isolated heteromultimeric Fc constructs described herein, wherein said first modified CH3 domain polypeptide includes amino acid modifications selected from L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A, and Y407V; and said second modified CH3 domain polypeptide includes amino acid modifications selected from T366V, T366I, T366L, T366M, N390D, N390E, K392L, K3921, K392D, K392E, K409F, K409W, T411D and T411E.

This application provides an isolated heteromultimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, wherein each modified CH3 domain polypeptide includes mutations of at least three amino acids, wherein one of said first and said second modified CH3 domain polypeptides comprises a mutation selected from T411E and T411D, wherein said first and second modified CH3 domain polypeptides preferably form a heterodimeric CH3 domain with a melting point (Tm) of at least about 70° C. and a purity of at least 90%. In one embodiment, an isolated heteromultimeric Fc construct is provided wherein said first modified CH3 domain polypeptide includes amino acid modifications at positions F405 and Y407 and said second modified CH3 domain polypeptide includes an amino acid modification at position T394. In one embodiment, an isolated heteromultimeric Fc construct is provided in which the first modified CH3 domain polypeptide includes an amino acid modification at position L351. In some embodiments, an isolated heteromultimer is provided as described herein, wherein said second modified CH3 domain polypeptide includes a modification at at least one of positions T366 and K392. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the modified CH3 domain has a melting point (Tm) of at least about 75° C. and is obtained at a purity of at least about 95%. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the at least one modified CH3 domain polypeptide includes at least one of the amino acid modifications N390R, S400E, and S400R. In some embodiments, an isolated heteromultimer is provided as described herein, wherein one of the first and second modified CH3 domain polypeptides includes an amino acid modification at position 347 and the other modified CH3 domain polypeptide includes an amino acid modification at position 360. In some embodiments, an isolated heteromultimer is provided. a heteromultimer described herein, wherein at least one of said first and second modified CH3 domain polypeptides includes a T350V amino acid modification. In specific embodiments, an isolated heteromultimer is provided as described herein, wherein said first modified CH3 domain polypeptide includes at least one amino acid modification selected from L351Y, F405A, and Y407V; and said second modified CH3 domain polypeptide includes at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In some embodiments described herein, an isolated heteromultimer is provided in which the first modified CH3 domain polypeptide includes amino acid modifications at positions D399 and Y407 and the second modified CH3 domain polypeptide includes amino acid modifications at positions K409 and T411. In some embodiments, an isolated heteromultimer is provided as described herein, wherein the first CH3 domain polypeptide includes an amino acid modification at position L351 and the second modified CH3 domain polypeptide includes amino acid modifications at positions T366 and K392. In specific embodiments, isolated heteromultimers are provided as described herein, wherein at least one of the first and second CH3 domain polypeptides includes the T350V amino acid modification. Some embodiments provide the isolated heteromultimers described herein, wherein the modified CH3 domain has a melting point (Tm) of at least about 750° C. or greater and is obtained at a purity of at least about 95%.

Some embodiments provide the isolated heteromultimeric Fc constructs described herein, wherein said first modified CH3 domain polypeptide includes amino acid modifications selected from L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A, and Y407V; and said second modified CH3 domain polypeptide includes amino acid modifications selected from T366V, T366I, T366L, T366M, N390D, N390E, K392L, K3921, K392D, K392E, K409F, K409W, T411D and T411E.

This application provides an isolated heteromultimeric Fc construct containing a modified CH3 domain containing a first modified CH3 domain polypeptide containing amino acid modifications L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide containing amino acid modifications T366I, K392M and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T366I, K392L and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T366L, K392M and T394W.

In some aspects, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T366L, K392L and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising the amino acid modifications T350V, L351Y, F405A and Y407V; and a second modified CH3 domain polypeptide containing amino acid modifications T350V, T366L, K392L and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T350V, T366L, K392M and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T350V, T366L, N390R, K392M and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided 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 containing amino acid modifications T350V, T366L, K392L and T394W.

In one aspect, an isolated heteromultimeric Fc construct is provided comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising the amino acid modifications T366V, K392L, K409F and T411E; and a second modified CH3 domain polypeptide containing amino acid modifications L351Y, D399R and Y407A.

In one aspect, an isolated heteromultimeric Fc construct is provided comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising the amino acid modifications T366V, K392LE K409F and T411E; and a second modified CH3 domain polypeptide containing amino acid modifications L351Y, D399R, S400R and Y407A.

In some embodiments, the Fc variant contains a CH2 domain. In some embodiments, the CH2 domain is a variant of the CH2 domain. In some embodiments, CH2 domain variants include asymmetric amino acid substitutions in the first and/or second polypeptide chain. In some embodiments, the heteromultimer includes asymmetric amino acid substitutions in the CH2 domain such that one chain of the heteromultimer selectively binds the Fc receptor.

In some embodiments, the heteromultimer selectively binds an Fc receptor. In some embodiments, the Fc receptor is a member of the Fcγ receptor family. In some embodiments, the receptor is selected from FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In one embodiment, the CH2 domain includes asymmetric amino acid modifications that promote selective binding of Fc-gamma receptors.

In some embodiments, the heteromultimer selectively binds to FcγRIIIa. In some embodiments, the heteromultimer includes asymmetric amino acid substitutions selected from S267D, K392D, and K409D. In some embodiments, the heteromultimer selectively binds to FcγRIIa. In some embodiments, the heteromultimer includes asymmetric amino acid substitutions selected from S239D, K326E, A330L, and I332E. In some embodiments, the heteromultimer selectively binds to FcγRIIb. In some embodiments, the heteromultimer includes asymmetric amino acid substitutions selected from S239D, D265S, E269K, and I332E. In some embodiments, the heteromultimer selectively binds to FcγRIIIa and FcγRIIa. In some embodiments, the heteromultimer includes asymmetric amino acid substitutions selected from S239D, D265S, and S298A. In some embodiments, the heteromultimer selectively binds to FcγRIIIa and FcγRIIb. In some embodiments, the heteromultimer includes asymmetric amino acid substitutions selected from S239D, S298A, K326E, A330L, and I332E. In some embodiments, the heteromultimer selectively binds to FcγRIIa and FcγRIIb. In some embodiments, the heteromultimer includes asymmetric amino acid substitutions selected from S239D, D265S, S298A, and I332E.

In some embodiments, a method is provided for developing a multifunctional drug containing a heteromultimer described herein. In some embodiments, a method for developing a bifunctional drug comprising an Fc heterodimer variant is provided. In some embodiments, a method is provided for engineering asymmetric mutations in the CH2 domain of a variant Fc heterodimer obtained by introducing mutations in the CH3 domain. In some embodiments, a method is provided for developing selectivity for various Fc-gamma receptors based on mutations in the asymmetric Fc. In some embodiments, a method is provided for engineering mutations that cause Fc gamma receptors to bind to one side of the Fc molecule. In some embodiments, a method is provided for developing polarity factors that cause Fcγ receptors to interact with only one side of the asymmetric Fc heteromultimer framework described herein.

In some embodiments, a polypeptide is provided comprising mutations in the CH2 domain of an asymmetric Fc that result in preferential Fc-gamma receptor selectivity profiles. In some embodiments, mutations in the CH3 domain result in preferential formation of a heterodimeric Fc.

In some embodiments, a method for developing bispecific therapeutic compounds based on the asymmetric Fc described herein is provided. In some embodiments, a method for developing multispecific therapeutic compounds based on the asymmetric Fc described herein is provided.

Monoclonal antibodies, such as IgG, are symmetrical molecules consisting of two equivalent heavy and two light polypeptide chains (Figure 14), each containing several immunoglobulin (Ig) structural domains. The IgG class of mAbs exists in four isoforms: IgG1, IgG2, IgG3 or IgG4. The heavy chain consists of four (VH, CH1, CH2 and CH3) and the light chain consists of two (VL and CL) Ig domains, respectively. The VH and CH1 domains of each heavy chain combine with the VL and CL domains of the light chain to form two Fab ("antigen-binding fragment") arms of the MAT. The CH3 and CH2 domains of the two heavy chains interact through protein-protein contacts along the CH3 domains and glycosylation in the CH2 domains to form a homodimeric Fc region (the “crystallizable fragment”). The linker region between the CH1 and CH2 domains of an antibody forms the hinge region of the antibody molecule. In addition to connecting the Fab and Fc regions of the MAT, the hinge also maintains disulfide bonds along the two heavy chains and holds them together. The number of amino acids and disulfide bonds in the hinge region varies significantly among the four IgG isotypes. The glycosylation pattern of IgG molecules can vary significantly; approximately 30 different carbohydrate molecules can be observed on IgG molecules [Arnold J.N.; Wormald M.R.; Sim R.B.; Rudd P.M. and Dwek R.A. (2007) Annual Reviews of Immunology 25, 21-50].

The symmetrical nature of the structure of monoclonal antibodies results in both Fab arms having the ability to bind antigen with an affinity ripe for recognition of the same epitope. On the other hand, the Fc region of an antibody molecule is involved in interactions with various receptor molecules on the surface of immune or "effector" cells, and some of these interactions mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and activation of complement. Typically, effector function induces immune responses, resulting in neutralization and elimination of pathogens or toxins, activation of complement, and a phagocytic response of the humoral immune system. Fcγ receptor (FcγR) molecules on the surface of effector cells contact the Fc of activated IgG antibody as part of the integral antibody-antigen immune complex to mediate and regulate the effector response. Optimizing the interaction of monoclonal antibody-based protein therapeutics with these Fcγ receptors may lead to improved efficacy of these drug candidates.

In humans, there are three known classes of FcγRs, with additional polymorphic types within each class. It is known that the Fc in the IgG1 molecule binds FcγRI (CD64) with dissociation constants in the nanomolar range, while the binding of FcγRII (CD32) and FcγRIII (CD 16) occurs with dissociation constants in the micromolar range [Bruhns P.; Iannascoli V.; England P.; Mancardi D.A.; Fernandez N.; Jorieux S. and Daeron M. (2009) Blood 113: 3716-25]. High-affinity FcγRI receptors can bind IgG in monomeric forms, whereas low-affinity FcγRII and FcγRIII receptors can only bind antigen-antibody immune complexes or IgG aggregates due to avidity effects. Different forms of IgG have different affinities for different FcγRs; in particular, IgG1 and IgG3 exhibit stronger activity. Fcγ receptors are extracellular domains of transmembrane proteins that also contain cytoplasmic domains involved in the regulation of cell signaling pathways. When clustered on the surface of an immune cell as a result of association with antibody-formed immune complexes, depending on the nature of the signal transduction compounds associated with FcγR on the cytoplasmic side 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 chromosome level, three genes encode FcγRI (FcγRIA, FcγRIB, FcγRIC) and FcγRII (FcγRIIA, FcγRIIB, FcγRIIC) and two genes encode FcγRIII (FcγRIIIA, FcγRIIIB). Among human IgG-binding Fcγ receptors, the FcγRIA, FcγRIC, and FcγRIIIA receptor types have been shown to associate with the membrane through a common γ-chain signaling adapter protein that contains a cytoplasmic immunoreceptor tyrosine activating motif (ITAM) that leads to activation of effector function. FcγRIIA and FcγRIIC also contain cytoplasmic ITAM, but without the common signaling adapter protein with the γ chain. At the same time, FcγRIIB is associated with the immunoreceptor tyrosine inhibitory motif (ITIM). Activation of FcγRIIB, resulting in phosphorylation of ITIM, results in inhibition of activation of the signaling cascade. FcγRIIIB, although it has none of the tyrosine immunomodulatory cytoplasmic tails, contains a glycosylphosphatidylinositol (GPI) anchor and has been shown to promote the activation of some granulocytes in the presence of FcγRIIA.

ITAM: Immunoreceptor tyrosine activating motif;

ITIM: Immunoreceptor tyrosine inhibitory motif;

GPI: Glycosylphosphatidylinositol

Although the functional role of the ITAM and ITIM motifs and their associated receptor molecules is known, the nature and mechanisms of modulation of signaling in combination are not well understood, especially in combination with the activity of other receptors on the surface of immune cells and host adapter molecules (for example, BCR , CD22, CD45, etc.) involved in signal transduction. In this context, the development of Fc-like molecules that can interact with these Fcγ receptors with perfect selectivity profiles is a useful platform for any attempt to deploy and modulate the actions of such receptor molecules with weak regulatory activities.

In the context of developing antibody molecules that can discriminate between Fcγ receptors, the work is complicated by the fact that the extracellular Fc binding regions of the FcγRII and FcγRIII type receptors have high sequence similarity (Figure 15), which may be due at least in part to duplications of ancestral gene fragments. The two main types of FcγRII receptors, A and B, share 69% sequence identity, while FcγRIIA and FcγRIIIA share approximately 44% sequence identity. FcγRIIB and FcγRIIC differ in only 2 residues in the extracellular region, but they differ significantly in the intracellular region, it is worth noting that they contain the ITIM and ITAM motifs, respectively. As a result, it can be expected that therapeutic antibody molecules that should bind one receptor can also potentially bind to other classes of receptors, possibly leading to unexpected therapeutic effects.

To further complicate matters, each receptor class contains multiple single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). The resulting diversity of receptors influences their affinity for IgG and their mechanism of action in different ways. These genetic variations may influence the affinity of specific IgG subclasses for Fcγ receptors, alter downstream effector events, or influence mechanisms that alter receptor expression levels, resulting in functionally relevant phenotypes, nonfunctional receptor variants, or receptor variants of unknown function (Bournazos S .; Woof J. M.; Hart S. P. and Dransfield I. (2009) Clinical and Experimental Immunology 157(2):244-54). They can potentially lead to complex effects that alter the balance between activating and inhibitory receptor signaling, resulting in disease-susceptible phenotypes.

Some of these allelic variants are listed in Table C. It is worth noting that the FcγRIIa variant with R131 exhibits a strong response to IgG1, while the alternative H131 variants showed more potent interactions with IgG2 and IgG3. In the case of FcγRIIIa, donors homozygous for V at position 158 showed increased NK cell activity compared to homozygous F/F158 individuals, due to the high affinity of the former allotype for human IgG1, IgG3 and IgG4. The NA1 and NA2 allelic variants of FcγRIIIb resulted from four amino acid substitutions, which in turn resulted in differences in receptor glycosylation. The NA1 allele causes increased binding to neutrophils and their phagocytosis of the immune complex. FcγRIIB has two known allelic variants, 232I and 232T. The 232T variant is known to have severely impaired negative regulatory activity. The frequencies of FcγR polymorphisms and their association with different susceptibility to infections or susceptibility to diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), vasculitis, idiopathic (autoimmune) thrombocytopenic purpura (ITP), myasthenia gravis, multiple sclerosis have been described. (MS) and immune neuropathies (Guillain-Barré syndrome (GBS)).

Copy number variation has been demonstrated at the FcγR gene locus, particularly for FcγRIIIB, FcγRIIC, and FcγRIIIA, and an additional correlation of these differences with the cell surface expression of these receptors has been noted. In contrast, no gene copy number variation was detected in FcγRIIa and FcγRIIb. It is known that low copy number of FcγRIIIb has been associated with glomerulonephritis in the autoimmune disease systemic lupus erythematosus (SLE) [Aitman TJ et al. (2006) Nature 16;439(7078):851-5]. This is of particular interest given that the non-signaling GPI module anchors the FcγRIIIb receptor. It can be assumed that these FcγRIIIb receptors could potentially act as competitive inhibitors of Fc interaction with other signaling FcγRs. Also of particular interest is the effect of copy number variations in FcγRIIc. The C/T single nucleotide polymorphism at position 202 in FcγRIIc converts the glutamine residue into a stop codon, preventing the formation of a functional protein. The functional open reading frame FcγRIIc is expressed in 9% of healthy individuals (in the white population), and there is a significant dominance (19%) of this allele in the ITP population, suggesting that these phenotypes predispose to ITP [Breunis WB et al. (2008) ) Blood 111 (3): 1029-38]. We demonstrated that in individuals expressing functional FcγRIIc on the surface of NK cells, the ADCC achieved is mediated by these receptors to a greater extent than by FcγRIIIa. This complexity associated with these polymorphisms and genetic variations highlights the need for personalized treatment strategies requiring highly specialized drugs.

Different effector cells differ in the presentation of these Fcγ receptors, as well as in their humoral and tissue distribution, which contributes to variation in their mechanism of activation and action [Table D]. Adjusting the selectivity of therapeutic antibodies for recognition of specific FcγR types and modulating the contribution of certain classes of effector cells leads to optimization of effector mechanisms for the treatment of specific diseases. This means selectively activating or inhibiting certain effector pathways depending on the disease being treated.

In addition, FcγRs are also expressed by follicular dendritic cells, endothelial cells, microglial cells, osteoclasts, and mesangial cells. To date, the functional significance of FcγR expression on these other cells is unknown.

High-affinity FcγRIs are composed of three immunoglobulin superfamily (IgSF) type C domains, whereas low-affinity FcγRII and FcγRIII are each composed of two IgSF type C domains. The structure of the receptor proteins FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb was revealed using crystallography. The two IgSF domains in these structures are located at an angle of 50-55 degrees relative to each other and are connected by a hinge.

The publicly available structure of the Fc-FcγR complex corresponds to the Fc-FcγRIIIb system, and the geometry of the FcγR in this complex remains 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 S. and Sun P. D. (2001) J Biol Chem 276, 16469-16477; Sondermann P. et al. Biochem Soc Trans. 2002 Aug; 30(4):481-6; Sondermann P, Oosthuizen V. Immunol Lett. 2002 Jun 3; 82(1-2):51-6; Radaev S, Sun P. Mo I Immunol. 2002 May; 38(14) :1073-83). (Figure 16). The strong similarity of sequences and structures between receptors provides the basis for comparative models of Fc bound to other receptors. On the other hand, the similarity of sequences and structures between these receptor molecules also makes it difficult to design Fcs with precise selectivity between receptors and their diverse isotypes.

Prior to structural evaluation of the Fc-FcγR complex based on crystallography, there were questions whether the 2-order symmetry axis in the Fc molecule indicated the presence of two potential binding sites and an effective 2:1 stoichiometry for Fc-FcγR association. Structural studies of Fc - FcγR interactions based on nuclear magnetic resonance (NMR) indicate that binding of an Fc to one FcγR on one side of the molecule causes 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 Biol 295(2):213-24). The geometry of the accessible cocrystallized Fc-FcγRIIIb complex confirms the association of FcγR with Fc in an asymmetric orientation with a 1:1 stoichiometry. Figure 16 shows that FcγR binds to a horseshoe-shaped pocket on one side of the Fc molecule and contacts CH2 domains on both strands.

Alanine scanning mutagenesis (Shields RL et al. (2001) JBC 276(9): 6591-604) provides insight into Fc residues that interact with different types of receptor and are therefore involved in Fc-FcγR interaction and recognition. Traditionally, optimization of therapeutic antibodies has centered around mutations that result in increased binding to activating FcγRIII receptors (US patent number 6,737,056) or decreased affinity for FcγRIIb (US 2009/0010920 A1). In all of these alternatives, mutations were introduced simultaneously into both chains.

Monoclonal antibodies often exhibit therapeutic activity by spatially localizing the target and the effector immune cell. A natural antibody accomplishes this localization by interacting with the target via Fab domains and with the effector cell via the Fc domain. They are able to bring the immune complex into proximity with the effector cell in such a way that a cell-mediated response can be evoked. The avidity effect required for FcγR signaling that occurs during the formation of immune complexes, which entails the targeting of multiple antibody molecules to a single target, provides another example of the importance of spatiotemporal organization in the functioning of the immune system.

There is also a spatiotemporal aspect of cell signaling that is induced as part of the effector activity of MAT molecules. Transmission of signals in the cell, for example, those arising from the activation of the FcγR molecule, involves the localization of the corresponding receptor molecules within regions of domains on the membrane called lipid rafts. Lipid rafts are enriched in glycosphingolipid and cholesterol and several classes of upstream signal transducers, including Src family kinases. When a cell is stimulated, various signaling molecules, adapter proteins and signal kinases, as well as phosphatases are attracted. The assembly of molecules in lipid rafts is important for signal transduction.

A synthetic design strategy that combines different antigen specificities and increased avidity to obtain better binding properties represents the basis for the development of bispecific therapeutics. Bispecific antibodies or other forms of bispecific or multifunctional protein drugs are designed to mediate interactions between a target and a variety of effector cells [ & Kontermann (2010) BioDrugs 24(2):89-98]. Multispecific therapeutic molecules are designed to retarget helper T cells or other immune effector cells to specific target cells.

In another embodiment, the present invention provides a method for computationally identifying Fc variant polypeptides based on calculated binding affinities for FcγRIIa, FcγRIIb and/or FcγRIIIa. In another embodiment, the method further includes computationally calculating the electrostatics, solvation, folding, packing density, hydrogen binding, and entropy effects of said Fc variant polypeptides. In yet another embodiment, the method of the present invention further comprises constructing Fc variant polypeptides and expressing said polypeptides as a therapeutic antibody, and further expressing said antibody in mammalian cells. In yet another embodiment, the method of the present invention involves constructing Fc variant polypeptides identified in silico by site-directed mutagenesis, PCR-based mutagenesis, cassette mutagenesis, or de novo synthesis.

Factors taken into account when designing a synthetic Fc scaffold include computer calculations of steric repulsion, internal surface area changes, relative contact density, relative solvation, and electrostatic effect. All resulting matrices were used to obtain an affinity index.

In one aspect of the present application, molecular engineering is described to achieve perfect FcγR selectivity profiles through the development of an asymmetric scaffold built on a heterodimeric Fc. This framework provides the ability to introduce asymmetric mutations into the CH2 domain to achieve a variety of new selectivity profiles. In addition, the scaffold has intrinsic properties for the development of multifunctional (bi-, tri-, tetra- or pentafunctional) therapeutic molecules based on it.

In some embodiments, the asymmetric scaffold is optimized for pH-dependent binding properties to the neonatal Fc receptor (FcRn) to provide better turnover of the molecule and increase its half-life and associated pharmacokinetic properties.

The asymmetric scaffold can be optimized for binding to functionally relevant FcγRI receptor allotypes. FcγRI is a known marker on macrophages that are involved in chronic inflammatory disorders such as rheumatoid arthritis, atopic dermatitis, psoriasis and a variety of lung diseases.

The asymmetric scaffold can be optimized for protein A binding. Protein A binding is often used to separate and purify antibody molecules. Mutations can be introduced into the asymmetric scaffold to avoid aggregation of the therapeutic during storage.

Therefore, it is particularly contemplated that the Fc variants of the present invention may include, but are not limited to, one or more additional amino acid residue substitutions, mutations and/or modifications that confer advantageous properties on the antibody, including, but not limited to: increased serum half-life, increased binding affinity, decreased immunogenicity, increased production, increased or decreased ADCC or CDC activity, altered glycosylation and/or disulfide bonds, and modified binding specificity.

It is contemplated that other properties may be altered in the Fc variants of the present invention, including increased in vivo half-life (eg, serum half-life) in a mammal, particularly a human; increased stability in vivo (eg, serum half-life) and/or in vitro (eg, storage time) and/or increased melting temperature (Tm), compared to a comparable molecule. In one embodiment, the Fc variant of the present invention has an in vivo half-life 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 , more than 4 months or more than 5 months. In another embodiment, the Fc variant of the present invention has an in vitro half-life (e.g., in a liquid or powder dosage form) greater than 15 days, greater than 30 days, greater than 2 months, greater than 3 months, greater than 6 months, or greater than 12 months , or more than 24 months, or more than 36 months, or more than 60 months.

It will also be apparent to one skilled in the art that the Fc variants of the present invention may have altered immunogenicity when administered to a subject. Accordingly, it is believed that a modified CH3 domain that minimizes the immunogenicity of the Fc variant is generally more desirable for use in therapy.

The Fc variants of the present invention can be combined with other Fc modifications, including, but not limited to, modifications that alter effector function. It is within the scope of the present invention to combine the Fc variant of the present invention with other Fc modifications to provide supportive, synergistic or novel properties to antibodies or Fc fusion proteins.

Such modifications may be present in the hinge, the CH1 or CH2 domains (or CH3, as long as they do not adversely affect the stability and purity of the modified CH3 domains of the present invention), or a combination of these domains. It is believed that the Fc variants of the present invention improve the properties of the modification with which they are combined. For example, if an Fc variant of the present invention is combined with a mutant that is known to bind FcγRIIIA with higher affinity than a comparable molecule containing a wild-type Fc region; combination with the mutant of the present invention results in a greater increase in affinity for FcγRIIIA.

In one embodiment, the Fc variants of the present invention can be combined with other known Fc variants, such as those described 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, ProcNatl. Acad Sci USA 92:1 1980-1 1984; Jefferis et al., 1995, Immunol Lett. 44:1 1 1 -1 17; Lund et al. 1995 Faseb J 9: 1 15-1 19; Jefferis et al., 1996, Immunol Lett 54:101-104; Lund et al. 1996, Immunol 157:4963-4969; Armor 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 Biol Chem 276:6591-6604; Jefferis et al. 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); in US patents No. 5624821; 5885573; 6194551; in US patent applications No. 60/601634 and 60/608852; in publications according to PCT Nos. WO 00/42072 and WO 99/58572.

It will be apparent to one skilled in the art that Fc variants of the present invention may have altered Fc ligand binding properties (e.g., FcγR, C1q) (examples of binding properties include, but are not limited to: binding specificity, equilibrium dissociation constant ( _KD ) , rates of dissociation and association (K _off and K _on , respectively), binding affinity and/or avidity), and that certain changes are more or less desirable. It is well known in the art that the equilibrium dissociation constant (K _D ) is calculated as k _off /k _on . In general, it will be apparent that a binding molecule (eg, antibody) with a low _KD is preferred over a binding molecule (eg, antibody) with a high _KD . However, in some cases, the value of k _on or k _off may be more important than the value of K _D . One skilled in the art can determine which kinetic parameter is most important for a given antibody application. For example, modified CH3 and/or CH2 that increase Fc binding to one or more positive regulators (eg, FcγRIIIA) while leaving unchanged or even decreasing Fc binding to the negative regulator FcγRIIB would be most useful for enhancing ADCC activity. Alternatively, modified CH3 and/or CH2 that reduce binding to one or more positive regulators and/or increase binding to FcγRIIB will be most useful in reducing ADCC activity. Accordingly, the ratio of binding affinities (eg, equilibrium dissociation constants (K _D )) can indicate whether the ADCC activity of the Fc variant is increased or decreased. For example, a decrease in the equilibrium dissociation constant ratio (K _D ) of FcγRIIIA/FcγRIIB will correlate with an increase in ADCC activity, while an increase in this ratio will correlate with a decrease in ADCC activity.

As part of the characterization of Fc variants, their binding affinity to FcγRIIIA (CD16a) and FcγRIIB(CD32b) was examined and reported as a ratio compared to wild-type IgG1 (see Example 4 and Table 5). In this case, it was possible to assess the effect of mutations in the CH3 domain on binding to these activating and inhibitory Fc receptors. In one embodiment, this application provides isolated heteromultimers containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, and wherein the modified CH3 domain has a melting point ( Tm) more than 70°C, while the binding of the heterodimer to CD16a is approximately the same as that of the wild-type homodimer. In some embodiments, binding of the heterodimer to CD16a is increased compared to the wild-type homodimer. In an alternative embodiment, binding of the heterodimer to CD16a is reduced compared to the wild-type homodimer.

In some embodiments, this application provides isolated heteromultimers containing a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, and wherein the modified CH3 domain has a melting point ( Tm) more than 70°C, while the binding of the heterodimer to CD32b is approximately the same as that of the wild-type homodimer. In some embodiments, binding of the heterodimer to CD32b is increased compared to the wild-type homodimer. In an alternative embodiment, binding of the heterodimer to CD32b is reduced compared to the wild-type homodimer.

It will be apparent to one skilled in the art that instead of representing the _KD of CD16a and CD32b binding as a comparison of the Fc variant to the wild-type homodimer, the _KD can be represented as the ratio of the binding of the Fc variant to CD16a to the binding of the Fc variant to CD32b (results not shown ). This ratio provides a measure of the effect of a mutation in the modified CH3 domain on ADCC (unchanged, increased or decreased compared to the wild type), which is described in more detail below.

The affinities and binding properties of the Fc variants of the present invention to the FcγR were initially determined using in vitro tests (biochemical or immunoassays) known in the art to determine Fc-FcγR interaction, i.e., specific binding of the Fc region to the FcγR, including but not limited to: enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance assays, immunoprecipitation assays (see below in the section entitled “Characteristic and Functional Assays”) 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 use a label on one or more components of interest 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 present invention can also be characterized by in vitro functional assays to determine one or more FcγR-mediated effector cell functions (see above in the section entitled "Characterization and Functional Assays"). In some embodiments, the molecules of the present invention have binding properties in in vivo models (such as those described and disclosed herein) similar to those in in vitro assays. However, the present invention does not exclude molecules of the present invention that do not exhibit the desired phenotype in in vitro assays but exhibit the desired phenotype in vivo.

The present invention includes Fc variants that bind FcγRIIIA (CD16a) with increased affinity compared to a similar molecule. In a specific embodiment, the Fc variants of the present invention bind FcγRIIIA with increased affinity and bind FcγRIIB (CD32b) with a binding affinity that is either unchanged or reduced compared to a similar molecule. In yet another embodiment, the Fc variants of the present invention have a lower equilibrium dissociation constant ratio (K _D ) with FcγRIIIA/FcγRIIB than that of a similar molecule.

Also included within the scope of the present invention are Fc variants that bind FcγRIIIA (CD16a) with reduced affinity compared to a similar molecule. In a specific embodiment, the Fc variants of the present invention bind FcγRIIIA with reduced affinity compared to a similar molecule and bind FcγRIIB with a binding affinity that is either unchanged or increased compared to a similar molecule.

In one embodiment, Fc variants bind to FcγRIIIA with increased affinity. In a specific embodiment, said Fc variants have an 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 at least 7-fold. 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times , or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times greater than that of a comparable molecule. In other embodiments, Fc variants have an affinity for FcγRIIIA that is increased by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by 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% compared to a similar molecule.

In another embodiment, the Fc variant has an equilibrium dissociation constant (K _D ) with an Fc ligand (e.g., FcγR, C1q) that is reduced by a factor of about 2 to 10, or by a factor of about 5 to 50, or by a factor of from about 25 to 250 times, or in the range from about 100 to 500 times, or in the range from about 250 to 1000 times compared to a similar molecule.

In another embodiment, said Fc variants have an equilibrium dissociation constant (K _D ) with FcγRIIIA that is reduced by at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, or at least 400 times, or by at least 600 times compared to a similar molecule. In another embodiment, Fc variants have an equilibrium dissociation constant (K _D ) with FcγRIIIA that is reduced by at least 10%, or by at least 20%, or by at least 30%, or by at least 40% , or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 150%, or by at least 200% compared to a similar 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 an affinity for FcγRIIB that is unchanged or reduced by at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20 times, or at least 50 times, or at least 100 times compared to a similar molecule. In other embodiments, the Fc variants have an affinity for FcγRIIB that is unchanged or reduced by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by 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 at least 150%, or at least 200% compared to a similar molecule.

In another embodiment, Fc variants have an equilibrium dissociation constant (K _D ) with 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 times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times compared to a similar molecule. In another specific embodiment, Fc variants have an equilibrium dissociation constant (K _D ) with FcγRIIB that is unchanged or increased by at least 10%, or by at least 20%, or by at least 30%, or by 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% compared to a similar molecule.

In another further embodiment, Fc variants bind FcγRIIIA with increased affinity compared to a similar molecule and bind FcγRIIB with an affinity that is unchanged or reduced compared to a similar molecule. In a specific embodiment, the Fc variants have an affinity for FcγRIIIA that is increased by at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 10-fold. 20 times, or at least 50 times, or at least 100 times compared to a similar molecule. In another specific embodiment, the Fc variants have an affinity for FcγRIIB that is either unchanged or reduced by at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold , or at least 10 times, or at least 20 times, or at least 50 times, or at least 100 times compared to a similar molecule. In other embodiments, Fc variants have an affinity for FcγRIIIA that is increased by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by 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 by at least 200% compared to a similar molecule, and said Fc variants have an affinity for FcγRIIB that is either unchanged or increased by at least 10%, or by at least 20%, or by 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% compared to a similar molecule.

In yet another embodiment, Fc variants have an equilibrium dissociation constant ratio (K _D ) with FcγRIIIA/FcγRIIB that is reduced compared to a similar molecule. In a specific embodiment, the Fc variants have an equilibrium dissociation constant ratio (K _D ) with FcγRIIIA/FcγRIIB that is reduced by at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10 times, or at least 20 times, or at least 50 times, or at least 100 times compared to a similar molecule. In another specific embodiment, Fc variants have an equilibrium dissociation constant ratio (K _D ) with FcγRIIIA/FcγRIIB that is reduced by at least 10%, or by at least 20%, or by at least 30%, or by 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% compared to a similar molecule.

In another embodiment, Fc variants bind FcγRIIIA with reduced affinity compared to a similar molecule. In a specific embodiment, said Fc variants have an affinity for FcγRIIIA that is reduced by at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20 times, or at least 50 times, or at least 100 times compared to a similar molecule. In other embodiments, the Fc variants have an affinity for FcγRIIIA that is reduced by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by 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% compared to a similar molecule.

In another further embodiment, Fc variants bind FcγRIIIA with reduced affinity and bind FcγRIIB with affinity that is either unchanged or increased compared to a similar molecule. In a specific embodiment, the Fc variants have an affinity for FcγRIIIA that is reduced by at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 10-fold. 20 times, or at least 50 times, or at least 100 times compared to a similar molecule. In another specific embodiment, Fc variants have an 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 at least 7-fold. 10 times, or at least 20 times, or at least 50 times, or at least 100 times greater than that of a comparable molecule. In other embodiments, Fc variants have an affinity for FcγRIIIA that is reduced by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by 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 by at least 200% compared to a similar molecule and said Fc variants have an affinity for FcγRIIB that is increased by at least 10%, or by at least 20%, or by at least 30%, or by 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% compared to a similar molecule.

In another further embodiment, Fc variants have an equilibrium dissociation constant (K _D ) with FcγRIIIA that is increased by at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold times, or at least 20 times, or at least 50 times compared to that of a comparable molecule. In a specific embodiment, said Fc variants have an equilibrium dissociation constant (K _D ) with FcγRIIB that is reduced by at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold times, or at least 10 times, or at least 20 times, or at least 50 times, or at least 100 times compared to a similar molecule.

CH2 options for FcγR selectivity

The protein-protein Fc-FcγR interaction in this complex indicates that the two chains in the Fc molecule interact with two different sites on the FcγR molecule. Although in natural Fc molecules the two heavy chains are symmetrical, the local environment of the FcγR near residues on one chain is different from the FcγR residues surrounding a residue at the same position on the opposite Fc chain. The two symmetrical positions interact with different samples of FcγR residues.

Given the asymmetry in Fc binding to FcγR, simultaneous mutations of the A and B chains of the Fc molecule do not affect interactions with FcγR in a symmetrical manner. When mutations are introduced to optimize interactions on one Fc chain with its local FcγR environment, in a homodimeric Fc structure, the corresponding mutation on the second chain may be favorable, unfavorable, or not conducive to the desired FcγR binding profile and selectivity.

Using a structured and computer-assisted approach, asymmetric mutations were engineered in the two Fc chains to overcome these limitations of traditional Fc development strategies in which the same mutations were introduced in both Fc chains. If the two Fc chains are independently optimized for increased binding to the corresponding side of the receptor molecule, better binding selectivity between receptors can be achieved.

For example, mutations at a particular position on one Fc chain can be designed so that they increase selectivity for a particular residue, realizing positive engineering, while a residue at the same position can be mutated so that it interacts unfavorably with the local environment in an alternative type of Fcγ receptor. implementation of negative development, thus achieving better selectivity between the two receptors. In some embodiments, a method is provided for designing asymmetric amino acid modifications in the CH2 domain that selectively bind one Fc-gamma receptor over a different Fc-gamma receptor (eg, selectively bind FcγRIIIa but not FcγRIIb). In some other embodiments, a method is provided for designing asymmetric amino acid modifications in the CH2 domain of a variant Fc heterodimer containing amino acid modifications in the CH3 domain to cause heterodimer formation. In another embodiment, a method is provided to achieve selectivity for various Fc-gamma receptors based on a variant Fc heterodimer containing asymmetric amino acid modifications in the CH2 domain. In yet another embodiment, a method is provided for developing asymmetric amino acid modifications that promote binding of Fc-gamma receptors to one side of the Fc molecule. In some other embodiments, a method is provided for developing polarity factors that cause Fc-gamma receptors to interact with only one side of a variant Fc heterodimer containing asymmetric amino acid modifications in the CH2 domain.

Asymmetrical development of mutations in the CH2 domain can be tailored to recognize FcγRs on one side of the Fc molecule. This side represents the productive side of the asymmetric Fc framework, while the opposite side tends to interact like the wild-type molecule without a developed selectivity profile and can be considered the non-productive side. A negative engineering strategy can be used to introduce mutations in the nonproductive side to block FcγR interactions with that side of the asymmetric Fc framework, thereby increasing the bias of Fcγ receptors toward the desired interaction.

The present invention also provides fusion polypeptides comprising a binding domain fused to an Fc region, wherein the Fc region comprises a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, and wherein the modified CH3 domain has a melting temperature (Tm) more than 70°C. In particular, it is contemplated that molecules containing a heterodimer containing a modified CH3 domain can be prepared using 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 having 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 receptor binding domain) , with Fc heterodimer variants. Alternatively, one skilled in the art can produce a variant Fc heterodimer by modifying the CH3 domain in the Fc region of a molecule containing an Fc region (eg, antibodies).

In one embodiment, the Fc variants are antibodies or Fc fusion proteins. In a specific embodiment, the present invention provides antibodies comprising an Fc region comprising a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, and wherein the modified CH3 domain has a melting temperature (Tm) greater than 70°C. Such antibodies include IgG molecules that naturally contain an Fc region containing a CH3 domain that can be modified to produce an Fc variant, or antibody derivatives that have artificially incorporated an Fc region containing a modified CH3 domain. Fc variants of the present invention include any antibody molecule that preferably binds specifically (i.e., successfully competes with non-specific binding, as determined by immunoassays well known in the art to assay specific antigen-antibody binding) to an antigen that includes an Fc region containing a modified CH3 domain. Such antibodies include, but are not limited to: polyclonal, monoclonal, monospecific, bispecific, multispecific, human, humanized, chimeric antibodies, single chain antibodies, Fab fragments, F(ab') ₂ fragments, disulfide-linked Fv and fragments containing any of VL or VH domains, or even a complementarity determining region (hypervariable region, CDR) that specifically binds an antigen, in some cases, into which an Fc heterodimer variant has been artificially incorporated or fused.

"Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which a secreted antibody bound to Fc receptors (FcRs) present on certain cytotoxic cells (eg, natural killer (NK) cells, neutrophils, and macrophages) allows These cytotoxic effector cells specifically bind to the antigen-bearing target cell and subsequently kill the target cell with cytotoxins. Specific high-affinity IgG antibodies directed to the surface of target cells target cytotoxic cells and are absolutely necessary for such killing. Lysis of the target cell is extracellular, requires direct cell-to-cell contact, and occurs without the participation of complement.

The ability of any particular antibody to mediate lysis of a target cell by ADCC can be analyzed. To assess ADCC activity, the antibody of interest is added to target cells in combination with immune effector cells that can be activated by antigen-antibody complexes, resulting in cytolysis of the target cell. Cytolysis is typically detected by the release of label (eg, radioactive substrates, fluorescent dyes, or natural intracellular proteins) from lysed cells. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) 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 in this application (see below in the section entitled "Characteristic and functional assays"). Alternatively or in addition, the ADCC activity of an antibody of interest can be assessed in vivo, for example, in an animal model such as that described in Clynes et al., 1998, PNAS USA 95:652.

It is contemplated that the Fc variants of the present invention can be characterized using in vitro functional assays to determine one or more FcγR-mediated effector cell functions. In specific embodiments, the molecules of the present invention have binding properties and effector cell functions in in vivo models (such as those described and disclosed herein) similar to those in in vitro assays. However, the present invention does not exclude molecules of the present invention that do not exhibit the desired phenotype in in vitro assays but exhibit the desired phenotype in vivo.

The present invention further provides Fc variants with enhanced CC function. In one embodiment, Fc variants have increased CDC activity. In one embodiment, 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 times greater than that of a comparable molecule. In another embodiment, 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 at least 10-fold times, or at least 20 times, or at least 50 times, or at least 100 times greater than that of a comparable molecule. In yet another embodiment, Fc variants have CDC activity that is increased by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by 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% compared to a similar molecule. In a specific embodiment, the Fc variants of the present invention bind C1q with increased affinity; have increased CDC activity and specifically bind to at least one antigen.

The present invention also provides Fc variants with reduced CDC function. In one embodiment, Fc variants have reduced CDC activity. In one embodiment, 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 times less than that of a comparable molecule. In another embodiment, the Fc variant binds C1q with an affinity that is reduced by at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20 times, or at least 50 times, or at least 100 times compared to a similar molecule. In another embodiment, Fc variants have CDC activity that is reduced by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50 %, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 150%, or at least 200% compared to a similar molecule. In a specific embodiment, Fc variants bind to C1q with reduced affinity, have reduced CDC activity, and specifically bind to at least one antigen.

In some embodiments, the Fc variants contain one or more engineered glycoforms, i.e., a carbohydrate composition that is covalently attached to a molecule containing an Fc region. The engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Engineered glycoforms can be produced by any method known to one skilled in the art, for example, by using engineered or altered expression strains, by co-expression with one or more enzymes, for example, β(1,4)-N-acetylglucosaminyltransferase III (GnT -IIII11), by expressing the Fc region-containing molecule in different organisms or cell lines from different organisms, or by modifying the carbohydrate(s) after expressing the Fc region-containing molecule. Methods for preparing engineered glycoforms are known in the art and include, but are not limited to, the glycoforms 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 Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) in US patent number 6602684; in US patent application number 10/277370; in US patent application number 10/113929; PCT WO 00/61739 A1; PCT WO 01/292246 A1; PCT WO 02/311140 A1; PCT WO 02/30954A1; in Potillegent™ technology (Biowa, Inc. Princeton, N.J.); in GlycoMAb™ glycosylation development technology (GLYCART biotechnology AG, Zurich, Switzerland). See, for example, WO 00061739; EA 01229125; US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.

Fc variants are believed to include antibodies containing a variable region and a heterodimeric Fc region, wherein the heterodimeric Fc region contains a modified CH3 domain containing amino acid mutations that promote the formation of a heterodimer with increased stability, and wherein the modified CH3 domain has a melting point (Tm ) more than 70°C. Fc variants, which are antibodies, can be produced "de novo" by joining a variable domain, or fragment thereof, that specifically binds at least one antigen to a heterodimeric Fc region containing a modified CH3 domain. Alternatively, Fc heterodimer variants can be generated by modifying the CH3 domain of an antibody containing the Fc region that binds the antigen.

Antibodies of the present invention may include, but are not limited to: synthetic antibodies, monoclonal antibodies, recombinant antibodies, intraantibodies, monospecific antibodies, multispecific antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single chain FvFc (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 regions of immunoglobulin molecules. The immunoglobulin molecules of the present invention may belong to any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ¹ and IgA ₂ ) or subclass immunoglobulin molecules.

The antibodies of the present invention can be of any animal origin, including birds and mammals (eg, human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken). In a specific embodiment, the antibodies are human or humanized monoclonal antibodies, particularly bispecific 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 usually defined as the pH at which the polypeptide carries zero net charge. It is known in the art that protein solubility is generally lowest when the pH of the solution is equal to the isoelectric point (pI) of the protein. Solubility can be optimized by changing the number and location of ionizable residues in the antibody molecule to adjust the pI. For example, the pI of a polypeptide can be adjusted by introducing suitable amino acid substitutions (eg, by replacing a charged amino acid, such as lysine, with an uncharged residue, such as alanine). Without being bound by any particular theory, amino acid substitutions in an antibody that result in changes in the pI of the antibody may improve the solubility and/or stability of the antibody. One of ordinary skill in the art will understand which amino acid substitutions will be most appropriate for a particular antibody to achieve the desired pI. The pI of a protein can be determined using 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 present invention is between pH 6.2 and pH 8.0. In another embodiment, the pI of the antibodies of the present invention is between pH 6.8 and pH 7.4. In one embodiment, substitutions resulting in changes to the pI of the Fc variant of the present invention will not significantly reduce its antigen binding affinity. It is assumed that a modified CH3 domain with increased stability can also be obtained by changing pI. In one embodiment, Fc heterodimer variants are specifically selected to have both increased stability and purity and any desired change in pI.

Antibodies of the present invention may be monospecific, bispecific, trispecific, or more multispecific. Multispecific antibodies can specifically bind to different epitopes of the desired target molecule or can specifically bind to both the target molecule and a heterologous epitope, such as a heterologous polypeptide or solid support material. See, for example, international publication numbers 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; US patents number; 4474893; 4714681; 4925648; 5573920 and 5601819 and Kostelny et al., 1992, J. Immunol, 148:1547).

Various implementations of multifunctional targeting molecules can be developed based on this asymmetric scaffold, as shown in Figure 20.

Multispecific antibodies have binding specificities for at least two different antigens. Although such molecules will typically bind only two antigens (ie, bispecific antibodies, BsAbs), antibodies with additional specificities, such as trispecific antibodies, are also within the scope of the present invention. Examples of BsAbs include, but are not limited to, those antibodies in which one arm is directed against a tumor cell antigen and the other arm is directed against a cytotoxic molecule, or both arms are directed against two different tumor cell antigens, or both arms are directed against two different soluble ligands, or one arm is directed against a soluble 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 producing bispecific antibodies are known in the art.

In an excellent approach, antibody variable domains with desired binding specificities (antibody-antigen binding sites) are fused to immunoglobulin constant domain sequences. The fusion can be performed with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. It is assumed that the first heavy chain constant region (CH1), containing the site required for light chain binding, is present in at least one of the fusion molecules. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into separate expression vectors that are co-transfected into a suitable host organism. This provides greater flexibility in adjusting the relative ratio of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used for construction produce optimal yields. See Example 1 and Table 2. However, it is possible to insert coding sequences for two or all three polypeptide chains into a single expression vector if expression of at least two polypeptide chains in equal ratios results in high yield or if the ratios are not particularly significant .

Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one of the antibodies in a heteroconjugate may be associated with avidin, and the other with biotin. Such antibodies, for example, have been proposed for targeting immune cells to unwanted cells (US patent number 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugated antibodies can be prepared using any convenient cross-linking technique. Suitable cross-linking agents are well known in the art and are described in US Pat. No. 4,676,980, along with a variety of cross-linking techniques.

The present invention provides antibodies with more than two valencies containing modified CH3 domains and the resulting Fc heterodimers. For example, trispecific antibodies can be obtained. See, for example, Tutt et al. J. Immunol. 147:60 (1991).

Antibodies of the present invention also include those antibodies that have a half-life (eg, serum half-life) in a mammal (eg, human) greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, more than 45 days, more than 2 months, more than 3 months, more than 4 months or more than 5 months. The increased half-life of the antibodies of the present invention in a mammal (e.g., a human) results in a higher titer of said antibodies or antibody fragments in the serum of the mammal and thus allows the frequency of administration of said antibodies or antibody fragments to be reduced and/or the concentration of said antibodies administered to be reduced. or antibody fragments. Antibodies having an extended half-life in vitro can be prepared using methods known to those skilled in the art. For example, antibodies with increased half-life in vivo can be obtained by modification (eg, substitution, deletion or insertion) of amino acid residues that are identified as being involved in the interaction between the Fc domain and the FcRn receptor (see, for example, International Publication Nos. WO 97/ 34631; WO 04/029207; US Patent No. 6737056 and US Patent Publication No. 2003/0190311).

In a specific embodiment, the modified CH3 domain-containing variant Fc heterodimer is a multispecific antibody (referred to herein as an antibody of the present invention), said antibody of the present invention specifically binding to an antigen of interest. In particular, the antibody of the present invention is a bispecific antibody. In one embodiment, an antibody of the present invention specifically binds a polypeptide antigen. In another embodiment, an antibody of the present invention specifically binds a non-polypeptide antigen. In yet another embodiment, administration of an antibody of the present invention to a mammal suffering from a disease or disorder may result in a therapeutic benefit for the mammal.

Virtually any molecule may be targeted and/or incorporated into the Fc heterodimer variant 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; growth hormone, including human growth hormone and bovine growth hormone; a factor that stimulates the release of growth hormone; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1 antitrypsin; Insulin A chain; Insulin B chain; proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VII, factor VIIIC, factor IX, tissue factor (TF), and von Willebrand factor; anticoagulants such as protein C; atrial natriuretic factor; pulmonary surfactant; a plasminogen activator such as urokinase, or from human urine, or tissue plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (a chemokine expressed and secreted by T cells upon activation); human macrophage inflammatory protein (MIP-1alpha); serum albumin such as human serum albumin; Mullerian inhibitory substance; Relaxin A chain; Relaxin B chain; prorelaxin; mouse gonadotropin-associated peptide; microbial protein such as beta-lactamase; DNase; IgE; cytotoxic T lymphocyte antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); hormone or growth factor receptors, such as, for example, EGFR, VEGFR; interferons such as interferon alpha (α-IFN), interferon beta (β-IFN) and interferon gamma (γ-IFN); protein A or D; rheumatoid factors; a neurotrophic factor such as brain-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), or 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-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25, CD33, CD34, CD40, CD40L, CD52, CD63, CD64, CD80 and CD147; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic protein (BMP); interferon such as interferon alpha, beta and gamma; colony stimulating factors (CSF) such as M-CSF, GM-CSF and G-CSF; interleukins (IL), for example IL-1 to IL-13; TNFα, superoxide dismutase; T cell receptors; surface membrane proteins; hemolysis stimulator; a viral antigen, such as, for example, part of the envelope of the AIDS virus, for example, gp120; transport proteins; homing receptors; addresses; regulatory proteins; cell adhesion molecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, ICAM-3 and VCAM, a4/p7 integrin, and (Xv/p3 integrin, including any of its subunits, integrin subunits alpha such as CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, alpha7, alpha8, alpha9, alphaD, CD11a, CD11b, CD51, CD11c, CD41, alphaIIb, alphaIELb; integrin beta subunits such as CD29, CD18, CD61, CD104, beta5, beta6, beta7 and beta8; combinations of integrin subunits, including, but not limited to, αVβ3, αVβ5 and α4β7; member of the apoptotic pathway; IgE; blood group antigens; flk2/flt3 receptor; obesity receptor (OB); mp1 receptor ; CTLA-4; protein C; Eph receptor such as EphA2, EphA4, EphB2, etc.; human leukocyte antigen (HLA) such as HLA-DR; complement proteins such as complement receptor CR1, C1Rq and other factors complement, such as C3 and C5; glycoprotein receptor, such as GpIbα, GPIIb/IIIa and CD200; and fragments of any of the above polypeptides.

Also provided are antibodies of the present invention that specifically bind cancer antigens, including, but not limited to: ALK receptor (pleiotrophin receptor), pleiotrophin, carcinoma pan-antigen KS 1/4; 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-38.13; CD 19; human B lymphoma antigen CD20; CD33; melanoma-specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3; tumor-specific transplantation antigen located on the cell surface (TSTA); virus-induced tumor antigens, including T-antigen of tumor-causing DNA viruses and envelope antigens of tumor-causing RNA viruses; oncofetal antigen alpha-fetoprotein such as colon CEA, oncofetal trophoblast glycoprotein 5T4 and oncofetal bladder tumor antigen; differentiation antigen such as human lung carcinoma antigens L6 and L20; fibrosarcoma antigens; human T-cell leukemia 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); APO-1 antigen of malignant human lymphocytes; differentiation antigen such as I antigen found in fetal red blood cells; Primary endoderm I antigen, found in adult erythrocytes; in embryos before implantation; I(Ma), found in gastric adenocarcinomas; M18, M39, found in mammary epithelium; SSEA-1, found in myeloid cells; VEP8; VEP9; Myl; Va4-D5; D ₁ 56-22, found in colorectal cancer; TRA-1-85 (blood type H); SCP-1, found in testicular and ovarian cancer; C14, found in colon adenocarcinoma; F3, found in lung adenocarcinoma; AH6, found in stomach cancer; Y-hapten; Ley, found in embryonal carcinoma cells; TL5 (blood type A); EGF receptor found in A431 cells; E series antigen ₁ (blood group B), found in pancreatic cancer; FC10.2, found in embryonal carcinoma cells; gastric adenocarcinoma antigen CO-514 (blood group Lea), found in adenocarcinoma; NS-10, found in adenocarcinomas; SO-43 (blood group Leb); G49, found in the EGF receptor of A431 cells; MH2 (blood type ALeb/Ley), found in colon adenocarcinoma; 19.9, found in colon cancer; stomach cancer mucins; T ₅ A ₇ found in myeloid cells; R ₂₄ , found in melanoma; 4.2, G _D3 , D1.1, OFA-1, G _M2 , OFA-2, G _D2 and M1:22:25:8 found in embryonal carcinoma cells, and SSEA-3 and SSEA-4 found in embryos at stages 4-8 cells; cutaneous T-cell lymphoma antigen; MART-1 antigen; Sialy Tn (STn) antigen; colon cancer antigen NY-CO-45; lung cancer antigen NY-LU-12, variant A; adenocarcinoma antigen ART1; paraneoplastic antigen associated with brain-testicular cancer (onconeuronal antigen MA2; paraneoplastic neuronal antigen); neurooncology ventral antigen 2 (NOVA2); hepatocellular carcinoma antigen 520 gene; tumor associated antigen CO-029; tumor associated antigens MAGE-C1 (testicular cancer antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4-a, MAGE-4-b and MAGE-X2 ; testicular cancer antigen (NY-EOS-1) and fragments of any of the above polypeptides.

In some embodiments, the heteromultimer described herein includes at least one therapeutic antibody. In some embodiments, the therapeutic antibody binds the target cancer antigen. In one embodiment, the therapeutic antibody may be selected from the group consisting of abagovomab, adalimumab, alemtuzumab, aurograb, bapineizumab, basiliximab, belimumab, bevacizumab, briakinumab, canakinumab, catumaxomab, certolizumab pegol, cetuximab, daclizumab, denosumab, ephaliz umaba, 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, ProxiniumTM, RencarexTM, ustekinumab, zalutumumab and any other antibodies.

Antibodies of the present invention include derivatives that have been modified (ie, by attaching any type of molecule to the antibody, eg, by covalent attachment). For example, but without limitation, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by introducing known protecting/blocking groups, proteolytic cleavage, attachment to a cellular ligand or other protein, etc. d. Any of a variety of chemical modifications can be accomplished using known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivative may contain one or more non-classical amino acids.

Antibodies or antibody fragments with increased half-life in vivo can be obtained by attaching polymer molecules, such as high molecular weight polyethylene glycol (PEG), to antibodies or antibody fragments. PEG can be attached to antibodies or antibody fragments with or without a multifunctional linker, either through site-specific conjugation of PEG to the N- or C-terminus of said antibodies or antibody fragments, or through epsilon amino groups present on lysine residues. Derivatization of linear or branched polymer should be used that results in minimal loss of biological activity. The degree of conjugation will be carefully monitored using EPAGE/SDS-PAGE and mass spectrometry to ensure proper conjugation of the PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates, for example, using size exclusion or ion exchange chromatography.

In addition, antibodies can be conjugated to albumin to produce an antibody or antibody fragment that is more stable in vivo or has a longer half-life in vivo. Such techniques are well known in the art, see, for example, international publications Nos. WO 93/15199, WO 93/15200 and WO 01/77137; and European patent number EP 413622. The scope of the present invention includes the use of antibodies or antibody fragments conjugated or fused to one or more molecules, including, but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimicking agents, synthetic drugs, inorganic molecules and organic molecules.

The present invention includes the use of antibodies or antibody fragments recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or fragment thereof, for example, a polypeptide consisting of at least 10, at least 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 produce fusion proteins. The fusion does not have to be direct; it can be accomplished through linker sequences. For example, antibodies can be used to target heterologous polypeptides to specific cell types, either in vitro or in vivo, by fusing or conjugating the antibodies with antibodies specific for specific cell surface receptors. Antibodies fused or conjugated to heterologous polypeptides can also be used in immunoassays and in vitro purification methods using methods known in the art. See, for example, international publication number WO 93/21232; European patent number EP 439095; Naramura et al., 1994, Immunol. Lett. 39:91-99; US patent number 5474981; Gillies et al. 1992, PNAS 89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446–2452.

The present invention further includes compositions containing heterologous proteins, peptides or polypeptides fused or conjugated to antibody fragments. For example, heterologous polypeptides can be fused or conjugated to a Fab fragment, an Fd fragment, an Fv fragment, an F(ab) ₂ fragment, a VH domain, a VL domain, a VH hypervariable region, a VL hypervariable region, or a fragment of the foregoing. Methods for fusing or conjugating polypeptides to antibody regions are well known in the art. See, for example, US patent numbers 5336603; 5622929; 5359046; 5349053; 5447851 and 5112946; European patents with numbers EP 307434 and EP 367166; international publications with numbers 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 Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337–11341.

Additional fusion proteins, for example with antibodies that specifically bind antigen (eg, see above), can be generated using gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling techniques (collectively referred to as “DNA shuffling”). DNA shuffling can be used to change the activities of antibodies of the present invention or fragments of these antibodies (eg, to produce antibodies or antibody fragments with higher affinities and lower dissociation rates). See generally US Pat. Nos. 5,605,793; 5811238; 5830721; 5834252 and 5837458, 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. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2): 308-313. Antibodies or antibody fragments, or encoded antibodies or antibody fragments, can be changed by performing nonspecific mutagenesis by PCR using imprecise polymerase, random insertion of nucleotides, or other methods, before recombination. One or more regions of a polynucleotide encoding an antibody or antibody fragment, from regions that specifically bind an antigen, can be recombined with one or more components, motifs, segments, portions, domains, fragments, etc. one or more heterologous molecules.

The present invention further includes the use of Fc heterodimer variants or fragments thereof conjugated to a therapeutic agent.

The antibody or antibody fragment can be conjugated to a therapeutic molecule, such as a cytotoxin, eg, a cytostatic or cell-destructive agent, a therapeutic agent, or a radioactive metal ion, eg, alpha emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include ribonuclease, monomethylaistatin E and F, paclitaksel, cytochalazine c, gramicyide d, bromide ethidium, emitin, mitomycin, ethoside, teenoside, vincristine, vinblastine, colchicin, doxorubicin, dowenorubicin, digidroxianthic, mitoxontin, mitramicin, mitramicin, mitramicin, mitramicin, mitramicin, mitramicin, mitramitsin, mitramicin, mitramicin, mitramicin, mitramicin, mitramicin, mitramicin, mitramicins Tinomicin d, 1-dehydrotestosterone , glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and analogs or homologues of the listed agents. Therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (eg, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BCNU), and lomustine (BCNU). CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and diaminedichloroplatinum(II) (DDP, cisplatin), anthracyclines (eg, daunorubicin (formerly called daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly called actinomycin), bleomycin , mithramycin and anthramycin (AMC)), and antimitotic agents (eg vincristine and vinblastine).A more extensive list of therapeutic molecules can be found in PCT publication WO 03/075957.

In addition, the antibody or antibody fragment can be conjugated to a therapeutic agent or drug molecule that modifies the underlying biological response. Therapeutic agents or drug molecules should not be considered as limited to classical chemical therapeutic agents. For example, the drug molecule may be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, onconase (or other cytotoxic RNase), Pseudomonas aeruginosa exotoxin, cholera toxin or diphtheria toxin; protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agent such as TNF-α, TNF-β, AIM I (see international publication number WO 97/33899), AIM II (see international publication number WO 97/34911), Fas ligand (Takahashi et al., 1994, J. Immunol., 6:1567), and VEGI (see international publication number WO 99/23105), a thrombotic agent or an antiangiogenic agent, for example 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"), granulocyte colony-stimulating factor ("G-CSF"), or a growth factor (eg, growth hormone ("GH")).

Moreover, the antibody can be conjugated to therapeutic molecules, such as radioactive materials or macrocyclic chelators suitable for conjugating radioactive metal ions (see above for examples of radioactive materials). In some 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 well known in the art and are 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. Biol. 26:943.

Methods for fusing or conjugating antibodies with polypeptide molecules are known in the art. See, for example, US patent numbers 5336603; 5622929; 5359046; 5349053; 5447851 and 5112946; EP 307434; EP 367166; publications according to PCT WO 96/04388 and WO 91/06570; Ashkenazi et al. 1991, PNAS USA 88:10535; Zheng et al 1995 J Immunol 154:5590; and Vil et al., 1992, PNAS USA 89:1 1337. Fusion of the antibody with the molecule does not have to be direct, but can also be accomplished through linker sequences. Such linker molecules are well known in the art and are 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 or a derivative, analog, or fragment thereof (eg, an antibody or fusion protein of the present invention) requires the construction of an expression vector containing a polynucleotide that encodes the Fc variant (eg, an antibody or fusion protein). Once a polynucleotide encoding an Fc variant (eg, antibody or fusion protein) has been produced, a vector for producing the Fc variant (eg, antibody or fusion protein) can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an Fc variant (eg, an antibody or fusion protein) are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding an Fc variant (eg, antibody or fusion protein) and suitable transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, in vivo synthesis techniques, and genetic recombination. The present invention therefore provides replicable vectors containing a nucleotide sequence encoding an Fc variant of the present invention operably linked to a promoter. Such vectors may include a nucleotide sequence encoding the constant region of an antibody molecule (see, for example, International Publication No. WO 86/05807; International Publication No. WO 89/01036; and US Pat. No. 5,122,464) and an antibody variable domain, or a polypeptide to produce a variant The Fc can be cloned into such a vector to express a full-length antibody chain (eg, heavy or light chain) or a complete Fc variant, including a fusion of a non-antibody polypeptide and an Fc region containing at least a modified CH3 domain.

The expression vector is transferred into a host cell using conventional techniques, and the transfected cells are then cultured using conventional techniques to produce the Fc variant of the present invention. Thus, the present invention includes host cells containing a polynucleotide encoding an Fc variant of the present invention operably linked to a heterologous promoter. In specific embodiments, for the expression of Fc variants containing double-chain antibodies, vectors encoding both the heavy and light chains can be co-expressed in a host cell to express the entire immunoglobulin molecule, as described in detail below.

A variety of host-expression vector systems can be used to express Fc variants of the present invention (eg, antibody or fusion protein molecules) (see, for example, US Pat. No. 5,807,715). Such host-expression vector systems provide a means by which coding sequences of interest can be obtained and then purified, and also provide cells that, when transformed or transfected with suitable nucleotide coding sequences, can express in situ the Fc variant of the present invention. Such systems include, but are not limited to: microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing sequences encoding an Fc variant; yeast (eg, Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing Fc variant coding sequences; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing sequences encoding an Fc variant; plant cell systems infected with recombinant viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) containing sequences encoding an Fc variant; or mammalian cell systems (eg, COS, CHO, BHK, 293, NS0 and 3T3 cells) bearing recombinant expression constructs containing promoters derived from the genome of mammalian cells (eg, the metallothionein promoter) or from mammalian viruses (eg, the late promoter adenovirus; vaccinia virus promoter 7.5K). In some embodiments, bacterial cells, such as Escherichia coli, or eukaryotic cells are used to express an Fc variant that is a recombinant antibody or fusion protein molecule. For example, mammalian cells, such as Chinese hamster ovary (CHO) cells, in combination with a vector, such as the major intermediate-early gene promoter element from human cytomegalovirus, provide an efficient antibody expression system (Foecking et al., 1986, Gene 45: 101; and Cockett et al., 1990, Bio/Technology 8:2). In a particular embodiment, the expression of nucleotide sequences encoding an Fc variant of the present invention (eg, an antibody or fusion protein) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

In bacterial systems, a variety of expression vectors can be successfully selected depending on the intended use of the Fc variant being expressed (eg, antibody or fusion protein). For example, if large quantities of such a protein are desired to produce pharmaceutical compositions of the Fc variant, vectors that direct expression of high levels of a fusion protein product that is easy to purify 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 sequence encoding the Fc variant can be ligated into the vector separately in the same reading frame as the encoding region lac Z, such that a lac Z fusion protein is produced; pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 24:5503-5509); and similar vectors. pGEX vectors can also be used to express foreign polypeptides as glutathione S-transferase (GST) fused proteins. Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a glutathione-agarose bead matrix followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from GST molecules.

In the insect system, Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector for the expression of foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence for the Fc variant (eg, antibody or fusion protein) can be cloned separately into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of the AcNPV promoter (eg, the polyhedrin promoter).

A variety of virus-based expression systems can be used in mammalian host cells. In cases where an adenovirus is used as an expression vector, the Fc variant coding sequence of interest (eg, antibody or fusion protein) can be ligated to an adenoviral transcription/translation control complex, eg, a late promoter and triple leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, the E1 or E3 region) will produce a recombinant virus that is viable and capable of expressing an Fc variant (eg, antibody or fusion protein) in infected hosts (eg, see Logan and Shenk, 1984, Proc Natl Acad Sci USA 81:355-359). Efficient translation of integrated antibody coding sequences may also require certain initiation signals. These signals include the ATG start codon and adjacent sequences. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and start codons can come from a variety of sources, both natural and synthetic. Expression efficiency can be increased by including suitable transcription enhancer elements, transcription terminators, etc. (See, eg, Bittner et al., 1987, Methods in Enzymol. 153:516-544).

Expression of an Fc variant (eg, antibody or fusion protein) can be controlled by any promoter or enhancer element known in the art. Promoters that can be used to control expression of a 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' position of the 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), metallothionein gene regulatory sequences (Brinster et al., 1982, Nature 296:39-42), tetracycline (Tet) promoter (Gossen et al., 1995, Proc. Nat. Acad. Sci. USA 89:5547 -5551); prokaryotic expression vectors such as the β-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); expression vectors in plants, including 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 bisphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310: 115-120); promoter elements from yeast or other fungi, such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, and the following transcriptional control regions in animals, which exhibit tissue specificity and are used in transgenic animals: gene regulatory region elastase I, which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987 , Hepatology 7:425-515); insulin gene regulatory region, which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene regulatory 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. Biol. 7: 1436-1444), a regulatory region of the mouse mammary tumor virus, which is active in testicular, lymphoid, mast cells and breast cells (Leder et al., 1986, Cell 45:485-495), the regulatory region of the albumin gene, which is active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), the regulatory region of the alpha-fetoprotein gene, which is active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235:53-58); the regulatory region of the alpha-1-antitrypsin gene, which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), the regulatory region of the beta-globin gene, which is active in myeloid cells (Mogram et al., 1985 , Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the regulatory region of the myelin basic protein gene, which is active in brain oligodendrocytes (Readhead et al., 1987, Cell 48:703-712); the regulatory region of the myosin light chain-2 gene, which is active in skeletal muscle (Sani, 1985, Nature 314:283-286); the regulatory region of the neuron-specific enolase (NSE) gene, which is active in nerve cells (Morelli et al., 1999, Gen. Virol. 50:571-83); the regulatory region of the brain-derived neurotrophic factor (BDNF) gene, which is active in nerve 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 Biol Res 32(5): 619-631; Morelli et al., 1999, Gen. Virol. 80:571- 83) and the regulatory region of the gonadotropin-releasing hormone gene, 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 present invention (e.g., an antibody or fusion protein) can be identified using three general approaches: (a) nucleic acid hybridization, (b) the 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 fusion protein in an expression vector can be detected by nucleic acid hybridization using probes containing sequences that are homologous to the inserted gene encoding a peptide, polypeptide, protein or fusion protein, respectively. In the second approach, the recombinant vector/host system can be identified and selected based on the presence or absence of certain "marker" gene functions (eg, thymidine kinase activity, antibiotic resistance, transformed phenotype, inclusion body formation in baculovirus, etc.) caused by insertion into the vector of a nucleotide sequence encoding an antibody or fusion protein. For example, if a nucleotide sequence encoding an Fc variant (eg, an antibody or fusion protein) is inserted into a vector marker gene sequence, recombinants containing the inserted gene encoding the antibody or fusion protein can be identified by the absence of marker gene function. In a third approach, recombinant expression vectors can be identified by analyzing the gene product (eg, antibody or fusion protein) expressed by the recombinant. Such assays may be based, for example, on the physical and functional properties of the fusion protein in in vitro assay systems, for example, binding to an antibody against a biologically active molecule.

In addition, a host cell strain may be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in a particular desired manner. Expression using some promoters can be increased in the presence of some inducers; thus, the expression of the genetically engineered fusion protein can be controlled. Moreover, different host cells have characteristic and specific mechanisms for translational and post-translational processing and modification (eg, glycosylation, phosphorylation) of proteins. Suitable cell lines or host systems can be selected to provide the desired modification and processing of the foreign protein being expressed. For example, expression in a bacterial system will produce a non-glycosylated 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 (eg, glycosylation and phosphorylation of the gene product) can be used. Such mammalian host cells include, but are not limited to: CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, NS0 and, in particular, neural cell lines, such as, for example, human neuroblastoma cells SK- N-AS, SK-N-FI, SK-N-DZ (Sugimoto et al., 1984, J. Natl. Cancer Inst. 73: 51-57), human neuroblastoma SK-N-SH (Biochim. Biophys. Acta , 1982, 704: 450-460), human cerebellar medulloblastoma Daoy (He et al., 1992, Cancer Res. 52: 1144-1148), glioblastoma cells DBTRG-05MG (Kruse et al., 1992, In Vitro Cell. Dev Biol. 28A: 609-614), human neuroblastoma IMR-32 (Cancer Res., 1970, 30: 2110-2118), human astrocytoma 1321N1 (Proc. Natl. Acad. Sci. USA, 1977, 74: 4816), human astrocytoma MOG-G-CCM (Br. J. Cancer, 1984, 49: 269), human glioblastoma-astrocytoma U87MG (Acta Pathol. Microbiol. Scand., 1968, 74: 465-486), human glioblastoma A172 (Olopade and al., 1992, Cancer Res. 52: 2523-2529), rat C6 glioma cells (Benda et al., 1968, Science 161: 370-371), mouse neuroblastoma Neuro-2a (Proc. Natl. Acad. Sci. USA, 1970, 65: 129-136), mouse neuroblastoma NB41A3 (Proc. Natl. Acad. Sci. USA, 1962, 48: 1184-1190), sheep choroid plexus SCP (Bolin et al., 1994, J. Virol. Methods 48: 211-221), G355-5, normal cat astrocytes PG-4 (Haapala et al., 1985, J. Virol. 53: 827-833), ferret brain Mpf (Trowbridge et al., 1982, In Vitro 18: 952-960) and normal cell lines, such as, for example, normal rat CTX TNA2 cells (Radany et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6467-6471), such as eg CRL7030 and Hs578Bst. Moreover, different vector/host expression systems may influence processing responses to varying degrees.

For long-term production of recombinant proteins in high yield, stable expression is often preferred. For example, cell lines that stably express an Fc variant of the present invention (eg, an antibody or fusion protein) can be developed. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by suitable expression regulatory elements (eg, promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.) and selectable marker. After introduction of foreign DNA, the engineered cells are allowed to grow for 1-2 days in enriched medium and then transferred to selective medium. A selectable marker in a recombinant plasmid confers resistance to selection and enables cells to stably integrate the plasmid into cell chromosomes and grow into foci, which in turn can be cloned and expanded into cell lines. This method can be successfully used to construct cell lines that express an Fc variant that specifically binds to antigen. Such engineered cell lines may be particularly useful for screening and evaluating compounds that affect the activity of an Fc variant (eg, a polypeptide or fusion protein) that specifically binds an antigen.

A variety of selection systems can be used, including but not limited to: herpes simplex virus thymidine kinase genes (Wigler et al., 1977, Cell 11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48 :2026) and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) in tk, hgprt or aprt cells, respectively. It is also possible to use antimetabolite resistance as a basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; 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 aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

Once an Fc variant (e.g., antibody or fusion protein) of the present invention has been produced by recombinant expression, it can be purified using any protein purification method known in the art, e.g., chromatography (e.g., ion exchange, affinity, especially affinity to a specific antigen after protein A, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard protein purification technique.

The Fc variant is typically obtained from the culture medium as a secreted polypeptide, although it can also be obtained from host cell lysate if obtained directly without a secretory signal. If the Fc variant is obtained membrane bound, it can be released from the membrane using a suitable detergent solution (eg Triton-X100).

If the Fc variant is produced in a recombinant cell other than a cell of human origin, it is completely free of proteins or polypeptides of human origin. However, it is necessary to purify the Fc variant from proteins or polypeptides of the recombinant cell in order to obtain preparations that are substantially homogeneous with respect to the Fc variant. In the first step, the culture medium or lysate is typically centrifuged to remove insoluble cell debris.

Fc heterodimers containing antibody constant domains can be conveniently purified by hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, with affinity chromatography being the preferred purification method. Other protein purification techniques are also available, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin-Sepharose chromatography, anion or cation exchange resin chromatography (e.g., polyaspartic acid column), chromatofocusing , EF in PAGE/SDS and precipitation with ammonium sulfate, depending on the polypeptide being purified. The suitability of protein A as an affinity ligand depends on the immunoglobulin Fc domain species and isotypes used. Protein A can be used to purify immunoglobulin Fc regions that are derived from human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (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 also available. Mechanically stable matrices such as controlled pore glass or poly(styrene divinyl)benzene allow higher flow rates and shorter processing times than can be achieved with agarose. The conditions for immunoadhesin binding to the protein A or G affinity column are entirely determined by the properties of the Fc domain; namely, its species and isotypes. As a rule, with the correct choice of ligand, effective binding occurs directly from the unprepared culture liquid. Bound Fc heterodimer variants can be efficiently eluted with either an acidic pH buffer (equal to or greater than 3.0) or a neutral pH buffer containing a mildly chaotropic salt. This affinity chromatography step will produce a drug of the Fc heterodimer variant that is >95% pure.

Expression levels of an Fc variant (eg, 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, if a marker in a vector system expressing an antibody or fusion protein is amplified, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is linked to the antibody gene, production of the antibody or fusion protein will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

A host cell can be co-transfected with two expression vectors of the present invention. For example, a first vector encodes a polypeptide designed on a heavy chain basis and a second vector encodes a polypeptide designed on a light chain basis. The two vectors may include identical selectable markers that enable the expression of heavy and light chain polypeptides in equal amounts. Alternatively, a separate vector may be used that encodes and is capable of expressing the fusion protein or both heavy and light chain polypeptides. The coding sequences for the fusion protein or heavy and light chains may include cDNA or genomic DNA.

Characteristic and functional analyzes

Fc variants (eg, antibodies or fusion proteins) according to the present invention can be characterized in a variety of ways. In one embodiment, the purity of Fc heterodimer variants is assessed using techniques well known in the art, including, but not limited to, SDS-PAGE, Western blotting, densitometry, or mass spectrometry. Protein stability can be characterized using a variety of techniques, including but not limited to: size exclusion chromatography, UV, visible light and circular dichroism spectroscopy, mass spectroscopy, differential light scattering, sample preparation stability analysis, freeze-thaw, in combination with other characterization techniques , differential scanning calorimetry, differential scanning fluorimetry, hydrophobic interaction chromatography, isoelectric focusing, receptor binding assays or relative protein expression levels. In an exemplary embodiment, the stability of Fc heterodimer variants is assessed by the melting temperature of the modified CH3 domain, compared to the wild-type CH3 domain, using techniques well known in the art, such as differential scanning calorimetry or differential scanning fluorimetry.

The ability of the Fc variants of the present invention to specifically bind to a ligand (eg, FcγRIIIA, FcγRIIB, C1q) can also be analyzed. Such analysis can be carried out in solution (for example, Houghten, Bio/Techniques, 13:412-421, 1992), on granules (Lam, Nature, 354:82-84, 1991), on microchips (Fodor, Nature, 364:555 -556, 1993), on bacteria (US patent number 5223409) 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. Biol., 222:301-310, 1991). Molecules found to be specifically bound to a ligand (eg FcγRIIIA, FcγRIIB, C1q or antigen) can then be subjected to ligand affinity analysis.

The Fc variants of the present invention can be assayed for specific binding to a molecule, such as an antigen (eg, cancer antigen and cross-reactivity with other antigens) or ligand (eg, FcγR), using any method known in the art. Immunoassays that can be used to analyze specific binding and cross-reactivity include, but are not limited to: competitive and noncompetitive assay systems using techniques such as Western blotting, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoprecipitation assays, precipitation reactions, gel diffuse precipitation reactions, immunodiffusion techniques, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescence immunoassays, protein A immunoassays, among others. Such assays are standard and well known in the art (see, for example, Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, volume 1, John Wiley & Sons, Inc., New York).

The binding affinity of Fc variants of the present invention to a molecule, such as an antigen or ligand (eg, FcγR), and the rate of dissociation of the interaction can be determined using competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising incubating a labeled ligand, such as an FcγR (e.g., labeled ZH or 1251), with a molecule of interest (e.g., Fc variants of the present invention) in the presence of increasing amounts of an unlabeled ligand, such as an FcγR, and detecting molecules bound to the labeled ligand. The affinity of the molecules of the present invention for the ligand and the rate of binding dissociation can be determined from saturation results using the Scatchard assay.

The kinetic parameters of the Fc variant can also be determined using surface plasma resonance (SPR) assays known in the art (eg, BIAcore kinetic assay). 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. In addition, any of the SPR devices and methods for measuring protein-protein interactions based on SPR described in US patent numbers 6373577; 6289286; 5322798; 5341215; 6268125 are provided in the methods of the present invention.

Fluorescence-excited cell sorting (FACS), using any of the techniques known to those skilled in the art, can be used to characterize the binding of Fc variants to a molecule expressed on the cell surface (eg, FcγRIIIA, FcγRIIB). Flow sorters are capable of rapidly screening large numbers of individual cells that contain inserts from libraries (eg, 10-100 million cells per hour) (Shapiro et al., Practical Flow Cytometry, 1995). Flow cytometers for sorting and studying biological cells are well known in the art. Known flow cytometers are described, for example, in US patent numbers 4347935; 5464581; 5483469; 5602039; 5643796; and 6211477. Other well-known flow cytometers are the FACS Vantage™ system from Becton Dickinson and Company and the COPAS™ system from Union Biometrica.

The Fc variants of the present invention can be characterized by their ability to mediate FcγR-mediated effector cell function. Examples of effector cell functions that may be analyzed include, but are not limited to: antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, opsonization, opsonophagocytosis, C1q binding, and complement-dependent cellular cytotoxicity (CDC). Any cell-based or cell-free assay known to those skilled in the art can be used to determine activation of effector cell function (for a review of effector cell assays, see Perussia et al., 2000, Methods Mol. Biol. 121: 179-92; Baggiolini and 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 Biol., 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 present invention can be assayed for FcγR-mediated ADCC activity in effector cells (eg, 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 Biol 121: 179-92). An example assay for determining the ADCC activity of molecules according to the present invention is based on a 51Cr release assay comprising: labeling target cells with [51 Cr]Na ₂ CrO ₄ (this cell membrane permeable molecule is often used for labeling because it binds cytoplasmic proteins and, although it is spontaneously released from cells with delayed kinetics, it is released en masse after necrosis of the target cell); opsonization of target cells with Fc variants according to the present invention; combining opsonized radiolabeled target cells with effector cells in a microtiter plate at a suitable ratio of target cells to effector cells; incubation of the cell mixture for 16-18 hours at 37°C; collection of supernatants; and radioactivity analysis. The cytotoxicity of the molecules of the present invention can then be determined, for example, using the following formula: % lysis = (experimental cpm - cpm leakage from target cells)/(cpm lysate after detergent treatment - cpm leakage from target cells) × 100%. Alternatively, % lysis = (ADCC-ANCC)/(maximum release spontaneous release). Specific lysis can be calculated using the formula: specific lysis = % lysis by molecules of the present invention - % lysis in the absence of molecules of the present invention. A graph can be constructed by varying either the target: effector cell ratio or the antibody concentration.

The method of describing the ability of Fc variants to bind C1q and mediate complement dependent cytotoxicity (CDC) is well known in the art. For example, to determine C1q binding, a C1q binding ELISA can be performed. An example analysis may be as described below. Assay plates can be coated overnight at 4C with the variant polypeptide or parent polypeptide (control) in coating buffer. The plates can then be washed and blocked. After washing, an aliquot of human C1q can be added to each well and incubated for 2 hours at room temperature. After an additional wash, 100 µl of peroxidase-conjugated sheep anti-complement C1q antibody can be added to each well and incubated for 1 hour at room temperature. The plate can be washed again with wash buffer and 100 µl of substrate buffer containing OPD (ortho-phenylenediamine dihydrochloride (Sigma)) can be added to each well. The oxidation reaction, observed by the appearance of a yellow color, can be allowed to proceed for 30 minutes and then stopped by adding 100 µl of 4.5 N H2SO4. The absorbance can then be read at 492-405 nm.

To assess complement activation, a complement-dependent cytotoxicity (CDC) assay can be performed (eg, as described in Gazzano-Santoro et al., 1996, J. Immunol. Methods 202:163). Briefly, various concentrations of Fc variant and human complement can be diluted in buffer. Cells that express the antigen to which the Fc variant is associated can be diluted to a concentration of approximately 1 x 106 cells/ml. Mixtures of the Fc variant, dilute human complement, and antigen-expressing cells can be added to a flat-bottomed 96-well culture plate and allowed to incubate for 2 hours at 37C and 5% CO2 to allow complement-mediated cell lysis to occur. 50 µl of Alamar Blue (Accumed International) can then be added to each well and incubated overnight at 37°C. Absorbance is measured using a 96-well photometer with excitation at 530 nm and emission at 590 nm. The results can be expressed as relative fluorescence units (RFU). Sample concentrations can be calculated from a standard curve, and the percentage of activity for the Fc variant of interest compared to a similar molecule (ie, a molecule containing an Fc region with an unmodified CH3 domain or a wild-type CH3 domain) can be reported.

Complement fixation assays can be performed on guinea pig, rabbit or human serum. Complement lysis of target cells can 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, which labels the target cells.

Methods

It is within the scope of the present invention to administer one or more Fc variants of the present invention (eg, antibodies) to an animal, particularly a mammal, especially a human, to prevent, treat or alleviate one or more symptoms associated with a disease, disorder or infection. The Fc variants of the present invention are particularly useful for treating or preventing a disease or disorder where altering the efficiency of effector cell function (eg, ADCC, CDC) is desired. Fc variants and compositions containing them are particularly useful for the treatment or prevention of primary or metastatic neoplastic disease (ie, cancer) and infectious diseases. The molecules of the present invention may be provided in pharmaceutically acceptable compositions known in the art or described herein. As detailed below, the molecules of the present invention may be used in methods of treating or preventing cancer (especially passive immunotherapy), autoimmune disease, inflammatory disorders, or infectious diseases.

The Fc variants of the present invention may also be usefully used in combination with other therapeutic agents known in the art for the treatment or prevention of cancer, autoimmune disease, inflammatory disorders or infectious diseases. In a specific embodiment, the Fc variants of the present invention can be used in combination with monoclonal or chimeric antibodies, lymphokines or hematopoietic growth factors (such as, for example, IL-2, IL-3 and IL-7), which, for example, are used to increasing the number or activity of effector cells that interact with molecules and increase the immune response. The Fc variants of the present invention can also be usefully used 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 antiviral agents.

Accordingly, the present invention provides methods for preventing, treating, or alleviating one or more symptoms associated with cancer and related conditions by administering one or more Fc variants of the present invention. Without being bound by any particular mechanism of action, an Fc variant of the present invention that binds FcγRIIIA and/or FcγRIIA with greater affinity than a comparable molecule, and further binds FcγRIIB with lower affinity than a comparable molecule, and/or said Fc variant, which has enhanced effector function, such as ADCC, KZC, phagocytosis, opsonization, etc., will lead to selective targeting and effective destruction of cancer cells.

It is further within the scope of the present invention to administer one or more Fc variants of the present 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 chemotherapy, hormonal therapies, biological treatments, immunotherapy, radiation treatments or surgery. In some embodiments, molecules of the present invention can be administered in combination with a therapeutically or prophylactically effective amount of one or more anticancer agents, therapeutic antibodies, or other agents known to those skilled in the art to treat and/or prevent cancer. Examples of dosage regimens and treatments that can be used in combination with the Fc variants of the present invention are well known in the art and have been described in detail in various publications (see, for example, PCT publications WO 02/070007 and WO 03/075957) .

Cancers and related disorders that may be treated or prevented by the methods and compositions of the present invention include, but are not limited to: leukemias, lymphomas, multiple myelomas, bone and connective tissue sarcomas, brain tumors, breast cancer, adrenal cancer , thyroid cancer, pancreatic cancer, pituitary cancer, eye cancer, vaginal cancer, vulvar cancer, cervical cancer, uterine cancer, ovarian cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gall cancer bladder, cholangiocarcinoma, lung cancer, testicular cancer, prostate cancer, penile cancer; oral cancer, salivary gland cancer, pharyngeal cancer, skin cancer, kidney cancer, bladder cancer (for a review of such disorders see Fishman et al., 1985, Medicine, 2nd 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 present invention further provides for the development of any of the antibodies known in the art for the treatment and/or prevention of cancer and related disorders such that the antibodies include a Pe region containing a modified CH3 domain according to the present invention.

In a specific embodiment, a molecule of the present invention (e.g., an antibody comprising a variant Fc heterodimer) inhibits or reduces primary tumor growth or cancer cell metastasis by at least 99%, at least 95%, at least 90%, at least 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% compared to primary tumor growth or metastasis observed in the absence of the specified molecule according to the present invention .

It is within the scope of the present invention to use one or more Fc variants of the present invention to prevent, treat, or control one or more symptoms associated with an inflammatory disorder in a subject. Without being tied to any specific mechanism of action, Fc variants with increased affinity for FcγRIIB will lead to inhibition of receptor activation and thus suppression of the immune response, and have therapeutic efficacy for the treatment and/or prevention of an autoimmune disorder. Moreover, antibodies that bind more than one target associated with an inflammatory disorder, such as bispecific antibodies containing an Fc heterodimer variant, may have a synergistic effect in monovalent therapy.

It is further within the scope of the present invention to administer the Fc variants of the present invention in combination with a therapeutically or prophylactically effective amount of one or more anti-inflammatory agents. The present invention also provides methods for preventing, treating, or controlling one or more symptoms associated with an autoimmune disease, further comprising administering to said subject an Fc variant of the present invention in combination with a therapeutically or prophylactically effective amount of one or more immunomodulatory agents. Examples of autoimmune disorders that can be treated by administration of Fc variants of the present invention include, but are not limited to: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune adrenal diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis , autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, dermatitis in gluten-sensitive celiac disease, chronic fatigue and immune dysfunction syndrome (CFIS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutination syndrome, Crohn's disease , discoid lupus, idiopathic mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus , Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 diabetes mellitus or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary tract cirrhosis liver, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, muscle stiffness syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, such as dermatitis herpetiformis, vitiligo and Wegener's granulomatosis. Examples of inflammatory disorders include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation caused by as a result of chronic viral or bacterial infections. Some autoimmune disorders are associated with an inflammatory condition, so the concepts of an autoimmune disorder and an inflammatory disorder overlap. Therefore, some autoimmune disorders can also be described as inflammatory disorders. Examples of inflammatory disorders that can be prevented, treated or controlled in accordance with the methods of the present invention include, but are not limited to, asthma, encephalitis, 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 bacterial infections.

The Fc variants of the present invention can also be used to reduce inflammation exhibited in animals, especially mammals suffering from inflammatory disorders. In a specific embodiment, the Fc variant of the present invention reduces 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 65%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, according at least 25%, at least 20%, or at least 10% compared to inflammation in an animal that was not administered the specified molecule.

The present invention further provides for the development of any antibodies known in the art for the treatment and/or prevention of an autoimmune disease or an inflammatory disease such that the antibodies contain an Fc heterodimer variant of the present invention.

Also included within the scope of the present invention are methods of 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 present invention. Infectious diseases that can be treated or prevented by the Fc variants of the present invention are caused by infectious agents, including, but not limited to, viruses, bacteria, fungi, protozoa and viruses.

Viral diseases that can be treated or prevented using the Fc variants of the present invention in combination with the methods of the present invention include, but are not limited to, diseases caused by hepatitis A, hepatitis B, hepatitis C, influenza, varicella, adenovirus , herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), rinderpest virus, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papovavirus, cytomegalovirus, echinovirus, arbovirus , hantavirus, coxsackie virus, mumps virus, measles virus, rubella virus, poliovirus, variola virus, Epstein-Barr virus, human immunodeficiency virus type I (HIV-1), human immunodeficiency virus type II (HIV-II) and viral agents diseases such as viral meningitis, encephalitis, dengue fever or smallpox.

Bacterial diseases that can be treated or prevented using the Fc variants of the present invention in combination with the methods of the present invention are caused by bacteria including, but not limited to: Mycobacteria, Rickettsia, Mycoplasma, Neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease ), Bacillus anthracis (anthrax), tetanus, streptococcus, staphylococcus, mycobacteria, tetanus, whooping cough, cholera, plague, diphtheria, chlamydia, S. aureus and legionella. Protozoal diseases that can be treated or prevented using the molecules of the present invention in combination with the methods of the present invention are caused by protozoa, including, but not limited to, leishmania, coccidia, trypanosome or malaria. Parasitic diseases that can be treated or prevented using the molecules of the present invention in combination with the methods of the present invention are caused by parasites containing, but not limited to, chlamydia and rickettsia.

In some embodiments, the Fc variants of the present invention may be administered in combination with a therapeutically or prophylactically effective amount of one or more additional therapeutic agents known to those skilled in the art for the treatment and/or prevention of an infectious disease. The present invention provides the use of the molecules of the present 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 antiviral agents.

The present invention provides methods and pharmaceutical compositions containing Fc variants of the present invention (eg, antibodies, polypeptides). The present invention also provides methods for treating, preventing, and reducing the severity 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 present invention or a pharmaceutical composition containing at least one Fc variant according to the present invention. In one aspect, the Fc variant is substantially purified (ie, substantially free of substances that limit its action or cause unwanted side effects, which include homodimers and other cellular material). In a specific embodiment, the subject is an animal, such as a mammal, including non-primate animals (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and primates (e.g., monkeys such as cynomolgus macaques and humans). In a particular embodiment, the subject is a human. In yet another specific embodiment, the antibody of the present invention is derived from the same species as the subject.

The route of administration of the composition depends on the condition being treated. For example, intravenous injection may be preferable for treating a systemic disorder such as cancer of the lymphatic system or a tumor that has metastasized. The dosage of the compositions to be administered can be determined by a person skilled in the art without undue experimentation in accordance with standard dose-response studies. Circumstances that should be considered in making such a decision include the condition or conditions being treated, the composition selected 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 to the patient orally, parenterally, intranasally, vaginally, rectally, lingually, sublingually, buccally, intrabuccally and/or transdermally.

Accordingly, compositions formulated for oral, lingual, sublingual, buccal and intrabuccal administration can be prepared without undue experimentation by means well known in the art, for example, the addition of an inert diluent or edible carrier. The composition can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral administration of a drug, excipients can be included in the pharmaceutical compositions of the present invention and administered in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like dosage forms.

Tablets, pills, capsules, lozenges and the like may also contain binders, excipients, disintegrating agents, 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 disintegrants include alginic acid, corn starch 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 to prepare these various compositions must be pharmaceutical grade and non-toxic in the quantities used.

The pharmaceutical compositions of the present invention can be administered parenterally, 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, fixed oils, polyethylene glycols, glycerin, propylene glycol and/or other synthetic solvents. Parenteral dosage forms may also include antibacterial agents, such as, for example, benzyl alcohol and/or methylparabens, 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 tonicity agents such as sodium chloride and dextrose may also be added. Compositions for parenteral administration can be contained in ampoules, disposable syringes and/or multi-dose vials made of glass or plastic. Rectal administration involves administering the composition into the rectum and/or colon. This can be done by using suppositories and/or enemas. Suppository dosage forms can be prepared using methods known in the art. Transdermal administration involves absorption of the composition through the skin. Transdermal dosage forms include patches, ointments, creams, gels, balms and the like. The compositions of the present invention can be administered nasally to a patient. As used herein, nasal administration 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 present invention can be used in accordance with the methods of the present invention to prevent, treat, or alleviate one or more symptoms associated with a disease, disorder, or infection. The pharmaceutical compositions of the present invention are intended to be sterile and in a suitable dosage form for administration to a subject.

In one embodiment, the compositions of the present invention are pyrogen-free dosage forms that are substantially free of endotoxins and/or similar pyrogenic substances.

Endotoxins include toxins that are contained within a microorganism and are released when the microorganism is destroyed or killed. Pyrogens also include heat-inducing, heat-stable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. These substances can cause fever, hypotension and shock when administered to humans. Due to the potential for adverse effects, it is preferable to remove even small amounts of endotoxins from intravenous solutions for pharmaceutical drugs. The Food and Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) per dose per kilogram of body weight over a one-hour period of intravenous drug administration (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1 ):223 (2000)). When therapeutic proteins are administered in quantities of several hundred or thousands of milligrams per kilogram of body weight, as may occur with monoclonal antibodies, it is preferable that even trace amounts of endotoxin be removed. In a particular embodiment, the levels of endotoxin and pyrogen in the composition are less than 10 EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU/mg, or less than 0.001 EU/mg.

The present invention provides methods for preventing, treating, or alleviating one or more symptoms associated with a disease, disorder, or infection, said methods 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 variants Fc, and (b) administering one or more subsequent doses of said Fc variants to maintain at a desired plasma concentration level (eg, from about 0.1 to about 100 μg/ml) of the Fc variant that continuously binds the antigen. In a specific embodiment, the plasma concentration of the Fc variant is maintained at 10 g/mL, 15 g/mL, 20 g/mL, 25 μg/mL, 30 μg/mL, 35 μg/mL, 40 μg/mL, 45 μg /ml or 50 µg/ml. In a specific embodiment, the specified effective amount of the Fc variant to be administered is in the range of at least 1 mg/kg to 8 mg/kg per dose. In another specific embodiment, the specified effective amount of the Fc variant to be administered is in the range of at least 4 mg/kg to 5 mg/kg per dose. In yet another specific embodiment, the specified effective amount of the Fc variant to be administered is in the range of 50 mg to 250 mg per dose. In yet another specific embodiment, the specified effective amount of the Fc variant to be administered is in the range of 100 mg to 200 mg per dose.

Also included within the scope of the present invention are protocols for the prevention, treatment, or amelioration of one or more symptoms associated with a disease, disorder, or infection, in which the Fc variant is used in combination with a therapy (eg, prophylactic or therapeutic agent) other than the Fc variant and/or or a fusion protein variant. The present invention is based, in part, on the recognition that the Fc variants of the present invention enhance and produce synergistic effects, increase efficacy, improve tolerability, and/or reduce side effects caused by other cancer treatments, including current standard and experimental chemotherapy. The combination therapy of the present invention has additive efficacy, additive therapeutic effect or synergistic effect. The combination therapy of the present invention allows for the use of lower dosages of a drug (eg, prophylactic or therapeutic agents) used in combination with Fc variants to prevent, treat, or alleviate one or more symptoms associated with a disease, disorder, or infection, and/or allows for less frequent administration of such prophylactic or therapeutic agents to a subject suffering from a disease, disorder or infection in order to improve the quality of life of said subject and/or to achieve a prophylactic or therapeutic effect. In addition, the combination therapy of the present invention reduces or avoids unwanted or harmful side effects associated with current monotherapy treatments and/or existing combination therapies, which in turn improves patient compliance with the treatment protocol. Many molecules that can be used in combination with the Fc variants of the present invention are well known in the art. See, for example, publications according to PCT WO 02/070007; WO 03/075957 and US Patent Publication 2005/064514.

The present invention provides kits containing one or more Fc variants with altered binding affinity to FcγR and/or C1q receptors and altered ADCC and/or CDC activity that specifically bind to an antigen conjugated or fused to a detectable agent, therapeutic agent or drug agent, in one or more containers, for use in controlling, diagnosing, preventing, treating or alleviating one or more symptoms associated with a disease, disorder or infection.

EXAMPLES

Examples are provided below to illustrate the implementation of the present invention. They are not intended to limit or define the entire scope of the present invention.

Example 1: Production of bivalent monospecific antibodies with heterodimeric Fc domains.

The genes encoding the antibody heavy and light chains were constructed by gene synthesis using codons optimized for human/mammalian expression. The Fab sequences were obtained from the known Her2/neu binding AT (Carter R. et al. (1992) Humanization of an anti PI 85 Her2 antibody for human cancer therapy. Proc Nati Acad Sci 89, 4285), and the Fc belonged to the IgG1 isotype (SEQ ID NO: 1). The final gene products were subcloned into the mammalian expression vector pTT5 (NRC-BRI, Canada) (Durocher, Y., Perret, S. and Kamen, A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human HER293 -EBNA1 cells. Nucleic acids research 30, E9 (2002)). Mutations in the CH3 domain were introduced by site-directed mutagenesis of the original pTT5 vectors. See Table 1, Table 6, and Table 7 for a list of mutations introduced into the modified CH3 domain.

To evaluate heterodimer formation and determine the ratio of homodimers to heterodimers, two heterodimer heavy chains with C-terminal extensions of different sizes were designed (specifically, chain A with a C-terminal histidine tag and chain B with a C-terminal mRFP plus StrepTagII). This difference in molecular weight allows homodimers to be distinguished from heterodimers by non-reducing SDS-PAGE, shown in Figure 25A.

HEK293 cells were transfected at exponential growth phase (1.5 to 2 million cells/ml) with aqueous 1 mg/ml 25 kDa polyethylenimine (PEI, Polysciences) at a PEI:DNA ratio of 2.5:1 (Raymond S. etc. A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications. Methods. 55(1):44-51 (2011)). To determine the optimal concentration range for heterodimer formation, DNA was transfected in three separate ratios of the two heavy chains. For example, transfection was carried out in a culture volume of 2 ml, and the transfected DNA consisted of 5% GFP, 45% salmon milk DNA, 25% light chain and 25% total heavy chains, with a heavy chain A plasmid (with a C-terminal histidine tag) and heavy chain plasmid B (with a C-terminal StrepTagII tag plus RFP) at 65%/55%/35% or 10%/20%/40%) were used in 3 different relative ratios (chain_A(His)/ chain_B(mRFP)), making up 10%/65%; 20%/55%; 40%/35% (the observed 1:1 ratio of expression of the WT_His/WT_mRFP heterodimer turned out to be close to the DNA ratio of 20%/55%). 4-48 hours after transfection in F17 serum-free medium (Gibco), TN1 peptone was added to a final concentration of 0.5%. The expressed antibody was analyzed by SDS-PAGE to determine the best ratio of heavy and light chains for optimal heterodimer formation (see Figure 25B and C).

A selected ratio of DNA, e.g. 50% light chain plasmid, 25% heavy chain A plasmid, 25% heavy chain B plasmid AZ33 and AZ34, with 5% GFP and 45% salmon milk DNA was used to transfect 150 ml cell culture , as described above. Transfected cells were harvested after 5-6 days, and the culture medium was collected after centrifugation at 4000 rpm and clarified using a 0.45-μm filter. See Table 2 below for a list of the percentages of light and heavy A and B chain plasmids used for scaled-up transfection assays for each of the CH3 mutant antibodies obtained for further analysis, including purity and melting point determination.

Example 2: Purification of bivalent monospecific antibodies with heterodimeric Fc domains.

The clarified culture medium was loaded onto a MabSelect SuRe protein A column (GE Healthcare) 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, and the resulting pooled fractions containing the antibody were neutralized with Tris at pH 11. Finally, the protein was desalted using a 10DG Econo-Pac column (Bio-Rad). The C-terminal mRFP tag was removed from heavy chain B by incubating the antibody with enterokinase (NEB) at 1:10,000 in PBS overnight 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) using an AKTA Express FPLC system 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.

The formation of heterodimers, in comparison with homodimers, was analyzed using EF-PAGE/SDS under non-reducing conditions and mass spectrometry. The protein A purified antibody was run on a 4-12% PAGE/SDS gradient gel under non-reducing conditions to determine the percentage of heterodimers formed before enterokinase (EK) treatment (see Figure 26). For mass spectrometry, all electrospray ionization-ion trap LC/MS time-of-flight (TOF-ESI) experiments were performed on an Agilent 1100 HPLC system coupled to a Waters Q-TOF2 mass spectrometer. Five micrograms of gel filtration purified antibody was injected into a Protein MicroTrap cartridge (1.0 by 8.0 mm), washed with 1% acetonitrile for 8 minutes, gradient from 1 to 20% acetonitrile/0.1% formic acid for 2 minutes. , and then eluted with a gradient of 20 to 60% acetonitrile/0.1% formic acid over 20 minutes. The eluate (30-50 μl/min) was sent to a spectrometer, which recorded a spectrum every second (mass to charge ratio (m/z) ranged from 800 to 4000) (see Figure 28). Variants forming more than 90% of heterodimers were selected for further analysis, with the exception of AZ12 and AZ14, which each formed more than 85% of heterodimers.

Example 3: Determination of the stability of bivalent monospecific antibodies with heterodimeric Fc domains using differential scanning calorimetry (DSC).

All DSC experiments were performed using a VP-Capillary GE device. The proteins were buffered with PBS (pH 7.4) and diluted to 0.4-0.5 mg/ml, of which 0.137 ml was loaded into the sample cuvette and measurements were carried out at a scanning speed of 1°C/min from 20 to 100°C. The results were analyzed using Origin software (GE Healthcare), PBS buffer background was subtracted (see Figure 27). See Table 3 for a list of the variants investigated and the melting points identified. See Table 4 for a list of options with a melting point of 70°C and above and Tm defined for each option.

Example 4: Assessment of Fc-gamma Receptor Binding Using Surface Plasma Resonance

All binding experiments were performed using a ProteOn XPR36 BioRad device at 25°C supplemented with 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA and 0.05% Tween 20 at pH 7.4. Recombinant HER-2/neu protein (p185, Erb-2 (eBiosciences, Inc.)) was mounted on an activated GLM sensor chip by injecting 4.0 μg/ml in 10 mM NaOAc (pH 4.5) at a flow rate of 25 μl/mL. min until approximately 3000 resonance units (RU) were immobilized, the remaining active groups were quenched, 40 μg/ml purified anti-HER-2/neu antibodies containing modified CH3 domains were captured by the sensor chip through binding to the Her-2/ protein neu when injected at a flow rate of 25 μL/min for 240 s (approximately 500 RU total), followed by injection of buffer to obtain a stable background. Fc-gamma receptor (CD16a (allotype f) and CD32b) at concentrations (6000, 2000, 667, 222 and 74.0 nM) were injected at a flow rate of 60 μl/min for 120 s. with a dissociation phase of 180 sec. to obtain a set of binding sensorgrams. The resulting K _D values were determined from binding isotherms using an equilibrium fitting model, with values reported as the average of three independent experiments. Comparisons were made with the Fc domain of wild-type IgG1 and binding was expressed as the ratio of K _D WT to Co variant (see Table 5).

Example 5; Rational design of Fc variants using the development of Fc_CH3 - frame 1 (1a and 1b) and the development of AZ17-62 and AZ133-AZ2438

To obtain AZ variants with high stability and purity, the structural and computational strategies described above were used (see Figure 24). For example, in-depth structure-function analysis of AZ8 provided in-depth insight into the significance of each of the introduced mutations in AZ8, L351Y_V397S_F405A_Y407V/K392V_T394W, compared to wild-type human IgG1, and showed that the important core mutations for the heterodimer were L351Y_F405A_Y407V/T394W, whereas V397S, K 392V not influenced the formation of the heterodimer. The mutations in the core (L351Y_F405A_Y407V/T394W) are referred to as “framework 1” mutations in this application. The analysis further revealed that important mutagenesis hotspots at the interface that were lost compared to wild-type (WT) homodimer formation were the F405-K409, Y407-T366 WT interactions and the Y407-Y407 and -F405 fold (see Figure 29 ). This was reflected in the folding, cavity and MD analysis, which revealed a large difference in conformation of the D399-S400-D401 loop region (see Figure 30) and the associated folded beta sheet at position K370. This resulted in the loss of K409-D399 interchain interactions (see Figure 30) and the weakening of the strong hydrogen bond of K370 to E357 (K370 was no longer directly in contact with S364 and E357, and was completely exposed to the solvent). In the CH3 domain of IgG1 WT, these regions bind the edge, and the edge protects interactions in the core from most solvent contacts and enhances the dynamic occurrence of favorable hydrophobic van der Waals interactions. The consequence was a smaller buried surface area of AZ8 compared to DT and higher solvent accessibility of the hydrophobic core. This allowed us to identify the most important factors for the reduced stability of AZ8 compared to the stability of DT, being a) the loss of the F405-K409 DT interaction and the F405 fold and b) the loss of the strong interaction of the Y407-Y407 and Y407-T366 fold. See Figure 29.

Therefore, the present inventors have discovered key sequence residues/motifs responsible for the low stability of AZ8 compared to WT. To improve the stability and specificity of the AZ8 heterodimer, subsequent positive development efforts, for this reason, were aimed specifically at stabilizing the loop conformation at positions 399-401 by obtaining a more “closed” conformation, like DT (see Figure 30), and adjusting at generally slightly weakened (loose) folding of the hydrophobic core at positions T366 and L368 (see Figure 29). To achieve this stabilization of the loop conformation at positions 399–401, the described computational approach was used to evaluate our various targeted design ideas. In particular, three different independent possibilities for the AZ8 Fc variant were analyzed to optimize the identified key regions to improve stability. First, the binding pocket located adjacent to positions K409 and F405A was assessed for better hydrophobic folding to protect the hydrophobic core as well as stabilize the loop conformation at positions 399-400 (see Figure 30). To do this, we introduced, not limited to those listed, additional point mutations at positions F405 and K392. Second, we assessed the possibility of improving the electrostatic interactions of positions 399–409 to stabilize the conformation of the 399–400 loop and protect the hydrophobic core. To do this, we introduced, not limited to those listed, additional point mutations at positions T411 and S400. Third, the binding pocket at core stacking positions T366, T394W, and L368 was assessed to improve hydrophobic core stacking (see Figure 29). To do this, we introduced, not limited to those listed, additional point mutations at positions T366 and L368. Various independent good design ideas were investigated computationally, and the expression and stability of some good variants identified by computational means (AZ17-AZ62) were tested empirically as described in Examples 1-4. See Table 4 for a list of some Fc-based heterodimer constructs developed using this design strategy with a melting point of 70°C or higher.

The AZ33 Fc variant is an example of the development of an Fc variant in which scaffold 1 was modified to produce scaffold 1a containing mutations to improve stability and purity. This Fc variant was designed from AZ8 to improve the hydrophobic folding at positions 392-394-409 and 366 to protect the hydrophobic core as well as stabilize the 399-400 loop conformation. This Fc variant, an AZ33 heterodimer, contained two additional point mutations distinct from those in the AZ8 core, K392M and T366I. The T366I_K392M_T394W/F405A_Y407V mutations are referred to as “framework 1a” mutations in this application. The K392M mutation was designed to improve folding in the cavity adjacent to positions K409 and F405A to protect the hydrophobic core and stabilize the conformation of loop 399-400 (see Figure 31). The T366I mutation was designed to improve the hydrophobic folding of the core and eliminate the formation of chain homodimers with T394W (see Figure 29). Experimental results with AZ33 showed a significant improvement in stability compared to other negatively designed Fc variants such as AZ8 (Tm 68°C), with AZ33 having a Tm of 74°C and heterodimer content of >98%. (See Figure 25C).

Development of Fc variants using scaffold 1 mutations in the third phase of Fc heterodimer variant development

Although AZ33 has substantially improved stability and specificity (or purity) compared to the parent AZ8 variant, our analysis showed that it is possible to achieve further improvements in the stability of the Fc heterodimer variant through additional amino acid modifications using the experimental results for AZ33 and the design methods described above. The expression and stability of variants developed using different design ideas were examined independently, but independent design ideas can be combined and the most successful heterodimer will be obtained using a combination of different design methods. Specifically, to optimize AZ8 folding, mutations in the cavity adjacent to positions K409-F405A-K392 were evaluated independently of mutations that optimize core folding at residues L366T-L368. These two sections 366-368 and 409-405-392 are distant from each other and are considered independent. The Fc variant AZ33, for example, was optimized for folding at positions 409-405-392, but not at positions 366-368, since these optimizing mutations were evaluated separately. Comparison of mutations at positions 366-368 suggests that T366L conferred improved stability compared to T366, as well as T366I, the latter point mutation used in the development of the Fc variant AZ33. Therefore, the presented experimental results directly suggest further optimization of AZ33 by introducing T366L, for example, instead of T366I. Therefore, amino acid mutations in the CH3 domain T366L_K392M_T394W/F405A_Y407V are referred to herein as “framework 1b” mutations.

Likewise, all experimental results were analyzed to identify point mutations that could be introduced to further improve the present AZ33 Fc heterodimer variant. These detected mutations were analyzed using the computational approach described above and ranked to produce a list of additional AZ33-based Fc heterodimer variants shown in Table 6.

Example 6: Rational design of Fc variants using Fc_CH3 development: frame 2 (a and b) and AZ63-101 and AZ2199-AZ2524 development

To improve the stability and purity of the original negative phase Fc variant, AZ15, the structural and computational strategies described above were used (see Figure 24). For example, in-depth structure-function analysis of the AZ15 Fc variant provided an in-depth understanding of each of the introduced mutations in AZ15, L351Y_Y407A/E357L_T366A_K409F_T411N, compared to wild-type (WT) human IgG1, and showed that the important mutations in the heterodimer core were L351Y_Y407A/T366A_K409F. then both E357L and T411N had no direct effect on heterodimer formation and stability. The core mutations (L351Y_Y407A/T366A_K409F) are referred to as “scaffold 2” mutations in this application. The analysis further revealed that important mutagenesis hotspots at the interface that are lost compared to the wild-type (WT) homodimer formed are the D399-K409 salt bridge, the Y407-T366 hydrogen bond, and the Y407-Y407 fold. The detailed analysis below describes how the present inventors improved the stability of the original Fc variant AZ15 and which amino acid modifications were introduced at which positions to produce these Fc variants with increased stability.

Development of Fc variants using framework 2 mutations and additional development of framework 2a mutations.

Computer analysis showed suboptimal folding of previously developed Fc variants, for example, the K409F_T366A_Y407A AZ15 mutations, and weakened folding of the hydrophobic core as a whole due to the loss of Y407-Y407 DT interactions. The heteromultimers described in this application are designed with more optimal stacking. Some of the positive development studies described in this application focused on point mutations to compensate for the folding deficiency of the parent AZ15 Fc variant. Target residues included positions T366, L351, and Y407. Various combinations of mutations at these positions were examined computationally, and the expression and stability of the best Fc variants identified by computational means (AZ63-AZ70) were tested empirically as described in Examples 1-4.

The AZ70 Fc variant is an example of the development of an Fc variant in which framework 2 was modified by introducing mutations to framework 2a to improve stability and purity. This Fc variant was developed based on AZ15 to achieve better folding of the hydrophobic core as described above. The AZ70 Fc variant contained the same mutations in core scaffold 2 (L351Y_Y407A/T366A_K409F) described above, except that T366 was mutated to T366V instead of T366A (Figure 33). The L351Y mutation improved the melting temperature of the 366A_409F/407A variant from 71.5°C to 74°C, and the additional change from 366A to 366V improved the Tm to 75.5°C (see AZ63, AZ64 and AZ70 in Table 4, with Tm being 71.5°C, 74°C and 75.5°C, respectively). The core mutations (L351Y_Y407A/T366V_K409F) are referred to herein as “framework 2a” mutations. Experimental results for the Fc variant AZ70 showed a significant improvement in stability compared to the original negative design Fc variant AZ15 (Tm 71°C), with AZ70 having a Tm of 75.5°C and heterodimer content of >90% (Figure 33 and 27).

Development of Fc variants using framework 2 mutations and additional development of framework 2b mutations.

Molecular dynamics (MD) simulations and folding analysis revealed a preferred, more “open” conformation of loop 399–400, which is likely due to the loss of the K409–D399 salt bridge in the DT. This also resulted in unsaturation of D399, which in turn favored a compensatory interaction with K392, resulting in a more “open” loop conformation. This more “open” loop conformation leads to a weakened fold as a whole and to increased solvent accessibility of residues at the core boundary of the CH3 domain, which, in turn, significantly destabilizes the heterodimeric complex. Therefore, one of the targeted positive development studies was to introduce a more “closed” DT-like loop conformation by introducing additional point mutations that compensate for the loss of the D399-K409 salt bridge and K409 folding interactions. Target residues included positions T411, D399, S400, F405, N390, K392, and combinations of these positions. Various folding, hydrophobic and electrostatic positive design strategies were computationally examined in relation to the above positions, and the expression and stability of the best Fc variants identified by computational means (AZ71-AZ101) were tested empirically as described in Examples 1-4.

The AZ94 Fc variant is an example of the development of an Fc variant in which framework 2 was modified by introducing mutations to framework 2b, along with the introduction of additional point mutations to improve stability and purity. This Fc variant was designed to give loop 399-400 a more “closed” DT-like conformation and compensate for the loss of the D399-K409 salt bridge as described above. The AZ94 Fc variant contained four additional point mutations in frame 2 (L351Y_Y407A/T366A_K409F) and replaced L351Y with wild-type L351, leaving (Y407A/T366A_K409F) as core mutations for this Fc variant. The core mutations Y407A/T366A_K409F are referred to herein as “framework 2b” mutations. The four additional AZ94 point mutations were K392E_T411E/D399R_S400R. The T411E/D399R mutations were introduced to form an additional salt bridge and compensate for the loss of the K409/D399 interaction (Figure 34). In addition, this salt bridge was designed to prevent homodimer formation by interfering with charge-charge interactions in both potential homodimers. The additional mutations K392E/S400R were intended to form a different salt bridge and therefore further give the 399_400 loop a more “closed” DT-like conformation (Figure 34). Experimental results for AZ94 showed improved stability and purity compared to the original negative design Fc variant AZ15 (71°C Tm, >90% purity), with the AZ94 Fc variant having a Tm of 74°C and heterodimer content or purity > 95%.

Development of Fc variants using scaffold 2 mutations in the third phase of Fc heterodimer variant development

The Fc variants AZ70 and AZ94 offer significant improvements in stability and purity over the original negatively designed Fc variants such as AZ15, but our analysis and head-to-head comparison of AZ70 and AZ94 suggests that unexpected improvements in the stability of this Fc heterodimer variant can be achieved with using additional amino acid modifications. For example, the Fc variants AZ70 and AZ94 were designed to modify two different non-optimized regions in the original AZ15 variant by improving the folding of the hydrophobic core and introducing mutations outside the core boundary residues, allowing for additional salt bridges and hydrogen bonds to stabilize the loop conformation at positions 399-401. The additional point mutations of the Fc variants AZ70 and AZ94 are distant from each other and, therefore, they are independent and can be transferred to other Fc variants developed from the same mutations in the core 2 framework, including the 2a and 2b mutations. Specifically, AZ70 carries only the optimized core mutations L351Y_Y407A/T366A_K409F, with no additional salt bridges, while AZ94 includes four additional electrostatic mutations (K392E_T411E/D399R_S400R) but contains one less mutation at the hydrophobic core interface (Y407A/T366A_K409F). These 2b framework mutations are less stable than those in AZ70 (see, for example, AZ63, which contains core mutations equivalent to AZ94 and has a Tm of 72°C), which is compensated for by introducing additional mutations K392E_T411E/D399R_S400R. The experimental results presented regarding stability and purity suggest that the combination of AZ70 mutations that optimize the hydrophobic core and AZ94 electrostatic mutations should further improve the stability and purity of Fc heterodimer variants. Similarly, all experimental results for Fc framework 2 variants (AZ63-101) were analyzed to identify point mutations that could be introduced to further improve the Fc heterodimer variants AZ70 and AZ94. These detected mutations were further analyzed using the computational approach described above and ranked to produce a list of additional Fc heterodimer variants based on AZ70 and AZ94 shown in Table 7.

Example 7: Effect of heterodimeric CH3 on FcγR binding

As an example of the prototype activity of the heterodimeric Fc towards FcγR, two variants of the antibody with the heterodimeric Fc region A:K409D_K392D/B:D399K_D356K (control 1 (het1 in Figure 35)) and A:Y349C_T366S_L368A_Y407V/B:S354C_T366W (control 4 ( het2 in Figure 35)) with Her2 Fab binding arms using the SPR assay described in Example 4 for FcγR binding. As shown in Figure 35, the present inventors found that both heterodimeric Fc regions bound different Fc-gamma receptors with the same relative strength as the wild-type IgG1 Fc region, but overall the heterodimeric Fc region bound each of the FcγRs slightly. better than wild type antibody. This suggests that mutations at the CH3 Fc interface may affect the binding strength of the Fc region to Fc-gamma receptors across all CH2 domains, as observed in molecular dynamics simulations and analyzes performed by the present inventors.

Example 8: Effect of Asymmetric Mutations in CH2 of Heterodimeric Fc on FcγR Binding

It is known that the replacement of serine at position 267 in the CH2 domain of the Fc region with aspartic acid (S267D) increases binding to the Fc-gamma receptors IIbF, IIbY and IIaR, if it is introduced in a homodimeric manner into both chains of the CH2 domain. This mutation can be introduced into only one of the CH2 domains in a heterodimeric Fc molecule to achieve an increase in binding strength of approximately twofold compared to the case when this mutation is introduced into a homodimeric CH2 Fc, as evidenced by the results presented in Figure 36A. On the other hand, the E269K mutation in the homodimeric CH2 domain of Fc prevents the binding of the Fc region to FcγR. The present inventors present a design for improved control of the binding strength of the Fc region to Fey receptors by asymmetrically introducing these favorable and unfavorable mutations into one of the two chains in the CH2 domain of the Fc. Introducing the E269K mutation in an asymmetric manner into one CH2 chain of a heterodimeric Fc results in polarity by blocking FcγR binding to the side where the mutation is present, while allowing the other Fc side to interact with the FcγR in the normal manner. The results of this experiment are presented in Figure 36A. The ability to selectively vary the binding strength of both Fc chains independently provides increased ability to control the binding strength and selectivity between the Fc and Fey receptors. Thus, this asymmetrical design of mutations in the CH2 domain allows us to implement positive and negative engineering strategies to promote or hinder certain binding patterns, providing an enhanced opportunity to introduce selectivity.

In a subsequent experiment, we modified the selectivity profile of the Fc core mutant S239D_D265S_I332E_S298A, which exhibited increased binding strength to the Fc-gamma receptors IIIaF and IIIaV, while continuing to exhibit weaker binding to the Fc-gamma receptors IIaR, IIbR, and IIbY. This is shown in the binding profile presented in Figure 36B. By introducing asymmetric mutations E269K into chain A and eliminating the I332E mutation from chain B, we were able to obtain a new FcγR binding profile that further reduces binding to receptors IIa and IIb and allows for more specific Fc binding to receptor IIIa.

In another example, shown in Figure 36C, asymmetric mutations are highlighted compared to the homodimeric Fc, including the S239D/K326E/A330L/I332E/S298A mutations in the CH2 domain. Compared with wild-type IgG1 Fc, this variant showed increased binding to the IIIa receptor, but it also binds the Ha and IIb receptors slightly more strongly than wild-type Fc. Introduction of these mutations in an asymmetric manner A:S239D/K326E/A330L/I332E and B:S298A not only reduces Ilia binding, but also increases binding to the Ha/Hb receptor, which leads to a loss of selectivity of this process. When introducing the asymmetric E269K mutation into this heterodimeric variant, i.e. A:S239D/K326E/A330L/I332E/E269K and B:S298A, IIa/IIb binding is reduced back to wild-type levels. This highlights the fact that the use of asymmetric mutations in the CH2 domain of the Fc may provide a significant opportunity to develop improved selectivity for Fc-gamma receptors.

The reagents used in the examples are commercially available or can be prepared using commercially available equipment, methods or reagents known in the art. The preceding examples have illustrated various aspects of the present invention and the implementation of the methods according to the present invention. The examples are not intended to provide an exhaustive description of the many different embodiments of the present invention. Thus, although the invention described above has been described in some detail by way of illustration and example for the purpose of clarity of understanding, it will be apparent to those of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Example 9: FcRn binding determined by SPR.

Binding to FcRn was determined by SPR in two different orientations.

1. Flow of the heterodimer variant through the immobilized FcRn. In this experiment, surfaces with a high immobilization density of approximately 5000 RU were prepared using a standard N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC) coupling combination. 100 nM of DT and each variant were injected in triplicate at a flow rate of 50 μl/min for 120 s with dissociation for 600 s in MES running buffer, pH6.

2. Flow of FcRn through indirectly captured heterodimer variants. In this SPR experiment, a surface immobilized with goat anti-human IgG antibodies was used to indirectly capture the antibodies (approximately 400 RU of each antibody), and then FcRn was injected at 3-fold dilutions (starting at a concentration of 6000 nM and below). The running buffer was 10 mM MES/150 mM NaCl/3.4 mM EDTA/0.05 Tween 20 at pH 6. No significant surface binding of FcRn was observed with immobilized goat polyclonal antibodies. Sensograms for all variants turned out to be similar to DT. Table 8 below shows the Kd determined by FcRn flow-mediated immobilization (2).

Example 10: Bispecific binding of the Fc heterodimer described herein

Bispecific binding was demonstrated using an Fc heterodimer with mutations in chain A: L351Y_F405A_Y407V, chain B: T366L_K392M_T394W, fused with scFv against HER2 (anti-HEP2) and HER3 (anti-HER3) at the N-terminus of chain A and chain B of the Fc heterodimer. The resulting variants, a bispecific HER2/HER3 variant and two monovalent-monospecific variants with HER2 or HER3, are shown in FIG. 40A. For the bispecific binding assay, the dose range of the two monovalent variants (monovalent anti-HER2 and monovalent anti-HER2, shown in Figure 40A) and the bispecific anti-HER2/HER3 heterodimer were incubated with MALME-M3 melanoma cells and then determined by FACS analysis. apparent binding affinity of each molecule (shown in Figure 40B). The assay system was set up according to the protocols described in: "Antitumor activity of a novel bispecific antibody that targets the ErbB2/ErbB3 oncogenic unit and inhibits heregulin-induced activation of ErbB3", McDonagh CF et al., Mol Cancer Ther. 11 (3):582-93 (2012).

Example 11: Expression and Purification of Bivalent Monospecific Antibodies with Heterodimeric Fc Domains and Quantitative Analysis of Purity by LC/MS

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 designs (AZ3000-AZ3021) were prepared and purified as described in Examples 1 and 2 In order to evaluate the stability of heterodimer formation and the effect of an excess of one of the heterodimer chains on heterodimer purity, selected heterodimers were transiently expressed using 3 different ratios of DNA of the two heavy chains A and B (for example, A:B ratio = 1: 1.5; 1:1; 1.5:1).

Genes encoding the heavy and light chains of the heterodimer were constructed by gene synthesis using columns optimized for human/mammalian expression as detailed in Example 1. Fab sequences were obtained from the known Her2/neu binding AT (Carter P. et al. (1992) Humanization of an anti PI 85 Her2 antibody for human cancer therapy. Proc Nati Acad Sci 89, 4285), and Fc belonged to the IgG1 isotype (SEQ ID NO: 1). This variant was expressed by transient co-expression as described in Examples 1-2, using 3 different ratios of heavy chain A to heavy chain B: 1:1.5, 1:1 and 1.5:1. Samples were purified using Protein A affinity chromatography and preparative gel filtration (see Example 2 for more details). Purified samples were deglycosylated using PNGase F overnight at 37°C. Before MS analysis, samples were applied to a Poros R2 column and eluted with a gradient of 20-90% acetonitrile, 0.2% MK over 3 minutes. The peak from the LC column was analyzed using an LTQ-Orbitrap XL mass spectrometer (cone voltage: 50 V, cylindrical lens: 215 V; PF resolution: 7500) and installed Promass software to obtain molecular weight profiles.

The relative peak heights of the heterodimer and homodimers were used to assess heterodimer purity (see Figure 39).

Example 12: Crystal structure of heteromultimers AZ3002 and AZ3003

The heterodimeric Fc constructs AZ3002 and AZ3003 were transiently expressed in CHO cells and purified to homogeneity by protein A and size exclusion chromatography. Purified Fc heterodimers were crystallized at 18°C after -24 hours of incubation using the hanging drop method via vapor diffusion at a 2:1 ratio above a stock solution of 5% (v/v) ethylene glycol, 18% (w/v) .) polyethylene glycol 3350 and 0.15 M ammonium iodide, using microseeds. The crystals were protected from low temperature by increasing the ethylene glycol concentration to 30% (v/v) and then flash cooled with liquid nitrogen. Diffraction data from both crystals were collected at 100 K, using 0.5 degree variations for a total of 200 degrees, and processed using XDS ¹ ( ^1. Kabsch, W. XDS. Acta Crystallogr D Biol Crystallogr 66, 125-132 (2010) .). The structure of AZ3002 was solved by molecular replacement using the Phaser program, using the structure from the International Protein Structure Bank PDB ID: 2J6E as query protein ² ( ^2. McCoy, A. J. Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr.D.Biol Crystallogr. 63, 32-41 (2007). The AZ3002 structure was then used to resolve AZ3003 in a similar manner. In order to ensure perfect inverse reciprocity of an Azymetric heterodimer present in a crystallographic asymmetric unit (e.g., the occupancy rate of molecule A can be equally described by molecule B, and vice versa), two possible heterodimer pairs were modeled, each with an atom occupancy rate of 0.5. using the Coot program, and refined the structures in the Refmac ^3.4 program ( ^3. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr.D.Biol.Crystallogr. 60, 2126-2132 (2004). ^4. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D. Biol. Crystallogr. 53, 240-255 (1997). Processing of diffraction results and structure refinement statistics are presented in Table 9.

An overlay of the crystal structures is shown in Figure 42. The crystal structure of the AZ3002 and AZ3003 heterodimers agrees very well with the computer models (all-atom RMSD=0.706 Å, backbone RMSD=0.659 Å CH3 domain) and confirms the predicted conformations of the most important residues in the core fold. .

Example 13: AZ3003 Glycosylation Assay

The AZ3003 heterodimer was expressed and purified as described in Example 11. Glycans were analyzed using the GlykoPrep™ rapid N-glycan platform with InstantAB™ (Prozyme) using the manufacturer's standard protocol.

The results are shown in Figure 43 and illustrate that AZ3003 exhibits a normal glycosylation pattern.

Example 14: Stability Assessment of AZ3003 under Accelerated Degradation Conditions

The stability of the AZ3003 heterodimer was assessed by incubation under forced degradation conditions. The stability of MAT under conditions of accelerated degradation may represent a good assessment of long-term stability and stability during formulation of the dosage form.

The purified heterodimer sample (which was expressed and purified as described in Example 11) was concentrated to a concentration of 100 mg/ml with no evidence of aggregation. The sample was diluted in a suitable buffer and evaluated under accelerated degradation conditions as described in Table 10 below. The treated samples were analyzed by EP-PAGE/SDS and HPLC-SEC.

EP in PAGE/SDS was carried out under reducing (R) and non-reducing (NR) conditions on ready-made gradient gels purchased from LONZA. Protein bands were visualized by staining with Coomassie brilliant blue G-250.

Analytical EC-HPLC was performed using either a BIOSEP-SEC-S4000, Phenomenex, or Bio-Sil TSK 4000, BioRad HPLC column at a flow rate of 0.8 mL/min with 10 mM sodium phosphate, 0.14 M Nad, 10 % isopropanol as a running buffer. This allowed for quantitative analysis of potential high and low molecular weight molecules.

The results are shown in Figure 44 and demonstrate that the AZ3003 heterodimer is stable and has a stability profile that matches that of standard commercial MAbs.

Example 15: Post-treatment evaluation of AZ3003

A production feasibility study of AZ3003 was conducted to evaluate the properties of AZ3003 using the industry-standard antibody purification process flow diagram shown in Figure 45. The process involved three column purification steps, including protein A affinity chromatography for product capture, followed by cation exchange chromatography ( HOC) to remove aggregates, leached protein A and host cell proteins (HCP), and finally anion exchange chromatography (AOX) in continuous flow mode to capture viruses, DNA and negatively charged contaminants. This assessment was used to identify potential manufacturing issues (eg, process stability, product stability and quality) with the drug candidate(s) during early research/development.

During the production feasibility assessment, the present inventors assessed chromatographic properties, protein stability and product quality using the industry standardized purification process shown in Figure 46. Table 11 (below) lists the main criteria used for the assessment, i.e. yield at different stages, content of high molecular weight aggregates (HMAs) and elution volume. High yields during the steps and low elution volumes during the purification process indicated the stability and good properties of the protein. Control of BMA content and their removal during the purification process is essential, since the presence of protein aggregates (BMA species) in the final product can lead to reduced activity, immunogenic reactions in the patient and/or the formation of particulate contaminants during the shelf life of the pharmaceutical product. Expression of MATs with minimal starting BMA content (<4%) is necessary as high levels of aggregate will require additional purification steps to remove them, hence increasing production time and cost.

Standard industrial purification processes were used to test the stability, chromatographic properties and quality of the AZ3003 product.

1.1 Protein A capture

The AZ3003 expressed culture medium was filtered through a 0.22 µm filter using a bottle neck filter (PES) from Millipore and applied to a Mab Select SuRe column (1.6 x 25 cm) equilibrated with 5 column volumes (CV). 20 mM Tris-HCl, 0.14 M NaCl, pH 7.5. After loading, the column was thoroughly washed with equilibration buffer until the absorbance at A280 reached a stable baseline level. AZ3003 was eluted with 0.1 M acetate buffer, pH 3.6, and immediately titrated to pH 5.2 by adding 1/10 volume of 1 M Tris base.

After the elution step, the column was washed with 0.1 M acetate, pH 3.0. Analysis by EPAGE/SDS-PAGE showed that all MATs were bound to the column, as no MATs were detected in the column effluent. Well purified MAbs were detected in the elution buffer at pH 3.6. An initial capture and purification step using Protein A affinity chromatography yielded product with >90% purity.

1.2 Low pH study

The next step in the product isolation and purification process was a low pH soak to inactivate the viruses. After elution from the protein A column, MAT (∼10 mg/mL, pH 4.0) was titrated to pH 3.6 with 10% acetic acid and incubated at room temperature with agitation for 90 min. The low pH stability of AZ3003 was assessed by SDS-PAGE, SEC-HPLC and by measuring turbidity at A410 nm. AZ3003 tolerated the low pH aging step well, showing no change in SDS-PAGE or SEC-HPLC. In addition, no increase in turbidity was detected after 90 min of incubation, indicating no formation of insoluble aggregates that could pose a problem during purification (i.e., clogging of filters and columns during purification, loss of product). The results showed that AZ30003 was stable during the low pH aging step.

1.3 Cation exchange chromatography (CEC)

DOC was studied as a 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: Pooled fractions from the Mab Select SuRe column (35 mg) were titrated to pH 5.2 by adding 10% (v/v) 1 M Tris base and then diluted in 2 times with equilibration buffer, 20 mM acetate, pH 5.2. These pooled fractions were applied to a Fractogel EMD S03 (M) column equilibrated with 5 OK 20 mM acetate, pH 5.2. The column was washed with equilibration buffer until the absorbance at A280 reached a stable baseline level. MAT was eluted from a 10 OK column with a linear salt gradient of 0 to 600 mM NaCl, pH 5.2. The remaining contaminants were removed from the column with 20 mM acetate, 1 M NaCl, pH 5.2, followed by treatment with 1 N NaOH. ESP/SDS-PAGE and SEC-HPLC analysis was performed to monitor BMA levels and their removal from the bulk MAT fraction from the column. The yield at this stage (based on A280 nm readout) was 73%.

SP HP, pH 5.2: Pooled fractions from the Mab Select SuRe column (50 mg) were titrated to pH 5.2 by adding 10% (v/v) 1 M Tris base and then diluted equally with equilibration buffer , 20 mM acetate, pH 5.2. These pooled fractions were applied to an SP HP column (1.6 x 2.5 cm/5 ml) equilibrated with 5 OK 20 mM acetate, pH 5.2 (Figure 21). The column was washed with equilibration buffer until the absorbance at A280 reached a stable baseline level. MAT was eluted from a 10 OK column with a linear salt gradient of 0 to 600 mM NaCl, pH 5.2. The remaining contaminants were removed from the column with 20 mM acetate, 1 M NaCl, pH 5.2, followed by treatment with 1 N NaOH. Analysis was carried out by EPAGE/SDS-PAGE and SEC-HPLC to monitor the levels of aggregation and their separation on this column. The yield at this stage (based on A280 nm readout) was 87%.

1.4 Anion exchange chromatography (AOX)

Anion exchangers (eg, quaternary amine, Q) are widely used for the purification of monoclonal antibodies. AOX media is used in continuous flow mode and MAbs are detected in the column flow while HCP, DNA, viruses and endotoxins are retained.

Pooled fractions from a Fractogel S03 M column at pH 5.2 (25 mg) were titrated to pH 7.0 by adding 1 M Tris base and applied to a 1 ml HiTrap Q FF column equilibrated with 5 OK 10 mM phosphate, pH 7.0 . The column was washed with equilibration buffer. However, in this case, a stable baseline was not achieved. Therefore, the column was washed with PBS to elute any residual bound MAbs. The column was then washed with 10 mM phosphate, 1 M NaCl, pH 7.0 to remove any associated contaminants. This step requires additional optimization to ensure that all MAT fractions are present in the column effluent. The yield at this stage (based on A280 nm reading) was 82%, with purity assessed by SEC-HPLC to be >98%.

1.5 Analysis by EF in PAGE/SDS purification stages

SDS-PAGE (Figure 35) was performed on eluates from each purification step to monitor the removal of contaminants throughout the process and to assess the quality and purity of the final product. Analysis of the gels showed the expected pattern of MAT movement under non-reducing conditions and under reducing conditions. Comparison of gels coated with combined fractions from two KOX resins did not reveal significant differences in product profiles. All final pooled fractions after DOC, ceramic hydroxyapatite chromatography (CHT), and hydrophobic interaction chromatography (HIC), pH 5.0 and pH 7.0, were similar with respect to purity and contaminating bands.

1.7 Purity assessment using EC-HPLC

Purified AZ3003 after three column chromatography steps: on protein A; KOKH; AOX in continuous flow mode was assessed using SEC-HPLC under native conditions (Figure 46).

AZ3003 eluted as a single peak within the expected 150 kDa region for native IgG1. Purity was assessed to be >98%.

1.8 Process outputs for cleaning AZ3003

Process yields were calculated for the further purification process and listed in Figure 46. AZ3003 yields across steps were typical of IgG purified using an industry-standardized three-column purification process.

AZ3003 was successfully purified from the culture medium using an industry-standardized purification process (Protein A affinity resin, KOX, and then AOX).

The results obtained showed that AZ3003 was comparable to standard MATs in overall product yield (see Kelley B. Biotechnol. Prog. 2007, 23, 995-1008) and the minimum level of aggregation observed. AZ3003 was also evaluated with respect to low pH storage and CIT (ceramic hydroxyapatite) and HIC (Phenyl HP pH 5 and pH 7) chromatography, obtaining good yield in the absence of aggregates. Figure 46 illustrates that the core heterodimer was successfully purified from the culture medium using an industry-standard purification process (Protein A affinity resin, KOX, and then AOX).

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference equally as if each individual publication, patent or patent application were specifically and separately identified as being incorporated herein by reference.

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LIST OF SEQUENCES

<110> ZIMWORKS INC.

SPRETER VON KREDENSTEIN, Thomas

DIXIT, Serjit Bimarao

LARIO, Paula Irene

PUN, David Kai Yuen

ESCOBAR-CABRERA, Eric

<120> DEVELOPMENT OF A STABLE HETERODIMERIC ANTIBODY WITH MUTATIONS IN

DOMAIN Fc

<130> 40252-702.702

<140> CA 2,815,266

<141> 2012-11-02

<150> PCT/CA2012/050780

<151> 2012-11-02

<150> 61/645,547

<151> 2012-05-10

<150> 61/557.262

<151> 2011-11-08

<150> 61/556,090

<151> 2011-11-04

<160> 7

<170> Patent In version 3.5

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<---

Claims (82)

1. A heterodimeric Fc construct for imparting thermostability to an antibody, wherein said antibody contains at least one antigen binding domain that specifically binds a cancer antigen or an immune antigen, the heterodimeric Fc construct contains a modified CH3 domain, wherein said modified CH3 domain contains: a first CH3 domain polypeptide and a second CH3 domain polypeptide, each of which contains amino acid modifications compared to a wild-type CH3 domain polypeptide; wherein the first CH3 domain polypeptide includes amino acid modifications at positions L351, F405 and Y407 and the second CH3 domain polypeptide includes amino acid modifications at positions T366, K392 and T394, wherein the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405S, F405T or F405V; the amino acid modification at position Y407 is Y407V; the amino acid modification at position T366 is T366I or T366L; the amino acid modification at position K392 is K392L or K392M and the amino acid modification at position T394 is T394W, wherein each of the first and second CH3 domain polypeptides further comprises the T350V amino acid modification; wherein the Fc construct is based on human IgG1; wherein the modified CH3 domain has a melting point (Tm) of at least about 74°C, and wherein the numbering of amino acid residues corresponds to the EU index described by Kabat. 2. A heat-stable antibody for binding a cancer antigen or an immune antigen, wherein the heat-stable antibody comprises: (i) one or more antigen binding domains, wherein at least one of the antigen binding domains specifically binds a cancer antigen or an immune antigen, and (ii) an Fc heterodimer construct comprising a modified CH3 domain, wherein said modified CH3 domain comprises: a first CH3 domain polypeptide and a second CH3 domain polypeptide, each of which contains amino acid modifications compared to a wild-type CH3 domain polypeptide; wherein the first CH3 domain polypeptide contains amino acid modifications at positions L351, F405 and Y407 and the second CH3 domain polypeptide contains amino acid modifications at positions T366, K392 and T394, wherein the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405S, F405T or F405V; the amino acid modification at position Y407 is Y407V; the amino acid modification at position T366 is T366I or T366L; the amino acid modification at position K392 is K392L or K392M and the amino acid modification at position T394 is T394W, wherein each of the first and second CH3 domain polypeptides further comprises the T350V amino acid modification; wherein the Fc construct is based on human IgG1; wherein the modified CH3 domain has a melting point (Tm) of at least about 74°C, and wherein the numbering of amino acid residues corresponds to the EU index described by Kabat. 3. The heterodimeric Fc construct of claim 1 or the thermostable antibody of claim 2, wherein the modified CH3 domain has a melting temperature (Tm) of about 75°C or higher, about 77°C or higher, or about 80°C or higher. 4. The heterodimeric Fc construct according to claim 1 or the thermostable antibody according to claim 2, wherein the amino acid modification at position F405 is F405A. 5. The heterodimeric Fc construct according to claim 1 or the thermostable antibody according to claim 2, wherein the first polypeptide of the CH3 domain contains the amino acid modification S400E or S400R. 6. A heterodimeric Fc construct according to claim 1 or a thermostable antibody according to claim 2, wherein the second CH3 domain contains the amino acid modification N390R. 7. The heterodimeric Fc construct according to claim 1 or the thermostable antibody according to claim 2, wherein the first CH3 domain polypeptide additionally contains the amino acid modification S400E and the second CH3 domain polypeptide additionally contains the amino acid modification N390R. 8. The heterodimeric Fc construct of claim 1 or the thermostable antibody of claim 2, wherein one of the first and second CH3 domain polypeptides further comprises the amino acid modification Q347R and the other CH3 domain polypeptide further contains the amino acid modification K360E. 9. A heterodimeric Fc construct for imparting thermostability to an antibody, wherein said antibody comprises at least one antigen binding domain that specifically binds a cancer antigen or an immune antigen, the heterodimeric Fc construct contains a modified CH3 domain, wherein said modified CH3 domain contains: a first CH3 domain polypeptide and a second CH3 domain polypeptide, each of which contains amino acid modifications relative to a wild-type CH3 domain polypeptide, wherein: (a) the first CH3 domain polypeptide contains the amino acid modifications T350V, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, K392M and T394W, or (b) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, K392M and T394W, or (c) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, K392L and T394W, or (d) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400R, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, K392M and T394W, or (e) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392M and T394W, or (f) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405V and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392M and T394W, or (g) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405T and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392M and T394W, or (h) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405S and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392M and T394W, or (i) the first CH3 domain polypeptide contains the amino acid modifications T350V, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392M and T394W, or (j) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, T366L, N390R, K392M and T394W, or (k) the first CH3 domain polypeptide contains the amino acid modifications Q347R, T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, K360E, T366L, N390R, K392M and T394W, or (l) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400R, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390D, K392M and T394W, or (m) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400R, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390E, K392M and T394W, or (n) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392L and T394W, or (o) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392F and T394W, or (p) the first CH3 domain polypeptide contains the amino acid modifications Y349C, T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, S354C, T366L, N390R, K392M and T394W, or (q) the first CH3 domain polypeptide contains the amino acid modifications Y349C, T350V, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, S354C, T366L, N390R, K392M and T394W, or (r) the first CH3 domain polypeptide contains the amino acid modifications Y349C, T350V, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, S354C, T366L, K392M and T394W, wherein the Fc construct is based on human IgG1; wherein the modified CH3 domain has a melting point (Tm) of at least about 74°C, and wherein the numbering of amino acid residues corresponds to the EU index described by Kabat. 10. A heat-stable antibody for binding a cancer antigen or immune antigen, containing: (i) one or more antigen binding domains, wherein at least one of the antigen binding domains specifically binds a cancer antigen or an immune antigen, and (ii) an Fc heterodimer construct comprising a modified CH3 domain, wherein said modified CH3 domain comprises: a first CH3 domain polypeptide and a second CH3 domain polypeptide, each of which contains amino acid modifications relative to the wild type CH3 domain polypeptide, wherein (a) the first CH3 domain polypeptide contains the amino acid modifications T350V, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, K392M and T394W, or (b) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, K392M and T394W, or (c) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, K392L and T394W, or (d) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400R, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, K392M and T394W, or (e) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392M and T394W, or (f) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405V and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392M and T394W, or (g) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405T and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392M and T394W, or (h) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405S and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392M and T394W, or (i) the first CH3 domain polypeptide contains the amino acid modifications T350V, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392M and T394W, or (j) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, T366L, N390R, K392M and T394W, or (k) the first CH3 domain polypeptide contains the amino acid modifications Q347R, T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, K360E, T366L, N390R, K392M and T394W, or (l) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400R, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390D, K392M and T394W, or (m) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400R, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390E, K392M and T394W, or (n) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392L and T394W, or (o) the first CH3 domain polypeptide contains the amino acid modifications T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, T366L, N390R, K392F and T394W, or (p) the first CH3 domain polypeptide contains the amino acid modifications Y349C, T350V, L351Y, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, S354C, T366L, N390R, K392M and T394W, or (q) the first CH3 domain polypeptide contains the amino acid modifications Y349C, T350V, S400E, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, S354C, T366L, N390R, K392M and T394W, or (r) the first CH3 domain polypeptide contains the amino acid modifications Y349C, T350V, F405A and Y407V and the second CH3 domain polypeptide contains the amino acid modifications T350V, S354C, T366L, K392M and T394W, wherein the Fc construct is based on human IgG1; wherein the modified CH3 domain has a melting point (Tm) of at least about 74°C, and wherein the numbering of amino acid residues corresponds to the EU index described by Kabat. 11. The heterodimeric Fc construct of claim 9, or the thermostable antibody of claim 10, wherein the antibody is a bispecific antibody or a multispecific antibody. 12. The heterodimeric Fc construct according to claim 1 or the thermostable antibody according to claim 2, wherein the antibody is a bispecific antibody or a multispecific antibody. 13. The heterodimeric Fc construct of claim 1 or 9 or the thermostable antibody of claim 2 or 10, wherein at least one antigen binding domain binds to a cancer antigen. 14. The heterodimeric Fc construct of claim 1 or 9 or the thermostable antibody of claim 2 or 10, wherein at least one antigen binding domain binds to an immune antigen. 15. The heterodimeric Fc construct of claim 1 or 9 or the thermostable antibody of claim 2 or 10, wherein at least one antigen binding domain is derived from the therapeutic antibody. 16. A heterodimeric Fc construct or a thermostable antibody according to claim 15, wherein the therapeutic antibody is selected from the group consisting of abagovomab, adalimumab, alemtuzumab, aurograb, bapineizumab, basiliximab, belimumab, bevacizumab, briakinumab, canakinumab, catumaxomab, certolizumab pegol, ce tuximab, daclizumab , denosumab, efalizumab, galiximab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lumiliximab, mepolizumab, motavizumab, muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab, ofatumumab, omalizum ba, palivizumab, panitumumab, pertuzumab, ranibizumab, reslizumab, rituximab , teplizumab, tocilizumab/atlizumab, tositumomab, trastuzumab, Proxinium ^TM , Rencarex ^TM , ustekinumab and zalutumumab. 17. The heterodimeric Fc construct of claim 1 or 9 or the thermostable antibody of claim 2 or 10, wherein the antibody comprises a first antigen binding domain and a second antigen binding domain. 18. A heterodimeric Fc construct according to claim 1 or 9 or a thermostable antibody according to claim 2 or 10, characterized in that the antibody is conjugated to a therapeutic agent.