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

Abstract

The present invention relates to Fc variants having enhanced binding to FcRn and extended half-life. In particular, the disclosure provides binding polypeptides (e.g., antibodies and immunoadhesins) comprising a modified Fc domain. The disclosure also provides nucleic acids encoding the binding polypeptides, recombinant expression vectors and host cells for making such binding polypeptides. Also provided are methods of treating disease using the binding polypeptides disclosed herein.

Description

[0001] This application is a divisional application of the invention application with a filing date of January 25, 2019, Chinese application number 201980015900.4, and invention name “Fc variants with enhanced binding to FcRn and extended half-life”.

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 622,468, filed on January 26, 2018, which is incorporated herein by reference. Background Art

[0004] The interaction of antibodies with the neonatal Fc receptor (FcRn) is a determinant of the maintenance and extension of the serum half-life of antibodies and other Fc-derived therapeutics. FcRn is a heterodimer of the class I MHC-like α-domain and the β2-macroglobulin (β2-m) subunit that recognizes a region on the antibody Fc heavy chain that is distinct from other Fcγ receptors (FcγRs). Although FcRn is expressed in a variety of tissues, it is thought to function primarily in the vascular endothelium, in the kidneys, and at the blood-brain barrier, where it serves to prevent IgG degradation, excretion, and initiation of an inflammatory response, respectively.

[0005] Antibody binding to FcRn is highly pH dependent, and the interaction occurs with high affinity (high nanomolar to low micromolar) only at low pH (pH < 6.5), but not at physiological pH (pH about 7.4). Upon acidification of endosomes to a pH less than 6.5, the interaction between IgG and FcRn becomes highly favorable and is directly responsible for inhibiting the degradation of FcRn-bound antibodies and promoting the recycling of FcRn-bound antibodies to the cell surface. An increase in pH weakens the interaction and promotes antibody release into the bloodstream.

[0006] Fc engineering using high-throughput mutagenesis methods has been widely used to identify variants that enhance FcRn binding affinity, as enhanced binding will likely lead to increased efficacy and reduced dosing frequency of therapeutic antibodies, a direct result of increased serum half-life compared to wild-type IgG antibodies. However, variants that enhance FcRn binding affinity can have unpredictable results. For example, certain IgG variants that show a substantial increase in FcRn affinity at pH 6.0, such as N434W or P257I / Q311I, etc., have wild-type or severely reduced serum half-lives in cynomolgus monkeys and human FcRn (hFcRn) transgenic mouse studies (see, e.g., Kuo et al. 2011 supra; Datta-Mannan et al. 2007, J. Biol. Chem. 282: 1709-1717; and Datta-Mannan et al. 2007, Metab. Dispos. 35: 86-94). The T250Q / M428L (QL) variant has shown IgG backbone-specific results in animal models (see, e.g., Datta-Mannan et al. 2007, J. Biol. Chem. 282: 1709-1717; and Hinton et al. 2006, J. Immunol. 176: 346-356). The M252Y / S254T / T256E (YTE, EU numbering) variant has shown a 10-fold enhancement in vitro, but exhibits reduced antibody-dependent cell-mediated cytotoxicity (ADCC) in vivo due to a 2-fold reduction in affinity for the FcγRIIIa receptor (see, e.g., Dall'Acqua et al. 2002 supra).

[0007] Therefore, there remains a need for alternative Fc variants with enhanced binding to FcRn and prolonged circulation half-life. Summary of the invention

[0008] The present invention is based on the discovery of novel IgG antibodies having 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.

[0009] Thus, in certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain comprising aspartic acid (D) or glutamic acid (E) at amino acid position 256, and / or tryptophan (W) or glutamine (Q) at amino acid position 307, wherein amino acid position 254 is not threonine (T), and further comprising phenylalanine (F) or tyrosine (Y) at amino acid position 434 or tyrosine (Y) at amino acid position 252, wherein the amino acid positions are numbered according to EU.

[0010] 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.

[0011] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0012] 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.

[0013] In certain exemplary embodiments, the separated binding polypeptide has an altered FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at an elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate.

[0014] 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.

[0015] In certain exemplary embodiments, the isolated binding polypeptide has an altered binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a reduced binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has an enhanced binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain.

[0016] In certain exemplary embodiments, the isolated binding polypeptide has about the same binding affinity for FcγRIIIa as a binding polypeptide comprising a wild-type Fc domain.

[0017] 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 having the triple amino acid substitutions M252Y / S254T / T256E according to EU numbering.

[0018] In certain exemplary embodiments, the separated binding polypeptide is an antibody, such as a monoclonal antibody. In certain exemplary embodiments, the separated antibody is a chimeric antibody, a humanized antibody or a human antibody. In certain exemplary embodiments, the separated antibody is a full-length antibody.

[0019] In certain exemplary embodiments, the isolated binding polypeptide specifically binds one or more human targets.

[0020] In other aspects, an isolated binding polypeptide is provided comprising a modified Fc domain comprising a combination of amino acid substitutions at positions selected from the group consisting of: a) a tyrosine (Y) at amino acid position 252 and an aspartic acid (D) at amino acid position 256, b) an aspartic acid (D) at amino acid position 256 and a phenylalanine (F) at amino acid position 434, c) an aspartic acid (D) at amino acid position 256 and a tyrosine (Y) at amino acid position 434, d) a tryptophan (W) at amino acid position 307 and a phenylalanine (F) at amino acid position 434, e) a tyrosine (Y) at amino acid position 252 and a tryptophan (W) at amino acid position 307, wherein the tyrosine (Y) is not at amino acid position 434, f) an aspartic acid (D) at amino acid position 256 and an amino acid position 307, 7, wherein a tyrosine (Y) is not at amino acid position 434, g) an aspartic acid (D) at amino acid position 256 and a glutamine (Q) at amino acid position 307, wherein the tyrosine (Y) is not at amino acid position 434, h) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, and a glutamine (Q) at amino acid position 307, wherein the tyrosine (Y) is not at amino acid position 434, 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 a threonine (T) is not at amino acid position 254, a histidine (H) is not at amino acid position 311, and a tyrosine (Y) is not at amino acid position 434, wherein the amino acid substitutions are according to EU numbering.

[0021] 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.

[0022] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0023] 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.

[0024] In certain exemplary embodiments, the separated binding polypeptide has an altered FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at an elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate. In certain exemplary embodiments, the separated binding polypeptide has a smaller FcRn binding affinity at non-acidic pH than a binding polypeptide comprising a modified Fc domain with a double amino acid substitution M428L / N434S according to EU numbering.

[0025] 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.

[0026] In certain exemplary embodiments, the isolated binding polypeptide has an altered binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a reduced binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has an enhanced binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain.

[0027] In certain exemplary embodiments, the isolated binding polypeptide has about the same binding affinity for FcγRIIIa as a binding polypeptide comprising a wild-type Fc domain.

[0028] 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 having the triple amino acid substitutions M252Y / S254T / T256E according to EU numbering.

[0029] In certain exemplary embodiments, the separated binding polypeptide is an antibody, such as a monoclonal antibody. In certain exemplary embodiments, the separated antibody is a chimeric antibody, a humanized antibody or a human antibody. In certain exemplary embodiments, the separated antibody is a full-length antibody.

[0030] In certain exemplary embodiments, the isolated binding polypeptide specifically binds one or more human targets.

[0031] In other aspects, an isolated binding polypeptide comprising a modified Fc domain is provided, the modified Fc domain comprising 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 a threonine (T) is not at amino acid position 254, a histidine (H) is not at amino acid position 256, 311, and tyrosine (Y) is not at amino acid position 434, or b) a triple amino acid substitution selected from M252Y / T256D / T307Q, M252Y / T256D / T307W, M252Y / T256E / T307Q and M252Y / T256E / T307W, wherein threonine (T) is not at amino acid position 254, histidine (H) is not at amino acid position 311, and tyrosine (Y) is not at amino acid position 434, wherein the amino acid substitutions are according to EU numbering.

[0032] 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.

[0033] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0034] 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.

[0035] In certain exemplary embodiments, the separated binding polypeptide has an altered FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at an elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate. In certain exemplary embodiments, the separated binding polypeptide has a smaller FcRn binding affinity at non-acidic pH than a binding polypeptide comprising a modified Fc domain with a double amino acid substitution M428L / N434S according to EU numbering.

[0036] 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.

[0037] In certain exemplary embodiments, the isolated binding polypeptide has an altered binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a reduced binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has an enhanced binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain.

[0038] In certain exemplary embodiments, the isolated binding polypeptide has about the same binding affinity for FcγRIIIa as a binding polypeptide comprising a wild-type Fc domain.

[0039] 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 having the triple amino acid substitutions M252Y / S254T / T256E according to EU numbering.

[0040] In certain exemplary embodiments, the separated binding polypeptide is an antibody, such as a monoclonal antibody. In certain exemplary embodiments, the separated antibody is a chimeric antibody, a humanized antibody or a human antibody. In certain exemplary embodiments, the separated antibody is a full-length antibody.

[0041] In certain exemplary embodiments, the isolated binding polypeptide specifically binds one or more human targets.

[0042] In certain aspects, an isolated binding polypeptide comprising a modified Fc domain is provided, wherein the modified Fc domain comprises aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307 according to EU numbering.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at an elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate.

[0047] 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.

[0048] In certain exemplary embodiments, the isolated binding polypeptide has altered binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain.

[0049] 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.

[0050] In certain exemplary embodiments, the isolated binding polypeptide specifically binds one or more human targets.

[0051] In certain aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding the isolated polypeptide.

[0052] In some aspects, a vector comprising the isolated nucleic acid molecule is provided. In some exemplary embodiments, the vector is an expression vector. In some aspects, an expression vector comprising the isolated nucleic acid molecule is provided.

[0053] In some aspects, a host cell comprising the vector is provided. In some aspects, a host cell comprising the expression vector is provided.

[0054] 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.

[0055] In certain aspects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.

[0056] In certain aspects, a pharmaceutical composition comprising the isolated antibody is provided.

[0057] In certain aspects, an isolated binding polypeptide comprising a modified Fc domain is provided, 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at an elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate.

[0062] 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.

[0063] In certain exemplary embodiments, the isolated binding polypeptide has altered binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain.

[0064] 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.

[0065] In certain exemplary embodiments, the isolated binding polypeptide specifically binds one or more human targets.

[0066] In certain aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding the isolated polypeptide.

[0067] In some aspects, a vector comprising the isolated nucleic acid molecule is provided. In some exemplary embodiments, the vector is an expression vector. In some aspects, an expression vector comprising the isolated nucleic acid molecule is provided.

[0068] In some aspects, a host cell comprising the vector is provided. In some aspects, a host cell comprising the expression vector is provided.

[0069] 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.

[0070] In certain aspects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.

[0071] In certain aspects, a pharmaceutical composition comprising the isolated antibody is provided.

[0072] In certain aspects, an isolated binding polypeptide comprising a modified Fc domain is provided, 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the separated binding polypeptide has an enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at an elevated non-acidic pH. In certain exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate.

[0077] 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.

[0078] In certain exemplary embodiments, the isolated binding polypeptide has altered binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain.

[0079] 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.

[0080] In certain exemplary embodiments, the isolated binding polypeptide specifically binds one or more human targets.

[0081] In certain aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding the isolated polypeptide.

[0082] In some aspects, a vector comprising the isolated nucleic acid molecule is provided. In some exemplary embodiments, the vector is an expression vector. In some aspects, an expression vector comprising the isolated nucleic acid molecule is provided.

[0083] In some aspects, a host cell comprising the vector is provided. In some aspects, a host cell comprising the expression vector is provided.

[0084] 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.

[0085] In certain aspects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.

[0086] In certain aspects, a pharmaceutical composition comprising the isolated antibody is provided.

[0087] In certain aspects, an isolated binding polypeptide comprising a modified Fc domain is provided, wherein the modified Fc domain comprises a combination of at least four amino acid substitutions comprising: aspartic acid (D) or glutamic acid (E) at amino acid position 256 and tryptophan (W) or glutamine (Q) at amino acid position 307, wherein amino acid position 254 is not threonine (T), and further comprises phenylalanine (F) or tyrosine (Y) at amino acid position 434; and tyrosine (Y) at amino acid position 252, wherein the amino acid positions are numbered according to EU.

[0088] In certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain having a combination of amino acid substitutions at positions selected from the group consisting of: a) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434; b) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434; c) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434; d) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434; or e) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434, wherein the amino acid substitutions are according to EU numbering.

[0089] In certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain comprising: a quadruple amino acid substitution 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.

[0090] 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.

[0091] 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 both human and rat FcRn binding affinity.

[0092] 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. In certain exemplary embodiments, the isolated binding polypeptide has an enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

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

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

[0095] In certain exemplary embodiments, the isolated binding polypeptide has an enhanced FcRn binding affinity at acidic pH and an 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 an enhanced FcRn binding affinity at acidic pH and an enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0096] In certain exemplary embodiments, the acidic pH is about 6.0. In certain exemplary embodiments, the non-acidic pH is about 7.4.

[0097] 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 reduced serum half-life compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a reduced serum half-life compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0098] In certain exemplary embodiments, the isolated binding polypeptide has an altered binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a reduced binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has a reduced binding affinity for FcγRIIIa compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0099] In certain exemplary embodiments, the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0100] In certain exemplary embodiments, the separated binding polypeptide is an antibody. In certain exemplary embodiments, the separated binding polypeptide is a monoclonal antibody. In certain exemplary embodiments, the separated antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain exemplary embodiments, the separated antibody is a full-length antibody.

[0101] In certain exemplary embodiments, the isolated binding polypeptide specifically binds one or more targets.

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

[0103] In certain aspects, a vector comprising the isolated nucleic acid molecule is provided.

[0104] In certain exemplary embodiments, the vector is an expression vector.

[0105] In certain aspects, a host cell comprising the vector is provided.

[0106] 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.

[0107] In certain aspects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.

[0108] In certain aspects, a pharmaceutical composition comprising the isolated antibody is provided.

[0109] In certain aspects, an isolated binding polypeptide is provided comprising 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.

[0110] In certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain comprising 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 according to EU numbering.

[0111] In certain aspects, an isolated binding polypeptide is provided comprising a modified Fc domain comprising 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 according to EU numbering.

[0112] In certain aspects, an isolated binding polypeptide is provided comprising 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 phenylalanine (F) at amino acid position 434 according to EU numbering.

[0113] In certain aspects, an isolated binding polypeptide is provided comprising 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.

[0114] 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.

[0115] In certain exemplary embodiments, the binding polypeptide has human FcRn binding affinity.

[0116] 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. In certain exemplary embodiments, the isolated binding polypeptide has a reduced serum half-life compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0117] In certain exemplary embodiments, the isolated binding polypeptide has an enhanced FcRn binding affinity at acidic pH and an 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 an enhanced FcRn binding affinity at acidic pH and an enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0118] In certain exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

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

[0120] In certain exemplary embodiments, the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising a wild-type Fc domain. In certain exemplary embodiments, the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0121] 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.

[0122] In certain exemplary embodiments, the isolated binding polypeptide specifically binds one or more targets.

[0123] In certain aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding the isolated polypeptide.

[0124] In certain aspects, an expression vector comprising the isolated nucleic acid molecule is provided.

[0125] In certain aspects, a host cell comprising the expression vector is provided.

[0126] In certain aspects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.

[0127] In certain aspects, a method of treating a disease or condition in a subject in need thereof is 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.

[0128] In certain exemplary embodiments, the disease or disorder is cancer. In certain exemplary embodiments, the cancer is a tumor.

[0129] In certain exemplary embodiments, the disease or disorder is an autoimmune disorder.

[0130] In certain aspects, a method of treating cancer in a subject in need thereof is 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.

[0131] In certain aspects, a method of treating an autoimmune disorder in a subject in need thereof is 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] 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.

[0133] Figure 1A to Figure 1B The structure of FcRn interacting with the IgG1 Fc region is depicted. Figure 1A Depicted is the interaction between hFcRn and IgG1 Fc (pdb:4n0u) showing one Fc monomer (dark grey ribbon), including glycosylation shown as a stick labeled “glycan” in complex with the α domain (grey) and β2-m (light grey) hFcRn subunits. Most antibody residues involved in the interaction with FcRn are located in the region adjacent to the C H 2-C H 3 interface (dash-dot line) in the loop directly adjacent and opposite to the glycosylation site. Figure 1B Depicts relative to Figure 1A Surface diagram of the IgG1 Fc crystal structure (pdb: 5d4q) rotated 75°. The FcRn binding interface contains C H 2 and C H3 domain. Saturation libraries were constructed at eleven positions shown as sticks, as shown: 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 (H310, H433, H435) responsible for pH dependence are clustered near the positions of interest and are shown in the figure.

[0134] FIG. 2A to FIG. 2D The Octet screening assay and results are depicted. Figure 2A The Octet screening assay is schematically presented. NiNTA biosensor captures histidine-tagged antigen and subsequently antibody variants for rat FcRn (rFcRn) binding kinetics. Figure 2B Depicted are rFcRn binding kinetics for wild type (solid line), T307A / E380A / N434A (AAA) variants (short dashes), LS (short dashes interspersed with a single dot), YTE (long dashes), H435A (long dashes interspersed with a single dot) and H310A / H435Q (long dashes interspersed with two dots) antibodies at pH 6.0, aligned with the start of the rFcRn association phase. H435A and H310A / H435Q variants show little to no FcRn binding. The YTE variant has the slowest FcRn dissociation rate detected in the Octet rFcRn binding assay. Figure 2C Graphically depicted mutant subsets obtained by Octet screening were normalized for FcRn binding kinetics at pH 6.0. Most mutants retained significant binding to rFcRn, but several mutants were similar to the analog control (dotted line), showing loss of all rFcRn binding (long dashed line, below the dotted line (analog)). Two variants (solid lines) had slower rFcRn dissociation rates than wild-type antibodies (thick long dashed line). Figure 2D Depicts a scatter plot analysis of the rFcRn off-rates of all point mutations, where rFcRn binding kinetics separated by residue position can be observed. Saturated variants fall into one of the following four rFcRn off-rate regimes: no binding (not shown), faster binding (black), wild-type-like binding (white), slower binding (grey). 18 mutants showed significantly slower off-rates from rFcRn than wild-type antibodies (black dot-dash lines).

[0135] Figure 3The Biacore kinetics of benchmark and wild-type variants and human and rat FcRn at pH 6.0 and pH 7.4 are depicted graphically. All FcRn binding curves of each human (first and third column) and rat (second and fourth column) FcRn for the wild-type (upper left), AAA variant (upper right), M428 / N434S (LS) variant (lower left) and M252Y / S254T / T256E (YTE) variant (lower right) of a series of concentrations are shown at pH 6.0 (first and third row) and pH 7.4 (second and fourth row). AAA, LS and YTE variants demonstrate a slower dissociation rate from FcRn than wild-type antibodies. In general, compared with wild-type, the antibody binds rFcRn with an affinity increase of about 10 times. The LS variant had the tightest affinity for hFcRn at pH 7.4 and the greatest residue binding at pH 7.4, whereas rFcRn bound the YTE variant most tightly.

[0136] Figure 4A The Biacore kinetics of the lead saturation variants with human and rat FcRn at pH 6.0 are graphically depicted. The FcRn binding kinetic traces of the concentration series of 18 lead saturation variants are shown. M252Y, T256D, T256E, N434F, N434P, N434Y, T307A, T307E, T307F, T307Q and T307W have slower dissociation rates from both human and rat FcRn. The remaining variants are specific only to rat FcRn.

[0137] Figure 4B Graphically depicted are the FcRn binding kinetics of WT, benchmark and lead single saturation variants to human FcRn at pH 6.0. FcRn binding sensorgrams of a range of concentrations of WT, LS, YTE and 18 saturation variants to human FcRn at pH 6.0. Single saturation variants used for combinatorial libraries are underlined and in bold.

[0138] FIG. 5A to FIG. 5D Data showing various variants with slower off-rates from both human and rat FcRn at pH 6.0 are depicted. Figure 5A and Figure 5B Biacore sensorgrams of various variants are depicted.

[0139] Figure 5A Depicted are the off-rates of human FcRn at pH 6.0 for the YTE variant (long dash interspersed with single dots), LS variant (long dash interspersed with two dots), wild-type (WT; dot-dash line), and lead saturation variant (lead; solid line of various shades). Figure 5A , depicted are normalized sensorgrams showing improved hFcRn dissociation rates compared to WT. Figure 5B Depicted are the off-rates of AAA variants (dashed line), LS variants (dash line interspersed with two dots), YTE variants (dash line interspersed with single dots), wild type (solid line), and lead saturation variants (dash lines of varying frequency and thickness) to rat FcRn at pH 6.0. For clarity, a representative injection of each of the 11 lead antibodies is shown. These lead single variants showed improved off-rate kinetics from human and rat FcRn compared to wild type. Figure 5C and Figure 5D Depicted are the association and dissociation rates of the lead saturated (white circles) and wild-type (black circles) antibody variants against human ( Figure 5C ) and rats ( Figure 5D ) Binding affinity graph for FcRn. Baseline variants are shown: AAA (diagonal line facing the lower right), LS (dashed line), and YTE (diagonal line facing the lower left). Despite the improvement in FcRn off-rate, most variants do not have tighter affinity for human or rat FcRn due to slower association kinetics. 11 variants have slower off-rates from both species FcRn.

[0140] FIG. 6A to FIG. 6D Depicted are data showing that combinations of lead saturation mutations further improve FcRn off-rate and binding affinity. Fig. 6A and Figure 6B Depicted are representative Biacore sensorgrams showing FcRn off-rates for human and rat FcRn, respectively. Fig. 6A Depicted are normalized sensorgrams of human FcRn for representative variants of single (dash line), double (light grey solid line), triple (grey solid line) and quadruple (black solid line) combination variants compared to wild type (dash-dot line) and LS variant (long dash line interspersed with two dots). Figure 6B Depicted are normalized sensorgrams of rat FcRn for representative variants of single (long dash interspersed with two dots), double (long dash interspersed with a single dot), triple (long dash) and quadruple (short dash) combination variants compared to wild type (dash dotted line) and YTE variant (solid line). Incorporation of multiple mutations reduced the off rate for FcRn and enhanced the binding affinity for FcRn to a greater extent than the benchmark variant. Figure 6C and Fig.6D Plotted showing association rate as a function of human ( Figure 6C ) or rats ( Fig.6D ) Combination saturation variants plotted as a function of off-rate to FcRn, revealing that most variants have enhanced binding to FcRn at pH 6.0 compared to the benchmark variant. The tightest binding variants for human and rat FcRn are quadruple and double combinations, respectively.

[0141] 7A to 7D Depicted are data showing that enhanced FcRn binding at pH 6.0 disrupts the pH dependence of the interaction. Fig. 7A and Figure 7B Depicted are the wild type (dotted line) and LS variant ( Fig. 7A , solid line) and the YTE variant ( Figure 7B Representative sensorgrams of Biacore FcRn binding kinetics of single (long dash interspersed with two dots), double (long dash interspersed with a single dot), triple (long dash) and quadruple (short dash) combination variants at pH 7.4 compared to (, solid line). Increasing the number of mutations that enhance FcRn binding results in greater residue binding at physiological pH, with most of the double, triple and quadruple variants showing robust binding to both FcRns. Figure 7C and Fig.7D All saturated variants at pH 7.4 are depicted for human ( Figure 7C ) or rats ( Fig.7D ) Homeostasis of FcRn As a function of binding affinity at pH 6.0. Figure 7C In , a comparison of the FcRn binding affinity of the residues at pH 7.4 and at pH 6.0 is shown. Lead combinations with improved FcRn binding properties occupy the lower left quadrant defined by the LS benchmark variants (diamonds). Fig.7D In the Figure, LS (diamonds) and YTE (triangles) variants were used as cutoffs for lead validation, respectively. These two variants had the tightest binding affinity at pH 6.0 and the greatest residue binding at pH 7.4 for human and rat FcRn, respectively. Figure 7C and Fig.7D In both figures, single (white circles), double (light grey circles), triple (dark grey circles) and quadruple (black circles) variants are shown as well as the YTE variant (triangles).

[0142] FIG. 8A to FIG. 8B Depicted are data obtained from FcRn affinity chromatography and differential scanning fluorimetry (DSF) of the benchmark variants. Fig. 8A Depicted are normalized elution curves for WT (solid black line), AAA (dash-dot line), LS (long dash line with two dots), YTE (long dash line with single dots), H435A (light grey solid line) and H310A / H435Q (AQ; dark grey solid line) variants. pH values ​​are shown at the top of the graph. Variants that are ineffective for FcRn binding (H435A, H310A / H435Q) do not bind to the column and elute in the effluent (<10 mL). AAA, LS and YTE variants elute at a higher pH than the WT antibody. Figure 8BDSF curves of WT (black), LS (grey) and YTE (dark grey) variants are depicted. YTE is less stable compared to WT and LS.

[0143] Figure 8C Depicted are the FcRn affinity column elution profiles for the seven lead single variants for the combinatorial variants compared to the WT and LS variants (vertical dashed lines). Two variants (N434F / Y) eluted at a higher pH than LS, suggesting a reduced pH dependence of the interaction with FcRn for the variants containing these mutations.

[0144] 9A to 9D Depicted are data showing that combinatorial variants significantly perturb pH dependence and thermal stability. Fig. 9A Representative FcRn affinity chromatograms of single (long dash interspersed with two dots), double (long dash interspersed with a single dot), triple (long dash) and quadruple variants (short dash) are depicted. Increasing the number of mutations that enhance FcRn binding shifts elution to higher pH values; LS variant (small vertical dashed line). Fig. 9B Depicted are box plots of elution pH for lead saturation and combinatorial variants, including single (white circles), double (horizontal lines), triple (vertical lines), and quadruple (squares) mutants, indicating a trend toward higher pH values ​​as the number of FcRn enhancing mutants increases. Fig. 9C A high correlation was shown between the elution pH from FcRn affinity chromatography and the hFcRn off-rate using Biacore (R 2 =0.94) reveals a loss of pH dependence of the antibody-FcRn interaction, which is accompanied by improved FcRn dissociation kinetics. The AAA (diagonal line facing the lower right), LS (dotted line), and YTE (diagonal line facing the lower left) variants have hFcRn dissociation rates and elution pH values ​​similar to the dual variant. Fig.9D The T obtained from the DSF of the combined saturated variants is depicted m Box plots reveal that additional FcRn binding enhancing mutations destabilize the antibody compared to WT, single or benchmark variants.

[0145] FIG. 10A to FIG. 10B Data obtained from FcRn affinity chromatography and DSF of seven lead variants are depicted. Fig. 10AFcRn affinity chromatography of M252Y (solid line), T256D (short dash line interspersed with a single dot), T256E (long dash line), T307Q (long dash line interspersed with a single dot), T307W (long dash line interspersed with two dots), N434F (dashed line) and N434Y (short dash line) variants is depicted. The chromatograms reveal an altered elution pH compared to the wild-type and LS antibodies (vertical dashed lines). N434F and N434Y have a higher elution pH (pH about 8.3) than the LS variants (vertical dashed lines). The pH at certain elution volumes is shown above the chromatograms for reference. Fig. 10B DSF curves for the 7 lead variants are depicted, showing that none of the 7 lead single variants destabilized the antibody to the same extent as the YTE variant (vertical dashed line). All variants except T307Q (long dashed line interspersed with single dots) were destabilized compared to WT (vertical dot-dash line).

[0146] FIG. 11A to FIG. 11B Depicted are data showing reduced FcγRIIIa binding in combination variants containing M252Y. Fig.11A FcγRIIIa binding sensorgrams of WT (black), LS (grey) and YTE (dark grey) variants are shown, revealing a reduced binding response by the YTE variant. Fig. 11B Box plots of FcγRIIIa binding responses of the baseline, single and combination variants as indicated are depicted. Variants with the M252Y mutation contained reduced binding responses to FcγRIIIa, including all quadruple variants. Combinations with N434F / Y generally showed enhanced responses to FcγRIIIa.

[0147] Fig. 11C Depicted are the FcγRIIIa binding responses of the seven lead single variants compared to WT and the YTE variant (dashed horizontal line). The M252Y mutation showed reduced FcγRIIIa binding compared to WT, while six variants showed WT-like or enhanced binding to the receptor.

[0148] FIG. 12A to FIG. 12D Depicted are data obtained from FcRn affinity chromatography, DSF, and FcγRIIIa binding of seven lead combinatorial variants. Fig. 12A Depicted are FcRn affinity chromatograms of 7 lead combinatorial variants compared to wild-type antibody and LS variants (vertical dashed and vertical solid lines, respectively). The elution pH of each lead variant is close to that of the LS variant. Fig. 12B DSF curves of lead combinatorial variants compared to YTE and wild-type variants (shown as vertical dashed lines) are shown. Six of the seven lead variants had similar or more unstable T values ​​than the YTE variant. m: MDWN (long dash line with two dots); YTWN (long dash line); YDTN (solid line); YETN (long dash line with a single dot); YDQN (dashed line); YEQN (short dash line with a single dot). DQ The N variant has a similar T m . Fig. 12C Depicted are Biacore sensorgrams of FcγRIIIa binding kinetics of seven lead variants compared to wild type (larger dashed line) and YTE variant (bold, long dashed line). Variants containing M252Y: YD TN (solid line), YQ N (a dash with a single dot), Y T W N (long dash), YE TN (long dash with a single dot) and YE QN (smaller dotted line), each of which has a reduced steady-state RU in a similar manner to YTE. Fig.12D Steady-state RU of 7 lead variants, wild type, and YTE variant are shown. DW N and M DQ The N variant has similar affinity for FcγRIIIa as the wild-type antibody.

[0149] FIG. 12E to FIG. 12H Depicted are data showing that 3 lead variants exhibit a range of key antibody attributes. Fig.12E FcRn affinity chromatography elution curves of DQ (solid line), DW (dashed line) and YD (strike line) variants are shown compared to WT and LS (vertical dashed lines). Each double variant shows an elution pH between WT and LS. Fig.12F DSF fluorescence curves of the three variants compared to the YTE and WT variants (vertical dash-dot lines) are plotted, revealing that YD (dashed line) and DW (dash-dot line) are slightly less stable compared to YTE, but DQ (solid line) is similar to WT. Figure 12G FcγRIIIa binding sensorgrams compared to WT and YTE (dashed horizontal lines) are depicted. YD (dashed lines) shows a similar binding response to YTE, while DQ (solid lines) and DW (dashed lines) show a slight decrease compared to WT. Fig.12H Depicted are data showing that homologous bridging RF ELISA revealed that the 3 lead variants and YTE exhibited significantly reduced or WT-like RF binding distinct from LS. **p<0.001, *p<0.01.

[0150] FIG. 13A to FIG. 13D Depicted are data showing comparison of FcRn binding kinetics of lead combinatorial variants at pH 6.0 and pH 7.4. Fig.13A and Fig. 13BShown are the comparisons with wild type (dashed line) and LS (hFcRn, Fig.13A , thick long dash) or YTE (rFcRn, Fig. 13B , thick long dash) compared to the lead combinatorial variants for human FcRn ( Fig.13A ) or rat FcRn( Fig. 13B ). Despite altered association and dissociation rates, each combinatorial variant had an overall tighter binding affinity to the corresponding FcRn. Fig. 13C and Fig.13D Biacore FcRn sensorgrams at pH 7.4 are shown. Each hFcRn lead variant had similar or decreased steady-state FcRn binding responses compared to the LS variant. DQ N and M DW The N variant showed less rFcRn binding than the YTE variant at pH 7.4.

[0151] Fig.14 is a table depicting Octet rFcRn binding dissociation rates for saturated libraries according to certain embodiments. Wild-type (WT) and wild-type-like (WT-like) species are represented by white rectangles; WT species are shown. Variants with little or no rFcRn binding compared to wild-type are represented by dark gray rectangles. Variants with faster rFcRn dissociation rates compared to wild-type are represented by light gray rectangles, and variants with slower rFcRn dissociation rates compared to wild-type are represented by black rectangles.

[0152] FIG. 15A to FIG. 15C Depicted is a new binding assay developed using the CM5 sensor chip. Fig.15A is a schematic diagram of the assay. Fig. 15B Direct immobilization of FcRn is shown. Fig. 15C Streptavidin capture of biotinylated FcRn is shown.

[0153] FIG. 16A to FIG. 16B FcRn binding of Antibody 2 at pH 6.0 is depicted. Fig.16A Human FcRn is depicted. Fig. 16B Mouse FcRn is depicted.

[0154] FIG. 17A to FIG. 17B FcRn binding of Antibody 2 at pH 7.4 is depicted. Fig.17A Human FcRn is depicted. Fig. 17B Mouse FcRn is depicted.

[0155] Fig.18The pH dependence of various Antibody 2 variants is graphically depicted. The lead variant maintains higher binding affinity at pH 6 and lower residue binding than LS at pH 7.4.

[0156] Fig.19 A comparison of the pH dependence of FcRn binding using the backbones of Antibody 1 and Antibody 2 is depicted.

[0157] Fig. 20 A comparison of thermal stability using the backbones of Antibody 1 and Antibody 2 is depicted.

[0158] Fig.21 A comparison of FcγRIIIa binding using the scaffolds of Antibody 1 and Antibody 2 is depicted.

[0159] Fig. 22 Depicted are multiple figures showing that the DQ, DW and YD variants can be transferred between IgG1 backbones. a to c depict normalized FcRn binding sensorgrams at pH 6.0 in three IgG1 backbones, where WT (light gray), LS (dark gray), DQ (solid black), DW (dashed line) and YD (dash line) variants show similar kinetics at low pH. The three variants, DQ, DW and YD, have slightly faster association and dissociation rates than the LS variant, but maintain tighter FcRn binding affinity. d to f depict FcRn binding sensorgrams at pH 7.4; LS benchmark variant (solid black). g to i depict comparisons of FcRn binding reactions at pH 7.4 with binding affinity at pH 6.0 for each antibody backbone with WT (gray), LS (dark gray), DQ (solid black), DW (open circles) and YD (open squares) variants. DQ, DW and YD showed improved FcRn characteristics with enhanced binding at pH 6.0 and minimal binding at pH 7.4.

[0160] FIG. 23A to FIG. 23C The three lead variants in the mAb2 backbone were shown to similarly improve binding to cynomolgus monkey FcRn. Fig.23A Depicted are normalized cFcRn binding sensorgrams of WT (grey), LS (dark grey), DQ (solid black), DW (dashed line) and YD (strike line) at pH 6.0, showing similar binding kinetics and affinity to hFcRn. Fig. 23B The cFcRn binding responses of the three variants are depicted to be significantly reduced at physiological pH; however LS (dark grey) shows stronger binding than WT (grey) in a similar manner to hFcRn. Fig.23CDepicted is the comparison of the residue cFcRn binding responses at pH 7.4 and cFcRn binding affinity at pH 6.0 for WT (grey), LS (dark grey), DQ (solid black), DW (open circles) and YD (open squares), revealing that all three variants maintain the improved FcRn binding properties observed for hFcRn.

[0161] FIG. 24A to FIG. 24B The lead variants were shown to extend the serum half-life of the antibody. Fig.24A ) and hFcRn transgenic mice ( Fig. 24B ) for WT (black circles with solid black lines), LS (white circles with black dashed lines), DQ (light gray circles with solid light gray lines), DW (dark gray circles with solid dark gray lines) and YD (black circles with dashed black lines) antibodies, pharmacokinetic curves of plasma antibody concentration as a function of time. All three lead variants extended antibody half-life compared to WT.

[0162] Fig.25 Describe the figure of the steady-state RU of all saturated variants for human FcRn as the function of binding affinity at pH 6.0 at pH 7.4.Show the comparison of residue FcRn binding affinity at pH 7.4 with FcRn at pH 6.0.The quadruple combination with improved FcRn binding properties at pH 6.0 and pH 7.4 is shown in the upper right quadrant of the figure with a square frame.Single (white circle), double (light gray circle), triple (dark gray circle) and quadruple (black circle) variants and benchmark AAA, LS and YTE variants (as shown in the figure) are shown.

[0163] Fig.26 Depicted is a schematic diagram of the BiotinCAPture method for capturing biotinylated FcRn.

[0164] Fig. 27 Depicted are graphs showing human FcRn binding kinetics at pH 6.0 for YTEKF benchmark and combinatorial variants as indicated.

[0165] FIG. 28A to FIG. 28B It is shown that at pH 6.0 ( Fig.28A ) and pH 7.4 ( Fig.28B ) FcRn binding kinetics of the combined variants compared to the YTEKF benchmark. The wild type is represented by a solid black line (WT) and the YTEKF benchmark is represented by a dashed line.

[0166] Fig.29Depicted is a graph of steady-state RU of selected variants for human FcRn at pH 7.4 as a function of binding affinity at pH 6.0 compared to the YTEKF benchmark. Several variants (lead quartet variants) showed enhanced binding affinity to human FcRn at both pH 6.0 and pH 7.4 relative to the YTEKF benchmark. DETAILED DESCRIPTION

[0167] The present disclosure provides binding polypeptides (e.g., antibodies) with altered Fc neo-receptor (FcRn) binding affinity. In certain embodiments, the binding polypeptide comprises a modified Fc domain, and the modified Fc domain enhances FcRn binding affinity compared to a binding polypeptide comprising a wild-type (e.g., non-modified) Fc domain. The present disclosure also provides nucleic acids encoding binding polypeptides, recombinant expression vectors, and host cells for preparing binding polypeptides, as well as pharmaceutical compositions comprising the binding polypeptides disclosed herein. Methods for treating diseases using the binding polypeptides disclosed herein are also provided.

[0168] 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 (e.g., binding of FcRn). Figure 1A As shown, the Fc domain contains a CH2 domain and a CH3 domain. Most of the residues involved in the interaction with FcRn are located at the C H 2-C H 3 Interface( Figure 1A , dashed line) in the loop directly adjacent to the glycosylation site. Figure 1B A surface schematic of the IgG1 Fc crystal structure (pdb: 5d4q) is shown, and the residues in the CH2 and CH3 domains that comprise the FcRn binding interface are shown. The present disclosure provides binding polypeptides comprising a modified Fc domain. The binding polypeptide comprising a modified Fc domain may be an antibody or an immunoadhesin or an Fc fusion protein.

[0169] In certain embodiments, the binding polypeptide may comprise a modified Fc domain comprising amino acid substitutions that change the antigen-independent effector function of the antibody, in particular, change the circulating half-life (e.g., serum half-life) of the binding polypeptide. In some embodiments, the binding polypeptide may comprise a modified Fc domain comprising amino acid substitutions that change 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, the binding polypeptide may comprise a modified 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, the binding polypeptide may comprise a modified Fc domain comprising amino acid substitutions that reduce the serum half-life of the binding polypeptide compared to a binding polypeptide comprising a wild-type (i.e., unmodified) Fc domain.

[0170] In certain embodiments, the binding polypeptide comprising a modified Fc domain that changes (i.e., increases or decreases) circulating half-life (e.g., serum half-life) further comprises one or more mutations in addition to one or more mutations that change circulating half-life. In certain embodiments, one or more mutations in addition to one or more mutations that change circulating half-life provide one or more desired biochemical characteristics, such as reduced or enhanced effector function, non-covalent dimerization ability, enhanced ability to localize at tumor sites, reduced serum half-life, increased serum half-life, etc., when compared to an intact, unaltered antibody with approximately the same immunogenicity.

[0171] When compared to binding polypeptides lacking these substitutions, the binding polypeptides described herein may show increased or decreased binding to neonatal Fc receptors (FcRn), and therefore have increased or decreased serum half-lives, respectively. Fc domains with improved FcRn affinity are expected to have longer serum half-lives, and such molecules have useful applications in methods for treating mammals, where the administered antibodies are required to have long half-lives, for example, to treat chronic diseases or conditions. On the contrary, Fc domains with reduced FcRn binding affinity are expected to have shorter serum half-lives, and such molecules may also be used, for example, for administration to mammals, where shortened circulation times may be advantageous, for example, for in vivo diagnostic imaging or when the initial antibody is present in the circulation for a long time with toxic side effects. Fc domains with reduced FcRn binding affinity are also less likely to cross the placenta, and therefore may also be used to treat diseases or conditions in pregnant women. In addition, other applications that may require reduced FcRn binding affinity include applications limited to the brain, kidneys and / or liver.

[0172] It is to be understood that the methods described in this disclosure are not limited to the specific methods and experimental conditions disclosed herein, as these methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0173] In addition, unless otherwise stated, the experiments described herein use conventional molecular and cell biology and immunological techniques within the technical scope of the art. Such techniques are well known to the technical staff and are fully explained in the literature. See, for example, Ausubel et al., editors, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including all supplements, MR Green and J. Sambrook edited Molecular Cloning: A Laboratory Manual (4th edition), and Harlow et al. Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0174] Unless otherwise defined, the scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. If there is any possible ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Unless otherwise specified, the use of "or" means "and / or". The use of the term "includes" and other forms such as "includes ("includes" and "included")" is not restrictive.

[0175] Generally, the terminology used for cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein is well known in the art and commonly used. The method and technology provided herein are usually carried out according to the conventional methods described in various general and more specific references as well known in the art and quoted and discussed in this specification, unless otherwise indicated. Enzymatic reaction and purification techniques are usually realized or carried out as described herein according to the manufacturer's instructions as in this area. The terminology and laboratory procedures and techniques for analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry as described herein are those terms, laboratory procedures and techniques well known in the art and commonly used. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, preparation and delivery and the treatment of patients.

[0176] In order that the present disclosure may be more readily understood, selected terms are defined below.

[0177] The term "polypeptide" refers to any polymer chain of amino acids and encompasses natural or artificial proteins, polypeptide analogs or variants of protein sequences or fragments thereof, unless the context otherwise contradicts. A polypeptide can be a monomer or a polymer. For example, a polypeptide fragment comprises at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, or at least about 20 consecutive amino acids.

[0178] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, by virtue of its origin or source of derivation, is not associated with naturally associated components with which it naturally accompanies it; is substantially free of other proteins from the same species; is expressed by cells from a different species; or is not found in nature. Thus, a protein or polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein or polypeptide may also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

[0179] As used herein, the term "binding protein" or "binding polypeptide" shall refer to a protein or polypeptide (e.g., an antibody or immunoadhesin) containing 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 antibody variable domains, ligand binding sites of receptors, or receptor binding sites of ligands. In some aspects, the binding protein or binding polypeptide includes multiple (e.g., two, three, four or more) binding sites. In some aspects, the binding protein or binding polypeptide is not a therapeutic enzyme.

[0180] The term "ligand" refers to any substance that can bind or be bound to another substance. Similarly, the term "antigen" refers to any substance that can produce antibodies. Although "antigen" is usually used to refer to antibody binding substrates, and "ligand" is often used when referring to receptor binding substrates, these terms are not distinguished from each other and cover a wide range of overlapping chemical entities. For the avoidance of doubt, antigen and ligand are used interchangeably in this article. Antigen / ligand can be a peptide, polypeptide, protein, aptamer, polysaccharide, sugar molecule, carbohydrate, lipid, oligonucleotide, polynucleotide, synthetic molecule, inorganic molecule, organic molecule and any combination thereof.

[0181] As used herein, the term "specifically binds" refers to an antibody or immunoadhesin that specifically binds to at most about 1 x 10 -6 M, about 1x10 -7 M, about 1x 10 -8 M, about 1x 10 -9 M, about 1x 10 -10 M, about 1x 10-11 M, about 1x 10 -12 The ability to bind to an antigen with a dissociation constant (Kd) of M or less, and / or the ability to bind to an antigen with an affinity that is at least about 2-fold greater than the affinity for a nonspecific antigen.

[0182] As used herein, the term "antibody" refers to an assembly (e.g., a complete antibody molecule, an immunoadhesin, or a variant thereof) that has a significant known specific immunoreactivity to an antigen of interest (e.g., a tumor-associated antigen). Antibodies and immunoglobulins comprise light chains and heavy chains with or without interchain covalent linkages between the light and heavy chains. The basic immunoglobulin structure in vertebrate systems is relatively clear.

[0183] As will be discussed in more detail below, the general term "antibody" includes five different classes of antibodies that can be distinguished biochemically. Although all five classes of antibodies are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With respect to IgG, immunoglobulins comprise 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. These four chains are connected via disulfide bonds in a "Y" configuration, with the light chain range starting at the mouth of the "Y" next to the heavy chain and continuing straight to the end of the variable region.

[0184] The light chain of immunoglobulin is classified as kappa (κ) or lambda (λ). Each heavy chain category can be combined with a κ or λ light chain. Generally, when immunoglobulin is generated by a hybridoma, a B cell or a genetically engineered host cell, the light chain and the heavy chain are covalently bonded to each other, and the "tail" parts of the two heavy chains are bonded to each other via covalent disulfide linkages or non-covalent linkages. In the heavy chain, the amino acid sequence extends from the N-terminus of the bifurcated end of the Y configuration to the C-terminus at the bottom of each chain. It will be understood by those skilled in the art that the heavy chain is classified as γ (γ), μ (μ), α (α), δ (δ) or ε (ε), with some subclasses (e.g., γl-γ4). The properties of the chain determine the "class" of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. The immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are fully characterized and known to confer functional specialization. Modified forms of each of these classes and isotypes are readily discernible to the skilled artisan upon timely disclosure and, therefore, are within the scope of the present disclosure.

[0185] Both the light chain and the heavy chain are divided into regions of structural and functional homology. The term "region" refers to a portion of an immunoglobulin or antibody chain ("part" or "portion"), and includes a constant region or a variable region, as well as more discrete fragments or portions of the region. For example, a light chain variable region includes "complementarity determining regions" or "CDRs" interspersed between "framework regions" or "FRs" as defined herein.

[0186] Regions of an immunoglobulin heavy or light chain can be defined as a "constant" (C) region or a "variable" (V) region, based on the relative lack of sequence variation within the region among members of multiple classes in the case of a "constant region," or based on the significant variation within the region among members of multiple classes in the case of a "variable region." The terms "constant region" and "variable region" can also be used with respect to function. In this regard, it will be understood that the variable region of an immunoglobulin or antibody determines antigen recognition and specificity. In contrast, the constant region of an immunoglobulin or antibody confers important effector functions, such as secretion, transplacental movement, Fc receptor binding, complement binding, and the like. The subunit structure and three-dimensional configuration of the constant regions of the various immunoglobulin classes are well known.

[0187] The constant region and variable region of immunoglobulin heavy chain and light chain are folded into domains. The term "domain" refers to the globular region of a heavy chain or light chain, which contains peptide loops (e.g., 3 to 4 peptide loops) stabilized, for example, via β-pleated sheets and / or intrachain disulfide bonds. The constant region on the light chain of an immunoglobulin is interchangeably referred to as a "light chain constant region domain," "CL region," or "CL domain." The constant domains on the heavy chain (e.g., hinge, CH1, CH2, or CH3 domains) are interchangeably referred to as "heavy chain constant region domains," "CH" region domains, or "CH domains." The variable domains on the light chain are interchangeably referred to as "light chain variable region domains," "VL region domains," or "VL domains." The variable domains on the heavy chain are interchangeably referred to as "heavy chain variable region domains," "VH region domains," or "VH domains."

[0188] By convention, the amino acid numbering of the variable constant region domains increases as they are away from the antigen binding site or amino terminus of the immunoglobulin or antibody. The N-terminus of each heavy and light chain immunoglobulin chain is the variable region, and the C-terminus is the constant region. The CH3 and CL domains contain the carboxyl termini of the heavy and light chains, respectively. Thus, the domains of the light chain immunoglobulins are arranged in a VL-CL orientation, while the domains of the heavy chains are arranged in a VH-CH1-hinge-CH2-CH3 orientation.

[0189] The amino acid assignments for each variable region domain are in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chain variable regions or between different light chain variable regions are assigned the same number. CDRs 1, 2, and 3 of the VL domain are also referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively. CDRs 1, 2, and 3 of the VH domain are also referred to herein as CDR-H1, CDR-H2, and CDR-H3, respectively. If so indicated, the assignment of CDRs may be in accordance with (Lefranc et al., Developmental & Comparative Immunology 27:55-77; 2003), not Kabat. The numbering of the heavy chain constant region is by the EU index as described in Kabat (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991).

[0190] As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" includes the amino terminal variable domain of an immunoglobulin light chain.

[0191] As used herein, the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain, which extends, for example, from about positions 114-223 in the Kabat numbering system (EU positions 118-215). The CH1 domain is adjacent to the amino-terminal end of the VH domain and the hinge region of the immunoglobulin heavy chain molecule and does not constitute a part of the Fc region of the immunoglobulin heavy chain.

[0192] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains about 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 different domains: the upper, middle and lower hinge domains (Roux et al. J. Immunol. 1998, 161: 4083).

[0193] As used herein, the term "CH2 domain" includes such a portion of a heavy chain immunoglobulin molecule that, for example, extends from about position 244-360 in the Kabat numbering system (EU position 231-340). The CH2 domain is unique because it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of a complete native IgG molecule. In one embodiment, the binding polypeptide of the present disclosure comprises a CH2 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0194] As used herein, the term "CH3 domain" includes such a portion of a heavy chain immunoglobulin molecule that extends from the N-terminus of the CH2 domain, for example, from about position 361-476 of the Kabat numbering system (EU position 341-445) for about 110 residues. The CH3 domain typically forms the C-terminal portion of the antibody. However, in some immunoglobulins, additional domains may extend from the CH3 domain to 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, the binding polypeptide of the present disclosure comprises a CH3 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0195] As used herein, the term "CL domain" includes the constant region domain of an immunoglobulin light chain, which, for example, extends 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 present disclosure comprises a CL domain derived from a kappa light chain (e.g., a human kappa light chain).

[0196] As used herein, the term "Fc region" is defined as the portion of the heavy chain constant region that begins at the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, taking 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 comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

[0197] As used herein, the term "native Fc" or "wild-type Fc" refers to a molecule comprising a sequence of a non-antigen binding fragment, the sequence of which is a monomer or polymer form, obtained by antibody digestion or produced by other means; and the term may include a hinge region. The original immunoglobulin source of natural Fc is usually of human origin, and may be any immunoglobulin, such as IgG1 and IgG2. Natural Fc molecules consist of monomeric polypeptides, which can be linked into dimer or multimer forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between the monomer subunits of natural Fc molecules ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of a natural Fc is a disulfide-bonded dimer produced by papain digestion of IgG. As used herein, the term "native Fc" is universal for monomeric, dimer, and multimer forms.

[0198] As used herein, the term "Fc variant" or "modified Fc" refers to a molecule or sequence that is modified from a native / wild-type Fc but still contains a binding site for FcRn. Thus, the term "Fc variant" may include molecules or sequences that are humanized from a non-human native Fc. In addition, a native Fc contains regions that can be removed because they provide structural features or biological activities that are 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 modified, wherein the one or more Fc sites or residues affect or participate in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed 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).

[0199] In certain exemplary embodiments, the specific Fc variants 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.

[0200] As used herein, the term "Fc domain" encompasses native / wild-type Fc as defined above as well as Fc variants and sequences. Like Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from whole antibodies or produced by other means.

[0201] As mentioned above, the variable region of an antibody allows it to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL domain and the VH domain of an antibody combine to form a variable region (Fv) that defines a three-dimensional antigen binding site. The quaternary antibody structure forms an antigen binding site at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each heavy chain and light chain variable region. As used herein, the term "antigen binding site" includes a site that specifically binds (immunoreacts with an antigen) an antigen (e.g., a cell surface or soluble antigen). The antigen binding site includes 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 a variable region, which is different between antibodies. The modified antibody of the present disclosure comprises at least one antigen binding site.

[0202] In certain embodiments, the binding polypeptide of the present disclosure comprises at least two antigen binding domains that provide the binding polypeptide and the antigen of selection. Antigen binding domains do not need to be derived from the same immunoglobulin molecule. In this regard, the variable region may be derived from or derived from any type of animal, which can be induced to produce a humoral response and produce an immunoglobulin for the desired antigen. Therefore, the variable region of the binding polypeptide can be, for example, of mammalian origin, such as humans, rodents, rats, goats, sheep, non-human primates (such as cynomolgus monkeys, macaques, etc.), wolves or camelids (e.g., from camels, llamas and related species).

[0203] In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, non-continuous amino acid sequences that are specifically positioned to form an antigen binding site because the antibody is assumed to assume its three-dimensional configuration in an aqueous environment. The rest of the heavy and light chain variable domains show less intermolecular variability in the amino acid sequence and are referred to as framework regions. The framework region primarily adopts a β-sheet conformation, and the CDRs form loops that connect the β-sheet structure and in some cases form a portion of the β-sheet structure. Therefore, these framework regions serve to form a scaffold that provides positioning of the six CDRs in the correct direction through interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope.

[0204] Exemplary binding polypeptides include antibody variants. As used herein, the term "antibody variant" includes synthetic and engineered forms of antibodies that are altered so that they are not naturally occurring, such as antibodies that include at least two heavy chain portions but not two complete heavy chains (such as domain-deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that are altered to bind to two or more different antigens or to bind to different epitopes on a single antigen; heavy chain molecules connected to scFv molecules, etc. In addition, the term "antibody variant" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three, four or more copies of the same antigen).

[0205] 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 a target molecule or specific site on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site may specifically bind to the same or different molecules (e.g., may bind to different ligands or different antigens, or bind to different epitopes on the same antigen). The subject binding polypeptide typically has at least one binding site specific for a human antigen molecule.

[0206] The term "specificity" refers to the ability to specifically bind to (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 a 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) parts of the same target. In certain embodiments, a binding polypeptide is specific for more than one target. Exemplary binding polypeptides (e.g., antibodies) comprising an antigen binding site that binds to an antigen expressed on a tumor cell are known in the art, and one or more CDRs from such antibodies can be included in antibodies as described herein.

[0207] As used herein, the term "antigen" or "target antigen" refers to a molecule or a portion of a molecule that can be bound by a binding site of a binding polypeptide. A target antigen may have one or more epitopes.

[0208] The term "about" or "approximately" means within about 20%, such as within about 10%, within about 5%, or within about 1% or less of a given value or range.

[0209] As used herein, "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance present in vitro (e.g., an isolated binding polypeptide provided herein) into a patient, such as by, but not limited to, 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 controlling or treating a disease or its symptoms, administration of the substance typically occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, administration of the substance typically occurs before the onset of the disease or its symptoms, and may be continued for a long period of time to delay or reduce the appearance or extent of disease-related symptoms.

[0210] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients (e.g., an isolated binding polypeptide provided herein), optionally in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0211] "Effective amount" refers to 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 active agent. The effective amount may vary between individuals depending on the health and physical condition of the individual to be treated, the classification group of the individual to be treated, the formulation of the composition, the individual's medical condition assessment, and other relevant factors.

[0212] 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., cattle, 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, untamed animals, farm animals, sports animals, and pets.

[0213] As used herein, the term "therapy" refers to any regimen, method and / or agent that can be used to prevent, manage, treat and / or improve a disease or symptoms associated therewith. In some embodiments, the term "therapy" refers to any regimen, method and / or agent that can be used to regulate the immune response to infection or symptoms associated therewith in a subject. In some embodiments, the terms "multiple therapies" and "therapy" refer to biological therapies, supportive therapies and / or other therapies known to those skilled in the art such as medical personnel that can be used to prevent, manage and / or improve a disease or symptoms associated therewith. In other embodiments, the terms "multiple therapies" and "therapy" refer to biological therapies, supportive therapies and / or other therapies known to those skilled in the art such as medical personnel that can be used to regulate the immune response to infection or symptoms associated therewith in a subject.

[0214] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or improvement in the progression, severity, and / or duration of a disease or symptoms associated therewith caused by the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents such as the isolated binding polypeptides provided herein). As used herein, the term "treat" may also refer to altering the course of a disease in a treated subject. The therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating one or more symptoms, reducing the direct or indirect pathological consequences of a disease, reducing the rate of disease progression, improving or slowing the disease state, and alleviating or improving prognosis.

[0215] Binding peptide

[0216] 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. Any antibody from any source or species can be used in the binding polypeptides disclosed herein. Suitable antibodies include, but are not limited to, human antibodies, humanized antibodies, or chimeric antibodies. Suitable antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, full-length antibodies, or single-chain antibodies.

[0217] Fc domains from any immunoglobulin class (e.g., IgM, IgG, IgD, IgA, and IgE) and species can be used in binding polypeptides disclosed herein. Chimeric Fc domains comprising partial 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. In the case of Fc domains of other species and / or Ig classes or isotypes, it will be appreciated by the skilled artisan that any amino acid substitutions described herein can be adjusted accordingly. 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 according to EU numbering, and any combination thereof. 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 triple amino acid substitution at any three 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 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 any combination thereof, wherein amino acid position N434 is not substituted (i.e., amino acid position N434 is wild type).

[0218] 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 according to EU numbering 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 according to EU numbering 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 quadruple amino acid substitution according to EU numbering 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, it may be desirable for the modified Fc domain to comprise an amino acid substitution at any amino acid position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N or Y436W 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 at any amino acid position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N or Y436W 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 at any amino acid position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N or Y436W 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).

[0219] In certain embodiments, the modified Fc domain may include amino acid substitutions selected from M252, T256, T307 or N434 according to EU numbering, and any combination thereof. In certain embodiments, the modified Fc domain may include 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 include 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 include 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 amino acid substitutions 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).

[0220] In an exemplary embodiment, the modified Fc domain may comprise an amino acid substitution according to EU numbering selected from the group consisting of: 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 and any combination thereof. In certain embodiments, the modified Fc domain may comprise a double amino acid substitution at any two amino acid positions according to EU numbering selected from the group consisting of: 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. In certain embodiments, the modified Fc domain may comprise a triple amino acid substitution at any three amino acid positions according to EU numbering, selected from the group consisting of: 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. In certain embodiments, the modified Fc domain may comprise a quadruple amino acid substitution at the following amino acid positions according to EU numbering, selected from the group consisting of: 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. In some embodiments, it may be desirable that the modified Fc domain comprises 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 that the modified Fc domain comprises 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 that the modified Fc domain comprises 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).

[0221] 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 that the modified Fc domain comprises 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 M252Y, wherein amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, it may be desirable that the modified Fc domain comprises 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 M252Y, 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 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 (i.e., amino acid position N434 is wild type).

[0222] 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 may comprise a polypeptide selected from the group consisting of 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 / T256D / N434Y The triple amino acid substitutions are 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.

[0223] 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.

[0224] In some embodiments, it may be desirable that the modified Fc domain comprises a wild-type amino acid at amino acid position N434 according to EU numbering. In some embodiments, it may be desirable that the Fc domain does not comprise a phenylalanine (F) or a tyrosine (Y) at amino acid position N434 according to EU numbering. In some embodiments, it may be desirable that the Fc domain does not comprise a tyrosine (Y) at amino acid position N434 according to EU numbering. In some embodiments, 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 the group consisting of M252Y / T256D / T307Q, M252Y / T256D / T307W, M252Y / T256E / T307Q, and M252Y / T256E / T307W according to EU numbering.

[0225] In one embodiment, the binding polypeptide with altered FcRn binding comprises an Fc domain with one or more amino acid substitutions as disclosed herein. In one embodiment, the binding polypeptide with enhanced FcRn binding affinity comprises an Fc domain with one or more amino acid substitutions as disclosed herein. In one embodiment, the binding polypeptide with enhanced FcRn binding affinity comprises an Fc domain with two or more amino acid substitutions as disclosed herein. In one embodiment, the binding polypeptide with enhanced FcRn binding affinity comprises an Fc domain with three or more amino acid substitutions as disclosed herein.

[0226] 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 considered to be cross-reactive between one or more different species. In one embodiment, the binding polypeptide may exhibit both human and rat FcRn binding affinity.

[0227] Neonatal Fc receptor (FcRn) interacts with the Fc region of the antibody to promote circulation by rescuing normal lysosomal degradation. This process is a pH-dependent process, which occurs in endosomes at acidic pH (e.g., pH less than 6.5) rather than at physiological pH conditions of the bloodstream (e.g., non-acidic pH). In some embodiments, compared with binding polypeptides comprising wild-type Fc domains, binding polypeptides of the present disclosure comprising modified Fc domains have enhanced FcRn binding affinity at acidic pH. In some embodiments, compared with binding polypeptides comprising wild-type Fc domains, binding polypeptides comprising modified Fc domains have enhanced FcRn binding affinity at pH less than 7, for example, at about pH 6.5, about pH 6.0, about pH 5.5, about pH 5.0. In some embodiments, the binding polypeptide comprises a modified Fc domain at a pH less than 7, for example, 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 an elevated non-acidic pH. The elevated non-acidic pH can be, for example, pH greater than 7, about pH 7, about pH 7.4, about pH 7.6, about pH 7.8, about pH 8.0, about pH 8.5, and about pH 9.0.

[0228] In certain embodiments, it may be expected that a binding polypeptide comprising a modified Fc domain exhibits 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 expected that a binding polypeptide comprising a modified Fc domain exhibits less FcRn binding affinity at non-acidic pH than a binding polypeptide comprising a modified Fc domain having the double amino acid substitutions M428L / N434S according to EU numbering. Thus, it may be expected that a binding polypeptide comprising a modified Fc domain exhibits minimal perturbation of pH-dependent FcRn binding.

[0229] In some embodiments, a binding polypeptide comprising a modified Fc domain having enhanced FcRn binding affinity at acidic pH has a reduced (i.e., slower) FcRn dissociation rate compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising a modified Fc domain having enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at elevated non-acidic pH has a slower FcRn dissociation rate at acidic pH compared to the FcRn dissociation rate of the binding polypeptide at elevated non-acidic pH.

[0230] In some embodiments, a binding polypeptide comprising a modified Fc domain is provided, the binding polypeptide exhibiting a higher FcRn binding affinity at a non-acidic pH compared to a binding polypeptide comprising a wild-type Fc. In some embodiments, a binding polypeptide comprising a modified Fc domain is provided, the binding polypeptide exhibiting a higher FcRn binding affinity at an acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising a modified Fc domain is provided, the binding polypeptide exhibiting a higher FcRn binding affinity at a non-acidic pH compared to a binding polypeptide comprising a wild-type Fc domain, and exhibiting a higher FcRn binding affinity at an acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. Therefore, in certain embodiments, a binding polypeptide comprising a modified Fc domain is provided, which exhibits a loss of pH-dependent FcRn binding.

[0231] Certain embodiments include antibodies that, in addition to the Fc mutations described herein that exhibit altered FcRn binding affinity, further comprise at least one amino acid in one or more constant region domains and / or at least one amino acid in one or more variable region domains that has been deleted or otherwise altered to provide the desired biochemical characteristics, such as reduced or enhanced effector function, non-covalent dimerization ability, enhanced ability to localize to tumor sites, decreased serum half-life, increased serum half-life, etc., when compared to the intact, unaltered antibody of substantially the same immunogenicity.

[0232] In certain other embodiments, the binding polypeptide comprises a constant region derived from a different antibody isotype (e.g., a constant region 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 a hinge portion of a hinge domain derived from a different antibody isotype, such as an upper hinge domain from an IgG4 molecule and an IgG1 middle hinge domain).

[0233] In certain embodiments, the Fc domain can be mutated to increase or decrease effector function using techniques known in the art. In some embodiments, the binding polypeptide of the present disclosure comprising a modified Fc domain has a changed binding affinity for Fc receptors. There are several different types of Fc receptors, which are classified based on the antibody types they recognize. For example, Fc-γ receptors (FcγR) bind to IgG class antibodies, Fc-α receptors (FcαR) bind to IgA class antibodies, and Fc-ε receptors (FcεR) bind to IgE class antibodies. The FcγR term includes several members such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb families. In some embodiments, compared with the binding polypeptide comprising a wild-type Fc domain, the binding polypeptide comprising a modified Fc domain has a changed FcγRIIIa binding affinity. In some embodiments, the binding polypeptide comprising a modified Fc domain has reduced binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, the binding polypeptide comprising a modified Fc domain has enhanced binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, the binding polypeptide comprising a modified Fc domain has approximately the same binding affinity for FcγRIIIa compared to a binding polypeptide comprising a wild-type Fc domain.

[0234] In other embodiments, the binding polypeptides used in the diagnostic and therapeutic methods described herein have a constant region, such as an IgG1 heavy chain constant region, that is altered to reduce or eliminate glycosylation. For example, a binding polypeptide (e.g., an antibody or immunoadhesin) comprising a modified Fc domain may further comprise an amino acid substitution that alters the glycosylation of the antibody Fc. For example, the modified Fc domain may have reduced glycosylation (e.g., N- or O-linked glycosylation).

[0235] Exemplary amino acid substitutions that confer reduced or altered glycosylation are disclosed in International PCT Publication No. WO05 / 018572, which is incorporated herein by reference in its entirety. In some embodiments, the binding polypeptide is modified to eliminate glycosylation. Such a binding polypeptide may be referred to as an "agly" binding polypeptide (e.g., an "agly" antibody). Although not bound by theory, it is believed that an "agly" binding polypeptide may have improved in vivo safety and stability. The agly binding polypeptide may have any isotype or subclass thereof, such as IgG1, IgG2, IgG3, or IgG4. Many art-recognized methods can be used to prepare "agly" antibodies or antibodies with altered polysaccharides. For example, genetically engineered host cells (e.g., modified yeast such as Pichia pastoris, or CHO cells) with modified glycosylation pathways (e.g., glycosyltransferase deletions) can be used to produce such antibodies.

[0236] In certain embodiments, the binding polypeptide may comprise an antibody constant region (e.g., an IgG constant region, such as a human IgG constant region, such as a human IgG1 constant region) that mediates one or more effector functions. For example, the binding of the C1 complex to the antibody constant region may activate the complement system. Activation of the complement system is important in the opsonization and lysis of cell pathogens. Activation of the complement system also stimulates inflammatory responses and may also be involved in autoimmune hypersensitivity reactions. In addition, antibodies bind to receptors on a variety of cells via the Fc domain (the Fc receptor binding site on the antibody Fc region binds to the Fc receptor (FcR) on the cell). There are many Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors), and IgM (mu receptors). The binding of antibodies to Fc receptors on the cell surface triggers many 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 (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In some embodiments, the binding polypeptide (e.g., antibody or immunoadhesin) binds to an Fcγ receptor. In alternative embodiments, the binding polypeptide may comprise a constant region that lacks one or more effector functions (e.g., ADCC activity) and / or is unable to bind to an Fcγ receptor.

[0237] Proteins (including antibodies) with low thermodynamic stability have an increased tendency to misfold and aggregate, and will limit or hinder the activity, efficacy, and potential of the protein as a useful therapeutic agent. In certain embodiments, the binding polypeptide comprising a modified Fc domain has about the same thermal stability as a binding polypeptide comprising a wild-type Fc domain. In some embodiments, the binding polypeptide comprising a modified Fc domain has about the same thermal stability as a binding polypeptide comprising a modified Fc domain having a triple amino acid substitution M252Y / S254T / T256E (YTE).

[0238] The resulting physiological characteristics, bioavailability, and other biochemical effects of the modifications (such as tumor localization, biodistribution, and serum half-life) can be readily measured and quantified without undue experimentation using well-known immunological techniques.

[0239] In certain embodiments, the binding polypeptides of the present disclosure may comprise an antigen-binding fragment of an antibody. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or an antibody that binds to an antigen or competes with an intact antibody (i.e., the intact antibody from which the polypeptide fragment of the immunoglobulin or antibody 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 exemplary embodiments, the binding polypeptides of the present disclosure comprise an antigen-binding fragment and a modified Fc domain.

[0240] In some embodiments, the binding polypeptide comprises a single chain variable region sequence (ScFv). The single chain variable region sequence comprises a single polypeptide having one or more antigen binding sites, such as a VL domain connected to a VH domain by a flexible linker. The ScFv molecule can be constructed in a VH-linker-VL direction or a VL-linker-VH direction. The flexible hinge connecting the VL and VH domains constituting the antigen binding site comprises about 10 to about 50 amino acid residues. Connecting peptides are known in the art. The binding polypeptide may comprise at least one scFv and / or at least one constant region. In one embodiment, the binding polypeptide of the present disclosure may comprise at least one scFv connected or fused to a modified Fc domain.

[0241] In some embodiments, the binding polypeptides of the present disclosure are multivalent (e.g., tetravalent) antibodies produced by fusing a DNA sequence encoding an antibody with a ScFv molecule (e.g., an altered ScFv molecule). For example, in one embodiment, these sequences are combined so that the ScFv molecule (e.g., an altered ScFv molecule) is connected to the Fc fragment of the antibody at its N-terminus or C-terminus via a flexible linker (e.g., a gly / ser linker). In another embodiment, the tetravalent antibodies of the present disclosure can be prepared by the following method: the ScFv molecule is fused to a connecting peptide, and the connecting peptide is fused to a modified Fc domain to construct a ScFv-Fab tetravalent molecule.

[0242] In another embodiment, the binding polypeptide of the present disclosure is a modified mini antibody. The modified mini antibody of the present disclosure is a dimeric molecule composed of two polypeptide chains, each of which comprises a ScFv molecule fused via a connecting peptide and a modified Fc domain. Mini antibodies can be prepared by constructing ScFv components and connecting peptide components using methods described in the art (see, for example, U.S. Patent No. 5,837,821 or WO94 / 09817A1). In another embodiment, a tetravalent mini antibody can be constructed. A tetravalent mini antibody can be constructed in the same manner as a mini antibody, except that two ScFv molecules are connected using a flexible joint. The connected scFv-scFv construct is then connected to a modified Fc domain.

[0243] In another embodiment, the binding polypeptides of the present disclosure comprise diabodies. Diabodies are dimeric tetravalent molecules, each of which has a polypeptide similar to a scFv molecule, but generally have a short (less than 10, e.g., about 1 to about 5) amino acid residue linker connecting the two variable domains, so that the VL and VH domains on the same polypeptide chain cannot interact with each other. Instead, the VL and VH domains of one polypeptide chain (respectively) interact with the VH and VL domains on the second polypeptide chain (see, e.g., WO 02 / 02781). The diabodies of the present disclosure comprise scFv-like molecules fused to a modified Fc domain.

[0244] In other embodiments, the binding polypeptide comprises a multispecific or multivalent antibody comprising one or more variable domains in tandem on the same polypeptide chain, such as a tandem variable domain (TVD) polypeptide. Exemplary TVD polypeptides include the "double-headed" or "double Fv" configuration described in U.S. Patent No. 5,989,830. In the double Fv configuration, the variable domains of two different antibodies are represented in a tandem orientation on two separate chains (one heavy chain and one light chain), wherein one polypeptide chain has two tandem VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains connected in tandem by a peptide linker (VL1-linker-VL2). In the crossover double-head configuration, the variable domains of two different antibodies are represented in a tandem orientation on two separate polypeptide chains (one heavy chain and one light chain), wherein one polypeptide chain has two tandem VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains connected in tandem in the opposite orientation by a peptide linker (VL2-linker-VL1). Additional antibody variants based on the "double Fv" format include dual variable domain IgG (DVD-IgG) bispecific antibodies (see U.S. Patent No. 7,612,181) and TBTI formats (see US2010 / 0226923 A1). In some embodiments, the binding polypeptide includes a multispecific or multivalent antibody comprising one or more variable domains tandemly connected on the same polypeptide chain fused to a modified Fc domain.

[0245] In another exemplary embodiment, the binding polypeptide comprises a crossover dual variable domain IgG (CODV-IgG) bispecific antibody based on a “two-head” configuration (see US20120251541 A1, which is herein incorporated by reference in its entirety).

[0246] In another exemplary embodiment, the binding polypeptide is an immunoadhesin. As used herein, "immunoadhesin" refers to a binding polypeptide comprising one or more binding domains (e.g., from a receptor, ligand, or cell adhesion molecule) connected to an immunoglobulin constant domain (i.e., Fc region) (see, e.g., Ashkenazi et al. 1995, Methods 8(2): 104–115, and Isaacs (1997) Brit. J. Rheum. 36: 305, which are incorporated herein by reference in their entirety. Immunoadhesins are identified by the suffix "-cept" in their international nonproprietary names (INN). Similar to antibodies, immunoadhesins have a long circulating half-life, are easily purified by affinity-based methods, and have an affinity advantage conferred by bivalency. Examples of commercially available therapeutic immunoadhesins include etanercept, Abatacept Linacip Aflibercept Belacept

[0247] 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), DARPins (see, e.g., Stumpp et al. (2008) Drug Discov. Today 13(15–16):695–701, which is incorporated herein by reference in its entirety), the Z domain of protein A (see Nygren et al. (2008) FEBS J. 275(11):2668–76, which is incorporated herein by reference in its entirety), lipocalin (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. (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).

[0248] 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 a target antigen, including both soluble factors (such as cytokines and membrane-bound factors) and transmembrane receptors.

[0249] The binding polypeptides of the present disclosure comprising the modified Fc domains described herein may include CDR sequences or variable domain sequences of known "parent" antibodies. In some embodiments, in addition to the modifications to the Fc domains disclosed herein, the parent antibody and the antibodies of the present disclosure may share similar or identical sequences.

[0250] Nucleic Acids and Expression Vectors

[0251] In one aspect, the invention provides polynucleotides encoding the binding polypeptides disclosed herein. Also provided are methods of making the binding polypeptides, the methods comprising expressing these polynucleotides.

[0252] Typically, polynucleotides encoding binding polypeptides disclosed herein are inserted into expression vectors for introduction into host cells that can be used to produce a desired amount of the claimed antibodies or immunoadhesins. Therefore, in certain aspects, the invention provides expression vectors comprising polynucleotides disclosed herein and host cells comprising these vectors and polynucleotides.

[0253] For the purpose of the specification and claims, the term "vector" or "expression vector" is used herein to refer to a vector used to introduce and express a desired gene in a cell. As known to those skilled in the art, such vectors can be easily selected from plasmids, bacteriophages, viruses and retroviruses. Typically, the vector will contain a selection marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0254] Many expression vector systems can be used.For example, a class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV) or SV40 virus.Other vector classes relate to the use of polycistronic systems with internal ribosome binding sites.In addition, the cell that DNA has been integrated into its chromosome can be selected by introducing one or more markers, and the one or more markers allow the selection of the host cell of transfection.The marker can provide the original nutrition, biocide resistance (for example antibiotic) or resistance to heavy metals such as copper to the nutrient deficiency subject.Selectable marker gene can be directly connected to the DNA sequence to be expressed, or introduced into the same cell by co-transformation.Additional elements may also be needed to optimally synthesize mRNA.These elements may include signal sequence, splicing signal and transcription promoter, enhancer and termination signal.In some embodiments, the variable region gene of the clone is inserted into the expression vector together with the heavy chain and light chain constant region gene (such as human gene) synthesized as described above.

[0255] In other embodiments, polycistronic constructs can be used to express binding polypeptides as described herein. In such expression systems, a variety of gene products of interest, such as heavy and light chains of antibodies, can be produced from a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible 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 effectively produce the full range of polypeptides disclosed in this application.

[0256] More generally, once a vector or DNA sequence encoding a binding polypeptide disclosed herein has been prepared, the expression vector can be introduced into an appropriate host cell. That is, the host cell can be transformed. The introduction of a plasmid into a host cell can be achieved by a variety of techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with envelope DNA, microinjection, and complete viral infection. See, for example, Ridgway, AAG "Mammalian Expression Vectors" Chapter 24.2, Pages 470-472 Vectors, Rodriguez and Denhardt, editors (Butterworths, Boston, MA 1988). The transformed cells are grown under conditions suitable for producing light and heavy chains, and heavy and / or light chain protein synthesis is determined. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence activated cell sorter analysis (FACS), immunohistochemistry, etc.

[0257] As used herein, the term "transformation" should be used in the broadest sense and refers to the introduction of DNA into a recipient host cell, which alters the genotype and thereby results in a change in the recipient cell.

[0258] Along the same lines, "host cells" refers to cells that have been transformed with a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. When describing a process for isolating a polypeptide from a recombinant host, the terms "cells" and "cell culture" are used interchangeably to refer to the source of the antibody, unless otherwise specifically indicated. In other words, recovery of the polypeptide from "cells" can mean recovery from whole cells pelleted by centrifugation, or from a cell culture containing both culture medium and suspended cells.

[0259] In one embodiment, the host cell line used to express the binding polypeptide is of eukaryotic or prokaryotic origin. In one embodiment, the host cell line used to express the binding polypeptide is of bacterial origin. In one embodiment, the host cell line used to express the binding polypeptide is of mammalian origin; those skilled in the art can determine the specific host cell line that is most suitable for expressing the desired gene product therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary line, DHFR-), HELA (human cervical cancer), CVI (monkey kidney line), COS (derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney). In one embodiment, the cell line provides for altered glycosylation of the antibody expressed thereby, e.g., afucosylation (e.g., PER.C6.RTM. (Crucell) or a FUT8 knockout CHO cell line (POTELLIGENT TM Cells) (Biowa, Princeton, NJ). In one embodiment, NS0 cells can be used. Host cell lines are generally available from commercial services, the American Tissue Culture Collection, or published literature.

[0260] In vitro production allows for scaling up to give a large amount of desired binding polypeptides. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include homogenous suspension cultures (e.g., in airlift reactors or continuous stirred reactors), or immobilized or embedded cell cultures on microbeads or ceramic cartridges on agarose (e.g., in hollow fibers, microcapsules). If necessary and / or desired, solutions of purified polypeptides can be obtained by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immuno) affinity chromatography.

[0261] One or more genes encoding binding polypeptides can also be expressed in non-mammalian cells such as bacteria or yeast or plant cells. In this regard, it should be understood that a variety of unicellular non-mammalian microorganisms such as bacteria, i.e., those microorganisms that can grow in culture or fermentation, can also be transformed. Bacteria that are easily transformed include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. It should also be understood that when expressed in bacteria, the polypeptide can become part of an inclusion body. The polypeptide must be isolated, purified, and then assembled into a functional molecule.

[0262] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of eukaryotic microorganisms, although many other strains are generally available. For expression in the genus Saccharomyces, plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157 (1980)) is generally used. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutant strains lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85: 12 (1977)). Then, the presence of trpl damage as a yeast host cell genomic feature provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0263] Treatment

[0264] In one aspect, the present invention provides a method for treating or diagnosing a patient in need thereof, comprising administering an effective amount of a binding polypeptide disclosed herein. In certain embodiments, the disclosure provides kits and methods for diagnosing and / or treating a disorder (e.g., a tumor disorder in a mammalian subject in need of such treatment). In certain exemplary embodiments, the subject is a human.

[0265] The binding polypeptides of the present disclosure can be used for many different applications. For example, in one embodiment, the subject binding polypeptide can be used to reduce or eliminate cells with epitopes recognized by the binding domain of the binding polypeptide. In another embodiment, the subject binding polypeptide effectively reduces the concentration of soluble antigens in the circulation or eliminates soluble antigens in the circulation. In another embodiment, the subject binding polypeptide is effective as a T cell engager. In one embodiment, the binding polypeptide can reduce tumor size, inhibit tumor growth and / or prolong the survival time of tumor-bearing animals. Therefore, the present disclosure also relates to methods for treating tumors in humans or other animals by administering an effective non-toxic amount of modified antibodies to such humans or animals.

[0266] In one embodiment, the subject binding polypeptides can be used to treat a disease or disorder. For example, the subject binding polypeptides can be used to treat an antibody-related disorder or an antibody-reactive disorder, condition, or disease. As used herein, the term "antibody-related disorder" or "antibody-reactive disorder" or "condition" or "disease" refers to or describes a disease or disorder that can be improved by administering a pharmaceutical composition comprising an antibody or binding polypeptide of the present disclosure.

[0267] In one embodiment, the subject binding polypeptides can be used to treat cancer. As used herein, the term "cancer" or "cancerous" refers to or describes a physiological condition that is typically characterized by uncontrolled 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 such 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.

[0268] In another embodiment, the subject binding polypeptides can be used to treat other conditions, including but not limited to infectious diseases, autoimmune conditions, inflammatory conditions, lung diseases, neuronal or neurodegenerative diseases, liver diseases, spinal diseases, uterine diseases, depression, etc. Non-limiting examples of infectious diseases include infectious diseases 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), etc.) or DNA viruses. Examples of infectious diseases also include but are not limited to bacterial infectious diseases caused by, for example, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus, Streptococcus, Escherichia coli, and other infectious diseases, including, for example, infectious diseases caused by Candida albicans. Other infectious diseases include, but are not limited to, malaria, SARS, yellow fever, Lyme borreliosis, 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). Thus, the present disclosure relates to a method of treating a variety of conditions that would benefit from the use of a subject binding polypeptide having, for example, an increased half-life.

[0269] By routine experimentation, those skilled in the art will be able to determine that an effective non-toxic amount of the modified binding polypeptide can be used for the purpose of treating malignant tumors. For example, the therapeutically active amount of the binding polypeptide disclosed herein can vary according to factors such as the subject's disease stage (e.g., stage I relative to stage IV), age, sex, medical complications (e.g., immunosuppressive conditions or diseases), and body weight, as well as the ability of the modified antibody to elicit a desired response in the subject. The dosage regimen can be adjusted to provide the best therapeutic response. For example, several separate doses can be administered daily, or the dosage can be reduced proportionally as indicated by the emergency state of the treatment situation.

[0270] In general, the compositions provided in the present disclosure can be used to prophylactically or therapeutically treat any tumor that contains an antigenic marker that allows for targeting of cancer cells by modified antibodies.

[0271] Pharmaceutical compositions and their administration

[0272] The method of preparing and administering the binding polypeptide of the present disclosure to the subject is well known or easily determined by those skilled in the art. The route of administration of the binding polypeptide of the present disclosure can be oral, parenteral, by inhalation or local. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Although all these forms of administration are obviously considered to be within the scope of the present disclosure, the form of administration will be for injection, particularly for intravenous or intraarterial injection or infusion of solution. Generally, suitable pharmaceutical compositions for injection may include buffer (e.g., acetate, phosphate or citrate buffer), surfactant (e.g., polysorbate), optional stabilizer (e.g., human albumin), etc. In some embodiments, the binding polypeptide can be delivered directly to the site of the bad cell population, thereby enhancing the exposure of the diseased tissue to the therapeutic agent.

[0273] Preparations 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 0.01-0.1M, such as 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oil. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents and inert gases, etc. More particularly, the pharmaceutical composition that can be suitable for injection includes sterile aqueous solution (water soluble) or dispersion, and the sterile powder for the temporary preparation of sterile injectable solution or dispersion. In this case, the composition must be sterile and should be the fluid of the degree that is easy to inject. It should be stable under manufacturing and storage conditions, and can usually prevent the contamination of microorganisms such as bacteria and fungi. Carrier can be a solvent or dispersion medium, and the solvent or dispersion medium contain for example water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol etc.) and its suitable mixture. For example, can be by using coating such as lecithin, by keeping required particle size and by using surfactant to keep suitable fluidity in the case of dispersion.

[0274] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isotonic agents, for example, sugars, polyols (e.g., 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.

[0275] In any case, a sterile injectable solution can be prepared by incorporating the active compound (e.g., a modified binding polypeptide itself or in combination with other active agents) in a desired amount into an appropriate solvent, followed by filtration sterilization, wherein the solvent has a combination of one or more components listed herein as required. Typically, a dispersion is prepared by incorporating the active compound into a sterile vehicle containing an alkaline dispersion medium and other components required from those listed above. In the case of sterile powders for preparing sterile injectable solutions, exemplary preparation methods include vacuum drying and freeze drying, which produce powders of active ingredients and any additional required ingredients from previously sterile filtered solutions. The preparations 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. In addition, the preparations can be packaged and sold in the form of a kit. Such products typically have labels or package inserts indicating that the relevant compositions can be used to treat subjects suffering from or susceptible to autoimmune or tumor disorders.

[0276] The effective dose of the disclosed compositions for treating the above-mentioned conditions varies according to many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is preventive or therapeutic. Typically, the patient is human, but non-human mammals including transgenic mammals can also be treated. Conventional methods known to those skilled in the art can be used to titrate the therapeutic dose to optimize safety and efficacy.

[0277] The binding polypeptide of the present disclosure can be administered multiple times. The intervals between single doses can be weekly, monthly or annual. 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, the dosage is adjusted to achieve a modified binding polypeptide concentration of about 1-1000 μg / ml in plasma, and in some methods the concentration is 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 vary according to the half-life of the antibody in the patient. Typically, humanized antibodies show the longest half-life, followed by chimeric antibodies and non-human antibodies.

[0278] The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, a composition containing an antibody of the present invention or its mixture is administered to a patient who is not yet in a disease state to enhance the patient's resistance. Such an amount is defined as a "preventive effective dose". In this use, the exact amount also depends on the patient's health and overall immunity, but is generally in the range of about 0.1 to about 25 mg per dose, especially about 0.5 to about 2.5 mg per dose. A relatively low dose is administered for a long time at relatively infrequent intervals. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer a relatively high dose (e.g., about 1 to 400 mg / kg antibody per dose, wherein a dose of about 5 to 25 mg is more commonly used for radioimmunoconjugates, and a higher dose is used for antibodies modified by cytotoxin-drugs) at relatively short intervals until disease progression is reduced or terminated, or until the patient shows partial or complete improvement of disease symptoms. Thereafter, a preventive regimen can be administered to the patient.

[0279] The binding polypeptides of the present disclosure may optionally be administered in combination with other agents effective for treating the disorder or condition for which treatment (e.g., prophylactic or therapeutic) is desired. 90 An effective single therapeutic dose (ie, a therapeutically effective amount) of the Y-labeled modified antibody ranges between about 5 and about 75 mCi, such as between about 10 and about 40 mCi. 131 An effective single therapeutic non-myeloablative dose range of the I modified antibody is between about 5 and about 70 mCi or between about 5 and about 40 mCi. 131 The effective single therapeutic ablative dose of I-labeled antibodies (i.e., autologous bone marrow transplantation may be required) ranges between about 30 and about 600 mCi, such as between about 50 and less than about 500 mCi. In conjunction with chimeric antibodies, due to the longer circulation half-life of murine antibodies, the effective single therapeutic non-bone marrow ablative dose of iodine-131 labeled chimeric antibodies ranges between about 5 and about 40 mCi, such as less than about 30 mCi. For example, 111 In-labeled imaging standards are typically less than about 5 mCi.

[0280] Although the binding polypeptide can be administered as described above, it must be emphasized that in other embodiments, the binding polypeptide can be administered to other healthy patients as a first-line therapy. In such embodiments, the binding polypeptide can be administered to patients with normal or average red bone marrow reserves and / or to patients who have not yet been treated and are not being treated. As used herein, the administration of modified antibodies or immunoadhesins together with adjuvant therapy or in combination means sequential, simultaneous, coexisting, concurrent, concomitant or concurrent administration or application of the therapy and disclosed antibodies. It will be appreciated by those skilled in the art that the various components of the combined therapeutic regimen can be administered or applied regularly to enhance the overall effectiveness of the treatment.

[0281] As previously mentioned, the binding polypeptides, immunoadhesins or recombinants thereof disclosed herein can be administered in a pharmaceutically effective amount for in vivo treatment of mammalian disorders. In this regard, it will be appreciated that the disclosed binding polypeptides will be formulated to facilitate administration of the active agent and enhance the stability of the active agent.

[0282] According to the pharmaceutical composition disclosed herein, a pharmaceutically acceptable non-toxic sterile carrier may be included, such as physiological saline, non-toxic buffer, preservative, etc. For the purpose of the present application, the pharmaceutically effective amount of the binding polypeptide, immunoadhesin or its recombinant conjugated or unconjugated with a therapeutic agent should be maintained, which means that it is enough to achieve effective combination with the antigen and enough to obtain the amount of benefit (for example, enough to improve the symptom of the disease or illness or detect the substance or cell). In the case of tumor cells, the modified binding polypeptide can interact with the immunoreactive antigen selected on the tumor cell or the immune response cell, and the increase of these cell deaths is provided. Of course, the pharmaceutical composition disclosed herein can be administered in a single dose or multiple doses to provide a modified binding polypeptide of a pharmaceutically effective amount.

[0283] In order to be consistent with the scope of the present disclosure, the binding polypeptides of the present disclosure can be administered to humans or other animals in an amount sufficient to produce a therapeutic or preventive effect according to the above-mentioned treatment methods. The binding polypeptides of the present disclosure 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 according to known techniques. Those skilled in the art will recognize that the form and characteristics of the pharmaceutically acceptable carrier or diluent depend on the amount of the active ingredient combined therewith, the route of administration and other well-known variables. Those skilled in the art will further understand that mixtures containing one or more binding polypeptides described in the present disclosure can prove to be particularly effective.

[0284] The contents of the articles, patents, and patent applications mentioned or cited herein, and all other documents and electronically available information 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.

[0285] Although the present invention has been described with reference to specific embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present 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 suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adopt specific circumstances, materials, composition of matter, processes, process steps or steps to achieve the objective spirit and scope of the present invention. It is contemplated that all such modifications are within the scope of the appended claims. Certain embodiments have now been described in detail and will be more clearly understood by reference to the following examples, which are intended to be illustrative only and not restrictive.

[0286] Example

[0287] The present invention is further illustrated by the following examples, which should not be construed as further limiting.

[0288] Example 1: Materials and Methods

[0289] Protein reagents:

[0290] 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); biotinylated cynomolgus 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 valine at position 158 (V158)). H435A and H310A / H435Q heavy chain variants were obtained from HEK293 conditioned medium. mAb2 variants were cloned by Evitria and purified from suspension CHO K1 conditioned medium using mAbSelect SuRe affinity columns (GE Healthcare), and the buffer was exchanged to phosphate buffered saline (PBS) at pH 7.4 for subsequent experiments.

[0291] Saturation library construction:

[0292] WT IgG1 mAb1 antibody heavy and light chains with leader DNA sequences were incorporated into pBH6414 and pBH6368 mammalian expression plasmids, respectively, using NcoI and HindIII restriction enzyme sites. A saturation library was created using the Lightning Site Directed Mutagenesis Kit (Agilent) and NNK (N=A / C / G / T, K=G / T) and WWC (W=A / T) primers (IDT Technologies) 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 variants AAA (T307A / E380A / N434A), LS (M428L / N434S) and YTE (M252Y / S254T / T256E) in the mAbl backbone were constructed into the pBH6414 vector by LakePharma.

[0293] Combinatorial saturation libraries were obtained by site-directed mutagenesis of mAb1 heavy chains in PCR reactions using Q5 mutagenesis kit (NEBiolabs) and T256D, T256E, T307Q, T307W, N434F and N434Y primers with WT and M252Y templates. Mutations were incorporated into the Ab3 backbone using Q5 mutagenesis kit (NEBiolabs) with M252Y, T256D, T307Q and T307W primers. The generation of all Fc variants was confirmed by Sanger sequencing (Genewiz, Inc.).

[0294] Recombinant Antibody Expression and Purification:

[0295] For conditioned medium screening, the DNA containing the mutant heavy chain and wild-type light chain of mAb1 is transfected into 1mL of Expi293 mammalian cells (Invitrogen) for expression according to the manufacturer's instructions. Cells are shaken and incubated at 900 rpm (RPM) in 2mL 96-well plates (Greiner Bio-One) at 37°C, 5% carbon dioxide and 80% humidity, and sealed with an inflatable membrane. Conditioned medium is collected five days after transfection and stored at -80°C until use. The lead variants of mAb1 and Ab3 skeletons are expressed with 30mL scale in a 125mL flask (Corning) with a 0.2 μm discharge cap. 125mL culture flasks are shaken with 125RPM throughout the expression duration. Conditioned medium is collected five days after transfection, and filtered by 0.22 μm, 50mL conical filters (Corning), and stored at 4°C until purification.

[0296] Separation of mAb1 and Ab3 was performed using a 1 mL mAbSelect SuRe HiTrap column (GE Healthcare). After a ten column volume wash step of PBS at pH 7.4, the antibodies were eluted with five column volumes of 0.1 M citric acid (Sigma) at pH 3.0 and neutralized with 0.5 mL of 1 M tris base (Sigma) at pH 9.0. The eluted antibodies were buffer exchanged in PBS at pH 7.4 and concentrated to >1 mg mL using a 30 kDa MWCO Amicon concentrator (Millipore). -1 The concentration of purified antibodies was determined by their UV absorbance at 280 nm (UV 280 ) and a suitable extinction coefficient.

[0297] Octet Conditioned Media Screening and Analysis:

[0298] Screening of conditioned media containing mAb1 variants was performed on an Octet QK 384 (PALL Life Sciences) with a Ni-NTA biosensor. His-tagged antigens were quantified at 15 μg mL in PBS, pH 7.4, 0.1% bovine serum albumin (BSA, Sigma), and 0.01% Tween-20 (Sigma) (PBST-BSA 7.4). -1 Capture 300sec, then wash with PBST-BSA of pH 7.4 for 20 seconds. Capture antibody 200sec in conditioned medium diluted 1:1 with PBST-BSA of pH 7.4. After the buffer wash step in pH 6.0 buffer, 200nM rFcRn is used to associate and FcRn binding kinetics are obtained with dissociation times of 150 and 200sec respectively at pH 6.0. In all steps during Octet screening, the temperature was 30°C and the shaking speed was 1000RPM. The rFcRn binding kinetic curve was corrected for the beginning of the FcRn association phase and modeled as a 1:1 binding model using Octet 7.1 analysis software.

[0299] FcRn binding kinetics:

[0300] FcRn binding kinetics at pH 6.0 and pH 7.4 were measured using a Biacore T200 instrument (GE Healthcare) using a modified protocol with either direct immobilization of FcRn or the biotin CAPture 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 (at a concentration of 20 μg mL -1 ) at 10 μL min -1 Immobilize in 10 mM sodium acetate (GE Healthcare) at pH 4.5 for 180 s to reach approximately 20 RU on the surface of the C1 sensor chip. In the case of the biotin CAPture kit, capture the CAPture reagent on the CAP chip surface to a binding RU>2,000 RU, followed by 0.1 μg mL -1 FcRn at 30uL min -1 Capture was performed in the appropriate channel for 24 s to a final binding RU of approximately 2 RU. The running buffer for FcRn binding kinetics experiments was PBS with 0.05% surfactant P-20 (PBS-P+, GE Healthcare) at pH 6.0 or 7.4. A 4-fold serial dilution from a concentration series of 1000 nM antibody was performed in quadruplicate for each variant (including 0 nM control). -1 Kinetic measurements were performed to obtain association and dissociation times of 180 and 300 sec, respectively, at a flow rate of 1.5 %. The C1 and CAP sensor chips were each incubated with 10 mM sodium tetraborate, 1 M NaCl, pH 8.5 (GE Healthcare) at 50 μL min -1 Regenerate for 30 sec or use 6 M guanidine hydrochloride, 250 mM sodium hydroxide (GE Healthcare) at 50 uL min -1 Regeneration was performed for 120 s, followed by an additional 60-90 sec stabilization step in PBS-P+, pH 6.0. Steady-state RU measurements were obtained in triplicate for all variants at 1000 nM at pH 7.4 using the same C1 or CAP sensor chip and kinetic parameters as described above, except that the capture level of FcRn was increased 10-20 fold for both methods.

[0301] Due to the avidity effect, the kinetic parameters of the concentration series at pH 6.0 were fitted to a bivalent model using Biacore T200 evaluation software. See, e.g., Suzuki et al., J. Immunol. (2010) 184: 1968-1976. Each concentration series was fitted independently to obtain the average association and dissociation rates and binding affinity. The apparent binding affinity was calculated based on the first association and dissociation rates of the bivalent model. Residue binding at pH 7.4 was measured in triplicate using 1000 nM of each antibody for reaction comparison. Each repeated steady-state reaction was averaged to obtain the mean and standard deviation.

[0302] FcRn affinity chromatography:

[0303] In one experiment, an FcRn affinity column was generated from a protocol adapted from Schlothauer et al. 2013, mAbs 5:576-586. A 1 mL Streptavidin HP HiTrap column (GE Healthcare) was charged with five column volumes of binding buffer (20 mM sodium phosphate (Sigma), 150 mM sodium chloride (NaCl; Sigma) at pH 7.4) at 1 mL min. -1 After equilibration, 4 mg of biotinylated cynoFcRn was injected. The column was washed with binding buffer and stored at 4°C until use.

[0304] The FcRn affinity column was equilibrated with five column volumes of low pH buffer (20 mM 2-(N-morpholino)ethanesulfonic acid (MES; Sigma, pH 5.5); 150 mM NaCl) and then 300 μg of each antibody was injected. The pH of the antibody solution was adjusted to pH 5.5 with the low pH buffer. After washing with the low pH buffer for ten column volumes, the column was washed with more than 30 column volumes of high pH buffer (20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (bistrispropane; Sigma, pH 9.5); 150 mM NaCl) at 1 mL min -1 The antibody was eluted in 1 mL fractions by a linear pH gradient and monitored by UV 280 The FcRn affinity column was re-equilibrated with ten column volumes of low pH buffer for subsequent runs or re-equilibrated with binding buffer for storage. All variants were performed in triplicate.

[0305] The FcRn affinity column elution curve for each antibody was modeled as a single Gaussian distribution using Equation 1 in Sigmaplot 11 (Systat Software, Inc.) to determine the UV 280 Elution volume at maximum.

[0306]

[0307] where x0 is the UV 280 The elution volume at the peak maximum, y0 is the baseline UV 280 Absorbance, and a and b are related to the full width at half maximum of the distribution. The pH of each fraction was measured by a Corning Pinnacle 540 pH meter and related to the elution volume using linear regression.

[0308] In another experiment, the FcRn affinity column was adapted from Schlothauer et al. 2013, mAbs 5:576-586, where biotinylated hFcRn was on a 1 mL Streptavidin HP HiTrap column (GE Healthcare). On an AKTAPure system (AKTA), 300 μg of each antibody in a low pH buffer (20 mM 2-(N-morpholino)ethanesulfonic acid (MES; Sigma at pH 5.5); 150 mM NaCl) was injected into the column. A linear pH gradient was generated at 0.5 mL min by using a low and high pH buffer (20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (bistrispropane; Sigma at pH 9.5); 150 mM NaCl) over 30 column volumes. -1 The antibody was eluted and the absorbance and pH were monitored. The column was re-equilibrated with low pH buffer for subsequent runs. All variants were performed in triplicate. The FcRn affinity column elution curve was fit to a single Gaussian distribution in Sigmaplot 11 (Systat Software, Inc.) to determine the UV 280 Elution volume and pH at maximum.

[0309] Differential Scanning Fluorescence Assay:

[0310] Differential scanning fluorimetry (DSF) experiments were performed in 20 μL reactions on a BioRad CFX96 Real-Time System Thermocycler (BioRad). Antibody samples and 5000x stock of Sypro Orange dye (Invitrogen) were diluted to 0.4 mg mL in PBS, pH 7.4. -1 The antibodies and Sypro Orange were mixed in a 1:1 ratio in a 96-well PCR plate and sealed with adhesive sealant (microseal, BioRad) to achieve a final concentration of 0.2 mg mL for each antibody and 5x Sypro Orange dye. -1. All antibody variants were run in triplicate. The thermal cycler program consisted of a 2-minute equilibration step at 20°C followed by a constant ramp rate of 0.5°C / 5sec to a final temperature of 100°C. Fluorescence measurements of each well were obtained using a FAM excitation wavelength (485nm) and ROX emission (625nm) detector suitable for Sypro Orange fluorescence (see, e.g., Biggar et al. 2012, Biotechniques 53:231-238). DSF fluorescence intensity curves and first derivatives were exported from BioRad CFX Manager and analyzed in Sigmaplot 11. T m Defined as the midpoint of the first transition in the fluorescence intensity curve.

[0311] FcγRIIIa binding kinetics:

[0312] 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 -1 Anti-HPC4 antibody (Roche) was added at 10 μl min -1 The surface of the CM5 sensor chip was coupled for 600 sec to a final density of >20,000 RU. The running buffer for FcγRIIIa binding kinetics experiments was HEPES-buffered saline at pH 7.4 with 0.05% surfactant P-20 (HBS-P+, GE Healthcare) and 2 mM calcium chloride (CaCl2, Fluka). -1 Capture 1.25 μg mL -1 Each kinetic trace was initialized with HPC4-labeled FcγRIIIa-V158 for 30 sec. The association and dissociation kinetics of each variant were measured at 300 nM, and each step of each variant was performed at 5 μl min -1 The measurement was performed for 120-180 sec. After the kinetic measurement was completed, 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+ with CaCl2 for 120 sec.

[0313] In one experiment, FcγRIIIa kinetics were analyzed in a similar manner as described for FcRn binding at pH 7.4. Kinetics were obtained for a series of 3-fold serial dilutions from 1000 nM for WT, benchmark, lead single and combination variants to determine binding affinity to FcγRIIIa. Steady-state RU was determined for each concentration and replicate, plotted as a function of antibody concentration and fitted to a steady-state model as shown in Equation 2.

[0314]

[0315] where the offset is the baseline RU of 0 nM antibody, R max is the steady-state RU at high antibody concentration, [antibody] is the concentration of antibody, and K D,app is the apparent binding affinity of the interaction between the variant and FcγRIIIa.

[0316] In another experiment, the FcγRIIIa kinetics experiment was analyzed in a similar manner as described for FcRn binding at pH 7.4 using the average steady-state binding response. For all variants, the steady-state RU of 300 nM antibody was determined in triplicate and averaged. The fold change of the response change relative to WT (response fold change) was determined for comparison between variants in each framework.

[0317] Isoelectric focusing

[0318] The isoelectric point (pI) of the lead variants was determined using capillary electrophoresis on Maurice C (Protein Simple). Each 200 μL sample contained 0.35% methylcellulose (Protein Simple), 4% ampholytes (pharmalyte) 3-10 (GE Healthcare), 10 mM arginine (Protein Simple), 0.2 mg mL -1 Antibodies and 4.05 and 9.99 pI markers (Protein Simple). The samples were loaded into the capillary at 1500 V for 1 minute, followed by a separation phase at 3000 V for 6 minutes and monitored using tryptophan fluorescence. The pI of each variant was determined using Maurice C software and defined as the pH at the fluorescence maximum of the major species.

[0319] Homogeneous bridging rheumatoid factor (RF) ELISA

[0320] EZ-Link Sulfo-NHS-LC-Biotin and Mix-n-Stain were used according to the manufacturer's instructions. TMAntibodies were biotinylated and labeled with digoxigenin using the Digoxigenin Antibody Labeling Kit (Biotium). For each variant, 4 μg mL -1 Stock solutions of biotinylated and digoxigenin-labeled antibodies were prepared and mixed with 300U / mL RF (Abcam) in a 1:1 ratio. After incubation for 20 hours at room temperature, 100μL 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 with 0.05% Tween-20, and 100μL of a 1:2000 dilution of HRP-conjugated anti-digoxigenin secondary antibody (Abcam) was added to each well. After incubation for 2 hours at room temperature, the wells were washed and treated with 100μL of TMB substrate (Abcam) for 15 minutes at room temperature. The reaction was stopped with 100μL of stop solution (Abcam) and the absorbance at 450nm was measured on a SpectraMax plate reader. Wells without antibody-RF mixture provided blank subtraction, and the experiment was repeated three times. P values ​​were determined using a Student's t-test.

[0321] In vivo pharmacokinetics

[0322] Pharmacokinetic studies were performed in cynomolgus monkeys and hFcRn transgenic mice (Tg32 strain, Jackson Laboratory, Bar Harbor, ME). In the monkey study, WT, LS, DQ, DW and YD variants in the mAb2 backbone were administered to the brachiocephalic vein of three untreated male cynomolgus monkeys at a single intravenous dose of 2.5 mg / kg, with a dose volume of 1.5 mL / kg. Blood samples (0.5 mL) were collected by venipuncture of the saphenous vein at 8 sampling times of 0.0035, 0.17, 1, 3, 7, 14, 21 and 28 days after administration. After collection, blood samples were centrifuged at 1500 g for 10 minutes at 4°C and stored at -80°C.

[0323] In hFcRn mice, the antibody variants were administered to the tail vein at a single intravenous dose of 2.5 mg / kg, with 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. The collected blood samples were transferred to microtubes and centrifuged at 1500 g for 10 minutes at 4°C. Plasma samples were collected, combined for each time point (6 mice / sample), and stored at -80°C before analysis.

[0324] All in vivo studies were performed in compliance with the Sanofi institutional animal care policy. Cynomolgus monkey and mouse studies were approved by the “Ministère de l'Enseignement Superieur et de la Recherche” in France and the “Regierungspraesidium Darmstadt” in Germany.

[0325] The concentration of each mAb2 variant at each time point was determined by bottom-up LC-MS / MS assay. After precipitation of plasma aliquots, the plasma precipitates were subjected to protein denaturation, reduction, alkylation, trypsin digestion, and solid phase extraction before analysis of surrogate peptides. mAb2 variants were expressed in 4% paraformaldehyde (100 μg / mL) at 1.00, 2.00, 5.00, 10.0, 20.0, 50.0, 100, 200, and 400 μg / mL -1 The calibration standards were prepared by spiking plasma. The HPLC was performed on a Waters Acquity UPLC system using a reverse phase XBridge BEHC18 column (2.1 x 150 mm, 3.5 μM, Waters) at 300 μL min -1 The flow rate of 0.1% formic acid in water and 0.1% formic acid in acetonitrile was used for peptide separation. For detection, a Sciex API5500 mass spectrometer was used in positive ion mode, wherein the source temperature was 700°C, the ion spray voltage was 5500V, the curtain and nebulizer gas were 40 and the collision gas was in the middle. The residence time was 20ms, and the entrance potential for each conversion was 10V. The multiple reaction monitoring transformation of two unique replacement peptides of mAb2 skeleton was used to determine the concentration relative to the standard and control using the peak area of ​​the MQIII integration algorithm from Analyst software. The clearance rate and serum half-life were obtained from the non-compartmental model of the antibody concentration as a function of time using Phoenix software (Certara). All time points showing a sharp decline in concentration were excluded from the average plasma concentration, because (without being bound by any theory) the assumed target-mediated drug disposal (TMDD) and / or anti-drug antibodies (ADA) interfered.

[0326] Example 2: Octet screening of saturation point mutations in conditioned medium

[0327] FcRn is a heterodimer of a class I MHC-like α domain and a β2 macroglobulin (β2-m) subunit (common to most Fc receptors). Figure 1A), and recognizes a region on the antibody Fc heavy chain that is distinct from other FcγRs (see, e.g., Oganesyan et al. 2014 J. Biol. Chem. 289:7812-7824; and Shields et al. 2001 supra).

[0328] To identify variants with slower FcRn off-rates than the WT antibody, a biolayer interferometry (BLI)-based assay was designed to screen antibody variants in conditioned media in a high-throughput manner ( Figure 2A The assay was developed using several benchmark variants that either enhanced (AAA, LS, and YTE) or decreased (H435A, H310A / H435Q) affinity for FcRn at pH 6.0 compared to the WT antibody. The NiNTA biosensor captured the his-tagged antigen and each antibody variant was subsequently captured at pH 7.4 to simulate conditioned media ( Figure 2A For each of the six variants, the binding kinetics to rat FcRn (rFcRn) at pH 6.0 were measured ( Figure 2B ), which has a dissociation rate from human IgG1 that is approximately 25 times slower than that of human FcRn (hFcRn) and is more suitable for Octet studies. H435A ( Figure 2B , long dash with single dots) and H310A / H435Q( Figure 2B , long dash with two dots) variants showed little to no FcRn binding kinetics (see also, e.g., Shields et al. 2001 supra; Medesan et al. 1997 supra; and Raghavan et al. 1995 supra). Figure 2B , solid line) compared to AAA( Figure 2B , dash), LS( Figure 2B , dashes with single dots) and YTE( Figure 2B , long dash) variants all exhibited slower dissociation kinetics, with FcRn dissociation rates reduced by 2-7.3 fold. This suggests that Octet screening is suitable for distinguishing variants with disrupted rFcRn dissociation kinetics.

[0329] The IgG1 antibody mAb1 was used as a model system to generate a saturation mutagenesis library to screen for mutants with reduced FcRn off-rates. Eleven positions in the Fc region of mAb1 were selected based on their proximity to or direct contribution to the FcRn interface ( Figure 1A and Figure 1B) (see, 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 in Expi293 cells for expression. Conditioned media screening of saturated library mutants was performed as described above. Normalized FcRn binding Octet sensorgrams for a subset of variants are shown in Figure 2C (long dash), and wild type ( Figure 2C , thick long dash) and the mock negative control ( Figure 2C Normalized FcRn binding Octet sensorgrams ( , dashed line). The mimics showed a lack of observable FcRn binding. Several mutants clearly disrupted rFcRn binding as little signal change was observed in the kinetic curves ( Figure 2C , long dash, below the dotted line (mock). The cutoff for variants with improved FcRn off-rate was defined as three standard deviations below the mean of the WT antibody. Figure 2C Among the subset of mutations shown in Figure 2C , solid line) has a significantly reduced off-rate compared to the wild-type antibody ( Figure 2C , thick long dash), while the remaining variants have similar ( Figure 2C , dashes with single dots) or faster ( Figure 2C , long dash line above the dotted line (mock)).

[0330] The rFcRn dissociation rates of all single-point mutations were Figure 2D and Fig.14 In is shown by position and mutation. Fig.14 In , data were classified into one of four categories according to the fold change in rFcRn off-rate compared to wild type, and the wild type category is represented by black squares.

[0331] exist Fig.14 In the saturated library, the fold changes of all possible substituted rFcRn off-rates at eleven positions are normalized to the mean of the WT antibody and color-coded. All mutants fall into one of four categories: little or no binding (dark gray), faster rFcRn off-rate (gray), WT-like rFcRn off-rate (horizontal line) and slower rFcRn off-rate (grid). Multiple variants have a slower rFcRn off-rate (grid) than the WT antibody.

[0332] Fig.14 The dark grey mutants are compared with the mock ( Figure 2C, dashed line) showed little to no binding to rFcRn in a similar manner 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 variants ( Figure 2D and Fig.14 , light grey rectangles) destabilize the interaction with rFcRn, with approximately 50% located at each C H 2 and C H 3 domains. Twenty-five mutants had dissociation rates similar to WT ( Figure 2D and Fig.14 , white rectangle), of which eight of the 11 positions had at least one WT-like mutation ( Fig.14 , white rectangle). Compared with the wild type, the following mutations have significantly reduced rFcRn dissociation rates ( Figure 2D and Fig.14 , black rectangles): 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 a dissociation rate more than two times slower than that of the WT antibody ( Figure 2D These mutants were expressed and purified by protein A chromatography for further in vitro FcRn kinetic characterization.

[0333] Example 3: Biacore FcRn binding kinetics at pH 6.0

[0334] AAA, LS and YTE variants were used as positive controls for both 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 wild-type, benchmark ( Figure 3 ) and the leading ( Figure 4A and Figure 4B ), and the binding curves of single injections of human and rat FcRn are shown in Figure 5A and Figure 5B The wild-type antibody had binding affinities of 2380±470 nM and 207±43 nM for human and rat FcRn, respectively (Table 1).

[0335] Table 1: In vitro characterization parameters for purified lead antibodies of mAbl.

[0336]

[0337]

[0338]

[0339] All data shown in Table 1 were obtained using the experimental techniques indicated at the top of each column.

[0340] As a comparison with the kinetic constants obtained from screening of conditioned medium, the rFcRn dissociation rate obtained by Octet using purified protein was measured. The elution pH was determined in triplicate (n=3) by FcRn affinity chromatography, and thermal stability was detected by DSF in triplicate (n=3). The FcRn binding kinetics to human and rat FcRn were obtained from Biacore in duplicate (n=2) using a series of antibody concentrations, and fitted independently. Using Biacore, the steady-state binding reaction (RU) of each variant to human and rat FcRn at pH 7.4 was measured in triplicate (n=3) with 1000nM antibody. The units measured each time are as follows: Octet pH 6.0 rFcRn dissociation rate (x10 -3 s -1 ); elution pH (unitless); DSF T m (℃); Biacore pH 6.0 hFcRn association rate (x10 4 M -1 s -1 ), dissociation rate (x 10 -1 s -1 ) and K D,app (x 10 9 M); Biacore pH 6.0 rFcRn association rate (x10 4 M -1 s -1 ), dissociation rate (x10 -3 s -1 ) and K D,app (x10 9 M); and Biacore pH 7.4 steady-state binding responses (RU).

[0341] exist Figure 5B In the graph, AAA (dashed line), LS (dash line with two dots) and YTE (dash line with a single dot) variants have 1.6 to 10.4 fold enhanced binding affinity compared to WT. The properties of the benchmark variants with the tightest FcRn affinity are species specific, as LS has the tightest affinity for hFcRn, while rFcRn has a tighter affinity for YTE (Table 2A).

[0342] Table 2A: In vitro characterization parameters for purified bi-combination antibodies of mAbl.

[0343]

[0344]

[0345]

[0346] The vast majority of lead variants of human and rat FcRn ( Figure 5A and 5B , solid lines with various shading) had significantly slower (>2-fold) association rates than WT or the benchmark variants (Table 1). The N434F and N434Y mutations were the only variants that exhibited increased association rates for both FcRns. Without being bound by any theory, the apparent binding affinity of the lead variants was generally weaker than WT due to slower association kinetics with hFcRn, which is consistent with the apparent binding affinity of rFcRn ( Figure 5C and Figure 5D , Table 1). The affinity for rFcRn is weaker than that of YTE ( Figure 5D , diagonal line facing the lower left corner, Table 2A). Without being bound by any theory, these results suggest that a single mutation is insufficient to enhance affinity over the LS and YTE variants. Sorting the FcRn off-rates (due to the weak binding affinity of the variants to hFcRn) revealed a subset of reduced off-rates for human and rat FcRn: M252Y, N434F / P / Y, T256D / E, and T307A / E / F / Q / W (Table 2A). Combinations of these variants are further of interest to further improve the FcRn binding capacity of the Fc region over the benchmark variants.

[0347] In vitro characterization parameters of the lead variants are shown in Table 2B.

[0348] Table 2B: In vitro characterization parameters of lead variants.

[0349]

[0350]

[0351] 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 to human and rat FcRn were obtained in quadruplicate and fitted independently. Unit: DSF T m (℃); FcγRIIIa binding (fold change relative to WT), Biacore pH 6.0 hFcRn association rate (x10 4 M -1 s -1 ), dissociation rate (x10 -1 s -1 ) and K D,app (x109 M); Biacore pH 6.0rFcRn K D,app (x10 9 M); Biacore pH 7.4 hFcRn and rFcRn steady state RU (RU).

[0352] Example 4: Combination variants further reduce FcRn binding and dissociation rates

[0353] Multiple lead mutations at a single position ( Fig.14 , black rectangles), such as T307 and N434, where six and three mutations were identified, respectively, that showed slower FcRn dissociation kinetics. Only mutations with the slowest FcRn dissociation rates from hFcRn at these positions were used to generate combinatorial variants. 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 variants. The combinatorial library consists of 54 variants in total, including seven lead singles, 18 doubles, 20 triples, 8 quadruple variants and the WT antibody. The nomenclature of these variants is as follows: the wild-type background contains M252, T256, T307 and N434, and is relabeled as MTTN. Thus, the triple variant Y T QY Contains M252 Y 、T307 Q and N434 Y mutation while maintaining the WT threonine at position 256.

[0354] As with the single mutations, FcRn binding kinetics using Biacore at pH 6.0 were used to determine which combinatorial variants had improved affinity. Fig. 6A and Figure 6B Representative FcRn binding kinetic traces for each of the single (long dash interspersed with two dots), double (long dash interspersed with a single dot), triple (long dash interspersed with a single dot) and quadruple (short dash interspersed with a single dot) are shown in Figure 1, as compared to WT (dashed line) and benchmark variants with the tightest affinity for their respective species FcRn (hFcRn: LS (long dash interspersed with two dots); rFcRn: YTE (solid line)). hFcRn association and dissociation rates ( Figure 6C ) revealed that two single, 15 double, 18 triple, and eight quadruple variants had enhanced binding affinity over the LS variant ( Figure 6C , dashed line). Similarly, all combinations except one triple variant had tighter affinity for rFcRn than YTE ( Fig.6D, diagonal line facing 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 for hFcRn were all quadruple variants ( Figure 6C , with squares), its binding affinity was about 500 times higher than that of the wild type. A similar phenomenon did not occur with rFcRn ( Fig.6D ), because the variant with the highest affinity is the double variant ( Fig.6D , horizontal line). Triple ( Fig.6D , vertical lines) and quadruple ( Fig.6D , squared) variants generally showed only a slight decrease in dissociation rate (less than 2-fold), but also exhibited a decreased association rate ( Fig.6D Without being bound by any theory, these results suggest that there may be a lower limit on the apparent binding affinity for FcRn that can be achieved with rFcRn (about 0.5 nM), which does not exist for hFcRn ( Figure 6B ). In total, more than 40 combinatorial variants had tighter affinities than the benchmark variant and require further characterization to select combinations with favorable properties for in vivo studies.

[0355] Example 5: Combination variants retain significant binding at physiological pH

[0356] Due to 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 perturbation of mutations on pH dependence. Variants H435A and H310A / H435Q with weak FcRn binding did not bind to the column regardless of pH ( Fig. 8A WT was eluted at around physiological pH (pH 7.37 ± 0.05), while AAA, LS, and YTE required a higher pH (Table 2B). All combinatorial variants and the seven lead single variants required a higher pH than WT to elute from the affinity column ( Fig. 8A and Figure 8C ). The N434F / Y variants eluted at a higher pH than the LS (Table 2B), which, without being bound by scientific theory, suggests that these variants (alone and in combination) disrupt the pH dependency. Representative chromatograms show a clear shift in elution pH to higher ( Fig. 9A and Fig. 9B ). Strong correlation between elution pH and hFcRn dissociation rate (R2 = 0.94) ( Fig. 9C ) indicated that the slower FcRn dissociation rate at pH 6.0 directly contributed to the increased elution pH of the FcRn variants.

[0357] FcRn binding kinetics experiments were performed using Biacore at pH 7.4 to measure residue binding activity under physiological conditions. Steady-state RU was used as a measure of residue FcRn binding affinity, as some variants showed unreliable kinetics and little to no binding at this pH. Fig. 7A and Figure 7B Representative kinetic traces of single (long dash interspersed with two dots), double (long dash interspersed with a single dot), triple (long dash) and quadruple (short dash) variants are shown in FIG. Fig. 7A , solid line) and YTE( Figure 7B , solid line). These two variants exhibited maximum residue binding to human and rat FcRn at pH 7.4, respectively. Most lead single variants had slightly elevated FcRn binding compared to WT (4.3 ± 1.0 RU), but it was less than AAA (13.1 ± 1.7 RU), LS (18.5 ± 2.6 RU) and YTE (13.1 ± 1.6 RU), except for N434F / Y mutations (Table 2A and Table 2B). Combination variants also had significant residue binding to FcRn of both species at pH 7.4, to an even greater extent than N434F / Y ( Fig. 7A and Figure 7B Without being bound by any theory, ideal candidates for in vivo studies are variants that have increased FcRn binding at low pH but maintain low levels of binding at elevated pH in a manner similar to WT (such as AAA, LS, and YTE variants). Figure 7C and Fig.7D In the figure shown, these combinations will occupy the lower left quadrant designated by the affinity of the LS and YTE variants for human and rat FcRn at each pH, ​​respectively.

[0358] Example 6: FcRn affinity chromatography

[0359] The combinatorial variants showed a moderate 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)( Figure 7C and Fig.7D)。Not bound by any theory, these results suggest that higher affinity at pH 6.0 generally translates to greater FcRn binding of residues at pH 7.4. These variants can remain bound to FcRn in the bloodstream and have a short serum half-life and / or promote its clearance, similar to the high FcRn affinity abdeg mutants (see, e.g., Swiercz et al. 2014 supra; and Vaccaro et al. 2005 supra). Since the antibody-FcRn interaction is pH-dependent and occurs only at low pH (<pH 6.5), saturation mutations can enhance the interaction through contributions from hydrophobicity or charge that may disrupt the deprotonation of key histidine residues ( Figure 1B , as shown) and weaken the interaction at physiological pH.

[0360] FcRn affinity chromatography uses a linear pH gradient to directly measure perturbations in the pH-dependence of FcRn interaction (see, e.g., Schlothauer et al. 2013 supra). FcRn affinity chromatography using the AAA, LS, YTE, H435A, and H310A / H435Q variants revealed that H435A ( Fig. 8A , light gray solid line) and H310A / H435Q ( Fig. 8A , AQ, dark gray solid line) do not bind to FcRn even at pH 5.5 and elute in the effluent. Wild-type antibody elutes near physiological pH (pH 7.37 ± 0.05), while AAA, LS, and YTE (which have slower dissociation rates and tighter FcRn binding affinities than wild-type by Octet ( Figure 2A-2D ) and Biacore ( Figure 3 )) require considerably higher pH to dissociate from the column (AAA: 7.94 ± 0.06; LS: 8.29 ± 0.03; YTE: 8.14 ± 0.03). The elution curves revealed that all variants in the combinatorial library require higher pH than wild-type to elute from the affinity column. Representative chromatograms at the average elution pH of single (long dash with two dots), double (long dash with one dot), triple (long dash), and quadruple (short dash) variants are shown in Fig. 9A . Compared to WT, seven lead single variants require higher pH to dissociate from the column ( Fig. 10A , Table 3), while those variants with similar wild-type kinetics for hFcRn (K288D / N, Y436H / H / W) all elute at a pH similar to wild-type.

[0361] Table 3: In vitro characterization parameters of lead antibody variants

[0362]

[0363]

[0364]

[0365] All data were obtained using the experimental technique at the top of each column. Elution pH was determined by FcRn affinity chromatography in triplicate (n=3), and thermal stability was probed by DSF in triplicate (n=3). FcRn binding kinetics to human and rat FcRn were obtained from Biacore using a range of antibody concentrations (n=4) and fit independently. The units for each measurement are as follows: Elution pH (unitless); DSF Tm (°C); Biacore pH6.0hFcRn association rate (x10 4 M -1 s -1 ), dissociation rate (x10 -1 s -1 ) and K D,app (x10 9 M); Biacore pH 6.0 rFcRn association rate (x10 4 M -1 s -1 ), dissociation rate (x10 -3 s -1 ) and K D,app (x10 9 M).

[0366] The N434F / Y variants all eluted at higher pH than the LS variant (N434F: 8.30 ± 0.05; N434Y: 8.46 ± 0.02) and showed considerable FcRn binding at pH 7.4 (Table 4). These results suggest that these variants alone can disrupt pH dependence. In general, the average elution pH increased with increasing number of mutations that enhanced FcRn binding ( Fig. 9B Without being bound by any theory, the hFcRn dissociation rate showed a strong correlation with the elution pH (R 2 =0.94)( Fig. 9C ) suggests that disruption of the pH dependence of the interaction directly contributes to the slower FcRn off-rate observed for the combinatorial library at pH 6.0.

[0367] Example 7: Thermal stability

[0368] Most proteins (including antibodies) with low thermodynamic stability have an increased tendency to misfold and aggregate and will limit or hinder their activity, efficacy and potential as new therapeutic agents. The thermal stability of each variant was determined using DSF and the reported melting temperature (T m) is defined as the midpoint of the first transition in the Sypro Orange fluorescence intensity curve. m Compared with WT at 69.0±0.2℃, the LS variant was similar to WT (68.5±0.3℃), and AAA and YTE were thermally unstable with a difference of about 8℃ (AAA: 61.3±0.6℃; YTE: 61.2±0.3℃)( Figure 8B , Fig. 9B and Fig. 10B ; and Table 2B, Table 3 and Table 4). m Compared with WT and LS at 69.0 ± 0.2 °C, the DSF, AAA and YTE variants had lower thermal stability, with a difference of about 8 °C.

[0369] Table 4: In vitro characterization parameters of benchmark and lead combinations

[0370]

[0371]

[0372] Mutations introduced into the wild-type backbone are indicated in bold and underlined. All data were obtained using the experimental technique indicated at the top of each column. Elution pH and T were determined in triplicate (n=3). m . 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 reactions at pH 7.4 were measured in triplicate at a single antibody concentration using Biacore. FcγRIIIa binding affinities were determined in duplicate from a range of antibody concentrations using Biacore. The units for each measurement are as follows: Elution pH (unitless); DSF Tm (°C); Biacore pH6.0hFcRn association rate (x10 5 M -1 s -1 ), dissociation rate (x10 -2 s -1 ) and K D,app (x10 9 M); Biacore pH 6.0 rFcRn association rate (x10 5 M -1 s -1 ), dissociation rate (x10 -3 s -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).

[0373] Compared with wild type, 12 of 18 lead saturation variants had reduced T m , and several T307 mutants (T307E / F / M / Q) showed a slight stabilizing effect (Table 4). The seven single variants used in combination ( Fig. 10B and Table 4) were not significantly unstable relative to YTE ( Figure 8B and Table 5). Compared with the single variant ( Fig.9D , white circles), compared to adding double ( Fig.9D , horizontal line), triple ( Fig.9D , vertical lines) and quadruple ( Fig.9D , with squares) variants resulted in a further decrease in overall thermal stability. Many variants showed T values ​​lower than that of AAA or YTE (61.2 ± 0.℃). m , of which >60% of these variants contained T307W. The quadruple variant ( Fig.9D , with squares) show a clear bimodal distribution of melting temperatures, with combinations containing T307Q having approximately 6°C higher thermal stability than those with T307W ( Fig.9D ).

[0374] Example 8: Fc variants alter binding interactions with FcγRIIIa

[0375] In addition to the interaction with FcRn, the Fc region hinge and C H The C2 domain is responsible for the interaction with other Fc receptors, including FcγRIIIa. H The YTE2 domains are located far from the interaction interface, and their ability to interact with these receptors may be impaired relative to wild type. FcγRIIIa binding was measured using Biacore in a similar manner to FcRn binding at pH 7.4, revealing that YTE( Fig.11A , dark grey) variants show similarity to wild type ( Fig.11A , black) decreased by about 50% compared to the binding response of YTE. Without being bound by any theory, the decreased FcγRIIIa binding of YTE is due to the M252Y mutation ( Fig. 11B , lowest white circle), because this variant alone has a significantly reduced affinity for the receptor. The other single mutations do not have this reduced affinity ( Fig. 11B , white circles), and the N434F / Y variant alone enhanced binding by 16%-40%. These effects were transferred to most but not all of their corresponding combinations. For example, combinations containing M252Y had a 17% to 72% reduction in FcγRIIIa binding (Table 5).

[0376] Table 5: Concentrations of saturating library variants in conditioned media

[0377]

[0378] A variant M DQF ( Fig. 11B , the highest in the triple variant class) showed a significant increase of 140% in FcγRIIIa binding. Thus, the combinatorial saturation library provides variants with a variety of Fc receptor functions that can be used to tailor therapeutic antibodies with specific effector functions.

[0379] Fig. 11C Shown are box plots of the FcγRIIIa binding responses of the seven lead single variants compared to WT and YTE variants.

[0380] Example 9: Seven lead combinations balance the pH dependence of FcRn interactions

[0381] Without being bound by any theory, candidate variants for further in vivo studies occupy Figure 7C and Fig.7D Seven variants met these criteria for hFcRn and included five double and two triple combinations (M DQ N, M DW N. YD TN, YE TN, Y T W N. YQ N and YEQ N) 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), where YQ N eluted at the highest pH of 8.51±0.14 ( Fig. 12A , Table 5), which showed only a slight perturbation in pH dependence and greater residue binding at pH 7.4 (Table 2A). One of the variants (M DQ N) had wild-type-like thermal stability, and six variants had similar or reduced T compared to the YTE variant. m ( Fig. 12B , Table 4). In the FcγRIIIa binding assay, five combination variants showed a similar reduction to YTE (Table 4). Further investigation of the single mutations revealed that M252Y significantly affected FcγRIIIa binding, and without being bound by any theory, this effect translated to the combination with this mutation. The remaining six single mutations were similar to WT or had slightly improved binding to the receptor.

[0382] Based on their FcRn binding properties, thermal stability, and FcγRIIIa binding, three combinatorial variants were selected for further study. DQ (T256D / T307Q), DW (T256D / T307W), and YD (M252Y / T256D) each provided the best FcRn binding properties (Table 2B) as LS variants ( Fig.12E Each variant provides a different range of thermal stability and FcγRIIIa binding properties ( Fig.12F and Figure 12G , Table 2B), which provides a range of functions. Fig.12H It is a diagram of a homogeneous bridged RF.

[0383] Respectively with LS( Fig.13A , thick long dash) and the YTE variant ( Fig. 13B , thick long dashed line), the enhancement in apparent binding affinity to human and rat FcRn at pH 6.0 is a compromise between association and dissociation rates ( Fig.13A and Fig. 13B , Table 4). In general, combinations with faster off-rates also have faster on-rates and vice versa. This observation was maintained between human and rat FcRn (Table 4). In addition, all of these variants had a higher affinity for hFcRn at pH 7.4 than the LS variant ( Fig. 13C , thick long dash) lower steady-state response. These results are inconsistent with rFcRn, because the five variants containing M252Y at pH 7.4 were significantly lower than those containing YTE. YD TN, YE TN, Y T W N. YQ N and YEQ N had increased FcRn binding (Figure 13, Table 5). DQ N and M DW The N variant was the only combination that cross-reacted between human and rat FcRn. In addition, these two variants did not perturb the interaction with FcγRIIIa to a similar extent as the variant containing M252Y ( Fig. 12C and Fig.12D And Table 5; M DQ N: 600 ± 4 nM; MDWN: 512 ± 30 nM; WT: 467 ± 99 nM). Thus, saturation and combinatorial mutagenesis at key FcRn interaction positions have led to the identification of lead variants that balance the pH dependence of the interaction, maintain function with the Fc receptor, can enhance FcRn function in vivo, and can extend the serum half-life of therapeutic antibodies.

[0384] Example 10: Rheumatoid Factor Binding Characteristics of Lead Combination Variants

[0385] The isoelectric point and RF binding of the lead variants were studied as these mutations may alter antibody surface charge and immunogenicity. More acidic antibodies have been thought to prolong antibody pharmacokinetics. All three leads resulted in a decrease in pI of approximately 0.2 pH units due to the T256D substitution compared to WT and LS controls. Mutations that enhance FcRn may simultaneously alter binding to host antibodies such as rheumatoid factor (RF) due to overlapping interaction interfaces. A homogenous bridging ELISA was suitable for measuring changes in RF binding of the lead variants. Interestingly, LS and YTE showed completely opposite changes in RF binding compared to WT ( Fig.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 decreased RF binding, while DQ produced a similar response to WT. Without being bound by any theory, these results suggest that DQ, DW and YD may provide an immunogenicity advantage over LS. The YD, DW and DQ variants represent a series of key antibody features that can be used in combination with improved FcRn binding properties relative to the benchmark YTE and LS variants.

[0386] Example 11: Lead combinatorial variants can be transferred to other antibodies

[0387] New binding assays were developed using the CM5 sensor chip, such as Fig.15A The binding assay included the steps of immobilizing streptavidin on a CM5 sensor chip to capture biotinylated FcRn to achieve approximately 30 RU (replenished as needed). Antibody binding kinetics were measured at pH 6.0 and 7.4 and regeneration at pH 8.5. Fig. 15B and Fig. 15C Direct immobilization of FcRn and streptavidin capture of biotinylated FcRn, respectively, are shown using the new binding assay.

[0388] FcRn binding of Antibody 2 at pH 6.0: In the case of mouse FcRn, the lead Antibody 2 variant showed a slower off-rate than the LS variant (underlined) and wild-type (black). Fig.16A For human FcRn, the lead variants all had faster association rates but similar dissociation rates to LS (underlined) ( Fig. 16B ).

[0389] FcRn binding of Antibody 2 at pH 7.4: All lead variants showed reduced human FcRn binding at pH 7.4 compared to LS (underlined). Fig.17A ). Same as Antibody 1 background, DW (M DW N) and DQ(M DQThe N) variant also showed binding to mouse (rat) FcRn at lower residues ( Fig. 17B ).

[0390] Compared to LS, the lead variants maintained higher binding affinity at pH 6.0 and lower residue binding at pH 7.4 ( Fig.18 Importantly, the variants were found to be transferable between different IgG1 backgrounds with little effect on FcRn binding. Fig.19 As shown, LS has a similar elution pH regardless of the background. WT, DQ, and DW in the Antibody 2 background showed a higher elution pH than in the Antibody 1 background, which may be the result of tighter binding at pH 6.0 in the Antibody 2 background.

[0391] The antibody 2 background variants all showed slightly increased thermal stability, e.g. Fig. 20 shown.

[0392] like Fig.21 As shown, similar to the background of antibody 1, YD( YD TN) shows the reduction in FcγRIIIa binding response (left) and affinity (right). DQ (light grey) and DW (dark grey) show FcγRIIIa binding properties similar to WT (black) in the context of Antibody 2. The effect on FcγRIIIa binding of LS is consistent between Antibody 1 and Antibody 2.

[0393] Thus, the lead variants in the Antibody 2 context did not significantly affect FcRn binding, pH dependence, thermal stability, or FcγRIIIa binding compared to the same lead variants in the Antibody 1 context.

[0394] In one embodiment, the DQ (T256D / T307Q), DW (T256D / T307W), and YD (M252Y / T256D) variants were incorporated into additional IgG1 antibodies and recombinant Fc fragments: mAb2 recognized a different antigen from mAb1, and Ab3 was an Fc fragment. In each case, in addition to elution pH, thermal stability, and FcγRIIIa binding affinity, pH-dependent FcRn binding kinetics ( Fig. 22 ) are also highly similar (Table 2B and Table 6). Without being bound by any theory, these results suggest that the DQ, DW and YD variants confer improved FcRn binding properties to proteins consisting of an Fc domain.

[0395] Table 6: Concentrations of saturating library variants in conditioned media

[0396]

[0397]

[0398] Example 12: Lead variants extend plasma antibody elimination half-life in vivo

[0399] The effects of the DQ, DW, and YD variants on antibody circulating half-life in cynomolgus monkeys and hFcRn transgenic mice (strain Tg32) were examined for pharmacokinetics (PK) compared to WT and LS controls (see, e.g., Avery et al. Mabs (2016) 8: 1064-1078). FcRn binding studies with cynomolgus monkey FcRn revealed similar binding affinity to hFcRn ( FIG. 23A to FIG. 23B ; Table 6). Each animal was injected intravenously with WT, LS, DQ, DW or YD variants, and the antibody concentration was quantified by mass spectrometry to determine the monkey ( Fig.24A ) and hFcRn transgenic mice ( Fig. 24B ) in the clearance rate and serum half-life. Obtain clearance rate and serum half-life as a function of time from a non-compartmental model of antibody concentration. In both cynomolgus monkeys and mice, all three lead variants and LS show a clearance rate significantly reduced compared to WT (p < 0.001). For monkeys and mice, the plasma half-life of WT antibodies is 9.9 ± 0.5 days and 11.7 days, respectively. In addition, compared with the wild-type in two species, the identified LS benchmarks and variants show a significant increase in elimination half-life (increased by 2.5 times and 1.7 times, respectively, in monkeys and mice) (Table 7). Compared with the LS benchmarks, DQ, DW and YD show similar half-life extensions (Table 7). The DQ, DW and YD mutations identified herein by saturation mutagenesis show that in mice and non-human primate models, the plasma half-life is significantly extended compared to its WT counterpart.

[0400] Table 7: Clearance and serum half-life of baseline and lead variants

[0401]

[0402] In Table 7, clearance and plasma half-life were determined using mAb2. Each clearance and half-life is an average of n=3 in the case of cynomolgus monkeys and a single assessment from a pool of n=6 hFcRn transgenic mice. The fold relative to WT and the fold relative to LS show the relative improvement in serum half-life compared to WT and LS, respectively. *n=2 due to ADA formation, **n=2 due to partial subcutaneous administration route

[0403] Example 13: Combinatorial variants with enhanced FcRn binding at pH 6.0 and pH 7.4

[0404] Based on the Octet screening (BLI-based screening) as described in Example 2, various single, double, triple and quadruple variants were generated and evaluated for binding to FcRn at pH 6.0 and pH 7.4 (Table 8).

[0405] Table 8: Binding affinity (pH 6.0) and steady-state binding (pH 7.4) of variants

[0406]

[0407]

[0408]

[0409]

[0410] In Table 8, the binding affinity to FcRn at pH 6.0 and the steady state binding to FcRn at pH 7.4 are shown for various single, double, triple and quadruple mutants as well as the benchmark variants (AAA, LS, YTE).

[0411] Plot these values ​​on Fig.25 middle. Fig.25 Comparison of binding affinity at pH 6.0 and RU at pH 7.4 is shown. As shown, the benchmark variant LS has the tightest binding affinity at pH 6.0, and the benchmark variants tested (AAA, LS, YTE) have the greatest residue binding at pH 7.4.

[0412] Sure Fig.25 Several combinatorial variants shown in show enhanced FcRn binding affinity at pH 6.0 and pH 7.4. To investigate whether any combinatorial variant shows tighter binding than the MST-HN variant (referred to herein as the "YTEKF benchmark", which contains mutations from Met252, Ser254, Thr256, His433, and Asn434 to Tyr252, Thr254, Glu256, Lys433, and Phe434) at pH 6.0 and pH 7.4, the following method was performed. Capture of biotinylated human, cynomolgus monkey, and mouse FcRn was performed via the biotin CAPture method (see Fig.26 Schematic diagram of the assay. For pH 6.0, a concentration series from 1000 nM was performed in duplicate (5 pts). For pH 7.4, a single concentration (1000 nM) injection was performed in triplicate (the capture level of each FcRn was increased 10-fold to observe binding at this pH). Association: 180 sec; Dissociation: 300 sec.

[0413] Fig. 27 The human FcRn binding kinetics of the YTEKF benchmark and various combinatorial variants at pH 6.0 are shown. Fig. 27As shown, all variants tested displayed two orders of magnitude tighter affinity to human FcRn compared to wild type (WT).

[0414] Fig.28A and Fig.28B It is shown that at pH 6.0 ( Fig.28A ) and pH 7.4 ( Fig.28B ) FcRn binding kinetics of the combined variants compared to the YTEKF benchmark. Fig.28A Most variants showed slower off-rates than the YTEKF benchmark and had similar or slower on-rates. Fig.28B Among them, YTEKF showed significant binding at pH 7.4, and the four variants showed higher binding of the residues.

[0415] Table 9: Binding affinity (pH 6.0) and steady-state binding (pH 7.4) of selected variants

[0416]

[0417] In Table 9, the binding affinity of select combinatorial variants as well as the YTEKF benchmark and WT to FcRn at pH 6.0 and the steady state binding to FcRn at pH 7.4 are shown.

[0418] Fig.29 A comparison of the binding affinity of selected combinatorial variants at pH 6.0 and RU at pH 7.4 is shown as shown in Table 9. Fig.29 As shown, it was found that the four quadruple variants had higher affinity for FcRn at pH 6.0 and pH 7.4 compared to the YTEKF benchmark. The four quadruple variants favored T256D, T307Q and N434Y mutations. Relative to WT and YTEKF, these quadruple variants showed an affinity (at pH 6.0) that was increased by approximately 500 times and 3 times, respectively.

[0419] Other characterization parameters such as thermal stability, binding to FcγRIIIa and elution pH were determined and are shown in Table 10.

[0420] Table 10: Additional characterization parameters of lead quartet variants

[0421]

[0422]

[0423] * Without being bound by theory, due to the presence of "YTE" in YTEKF, thermal stability and FcγRIIIa binding are expected to be similar to the lead quadruple variant.

[0424] As shown in Table 10, all lead quadruple variants were found to be thermolabile and displayed reduced FcγRIIIa binding ability.

[0425] The present disclosure relates to the following embodiments:

[0426] 1. An isolated binding polypeptide comprising a modified Fc domain, the modified Fc domain comprising:

[0427] aspartic acid (D) or glutamic acid (E) at amino acid position 256, and / or tryptophan (W) or glutamine (Q) at amino acid position 307, wherein amino acid position 254 is not threonine (T), and further comprising:

[0428] Phenylalanine (F) or Tyrosine (Y) at amino acid position 434; or

[0429] Tyrosine (Y) at amino acid position 252,

[0430] The amino acid positions are numbered according to EU.

[0431] 2. An isolated binding polypeptide comprising a modified Fc domain comprising a combination of amino acid substitutions at positions selected from:

[0432] a) tyrosine (Y) at amino acid position 252 and aspartic acid (D) at amino acid position 256;

[0433] b) aspartic acid (D) at amino acid position 256 and phenylalanine (F) at amino acid position 434;

[0434] c) aspartic acid (D) at amino acid position 256 and tyrosine (Y) at amino acid position 434;

[0435] d) tryptophan (W) at amino acid position 307 and phenylalanine (F) at amino acid position 434;

[0436] e) a tyrosine (Y) at amino acid position 252 and a tryptophan (W) at amino acid position 307, wherein the tyrosine (Y) is not at amino acid position 434;

[0437] f) aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434;

[0438] g) aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434;

[0439] h) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434; and

[0440] i) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, and glutamine (Q) at amino acid position 307, wherein threonine (T) is not at amino acid position 254, histidine (H) is not at amino acid position 311, and tyrosine (Y) is not at amino acid position 434;

[0441] The amino acid substitutions are according to EU numbering.

[0442] 3. An isolated binding polypeptide comprising a modified Fc domain comprising:

[0443] a) a double amino acid substitution selected from M252Y / T256D, M252Y / T256E, M252Y / T307Q, M252Y / T307W, T256D / T307Q, T256D / T307W, T256E / T307Q, and T256E / T307W, wherein the threonine (T) is not at amino acid position 254, the histidine (H) is not at amino acid position 311, and the tyrosine (Y) is not at amino acid position 434; or

[0444] 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 the threonine (T) is not at amino acid position 254, the histidine (H) is not at amino acid position 311, and the tyrosine (Y) is not at amino acid position 434;

[0445] The amino acid substitutions are according to EU numbering.

[0446] 4. An isolated binding polypeptide according to any one of items 1-3, wherein the modified Fc domain is a modified human Fc domain.

[0447] 5. The isolated binding polypeptide according to any one of items 1-4, wherein the modified Fc domain is a modified IgG1 Fc domain.

[0448] 6. The isolated binding polypeptide according to any one of items 1-5, wherein the binding polypeptide has human FcRn binding affinity.

[0449] 7. The isolated binding polypeptide according to any one of items 1-5, wherein the binding polypeptide has rat FcRn binding affinity.

[0450] 8. An isolated binding polypeptide according to any one of items 1-7, wherein the binding polypeptide has binding affinity to human and rat FcRn.

[0451] 9. An isolated binding polypeptide according to any one of items 2-8, wherein the isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0452] 10. An isolated binding polypeptide according to claim 9, wherein the isolated binding polypeptide has an increased serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0453] 11. An isolated binding polypeptide according to any one of items 1-8, wherein the isolated binding polypeptide has altered FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0454] 12. An isolated binding polypeptide according to claim 11, wherein the isolated binding polypeptide has enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0455] 13. An isolated binding polypeptide according to any one of items 1-12, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain.

[0456] 14. An isolated binding polypeptide according to any one of items 1 to 13, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at an elevated non-acidic pH.

[0457] 15. An isolated binding polypeptide according to any one of items 12-14, wherein the enhanced FcRn binding affinity comprises a decreased FcRn binding off-rate.

[0458] 16. An isolated binding polypeptide according to any one of items 13-15, wherein the acidic pH is about 6.0.

[0459] 17. An isolated binding polypeptide according to any one of items 13-16, wherein the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0460] 18. An isolated binding polypeptide according to any one of items 1 to 17, wherein the isolated binding polypeptide has altered FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0461] 19. An isolated binding polypeptide according to any one of items 1 to 18, wherein the isolated binding polypeptide has reduced FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0462] 20. An isolated binding polypeptide according to any one of items 1-18, wherein the isolated binding polypeptide has enhanced FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0463] 21. An isolated binding polypeptide according to any one of items 1-18, wherein the isolated binding polypeptide has about the same FcγRIIIa binding affinity as a binding polypeptide comprising a wild-type Fc domain.

[0464] 22. An isolated binding polypeptide according to any one of items 1-21, wherein the isolated binding polypeptide has approximately the same thermal stability as a binding polypeptide comprising a wild-type Fc domain.

[0465] 23. An isolated binding polypeptide according to any one of items 1-21, wherein the isolated binding polypeptide has approximately the same thermal stability as a binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain has a triple amino acid substitution M252Y / S254T / T256E according to EU numbering.

[0466] 24. An isolated binding polypeptide according to any one of items 1-23, wherein the isolated binding polypeptide is an antibody.

[0467] 25. The isolated binding polypeptide according to any one of items 1-24, wherein the isolated binding polypeptide is a monoclonal antibody.

[0468] 26. An isolated binding polypeptide according to any one of items 24-25, wherein the isolated antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0469] 27. An isolated binding polypeptide according to any one of items 24-26, wherein the isolated antibody is a full-length antibody.

[0470] 28. An isolated binding polypeptide according to any one of items 1-27, wherein the isolated binding polypeptide specifically binds to one or more human targets.

[0471] 29. An isolated nucleic acid molecule comprising a nucleic acid encoding the isolated polypeptide according to any one of items 1-28.

[0472] 30. A vector comprising the isolated nucleic acid molecule according to item 29.

[0473] 31. A vector according to claim 30, wherein the vector is an expression vector.

[0474] 32. A host cell comprising the vector according to any one of items 30-31.

[0475] 33. A host cell according to item 32, wherein the host cell is of eukaryotic or prokaryotic origin.

[0476] 34. A host cell according to any one of items 32-33, wherein the host cell is of mammalian origin.

[0477] 35. A host cell according to any one of items 32-33, wherein the host cell is of bacterial origin.

[0478] 36. A pharmaceutical composition comprising the isolated binding polypeptide according to any one of items 1-28.

[0479] 37. A pharmaceutical composition comprising the isolated antibody according to any one of items 24-27.

[0480] 38. An isolated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307 according to EU numbering.

[0481] 39. An isolated binding polypeptide 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.

[0482] 40. An isolated binding polypeptide 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.

[0483] 41. An isolated binding polypeptide according to any one of items 38-40, wherein the modified Fc domain is a modified human Fc domain.

[0484] 42. An isolated binding polypeptide according to any one of items 38-41, wherein the modified Fc domain is a modified IgG1 Fc domain.

[0485] 43. An isolated binding polypeptide according to any one of items 38-42, wherein the binding polypeptide has human FcRn binding affinity.

[0486] 44. An isolated binding polypeptide according to any one of items 38-42, wherein the binding polypeptide has rat FcRn binding affinity.

[0487] 45. An isolated binding polypeptide according to any one of items 38-44, wherein the isolated binding polypeptide has an increased serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0488] 46. ​​An isolated binding polypeptide according to any one of items 38-44, wherein the isolated binding polypeptide has enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0489] 47. An isolated binding polypeptide according to any one of items 38-44, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain.

[0490] 48. An isolated binding polypeptide according to any one of items 38-47, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at an elevated non-acidic pH.

[0491] 49. An isolated binding polypeptide according to any one of items 46-48, wherein the enhanced FcRn binding affinity comprises a reduced FcRn binding dissociation rate.

[0492] 50. An isolated binding polypeptide according to item 47 or 48, wherein the acidic pH is about 6.0.

[0493] 51. An isolated binding polypeptide according to item 47, wherein the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0494] 52. An isolated binding polypeptide according to claim 48, wherein the acidic pH is about 6.0.

[0495] 53. An isolated binding polypeptide according to any one of items 38-52, wherein the isolated binding polypeptide has altered FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0496] 54. An isolated binding polypeptide according to any one of items 38-53, wherein the isolated binding polypeptide is a monoclonal antibody.

[0497] 55. An isolated binding polypeptide according to any one of items 38-54, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0498] 56. An isolated binding polypeptide according to any one of items 38-55, wherein the isolated binding polypeptide specifically binds to one or more human targets.

[0499] 57. An isolated nucleic acid molecule comprising a nucleic acid encoding an isolated polypeptide according to any one of items 38-56.

[0500] 58. An expression vector comprising the isolated nucleic acid molecule according to item 57.

[0501] 59. A host cell comprising the expression vector according to item 58.

[0502] 60. A pharmaceutical composition comprising the isolated binding polypeptide according to any one of items 38-58.

[0503] 61. An isolated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises a combination of at least four amino acid substitutions comprising:

[0504] aspartic acid (D) or glutamic acid (E) at amino acid position 256 and tryptophan (W) or glutamine (Q) at amino acid position 307, wherein amino acid position 254 is not threonine (T), and further comprising:

[0505] Phenylalanine (F) or tyrosine (Y) at amino acid position 434; and

[0506] Tyrosine (Y) at amino acid position 252,

[0507] The amino acid positions are numbered according to EU.

[0508] 62. An isolated binding polypeptide comprising a modified Fc domain having a combination of amino acid substitutions at positions selected from:

[0509] a) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434;

[0510] b) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434;

[0511] c) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434;

[0512] d) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434; or

[0513] e) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434,

[0514] The amino acid substitutions are according to EU numbering.

[0515] 63. An isolated binding polypeptide comprising a modified Fc domain comprising:

[0516] a quadruple amino acid substitution 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,

[0517] The amino acid substitutions are according to EU numbering.

[0518] 64. An isolated binding polypeptide according to any one of items 61-63, wherein the modified Fc domain is a modified human Fc domain.

[0519] 65. An isolated binding polypeptide according to any one of items 61-64, wherein the modified Fc domain is a modified IgG1 Fc domain.

[0520] 66. An isolated binding polypeptide according to any one of items 61-65, wherein the binding polypeptide has human FcRn binding affinity.

[0521] 67. An isolated binding polypeptide according to any one of items 61-65, wherein the binding polypeptide has rat FcRn binding affinity.

[0522] 68. An isolated binding polypeptide according to any one of items 61-67, wherein the binding polypeptide has binding affinity to human and rat FcRn.

[0523] 69. An isolated binding polypeptide according to any one of items 61-68, wherein the isolated binding polypeptide has an altered FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0524] 70. An isolated binding polypeptide according to items 61-69, wherein the isolated binding polypeptide has enhanced FcRn binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0525] 71. An isolated binding polypeptide according to any one of items 61-70, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain.

[0526] 72. An isolated binding polypeptide according to any one of items 61-71, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0527] 73. An isolated binding polypeptide according to any one of items 61-72, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising a wild-type Fc domain.

[0528] 74. An isolated binding polypeptide according to any one of items 61-73, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at non-acidic pH compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0529] 75. An isolated binding polypeptide according to any one of items 61-74, wherein 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.

[0530] 76. An isolated binding polypeptide according to any one of items 61-75, wherein 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.

[0531] 77. An isolated binding polypeptide according to any one of items 61-76, wherein the acidic pH is about 6.0.

[0532] 78. An isolated binding polypeptide according to any one of items 61-77, wherein the non-acidic pH is about 7.4.

[0533] 79. An isolated binding polypeptide according to any one of items 61-78, wherein the isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0534] 80. An isolated binding polypeptide according to any one of items 61-79, wherein the isolated binding polypeptide has a reduced serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0535] 81. An isolated binding polypeptide according to any one of items 61-80, wherein the isolated binding polypeptide has a reduced serum half-life compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0536] 82. An isolated binding polypeptide according to any one of items 61-81, wherein the isolated binding polypeptide has altered FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0537] 83. An isolated binding polypeptide according to any one of items 61-82, wherein the isolated binding polypeptide has reduced FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0538] 84. An isolated binding polypeptide according to any one of items 61-83, wherein the isolated binding polypeptide has reduced FcγRIIIa binding affinity compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0539] 85. An isolated binding polypeptide according to any one of items 61-84, wherein the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising a wild-type Fc domain.

[0540] 86. An isolated binding polypeptide according to any one of items 61-85, wherein the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0541] 87. An isolated binding polypeptide according to any one of items 61-86, wherein the isolated binding polypeptide is an antibody.

[0542] 88. An isolated binding polypeptide according to any one of items 61-87, wherein the isolated binding polypeptide is a monoclonal antibody.

[0543] 89. An isolated binding polypeptide according to any one of items 61-88, wherein the isolated antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0544] 90. An isolated binding polypeptide according to any one of items 61-89, wherein the isolated antibody is a full-length antibody.

[0545] 91. An isolated binding polypeptide according to any one of items 61-90, wherein the isolated binding polypeptide specifically binds to one or more targets.

[0546] 92. An isolated nucleic acid molecule comprising a nucleic acid encoding an isolated polypeptide according to any one of items 61-91.

[0547] 93. A vector comprising the isolated nucleic acid molecule according to item 92.

[0548] 94. A vector according to item 93, wherein the vector is an expression vector.

[0549] 95. A host cell comprising a vector according to any one of items 93-94.

[0550] 96. A host cell according to item 95, wherein the host cell is of eukaryotic or prokaryotic origin.

[0551] 97. A host cell according to any one of items 95-96, wherein the host cell is of mammalian origin.

[0552] 98. A host cell according to any one of items 95-96, wherein the host cell is of bacterial origin.

[0553] 99. A pharmaceutical composition comprising the isolated binding polypeptide according to any one of items 61-92.

[0554] 100. A pharmaceutical composition comprising the isolated antibody according to any one of items 87-90.

[0555] 101. An isolated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises 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.

[0556] 102. An isolated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises 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 according to EU numbering.

[0557] 103. An isolated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises 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 according to EU numbering.

[0558] 104. An isolated binding polypeptide comprising a modified Fc domain comprising tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434 according to EU numbering.

[0559] 105. An isolated binding polypeptide comprising 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.

[0560] 106. An isolated binding polypeptide according to any one of items 101-105, wherein the modified Fc domain is a modified human Fc domain.

[0561] 107. An isolated binding polypeptide according to any one of items 101-106, wherein the modified Fc domain is a modified IgG1 Fc domain.

[0562] 108. An isolated binding polypeptide according to any one of items 101-107, wherein the binding polypeptide has human FcRn binding affinity.

[0563] 109. An isolated binding polypeptide according to any one of items 101-108, wherein the isolated binding polypeptide has a reduced serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

[0564] 110. An isolated binding polypeptide according to any one of items 101-109, wherein the isolated binding polypeptide has a reduced serum half-life compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0565] 111. An isolated binding polypeptide according to any one of items 101-110, wherein 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.

[0566] 112. An isolated binding polypeptide according to any one of items 101-111, wherein 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.

[0567] 113. An isolated binding polypeptide according to item 111 or 112, wherein the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0568] 114. An isolated binding polypeptide according to any one of items 101-113, wherein the isolated binding polypeptide has reduced FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0569] 115. An isolated binding polypeptide according to any one of items 101-114, wherein the isolated binding polypeptide has reduced FcγRIIIa binding affinity compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0570] 116. An isolated binding polypeptide according to any one of items 101-115, wherein the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising a wild-type Fc domain.

[0571] 117. An isolated binding polypeptide according to any one of items 101-116, wherein the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0572] 118. An isolated binding polypeptide according to any one of items 101-117, wherein the isolated binding polypeptide is a monoclonal antibody.

[0573] 119. An isolated binding polypeptide according to item 118, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0574] 120. An isolated binding polypeptide according to any one of items 101-119, wherein the isolated binding polypeptide specifically binds to one or more targets.

[0575] 121. An isolated nucleic acid molecule comprising a nucleic acid encoding an isolated polypeptide according to any one of items 101-120.

[0576] 122. An expression vector comprising the isolated nucleic acid molecule according to item 121.

[0577] 123. A host cell comprising the expression vector according to item 122.

[0578] 124. A pharmaceutical composition comprising the isolated binding polypeptide according to any one of items 101-120.

[0579] 125. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated binding polypeptide according to any one of items 1-28, 38-58, 61-91 and 101-120, or administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of items 36-37, 60, 99-100 and 124.

[0580] 126. A method according to item 125, wherein the disease or condition is cancer.

[0581] 127. The method of claim 126, wherein the cancer is a tumor.

[0582] 128. A method according to item 125, wherein the disease or disorder is an autoimmune disorder.

[0583] 129. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated binding polypeptide according to any one of items 1-28 and 38-58, or administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of items 36, 37 and 60.

[0584] 130. A method of treating an autoimmune disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated binding polypeptide according to any one of items 61-91 and 101-120, or administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of items 99, 100 and 124.

Claims

1. An isolated binding polypeptide comprising a modified Fc domain, the modified Fc domain comprising: aspartic acid (D) or glutamic acid (E) at amino acid position 256, and / or tryptophan (W) or glutamine (Q) at amino acid position 307, wherein amino acid position 254 is not threonine (T), and further comprising: Phenylalanine (F) or Tyrosine (Y) at amino acid position 434; or Tyrosine (Y) at amino acid position 252, The amino acid positions are numbered according to EU.

2. An isolated binding polypeptide comprising a modified Fc domain comprising a combination of amino acid substitutions at positions selected from: a) tyrosine (Y) at amino acid position 252 and aspartic acid (D) at amino acid position 256; b) aspartic acid (D) at amino acid position 256 and phenylalanine (F) at amino acid position 434; c) aspartic acid (D) at amino acid position 256 and tyrosine (Y) at amino acid position 434; d) tryptophan (W) at amino acid position 307 and phenylalanine (F) at amino acid position 434; e) a tyrosine (Y) at amino acid position 252 and a tryptophan (W) at amino acid position 307, wherein the tyrosine (Y) is not at amino acid position 434; f) aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434; g) aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434; h) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307, wherein tyrosine (Y) is not at amino acid position 434; as well as i) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, and glutamine (Q) at amino acid position 307, wherein threonine (T) is not at amino acid position 254, Histidine (H) is not at amino acid position 311, and tyrosine (Y) is not at amino acid position 434; The amino acid substitutions are according to EU numbering.

3. An isolated binding polypeptide comprising a modified Fc domain comprising: a) a double amino acid substitution selected from M252Y / T256D, M252Y / T256E, M252Y / T307Q, M252Y / T307W, T256D / T307Q, T256D / T307W, T256E / T307Q, and T256E / T307W, wherein the threonine (T) is not at amino acid position 254, the histidine (H) is not at amino acid position 311, and the tyrosine (Y) is not 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 the threonine (T) is not at amino acid position 254, the histidine (H) is not at amino acid position 311, and the tyrosine (Y) is not at amino acid position 434; The amino acid substitutions are according to EU numbering.

4. The isolated binding polypeptide according to any one of claims 1-3, wherein the modified Fc domain is a modified human Fc domain.

5. The isolated binding polypeptide according to any one of claims 1-4, wherein the modified Fc domain is a modified IgG1 Fc domain.

6. The isolated binding polypeptide according to any one of claims 1-5, wherein the binding polypeptide has human FcRn binding affinity.

7. The isolated binding polypeptide of any one of claims 1-5, wherein the binding polypeptide has rat FcRn binding affinity.

8. The isolated binding polypeptide of any one of claims 1-7, wherein the binding polypeptide has both human and rat FcRn binding affinity.

9. The isolated binding polypeptide of any one of claims 2-8, wherein the isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

10. The isolated binding polypeptide of claim 9, wherein the isolated binding polypeptide has an increased serum half-life compared to a binding polypeptide comprising a wild-type Fc domain.

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