Fc variants with enhanced binding to FcRn and extended half-lives

Abstract

Binding polypeptides (eg, antibodies and immunoadhesins) comprising an altered Fc domain are provided. [Solution] Provided are isolated binding polypeptides comprising modified Fc domains having specific sequences. Also provided are nucleic acids encoding the binding polypeptides, recombinant expression vectors, and host cells for producing the binding polypeptides. Also provided are methods of using the binding polypeptides disclosed herein to treat disease.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 622,468 (filed January 26, 2018), the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] background Antibody interaction with the neonatal Fc receptor (FcRn) is a determinant in maintaining and extending the serum half-life of antibodies and other Fc-derived therapeutics. FcRn is a heterodimer of an MHC class-I-like α-domain and a β2-macroglobulin (β2-m) subunit that recognizes regions on the antibody Fc heavy chain distinctly from other Fcγ receptors (FcγRs). FcRn is expressed in various tissues, but it is thought to act primarily in the vascular endothelium, kidney, and blood-brain barrier, where it prevents IgG degradation, clearance, and triggering of inflammatory responses, respectively.

[0003] Antibody binding to FcRn is highly pH-dependent, and the interaction occurs with high affinity (high nanomolar to low micromolar concentrations) only at low pH (pH < 6.5), but not at physiological pH (approximately pH 7.4). Acidifying endosomes to a pH below 6.5 highly favors the interaction between IgG and FcRn and directly accounts for the inhibition of degradation of FcRn-bound antibodies and their recycling to the cell surface. Increasing the pH weakens the interaction and promotes the release of antibodies into the bloodstream.

[0004] Because enhanced binding likely leads to increased efficacy and reduced dosing frequency for therapeutic antibodies as a direct result of extended serum half-life compared to wild-type IgG antibodies, Fc engineering using high-throughput mutagenesis approaches has been widely pursued to identify mutants that enhance FcRn binding affinity. However, mutants that enhance FcRn binding affinity may have unexpected consequences. For example, certain IgG mutants that exhibit significantly increased FcRn affinity at pH 6.0, such as N434W or P257I / Q311I, among others, have wild-type or severely reduced serum half-lives in cynomolgus monkey and human FcRn (hFcRn) transgenic mouse studies (see, e.g., Kuo et al., 2011, supra; Non-Patent Document 1; and Non-Patent Document 2). The T250Q / M428L (QL) mutant showed IgG backbone-specific results in animal models (see, e.g., Non-Patent Document 1; and Non-Patent Document 3). The M252Y / S254T / T256E (YTE, EU numbering) mutant exhibits a 10-fold enhancement in vitro but reduced antibody-dependent cell-mediated cytotoxicity (ADCC) in vivo due to a two-fold reduction in affinity for the FcγRIIIa receptor (see, e.g., Dall'Acqua et al. 2002 supra). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Datta-Mannan et al.2007, J.Biol.Chem.282:1709-1717 [Non-patent document 2] Datta-Mannan et al.2007, Metab.Dispos.35:86-94 [Non-patent document 3] Hinton et al.2006, J.Immunol.176:346-356 Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there remains a need for alternative Fc variants with enhanced binding to FcRn and extended circulating half-lives. [Means for solving the problem]

[0007] Abstract The present invention is based on the discovery of novel IgG antibodies that have one or more of the following characteristics: increased serum half-life, enhanced FcRn binding affinity, enhanced FcRn binding affinity at acidic pH, enhanced FcγRIIIa binding affinity, and similar thermal stability compared to wild-type IgG antibodies.

[0008] Thus, in certain aspects, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising an aspartic acid (D) or glutamic acid (E) at amino acid position 256, and / or a tryptophan (W) or glutamine (Q) at amino acid position 307, wherein amino acid position 254 is not threonine (T), and further comprising a phenylalanine (F) or tyrosine (Y) at amino acid position 434, or a tyrosine (Y) at amino acid position 252, wherein the amino acid positions are according to EU numbering.

[0009] In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0010] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0011] In certain exemplary embodiments, the isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0012] In certain exemplary embodiments, the isolated binding polypeptide has altered FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at non-acidic high pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding off-rate.

[0013] In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0014] In certain exemplary embodiments, the isolated binding polypeptide has an altered FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a decreased FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a decreased FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. and has enhanced FcγRIIIa binding affinity compared to a binding polypeptide comprising:

[0015] In certain exemplary embodiments, the isolated binding polypeptide has approximately the same FcγRIIIa binding affinity as a binding polypeptide comprising a wild-type Fc domain.

[0016] In certain exemplary embodiments, the isolated binding polypeptide has about the same thermal stability as a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has about the same thermal stability as a binding polypeptide comprising a modified Fc domain with the triple amino acid substitutions M252Y / S254T / T256E, according to EU numbering.

[0017] In certain exemplary embodiments, the isolated binding polypeptide is an antibody, e.g., a monoclonal antibody. In certain exemplary embodiments, the isolated antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain exemplary embodiments, the isolated antibody is a full-length antibody.

[0018] In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more human targets.

[0019] In other aspects, the present invention provides a method for the preparation of a tyrosine (Y) at amino acid position 252 and an aspartic acid (D) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a phenylalanine (F) at amino acid position 434, a tryptophan (W) at amino acid position 307, and a tyrosine (Y ... tryptophan (W) at amino acid position 307, and a tryptophan (W) at amino acid position 307, and a tryptophan (W) at amino acid position 307, and a tryptophan (W) at amino acid position 434, and a tryptophan (W) at amino acid position 307, and a tryptophan (W) at amino acid position 434, and a tryptophan (W) at amino acid position 256, and a tryp and i) a tyrosine (Y) at amino acid position 252, a glutamic acid (E) at amino acid position 256, and a glutamine (Q) at amino acid position 307, wherein threonine (T) is absent at amino acid position 254, histidine (H) is absent at amino acid position 311, and tyrosine (Y) is absent at amino acid position 434, wherein the amino acid substitutions are according to EU numbering.

[0020] In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0021] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0022] In certain exemplary embodiments, the isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide comprising a wild-type Fc domain, hi certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0023] In certain exemplary embodiments, the isolated binding polypeptide has altered FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at non-acidic high pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding dissociation rate. In certain exemplary embodiments, the isolated binding polypeptide has lower FcRn binding affinity at non-acidic pH than a binding polypeptide comprising a modified Fc domain with the double amino acid substitution M428L / N434S, according to EU numbering.

[0024] In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0025] In certain exemplary embodiments, the isolated binding polypeptide has altered FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has decreased FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0026] In certain exemplary embodiments, the isolated binding polypeptide has approximately the same FcγRIIIa binding affinity as a binding polypeptide comprising a wild-type Fc domain.

[0027] In certain exemplary embodiments, the isolated binding polypeptide has about the same thermal stability as a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has about the same thermal stability as a binding polypeptide comprising a modified Fc domain with the triple amino acid substitutions M252Y / S254T / T256E according to EU numbering.

[0028] In certain exemplary embodiments, the isolated binding polypeptide is an antibody, e.g., a monoclonal antibody. In certain exemplary embodiments, the isolated antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain exemplary embodiments, the isolated antibody is a full-length antibody.

[0029] In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more human targets.

[0030] In other aspects, a) a double amino acid substitution selected from the group consisting of M252Y / T256D, M252Y / T256E, M252Y / T307Q, M252Y / T307W, T256D / T307Q, T256D / T307W, T256E / T307Q, and T256E / T307W, [wherein threonine (T) is absent at amino acid position 254 and histidine is absent at amino acid position 256] a) a triple amino acid substitution selected from the group consisting of M252Y / T256D / T307Q, M252Y / T256D / T307W, M252Y / T256E / T307Q, and M252Y / T256E / T307W, wherein threonine (T) is not present at amino acid position 311 and tyrosine (Y) is not present at amino acid position 434; or b) a triple amino acid substitution selected from the group consisting of M252Y / T256D / T307Q, M252Y / T256D / T307W, M252Y / T256E / T307Q, and M252Y / T256E / T307W, wherein threonine (T) is not present at amino acid position 311 and tyrosine (Y) is not present at amino acid position 434; is absent at amino acid position 254, histidine (H) is absent at amino acid position 311, and tyrosine (Y) is absent at amino acid position 434, wherein the amino acid substitutions are according to EU numbering.

[0031] In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is an IgG1 Fc domain.

[0032] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0033] In certain exemplary embodiments, the isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0034] In certain exemplary embodiments, the isolated binding polypeptide has altered FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at high non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding dissociation rate. In certain exemplary embodiments, the isolated binding polypeptide has lower FcRn binding affinity at non-acidic pH than a binding polypeptide comprising a modified Fc domain with the double amino acid substitution M428L / N434S, according to EU numbering.

[0035] In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0036] In certain exemplary embodiments, the isolated binding polypeptide has altered FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has decreased FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0037] In certain exemplary embodiments, the isolated binding polypeptide has approximately the same FcγRIIIa binding affinity as a binding polypeptide comprising a wild-type Fc domain.

[0038] In certain exemplary embodiments, the isolated binding polypeptide has about the same thermal stability as a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has about the same thermal stability as a binding polypeptide comprising a modified Fc domain with the triple amino acid substitutions M252Y / S254T / T256E, according to EU numbering.

[0039] In certain exemplary embodiments, the isolated binding polypeptide is an antibody, e.g., a monoclonal antibody. In certain exemplary embodiments, the isolated antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain exemplary embodiments, the isolated antibody is a full-length antibody.

[0040] In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more human targets.

[0041] In a particular aspect, an isolated binding polypeptide is provided comprising a modified Fc domain, wherein the modified Fc domain comprises an aspartic acid (D) at amino acid position 256 and a glutamine (Q) at amino acid position 307, according to EU numbering.

[0042] In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0043] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity or rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0044] In certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0045] In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at non-acidic high pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding dissociation rate.

[0046] In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0047] In certain exemplary embodiments, the isolated binding polypeptide has an altered FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0048] In certain exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In certain exemplary embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0049] In certain exemplary embodiments, the isolated binding polypeptide specifically binds to one or more human targets.

[0050] In certain aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding an isolated polypeptide.

[0051] In certain aspects, a vector is provided that comprises the isolated nucleic acid molecule. In certain exemplary embodiments, the vector is an expression vector. In certain aspects, an expression vector is provided that comprises the isolated nucleic acid molecule.

[0052] In certain aspects, a host cell is provided that comprises the vector. In certain aspects, a host cell is provided that comprises the expression vector.

[0053] In certain exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In certain exemplary embodiments, the host cell is of mammalian origin. In certain exemplary embodiments, the host cell is of bacterial origin.

[0054] In certain aspects, pharmaceutical compositions comprising the isolated binding polypeptides are provided.

[0055] In certain aspects, pharmaceutical compositions comprising the isolated antibodies are provided.

[0056] In certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain, wherein the modified Fc domain comprises an aspartic acid (D) at amino acid position 256 and a tryptophan (W) at amino acid position 307, according to EU numbering.

[0057] In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0058] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity or rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0059] In certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0060] In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at non-acidic high pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding dissociation rate.

[0061] In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0062] In certain exemplary embodiments, the isolated binding polypeptide has an altered FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0063] In certain exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In certain exemplary embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0064] In certain exemplary embodiments, the isolated binding polypeptides specifically bind to one or more human targets.

[0065] In certain aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding an isolated polypeptide.

[0066] In certain aspects, a vector is provided that comprises the isolated nucleic acid molecule. In certain exemplary embodiments, the vector is an expression vector. In certain aspects, an expression vector is provided that comprises the isolated nucleic acid molecule.

[0067] In certain aspects, a host cell is provided that comprises the vector. In certain aspects, a host cell is provided that comprises the expression vector.

[0068] In certain exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In certain exemplary embodiments, the host cell is of mammalian origin. In certain exemplary embodiments, the host cell is of bacterial origin.

[0069] In certain aspects, pharmaceutical compositions comprising the isolated binding polypeptides are provided.

[0070] In certain aspects, pharmaceutical compositions comprising the isolated antibodies are provided.

[0071] In certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain, wherein the modified Fc domain comprises a tyrosine (Y) at amino acid position 252 and an aspartic acid (D) at amino acid position 256, according to EU numbering.

[0072] In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0073] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity or rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0074] In certain exemplary embodiments, the isolated binding polypeptide has an increased serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0075] In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at non-acidic high pH. In certain exemplary embodiments, the enhanced FcRn binding affinity comprises a decreased FcRn binding dissociation rate.

[0076] In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0077] In certain exemplary embodiments, the isolated binding polypeptide has an altered FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0078] In certain exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In certain exemplary embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0079] In certain exemplary embodiments, the isolated binding polypeptides specifically bind to one or more human targets.

[0080] In certain aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding an isolated polypeptide.

[0081] In certain aspects, a vector is provided that comprises the isolated nucleic acid molecule. In certain exemplary embodiments, the vector is an expression vector. In certain aspects, an expression vector is provided that comprises the isolated nucleic acid molecule.

[0082] In certain aspects, a host cell is provided that comprises the vector. In certain aspects, a host cell is provided that comprises the expression vector.

[0083] In certain exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In certain exemplary embodiments, the host cell is of mammalian origin. In certain exemplary embodiments, the host cell is of bacterial origin.

[0084] In certain aspects, pharmaceutical compositions comprising the isolated binding polypeptides are provided.

[0085] In certain aspects, pharmaceutical compositions comprising the isolated antibodies are provided.

[0086] In certain aspects, an isolated binding polypeptide is provided comprising an altered Fc domain, wherein the altered Fc domain comprises a combination of at least four amino acid substitutions: an aspartic acid (D) or glutamic acid (E) at amino acid position 256, and a tryptophan (W) or glutamine (Q) at amino acid position 307, where amino acid position 254 is not threonine (T), and further comprises: a phenylalanine (F) or tyrosine (Y) at amino acid position 434; and a tyrosine (Y) at amino acid position 252, where amino acid positions are according to EU numbering.

[0087] In particular aspects, a) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; b) a tyrosine (Y) at amino acid position 252, a glutamic acid (E) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; c) a tyrosine (Y) at amino acid position 252, a glutamic acid (E) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; d) an aspartic acid (D) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a tyrosine (Y) at amino acid position 434. or e) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a phenylalanine (F) at amino acid position 434, wherein the amino acid substitutions are according to EU numbering.

[0088] In certain aspects, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising quadruple amino acid substitutions selected from the group consisting of M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307W / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307Q / N434F, and M252Y / T256D / T307W / N434Y, wherein the amino acid substitutions are according to EU numbering.

[0089] In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0090] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity. In certain exemplary embodiments, the binding polypeptide has rat FcRn binding affinity. In certain exemplary embodiments, the binding polypeptide has human and rat FcRn binding affinity.

[0091] In certain exemplary embodiments, the isolated binding polypeptide has an altered FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain, hi certain exemplary embodiments, the isolated binding polypeptide has an enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0092] In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0093] In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0094] In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0095] In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the non-acidic pH is about 7.4.

[0096] In certain exemplary embodiments, the isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a decreased serum half-life compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a decreased serum half-life compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0097] In certain exemplary embodiments, the isolated binding polypeptide has altered FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has decreased FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has decreased FcγRIIIa binding affinity compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0098] In certain exemplary embodiments, the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising a wild-type Fc domain, hi certain exemplary embodiments, the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0099] In certain exemplary embodiments, the isolated binding polypeptide is an antibody. In certain exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In certain exemplary embodiments, the isolated antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain exemplary embodiments, the isolated antibody is a full-length antibody.

[0100] In certain exemplary embodiments, the isolated binding polypeptides specifically bind to one or more targets.

[0101] In certain aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding an isolated polypeptide.

[0102] In certain aspects, a vector comprising the isolated nucleic acid molecule is provided.

[0103] In certain exemplary embodiments, the vector is an expression vector.

[0104] In certain aspects, a host cell containing the vector is provided.

[0105] In certain exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In certain exemplary embodiments, the host cell is of mammalian origin. In certain exemplary embodiments, the host cell is of bacterial origin.

[0106] In certain aspects, pharmaceutical compositions comprising the isolated binding polypeptides are provided.

[0107] In certain aspects, pharmaceutical compositions comprising the isolated antibodies are provided.

[0108] In certain aspects, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a tyrosine (Y) at amino acid position 434, according to EU numbering.

[0109] In a particular aspect, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising, according to EU numbering, a tyrosine (Y) at amino acid position 252, a glutamic acid (E) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a tyrosine (Y) at amino acid position 434.

[0110] In a particular aspect, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising, according to EU numbering, a tyrosine (Y) at amino acid position 252, a glutamic acid (E) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a tyrosine (Y) at amino acid position 434.

[0111] In certain aspects, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising, according to EU numbering, a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a phenylalanine (F) at amino acid position 434.

[0112] In certain aspects, an isolated binding polypeptide is provided that comprises a modified Fc domain comprising a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a tyrosine (Y) at amino acid position 434, according to EU numbering.

[0113] In certain exemplary embodiments, the modified Fc domain is a modified human Fc domain. In certain exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0114] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity.

[0115] In certain exemplary embodiments, the isolated binding polypeptide has a reduced serum half-life compared to a binding polypeptide comprising a wild-type Fc domain, hi certain exemplary embodiments, the isolated binding polypeptide has a reduced serum half-life compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0116] In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0117] In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0118] In certain exemplary embodiments, the isolated binding polypeptide has reduced FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain, hi certain exemplary embodiments, the isolated binding polypeptide has reduced FcγRIIIa binding affinity compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0119] In certain exemplary embodiments, the isolated binding polypeptide has reduced thermal stability as a binding polypeptide comprising a wild-type Fc domain, hi certain exemplary embodiments, the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0120] In certain exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In certain exemplary embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0121] In certain exemplary embodiments, the isolated binding polypeptides specifically bind to one or more targets.

[0122] In certain aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding an isolated polypeptide.

[0123] In certain aspects, an expression vector comprising the isolated nucleic acid molecule is provided.

[0124] In certain aspects, a host cell comprising the expression vector is provided.

[0125] In certain aspects, pharmaceutical compositions comprising the isolated binding polypeptides are provided.

[0126] In certain aspects, methods are provided for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide or administering to the subject a therapeutically effective amount of the pharmaceutical composition.

[0127] In certain exemplary embodiments, the disease or disorder is cancer. In certain exemplary embodiments, the cancer is a tumor.

[0128] In certain exemplary embodiments, the disease or disorder is an autoimmune disorder.

[0129] In certain aspects, methods of treating cancer in a subject in need thereof are provided, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide or administering to the subject a therapeutically effective amount of the pharmaceutical composition.

[0130] In certain aspects, methods are provided for treating an autoimmune disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide or administering to the subject a therapeutically effective amount of the pharmaceutical composition.

[0131] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0132] [Figure 1] Figures 1A-1B show the structure of FcRn interacting with the IgG1 Fc region. Figure 1A shows the interaction between hFcRn and IgG1 Fc (pdb:4n0u), showing one Fc monomer (dark gray ribbon), including glycosylation shown as a stick labeled "glycan," in complex with the α-domain (gray) and β2-m (light gray) hFcRn subunits. Most of the antibody residues involved in the interaction with FcRn are located in the loop opposite the glycosylation site, immediately adjacent to the CH2-CH3 interface (dotted line). Figure 1B shows a surface depiction of the IgG1 Fc crystal structure (pdb:5d4q) rotated 75° relative to Figure 1A. The FcRn-binding interface is composed of residues from the CH2 and CH3 domains. As shown, saturation libraries were constructed at 11 positions shown as sticks: M252; I253; S254; T256; K288; T307; K322; E380; L432; N434, and Y436. All of these residues are in close proximity to or in direct contact with FcRn. The surfaces of the key histidine residues responsible for pH dependence (H310, H433, and H435) are clustered near the target positions, as shown. [Figure 2-1]Figures 2A-2D show the Octet screening assay and results. Figure 2A shows a schematic of the Octet screening assay. A NiNTA biosensor captures histidine-tagged antigen and subsequently captures antibody mutants for rat FcRn (rFcRn) binding kinetics. Figure 2B shows the rFcRn binding kinetic profiles at pH 6.0 of wild-type (solid line), T307A / E380A / N434A (AAA) mutant (short-dashed line), LS (short-dashed line), YTE (long-dashed line), H435A (long-dashed line), and H310A / H435Q (long-dashed line) antibodies aligned to the onset of the rFcRn association step. The H435A and H310A / H435Q mutants showed little to no FcRn binding. The YTE variants have the slowest FcRn dissociation rates tested in the Octet rFcRn binding assay. Figure 2C shows a schematic of the normalization of FcRn binding kinetics at pH 6.0 by a subset of variants obtained from the Octet screen. While most variants retained significant binding to rFcRn, some were similar to the mock control (dotted line) and showed a total loss of rFcRn binding (long dashed line, located below the dotted line (mock)). Two variants (solid lines) had slower rFcRn dissociation rates than the wild-type antibody (thick long dashed line). Figure 2D shows a scatterplot analysis of rFcRn dissociation rates for all point mutations, with observable rFcRn binding kinetics separated by residue position. The saturable mutants were classified into one of four rFcRn dissociation rate types: no binding (not shown), faster binding (black), wild-type-like binding (white), and slower binding (gray). Eighteen mutants exhibited rFcRn dissociation rates significantly slower than the wild-type antibody (dashed black line). [Figure 2-2] Continued from Figure 2-1. [Figure 3]Figure 3 graphically shows the Biacore kinetics of the benchmark and wild-type mutants with human and rat FcRn at pH 6.0 and pH 7.4. All FcRn binding curves for a range of concentrations of the wild-type (top left), AAA mutant (top right), M428 / N434S (LS) mutant (bottom left), and M252Y / S254T / T256E (YTE) mutant (bottom right) are shown at pH 6.0 (first and third rows) and pH 7.4 (second and fourth rows) for human (first and third columns) and rat (second and fourth columns) FcRn, respectively. The AAA, LS, and YTE mutants exhibited slower dissociation rates from FcRn than the wild-type antibody. Overall, the antibodies bind to rFcRn with approximately 10-fold increased affinity compared to the wild-type. The LS variant had the tightest affinity at pH 7.4 and the greatest residual binding to hFcRn at pH 7.4, whereas rFcRn bound most tightly to the YTE variant. [Figure 4A] Figure 4A graphically depicts the Biacore kinetics of lead saturation mutants with human and rat FcRn at pH 6.0. FcRn binding rate traces are shown for a concentration series of 18 lead saturation mutants. M252Y, T256D, T256E, N434F, N434P, N434Y, T307A, T307E, T307F, T307Q, and T307W had slower dissociation rates from both human and rat FcRn. The remaining mutants were specific for rat FcRn only. [Figure 4B] Figure 4B shows a schematic representation of the FcRn binding kinetics of WT, benchmark, and lead single saturation mutants to human FcRn at pH 6.0. FcRn binding sensorgrams at pH 6.0 with a concentration series of WT, LS, YTE, and 18 saturation mutants. The single saturation mutants used in the combinatorial library are bold and underlined. [Figure 5-1]Figures 5A-5D present data showing several mutants with slower dissociation rates from both human and rat FcRn at pH 6.0. Figures 5A and 5B show Biacore sensorgrams of various mutants. Figure 5A shows the dissociation rates of human FcRn at pH 6.0 for the YTE mutant (long dash-dot line), LS mutant (long dash-double dot line), wild-type (WT; dotted line), and lead saturation mutant (lead; solid lines of various intensities). In Figure 5A, normalized sensorgrams are shown showing improved hFcRn dissociation rates compared to WT. Figure 5B shows the dissociation rates of rat FcRn at pH 6.0 for the AAA mutant (dotted line), LS mutant (dash-double dot line), YTE mutant (dashed dot line), wild-type (solid line), and lead saturation mutant (various frequencies and intensities). Representative injections of each of the 11 lead antibodies are shown for clarity. These lead single mutants showed improved dissociation rates from human and rat FcRn compared to wild-type. Figures 5C and 5D show binding affinity plots using the association and dissociation rates obtained from Biacore kinetic measurements for human (Figure 5C) and rat (Figure 5D) FcRn for the lead saturation (open circles) and wild-type (filled circles) antibody mutants. Benchmark mutants are shown: AAA (downward right diagonal line), LS (dotted line), and YTE (downward left diagonal line). Despite improvements in FcRn dissociation rates, the majority of the mutants did not have tighter affinity for human or rat FcRn due to slower binding kinetics. Eleven mutants had slower dissociation rates from the FcRn of both species. [Figure 5-2] Continued from Figure 5-1. [Figure 6-1]Figures 6A-6D present data demonstrating that combinations of lead saturation mutations further improved FcRn dissociation rates and binding affinity. Figures 6A and 6B show Biacore sensorgrams showing FcRn dissociation rates for human and rat FcRn, respectively. Figure 6A shows normalized sensorgrams for human FcRn of representative mutants of single (dashed line), double (light gray solid line), triple (gray solid line), and quadruple (black solid line) combination mutants compared to the wild-type (dotted line) and the LS mutant (long dashed-dotted line). Figure 6B shows normalized sensorgrams for rat FcRn of representative mutants of single (long dashed-dotted line), double (long dashed-dotted line), triple (long dashed line), and quadruple (short dashed line) combination mutants compared to the wild-type (dotted line) and the YTE mutant (solid line). Incorporation of multiple mutations reduced the dissociation rate for FcRn to a greater extent than the benchmark mutants and enhanced binding affinity. Figures 6C and 6D show plots of the combination saturation mutants showing the binding rate as a function of the dissociation rate for human (Figure 6C) or rat (Figure 6D) FcRn, revealing that the majority of the mutants have enhanced binding to FcRn at pH 6.0 compared to the benchmark mutants. The tightest binding mutants for human and rat FcRn were the quadruple and triple combinations, respectively. [Figure 6-2] Continued from Figure 6-1. [Figure 7-1]Figures 7A-7D present data demonstrating that enhanced FcRn binding at pH 6.0 disrupted the pH dependence of the interaction. Figures 7A and 7B show representative sensorgrams of Biacore FcRn binding kinetics at pH 7.4 for single (long dashed-dotted line), double (long dashed-dotted line), triple (long dashed line), and quadruple (short dashed line) combination mutants compared to the wild-type (dotted line), and the LS (Figure 7A, solid line) and YTE (Figure 7B, solid line) mutants. Increasing the number of FcRn-binding-enhancing mutations resulted in higher residual binding at physiological pH, with most double, triple, and quadruple mutants exhibiting robust binding to FcRn of both species. Figures 7C and 7D show the steady-state RU at pH 7.4 for all saturation mutants against human (Figure 7C) or rat (Figure 7D) FcRn.

number

[0133] Detailed Description The present disclosure provides binding polypeptides (e.g., antibodies) with altered Fc neonatal receptor (FcRn) binding affinity. In certain embodiments, the binding polypeptides comprise an altered Fc domain that enhances FcRn binding affinity compared to binding polypeptides comprising a wild-type (e.g., unmodified) Fc domain. The present disclosure also provides nucleic acids encoding the binding polypeptides, recombinant expression vectors and host cells for producing the binding polypeptides, and pharmaceutical compositions comprising the binding polypeptides disclosed herein. Methods of using the binding polypeptides of the present disclosure to treat disease are also provided.

[0134] The Fc domain of an immunoglobulin is involved in non-antigen binding functions and has several effector functions mediated by the binding of effector molecules, such as FcRn. As illustrated in Figure 1A, the Fc domain is composed of a CH2 domain and a CH3 domain. Most of the residues involved in the interaction with FcRn are located in the C H 2-C H The modified Fc domain is located in the loop immediately adjacent to the CH3 interface (FIG. 1A, dotted line) and opposite the glycosylation site. FIG. 1B illustrates a surface representation of the IgG1 Fc crystal structure (pdb:5d4q) and shows residues in the CH2 and CH3 domains that make up the FcRn-binding interface. The present disclosure provides binding polypeptides comprising the modified Fc domain. The binding polypeptides comprising the modified Fc domain can be antibodies, immunoadhesins, or Fc fusion proteins.

[0135] In certain embodiments, a binding polypeptide may comprise an altered Fc domain comprising amino acid substitutions that alter the antigen-dependent effector functions of the antibody, particularly the circulating half-life (e.g., serum half-life) of the binding polypeptide. In some embodiments, a binding polypeptide may comprise an altered Fc domain comprising amino acid substitutions that alter the serum half-life of the binding polypeptide compared to a binding polypeptide comprising a wild-type (i.e., unmodified) Fc domain. In some embodiments, a binding polypeptide may comprise an altered Fc domain comprising amino acid substitutions that increase the serum half-life of the binding polypeptide compared to a binding polypeptide comprising a wild-type (i.e., unmodified) Fc domain. In some embodiments, a binding polypeptide may comprise an altered Fc domain comprising amino acid substitutions that decrease the serum half-life of the binding polypeptide compared to a binding polypeptide comprising a wild-type (i.e., unmodified) Fc domain.

[0136] In certain embodiments, a binding polypeptide comprising a modified Fc domain that alters (i.e., increases or decreases) circulating half-life (e.g., serum half-life) contains one or more mutations in addition to the mutations that alter circulating half-life. In certain embodiments, the one or more mutations in addition to the mutations that alter circulating half-life include one or more The modified antibody results in one or more of the following desirable biochemical characteristics, for example, decreased or enhanced effector function, ability to non-covalently dimerize, increased ability to localize to tumor sites, decreased serum half-life, increased serum half-life, and the like, when compared to an unmodified whole antibody of about the same immunogenicity.

[0137] The binding polypeptides described herein may exhibit either increased or decreased binding to the neonatal Fc receptor (FcRn) when compared to binding polypeptides lacking these substitutions, and therefore have increased or decreased serum half-lives, respectively. Fc domains with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules have utility in methods of treating mammals where a long half-life of the administered antibody is desired, e.g., to treat chronic diseases or disorders. In contrast, Fc domains with decreased FcRn binding affinity are expected to have shorter serum half-lives, and such molecules are also useful for administration to mammals where, for example, a shortened circulation time would be advantageous, e.g., for in vivo diagnostic imaging, or in situations where the starting antibody has toxic side effects if present in the circulation for an extended period of time. Fc domains with decreased FcRn binding affinity are also less likely to cross the placenta and therefore are useful in treating diseases or disorders in pregnant women. Additionally, other applications in which reduced FcRn binding affinity may be desirable include applications localized to the brain, kidney, and / or liver.

[0138] It is understood that the methods described in this disclosure are not limited to the particular methods and experimental conditions disclosed herein as such methods and conditions may vary, and it is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0139] Furthermore, the experiments described herein, unless otherwise indicated, employ conventional molecular and cell biology and immunological techniques within the skill of one in the art. Such techniques are well known to those skilled in the art and are fully explained in the literature. See, for example, Ausubel, et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008) (including all supplements); Molecular Cloning: A Laboratory Manual (Fourth Edition) by M.R. Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2 nd See the 2011 edition.

[0140] Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the event of potential ambiguity, definitions provided herein supersede any dictionary or extrinsic definition. Unless otherwise required by context, singular forms shall include plural forms and plural forms shall include the singular. Unless otherwise stated, "or" means "and / or." Use of the term "comprising," as well as other forms such as "includes" and "included," is not limiting.

[0141] Generally, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein is well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and, unless otherwise specified, are as described in the various general and more specific references cited and discussed throughout the specification. Enzymatic reactions and purification techniques are generally performed according to conventional methods well known in the art. Manufacturer's specifications are used, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0142] In order that this disclosure may be more readily understood, selected terms are defined below.

[0143] The term "polypeptide" refers to a polymeric chain of amino acids and, unless contradicted by context, includes natural or artificial proteins, polypeptide analogs, or protein sequence variants, or fragments thereof. Polypeptides can be monomeric or polymeric. A polypeptide fragment contains, for example, at least about 5 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, or at least about 20 contiguous amino acids.

[0144] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, by virtue of its origin or source, is free from associated naturally occurring components with which it is naturally associated; is substantially free from other proteins from the same species or is expressed by cells from a different species; or is non-naturally occurring. Thus, a protein or polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates would be "isolated" from its naturally associated components. A protein or polypeptide can also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

[0145] As used herein, the term "binding protein" or "binding polypeptide" is intended to refer to a protein or polypeptide (e.g., an antibody or immunoadhesin) that contains at least one binding site that is responsible for selectively binding to a target antigen of interest (e.g., a human target antigen). Exemplary binding sites include an antibody variable domain, a ligand binding site of a receptor, or a receptor binding site of a ligand. In certain aspects, a binding protein or binding polypeptide comprises multiple (e.g., two, three, four, or more) binding sites. In certain aspects, a binding protein or binding polypeptide is not a therapeutic enzyme.

[0146] The term "ligand" refers to a substance that can bind to or be bound by another substance. Similarly, the term "antigen" refers to any substance against which an antibody can be generated. Although "antigen" is commonly used in reference to an antibody-binding substance, and "ligand" is often used in reference to a receptor-binding substance, these terms are not distinguishable from one another and encompass a wide range of overlapping chemical entities. For the avoidance of doubt, antigen and ligand are used interchangeably throughout this specification. Antigens / ligands may be peptides, polypeptides, proteins, aptamers, polysaccharides, sugar molecules, carbohydrates, lipids, oligonucleotides, polynucleotides, synthetic molecules, inorganic molecules, organic molecules, and any combination thereof.

[0147] As used herein, the term "specifically binds" means to bind to at most about 1 x 10 -6 M, about 1x10 -7 M, about 1x10 -8 M, about 1x10 -9 M, about 1x10 -10 M, about 1x10 -11 M, about 1x10 -12 Binds to antigen with a dissociation constant (Kd) of M or less and / or non-specific Refers to the ability of an antibody or immunoadhesin to bind to an antigen with an affinity that is at least about two-fold greater than its affinity for the antigen.

[0148] As used herein, the term "antibody" refers to an aggregate (e.g., intact antibody molecule) that has significant, known specific immunoreactive activity against an antigen of interest (e.g., a tumor-associated antigen). , immunoadhesin, or variants thereof). Antibodies and immunoglobulins comprise light and heavy chains with or without covalent interchain bonds between them. Basic immunoglobulin structure in vertebrate systems is relatively well understood.

[0149] As discussed in more detail below, the generic term "antibody" includes five different biochemically distinguishable classes of antibodies. While all five classes of antibodies are expressly within the scope of this disclosure, the following discussion generally relates to immunoglobulin molecules of the IgG class. With respect to IgG, immunoglobulins contain two identical light chains with a molecular weight of approximately 23,000 daltons and two identical heavy chains with a molecular weight of 53,000-70,000. The four chains are joined by disulfide bonds in a "Y" configuration, where the light chains surround the heavy chains at the mouth of the "Y" and continue through the variable region.

[0150] Immunoglobulin light chains are classified as either kappa (κ) or lambda (λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked by covalent disulfide bonds, or noncovalently when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. In the heavy chain, the amino acid sequence runs from the N-terminus at the forked ends in a Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε), within which there are several subclasses (e.g., γl-γ4). It is the nature of this chain that determines the "class" of the antibody: IgG, IgM, IgA IgG, or IgE, respectively. Immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to have specialized functions. Modified versions of each of these classes and isotypes will be readily discernible to the skilled artisan in light of the present disclosure, and accordingly, are within the scope of the present disclosure.

[0151] Both light and heavy chains are divided into structural and functional homologies. The term "region" refers to a portion or part of an immunoglobulin or antibody chain and includes the constant or variable region as well as more distinct portions of said regions. For example, a light chain variable region contains "complementarity-determining regions" or "CDRs" interspersed between "framework regions" or "FRs" as defined herein.

[0152] Regions of an immunoglobulin heavy or light chain may be defined as "constant" (C) regions or "variable" (V) regions, based on the relative lack of sequence variation among different class members, in the case of "constant regions," or on the significant variation among different class members, in the case of "variable regions." The terms "constant region" and "variable region" may also be used functionally. In this context, it will be understood that the variable region of an immunoglobulin or antibody determines antigen recognition and specificity. Conversely, the constant region of an immunoglobulin or antibody confers important effector functions such as secretion, transplacental movement, Fc receptor binding, complement fixation, and the like. The subunit structures and three-dimensional configurations of the various immunoglobulin classes are well known.

[0153] The constant and variable regions of immunoglobulin heavy and light chains are folded into domains. The term "domain" refers to a globular region of a heavy or light chain that includes, for example, a β-pleated sheet and / or peptide loops (e.g., 3-4 peptide loops) stabilized by interchain disulfide bonds. The constant region domain on the light chain of an immunoglobulin is interchangeably referred to as a "light chain constant region," "CL region," or "CL domain." The constant domain on the heavy chain (e.g., hinge, CH1, CH2, or CH3 domain) is interchangeably referred to as a "heavy chain constant region domain," "CH" region domain, or "CH domain." The variable domain on the light chain is interchangeably referred to as a "light chain variable region domain," "VL region domain," or "VL domain." The variable domains on the heavy chain are referred to interchangeably as "heavy chain variable region domains," "VH region domains," or "VH domains."

[0154] By convention, the numbering of the amino acids in the variable constant region increases as they become more distant from the antigen-binding site or the amino terminus of the immunoglobulin or antibody. The N-terminus of each heavy and light immunoglobulin chain is the variable region, and the C-terminus is the constant region. The CH3 and CL domains comprise the carboxy termini of the heavy and light chains, respectively. Thus, the domains of the light chain immunoglobulin are arranged in a VL-CL orientation, while the domains of the heavy chain are arranged in a VH-CH1-hinge-CH2-CH3 orientation.

[0155] The assignment of each variable region domain is based on Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and The definition of CDRs follows that of Kabat (1991). Kabat also provides a widely used numbering system (Kabat numbering), in which corresponding residues between different heavy chain variable regions or different light chain variable regions are assigned the same number. CDRs 1, 2, and 3 of a VL domain are also referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively. CDRs 1, 2, and 3 of a VH domain are also referred to herein as CDR-H1, CDR-H2, and CDR-H3, respectively. When so indicated, CDR assignments are based on IMGT instead of Kabat. (R) (Lefranc et al., D The numbering of the heavy chain constant region is according to Kabat (Kabat, Sequences of Proteins of Immunological (Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991).

[0156] As used herein, the term "VH domain" comprises the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" comprises the amino-terminal variable domain of an immunoglobulin light chain.

[0157] As used herein, the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain, e.g., extending from about positions 114-223 in the Kabat numbering system (EU positions 118-215). The CH1 domain is adjacent to the VH domain and amino-terminally adjacent to the hinge region of the immunoglobulin heavy chain molecule and does not form part of the Fc region of the immunoglobulin heavy chain.

[0158] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al. J. Immunol. 1998, 161:4083).

[0159] The term "CH2 domain," as used herein, includes, for example, a portion of a heavy chain immunoglobulin molecule extending from about position 244 to 360 in the Kabat numbering system (EU positions 231 to 340). The CH2 domain is unique in that it is not tightly paired with another domain. Rather, two N-linked branched hydrocarbon chains are located between the two CH2 domains in an intact, native IgG molecule. In one embodiment, a binding polypeptide of the disclosure comprises a CH2 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0160] As used herein, the term "CH3 domain" includes a portion of a heavy chain immunoglobulin molecule extending approximately 110 residues from the N-terminus of the CH2 domain, e.g., approximately positions 361-476 in the Kabat numbering system (EU positions 341-445). The CH3 domain typically forms the C-terminal portion of an antibody. However, in some immunoglobulins, additional domains may extend from the CH3 domain and form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ chain of IgM and the e chain of IgE). In one embodiment, a binding polypeptide of the disclosure comprises a CH3 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0161] The term "CL domain" as used herein includes, for example, the immunoglobulin constant region domain in the light chain extending from about Kabat position 107A to about Kabat position 216. The CL domain is adjacent to the VL domain. In one embodiment, a binding polypeptide of the disclosure includes a CL domain derived from a kappa light chain (e.g., a human kappa light chain).

[0162] As used herein, the term "Fc region" is defined as the portion of the heavy chain constant region that begins at the hinge region immediately upstream of the papain cleavage site (i.e., residue 216 of IgG, considering the first residue of the heavy chain constant region as 114) and ends at the C-terminus of the antibody. Thus, a complete Fc region includes at least the hinge, CH2, and CH3 domains.

[0163] As used herein, the term "native Fc" or "wild-type Fc" refers to a molecule resulting from antibody digestion or produced by other means, containing the sequence of a non-antigen-binding fragment, which may contain a hinge region, in a monomeric or multimeric form. The original immunoglobulin source of a native Fc is typically human and may be any immunoglobulin, such as IgG1 or IgG2. Native Fc molecules are made up of monomeric polypeptides that can be linked by covalent (i.e., disulfide) and non-covalent bonds to form dimers or multimers. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from one to four, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. As used herein, the term "native Fc" refers collectively to the monomeric, dimeric, and multimeric forms.

[0164] As used herein, the term "Fc variant" or "altered Fc" refers to a molecule or sequence that has been altered from a native / wild-type Fc but still contains a binding site for FcRn. Thus, the term "Fc variant" can include a molecule or sequence that has been humanized from a non-human native Fc. Furthermore, a native Fc contains regions that can be removed because they confer structural features or biological activity not required for the antibody-like binding polypeptides described herein. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues, or in which one or more Fc sites or residues have been altered, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0165] In certain exemplary embodiments, the Fc variants characterized herein have one or more of increased serum half-life, enhanced FcRn binding affinity, enhanced FcRn binding affinity at acidic pH, enhanced FcγRIIIa binding affinity, and / or similar thermal stability compared to an IgG antibody comprising a wild-type Fc.

[0166] As used herein, the term "Fc domain" encompasses native / wild-type Fc and Fc variant sequences as defined above. As with Fc variant and native Fc molecules, the term "Fc domain" includes the molecule in monomeric or multimeric form, whether digested from a whole antibody or produced by other means.

[0167] As noted above, the variable region of an antibody enables the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL and VH domains of an antibody combine to form a variable region (Fv) that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by the complementarity-determining regions (CDRs) on each of the heavy and light chain variable regions. As used herein, the term "antigen-binding site" includes the site that specifically binds (immunoreacts with) an antigen (e.g., a cell-surface or soluble antigen). An antigen-binding site comprises immunoglobulin heavy and light chain variable regions, and the binding site formed by these variable regions determines the specificity of the antibody. The antigen-binding site is formed by variable regions that differ from antibody to antibody. The modified antibodies of the present disclosure contain at least one antigen-binding site.

[0168] In certain embodiments, binding polypeptides of the present disclosure comprise at least two antigen-binding domains that confer association of the binding polypeptide with a selected antigen. The antigen-binding domains need not necessarily be derived from the same immunoglobulin molecule. In this regard, the variable regions can be or are derived from any type of animal that can be introduced to mount a humoral response and generate immunoglobulins against a desired antigen. As such, the variable regions of the binding polypeptides can be, for example, of mammalian origin, such as human, mouse, rat, goat, sheep, non-human primate (e.g., cynomolgus monkeys, macaques, etc.), lupine, or camelid (e.g., camel, llama, and related species).

[0169] In naturally occurring antibodies, the six CDRs present in each monomeric antibody are short, discontinuous sequences of amino acids specifically positioned to form the antigen-binding site as the antibody assumes its three-dimensional structure in an aqueous environment. The remainder of the heavy and light chain variable domains exhibit less intermolecular variability in amino acid sequence and are called framework regions. The framework regions largely adopt a β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, these framework regions act as a scaffold that positions the six CDRs in the correct orientation through interchain noncovalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to an epitope on the immunoreactive antigen. This complementary surface promotes noncovalent binding of the antibody to the immunoreactive antigen epitope.

[0170] Exemplary binding polypeptides include antibody variants. As used herein, the term "antibody variant" includes synthetic and engineered forms of antibodies that have been altered so that they are not naturally occurring, such as antibodies that contain at least two heavy chain portions but not two complete heavy chains (e.g., domain-deleted antibodies or minibodies); multispecific forms (e.g., bispecific, trispecific, etc.) that have been altered to bind to two or more different antibodies or to different epitopes on a single antigen; heavy chain molecules joined to scFv molecules; and the like. Furthermore, the term "antibody variant" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc.), antibodies that bind three, four, or more copies of the same antigen.

[0171] As used herein, the term "valency" refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to one target molecule or a specific site on a target molecule. If a polypeptide contains more than one target binding site, each target binding site may specifically bind to the same or a different molecule (e.g., a different ligand or a different antigen). (They may bind to different epitopes on the same antigen, or different epitopes on the same antigen). The subject binding polypeptides typically have at least one binding site specific for a human antigen molecule.

[0172] The term "specificity" refers to the ability to specifically bind (e.g., immunoreact with) a given target antigen (e.g., a human target antigen). A binding polypeptide can be monospecific and contain one or more binding sites that specifically bind to a target, or the polypeptide can be multispecific and contain two or more binding sites that specifically bind to the same or different targets. In certain embodiments, a binding polypeptide is specific for two different (e.g., non-overlapping) portions of the same target. In certain embodiments, a binding polypeptide is specific for more than one target. Exemplary binding polypeptides (e.g., antibodies) that contain an antigen-binding site that binds to an antigen expressed on tumor cells are known in the art, and one or more CDRs from such antibodies can be included in the antibodies described herein.

[0173] As used herein, the term "antigen" or "target antigen" refers to a molecule or portion of a molecule capable of being bound by the binding site of a binding polypeptide. A target antigen may have one or more epitopes.

[0174] The terms "about" or "approximately" mean within about 20%, for example, within about 10%, within about 5%, or within about 1% or less of a given value or range.

[0175] As used herein, "administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., an isolated binding polypeptide provided herein) present outside the body into a patient, for example, but not limited to, by pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When a disease or a symptom thereof is being managed or treated, administration of the substance typically occurs after the onset of the disease or its symptoms. When a disease or a symptom thereof is being prevented, administration of the substance typically occurs before the onset of the disease or its symptoms and may be continued over time to postpone or reduce the appearance or magnitude of disease-related symptoms.

[0176] As used herein, the term "composition" is intended to encompass an article of manufacture containing the specified ingredients (e.g., the isolated binding polypeptides provided herein), optionally in specified amounts, as well as any products resulting directly or indirectly from the combination of the specified ingredients, optionally in specified amounts.

[0177] "Effective amount" means an amount of an active agent (e.g., an isolated binding polypeptide of the present disclosure) sufficient to achieve a desired physiological result in an individual in need of the agent. The effective amount may vary from individual to individual depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, an assessment of the individual's medical condition, and other relevant factors.

[0178] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, the term "subject" as used herein refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, feral animals, farm animals, sport animals, and pets.

[0179] As used herein, the term "treatment" refers to the prevention, management, or treatment of a disease or its associated symptoms. In some embodiments, the term "therapy" refers to any protocol, method, and / or agent that can be used in the prevention, management, treatment, and / or amelioration of a disease or a symptom associated therewith in a subject. In some embodiments, the term "therapy" refers to any protocol, method, and / or agent that can be used in the prevention, management, treatment, and / or amelioration of a disease or a symptom associated therewith known to one of skill in the art, such as a healthcare professional. In other embodiments, the term "therapy" refers to any biological therapy, supportive care, and / or other therapy that can be used in the prevention, management, treatment, and / or amelioration of a disease or a symptom associated therewith in a subject known to one of skill in the art, such as a healthcare professional.

[0180] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or its associated symptoms resulting from the administration of one or more therapies (including, but not limited to, one or more prophylactic or therapeutic agents, such as the isolated binding polypeptides provided herein). As used herein, the term "treating" can also refer to altering the course of the disease in the subject being treated. The therapeutic effect of treatment includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, reducing the rate of disease progression, remission or palliation of the disease state, and remission or improved prognosis.

[0181] Binding Polypeptides In one aspect, the present disclosure provides binding polypeptides (e.g., antibodies, immunoadhesins, antibody variants, and fusion proteins) comprising a modified Fc domain. The binding polypeptides disclosed herein encompass any binding polypeptide comprising a modified Fc domain. In certain embodiments, the binding polypeptide is an antibody, or an immunoadhesin, or a derivative thereof. Antibodies from any source or species can be used in the binding polypeptides disclosed herein. Suitable antibodies include, without limitation, human antibodies, humanized antibodies, or chimeric antibodies. Suitable antibodies include, without limitation, monoclonal antibodies, polyclonal antibodies, full-length antibodies, or single-chain antibodies.

[0182] Fc domains from any immunoglobulin class (e.g., IgM, IgG, IgD, IgA, and IgE) and species can be used in the binding polypeptides disclosed herein. Chimeric Fc domains comprising portions of Fc domains from different species or Ig classes can also be used. In certain embodiments, the Fc domain is a human Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain. In other embodiments, the Fc domain is an IgG4 Fc domain. In some embodiments, the Fc domain is a human IgG1 or IgG4 Fc domain. In some embodiments, the Fc domain is a human IgG1 Fc domain. For Fc domains of other species and / or Ig classes or isotypes, it will be apparent to those skilled in the art that any of the amino acid substitutions described herein can be adapted as appropriate. In some embodiments, the modified Fc domain may comprise an amino acid substitution selected from M252, 1253, S254, T256, K288, T307, K322, E380, L432, N434, or Y436, and combinations thereof, according to EU numbering. In some embodiments, the modified Fc domain may comprise a double amino acid substitution at any two amino acid positions selected from M252, 1253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436, according to EU numbering. In some embodiments, the modified Fc domain may comprise a double amino acid substitution at any two amino acid positions selected from M252, 1253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436, according to EU numbering. The modified Fc domain may comprise a triple amino acid substitution at any three amino acid positions selected from E380, L432, N434, and Y436. In some embodiments, the modified Fc domain may comprise a quadruple amino acid substitution at any four amino acid positions selected from M252, 1253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436 according to EU numbering. In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution at any amino acid position selected from M252, 1253, S254, T256, K288, T307, K322, E380, L432, or Y436 according to EU numbering, and combinations thereof, wherein amino acid position N434 is not substituted (i.e., amino acid position N434 is wild-type).

[0183] In some embodiments, the modified Fc domain may comprise an amino acid substitution selected from M252Y (i.e., tyrosine at amino acid position 252), T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, N434F, N434P, N434Y, Y436H, Y436N, or Y436W, and any combination thereof, according to EU numbering. In some embodiments, the modified Fc domain may comprise a double amino acid substitution selected from M252 (wherein the substitution is M252Y); T256 (wherein the substitution is T256D or T256E); K288 (wherein the substitution is K288D or K288N); T307 (wherein the substitution is T307A, T307E, T307F, T307M, T307Q, or T307W); E380 (wherein the substitution is E380C); N434 (wherein the substitution is N434F, N434P, or N434Y); Y436 (wherein the substitution is Y436H, Y436N, or Y436W). In some embodiments, the modified Fc domain may comprise a triple amino acid substitution selected from, according to EU numbering, M252 (wherein the substitution is M252Y); T256 (wherein the substitution is T256D or T256E); K288 (wherein the substitution is K288D or K288N); T307 (wherein the substitution is T307A, T307E, T307F, T307M, T307Q, or T307W); E380 (wherein the substitution is E380C); N434 (wherein the substitution is N434F, N434P, or N434Y); Y436 (wherein the substitution is Y436H, Y436N, or Y436W).In some embodiments, the modified Fc domain may comprise a quadruple amino acid substitution selected from, according to EU numbering, M252 (wherein the substitution is M252Y); T256 (wherein the substitution is T256D or T256E); K288 (wherein the substitution is K288D or K288N); T307 (wherein the substitution is T307A, T307E, T307F, T307M, T307Q, or T307W); E380 (wherein the substitution is E380C); N434 (wherein the substitution is N434F, N434P, or N434Y); Y436 (wherein the substitution is Y436H, Y436N, or Y436W). In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution at any of amino acid positions selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W, and any combination thereof, according to EU numbering, wherein amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution at an amino acid position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W, and any combination thereof, according to EU numbering, wherein amino acid position N434 is not substituted with tyrosine (Y). In some embodiments, the modified Fc domain comprises M252Y, T256D, T256E, K288D, K288N, T307A, according to EU numbering. It may be desirable to include an amino acid substitution at any of amino acid positions selected from T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W, and any combination thereof, and wherein amino acid position N434 is not substituted (i.e., amino acid position N434 is wild type).

[0184] In certain embodiments, the modified Fc domain may comprise amino acid substitutions selected from M252, T256, T307, or N434, and any combination thereof, according to EU numbering. In certain embodiments, the modified Fc domain may comprise double amino acid substitutions at any two amino acid positions selected from M252, T256, T307, and N434, according to EU numbering. In certain embodiments, the modified Fc domain may comprise triple amino acid substitutions at any three amino acid positions selected from M252, T256, T307, and N434, according to EU numbering. In certain embodiments, the modified Fc domain may comprise quadruple amino acid substitutions at amino acid positions M252, T256, T307, and N434, according to EU numbering. In some embodiments, it may be desirable for the modified Fc domain to include an amino acid substitution selected from M252, T256, or T307, according to EU numbering, and any combination thereof, wherein amino acid position N434 is not substituted (i.e., amino acid position N434 is wild-type).

[0185] In exemplary embodiments, the modified Fc domain may comprise an amino acid substitution selected from M252 (wherein the substitution is M252Y); T256 (wherein the substitution is T256D or T256E); T307 (wherein the substitution is T307Q or T307W); or N434 (wherein the substitution is N434F or N434Y), according to EU numbering. In certain embodiments, the modified Fc domain may comprise a double amino acid substitution at any two amino acid positions selected from M252 (wherein the substitution is M252Y); T256 (wherein the substitution is T256D or T256E); T307 (wherein the substitution is T307Q or T307W); or N434 (wherein the substitution is N434F or N434Y), according to EU numbering. In certain embodiments, the modified Fc domain may comprise a triple amino acid substitution at any three amino acid positions selected from M252 (wherein the substitution is M252Y); T256 (wherein the substitution is T256D or T256E); T307 (wherein the substitution is T307Q or T307W); or N434 (wherein the substitution is N434F or N434Y), according to EU numbering. In certain embodiments, the modified Fc domain may comprise a quadruple amino acid substitution at an amino acid position selected from M252 (wherein the substitution is M252Y); T256 (wherein the substitution is T256D or T256E); T307 (wherein the substitution is T307Q or T307W); or N434 (wherein the substitution is N434F or N434Y), according to EU numbering. In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution selected from M252Y, T256D, T256E, T307Q, or T307W, according to EU numbering, and any combination thereof, wherein amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y).In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution selected from M252Y, T256D, T256E, T307Q, or T307W, according to EU numbering, and any combination thereof, wherein amino acid position N434 is not substituted with tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution selected from M252Y, T256D, T256E, T307Q, or T307W, according to EU numbering, and any combination thereof, wherein amino acid position N434 is not substituted (i.e., amino acid position N434 is wild type).

[0186] In certain embodiments, the modified Fc domain may comprise an amino acid substitution selected from T256D, or T256E, and / or T307W, or T307Q, according to EU numbering, and further comprises an amino acid substitution selected from N434F, or N434Y, or M252Y. In some embodiments, it may be desirable for the modified Fc domain to comprise an amino acid substitution selected from T256D, or T256E, and / or T307W, or T307Q, according to EU numbering, and further comprise the amino acid substitution M252Y, wherein amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, the modified Fc domain comprises an amino acid substitution selected from T256D, or T256E, and / or T307W, or T307Q, according to EU numbering, and may further desirably comprise the amino acid substitution M252Y, wherein amino acid position N434 is not substituted with tyrosine (Y). In some embodiments, the modified Fc domain comprises an amino acid substitution selected from T256D, or T256E, and / or T307W, or T307Q, according to EU numbering, and may further desirably comprise the amino acid substitution M252Y, wherein amino acid position N434 is not substituted (i.e., amino acid position N434 is wild type).

[0187] In some embodiments, the modified Fc domain may comprise a double amino acid substitution selected from M252Y / T256D, M252Y / T256E, M252Y / T307Q, M252Y / T307W, M252Y / N434F, M252Y / N434Y, T256D / T307Q, T256D / T307W, T256D / N434F, T256D / N434Y, T256E / T307Q, T256E / T307W, T256E / N434F, T256E / N434Y, T307Q / N434F, T307Q / N434Y, T307W / N434F, and T307W / N434Y, according to EU numbering. In some embodiments, the modified Fc domain is M252Y / T256D / T307Q, M252Y / T256D / T307W, M252Y / T256D / N434F, M252Y / T256D / N434Y, M252Y / T256E / T307Q, M252Y / T256E / T307W, M252Y / T256E / N434F, M252Y / T256E / N434Y, M252Y / T307Q / N434F, M252Y / T307Q / N434Y, M252Y / T307W / N434F, M252T / T307W / N434Y, T256D / 307Q / N434F, T256D / 307W / N434F, T256D / 307Q / N434Y, T256D / 307W / N434Y, T256E / 307Q / N434F, T256E / 307W / N434F, T256E / 307Q / N434Y, and T256E / 307W / N434Y.

[0188] In some embodiments, the modified Fc domain may comprise a quadruple amino acid substitution selected from M252Y / T256D / T307Q / N434F, M252Y / T256E / T307Q / N434F, M252Y / T256D / T307W / N434F, M252Y / T256E / T307W / N434F, M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307W / N434Y, and M252Y / T256E / T307W / N434Y, according to EU numbering.

[0189] In some embodiments, it may be desirable for the modified Fc domain to comprise a wild-type amino acid at amino acid position N434 according to EU numbering. In some embodiments, it may be desirable for the Fc domain to not comprise a phenylalanine (F) or tyrosine (Y) at amino acid position N434 according to EU numbering. In some embodiments, it may be desirable for the Fc domain to not comprise a tyrosine (Y) at amino acid position N434 according to EU numbering. In some embodiments, the modified Fc domain may comprise a wild-type amino acid at amino acid position N434 according to EU numbering. The modified Fc domain may comprise a double amino acid substitution selected from M252Y / T256D, M252Y / T256E, M252Y / T307Q, M252Y / T307W, T256D / T307Q, T256D / T307W, T256E / T307Q, and T256E / T307W according to EU numbering. In some embodiments, the modified Fc domain may comprise a triple amino acid substitution selected from M252Y / T256D / T307Q, M252Y / T256D / T307W, M252Y / T256E / T307Q, and M252Y / T256E / T307W according to EU numbering.

[0190] In one embodiment, a binding polypeptide with altered FcRn binding comprises an Fc domain with one or more amino acid substitutions disclosed herein. In one embodiment, a binding polypeptide with enhanced FcRn binding affinity comprises an Fc domain with one or more amino acid substitutions disclosed herein. In one embodiment, a binding polypeptide with enhanced FcRn binding affinity comprises an Fc domain with two or more amino acid substitutions disclosed herein. In one embodiment, a binding polypeptide with enhanced FcRn binding affinity comprises an Fc domain with three or more amino acid substitutions disclosed herein.

[0191] In some embodiments, the binding polypeptide may exhibit species-specific FcRn binding affinity. In one embodiment, the binding polypeptide may exhibit human FcRn binding affinity. In one embodiment, the binding polypeptide may exhibit rat FcRn binding affinity. In some embodiments, the binding polypeptide may exhibit cross-species FcRn binding affinity. Such binding polypeptides are said to be cross-reactive across one or more different species. In one embodiment, the binding polypeptide may exhibit both human and rat FcRn binding affinity.

[0192] The neonatal Fc receptor (FcRn) interacts with the Fc region of antibodies to promote their recycling by rescuing normal lysosomal degradation. This process is pH-dependent, occurring in endosomes at acidic pH (e.g., pH less than 6.5) but not under physiological pH conditions of the bloodstream (e.g., non-acidic pH). In some embodiments, binding polypeptides of the present disclosure comprising an altered Fc domain have enhanced FcRn binding affinity at acidic pH compared to binding polypeptides comprising a wild-type Fc domain. In some embodiments, binding polypeptides comprising an altered Fc domain have enhanced FcRn binding affinity at a pH less than 7, e.g., at about pH 6.5, about pH 6.0, about pH 5.5, or about pH 5.0, compared to binding polypeptides comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising an altered Fc domain has enhanced FcRn-binding affinity at a pH below 7, e.g., about pH 6.5, about pH 6.0, about pH 5.5, about pH 5.0, compared to the FcRn-binding affinity of the binding polypeptide at a non-acidic high pH. A non-acidic high pH can be, for example, a pH above 7, about pH 7, about pH 7.4, about pH 7.6, about pH 7.8, about pH 8.0, about pH 8.5, about pH 9.0.

[0193] In certain embodiments, it may be desirable for a binding polypeptide comprising an altered Fc domain to exhibit approximately the same FcRn-binding affinity at non-acidic pH as a binding polypeptide comprising a wild-type Fc domain. In some embodiments, it may be desirable for a binding polypeptide comprising an altered Fc domain to exhibit a lower FcRn-binding affinity at non-acidic pH than a binding polypeptide comprising an altered Fc domain with the double amino acid substitution M428L / N434S according to EU numbering. Thus, it may be desirable for a binding polypeptide comprising an altered Fc domain to exhibit minimal perturbation to pH-dependent FcRn binding.

[0194] In some embodiments, a binding polypeptide comprising an altered Fc domain that has enhanced FcRn binding affinity at acidic pH exhibits a decreased (i.e., slower) FcRn dissociation rate compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising an altered Fc domain that has enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at non-acidic high pH exhibits a slower FcRn dissociation rate at acidic pH compared to the FcRn dissociation rate of the binding polypeptide at non-acidic high pH.

[0195] In some embodiments, binding polypeptides comprising modified Fc domains are provided that exhibit higher FcRn binding affinity at non-acidic pH compared to binding polypeptides comprising wild-type Fc domains. In some embodiments, binding polypeptides comprising modified Fc domains are provided that exhibit higher FcRn binding affinity at acidic pH compared to binding polypeptides comprising wild-type Fc domains. In some embodiments, binding polypeptides comprising modified Fc domains are provided that exhibit higher FcRn binding affinity at non-acidic pH compared to binding polypeptides comprising wild-type Fc domains, and higher FcRn binding affinity at acidic pH compared to binding polypeptides comprising wild-type Fc domains. Thus, in certain embodiments, binding polypeptides comprising modified Fc domains that exhibit loss of pH-dependent FcRn binding are provided.

[0196] Certain embodiments include antibodies that, in addition to the Fc mutations exhibiting altered FcRn binding affinity described herein, comprise at least one amino acid in one or more of the constant region domains and / or at least one amino acid in one or more of the variable region domains that has been deleted or otherwise altered to provide desired biochemical characteristics such as, for example, decreased or enhanced effector function, ability to non-covalently dimerize, increased ability to localize to tumor sites, decreased serum half-life, increased serum half-life, and the like, when compared to an unaltered whole antibody of approximately the same immunogenicity.

[0197] In certain other embodiments, the binding polypeptide comprises constant regions derived from different antibody isotypes (e.g., constant regions from two or more of human IgG1, IgG2, IgG3, or IgG4). In other embodiments, the binding polypeptide comprises a chimeric hinge (i.e., a hinge comprising hinge portions derived from hinge domains of different antibody isotypes, e.g., an upper hinge domain from an IgG4 molecule and an IgG1 middle hinge domain).

[0198] In certain embodiments, the Fc domain can be mutated to increase or decrease effector function using techniques known in the art. In some embodiments, binding polypeptides of the present disclosure comprising an altered Fc domain have altered binding affinity to an Fc receptor. There are several different types of Fc receptors, which are classified based on the type of antibody they recognize. For example, Fc-gamma receptors (FcγR) bind to IgG class antibodies, Fc-alpha receptors (FcαR) bind to IgA class antibodies, and Fc-epsilon receptors (FcεR) bind to IgE class antibodies. FcγR belongs to a family that includes several members, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb. In some embodiments, binding polypeptides comprising an altered Fc domain have altered FcγRIIIa binding affinity compared to binding polypeptides comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising an altered Fc domain has reduced FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising an altered Fc domain has enhanced FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising an altered Fc domain has , have approximately the same FcγRIIIa binding affinity as compared to a binding polypeptide comprising a wild-type Fc domain.

[0199] In other embodiments, binding polypeptides for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgG1 heavy chain constant region, that has been altered to reduce or eliminate glycosylation. For example, a binding polypeptide (e.g., an antibody or immunoadhesin) that comprises a modified Fc domain can further comprise amino acid substitutions that alter the glycosylation of the antibody Fc. For example, the modified Fc domain can have reduced glycosylation (e.g., N- or O-linked glycosylation).

[0200] Exemplary amino acid substitutions resulting in reduced or altered glycosylation are described in International PCT Publication No. WO 05 / 018572, which is incorporated herein by reference in its entirety. In some embodiments, binding polypeptides are modified to remove glycosylation. Such binding polypeptides may be referred to as "agly" binding polypeptides (e.g., "agly antibodies"). Without being bound by theory, it is believed that "agly" binding polypeptides may have improved safety and stability profiles in vivo. Agly binding polypeptides may be of any isotype or subclass, e.g., IgG1, IgG2, IgG3, or IgG4. Numerous art-recognized methods are available for producing "agly" antibodies or antibodies with altered glycans. For example, genetically engineered host cells (e.g., modified yeast, e.g., Picchia, or CHO cells) with altered glycosylation pathways (e.g., glycosyltransferase deletions) can be used to produce such antibodies.

[0201] In certain embodiments, the binding polypeptide can comprise an antibody constant region (e.g., an IgG constant region, e.g., a human IgG constant region, e.g., a human IgG1 constant region) that mediates one or more effector functions. For example, binding of the C1-complex to an antibody constant region can activate the complement system. Activation of the complement system is important in the opsonization and lysis of cellular pathogens. Activation of the complement system also stimulates inflammatory responses and is also involved in autoimmune hypersensitivity. Furthermore, antibodies bind to receptors on various cells via their Fc domains (the Fc receptor binding site on an antibody Fc region binds to an Fc receptor (FcR) on a cell). There are numerous Fc receptors specific for different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors), and IgM (mu receptors). Binding of an antibody to an Fc receptor on the cell surface elicits a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, transplacental transfer, and control of immunoglobulin production. In some embodiments, a binding polypeptide (e.g., an antibody or immunoadhesin) binds to an Fc-gamma receptor. In alternative embodiments, the binding polypeptide may lack one or more effector functions (e.g., ADCC activity) and / or comprise a constant region incapable of binding to an Fcγ receptor.

[0202] Proteins, including antibodies, with low thermodynamic stability have an increased tendency to misfold and aggregate, which may limit or hinder the protein's activity, efficacy, and potential as a useful therapeutic agent. In certain embodiments, binding polypeptides comprising an altered Fc domain have about the same thermostability as binding polypeptides comprising a wild-type Fc domain. In some embodiments, binding polypeptides comprising an altered Fc domain have about the same thermostability as binding polypeptides comprising an altered Fc domain with the triple amino acid substitution M252Y / S254T / T256E (YTE).

[0203] The resulting physiological profile, bioavailability and other bioavailability Chemical effects, such as tumor localization, biodistribution and serum half-life, can be readily measured and quantified using well-known immunological techniques without undue experimentation.

[0204] In certain embodiments, binding polypeptides of the present disclosure may comprise antigen-binding fragments of antibodies. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with the intact antibody (i.e., the intact antibody from which it is derived) for antigen binding (i.e., specific binding). Antigen-binding fragments can be produced by recombinant or biochemical methods well known in the art. Exemplary antigen-binding fragments include Fv, Fab, Fab', and (Fab')2. In an exemplary embodiment, binding polypeptides of the present disclosure comprise an antigen-binding fragment and a modified Fc domain.

[0205] In some embodiments, the binding polypeptide comprises a single-chain variable region sequence (ScFv). A single-chain variable region sequence comprises a single polypeptide having one or more antigen-binding sites, for example, a VL domain linked to a VH domain by a flexible linker. ScFv molecules can be constructed in a VH-linker-VL configuration or a VL-linker-VH configuration. The flexible hinge connecting the VL and VH domains that make up the antigen-binding site comprises about 10 to about 50 amino acid residues. Peptide connections are known in the art. A binding polypeptide can comprise at least one scFv and / or at least one constant region. In one embodiment, a binding polypeptide of the present disclosure can comprise at least one scFv linked to or fused to an engineered Fc domain.

[0206] In some embodiments, binding polypeptides of the disclosure are multivalent (e.g., tetravalent) antibodies produced by fusing DNA sequences encoding antibodies with ScFv molecules (e.g., modified ScFv molecules). For example, in one embodiment, these sequences are combined such that the ScFv molecules (e.g., modified ScFv molecules) are linked at their N- or C-termini to the Fc fragment of an antibody via a flexible linker (e.g., a gly / ser linker). In another embodiment, a tetravalent antibody of the disclosure can be produced by fusing an ScFv molecule to a connecting peptide, which is then fused to an engineered Fc domain to construct an ScFv-Fab tetravalent molecule.

[0207] In another embodiment, the binding polypeptide of the present disclosure is an engineered minibody. The engineered minibody of the present disclosure is a dimeric molecule constructed from two polypeptide chains, each containing an ScFv molecule, fused to an engineered Fc domain via a connecting peptide. Minibodies can be produced by constructing the ScFv components and connecting the peptide components using methods described in the art (see, e.g., U.S. Pat. No. 5,837,821 or WO 94 / 09817 A1). In another embodiment, tetravalent minibodies can be constructed in the same manner as minibodies, except that two ScFv molecules are linked using a flexible linker. The linked scFv-scFv construct is then conjugated to an engineered Fc domain.

[0208] In another embodiment, binding polypeptides of the present disclosure include diabodies. Diabodies are dimeric tetravalent molecules, each comprising a polypeptide similar to an scFv molecule, but with a typically short (fewer than 10, e.g., about 1 to about 5) amino acid residue linker connecting both variable domains, such that the VL and VH domains on the same polypeptide chain cannot interact. Instead, the VL and VH domains of one polypeptide chain interact with the VH and VL domains (respectively) on a second polypeptide chain (see, e.g., WO 02 / 02781). Bispecific antibodies of the present disclosure comprise scFv-like molecules fused to an engineered Fc domain.

[0209] In other embodiments, the binding polypeptide comprises a multivalent antibody comprising one or more variable domains contiguously on the same polypeptide chain, e.g., a tandem variable domain (TVD) polypeptide. Exemplary TVD polypeptides include the "double head" or "dual-Fv" configuration described in U.S. Patent No. 5,989,830. The variable domains of two different antibodies are expressed in tandem on two separate chains (one heavy and one light), where one polypeptide chain comprises two consecutive VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains connected contiguously by a peptide linker (VL1-linker-VL2). In the crossover double-head configuration, the variable domains of two different antibodies are expressed in tandem on two separate polypeptide chains (one heavy chain and one light chain), where one polypeptide chain has two VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains connected in opposite orientation by a consecutive peptide linker (VL2-linker-VL1). Additional antibody variants based on the "dual-Fv" format include dual-variable-domain IgG (DVD-IgG) bispecific antibodies (see U.S. Pat. No. 7,612,181) and the TBTI format (see U.S. 2010 / 0226923 A1). In some embodiments, the binding polypeptide comprises a multispecific or multivalent antibody comprising one or more variable domains consecutively on the same polypeptide fused to an altered Fc domain.

[0210] In another exemplary embodiment, the binding polypeptide is a crossover dual variable domain IgG (CODV-IgG) bispecific antibody based on a "double-headed" configuration (see US20120251541 A1, which is incorporated herein by reference in its entirety).

[0211] In another exemplary embodiment, the binding polypeptide is an immunoadhesin. As used herein, "immunoadhesin" refers to a binding polypeptide (e.g., derived from a receptor, ligand, or cell adhesion molecule) comprising one or more binding domains linked to an immunoglobulin constant domain (i.e., Fc region) (see, e.g., Ashkenazi et al., Immunoadhesin). (See, e.g., Isaacs (1997) Brit. J. Rheum. 36:305, which are incorporated herein by reference in their entireties. Immunoadhesins are identified by the suffix "-cept" in their International Nonproprietary Names (INN). Like antibodies, immunoadhesins have long circulating half-lives, are easily purified by affinity-based methods, and have the advantage of avidity afforded by bivalency. Examples of commercially available therapeutic immunoadhesins include etanercept (ENBREL) and erythropoietin (ERT). (R) ), abatacept (ORENCIA (R) ), rilonacept (ARCALYST (R) ), aflibercept (ZALTRAP (R) / EYLEA (R) ), and belatacept (NULOJIX (R) ) are mentioned. can be.

[0212] In certain embodiments, the binding polypeptide comprises an immunoglobulin-like domain. Suitable immunoglobulin-like domains include, but are not limited to, fibronectin domains (see, e.g., Koide et al. (2007) Methods Mol. Biol. 352:95-109, which is incorporated herein by reference in its entirety), DARPin (see, e.g., Stumpp et al. (2008) Drug Discov. Today 13 (15-16):695-701, which is incorporated herein by reference in its entirety), Protein A Z domain (Nygren et al. (2008) FEBS J. 275 (11):2668-7 6, which is incorporated herein by reference in its entirety), lipocalins (see, e.g., Skerra et al. (2008) FEBS J. 275 (11):2677-83, which is incorporated herein by reference in its entirety), affilins (see, e.g., Ebersbach et al. (2007) J. Mol. Biol. 372 (1):172-85, which is incorporated herein by reference in its entirety), affitins (see, e.g., Krehenbrink et al. (2008). J. Mol. Biol. 383 (5):1058-68, which is incorporated herein by reference in its entirety), avimers (see, e.g., Silverman et al. (2008). J. Mol. Biol. 383 (5):1058-68, which is incorporated herein by reference in its entirety), and the like. al. (2005) Nat. Biotechnol. 23 (12):1556-61, which is incorporated herein by reference in its entirety), Fynomers (see, e.g., Grabulovski et al. (2007) J Biol Chem 282 (5):3196-3204, which is incorporated herein by reference in its entirety), and Kunitz domain peptides (see, e.g., Nixon et al. (2006) Curr Opin Drug Discov Devel 9 (2):261-8, which is incorporated herein by reference in its entirety).

[0213] For the binding polypeptides and immunoadhesins of the present disclosure, virtually any antigen can be targeted by the binding polypeptide, including, but not limited to, proteins, subunits, domains, motifs, and / or epitopes of the target antigen, including both soluble and membrane-bound factors such as cytokines, and transmembrane receptors.

[0214] Binding polypeptides of the disclosure comprising the modified Fc domains described herein may comprise the CDR or variable domain sequences of a known "parent" antibody. In some embodiments, the parent antibody and the antibody of the disclosure may share similar or identical sequences except for the modifications to the Fc domain disclosed herein.

[0215] Nucleic acids and expression vectors In one aspect, the present invention provides polynucleotides encoding the binding polypeptides disclosed herein. Also provided are methods for producing the binding polypeptides comprising expressing these polynucleotides.

[0216] Polynucleotides encoding the binding polypeptides disclosed herein are typically inserted into expression vectors for introduction into host cells which can be used to produce desired quantities of the claimed antibodies or immunoadhesins. Thus, in certain aspects, the invention provides expression vectors comprising the polynucleotides disclosed herein, and host cells comprising these vectors and polynucleotides.

[0217] The term "vector" or "expression vector" is used herein, for purposes of the specification and claims, to mean a vector used to introduce and express a desired gene into a cell. As known to those skilled in the art, such vectors can be easily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, a vector contains a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0218] Numerous expression vector systems can be used. For example, one class of vectors is composed of bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, and the like. DNA elements derived from viruses, retroviruses (RSV, MMTV, or MOMLV), or animal viruses such as SV40 are utilized. Others include the use of polycistronic systems with internal ribosome binding sites. Furthermore, cells that have integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for selection of transfected host cells. Markers can provide for prototrophy to auxotrophic hosts, resistance to biocides (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with heavy and light chain constant region genes (e.g., human genes) synthesized as discussed above.

[0219] In other embodiments, the binding polypeptides described herein can be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as antibody heavy and light chains, can be produced from a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRES) to generate relatively high levels of polypeptides in eukaryotic host cells. Suitable IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference. Those skilled in the art will appreciate that such expression systems can be used to efficiently produce the full range of polypeptides disclosed in the present application.

[0220] More generally, once a vector or DNA sequence encoding a binding polypeptide of the disclosure has been prepared, the expression vector can be introduced into a suitable host cell; i.e., the host cell can be transformed. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion using enveloped DNA, microinjection, and infection with intact virus. See, e.g., Ridgway, AAG, "Mammalian Expression Vectors," Chapter 24.2, pp. 470-472, Rodriguez and Denhardt, Eds. (Butterworths, Boston, MA 1988). Transformed cells are grown under conditions appropriate for the production of the light and heavy chains, and assayed for heavy and / or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0221] As used herein, the term "transformation" is intended to be used broadly to refer to the introduction of DNA into a recipient host cell, altering the genotype and resulting in a change in the recipient cell.

[0222] Similarly, a "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA techniques and encoding at least one heterologous gene. In describing processes for the isolation of polypeptides from recombinant hosts, the terms "cells" and "cell culture" are used interchangeably to indicate the source of the antibody, unless otherwise clearly specified. In other words, recovery of polypeptide from the "cells" can mean either from centrifuged whole cells or from the cell culture containing both the medium and the suspended cells.

[0223] In one embodiment, the host cell line used for expression of the binding polypeptide is of eukaryotic or prokaryotic origin. In one embodiment, the host cell line used for expression of the binding polypeptide is of bacterial origin. In one embodiment, the host cell line used for expression of the binding polypeptide is of mammalian origin; one of skill in the art can determine the particular host cell line that is most suitable for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary line, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (CVI-derived cells with SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). In one embodiment, cell lines are designed to have altered glycosylation of antibodies expressed therefrom, e.g., afucosylation (e.g., PER.C6.RTM. (Crucell) or a FUT8-knockout CHO cell line (POTELLIGENT TM In one embodiment, NSO cells can be used. Host cell lines are typically obtained from commercial services, such as American Available from the Tissue Culture Collection or published literature.

[0224] In vitro production allows for scale-up to obtain large quantities of the desired binding polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogeneous suspension cultures in airlift reactors or continuous stirred reactors, or immobilized or entrapped cell cultures in, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immuno-)affinity chromatography.

[0225] One or more genes encoding binding polypeptides can also be expressed in non-mammalian cells, such as bacteria, yeast, or plant cells. In this regard, it will be appreciated that various unicellular non-mammalian microorganisms, such as bacteria, can also be transformed; i.e., grown in culture or fermentation. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. It will also be appreciated that when expressed in bacteria, the polypeptides can become part of inclusion bodies. The polypeptides must be isolated, purified, and then assembled into functional molecules.

[0226] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or commonly known as baker's yeast, is the most widely used eukaryotic microorganism, although numerous other strains are commonly available. For expression in yeast, the plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow on tryptophan, ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The yeast host cell genome is then characterized and the vector is then inserted into the yeast host cell. The trpl lesion as a marker provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0227] Treatment method In one aspect, the present invention provides a method of treating or diagnosing a patient in need thereof, comprising administering an effective amount of a binding polypeptide disclosed herein. In certain embodiments, the present disclosure provides kits and methods for diagnosing and / or treating disorders, e.g., neoplastic disorders, in mammalian subjects in such treatments. In certain exemplary embodiments, the subject is human.

[0228] The binding polypeptides of the present disclosure are useful in a number of different applications. For example, in one embodiment, the subject binding polypeptides are useful for reducing or eliminating cells bearing the epitope recognized by the binding domain of the binding polypeptide. In another embodiment, the subject binding polypeptides are effective in reducing the concentration of circulating soluble antigens or in eliminating soluble antigens. In another embodiment, the subject binding polypeptides are T-cell engagers. In one embodiment, the binding polypeptides can reduce tumor size, inhibit tumor growth, and / or prolong the survival of tumor-bearing animals. Accordingly, the present disclosure also relates to methods of treating tumors in humans or other animals by administering an effective, non-toxic amount of the modified antibodies to the human or other animal.

[0229] In one embodiment, the subject binding polypeptides are useful for the treatment of a disease or disorder. For example, the subject binding polypeptides are useful for the treatment of an antibody-associated disorder or an antibody-responsive disorder, condition, or disease. As used herein, the term "antibody-associated disorder" or "antibody-responsive disorder" or "condition" or "disease" refers to or describes a disease or disorder that can be ameliorated by administration of a pharmaceutical composition comprising an antibody or binding polypeptide of the disclosure.

[0230] In one embodiment, the subject binding polypeptides are useful for the treatment of cancer. As used herein, the terms "cancer" or "cancerous" refer to a physiological condition that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of the above cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, biliary tract tumors, and head and neck cancer.

[0231] In other embodiments, the subject binding polypeptides are useful in treating other disorders, including, but not limited to, infectious diseases, autoimmune disorders, inflammatory disorders, pulmonary diseases, neurological or neurodegenerative diseases, liver diseases, spinal diseases, uterine diseases, depressive disorders, and the like. Non-limiting examples of infectious diseases include those caused by RNA viruses (e.g., orthomyxoviruses (e.g., influenza), paramyxoviruses (e.g., respiratory syncytial virus, parainfluenza virus, metapneumovirus), rhabdoviruses (e.g., rabies virus), coronaviruses, alphaviruses (e.g., chikungunya virus), lentiviruses (e.g., HIV), and the like) or DNA viruses. Examples of infectious diseases also include, but are not limited to, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and the like. Examples of infectious diseases include bacterial infections caused by bacteria such as Escherichia coli, Streptococcus, and Candida albicans. Other infectious diseases include, but are not limited to, malaria, SARS, yellow fever, Lyme disease, leishmaniasis, anthrax, and meningitis. Exemplary autoimmune disorders include, but are not limited to, psoriasis, rheumatoid arthritis, Sjogren's syndrome, transplant rejection, Graves' disease, myasthenia gravis, and lupus (e.g., systemic lupus erythematosus). Accordingly, the present disclosure relates to methods of treating a variety of conditions that would benefit from the use of the subject binding polypeptides having, for example, enhanced half-lives.

[0232] Those skilled in the art can determine, by routine experimentation, an effective, non-toxic amount of modified binding polypeptide that would be sufficient for the purpose of treating a malignancy. For example, a therapeutically active amount of a binding polypeptide of the present disclosure may vary according to factors such as the stage of the disease (e.g., stage I vs. stage IV), age, sex, medical complications (e.g., immunosuppressive conditions or diseases), and weight of the subject, as well as the ability of the modified antibody to elicit a desired response in the subject. Dosage regimens may be adjusted to produce the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0233] In general, the compositions provided in this disclosure can be used to treat any neoplasm prophylactically or therapeutically and contain antigenic markers that allow for targeting of cancerous cells with engineered antibodies.

[0234] Pharmaceutical Compositions and Their Administration Methods for preparing and administering binding polypeptides of the present disclosure to a subject are well known or readily determined by those of skill in the art. Routes of administration of binding polypeptides of the present disclosure can be oral, parenteral, by inhalation, or topical. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all of these modes of administration are expressly contemplated as being within the scope of the present disclosure, the administration mode will be an injectable solution, particularly for intravenous or intraarterial injection or infusion. Typically, pharmaceutical compositions suitable for injection may include buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), and optionally stabilizers (e.g., human albumin). In some embodiments, binding polypeptides can be delivered directly to the site of harmful cell populations, thereby increasing the exposure of affected tissues to the therapeutic agent.

[0235] Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. In the compositions and methods of the present disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, e.g., 0.05 M phosphate buffer, or 0.8% saline. Other common daily oral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, e.g., those based on Ringer's dextrose, and the like. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present. More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and typically preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.

[0236] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Often, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, are included in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0237] In either case, sterile injectable solutions can be prepared by incorporating the required amount of the active compound (e.g., the modified binding polypeptide by itself or in combination with other active agents) in an appropriate solvent with one or a combination of ingredients enumerated herein, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary preparation methods include vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof. Formulations for injection are processed, filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under aseptic conditions according to methods known in the art. Furthermore, formulations can be packaged and sold in kit form. Such products typically have a label or package insert indicating that the accompanying composition is useful for treating a subject suffering from or susceptible to an autoimmune or oncological disorder.

[0238] The effective dose of the compositions of the present disclosure for treating the above conditions will vary depending on many different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other administered drugs, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0239] The binding polypeptides of the present disclosure can be administered on multiple occasions. The interval between single doses can be weekly, monthly, or yearly. The intervals can also be irregular, as indicated by measuring the blood levels of the modified binding polypeptide or antigen in the patient. In some methods, dosage is adjusted to achieve a plasma concentration of the modified binding polypeptide of about 1-1000 μg / ml, and in some methods about 25-300 μg / ml. Alternatively, the binding polypeptide can be administered as a sustained-release formulation, in which case less frequent administration is required. For antibodies, dosage and frequency will vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit the longest half-life, followed by chimeric and non-human antibodies.

[0240] Dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance. Such an amount is defined as a "prophylactically effective dose." In this use, the precise amount again depends on the patient's health and general immunity, but generally ranges from about 0.1 to about 25 mg per dose, particularly from about 0.5 to about 2 mg per dose. Relatively low dosages range from about 1 to 400 mg / kg of antibody per dose, about 5 to 25 mg for radioimmunoconjugates, and higher doses are more commonly used for cytotoxin-drug modified antibodies. In therapeutic applications, relatively high dosages (e.g., about 1 to 400 mg / kg of antibody per dose, about 5 to 25 mg for radioimmunoconjugates, and higher doses for cytotoxin-drug modified antibodies) may be required at relatively short intervals until disease progression is reduced or terminated, or until the patient shows partial or complete remission of disease symptoms. Thereafter, the patient may be administered a prophylactic regimen.

[0241] The binding polypeptides of the disclosure can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (eg, prophylactic or therapeutic). 90 An effective single treatment dosage (ie, a therapeutically effective amount) of a Y-labeled modified antibody of the present disclosure is between about 5 and about 75 mCi, for example, between about 10 and about 40 mCi. 131 I-Modified Antibodies Effective single treatment non-marrow ablative dosages range between about 5 and about 70 mCi, or between about 5 and about 40 mCi. 131 I-labeled antibody Effective single-treatment ablative dosages (i.e., which may require autologous bone marrow transplantation) range between about 30 and about 600 mCi, e.g., between about 50 and about 500 mCi. In conjunction with chimeric antibodies, due to their longer circulating half-lives compared to murine antibodies, effective single-treatment non-marrow ablative dosages of iodine-131 labeled chimeric antibodies range between about 5 and about 40 mCi, such as less than about 30 mCi. For example, 111 Imaging criteria for In labeling are typically Typically less than about 5 mCi.

[0242] It should be emphasized that while the binding polypeptides can be administered as described immediately above, in other embodiments, the binding polypeptides can be administered as a first-line treatment to otherwise healthy patients. In such embodiments, the binding polypeptides can be administered to patients with normal or average red marrow reserves and / or to treatment-naive and treatment-naive patients. As used herein, administration of a modified antibody or immunoadhesin in conjunction with or in combination with adjunctive therapy refers to sequential, simultaneous, coextensive, concurrent, concomitant, or contemporaneous administration or application of the therapy and the disclosed antibody. Those skilled in the art will appreciate that the administration or application of the various components of a combined therapeutic regimen can be timed to enhance the overall effectiveness of the treatment.

[0243] As previously discussed, the disclosed binding polypeptides, immunoadhesins, or combinations thereof can be administered in pharmaceutically effective amounts for the in vivo treatment of mammalian disorders. In this regard, it will be understood that the disclosed binding polypeptides will be formulated to facilitate administration and promote stability of the active agent.

[0244] Pharmaceutical compositions according to the present disclosure may include pharmaceutically acceptable, non-toxic, sterile carriers such as saline, non-toxic buffers, preservatives, and the like. For purposes of this application, a pharmaceutically effective amount of a binding polypeptide, immunoadhesin, or combination thereof, conjugated or unconjugated to a therapeutic agent, shall be considered to mean an amount sufficient to achieve effective binding to the antigen and achieve a benefit, e.g., ameliorating symptoms of a disease or disorder, or to detect a substance or cell. In the case of tumor cells, the modified binding polypeptides can interact with immunoreactive antigens on neoplastic or immunoreactive cells and result in increased killing of those cells. Of course, the pharmaceutical compositions of the present disclosure can be administered in a single dose or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

[0245] In accordance with the scope of the present disclosure, the disclosed binding polypeptides can be administered to humans or other animals in amounts sufficient to produce a therapeutic or prophylactic effect according to the treatment methods described above. The binding polypeptides can be administered to such humans or other animals in conventional dosage forms prepared by combining the antibodies of the present disclosure with conventional pharmaceutically acceptable carriers or diluents in accordance with known techniques. It will be recognized by those of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent will be dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables. It will further be appreciated by those of skill in the art that cocktails comprising one or more of the binding polypeptides described in this disclosure may prove particularly effective.

[0246] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0247] While the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made, and equivalents may be substituted, without departing from the spirit and scope of the invention. It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made, using appropriate equivalents, without departing from the scope of the embodiments disclosed herein. Furthermore, many modifications may be made to adapt a particular situation, material, composition of matter, method, process step(s), to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. While specific embodiments have been described in detail, these will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting. [Example]

[0248] This invention is further illustrated by the following examples, which should not be construed as further limiting.

[0249] Example 1: Materials and Methods Protein Reagents: The following proteins were expressed and isolated: antigen with a C-terminal 8x histidine tag; rFcRn (UniProt: P1359, p51 subunit: residues 23-298; UniProt: P07151, β2-m: residues 21-119); and biotinylated cynomolgus monkey FcRn. (UniProt: Q8SPV9, p51 subunit: residues 24-297 with a C-terminal Avi-tag; UniProt: Q8SPW0, β2-m: residues 21-119); biotinylated hFcRn (UniProt: P55899, p51 subunit: residues 24-297 with a C-terminal Avi-tag; UniProt: P61769, β2-m: residues 21-119); human CD16a (UniProt: P08637, FcγRIIIa: residues 17-208 with a C-terminal HPC4 tag and a valine at position 158 (V158)). The H435A and H310A / H435Q heavy chain mutants were obtained from HEK293 conditioned medium. mAb2 variants were cloned by Evitria and purified from suspension CHO K1 conditioned medium using a mAbSelect SuRe affinity column (GE Healthcare), and buffer exchanged into phosphate-buffered saline (PBS) pH 7.4 for subsequent experiments.

[0250] Saturation library construction: The WT IgG1 mAb1 antibody heavy and light chains with leader DNA sequences were engineered into pBH6414 and pBH6368 mammalian expression plasmids, respectively, using the NcoI and HindIII restriction enzyme sites. The saturation library was engineered using Lightning site specific A heterologous mutagenesis kit (Agilent) and NNK (N = A / C / G / T, K = G / T) and WWC (W = A / T) primers (IDT Technologies) were used to introduce all possible amino acids at the following positions: M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436 (EU numbering). The heavy chain DNA sequences of three control mutants in the mAb1 backbone, AAA (T307A / E380A / N434A), LS (M428L / N434S), and YTE (M252Y / S254T / T256E), were constructed in the pBH6414 vector (LakePharma).

[0251] A combinatorial saturation library was generated by site-directed mutagenesis of the mAb1 heavy chain using the Q5 mutagenesis kit (NEBiolabs) and primers T256D, T256E, T307Q, T307W, N434F, and N434Y in PCR reactions with WT and M252Y templates. Mutations were incorporated into the Ab3 backbone using the Q5 mutagenesis kit (NEBiolabs) with primers M252Y, T256D, T307Q, and T307W. The construction of all Fc variants was confirmed by Sanger sequencing (Genewiz, Inc.).

[0252] Recombinant antibody expression and purification: For conditioned medium screening, DNA containing the mutant heavy chain and wild-type light chain of mAb1 was transfected into 1 mL of Expi293 mammalian cells (Invitrogen) for expression according to the manufacturer's instructions. Cells were incubated in 2 mL 96-well plates (Greiner Bio-One) sealed with a vented membrane at 37°C, 5% carbon dioxide, and 80% humidity, shaking at 900 revolutions per minute (RPM). Conditioned medium was collected 5 days after transfection and stored at -80°C until use. Lead variants in the mAb1 and Ab3 backbones were expressed in 125 mL flasks equipped with 0.2 μm vented caps (Corning) at a 30 mL scale. The 125 mL culture flasks were shaken at 125 RPM throughout the entire expression period. Conditioned medium was collected 5 days after transfection, filtered through a 0.22 μm, 50 mL conical filter (Corning), and stored at 4°C until purification.

[0253] Isolation of mAb1 and Ab3 was performed using a 1 mL mAbSelect SuRe HiTrap column (GE Healthcare). After a wash step with 10 column volumes of PBS pH 7.4, the antibodies were eluted with 5 column volumes of 0.1 M citric acid pH 3.0 (Sigma) and neutralized with 0.5 mL of 1 M tris base pH 9.0 (Sigma). The eluted antibodies were buffer exchanged into PBS pH 7.4 and collected at >1 mg mL for subsequent testing using 30 kDa MWCO Amicon concentrators (Millipore). -1 The concentrations of the purified antibodies were determined by measuring their UV absorbance at 280 nm (UV 280 ) using the appropriate extinction coefficient.

[0254] Octet Conditioned Media Screening and Analysis: Screening of conditioned media containing mAb1 variants was performed using an Octet QK 384 (PALL Life Sciences) equipped with a Ni-NTA biosensor. His-tagged antigen was added at 15 μg mL -1The antibodies were captured in PBS, 0.1% bovine serum albumin (BSA, Sigma), and 0.01% Tween-20 (Sigma) pH 7.4 (PBST-BSA 7.4) for 300 seconds at RT, followed by a 20-second wash with PBST-BSA pH 7.4. The antibodies were captured for 200 seconds in conditioned medium diluted 1:1 with PBST-BSA pH 7.4. After a buffer wash step in pH 6.0 buffer, FcRn binding kinetics were obtained using 200 nM rFcRn for association and dissociation times of 150 and 200 seconds, respectively, at pH 6.0. The temperature was 30°C and the shaking speed was 1000 RPM during all steps during the Octet screening. rFcRn binding was observed. The combined kinetic profiles were corrected for the onset of the FcRn binding phase and modeled to a 1:1 binding model using Octet 7.1 analysis software.

[0255] FcRn binding kinetics: FcRn binding kinetics at pH 6.0 and pH 7.4 were measured using a Biacore T200 instrument (GE Healthcare) using either direct immobilization of FcRn or a modified protocol with the BiotinCAPture kit (GE Healthcare) (see, e.g., Abdiche et al., MAbs (2015) 7:331-343; Karlsson et al., Anal. Biochem. (2016) 502:53-63). For direct immobilization, biotinylated FcRn was added at a concentration of 20 μg mL. -1 10 μL for 180 seconds (s) -1 The CAPture reagent was immobilized via amine coupling chemistry (GE Healthcare) to approximately 20 RU on the surface of a C1 sensor chip in 10 mM sodium acetate pH 4.5 (GE Healthcare) at 2000 rpm. The Biotin CAPture kit was used to capture the CAPture reagent onto the CAP chip surface to a bound RU of >2,000 RU, followed by addition of 0.1 μg mL -1 FcRn in the appropriate channel for 24 seconds, 30 μL -1The antibodies were captured at 1000 nM with a final binding RU of approximately 2 RU. The running buffer for FcRn binding kinetics experiments was PBS, pH 6.0 or 7.4, containing 0.05% surfactant P-20 (PBS-P+, GE Healthcare). A series of 4-fold serial dilutions from 1000 nM antibody was performed in quadruplicate for each variant, including a 0 nM control. Kinetic measurements were performed at a flow rate of 10 μL min with association and dissociation times of 180 and 300 s, respectively. -1 The C1 and CAP sensor chips were each washed with 10 mM sodium tetraborate, 1 M NaCl pH 8.5 (GE Healthcare) for 30 seconds in 50 μL portions. -1 or 6M guanidine hydrochloride, 250mM sodium hydroxide (GE Healthcare) for 120s, 50uL min -1 The regeneration was performed using 1000 nM of 1000 nM HCl followed by an additional 60-90 s stabilization step in PBS-P+ pH 6.0. pH 7.4 steady-state RU measurements were obtained in triplicate for all variants at 1000 nM using the same Cl or CAP sensor chips and kinetic parameters as above, except that the level of FcRn capture increased 10- to 20-fold for both methods.

[0256] Kinetic parameters for the concentration series at pH 6.0 were fitted to a bivalent model with avidity effects using Biacore T200 evaluation software. See, for example, Suzuki et al., J. Immunol. (2010) 184:1968-1976. Each concentration series was fitted independently to obtain average binding and dissociation rates and binding affinity. Apparent binding affinity was calculated from the first binding and dissociation rates from the bivalent model. Residual binding at pH 7.4 was measured using 1000 nM of each antibody in triplicate for response comparison. The steady-state responses of each replicate were averaged to obtain the mean and standard deviation.

[0257] FcRn affinity chromatography: In one embodiment, an FcRn affinity column was prepared from a protocol adapted from Schlothauer et al. 2013, mAbs 5:576-586. A 1 mL Streptavidin HP HiTrap column (GE Healthcare) was loaded with 1 mL of binding buffer (20 mM sodium phosphate (Sigma) pH 7.4, 150 mM sodium chloride (NaCl; Sigma)). -1 The column was equilibrated with 5 column volumes at 200 rpm, followed by injection of 4 mg of biotinylated cynoFcRn. The column was washed with binding buffer and stored at 4°C until use.

[0258] The FcRn affinity column was loaded onto five columns of low pH buffer (20 mM 2-(N-morpholino)ethanesulfonic acid (MES; Sigma) pH 5.5; 150 mM NaCl). After equilibration with 10 column volumes of low-pH buffer, 300 μg of each antibody was injected. The pH of the antibody solution was adjusted to pH 5.5 using a low-pH buffer. After washing with 10 column volumes of low-pH buffer, the antibody was eluted in 1 mL min over 30 column volumes using a linear pH gradient with high-pH buffer (20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (bistrispropane; Sigma) pH 9.5; 150 mM NaCl). -1 Elute in 1 mL fractions at 1000 kJ / min and UV 280 The FcRn affinity column was then The column was re-equilibrated with 10 column volumes of low pH buffer for subsequent runs or with binding buffer for storage. All variants were performed in triplicate.

[0259] The FcRn affinity column elution profile for each mutant was modeled into a single Gaussian distribution using equation 1 in Sigmaplot 11 (Systat Software, Inc.) and UV 280 The elution volume at the maximum was determined.

number

[0260] In a separate experiment, an FcRn affinity column was adapted from Schlothauer et al. 2013, mAbs 5:576-586 using biotinylated hFcRn on a 1 mL Streptavidin HP HiTrap column (GE Healthcare). 300 μg of each antibody in low pH buffer (20 mM 2-(N-morpholino)ethanesulfonic acid (MES; Sigma) pH 5.5; 150 mM NaCl) was injected onto the column using an AKTA Pure System (AKTA). A linear pH gradient was generated using low and high pH buffers (20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (bistrispropane; Sigma) pH 9.5; 150 mM NaCl) to separate the antibody in 0.5 mL increments over 30 column volumes. -1 The antibody was eluted at 100°C, monitoring absorbance and pH. The column was re-equilibrated with a low pH buffer for subsequent runs. All variants were run in triplicate. The FcRn affinity column elution profile was fitted to a single Gaussian distribution in Sigmaplot 11 (Systat Software, Inc.) to calculate the UV distribution. 280 The pH of the elution volume from the maximum was determined. .

[0261] Differential scanning fluorimetry: Differential scanning fluorimetry (DSF) experiments were performed in a BioRad CFX96 real-time thermal cycler (BioRad) using 20 μL reactions. Antibody samples and 5000x stocks of Sypro Orange dye (Invitrogen) were diluted to 0.4 mg mL each in PBS pH 7.4. -1Antibodies and Sypro Orange were mixed in a 1:1 ratio in a 96-well PCR plate and sealed with adhesive microseal (BioRad) to give a final concentration of 0.2 mg mL of each antibody. -1 and 5x Sypro The antibody variants were run in triplicate. The thermal cycler program consisted of a 2-minute equilibration step at 20°C, followed by a constant temperature ramp of 0.5°C / 5 seconds to a final temperature of 100°C. Fluorescence measurements of each well were obtained using a FAM excitation wavelength (485 nm) and ROX emission (625 nm) detector appropriate for Sypro orange fluorescence (see, e.g., Biggar et al. 2012, Biotechniques 53:231-238). DSF fluorescence intensity profiles and first derivatives were exported from BioRad CFX Manager and analyzed using Sigma-Aldrich. Analysis was performed at t 11. m is defined as the midpoint of the first transition in the fluorescence intensity profile was done.

[0262] FcγRIIIa binding kinetics: Binding kinetics and affinity were measured using a Biacore T200 instrument (GE Healthcare) (Zhou et al. 2008 Biotechnol. Bioeng. 99:652-665). 50 μg mL in acetate pH 4.5 -1 Anti-HPC4 antibody (Roche) was applied to the surface of a CM5 sensor chip for 600 seconds in 10 μl. -1 The running buffer for FcγRIIIa binding kinetics experiments was HEPES-buffered saline pH 7.4 containing 0.05% surfactant P-20 (HBS-P+, GE Healthcare) and 2 mM calcium chloride (CaCl2, Fluka). Therapeutic follow-up was performed at 1.25 μg mL -1 Capture of HPC4-tagged FcγRIIIa-V158 for 30 seconds (5 μl) -1The binding and dissociation kinetics of each mutant at 300 nM were measured for 120–180 s at 5 μl min for each step. Upon completion of the kinetic measurements, the CM5 chip was regenerated with HBS-P+ buffer supplemented with 10 mM EDTA (Ambion). Before the next kinetic measurement, the CM5 chip was washed with HBS-P+ containing CaCl2 for 120 s.

[0263] In one experiment, FcγRIIIa kinetics was analyzed at pH 7.4 in a manner similar to that described for FcRn binding. Kinetics were obtained for a series of 3-fold serial dilutions from 1000 nM for WT, benchmark, lead single, and combination mutants to determine binding affinity to FcγRIIIa. The steady-state RU at each concentration and replicate was determined, plotted as a function of antibody concentration, and fitted to a steady-state model as shown in Equation 2.

number

[0264] In a separate experiment, FcγRIIIa kinetics experiments were analyzed using the average steady-state binding response in a manner similar to that described for FcRn binding at pH 7.4. For all variants, the steady-state RU at 300 nM antibody was determined in triplicate and averaged. The fold change in response compared to WT (response fold change) was determined for comparison between variants in each backbone.

[0265] Isoelectric focusing The isoelectric point (pI) of the lead variant was determined using capillary electrophoresis on Maurice C (Protein Simple). Each 200 μL sample was diluted in 0.35% methylcellulose (Protein Simple), 4% pharmalyte 3-10 (GE Healthcare), 10 mM arginine (Protein Simple), 0.2 mg mL -1 The antibody and 4.05 and 9.99 pI markers (Protein Simple) were included. Samples were loaded into the capillary at 1500 V for 1 min, followed by a 6 min separation phase at 3000 V and monitored using tryptophan fluorescence. The pI for each variant was determined using Maurice C software and defined as the pH at the fluorescence maximum for the major species.

[0266] Homogeneous bridging rheumatoid factor (RF) ELISA Antibodies were biotinylated and mixed with EZ-Link Sulfo-NHS-LC-Biotin and Mix-n-Stain. TM Digoxigen labeling was performed using a digoxigenin antibody labeling kit (Biotium) according to the manufacturer's instructions. Biotinylated and digoxigenin-labeled antibodies were used at 4 μg mL. -1A stock solution containing 1:1 was prepared for each mutant and mixed with 300 U / mL RF (Abcam) at a 1:1 ratio. After 20 hours of incubation at room temperature, 100 μL of each antibody-RF mixture was added to a Streptawell plate (Sigma-Aldrich) and incubated for 2 hours at room temperature. The plate was washed three times with PBS pH 7.4 containing 0.05% Tween-20, and 100 μL of a 1:2000 dilution of HRP-conjugated anti-digoxigenin secondary antibody (Abcam) was added to each well. After 2 hours of incubation at room temperature, the wells were washed and treated with 100 μL of TMB substrate (Abcam) for 15 minutes at room temperature. The reaction was stopped with 100 μL of stop solution (Abcam), and absorbance was measured at 450 nm using a SpectraMax plate reader. Wells without the antibody-RF mixture provided blank subtraction, and experiments were repeated three times. P values ​​were determined using a Student's t-test.

[0267] In vivo pharmacokinetics Pharmacokinetic studies were performed in cynomolgus monkeys and hFcRn transgenic mice (Tg32 strain, Jackson Laboratory, Bar Harbor, ME). In the monkey studies, the WT, LS, DQ, DW, and YD variants in the mAb2 backbone were administered as a single intravenous dose of 2.5 mg / kg into the brachiocephalic vein at a dose of 1.5 mL / kg to three naive male cynomolgus monkeys. Blood samples (0.5 mL) were collected by venipuncture of the saphenous vein at eight sampling times: 0.0035, 0.17, 1, 3, 7, 14, 21, and 28 days after dosing. Once collected, blood samples were centrifuged at 1500 g for 10 minutes at 4°C and stored at -80°C.

[0268] In hFcRn mice, the antibody variants were administered as a single intravenous dose of 2.5 mg / kg into the tail vein at a dose volume of 5 ml / kg. At each time point, 20 μl of blood was collected from the saphenous vein using a pre-filled heparin capillary. Collected blood samples were transferred to microtubes and centrifuged at 1500 g for 10 minutes at 4°C. Plasma samples were collected, pooled for each time point (6 mice / sample), and stored at -80°C before analysis.

[0269] All in vivo studies were conducted in compliance with the Sanofi Research Institutional Animal Care Guidelines. Monkey and mouse studies were conducted in accordance with the French Ministry of Animal Care and Use. t Superieur et de la Recherche" and the German "Regierungspraesidium Darmstadt." The concentration of each mAb2 variant at each time point was determined by a bottom-up LC-MS / MS assay. After precipitation of plasma aliquots, the plasma pellet was subjected to protein denaturation, reduction, alkylation, trypsin digestion, and solid-phase extraction before analyzing the surrogate peptides. Calibration standards were prepared by dissolving the mAb2 variants in plasma at concentrations of 1.00, 2.00, 5.00, 10.0, 20.0, 50.0, 100, 200, and 400 μg mL−1. -1 Peptide separation was performed on a Waters Acquity UPLC system using a reversed-phase XBridge BEH C18 column (2.1 x 150 mm, 3.5 μM, 300 Å, Waters) at a flow rate of 300 μL min . -1 The analysis was performed with a stepwise gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For detection, a Sciex API5500 mass spectrometer was used in positive ion mode with a source temperature of 700°C, an ion spray voltage of 5500V, a curtain and nebulizer gas of 40, and a collision gas of mid. Data acquisition times (dwell times) were 20 ms, and the entrance potential was 10V for each transition. Two unique alternative peptides of the mAb2 scaffold were analyzed. All multiple reaction monitoring transitions were used to determine concentrations using peak areas from the MQIII integrated algorithm in Analyst software compared to standards and controls. Clearance rates and serum half-lives were obtained from a non-compartmental model of antibody concentration as a function of time using Phoenix software (Certara). Without being bound by any theory, all time points showing a rapid decrease in concentration were excluded from the mean plasma concentrations due to speculation of target-mediated drug disposition (TMDD) and / or anti-drug antibody (ADA) interference.

[0270] Example 2: Octet screening for saturation point mutations in conditioned media FcRn is a heterodimer of an MHC class-I-like α-domain and a β2-macroglobulin (β2-m) subunit (Figure 1A), which is common to the majority of Fc receptors, and regions on the antibody Fc heavy chain that clearly distinguish it from other FcγRs (see, e.g., Oganesyan et al. 2014 J. Biol. Chem. 289:7812-7824; and Shields et al. 2001 supra).

[0271] To identify variants with slower FcRn dissociation rates than the WT antibody, a biolayer interferometry (BLI)-based assay was designed to screen antibody variants in conditioned medium in a high-throughput manner (Figure 2A). This assay was developed using several benchmark variants that either enhance (AAA, LS, and YTE) or decrease (H435A, H310A / H435Q) affinity for FcRn at pH 6.0 compared to the WT antibody. A NiNTA biosensor captured the His-tagged antigen, followed by capture of each antibody variant at pH 7.4 to mimic conditioned medium (Figure 2A). The binding kinetics of each of the six variants to rat FcRn (rFcRn), which has a 25-fold slower dissociation rate from human IgG1 and is more suitable for Octet studies than human FcRn (hFcRn), at pH 6.0 was measured (Figure 2B). The H435A (Figure 2B, long dashed-dotted line) and H310A / H435Q (Figure 2B, long dashed-double-dot line) mutants exhibited little or no FcRn binding kinetics (see, e.g., Shields et al., 2001, supra; Medesan et al., 1997, supra; and Raghavan et al., 1995, supra). The AAA (Figure 2B, short dashed line), LS (Figure 2B, short dashed-dotted line), and YTE (Figure 2B, long dashed line) mutants all exhibited slower dissociation rates compared with WT (Figure 2B, solid line), with FcRn dissociation rates reduced between 2- and 7.3-fold. This demonstrated that the Octet screen is suitable for distinguishing mutants with perturbed rFcRn dissociation kinetics.

[0272] The IgG1 antibody, mAb1, served as a model system for generating a saturation mutagenesis library to screen for mutants with reduced FcRn dissociation rates. Eleven positions in the Fc region of mAb1 were selected based on their proximity to or direct contribution to the FcRn interface (Figures 1A and 1B) (e.g., Oganesyan et al., 2014, supra; and Shields et al., 2001, supra). All point mutations at these positions were constructed using site-directed mutagenesis and transfected into Expi293 cells for expression. Conditioned medium screening was performed as described above for the saturation library mutants. Normalized FcRn-binding Octet sensorgrams for a subset of mutants are shown in Figure 2C (long-dashed line) along with the wild-type (Figure 2C, thick long-dashed line) and mock negative controls (Figure 2C, dotted line). The mock showed no observable FcRn binding. Some mutants clearly disrupted rFcRn binding, as they showed little or no signal change in the rate profile (Fig. 2C, long dashed line, located below the dotted line (false)). The cutoff for mutants with improved FcRn dissociation rates was defined as three standard deviations below the mean of the WT antibody. In the subset of mutations shown in Fig. 2C, two (Fig. 2C, solid line) had improved dissociation rates compared to the wild-type antibody. The rFcRn dissociation rate was significantly reduced (Fig. 2C, thick long-dashed line), whereas the remaining mutants had similar (Fig. 2C, short dash-dotted line) or faster (Fig. 2C, long-dashed line above dotted line (false)) rFcRn dissociation rates.

[0273] The rFcRn dissociation rates for all of the single point mutations are shown for each point and mutation in Figure 2D and Figure 14. In Figure 14, the data are classified into one of four categories depending on the fold change in rFcRn dissociation rate compared to the wild type, with the wild-type species indicated by a black box.

[0274] In Figure 14, the fold change in rFcRn dissociation rate for all possible substitutions at 11 positions in the saturation library was normalized to the average for the WT antibody and color-coded. All mutants fall into one of four categories: little to no binding (dark gray), faster rFcRn dissociation rate (gray), WT-like rFcRn dissociation rate (horizontal line), and slower rFcRn dissociation rate (grid). Several mutants had slower rFcRn dissociation rates than the WT antibody (grid).

[0275] The dark grey mutants in Figure 14 showed little or no binding to rFcRn in a manner similar to mock (Figure 2C, dotted lines) and were localized to the M252, I253, and S254 loops. The only mutations at I253 were methionine and valine, and both significantly increased the rFcRn dissociation rate, further supporting the importance of I253 for FcRn interaction. Another 120 mutants (Figure 2D and Figure 14, light grey boxes) showed significant binding to each C H 2 and C H The interaction with rFcRn located in the 3 domains was destabilized by approximately 50%. 25 mutants had WT-like dissociation rates (Figure 2D and Figure 14, open boxes). Eight of the 11 positions had at least one WT-like mutation (Figure 14, open boxes). The following mutations had significantly reduced rFcRn dissociation rates compared to the wild-type (Figure 2D and Figure 14, closed boxes): M252Y, T256D / E, K288D / N, T307A / E / F / M / Q / W, E380C, N434F / P / Y, and Y436H / N / W. The M252Y, N434F, and N434Y mutations had dissociation rates that were more than half that of the WT antibody (Figure 2D). These mutants were expressed and purified using protein A chromatography for further in vitro FcRn kinetic characterization.

[0276] Example 3: Biacore FcRn binding kinetics at pH 6.0 The AAA, LS, and YTE variants served as positive controls for human and rat FcRn at pH 6.0 in FcRn binding kinetics measurements using Biacore. Concentration-dependent binding to FcRn was observed for all variants, including the wild-type, benchmark (Figure 3), and lead (Figures 4A and 4B), and the single-injection binding profiles for human and rat FcRn are shown in Figures 5A and 5B, respectively. The wild-type antibody had binding affinities of 2380 ± 470 nM and 207 ± 43 nM for human and rat FcRn, respectively (Table 1).

[0277] [Table 1] [Table 2]

[0278] All data shown in Table 1 were obtained using the experimental technique indicated at the top of each column.

[0279] The rFcRn dissociation rate by Octet using purified protein was measured relative to the rate constant obtained from screening in conditioned medium. The elution pH was determined in triplicate (n = 3) by FcRn affinity chromatography, and DSF was investigated for thermal stability in triplicate (n = 3). FcRn binding kinetics for human and rat FcRn were obtained in duplicate (n = 2) from Biacore using a range of antibody concentrations and fitted independently. The steady-state binding response (RU) for each variant with human and rat FcRn at pH 7.4 was measured in triplicate (n = 3) using Biacore with 1000 nM antibody. The units for each measurement are as follows: Octet pH 6.0 rFcRn dissociation rate (x 10 -3 seconds -1 ); Dissolution pH (unitless); DSF T m (℃ );Biacore pH6.0 hFcRn binding rate (x10 4 M -1 seconds-1 ), dissociation rate (x10 -1 seconds -1 ) and K D,app (x10 9 M);Biacore pH6.0 rFcRn binding rate (x10 4 M -1 seconds -1 ), dissociation rate (x10 -3 seconds -1 ) and K D,app (x10 9 M); and Biacore pH 7.4 steady-state binding response (RU).

[0280] In Figure 5B, the AAA (dotted line), LS (dashed-dotted line), and YTE (dashed-dotted line) mutants had binding affinities that were between 1.6- and 10.4-fold enhanced compared to WT. The AAA mutants had the tightest affinity for hFcRn, whereas rFcRn had even tighter affinity for YTE, indicating that the identity of the benchmark mutants with the tightest FcRn affinity was species-specific (Table 2A).

[0281] [Table 3] [Table 4]

[0282] The majority of lead mutants for both human and rat FcRn (Figures 5A and 5B, solid lines of various hues) had significantly slower binding rates (<2-fold) than either WT or the benchmark mutants (Table 1). Only the N434F and N434Y mutations showed enhanced binding rates for both species of FcRn. Without being bound by theory, as a result of the slower binding kinetics with hFcRn, the apparent binding affinities of the lead mutants, unlike rFcRn, were generally weaker than WT (Figures 5C and 5D, Table 1). The affinity for rFcRn was weaker than YTE (Figure 5D, diagonal line to the lower left, Table 2A). Without being bound by any theory, these results indicated that no single mutation was sufficient to enhance affinity over the LS and YTE mutants. Ranking of FcRn dissociation rates (due to the weak binding affinity of the variants for hFcRn) revealed a subset with reduced dissociation rates for human and rat FcRn: M252Y, N434F / P / Y, T256D / E, and T307A / E / F / Q / W (Table 2A). These variants are of further interest in combination to further improve the FcRn-binding ability of the Fc region over the benchmark variants.

[0283] In vitro characterization parameters for the lead mutants are shown in Table 2B.

[0284] [Table 5]

[0285] In Table 2B, all data were obtained using the experimental technique at the top of each column. FcRn affinity chromatography, DSF, and FcγRIIIa binding were performed in triplicate (n=3). FcRn binding kinetics for human and rat FcRn were obtained in quadruplicate and fitted independently. Units: DSF T m (℃);FcγRIIIa binding (fold change compared to WT), Biacore pH 6.0 hFcRn binding rate (x10 4 M -1 seconds-1 ), dissociation rate (x10 -1 seconds -1 ) and K D,app (x10 9 M);Biacore pH 6.0 rFcRn K D,app (x10 9 M); Biacore pH 7.4 hF cRn and rFcRn steady-state RU (RU).

[0286] Example 4: Combination mutants further decrease FcRn binding dissociation rates Multiple lead mutations were located at a single position, such as T307 and N434 (Figure 14, black boxes), where six and three mutations, respectively, were identified that exhibited slower FcRn dissociation kinetics. Only the mutations with the slowest FcRn dissociation rates from hFcRn at these positions were used to generate combinatorial mutants. In this case, T307Q, T307W, N434F, and N434Y were mixed with M252Y, T256D, and T256E using mixed-primer PCR and site-directed mutagenesis to obtain double, triple, and quadruple mutants. In total, the combinatorial library consisted of 54 mutants, including seven lead single, 18 double, 20 triple, and eight quadruple mutants, as well as the WT antibody. The nomenclature of these mutants is as follows: the wild-type background contains M252, T256, T307, and N434, and is reclassified as MTTN. Therefore, the triple mutants Y T QY is M252 Y , T307 Q and N434 Y Contains the mutation but maintains the WT threonine at position 256.

[0287] Using single mutations, we determined which combination mutants had improved affinity by measuring FcRn binding kinetics at pH 6.0 using Biacore. Representative FcRn binding rate traces for each of the single (long dashed-dotted line), double (long dashed-dotted line), triple (long dashed line), and quadruple (short dashed line) mutants are shown in Figures 6A and 6B, compared to the benchmark mutants with the tightest affinity for each species of FcRn:WT (dotted line) and FcRn (hFcRn:LS (long dashed-dotted line); rFcRn:YTE (solid line)). hFcRn binding and dissociation rates (Figure 6C) revealed that two single, 15 double, 18 triple, and 8 quadruple mutants had enhanced binding affinity compared to the LS mutant (Figure 6C, dotted line). Similarly, all combinations except for one triple mutant had tighter affinity for rFcRn than YTE (Figure 6D, diagonal line to the lower left). In the case of hFcRn, additional FcRn-enhancing mutations further increased binding affinity (Figure 6C). The five combinations with the tightest affinity to hFcRn were all quadruple mutants (Figure 6C, checkerboard), with binding affinities approximately 500-fold higher than those of the wild type. A similar phenomenon did not occur with rFcRn (Figure 6D, horizontal lines), as the mutants with the highest affinity were double mutants (Figure 6D, horizontal lines). Triple (Figure 6D, vertical lines) and quadruple (Figure 6D, checkerboard) mutants typically showed only a slight decrease in dissociation rate (less than 2-fold) but also a decreased association rate (Figure 6D). Without being bound by any theory, these results suggest that there is likely a lower limit in FcRn apparent binding affinity reached by rFcRn (approximately 0.5 nM), but not by hFcRn (Figure 6B). In total, more than 40 combination mutants had closer affinity than the benchmark mutants, and further characterization is required to select combinations with the most favorable properties for in vivo studies.

[0288] Example 5: Combination mutants retain significant binding at physiological pH As a result of the significantly improved FcRn affinity at pH 6.0, the effect on pH dependence was investigated using FcRn affinity chromatography and Biacore steady-state measurements at pH 7.4. FcRn affinity chromatography uses a linear pH gradient to directly measure the pH-dependent perturbation by mutations. H435A and H310A / H435Q, which have weak FcRn binding, did not bind to the column regardless of pH (Figure 8A). WT eluted near physiological pH (pH 7.37 ± 0.05), while AAA, LS, and YTE required higher pH (Table 2B). All combination mutants and the seven lead single mutants required a higher pH than WT to elute from the affinity column (Figures 8A and 8C). The N434F / Y mutant eluted at a higher pH than LS (Table 2B), indicating that, without intending to be bound by scientific theory, these mutants disrupted the pH dependence, both individually and in combination. Representative chromatograms showed a clear shift to higher elution pH with the number of mutations (Figures 9A and 9B). The strong correlation between elution pH and hFcRn dissociation rate (R = 0.94) (Figure 9C) indicates that the slower FcRn dissociation rate at pH 6.0 directly contributed to the increased elution pH for the FcRn mutants.

[0289] To measure residual binding activity under physiological conditions, FcRn binding kinetics experiments were performed at pH 7.4 using Biacore. Because some mutants exhibited unreliable kinetics and little or no binding at this pH, steady-state RU was used as a measure of residual FcRn binding affinity. Representative kinetic traces of the single (long dashed-dot line), double (long dashed-dot line), triple (long dashed line), and quadruple (short dashed line) mutants are shown in Figures 7A and 7B in comparison with LS (Figure 7A, solid line) and YTE (Figure 7B, solid line). These two mutants exhibited the highest residual binding to human and rat FcRn, respectively, at pH 7.4. Most of the lead single mutants had slightly elevated FcRn binding compared to WT (4.3 ± 1.0 RU), except for the N434F / Y mutation, AAA (13.1 ± 1.7 RU), L (18.5 ± 2.6 RU), and YTE (13.1 ± 1.6 RU). The binding affinities of the LS and YTE mutants were lower (Tables 2A and 2B). The combination mutants also had significant residual binding to both species of FcRnn at pH 7.4, to a degree comparable to that of N434F / Y (Figures 7A and 7B). Without being bound by any theory, ideal candidates for in vivo studies would be mutants that have increased FcRn binding at low pH (e.g., the AAA, LS, and YTE mutants) but maintain low levels of binding at high pH, ​​in a manner similar to that of WT. In the plots shown in Figures 7C and 7D, these combinations would occupy the lower left quadrant designated by the affinities of the LS and YTE mutants for human and rat FcRn, respectively, at each pH.

[0290] Example 6: FcRn affinity chromatography The combination mutants showed a moderately positive correlation between apparent binding affinity at pH 6.0 and steady-state RU at pH 7.4 (hFcRn:R 2 =0.69, rFcRn:R 2= 0.71) (FIGS. 7C and 7D). Without being bound by any theory, these results suggest that higher affinity at pH 6.0 typically leads to higher residual FcRn binding at pH 7.4. These variants may remain bound to FcRn in the bloodstream and, like the high FcRn affinity abdeg mutants, have a short serum half-life and / or promote their clearance (e.g., Swiercz et al., supra 2014; and Vaccaro et al., supra 2005). Antibody-FcRn interactions are pH-dependent and occur only at low pH (<pH 6.5), so saturated mutants may enhance interactions due to hydrophobic or charge-derived contributions, which may interfere with deprotonation of important histidine residues (as shown in FIG. 1B), and weaken this interaction at physiological pH.

[0291] FcRn affinity chromatography uses a linear pH gradient to directly measure pH-dependent perturbations of FcRn interactions (see, e.g., Schlothauer et al., 2013, supra). FcRn affinity chromatography with the AAA, LS, YTE, H435A, and H310A / H435Q mutants revealed that H435A (Figure 8A, light gray solid line) and H310A / H435Q (Figure 8A, AQ, dark gray solid line) did not bind to FcRn even at pH 5.5 and eluted with flow-through. The wild-type antibody eluted near physiological pH (pH 7.37 ± 0.05), whereas AAA, LS, and YTE, which have slower dissociation rates and tighter FcRn-binding affinities than the wild-type by Octet (Figure 2) and Biacore (Figure 3), required significantly higher pHs to dissociate from the column (AAA: 7.94 ± 0.06; LS: 8.29 ± 0.03; YTE: 8.14 ± 0.03). The elution profiles revealed that all of the variants in the combinatorial library required a higher pH than the wild-type to elute from the affinity column. Representative average elution pHs for the single (long dashed double-dot line), double (long dashed single-dot line), triple (long dashed line), and quadruple (short dashed line) variants are shown in Figure 9A. Although the seven lead single mutants required a higher pH to dissociate from the column compared to the WT (Fig. 10A, Table 3), those with wild-type-like kinetics for hFcRn (K288D / N, Y436H / H / W) all eluted at a similar pH to the wild type.

[0292] [Table 6]

[0293] All data were obtained using the experimental technique at the top of each column. Elution pH was determined in triplicate (n=3) by FcRn affinity chromatography, and thermal stability was examined in triplicate (n=3) by DSF. FcRn binding kinetics for human and rat FcRn were obtained from Biacore using a range of antibody concentrations (n=4) and fitted independently. Units for each measurement are as follows: elution pH (unitless); DSF T m (℃);Biacore pH6.0 hFcRn binding rate (x10 4 M -1 seconds -1 ), dissociation rate (x10 -1 seconds -1 ) and K D,app (x10 9 M);Biacore pH 6.0 rFcRn binding rate (x10 4 M -1 seconds -1 ), dissociation rate (x10 -3 seconds -1 ) and K D,app (x10 9 M).

[0294] Both N434F / Y mutants eluted at a higher pH than the LS mutant (N434F: 8.30±0.05; N434Y: 8.46±0.02) and showed significant FcRn binding at pH 7.4 (Table 4). These results indicate that these mutants alone can disrupt the pH dependence. In general, the mean elution pH increased with increasing number of FcRn-binding-enhancing mutations (Figure 9B). A strong correlation (R 2 = 0.94) is the hFcRn dissociation rate While not being bound by any theory, the interaction p These results indicate that H-dependent disruption directly contributes to the slower FcRn dissociation rate observed for the combinatorial library at pH 6.0.

[0295] Example 7: Thermal Stability Most proteins, including antibodies, with low thermodynamic stability have an increased tendency to misfold and aggregate, limiting or hindering their activity, efficacy, and potential as novel therapeutic agents. The thermal stability of each mutant was determined using DSF and the reported melting temperature (T m ) in the Sypro Orange fluorescence intensity profile Defined as the midpoint of the first transition. T of 69.0 ± 0.2 °C m Compared to WT with L The S mutant is similar to WT (68.5 ± 0.3 °C), and AAA and YTE are thermally destabilized by approximately 8 °C (AAA: 61.3 ± 0.6 °C; YTE: 61.2 ± 0.3 °C) (Figures 8B, 9B, and 10B; and Tables 2B, 3, and 4). m Compared to WT and LS, which have had a lower thermal stability.

[0296] [Table 7]

[0297] Mutations introduced into the wild-type backbone are bold and underlined. All data were obtained using the experimental techniques indicated at the top of the columns. Elution pH and T m was determined in triplicate (n=3) FcRn binding kinetics at pH 6.0 for human and rat FcRn were obtained from Biacore (n=4) and fitted independently. Steady-state FcRn binding responses at pH 7.4 were measured in triplicate at a single antibody concentration using Biacore. FcγRIIIa binding affinity was determined in duplicate from a range of antibody concentrations using Biacore. Units for each measurement are as follows: elution pH (unitless); DSF Tm (°C); Biacore pH 6.0 hFcRn binding rate (x10 5 M -1 seconds -1 ), dissociation rate ( x10 -2 seconds-1 ) and K D,app (x10 9 M);Biacore pH6.0 rFcRn binding rate (x10 5 M -1 seconds -1 ), dissociation rate (x10 -3 seconds -1 ) and K D,app (x10 9 M); Biacore pH 7.4 steady-state binding response (RU) and FcγRIIIa K D,app (x10 9 M).

[0298] Twelve of the 18 read saturation mutants showed reduced T compared to the wild type. m It has And some of the T307 mutants (T307E / F / M / Q) showed slight stability (Table 4). One of the seven single mutants used in the combination (Figure 10B and Table 4) was significantly destabilized compared to YTE (Figure 8B and Table 5). The addition of double (Figure 9D, horizontal line), triple (Figure 9D, vertical line), and quadruple (Figure 9D, checkerboard pattern) mutants resulted in a further decrease in overall thermal stability compared to the single mutant (Figure 9D, open circle). Multiple mutants exhibited lower T than either AAA or YTE. m (61.2±0.℃), >60% of these mutants contained T307W. The quadruple mutant (Fig. 9D, checkerboard) showed a unique bimodal distribution of melting temperatures, with combinations containing T307Q having approximately 6°C higher thermal stability than those with T307W (Fig. 9D).

[0299] Example 8: Fc variants alter binding interactions with FcγRIIIa In addition to interacting with FcRn, the Fc region hinge and C H 2 domain, FcγRII Five of the seven single mutants used for the combinatorial saturation library were C H These receivers are located in two domains. Their ability to interact with the receptor may be impaired compared to wild-type, despite their location far from the interaction interface. Using Biacore to measure FcγRIIIa binding in a manner similar to FcRn binding at pH 7.4 revealed that the YTE (Figure 11A, dark gray) mutants exhibited approximately a 50% reduction in binding response compared to wild-type (Figure 11A, black). Without being bound by any theory, the reduced FcγRIIIa binding for YTE is a result of the M252Y mutation, as the M252Y mutant alone has significantly reduced affinity for this receptor (Figure 11B, bottom open circle). Other single mutations did not share this reduced affinity (Figure 11B, open circle), and the N434F / Y mutant alone enhanced binding by 16–40%. These effects were transferred to most, but not all, of their corresponding combinations. For example, the M252Y-containing combination had between a 17% and 72% reduction in FcγRIIIa binding (Table 5).

[0300] [Table 8]

[0301] One mutant M DQF (Figure 11B, the highest in the triple mutant category) showed a dramatic 140% increase in FcγRIIIa binding. Thus, the combinatorial saturation library provided mutants with a wide range of Fc receptor functions that can be utilized to tailor therapeutic antibodies with specific effector functions.

[0302] FIG. 11C shows box plots of the FcγRIIIa binding responses of the seven lead single mutants compared to the WT and YTE mutants.

[0303] Example 9: Seven lead combinations balance the pH dependence of FcRn interactions Without being bound by any theory, candidate mutants for further in vivo study occupied the lower left quadrant of the plots shown in Figures 7C and 7D. Seven mutants met these criteria for hFcRn, including five double and two triple combinations (M DQ N, M DW N, YD T.N., YE T.N., Y T W N, YDQ N and YEQ Each of these combinations contained a nucleotide sequence identical to that of the FcRn nucleotide sequence (nucleotide sequence N434) and did not contain a mutation at position N434 (Table 3). Each of these combinations eluted from the FcRn affinity column between AAA (pH 7.94±0.06) and LS (pH 8.29±0.03), YDQ N eluted at the highest pH of 8.51 ± 0.14 (Figure 12A, Table 5), showed only a slight perturbation in pH dependence, and higher residual binding at pH 7.4 (Table 2A). DQ N) have wild-type-like thermostability, and six have similar or reduced T compared to the YTE mutant. m (Fig. 12B, Table 4). In the FcγRIIIa binding assay, five combination mutants showed a similar decrease as YTE (Table 4). Further investigation with single mutations revealed that M252Y significantly affected FcγRIIIa binding and, without being bound by any theory, transferred this effect to combinations containing this mutation. The remaining six single mutations were WT-like or had slightly improved binding to this receptor.

[0304] Three combination mutants were selected for further study based on their FcRn-binding properties, thermal stability, and FcγRIIIa binding. DQ (T256D / T307Q), DW (T256D / T307W), and YD (M252Y / T256D) each provided optimal FcRn-binding properties (Table 2B), similar to the LS mutant (Figure 12E). Each mutant offers different thermal stability and FcγRIIIa-binding properties, resulting in a range of functionalities (Figures 12F and 12G, Table 2B). Figure 12H is a plot of the uniform bridging RF.

[0305] The enhancement of apparent binding affinity at pH 6.0 for both human and rat FcRn compared to the LS (Figure 13A, thick long-dashed line) and YTE mutants (Figure 13B, thick long-dashed line), respectively, represents a compromise between on-rate and off-rate (Figures 13A and 13B, Table 4). Typically, combinations with faster off-rates also have faster on-rates, and vice versa. This observation held true between human and rat FcRn (Table 4). Furthermore, all of these mutants had a lower steady-state response to hFcRn at pH 7.4 than the LS mutant (Figure 13C, thick long-dashed line). These results were inconsistent with rFcRn, where five M252Y-containing mutants, YD T.N., YE T.N., Y T W N, YDQ N and YEQ N had increased FcRn binding at pH 7.4 compared to YTE (Figure 13, Table 5). DQ N and M DW The N mutant was the only combination that was cross-reactive between human and rat FcRn. Furthermore, these two mutants did not perturb the interaction with FcγRIIIa to the same extent as the M252Y-containing mutant (Figures 12C and 12D and Table 5; M DQN: 600±4 nM; MDWN: 512±30 nM; WT: 467±99 nM). Thus, saturation and combinatorial mutagenesis at key FcRn-interacting sites led to the identification of lead mutants that balance the pH dependence of the interaction, maintain functionality at the Fc receptor, can enhance FcRn functionality in vivo, and extend the serum half-life of therapeutic antibodies.

[0306] Example 10: Rheumatoid factor binding characteristics of lead combination mutants Because these mutations may alter antibody surface charge and immunogenicity, the isoelectric point and RF binding of the lead mutants were examined. A more acidic antibody was thought to prolong antibody pharmacokinetics. Compared to the WT and LS controls, all three leads experienced a decrease in pI of approximately 0.2 pH units as a result of the T256D substitution. FcRn-enhancing mutations simultaneously alter binding to host antibodies such as rheumatoid factor (RF) due to overlapping interaction interfaces. A homogeneous bridging ELISA was adapted to measure changes in RF binding for the lead mutants. Interestingly, LS and YTE showed completely opposite shifts in RF binding compared to WT (Figure 12H). LS significantly increased RF binding, while YTE showed a significant decrease (p<0.001). YD (p<0.001) and DW (p<0.01) also significantly reduced RF binding, while DQ produced a response similar to WT. Without being bound by any theory, these results indicate that DQ, DW, and YD may confer an immunogenicity advantage compared to LS. The YD, DW, and DQ variants represent a range of key antibody characteristics that can be influenced in conjunction with improved FcRn binding properties over the benchmark YTE and LS variants.

[0307] Example 11: Lead combination mutants are transferable to other antibodies As shown in Figure 15A, a new binding assay was developed using a CM5 sensor chip. The binding assay involves immobilizing streptavidin to approximately 30 RU on a CM5 sensor chip to capture biotinylated FcRn, which is replenished as needed. Antibody binding kinetics were measured at pH 6.0 and 7.4, followed by regeneration at pH 8.5. Figures 15B and 15C show direct immobilization of FcRn and streptavidin capture of biotinylated FcRn using the new binding assay.

[0308] Antibody-2 FcRn binding at pH 6.0: For mouse FcRn, the lead antibody-2 mutants show slower dissociation rates than the LS mutant (dashed line) and wild-type (black) (Figure 16A). For human FcRn, the lead mutants all have faster binding rates but similar dissociation rates to LS (dashed line) (Figure 16B).

[0309] Antibody-2 FcRn binding at pH 7.4: All mutants showed reduced human binding at pH 7.4 compared to LS (dashed line) (Figure 17A). Similar to the Antibody-1 background, DW (M DW N) and DQ(M DQ N) mutant also showed lower residual binding to mouse (rat) FcRn at pH 7.4 (Figure 17B).

[0310] The lead variant maintained higher binding affinity at pH 6.0 and lower residual binding at pH 7.4 compared to LS (Figure 18). Importantly, the variants were found to be transferable between different IgG1 backgrounds with little effect on FcRn binding. As shown in Figure 19, LS had similar elution pH regardless of background. In the Antibody-2 background, WT, DQ, and DW exhibited higher elution pH than in the Antibody-1 background, likely as a result of tighter binding at pH 6.0 in the Antibody-2 background.

[0311] All of the Antibody-2 background mutants showed slightly increased thermostability, as shown in FIG.

[0312] As shown in Figure 21, similar to the antibody-1 background, YD( YD TN) showed a decrease in FcγRIIIa binding response (left) and affinity (right). DQ (light gray) and DW (dark gray) showed FcγRIIIa binding properties similar to WT (black) in the Antibody-2 background. The effect of LS on FcγRIIIa binding was consistent between Antibody-1 and Antibody-2.

[0313] Thus, the lead mutant in the antibody-2 background does not significantly affect FcRn binding, pH dependency, thermostability, or FcγRIIIa binding compared to the same lead mutant in the antibody-1 background.

[0314] In one embodiment, the DQ (T256D / T307Q), DW (T256D / T307W) and YD (M252Y / T256D) mutants were incorporated into additional IgG1 antibodies and recombinant Fc fragments: mAb2 recognizes a different antigen than mAb1, and Ab3 is an Fc fragment. In each case, the pH-dependent FcRn binding kinetics (Figure 1) were analyzed. 22) were highly similar in elution pH, thermal stability, and FcγRIIIa binding affinity (Tables 2B and 6). Without being bound by any theory, these results indicate that the DQ, DW, and YD mutants confer their improved FcRn binding properties to proteins consisting of an Fc domain.

[0315] [Table 9]

[0316] Example 12: Lead mutants extended in vivo plasma antibody elimination half-life The pharmacokinetics (PK) of the DQ, DW, and YD variants was investigated in cynomolgus monkeys and hFcRn transgenic mice (strain Tg32) compared with WT and LS controls, and their antibody circulating half-lives were examined (see, e.g., Avery et al. Mabs (2016) 8:1064-1078). FcRn binding studies using cynomolgus monkey FcRn revealed binding affinities similar to hFcRn (Figures 23A-23B; Table 6). Each animal was intravenously injected with WT, LS, DQ, DW, or YD variants, and antibody concentrations were quantified using a mass spectrometry approach to determine clearance rates and serum half-lives in monkeys (Figure 24A) and hFcRn transgenic mice (Figure 24B). Clearance rates and serum half-lives were obtained from a non-compartmental model of antibody concentration as a function of time. All three lead mutants and LS showed significantly reduced clearance rates compared to WT in both monkeys and mice (p<0.001). The plasma half-lives of the WT antibody were 9.9±0.5 days and 11.7 days in monkeys and mice, respectively. Furthermore, the LS benchmark and identified mutants showed significantly increased elimination half-lives compared to the wild type in both species (2.5-fold and 1.7-fold increases in monkeys and mice, respectively) (Table 7). DQ, DW, and YD showed similar increases in half-life compared to the LS benchmark (Table 7). The DQ, DW, and YD mutants identified herein by saturation mutagenesis showed significantly increased plasma half-lives compared to their WT counterparts in both mice and non-human primate animal models.

[0317] [Table 10]

[0318] In Table 7, clearance rates and plasma half-lives were determined using mAb2. Each clearance rate and half-life is the mean of n=3 for cynomolgus monkeys and a single assessment from a pool of n=6 hFcRn transgenic mice. Fold over WT and fold over LS indicate relative improvement compared to WT and LS, respectively. *n=2 for ADA formation, **n=2 for partial subcutaneous administration route

[0319] Example 13: Combination variants with enhanced FcRn binding at pH 6.0 and pH 7.4 Based on the Octet screen (BLI-based screen) as described in Example 2, various single, double, triple, and quadruple mutants were generated and their binding to FcRn at pH 6.0 and pH 7.4 was evaluated (Table 8).

[0320] [Table 11] [Table 12]

[0321] Table 8 shows the binding affinity to FcRn at pH 6.0 and steady-state binding to FcRn at pH 7.4 for various single, double, triple, and quadruple mutants as well as benchmark mutants (AAA, LS, YTE).

[0322] These values ​​are plotted in Figure 25, which shows the binding affinity at pH 6.0 and the RU at pH 7.4. As shown, the benchmark variant LS had the tightest binding affinity at pH 6.0 and the greatest residual binding at pH 7.4 among the benchmark variants tested (AAA, LS, YTE).

[0323] It was determined that several combination mutants, shown in Figure 25, exhibited enhanced FcRn binding affinity at pH 6.0 and pH 7.4. To determine whether any of the combination mutants exhibited tighter binding than the MST-HN mutant (referred to herein as the "YTEKF benchmark" and containing mutations at Met252, Ser254, Thr256, His433, and Asn434 to Tyr252, Thr254, Glu256, Lys433, and Phe434) at both pH 6.0 and pH 7.4, both of the following methodologies were performed: Capture of biotinylated human, cynomolgus monkey, and mouse FcRn was performed by the Biotin CAPture method (see Figure 26 for a schematic). At pH 6.0, a concentration series (5 points) starting from 1000 nM was performed in duplicate. For pH 7.4, triplicate injections of a single concentration (1000 nM) were performed (a 10-fold increase in capture level for each FcRn was observed for binding at this pH). Association: 180 sec; Dissociation: 300 sec.

[0324] The human FcRn binding kinetics of the YTEKF benchmark and various combination mutants at pH 6.0 are shown in Figure 27. As shown in Figure 27, all tested mutants exhibited two orders of magnitude tighter affinity for human FcRn compared to the wild type (WT).

[0325] Figures 28A and 28B show the FcRn binding kinetics of the combination mutants compared to the YTEKF benchmark at pH 6.0 (Figure 28A) and pH 7.4 (Figure 28B). In Figure 28A, the majority of the mutants exhibited slower dissociation rates than the YTEKF benchmark and had similar or slower binding rates. In Figure 28B, YTEKF exhibited significant binding at pH 7.4, and four mutants exhibited higher residual binding.

[0326] [Table 13]

[0327] Table 9 shows the binding affinity to FcRn at pH 6.0 and steady-state binding to FcRn at pH 7.4 for selected combination mutants as well as YTEKF benchmark and WT.

[0328] Figure 29 shows a comparison of binding affinity at pH 6.0 and RU at pH 7.4 for select combination antibodies shown in Table 9. As shown in Table 9 and Figure 29, four quadruple mutants were found to have higher affinity for FcRn at pH 6.0 and pH 7.4 compared to the YTEKF benchmark. The four quadruple mutants were favored with the T256D, T307Q, and N434Y mutations. These quadruple mutants showed approximately a 500-fold and 3-fold improvement in affinity (pH 6.0) over WT and YTEKF, respectively.

[0329] Other characterization parameters, such as thermal stability, binding to FcγRIIIa, and elution pH, were determined and are shown in Table 10.

[0330] [Table 14]

[0331] As shown in Table 10, all lead quadruple mutants were found to be thermally destabilized and exhibited reduced FcγRIIIa binding ability.

Claims

1. An isolated binding polypeptide containing a modified Fc domain, which, according to EU numbering, has an aspartic acid (D) at amino acid position 256 and a glutamine (Q) at amino acid position 307, thereby increasing the FcRn binding affinity of the modified Fc domain.

2. Isolated conjugated polypeptide comprising a modified Fc domain, wherein the only modification of the modified Fc domain compared to the wild-type Fc domain is, according to EU numbering, aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307.

3. The isolated conjugated polypeptide according to claim 1 or 2, wherein the isolated conjugated polypeptide has an increased serum half-life compared to the conjugated polypeptide containing a wild-type Fc domain.

4. The isolated bound polypeptide according to any one of claims 1 to 3, wherein the isolated bound polypeptide has enhanced FcRn binding affinity compared to the bound polypeptide containing a wild-type Fc domain, and optionally the FcRn is human FcRn.

5. The isolated bound polypeptide according to claim 4, wherein the isolated bound polypeptide has enhanced FcRn binding affinity at an acidic pH compared to the bound polypeptide containing the wild-type Fc domain, and optionally the acidic pH is 6.

0.

6. The isolated bound polypeptide has a higher FcRn binding affinity at an acidic pH compared to the FcRn binding affinity of the bound polypeptide at a non-acidic pH, wherein the acidic pH is 6.0 and optionally the non-acidic pH is 7.4, any one of claims 1 to 5. Isolated bound polypeptides as described in the section.

7. The isolated conjugated polypeptide according to any one of claims 1 to 6, wherein the isolated conjugated polypeptide has a modified FcγRIIIa binding affinity compared to the conjugated polypeptide containing a wild-type Fc domain, and optionally FcγRIIIa is human FcγRIIIa.

8. The isolated conjugated polypeptide according to any one of claims 1 to 7, wherein the modified Fc domain is a modified human Fc domain.

9. The isolated conjugated polypeptide according to any one of claims 1 to 8, wherein the modified Fc domain is a modified IgGFc domain.

10. The isolated conjugated polypeptide according to any one of claims 1 to 9, wherein the modified Fc domain is a modified human IgG1 Fc domain.

11. The isolated bound polypeptide according to any one of claims 1 to 10, wherein the isolated bound polypeptide has human FcRn binding affinity and, optionally, rat FcRn binding affinity.

12. The isolated conjugated polypeptide according to any one of claims 1 to 11, wherein the isolated conjugated polypeptide specifically binds to one or more human targets.

13. The isolated conjugated polypeptide according to any one of claims 1 to 12, wherein the isolated conjugated polypeptide is a monoclonal antibody.

14. The isolated conjugated polypeptide according to claim 13, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

15. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an isolated bound polypeptide according to any one of claims 1 to 14.

16. An expression vector comprising an isolated nucleic acid molecule as described in claim 15.

17. A host cell comprising the expression vector according to claim 16.

18. The host cell according to claim 17, wherein the host cell is a mammalian cell.

19. A step of culturing the host cells according to claim 17 or 18 under conditions that enable the expression of the bound polypeptide, and Steps to separate the bound polypeptide from the culture. A method for producing isolated bound polypeptides containing [a specific substance].

20. A pharmaceutical composition comprising an isolated bound polypeptide according to any one of claims 1 to 14, and a pharmaceutically acceptable carrier.

21. A method for treating a disease or disorder in a subject requiring such treatment, comprising administering to the subject a therapeutically effective amount of an isolated bound polypeptide according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 20.

22. A method for treating cancer in a subject requiring such treatment, comprising administering to the subject a therapeutically effective amount of an isolated bound polypeptide according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 20.

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