Fc variants with enhanced binding to fc rn and extended half-life

Abstract

The present invention relates to Fc variants with enhanced binding to FcRn and prolonged half-life. In particular, the present invention provides binding polypeptides (e.g., antibodies and immunoadhesins) comprising modified Fc domains. The present invention also provides nucleic acids encoding the binding polypeptides, recombinant expression vectors, and host cells for making such binding polypeptides. The present invention also provides methods of using the binding polypeptides disclosed herein to treat disease.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201980015900.4, filed on January 25, 2019, entitled "Fc variant having enhanced binding with 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 January 26, 2018, which is incorporated herein by reference. Technical Field

[0004] This invention relates to Fc variants having enhanced binding to FcRn and an extended half-life. Specifically, the invention provides binding peptides (e.g., antibodies and immunoadhesins) comprising modified Fc domains; provides nucleic acids encoding the binding peptides, recombinant expression vectors, and host cells for preparing such binding peptides; and provides methods for treating diseases using the binding peptides disclosed in this invention. Background Technology

[0005] The interaction between antibodies and nascent Fc receptors (FcRn) is a key determinant of maintaining and prolonging the serum half-life of antibodies and other Fc-derived therapeutics. FcRn is a heterodimer of a class I MHC-like α-domain and a β2-macroglobulin (β2-m) subunit, which recognizes regions on the antibody Fc heavy chain that differ from other Fcγ receptors (FcγR). Although FcRn is expressed in a variety of tissues, it is thought to function primarily in the vascular endothelium, kidneys, and at the blood-brain barrier, where it is responsible for inhibiting IgG degradation, excretion, and initiating inflammatory responses, respectively.

[0006] Antibodies binding to FcRn are highly pH-dependent, and this interaction occurs only at low pH (pH < 6.5) with high affinity (high nanomolar to low micromolar), but not at physiological pH (pH approximately 7.4). When endosomes are acidified to pH less than 6.5, the interaction between IgG and FcRn becomes highly favorable and directly inhibits the degradation of FcRn-bound antibodies and promotes their recycling to the cell surface. Increasing pH weakens the interaction and promotes antibody release into the bloodstream.

[0007] Fc engineering using high-throughput mutagenesis has been widely used to identify variants that enhance FcRn binding affinity, as enhanced binding can lead to increased efficacy and reduced dose-rate of therapeutic antibodies, a direct result of prolonged serum half-life compared to wild-type IgG antibodies. However, variants with enhanced FcRn binding affinity can have unpredictable outcomes. For example, some IgG variants, such as N434W or P257I / Q311I, which show a significant increase in FcRn affinity at pH 6.0, have exhibited wild-type or severely reduced serum half-lives in cynomolgus monkeys and human FcRn (hFcRn) transgenic mice (see, for example, Kuo et al. 2011, ibid.; 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, for example, 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 number) variant has shown 10-fold enhancement in vitro, but exhibits antibody-dependent cell-mediated reduced cytotoxicity (ADCC) in vivo due to a 2-fold decrease in affinity for the FcγRIIIa receptor (see, for example, Dall'Acqua et al. 2002, ibid.).

[0008] Therefore, there is still a need for alternative Fc variants that have enhanced binding with FcRn and extended cyclic half-life. Summary of the Invention

[0009] This 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.

[0010] Therefore, in some aspects, it is provided to provide a separated binding polypeptide 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 position is in accordance with EU numbering.

[0011] In some exemplary embodiments, the modified Fc domain is a modified human Fc domain. In some exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0012] In some exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0013] In some exemplary embodiments, the isolated binding peptide has a modified serum half-life compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an increased serum half-life compared to a binding peptide containing a wild-type Fc domain.

[0014] In some exemplary embodiments, the isolated binding peptide has a modified FcRn binding affinity compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcRn binding affinity compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcRn binding affinity at acidic pH compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcRn binding affinity at acidic pH compared to the binding peptide's FcRn binding affinity at elevated non-acidic pH. In some exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate.

[0015] In some exemplary embodiments, the acidic pH is about 6.0. In some exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0016] In some exemplary embodiments, the isolated binding peptide has a modified FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has a reduced FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain.

[0017] In some exemplary embodiments, the isolated binding polypeptide has approximately the same FcγRIIIa binding affinity as the binding polypeptide containing the wild-type Fc domain.

[0018] In some exemplary embodiments, the isolated binding peptide has substantially the same thermal stability as a binding peptide comprising a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has substantially the same thermal stability as a binding peptide comprising a modified Fc domain having triple amino acid substitutions M252Y / S254T / T256E according to EU numbers.

[0019] In some exemplary embodiments, the isolated binding polypeptide is an antibody, such as a monoclonal antibody. In some exemplary embodiments, the isolated antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some exemplary embodiments, the isolated antibody is a full-length antibody.

[0020] In some exemplary embodiments, the isolated binding peptide specifically binds to one or more human targets.

[0021] In other aspects, a separated binding polypeptide comprising a modified Fc domain comprising a combination of amino acid substitutions selected from the following positions: 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) tyrosine (Y) at amino acid position 252 and tryptophan (W) at amino acid position 307, wherein 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) h) 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; i) 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 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, wherein the amino acid substitutions are in accordance with EU designations.

[0022] In some exemplary embodiments, the modified Fc domain is a modified human Fc domain. In some exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0023] In some exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0024] In some exemplary embodiments, the isolated binding peptide has a modified serum half-life compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an increased serum half-life compared to a binding peptide containing a wild-type Fc domain.

[0025] In some exemplary embodiments, the isolated binding peptide has a modified FcRn binding affinity compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcRn binding affinity compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcRn binding affinity at acidic pH compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcRn binding affinity at acidic pH compared to the binding peptide's FcRn binding affinity at elevated non-acidic pH. In some exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate. In some exemplary embodiments, the isolated binding peptide has a smaller FcRn binding affinity at non-acidic pH than a binding peptide containing a modified Fc domain having a double amino acid substitution M428L / N434S according to EU designation.

[0026] In some exemplary embodiments, the acidic pH is about 6.0. In some exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0027] In some exemplary embodiments, the isolated binding peptide has a modified FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has a reduced FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain.

[0028] In some exemplary embodiments, the isolated binding polypeptide has approximately the same FcγRIIIa binding affinity as the binding polypeptide containing the wild-type Fc domain.

[0029] In some exemplary embodiments, the isolated binding peptide has substantially the same thermal stability as a binding peptide comprising a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has substantially the same thermal stability as a binding peptide comprising a modified Fc domain having triple amino acid substitutions M252Y / S254T / T256E according to EU numbers.

[0030] In some exemplary embodiments, the isolated binding polypeptide is an antibody, such as a monoclonal antibody. In some exemplary embodiments, the isolated antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some exemplary embodiments, the isolated antibody is a full-length antibody.

[0031] In some exemplary embodiments, the isolated binding peptide specifically binds to one or more human targets.

[0032] In other respects, a separated 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 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, or b) The amino acid substitutions are 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, and the amino acid substitutions are in accordance with EU numbers.

[0033] In some exemplary embodiments, the modified Fc domain is a modified human Fc domain. In some exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0034] In some exemplary embodiments, the binding polypeptide has human FcRn binding affinity, rat FcRn binding affinity, or both human and rat FcRn binding affinity.

[0035] In some exemplary embodiments, the isolated binding peptide has a modified serum half-life compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an increased serum half-life compared to a binding peptide containing a wild-type Fc domain.

[0036] In some exemplary embodiments, the isolated binding peptide has a modified FcRn binding affinity compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcRn binding affinity compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcRn binding affinity at acidic pH compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcRn binding affinity at acidic pH compared to the binding peptide's FcRn binding affinity at elevated non-acidic pH. In some exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate. In some exemplary embodiments, the isolated binding peptide has a smaller FcRn binding affinity at non-acidic pH than a binding peptide containing a modified Fc domain having a double amino acid substitution M428L / N434S according to EU designation.

[0037] In some exemplary embodiments, the acidic pH is about 6.0. In some exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0038] In some exemplary embodiments, the isolated binding peptide has a modified FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has a reduced FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain.

[0039] In some exemplary embodiments, the isolated binding polypeptide has approximately the same FcγRIIIa binding affinity as the binding polypeptide containing the wild-type Fc domain.

[0040] In some exemplary embodiments, the isolated binding peptide has substantially the same thermal stability as a binding peptide comprising a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has substantially the same thermal stability as a binding peptide comprising a modified Fc domain having triple amino acid substitutions M252Y / S254T / T256E according to EU numbers.

[0041] In some exemplary embodiments, the isolated binding polypeptide is an antibody, such as a monoclonal antibody. In some exemplary embodiments, the isolated antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some exemplary embodiments, the isolated antibody is a full-length antibody.

[0042] In some exemplary embodiments, the isolated binding peptide specifically binds to one or more human targets.

[0043] In some aspects, a separated 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.

[0044] In some exemplary embodiments, the modified Fc domain is a modified human Fc domain. In some exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0045] In some exemplary embodiments, the binding polypeptide has human FcRn binding affinity or rat FcRn binding affinity or both human and rat FcRn binding affinity.

[0046] In some exemplary embodiments, the isolated binding peptide has an increased serum half-life compared to binding peptides containing a wild-type Fc domain.

[0047] In some exemplary embodiments, the isolated binding peptide has enhanced FcRn binding affinity compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has enhanced FcRn binding affinity at acidic pH compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has enhanced FcRn binding affinity at acidic pH compared to the binding peptide's FcRn binding affinity at elevated non-acidic pH. In some exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate.

[0048] In some exemplary embodiments, the acidic pH is about 6.0. In some exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0049] In some exemplary embodiments, the isolated binding peptide has a modified FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain.

[0050] In some exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In some exemplary embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0051] In some exemplary embodiments, the isolated binding peptide specifically binds to one or more human targets.

[0052] In some respects, an isolated nucleic acid molecule is provided, which contains a nucleic acid encoding the isolated polypeptide.

[0053] 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.

[0054] In some aspects, a host cell comprising the vector is provided. In other aspects, a host cell comprising the expression vector is provided.

[0055] In some exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In some exemplary embodiments, the host cell is of mammalian origin. In some exemplary embodiments, the host cell is of bacterial origin.

[0056] In some respects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.

[0057] In some respects, a pharmaceutical composition comprising the isolated antibody is provided.

[0058] In some aspects, a separated binding polypeptide comprising a modified Fc domain is provided, wherein the modified Fc domain comprises aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307 according to EU numbering.

[0059] In some exemplary embodiments, the modified Fc domain is a modified human Fc domain. In some exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0060] In some exemplary embodiments, the binding polypeptide has human FcRn binding affinity or rat FcRn binding affinity or both human and rat FcRn binding affinity.

[0061] In some exemplary embodiments, the isolated binding peptide has an increased serum half-life compared to binding peptides containing a wild-type Fc domain.

[0062] In some exemplary embodiments, the isolated binding peptide has enhanced FcRn binding affinity compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has enhanced FcRn binding affinity at acidic pH compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has enhanced FcRn binding affinity at acidic pH compared to the binding peptide's FcRn binding affinity at elevated non-acidic pH. In some exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate.

[0063] In some exemplary embodiments, the acidic pH is about 6.0. In some exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0064] In some exemplary embodiments, the isolated binding peptide has a modified FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain.

[0065] In some exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In some exemplary embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0066] In some exemplary embodiments, the isolated binding peptide specifically binds to one or more human targets.

[0067] In some respects, an isolated nucleic acid molecule is provided, which contains a nucleic acid encoding the isolated polypeptide.

[0068] 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.

[0069] In some aspects, a host cell comprising the vector is provided. In other aspects, a host cell comprising the expression vector is provided.

[0070] In some exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In some exemplary embodiments, the host cell is of mammalian origin. In some exemplary embodiments, the host cell is of bacterial origin.

[0071] In some respects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.

[0072] In some respects, a pharmaceutical composition comprising the isolated antibody is provided.

[0073] In some aspects, a separated binding polypeptide comprising a modified Fc domain is provided, wherein the modified Fc domain comprises tyrosine (Y) at amino acid position 252 and aspartic acid (D) at amino acid position 256 according to EU numbering.

[0074] In some exemplary embodiments, the modified Fc domain is a modified human Fc domain. In some exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0075] In some exemplary embodiments, the binding polypeptide has human FcRn binding affinity or rat FcRn binding affinity or both human and rat FcRn binding affinity.

[0076] In some exemplary embodiments, the isolated binding peptide has an increased serum half-life compared to binding peptides containing a wild-type Fc domain.

[0077] In some exemplary embodiments, the isolated binding peptide has enhanced FcRn binding affinity compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has enhanced FcRn binding affinity at acidic pH compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has enhanced FcRn binding affinity at acidic pH compared to the binding peptide's FcRn binding affinity at elevated non-acidic pH. In some exemplary embodiments, the enhanced FcRn binding affinity includes a reduced FcRn binding dissociation rate.

[0078] In some exemplary embodiments, the acidic pH is about 6.0. In some exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0079] In some exemplary embodiments, the isolated binding peptide has a modified FcγRIIIa binding affinity compared to a binding peptide containing a wild-type Fc domain.

[0080] In some exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In some exemplary embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0081] In some exemplary embodiments, the isolated binding peptide specifically binds to one or more human targets.

[0082] In some respects, an isolated nucleic acid molecule is provided, which contains a nucleic acid encoding the isolated polypeptide.

[0083] 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.

[0084] In some aspects, a host cell comprising the vector is provided. In other aspects, a host cell comprising the expression vector is provided.

[0085] In some exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In some exemplary embodiments, the host cell is of mammalian origin. In some exemplary embodiments, the host cell is of bacterial origin.

[0086] In some respects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.

[0087] In some respects, a pharmaceutical composition comprising the isolated antibody is provided.

[0088] In some aspects, a separated 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 comprising phenylalanine (F) or tyrosine (Y) at amino acid position 434; and tyrosine (Y) at amino acid position 252, wherein the amino acid positions are in accordance with EU numbers.

[0089] In some aspects, a separated binding polypeptide comprising a modified Fc domain having a combination of amino acid substitutions 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, glutamine (Q) 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 in accordance with EU designations.

[0090] In some aspects, a separated binding polypeptide comprising a modified Fc domain is provided, the modified Fc domain comprising a quadruple amino acid substitution selected from 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 substitution is in accordance with EU designations.

[0091] In some exemplary embodiments, the modified Fc domain is a modified human Fc domain. In some exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0092] In some exemplary embodiments, the binding polypeptide has human FcRn binding affinity. In some exemplary embodiments, the binding polypeptide has rat FcRn binding affinity. In some exemplary embodiments, the binding polypeptide has both human and rat FcRn binding affinity.

[0093] In some exemplary embodiments, the isolated binding peptide has a modified FcRn binding affinity compared to a binding peptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has an enhanced FcRn binding affinity compared to a binding peptide containing a wild-type Fc domain.

[0094] In some exemplary embodiments, the isolated binding peptide exhibits enhanced FcRn binding affinity at acidic pH compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide exhibits enhanced FcRn binding affinity at acidic pH compared to binding peptides containing M252Y / S254T / T256E / H433K / N434F.

[0095] In some exemplary embodiments, the isolated binding peptide exhibits enhanced FcRn binding affinity at non-acidic pH compared to binding peptides containing wild-type Fc domains. In some exemplary embodiments, the isolated binding peptide exhibits enhanced FcRn binding affinity at non-acidic pH compared to binding peptides containing M252Y / S254T / T256E / H433K / N434F.

[0096] In some exemplary embodiments, the isolated binding peptide exhibits enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH, compared to binding peptides containing wild-type Fc domains. In some exemplary embodiments, the isolated binding peptide exhibits enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH, compared to binding peptides containing M252Y / S254T / T256E / H433K / N434F.

[0097] In some exemplary embodiments, the acidic pH is about 6.0. In some exemplary embodiments, the non-acidic pH is about 7.4.

[0098] In some exemplary embodiments, the isolated binding peptide has a modified serum half-life compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has a reduced serum half-life compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has a reduced serum half-life compared to binding peptides containing M252Y / S254T / T256E / H433K / N434F.

[0099] In some exemplary embodiments, the isolated binding peptide has a modified FcγRIIIa binding affinity compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has a reduced FcγRIIIa binding affinity compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has a reduced FcγRIIIa binding affinity compared to binding peptides containing M252Y / S254T / T256E / H433K / N434F.

[0100] In some exemplary embodiments, the isolated binding peptide has reduced thermal stability compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has reduced thermal stability compared to binding peptides containing M252Y / S254T / T256E / H433K / N434F.

[0101] In some exemplary embodiments, the isolated binding polypeptide is an antibody. In some exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In some exemplary embodiments, the isolated antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some exemplary embodiments, the isolated antibody is a full-length antibody.

[0102] In some exemplary embodiments, the isolated binding peptide specifically binds to one or more targets.

[0103] In some respects, an isolated nucleic acid molecule is provided, which contains a nucleic acid encoding the isolated polypeptide.

[0104] In some respects, a carrier containing the isolated nucleic acid molecules is provided.

[0105] In some exemplary embodiments, the vector is an expression vector.

[0106] In some respects, a host cell comprising the vector is provided.

[0107] In some exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In some exemplary embodiments, the host cell is of mammalian origin. In some exemplary embodiments, the host cell is of bacterial origin.

[0108] In some respects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.

[0109] In some respects, a pharmaceutical composition comprising the isolated antibody is provided.

[0110] In some aspects, a separated binding polypeptide comprising a modified Fc domain is provided, said 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 tyrosine (Y) at amino acid position 434 according to EU numbers.

[0111] In some aspects, a separated binding polypeptide comprising a modified Fc domain is provided, said modified Fc domain comprising 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 according to EU numbers.

[0112] In some aspects, a separated binding polypeptide comprising a modified Fc domain is provided, said modified Fc domain comprising 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 according to EU numbers.

[0113] In some aspects, a separated binding polypeptide comprising a modified Fc domain is provided, said 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 numbers.

[0114] In some aspects, a separated binding polypeptide comprising a modified Fc domain is provided, said modified Fc domain comprising 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 according to EU numbers.

[0115] In some exemplary embodiments, the modified Fc domain is a modified human Fc domain. In some exemplary embodiments, the modified Fc domain is a modified IgG1 Fc domain.

[0116] In some exemplary embodiments, the binding polypeptide has human FcRn binding affinity.

[0117] In some exemplary embodiments, the isolated binding peptide has a reduced serum half-life compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has a reduced serum half-life compared to binding peptides containing M252Y / S254T / T256E / H433K / N434F.

[0118] In some exemplary embodiments, the isolated binding peptide exhibits enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH, compared to binding peptides containing wild-type Fc domains. In some exemplary embodiments, the isolated binding peptide exhibits enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH, compared to binding peptides containing M252Y / S254T / T256E / H433K / N434F.

[0119] In some exemplary embodiments, the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0120] In some exemplary embodiments, the isolated binding peptide has reduced FcγRIIIa binding affinity compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has reduced FcγRIIIa binding affinity compared to binding peptides containing M252Y / S254T / T256E / H433K / N434F.

[0121] In some exemplary embodiments, the isolated binding peptide has reduced thermal stability compared to binding peptides containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding peptide has reduced thermal stability compared to binding peptides containing M252Y / S254T / T256E / H433K / N434F.

[0122] In some exemplary embodiments, the isolated binding polypeptide is a monoclonal antibody. In some exemplary embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0123] In some exemplary embodiments, the isolated binding peptide specifically binds to one or more targets.

[0124] In some respects, an isolated nucleic acid molecule is provided, which contains a nucleic acid encoding the isolated polypeptide.

[0125] In some respects, an expression vector comprising the isolated nucleic acid molecule is provided.

[0126] In some respects, a host cell comprising the expression vector is provided.

[0127] In some respects, a pharmaceutical composition comprising the isolated binding polypeptide is provided.

[0128] In some respects, a method of treating a disease or condition in a subject with such need 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.

[0129] In some exemplary embodiments, the disease or condition is cancer. In some exemplary embodiments, the cancer is a tumor.

[0130] In some exemplary embodiments, the disease or condition is an autoimmune disease.

[0131] In some aspects, a method of treating cancer in a subject with this need 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.

[0132] In some aspects, a method for treating an autoimmune disease in a subject with such need 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. Attached Figure Description

[0133] The foregoing and other features and advantages of the invention will be more fully understood from the following detailed description of illustrative embodiments, taken in conjunction with the accompanying drawings.

[0134] Figure 1 The structure of FcRn that interacts with the IgG1 Fc region was depicted. Figure 1 A depicts the interaction between hFcRn and IgG1 Fc (pdb: 4n0u) showing a single Fc monomer (dark gray band), including glycosylation of a bar labeled as “glycan” in the complex with an α-domain (gray) and a β2-m (light gray) hFcRn subunit. Most of the antibody residues involved in the interaction with FcRn are located at C H 2-C H 3. The interface (dotted line) is directly adjacent to the ring and opposite to the glycosylation site. Figure 1 B describes the relative Figure 1 Figure A (left) is a schematic diagram of the surface of an IgG1 Fc crystal structure (pdb: 5d4q) rotated 75°. The FcRn binding interface contains C... H 2 and C HResidues in the 3-domain structure. The saturated library was constructed at eleven sites, shown as bars, as illustrated: M252; I253; S254; T256; K288; T307; K322; E380; L432; N434; and Y436. All of these residues are close to or in direct contact with the FcRn. The key histidine residues responsible for pH dependence (H310, H433, H435) aggregate on the surface near the sites of interest, as shown in the figure.

[0135] Figures 2A to 2D The Octet screening assay and results are described. Figure 2A The Octet screening assay is illustrated schematically. The NiNTA biosensor captures histidine-tagged antigens and subsequently captures antibody variants for use in rat FcRn (rFcRn) binding kinetics. Figure 2B The rFcRn binding kinetics of wild-type (solid line), T307A / E380A / N434A (AAA) variant (dash), LS (dash with single dot), YTE (dash), H435A (dash with single dot), and H310A / H435Q (dash with two dot) antibodies at pH 6.0 are plotted, aligned with the start of the rFcRn association phase. The H435A and H310A / H435Q variants show little or no FcRn binding. The YTE variant was detected as having the slowest FcRn dissociation rate in the Octet rFcRn binding assay. Figure 2C The diagram illustrates the normalization of FcRn binding kinetics at pH 6.0 for a subset of mutants obtained through Octet screening. Most mutants retained significant binding to rFcRn, but several mutants, similar to the mimic control (dotted lines), showed a complete loss of rFcRn binding (long dashed lines, below the dotted lines (mimic)). Two variants (solid lines) exhibited slower rFcRn dissociation rates than the wild-type antibody (thick long dashed lines). Figure 2D A scatter plot analysis depicting the rFcRn dissociation rates for all point mutations reveals rFcRn binding kinetics separated by residue positions. Saturated variants fall into one of four rFcRn dissociation rate schemes: no binding (not shown), faster binding (black), wild-type-like binding (white), and slower binding (grey). Eighteen mutants show significantly slower dissociation rates from rFcRn compared to the wild-type antibody (black dashed lines).

[0136] Figure 3The Biacore kinetics of baseline and wild-type variants with human and rat FcRn are plotted graphically at pH 6.0 and pH 7.4. Binding curves for all FcRn at a range of concentrations for wild-type (top left), AAA variant (top right), M428 / N434S(LS) variant (bottom left), and M252Y / S254T / T256E(YTE) variant (bottom right) are shown at pH 6.0 (first and third rows) and pH 7.4 (second and fourth rows). The AAA, LS, and YTE variants show a slower dissociation rate from FcRn than the wild-type antibody. Generally, the antibody binds rFcRn with approximately 10-fold increased affinity compared to the wild-type antibody. The LS variant exhibits the strongest affinity for hFcRn at pH 7.4 and the largest residue binding at pH 7.4, while rFcRn binds most tightly to the YTE variant.

[0137] Figure 4A The Biacore kinetics of lead saturated variants with human and rat FcRn at pH 6.0 are illustrated. FcRn binding kinetic trajectories for concentration series of 18 lead saturated variants are shown. M252Y, T256D, T256E, N434F, N434P, N434Y, T307A, T307E, T307F, T307Q, and T307W exhibit slower dissociation rates from both human and rat FcRn. The remaining variants are specific only for rat FcRn.

[0138] Figure 4B The binding kinetics of human FcRn with single saturated variants of WT, baseline, and lead at pH 6.0 are illustrated. FcRn binding sensing plots of human FcRn with a range of concentrations of WT, LS, YTE, and 18 saturated variants at pH 6.0 are also presented. Single saturated variants used for the combined library are indicated by underlining and bold.

[0139] Figures 5A to 5D Data were depicted for several variants that exhibited slower dissociation rates from both human and rat FcRn at pH 6.0. Figure 5A and Figure 5B Various variations of Biacore sensor maps were depicted. Figure 5A The dissociation rates of human FcRn in the YTE variant (long streaks with single dots), LS variant (long streaks with two dots), wild type (WT; dotted lines), and leader-saturated variant (leader; solid lines with various shades) at pH 6.0 are depicted. Figure 5A The normalized sensor plot is depicted, showing the improved hFcRn dissociation rate compared to WT. Figure 5BThe dissociation rates of rat FcRn for the AAA variant (dashed line), LS variant (dash line with two dots), YTE variant (dash line with one dot), wild type (solid line), and leader saturated variants (dash lines of different frequencies and thicknesses) at pH 6.0 are depicted. For clarity, representative injections of each of the 11 leader antibodies are shown. These single leader variants exhibit improved dissociation rate kinetics from human and rat FcRn compared to the wild type. Figure 5C and Figure 5D The association and dissociation rates of the antibody variants against human (lead saturation (white circle) and wild-type (black circle) antibodies were depicted using association and dissociation rates obtained from Biacore kinetic measurements. Figure 5C ) and rats ( Figure 5D Binding affinity plot of FcRn. Benchmark variants are shown: AAA (lower right diagonal), LS (dashed line), and YTE (lower left diagonal). Although FcRn dissociation rates are improved, most variants do not have a stronger affinity for human or rat FcRn due to slower association kinetics. Eleven variants exhibit slower dissociation rates from FcRn in both species.

[0140] Figures 6A to 6D Data depicting the combined effects of the leader saturation mutation further improve the FcRn dissociation rate and binding affinity. Figure 6A and Figure 6B Representative Biacore sensor plots depicting the dissociation rates of FcRn in humans and rats were depicted. Figure 6A Normalized sensor maps of human FcRn for representative variants of single (dash), double (light gray solid line), triple (gray solid line), and quadruple (black solid line) combination variants are depicted compared to wild type (dash line) and LS variant (long dash line with two dots). Figure 6B Normalized sensory maps of rat FcRn in representative variants of single (long dashed line with two dots), double (long dashed line with one dot), triple (long dashed line), and quadruple (short dashed line) combination variants were depicted compared to wild-type (dotted line) and YTE variants (solid line). The incorporation of multiple mutations significantly reduced the dissociation rate of FcRn and enhanced its binding affinity compared to the baseline variant. Figure 6C and Figure 6D The association rate was depicted as a human ( Figure 6C ) or rats ( Figure 6D The plot shows the combined saturated variants as a function of the dissociation rate of FcRn, revealing that most variants exhibit enhanced binding to FcRn at pH 6.0 compared to the baseline variant. The most tightly bound variants for human and rat FcRn are the tetrad and dual-combinations, respectively.

[0141] Figures 7A to 7D Data depicting the pH dependence of the interaction, showing that enhanced FcRn binding at pH 6.0 disrupts the interaction. Figure 7A and Figure 7B Depicting the wild type (dotted line) and LS variant ( Figure 7A (solid line) and YTE variant ( Figure 7B Representative sensor plots of Biacore FcRn binding kinetics for single (long dashed line with two dots), double (long dashed line with one dot), triple (long dashed line), and quadruple (short dashed line) combination variants at pH 7.4, compared to solid lines. Increasing the number of mutations enhancing FcRn binding results in greater residue binding at physiological pH, with most double, triple, and quadruple variants showing robust binding to both FcRn types. Figure 7C and Figure 7D The effects of all saturated variants on humans at pH 7.4 were described. Figure 7C ) or rats ( Figure 7D The steady-state RU of FcRn ( Equation 2 is plotted as a function of binding affinity at pH 6.0. Figure 7C The diagram shows a comparison of FcRn binding affinity at pH 7.4 versus pH 6.0. The lead combination with improved FcRn binding properties occupies the lower left quadrant defined by the LS baseline variant (diamond). Figure 7D In the study, the LS (rhomboid) and YTE (triangular) variants were used as cutoff values ​​for lead validation. These two variants exhibited the tightest binding affinity at pH 6.0 and the largest residue binding for human and rat FcRn, respectively, at pH 7.4. Figure 7C and Figure 7D The two figures show single (white circle), double (light gray circle), triple (dark gray circle), and quadruple (black circle) variants, as well as the YTE variant (triangle).

[0142] Figures 8A to 8B Data obtained from FcRn affinity chromatography and differential scanning fluorescence (DSF) assays of the baseline variant are depicted. Figure 8A Normalized elution curves were plotted for the WT (solid black line), AAA (dotted line), LS (long dashed line with two dots), YTE (long dashed line with a single dot), H435A (light gray solid line), and H310A / H435Q (AQ; dark gray solid line) variants. pH values ​​are shown at the top of the graph. Variants ineffective against FcRn binding (H435A, H310A / H435Q) did not bind to the column and eluted in the eluent (< 10 mL). AAA, LS, and YTE variants eluted at higher pH levels than the WT antibody. Figure 8BDSF curves were plotted for the WT (black), LS (grey), and YTE (dark gray) variants. Compared to WT and LS, YTE is unstable.

[0143] Figure 8C FcRn affinity column elution profiles for the seven lead single variants used in the combined variants were plotted compared to the WT and LS variants (vertical dashed lines). Two variants (N434F / Y) eluted at higher pH levels than LS, indicating a reduced pH dependence of FcRn interaction for variants containing these mutations.

[0144] Figures 9A to 9D Data depicting the combined variants significantly disrupted pH dependence and thermal stability were presented. Figure 9A Representative FcRn affinity chromatograms are depicted for single (long streaks with two dots), double (long streaks with a single dot), triple (long streaks), and quadruple variants (short streaks). Increasing the number of enhanced FcRn binding mutations shifts the elution towards higher pH values; LS variants (small vertical dashed lines). Figure 9B Box plots depicting elution pH for lead saturation and combined variants (including single (white circles), double (horizontal lines), triple (vertical lines), and quadruple (square) mutants) indicate a trend toward higher pH values ​​as the number of FcRn-enhancing mutants increases. Figure 9C The study showed a high correlation (R0) between the elution pH from FcRn affinity chromatography and the dissociation rate of hFcRn using Biacore. 2 = 0.94) revealed the loss of pH-dependent antibody-FcRn interaction, accompanied by improved FcRn dissociation kinetics. The AAA (lower right diagonal), LS (dotted line), and YTE (lower left diagonal) variants exhibited similar hFcRn dissociation rates and elution pH values ​​to the dual variants. Figure 9D The T values ​​obtained from the DSF of the combined saturated variants were depicted. m The box plots revealed that, compared to WT, single, or baseline variants, additional mutations that enhance FcRn binding destabilize the antibody.

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

[0146] Figures 11A to 11B Data depicting reduced FcγRIIIa binding in the combined variant containing M252Y are presented. Figure 11A The FcγRIIIa binding sensing maps shown for the WT (black), LS (grey), and YTE (dark gray) variants reveal a reduced binding response generated by the YTE variant. Figure 11B Box plots depicting the FcγRIIIa binding responses of baseline, single, and combined variants are shown. Variants with the M252Y mutation exhibit reduced binding responses to FcγRIIIa, including all tetrodovarians. Combinations with N434F / Y generally show enhanced responses to FcγRIIIa.

[0147] Figure 11C The FcγRIIIa binding response of seven single-lead variants was depicted compared to the WT and YTE variants (horizontal dashed lines). The M252Y mutation showed reduced FcγRIIIa binding compared to WT, while six variants showed similar or enhanced binding to the receptor as WT.

[0148] Figures 12A to 12D Data obtained from FcRn affinity chromatography, DSF, and FcγRIIIa binding of seven lead combination variants are depicted. Figure 12A FcRn affinity chromatography plots of seven lead combination variants were depicted compared to the wild-type antibody and the LS variant (vertical dashed and vertical solid lines, respectively). The elution pH of each lead variant was close to that of the LS variant. Figure 12B The DSF curves of the lead variants are shown compared to the YTE and wild-type variants (as indicated by the vertical dashed line). Six of the seven lead variants have T values ​​similar to or less stable than the YTE variant. mMDWN (long dash with two dots); YTWN (long dash); YDTN (solid line); YETN (long dash with a single dot); YDQN (dashed line); YEQN (short dash with a single dot). M DQ The N variant has similar T to the wild-type antibody (underlined). m . Figure 12C Biacore sensing plots depict the FcγRIIIa binding dynamics of seven leader variants compared to the wild type (larger dashed line) and the YTE variant (thick dashed line). Variants containing M252Y: YD TN (solid line) YDQ N (a short dash with a single dot) Y T W N (long dash) YE TN (long dash with interspersed dots) and YE QN (smaller dotted lines), where each has a reduced steady-state RU in a similar manner to YTE. Figure 12D Steady-state RUs for seven leader variants, wild-type, and YTE variants are shown. Only M... DW N and M DQ The N variant has a similar affinity for FcγRIIIa as the wild-type antibody.

[0149] Figures 12E to 12H Data depicting three lead variants showcasing a range of key antibody properties is presented. Figure 12E The FcRn affinity chromatography elution profiles for the DQ (solid line), DW (dashed line), and YD (stripe line) variants are shown compared to WT and LS (vertical dashed lines). Elution pH values ​​for each dual variant are shown between WT and LS. Figure 12F DSF fluorescence curves of the three variants compared to YTE and WT (vertical dashed line) revealed that YD (dash line) and DW (dash line) were slightly unstable compared to YTE, but DQ (solid line) was similar to WT. Figure 12G The binding sensing plots of FcγRIIIa are depicted compared to WT and YTE (horizontal dashed lines). YD (dash line) shows a binding response similar to YTE, while DQ (solid line) and DW (dashed line) show a slight decrease compared to WT. Figure 12H The following data are presented, showing that homobridged RF ELISA revealed three lead variants and YTE showed significantly reduced or WT-like RF binding, unlike LS. **p < 0.001, *p < 0.01.

[0150] Figures 13A to 13D Data depicting comparative FcRn binding kinetics of the lead conjugate variants at pH 6.0 and pH 7.4 are presented. Figure 13Aand Figure 13B The results at pH 6.0 show the comparison with wild type (dashed line) and LS (hFcRn). Figure 13A (thick long dash) or YTE (rFcRn, Figure 13B Compared to (thick underline), the lead combination variant is more effective for human FcRn ( Figure 13A ) or rat FcRn ( Figure 13B Biacore FcRn binding sensing plot. Despite altered association and dissociation rates, each combination variant exhibits a generally tighter binding affinity to the corresponding FcRn. Figure 13C and Figure 13D A Biacore FcRn sensing plot at pH 7.4 is shown. Each hFcRn lead variant exhibits similar or reduced steady-state FcRn binding reactivity compared to the LS variant. Only M... DQ N and M DW The N variant showed less rFcRn binding at pH 7.4 than the YTE variant.

[0151] Figure 14 This is a table depicting the Octet rFcRn binding and dissociation rates of saturated libraries according to certain implementation schemes. Wild-type (WT) and wild-type-like (WT-like) species are represented by white rectangles; WT species are shown in the figure. 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] Figures 15A to 15C A novel combined measurement developed using the CM5 sensor chip is described. Figure 15A This is a schematic diagram of the measurement. Figure 15B The direct fixation of FcRn is shown. Figure 15C Streptavidin capture of biotinylated FcRn is shown.

[0153] Figures 16A to 16B The binding of antibody 2 to FcRn at pH 6.0 was depicted. Figure 16A It depicts the human FcRn. Figure 16B The mouse FcRn was described.

[0154] Figures 17A to 17B The binding of antibody 2 to FcRn at pH 7.4 was depicted. Figure 17A It depicts the human FcRn. Figure 17B The mouse FcRn was described.

[0155] Figure 18The pH dependence of various antibody 2 variants is illustrated. The lead variant maintains higher binding affinity at pH 6 and lower residue binding than LS at pH 7.4.

[0156] Figure 19 A comparison of pH-dependent FcRn binding using the backbones of antibody 1 and antibody 2 was depicted.

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

[0158] Figure 21 A comparison of FcγRIIIa binding using the backbones of antibody 1 and antibody 2 is depicted.

[0159] Figure 22 Several plots were depicted showing the transfer of DQ, DW, and YD variants between IgG1 backbones. a through c depict normalized FcRn binding sensing plots at pH 6.0 in the three IgG1 backbones, where the WT (light gray), LS (dark gray), DQ (solid black), DW (dashed line), and YD (stripe) variants show similar kinetics at low pH. The DQ, DW, and YD variants have slightly faster association and dissociation rates than the LS variant, but maintain a tighter FcRn binding affinity. d through f depict FcRn binding sensing plots at pH 7.4; LS is the baseline variant (solid black). g through i depict a comparison of FcRn binding responses 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 (hollow circle), and YD (hollow square) variants. DQ, DW, and YD exhibited improved FcRn characteristics, with enhanced binding at pH 6.0 and minimal binding at pH 7.4.

[0160] Figures 23A to 23C The study showed that three leader variants in the mAb2 skeletal framework similarly improved binding to cynomolgus monkey FcRn. Figure 23A Normalized cFcRn binding sensitivities for WT (grey), LS (dark gray), DQ (solid black), DW (dashed line), and YD (stretched line) at pH 6.0 were plotted, showing binding kinetics and affinity similar to hFcRn. Figure 23B The cFcRn binding response of the three variants was depicted as significantly reduced at physiological pH; however, LS (dark gray) showed stronger binding than WT (gray) in a similar manner to hFcRn. Figure 23CThe cFcRn binding affinity of residues in WT (gray), LS (dark gray), DQ (solid black), DW (hollow circle), and YD (hollow cube) at pH 7.4 was compared with that at pH 6.0, revealing that all three variants maintained the improved FcRn binding properties observed for hFcRn.

[0161] Figures 24A to 24B The lead variant was shown to extend the serum half-life of the antibody. In cynomolgus monkeys (… Figure 24A ) and hFcRn transgenic mice ( Figure 24B In the pharmacokinetic curves of plasma antibody concentration as a function of time, for WT (black circle with a solid black line), LS (white circle with a dashed black line), DQ (light gray circle with a solid light gray line), DW (dark gray circle with a solid dark gray line), and YD (black circle with a dashed black line), the plasma antibody concentrations were plotted. Compared to WT, all three lead variants prolonged the antibody half-life.

[0162] Figure 25 A graph depicts the steady-state RU of all saturated variants for human FcRn as a function of binding affinity at pH 6.0. A comparison of FcRn binding affinity at pH 7.4 and at pH 6.0 is shown. Quadruple combinations exhibiting improved FcRn binding properties at both pH 6.0 and pH 7.4 are shown in boxes in the upper right quadrant of the graph. Single (white circle), dual (light gray circle), triple (dark gray circle), and quadruple (black circle) variants, as well as the baseline AAA, LS, and YTE variants, are shown (as illustrated).

[0163] Figure 26 A schematic diagram of the biotin CAPture method for capturing biotinylated FcRn is depicted.

[0164] Figure 27 A graph depicting the human FcRn binding kinetics at pH 6.0 for the YTEKF baseline and combined variants as shown is presented.

[0165] Figures 28A to 28B It was shown at pH 6.0 ( Figure 28A ) and pH 7.4 ( Figure 28B FcRn binding kinetics of the hybrid variant compared to the YTEKF baseline. Wild type is represented by a solid black line (WT), and the YTEKF baseline is represented by a dashed line.

[0166] Figure 29A graph was plotted showing the steady-state RU of the selected variants for human FcRn at pH 7.4 as a function of binding affinity at pH 6.0, compared to the YTEKF baseline. Several variants (lead quartet variants) showed enhanced binding affinity for human FcRn at both pH 6.0 and pH 7.4 relative to the YTEKF baseline. Detailed Implementation

[0167] This disclosure provides binding peptides (e.g., antibodies) having modified Fc neogenic receptor (FcRn) binding affinity. In some embodiments, the binding peptide comprises a modified Fc domain that enhances FcRn binding affinity compared to binding peptides comprising a wild-type (e.g., unmodified) Fc domain. This disclosure also provides nucleic acids encoding the binding peptide, recombinant expression vectors, and host cells for preparing the binding peptide, as well as pharmaceutical compositions comprising the binding peptides disclosed herein. Methods for treating diseases using the binding peptides of this disclosure are also provided.

[0168] The Fc domain of immunoglobulins participates in non-antigen binding functions and possesses several effector functions mediated by the binding of effector molecules (e.g., FcRn binding). Figure 1 As shown in Figure A, the Fc domain contains both a CH2 domain and a CH3 domain. Most residues involved in the interaction with FcRn are located at the C... H 2-C H 3-interface ( Figure 1 A (dashed line) is in the ring directly adjacent to and opposite to the glycosylation site. Figure 1 B shows a surface schematic of the IgG1 Fc crystal structure (pdb: 5d4q) and illustrates residues in the CH2 and CH3 domains containing the FcRn binding interface. This disclosure provides binding peptides containing modified Fc domains. The binding peptides containing modified Fc domains can be antibodies, immunoadhesins, or Fc fusion proteins.

[0169] In some embodiments, the binding peptide may comprise a modified Fc domain containing amino acid substitutions, said amino acid substitutions altering the antigen-independent effector function of the antibody, particularly altering the circulating half-life (e.g., serum half-life) of the binding peptide. In some embodiments, the binding peptide may comprise a modified Fc domain containing amino acid substitutions, said amino acid substitutions altering the serum half-life of the binding peptide compared to a binding peptide comprising a wild-type (i.e., unmodified) Fc domain. In some embodiments, the binding peptide may comprise a modified Fc domain containing amino acid substitutions, said amino acid substitutions increasing the serum half-life of the binding peptide compared to a binding peptide comprising a wild-type (i.e., unmodified) Fc domain. In some embodiments, the binding peptide may comprise a modified Fc domain containing amino acid substitutions, said amino acid substitutions decreasing the serum half-life of the binding peptide compared to a binding peptide comprising a wild-type (i.e., unmodified) Fc domain.

[0170] In some embodiments, the binding polypeptide comprising a modified Fc domain that alters (i.e., increases or decreases) the circulating half-life (e.g., serum half-life) further comprises one or more mutations in addition to the one or more mutations that alter the circulating half-life. In some embodiments, the one or more mutations in addition to the one or more mutations that alter the 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 to tumor sites, reduced serum half-life, increased serum half-life, etc., compared to an intact antibody with substantially the same immunogenicity.

[0171] When compared to binding peptides lacking these substitutions, the binding peptides described herein may exhibit increased or decreased binding to nascent Fc receptors (FcRn), and thus 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 a long half-life of the administered antibody is desired, for example, to treat chronic diseases or conditions. Conversely, Fc domains with decreased FcRn binding affinity are expected to have shorter serum half-lives, and such molecules can also be used, for example, for administration to mammals where a shortened circulation time may be advantageous, for example, for in vivo diagnostic imaging or in cases where there are toxic side effects when the initiating antibody is present in circulation for an extended period. Fc domains with decreased 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. Furthermore, other applications that may require decreased FcRn binding affinity include applications limited to the brain, kidney, and / or liver.

[0172] It should 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 can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0173] Furthermore, unless otherwise stated, the experiments described herein utilize conventional molecular and cell biological and immunological techniques within the scope of the art. Such techniques are well known to those skilled in the art and are well explained in the literature. See, for example, Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987–2008), including all supplements; Molecular Cloning: A Laboratory Manual (4th edition), eds., MR Green and J. Sambrook; 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 one of ordinary skill in the art. In the event of any ambiguity, the definitions provided herein take precedence over any dictionary or external definition. 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 “including” and other forms such as “includes” and “included” is not restrictive.

[0175] Generally, the terminology used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well-known in the art and described in various general and more specific references cited and discussed herein, unless otherwise stated. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions as is commonly practiced in the art or as described herein. The terminology, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0176] To make this disclosure easier to understand, the selected terms are defined as follows.

[0177] The term "peptide" refers to any aggregate chain of amino acids and encompasses natural or artificial proteins, peptide analogs, or variants or fragments of protein sequences, unless the context otherwise implies. Peptides can be monomers or polymers. For example, a peptide fragment may contain 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 terms "isolated protein" or "isolated polypeptide" refer to a protein or polypeptide that, due to its origin or derivative, is not associated with its naturally associated components in its native state; is substantially free of other proteins from the same species; is expressed by cells from a different species; or is not present in nature. Therefore, proteins or polypeptides synthesized chemically or in cellular systems different from their natural source will be "isolated" from their naturally associated components. Isolation can also be achieved by using protein purification techniques well-known in the art, making the protein or polypeptide substantially free of its natural associated components.

[0179] As used herein, the terms "binding protein" or "binding polypeptide" refer to a protein or polypeptide (e.g., an antibody or immunoadhesin) containing at least one binding site 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, a binding protein or binding polypeptide includes multiple (e.g., two, three, four, or more) binding sites. In some aspects, a binding protein or binding polypeptide is not a therapeutic enzyme.

[0180] The term "ligand" refers to any substance that can bind to or be bound to another substance. Similarly, the term "antigen" refers to any substance that can produce antibodies. Although "antigen" is commonly used to refer to antibody-binding substrates, and "ligand" is frequently used when referring to receptor-binding substrates, these terms are not distinguished from each other and cover a wide range of overlapping chemical entities. To avoid ambiguity, antigen and ligand are used interchangeably herein. Antigen / ligand can be peptides, polypeptides, proteins, aptamers, polysaccharides, sugar molecules, carbohydrates, lipids, oligonucleotides, polynucleotides, synthetic molecules, inorganic molecules, organic molecules, and any combination thereof.

[0181] As used in this article, "specific binding" refers to antibodies or immunoadhesins with a binding rate of up to approximately 1 x 10⁻⁶. -6 M, approximately 1 x 10 -7 M, approximately 1 x 10 -8 M, approximately 1 x 10 -9M, approximately 1 x 10 -10 M, approximately 1 x 10 -11 M, approximately 1 x 10 -12 It binds to antigens with a dissociation constant (M) or lower (Kd), and / or has the ability to bind to antigens with an affinity at least about 2 times greater than that for nonspecific antigens.

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

[0183] As will be discussed in more detail below, the general term "antibody" includes five distinct classes of antibodies that can be distinguished biochemically. Although all five classes of antibodies are obviously within the scope of this disclosure, the following discussion will generally refer to the IgG class of immunoglobulin molecules. Regarding IgG, immunoglobulins consist of 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 Daltons. These four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain extends from the opening of the "Y" next to the heavy chain and continues to the end of the variable region.

[0184] Immunoglobulins are classified into light chains as kappa (κ) or lambda (λ). Each heavy chain class can bind to either a κ or λ light chain. Typically, when immunoglobulins are generated from hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bonded to each other, and the “tail” portions 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 Y-configuration to the C-terminus at the bottom of each chain. Those skilled in the art will understand that heavy chains are classified as γ (γ), μ (μ), α (α), δ (δ), or ε (ε), among which there are several subclasses (e.g., γ1-γ4). The properties of this chain, respectively, determine the “class” of the antibody as IgG, IgM, IgA, IgG, or IgE. Immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to confer functional specialization. Because of the timely disclosure, the modifications of each of these categories and of the same type are readily identifiable to those skilled in the art, and therefore fall within the scope of this disclosure.

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

[0186] Regions of the heavy or light chains of immunoglobulins can be defined as "constant" (C) regions or "variable" (V) regions. In the case of a "constant region," it is based on the relative lack of sequence variation within the region across multiple class members, while in the case of a "variable region," it is based on significant variation within the region across multiple class members. The terms "constant region" and "variable region" can also be used in relation to function. In this respect, it should be understood that the variable regions of immunoglobulins or antibodies determine antigen recognition and specificity. Conversely, the constant regions of immunoglobulins or antibodies confer important effector functions such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. The subunit structures and three-dimensional conformations of the constant regions of various immunoglobulin classes are well known.

[0187] The constant and variable regions of the immunoglobulin heavy and light chains are folded into domains. The term "domain" refers to a globular region of the heavy or light chain containing, for example, peptide rings (e.g., containing 3 to 4 peptide rings) stabilized via β-sheets and / or intrachain disulfide bonds. The constant regions on the light chains of immunoglobulins are interchangeably referred to as "light chain constant region domains," "CL regions," or "CL domains." The constant domains on the heavy chains (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 chains are interchangeably referred to as "light chain variable region domains," "VL region domains," or "VL domains." The variable domains on the heavy chains are interchangeably referred to as "heavy chain variable region domains," "VH region domains," or "VH domains."

[0188] By convention, the amino acid numbers of the variable constant region domains increase as they move further away from the antigen-binding site or N-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 C-termini of the heavy and light chains, respectively. Therefore, the domains of the light chain immunoglobulin are aligned in the VL-CL direction, while the domains of the heavy chain are aligned in the VH-CH1-hinge-CH2-CH3 direction.

[0189] The amino acid assignments for each variable domain follow the definitions in 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. The CDRs 1, 2, and 3 of the VL domain are also referred to as CDR-L1, CDR-L2, and CDR-L3, respectively, in this paper. The CDRs 1, 2, and 3 of the VH domain are also referred to as CDR-H1, CDR-H2, and CDR-H3, respectively, in this paper. If indicated otherwise, the CDR assignments may follow IMGT® (Lefranc et al., Developmental & Comparative Immunology 27:55-77; 2003) rather than Kabat. The heavy chain constant region is numbered via 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 N-terminal variable domain of the immunoglobulin heavy chain, and the term "VL domain" includes the N-terminal variable domain of the immunoglobulin light chain.

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

[0192] As used herein, the term "hinge region" refers to the portion of a heavy-chain molecule that connects the CH1 and CH2 domains. The hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be further subdivided into three distinct 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, extending, for example, from approximately positions 244-360 (EU positions 231-340) in the Kabat numbering system. The CH2 domain is unique because it does not pair tightly with another domain. Instead, two N-linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. In one embodiment, the binding polypeptide of this 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 a portion of a heavy chain immunoglobulin molecule that extends approximately 110 residues from the N-terminus of the CH2 domain, for example, from approximately positions 361-476 (EU positions 341-445) in the Kabat numbering system. The CH3 domain typically forms the C-terminal portion of an 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 this 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 a constant region domain of an immunoglobulin light chain, which extends, for example, from approximately Kabat position 107A to approximately Kabat position 216. The CL domain is adjacent to the VL domain. In one embodiment, the binding polypeptide of this disclosure comprises a CL domain derived from a κ light chain (e.g., a human κ light chain).

[0196] As used herein, the term "Fc region" is defined as a portion of the heavy chain constant region that begins just upstream of the papain cleavage site (i.e., residue 216 in IgG, where the first residue of the heavy chain constant region is 114) and ends at the C-terminus of the antibody. Therefore, a complete Fc region contains at least a hinge domain, a CH2 domain, and a CH3 domain.

[0197] As used herein, the term "natural Fc" or "wild-type Fc" refers to a molecule containing a sequence of a non-antigen-binding fragment, which may be in monomeric or multimeric form, obtained by antibody digestion or otherwise produced; and the term may include a hinge region. The original immunoglobulin source of natural Fc is typically human and can be any immunoglobulin, such as IgG1 and IgG2. Natural Fc molecules consist of monomeric polypeptides that can be linked together in dimer or multimeric form via covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between the monomeric subunits of a natural Fc molecule 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 natural Fc is a disulfide-bonded dimer produced by the digestion of IgG with papain. As used herein, the term "natural Fc" is used generally for monomeric, dimer, and multimeric forms.

[0198] As used herein, the term “Fc variant” or “modified Fc” refers to a molecule or sequence modified from a natural / wild-type Fc but still containing a binding site for FcRn. Therefore, the term “Fc variant” can include molecules or sequences humanized from a non-human natural Fc. Furthermore, natural Fc contains regions that can be removed because they provide structural features or biological activities not required by the antibody-like binding peptides described herein. Therefore, the term “Fc variant” includes molecules or sequences lacking one or more natural Fc sites or residues, or where one or more Fc sites or residues have been modified to affect or participate in: (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity when expressed in selected host cells, (4) glycosylation, (5) complement interaction, (6) binding to Fc receptors other than rescue receptors, or (7) antibody-dependent cytotoxicity (ADCC).

[0199] In some exemplary embodiments, the Fc variants described herein have one or more of the following characteristics compared to IgG antibodies containing wild-type Fc: increased serum half-life, enhanced FcRn binding affinity, enhanced FcRn binding affinity at acidic pH, enhanced FcγRIIIa binding affinity, and / or similar thermal stability.

[0200] As used herein, the term "Fc domain" encompasses both natural / wild-type Fc as defined above, as well as Fc variants and sequences. Similar to Fc variants and natural Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether derived from whole antibody digestion or otherwise produced.

[0201] As described above, the variable region of an antibody allows it to selectively recognize and specifically bind to epitopes on antigens. That is, the VL and VH domains of the antibody combine to form a variable region (Fv) defining a three-dimensional antigen-binding site. This quaternary antibody structure forms an antigen-binding site at the end of each arm of the Y. More specifically, this antigen-binding site is defined by three complementarity-determining regions (CDRs) on each heavy and light chain variable region. As used herein, the term "antigen-binding site" includes a site that specifically binds (in response to an antigen) to an antigen (e.g., a cell surface or soluble antigen). Antigen-binding sites comprise variable regions of the immunoglobulin heavy and light chains, and the binding sites formed by these variable regions determine the specificity of the antibody. Antigen-binding sites are formed by variable regions that vary between antibodies. The modified antibody of this disclosure contains at least one antigen-binding site.

[0202] In some embodiments, the binding polypeptide of this disclosure comprises at least two antigen-binding domains that provide binding of the binding polypeptide to a selected antigen. The antigen-binding domains do not need to be derived from the same immunoglobulin molecule. In this respect, the variable region may be derived from or derived from any type of animal that can be induced to produce a humoral response and generate immunoglobulins against the desired antigen. Therefore, the variable region of the binding polypeptide can be of mammalian origin, for example, from humans, mice, 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 chain-receptor domains (CDRs) present on each monomeric antibody are short, discontinuous amino acid sequences that are specifically localized to form antigen-binding sites, assuming the antibody exhibits its three-dimensional conformation in an aqueous environment. The remainder of the heavy and light chain variable domains exhibits less intermolecular variability in the amino acid sequence and is referred to as framework regions. Framework regions primarily adopt a β-sheet conformation, and the CDRs form loops that connect to the β-sheet structure and, in some cases, form part of the β-sheet structure. Thus, these framework regions act as a scaffold, providing the six CDRs with the correct orientation through interchain non-covalent interactions. The antigen-binding domain formed by the localized CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to the immunoreactive antigen epitope.

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

[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 one target molecule or specific site on a target molecule. When a polypeptide contains more than one target binding site, each target binding site may specifically bind to the same or different molecules (e.g., it may bind to different ligands or different antigens, or bind to different epitopes on the same antigen). Subject-binding polypeptides typically have at least one binding site specific to human antigen molecules.

[0206] The term "specificity" refers to the ability to specifically bind to (e.g., to, an immune response to) a given target antigen (e.g., a human target antigen). A binding peptide may be monospecific and contain one or more binding sites that specifically bind to a target, or a peptide may be multispecific and contain two or more binding sites that specifically bind to the same or different targets. In some embodiments, the binding peptide is specific for two different (e.g., non-overlapping) portions of the same target. In some embodiments, the binding peptide is specific for more than one target. Exemplary binding peptides (e.g., antibodies) containing antigen-binding sites that bind to antigens expressed on tumor cells are known in the art, and one or more CDRs from such antibodies may be included in antibodies as described herein.

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

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

[0209] As used herein, “administer” (“administer” or “administration”) means the act of injecting or otherwise physically delivering a substance present outside the body (e.g., the isolated binding peptides provided herein) to a patient, such as by, but not limited to, the terms 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, the administration of the substance typically occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the administration of the substance typically occurs before the onset of the disease or its symptoms and may continue for a prolonged period to delay or reduce the onset or severity of disease-related symptoms.

[0210] As used herein, the term "composition" is intended to cover products containing optionally specified amounts of specified ingredients (e.g., isolated binding polypeptides provided herein), and any product produced directly or indirectly from combinations of optionally specified amounts of specified ingredients.

[0211] "Effective amount" refers to the amount of the active pharmaceutical agent (e.g., the isolated binding polypeptide of this disclosure) sufficient to achieve the desired physiological outcome in an individual requiring the active pharmaceutical agent. The effective amount can vary between individuals depending on the health and physical condition of the individual to be treated, the individual's classification group, 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., a cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey and a human). In some embodiments, as used herein, the term “subject” 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, sporting animals, and pets.

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

[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 related symptoms 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 isolated binding peptides provided herein). As used herein, the term "treatment" may also refer to altering the course of a disease in a treated subject. The therapeutic effects of a treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating one or more symptoms, reducing the direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or slowing the disease state, and alleviating or improving prognosis.

[0215] binding peptides

[0216] In one aspect, this disclosure provides binding peptides (e.g., antibodies, immunoadhesins, antibody variants, and fusion proteins) comprising a modified Fc domain. The binding peptides disclosed herein encompass any binding peptide comprising a modified Fc domain. In some embodiments, the binding peptide is an antibody or an immunoadhesin or a derivative thereof. Any antibody from any source or species may be used in the binding peptides 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 the binding peptides disclosed herein. Chimeric Fc domains comprising portions of the Fc domain from different species or Ig classes can also be employed. In some 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 exemplary 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 from other species and / or Ig classes or isotypes, those skilled in the art will understand that any amino acid substitutions described herein can be modified accordingly. In some embodiments, the modified Fc domain may contain amino acid substitutions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, or Y436 according to EU designations, and any combination thereof. In some embodiments, the modified Fc domain may contain double amino acid substitutions at any two amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436 according to EU designations. In some embodiments, the modified Fc domain may contain triple amino acid substitutions at any three amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436 according to EU designations. In some embodiments, the modified Fc domain may contain tetranucleotide substitutions at any four amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436 according to EU designations. In some embodiments, it may be necessary for the modified Fc domain to contain amino acid substitutions at any amino acid position selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, or Y436 and any combination thereof according to EU designations, wherein amino acid position N434 is unsubstituted (i.e., amino acid position N434 is wild-type).

[0218] In some embodiments, the modified Fc domain may contain 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. In some embodiments, the modified Fc domain may contain double amino acid substitutions according to EU designations, wherein the double amino acid substitutions are 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 include a triple amino acid substitution according to EU designations, wherein the triple amino acid substitution is 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 include a tetraamino acid substitution according to EU designations, wherein the tetraamino acid substitution is 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 contain an amino acid substituted at any amino acid position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W and any combination thereof, 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 contain an amino acid substitution at any position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W 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 contain an amino acid substitution at any position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W and any combination thereof, wherein amino acid position N434 is not substituted (i.e., amino acid position N434 is wild-type).

[0219] In some embodiments, the modified Fc domain may contain amino acid substitutions selected from M252, T256, T307, or N434 according to EU designations, and any combination thereof. In some embodiments, the modified Fc domain may contain double amino acid substitutions at any two amino acid positions selected from M252, T256, T307, and N434 according to EU designations. In some embodiments, the modified Fc domain may contain triple amino acid substitutions at any three amino acid positions selected from M252, T256, T307, and N434 according to EU designations. In some embodiments, the modified Fc domain may need to contain amino acid substitutions selected from M252, T256, or T307 according to EU designations, and any combination thereof, wherein amino acid position N434 is unsubstituted (i.e., amino acid position N434 is wild-type).

[0220] In exemplary embodiments, the modified Fc domain may contain amino acid substitutions selected from the following according to EU numbers: 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 some embodiments, the modified Fc domain may contain double amino acid substitutions at any two amino acid positions selected from the following according to EU numbers: 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, the modified Fc domain may contain triple amino acid substitutions at any three amino acid positions according to EU numbers, wherein the three amino acid positions are selected from: M252, wherein the substitution is M252Y; T256, wherein the substitution is T256D or T256E; T307, wherein the substitution is T307Q or T307W; or N434, wherein the substitution is N434F or N434Y. In some embodiments, the modified Fc domain may contain quadruple amino acid substitutions at the following amino acid positions according to EU numbers, wherein the amino acid positions are selected from: M252, wherein the substitution is M252Y; T256, wherein the substitution is T256D or T256E; T307, wherein the substitution is T307Q or T307W; or N434, wherein the substitution is N434F or N434Y. In some embodiments, it may be desirable for the modified Fc domain to contain amino acid substitutions selected from M252Y, T256D, T256E, T307Q, or T307W according to EU designations, 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 contain amino acid substitutions selected from M252Y, T256D, T256E, T307Q, or T307W according to EU designations, 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 contain amino acid substitutions selected from M252Y, T256D, T256E, T307Q, or T307W according to EU designations, wherein amino acid position N434 is not substituted (i.e., amino acid position N434 is wild-type).

[0221] In some embodiments, the modified Fc domain may contain amino acid substitutions selected from T256D or T256E and / or T307W or T307Q according to EU designations, and further contain amino acid substitutions selected from N434F or N434Y or M252Y. In some embodiments, it may be desirable for the modified Fc domain to contain amino acid substitutions selected from T256D or T256E and / or T307W or T307Q according to EU designations, and further contain 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 for the modified Fc domain to contain amino acid substitutions selected from T256D or T256E and / or T307W or T307Q according to EU designations, and further contain 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 contain an amino acid substitution selected from T256D or T256E and / or T307W or T307Q according to EU designations, and further to contain an 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 contain 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 designations. In some implementations, the modified Fc domain may include a selection from the EU designations 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 / T256E / N434Y, M252Y / T307Q / N434F, and M25 Triple amino acid substitutions of 2Y / 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 include a tetraamino 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 designations.

[0224] In some embodiments, it may be desirable for the modified Fc domain to contain a wild-type amino acid at amino acid position N434 according to EU designation. In some embodiments, it may be desirable for the Fc domain to not contain phenylalanine (F) or tyrosine (Y) at amino acid position N434 according to EU designation. In some embodiments, it may be desirable for the Fc domain to not contain tyrosine (Y) at amino acid position N434 according to EU designation. In some embodiments, the modified Fc domain may contain 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 designation. In some embodiments, the modified Fc domain may contain a triple amino acid substitution selected from M252Y / T256D / T307Q, M252Y / T256D / T307W, M252Y / T256E / T307Q and M252Y / T256E / T307W according to EU designations.

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

[0226] In some embodiments, the binding peptide may exhibit species-specific FcRn binding affinity. In one embodiment, the binding peptide may exhibit human FcRn binding affinity. In one embodiment, the binding peptide may exhibit rat FcRn binding affinity. In some embodiments, the binding peptide may exhibit cross-species FcRn binding affinity. Such a binding peptide is considered to have cross-reactivity between one or more different species. In one embodiment, the binding peptide may exhibit both human and rat FcRn binding affinity.

[0227] The interaction between the nascent Fc receptor (FcRn) and the Fc region of an antibody promotes circulation by rescuing normal lysosomal degradation. This process is pH-dependent and occurs in endosomes at acidic pH (e.g., pH less than 6.5) rather than under the physiological pH conditions of blood flow (e.g., non-acidic pH). In some embodiments, the binding peptide of this disclosure containing a modified Fc domain exhibits enhanced FcRn binding affinity at acidic pH compared to binding peptides containing a wild-type Fc domain. In some embodiments, the binding peptide containing a modified Fc domain exhibits enhanced FcRn binding affinity at pH less than 7, for example, at about pH 6.5, about pH 6.0, about pH 5.5, and about pH 5.0, compared to binding peptides containing a wild-type Fc domain. In some embodiments, the binding peptide exhibits enhanced FcRn binding affinity at pH less than 7, such as about pH 6.5, about pH 6.0, about pH 5.5, and about pH 5.0, compared to the FcRn binding affinity of binding peptides containing modified Fc domains at elevated non-acidic pH. 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 some embodiments, it is expected that binding peptides comprising modified Fc domains will exhibit substantially the same FcRn binding affinity at non-acidic pH as binding peptides comprising wild-type Fc domains. In some embodiments, it is expected that binding peptides comprising modified Fc domains will exhibit a lower FcRn binding affinity at non-acidic pH than binding peptides comprising modified Fc domains having dual amino acid substitutions M428L / N434S according to EU designations. Therefore, it is expected that binding peptides comprising modified Fc domains will exhibit minimal perturbation to pH-dependent FcRn binding.

[0229] In some embodiments, binding peptides containing modified Fc domains, which exhibit enhanced FcRn binding affinity at acidic pH, have a reduced (i.e., slower) FcRn dissociation rate compared to binding peptides containing wild-type Fc domains. In some embodiments, binding peptides containing modified Fc domains (which exhibit enhanced FcRn binding affinity at acidic pH compared to their FcRn binding affinity at elevated non-acidic pH) have a slower FcRn dissociation rate at acidic pH compared to their FcRn dissociation rate at elevated non-acidic pH.

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

[0231] Some implementations include antibodies that, in addition to the Fc mutations described herein exhibiting altered FcRn binding affinity, also contain 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, wherein the at least one amino acid has been deleted or otherwise altered to provide desired biochemical characteristics, such as reduced or enhanced effector function, nonvalent dimerization ability, enhanced ability to localize to tumor sites, reduced serum half-life, increased serum half-life, etc., when compared to intact antibodies with substantially the same immunogenicity.

[0232] In some other embodiments, the binding peptide comprises constant regions derived from different antibody isotypes (e.g., constant regions from two or more of human IgG1, IgG2, IgG3, or IgG4). In other embodiments, the binding peptide comprises a chimeric hinge (i.e., a hinge comprising hinge portions of hinge domains derived from different antibody isotypes, such as the upper hinge domain of an IgG4 molecule and the middle hinge domain of an IgG1 molecule).

[0233] In some embodiments, techniques known in the art can be used to mutate the Fc domain to increase or decrease effector function. In some embodiments, binding peptides of this disclosure comprising a modified Fc domain have altered binding affinity for Fc receptors. Several different types of Fc receptors exist, classified based on the types of antibodies they recognize. For example, Fc-γ receptors (FcγR) bind to IgG antibodies, Fc-α receptors (FcαR) bind to IgA antibodies, and Fc-ε receptors (FcεR) bind to IgE antibodies. The term FcγR includes several families of members such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb. In some embodiments, binding peptides comprising a modified Fc domain have altered FcγRIIIa binding affinity compared to binding peptides comprising a wild-type Fc domain. In some embodiments, binding peptides containing modified Fc domains have reduced FcγRIIIa binding affinity compared to binding peptides containing wild-type Fc domains. In some embodiments, binding peptides containing modified Fc domains have enhanced FcγRIIIa binding affinity compared to binding peptides containing wild-type Fc domains. In some embodiments, binding peptides containing modified Fc domains have substantially the same FcγRIIIa binding affinity compared to binding peptides containing wild-type Fc domains.

[0234] In other embodiments, the binding peptides used in the diagnostic and therapeutic methods described herein have constant regions, such as the IgG1 heavy chain constant region, which are modified to reduce or eliminate glycosylation. For example, binding peptides comprising a modified Fc domain (e.g., antibodies or immunoadhesins) may further comprise amino acid substitutions that alter 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 imparting 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 peptide is modified to eliminate glycosylation. Such a binding peptide may be referred to as an “agly” binding peptide (e.g., an “agly” antibody). While not bound by theory, it is believed that “agly” binding peptides may have improved in vivo safety and stability. Agly binding peptides may have any of their isotypes or subclasses, such as IgG1, IgG2, IgG3, or IgG4. Many methods recognized in the art can be used to prepare “agly” antibodies or antibodies with modified glycans. For example, genetically engineered host cells (e.g., modified yeast such as Pichia pastoris, or CHO cells) with modified glycosylation pathways (e.g., glycosyltransferase deficiency) can be used to produce such antibodies.

[0236] In some embodiments, the binding peptide may include 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, binding of the C1 complex to the antibody constant region can activate the complement system. Activation of the complement system is important in the opsonization and lysis of cellular pathogens. Activation of the complement system also stimulates inflammatory responses and may also be involved in autoimmune hypersensitivity reactions. Furthermore, antibodies bind to various cellular receptors via their Fc domains (Fc receptor binding sites on the antibody Fc region bind to cellular Fc receptors (FcRs)). Many Fc receptors exist that are specific to different classes of antibodies, including IgG (γ receptor), IgE (ε receptor), IgA (α receptor), and IgM (mu receptor). The binding of antibodies to Fc receptors on cell surfaces triggers a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, cytotoxic cell lysis of antibody-coated target cells (a process 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 peptide (e.g., an antibody or immunoadhesin) binds to the Fcγ receptor. In alternative embodiments, the binding peptide may include a constant region that lacks one or more effector functions (e.g., ADCC activity) and / or cannot bind to the Fcγ receptor.

[0237] Proteins (including antibodies) with low thermodynamic stability exhibit increased tendency to misfold and aggregate, which can limit or hinder their activity, efficacy, and potential as useful therapeutic agents. In some embodiments, binding peptides comprising modified Fc domains have substantially the same thermostability as binding peptides comprising wild-type Fc domains. In some embodiments, binding peptides comprising modified Fc domains have substantially the same thermostability as binding peptides comprising modified Fc domains having triple amino acid substitutions M252Y / S254T / T256E (YTE).

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

[0239] In some embodiments, the binding polypeptide of this disclosure may comprise an antigen-binding fragment of an antibody. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes for antigen binding with an intact antibody (i.e., the intact antibody from which the polypeptide fragment of the immunoglobulin or antibody originates) (i.e., specific binding). Antigen-binding fragments can be generated 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 polypeptide of this disclosure comprises an antigen-binding fragment and a modified Fc domain.

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

[0241] In some embodiments, the binding peptide of this disclosure is a multivalent (e.g., tetravalent) antibody generated by fusing a DNA sequence encoding an antibody with an ScFv molecule (e.g., a modified ScFv molecule). For example, in one embodiment, these sequences are combined such that the ScFv molecule (e.g., a modified ScFv molecule) is linked to the Fc fragment of the antibody via a flexible linker (e.g., a gly / ser linker) at its N-terminus or C-terminus. In another embodiment, the tetravalent antibody of this disclosure can be prepared by fusing an ScFv molecule with a linker peptide, which is then fused with a modified Fc domain to construct an ScFv-Fab tetravalent molecule.

[0242] In another embodiment, the binding polypeptide of this disclosure is a modified microantibody. The modified microantibody of this disclosure is a dimer molecule composed of two polypeptide chains, each polypeptide chain containing an ScFv molecule fused to a modified Fc domain via a linker peptide. The microantibody can be prepared by constructing the ScFv component and the linker peptide component using methods described in the art (see, for example, U.S. Patent 5,837,821 or WO94 / 09817A1). In another embodiment, a tetravalent microantibody can be constructed. The tetravalent microantibody can be constructed in the same manner as the microantibody, except that two ScFv molecules are linked using a flexible linker. The linked scFv-scFv construct is then linked to the modified Fc domain.

[0243] In another embodiment, the binding peptide of this disclosure comprises a biantibody. The biantibody is a dimeric tetravalent molecule, each having a polypeptide similar to an scFv molecule, but typically having a short (less than 10, e.g., about 1 to about 5) amino acid residue linker connecting two variable domains, such that the VL and VH domains on the same polypeptide chain do not interact. Instead, the VL and VH domains of one polypeptide chain interact (respectively) with the VH and VL domains on a second polypeptide chain (see, for example, WO 02 / 02781). The biantibody of this disclosure comprises an scFv-like molecule fused to a modified Fc domain.

[0244] In other embodiments, the binding peptide comprises a multispecific or multivalent antibody that includes one or more variable domains, such as tandem variable domain (TVD) peptides, tandemly linked on the same polypeptide chain. Exemplary TVD peptides 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 tandem 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 tandemly linked by a peptide linker (VL1-linker-VL2). In a cross-headed configuration, the variable domains of two different antibodies are represented in tandem on two separate polypeptide chains (one heavy chain and one light chain), one polypeptide chain having two tandem VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consisting of complementary VL domains tandemly linked in opposite directions via a peptide linker (VL2-linker-VL1). Additional antibody variants based on the “double Fv” form include bispecific antibodies with dual variable domains (DVD-IgG) (see U.S. Patent No. 7,612,181) and TBTI forms (see US 2010 / 0226923 A1). In some embodiments, the binding polypeptide comprises a multispecific or multivalent antibody containing one or more variable domains tandemly on the same polypeptide chain fused with a modified Fc domain.

[0245] In another exemplary embodiment, the binding peptide comprises a bispecific antibody against a cross-linked dual variable domain IgG (CODV-IgG) based on a “two-headed” configuration (see US 20120251541 A1, which is incorporated herein by reference in its entirety).

[0246] In another exemplary embodiment, the binding polypeptide is an immunoadhesin. As used herein, “immunoadhesin” means a binding polypeptide comprising one or more binding domains (e.g., derived from receptors, ligands, or cell adhesion molecules) linked to an immunoglobulin constant domain (i.e., the Fc region) (see, for example, 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). Immunoadhesin is referred to in its International Nonproprietary Name. Immunoadhesives (INNs) are identified by the suffix "-cept". Similar to antibodies, immunoadhesives have long circulating half-lives, are easily purified using affinity-based methods, and possess an affinity advantage conferred by divalent antibodies. Examples of commercially available therapeutic immunoadhesives include etanercept (ENBREL®), abatacept (ORENCIA®), linalcept (ARCALYST®), aflibercept (ZALTRAP® / EYLEA®), and beraccept (NULOJIX®).

[0247] In some embodiments, the binding peptide comprises an immunoglobulin-like domain. Suitable immunoglobulin-like domains include, but are not limited to, fibronectin domains (see, for example, Koide et al. (2007), Methods Mol. Biol. 352: 95–109, which is incorporated herein by reference in its entirety), DARPin (see, for example, 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, for example, Nygren et al. (2008) FEBS J. 275 (11): 2668–76, which is incorporated herein by reference in its entirety), lipid transport proteins (see, for example, Skerra et al. (2008) FEBS J. 275 (11): 2677–83, which is incorporated herein by reference in its entirety), and affilin (see, for example, Ebersbach et al. (2007) J. Mol. Biol. 372). (1):172–85, which is incorporated herein by reference in its entirety), Affitins (see, for example, Krehenbrink et al. (2008). J. Mol. Biol. 383 (5): 1058–68, which is incorporated herein by reference in its entirety), Avimer (see, for example, Silverman et al. (2005) Nat. Biotechnol. 23 (12): 1556–61, which is incorporated herein by reference in its entirety), Fynomer (see, for example, 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, for example, Nixon et al. (2006) Curr OpinDrug Discov Devel 9 (2): 261–8, which is incorporated herein by reference in its entirety).

[0248] For the binding peptides and immunoadhesins disclosed herein, virtually any antigen can be targeted by the binding peptides, including but not limited to the proteins, subunits, domains, motifs and / or epitopes of the target antigen, said target antigen including both soluble factors (such as cytokines and membrane-bound factors) and transmembrane receptors.

[0249] The binding peptides of this disclosure, which include the modified Fc domain described herein, may include a known CDR sequence or a variable domain sequence of a “parental” antibody. In some embodiments, the parental antibody and the antibody of this disclosure may share similar or identical sequences, except for the modification of the Fc domain disclosed herein.

[0250] Nucleic acids and expression vectors

[0251] In one aspect, the present invention provides polynucleotides encoding the binding polypeptides disclosed herein. A method for preparing the binding polypeptides is also provided, the method comprising expressing these polynucleotides.

[0252] Typically, polynucleotides encoding the binding polypeptides disclosed herein are inserted into expression vectors to be introduced into host cells that can be used to generate desired amounts of claimed antibodies or immunoadhesins. Therefore, in some aspects, the present invention provides expression vectors comprising the polynucleotides disclosed herein, and host cells comprising these vectors and the polynucleotides.

[0253] For the purposes of this specification and claims, the term "vector" or "expression vector" is used herein to mean a vector for introducing and expressing a desired gene in a cell. As will be known to those skilled in the art, such vectors can be readily selected from plasmids, bacteriophages, viruses, and retroviruses. Typically, a vector will contain selection markers, appropriate restriction sites to facilitate the 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, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Other vector classes involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells with DNA integrated into their chromosomes can be selected by introducing one or more markers, which allow for selection of the host cells to be transfected. These markers can provide resistance to auxotrophic hosts, biocidal agents (e.g., antibiotics), or heavy metals such as copper. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include signal sequences, splicing signals, and transcription promoters, enhancers, and termination signals. In some embodiments, a cloned variable region gene is inserted into an expression vector along with heavy and light chain constant region genes (e.g., human genes) synthesized as described above.

[0255] In other embodiments, polycistronic constructs can be used to express binding peptides as described herein. In such expression systems, a variety of gene products of interest, such as heavy and light chains of antibodies, can be generated from a single polycistronic construct. These systems advantageously utilize internal ribosome entry sites (IRES) to provide relatively high levels of peptides in eukaryotic host cells. A compatible IRES sequence is disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference. Those skilled in the art will understand that such expression systems can be used to efficiently generate the full range of peptides disclosed in this application.

[0256] More generally, once a vector or DNA sequence encoding the disclosed binding polypeptide has been prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. The introduction of the plasmid into the host cell can be achieved using 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 enveloped DNA, microinjection, and full viral infection. See, for example, Ridgway, AAG, “Mammalian Expression Vectors,” Chapter 24.2, pp. 470-472, Vectors, edited by Rodriguez and Denhardt (Butterworths, Boston, MA 1988). The transformed cells are grown under conditions suitable for the production of light and heavy chains, and heavy and / or light chain protein synthesis is measured. Exemplary assays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), immunohistochemistry, etc.

[0257] As used in this article, the term “transformation” should be used in a broad sense, referring to the introduction of DNA into a recipient host cell, which alters the genotype and thus causes changes in the recipient cell.

[0258] Following the same line of thought, "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. In describing the process for isolating peptides from a recombinant host, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the antibody, unless otherwise explicitly stated. In other words, recovering peptides from "cells" can mean recovering them from whole cells precipitated 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 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 (a 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), and 293 (human kidney). In one implementation, the cell line provides altered glycosylation of the antibody it expresses, such as non-fucosylation (e.g., PER.C6.RTM. (Crucell)) or the FUT8 knockout CHO cell line (POTELLIGENT). TM (Biowa, Princeton, NJ)). In one implementation, NSO cells may be used. The host cell line is typically available from commercial services, the American Tissue Culture Collection, or publicly available literature.

[0260] In vitro production allows for scale-up to yield large quantities of the desired binding peptide. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include homogeneous suspension culture (e.g., in an airlift reactor or a continuous stirred reactor) or cell culture immobilized or embedded in agarose beads or ceramic casks (e.g., in hollow fibers, microcapsules). Solutions of the peptide can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immuno)affinity chromatography, if necessary and / or required.

[0261] One or more genes encoding the binding polypeptide can also be expressed in non-mammalian cells such as bacteria, yeast, or plant cells. In this respect, it should be understood that a variety of single-celled non-mammalian microorganisms, such as bacteria—those microorganisms capable of growth in culture or fermentation—can also be transformed. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of *Escherichia coli* or *Salmonella*; members of the Bacillus family, such as *Bacillus subtilis*; members of the *Streptococcus* genus; and *Haemophilus influenzae*. It should also be understood that when expressed in bacteria, the polypeptide can become part of an integrity. The polypeptide must be isolated, purified, and then assembled into a functional molecule.

[0262] Besides prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae or Bacillus oryzae are the most commonly used eukaryotic microorganisms, although many other strains are generally available. For expression in the genus *Saccharomyces*, plasmids such as YRp7 (Stinchcomb et al., *Nature*, 282:39 (1979); Kingsman et al., *Gene*, 7:141 (1979); Tschemper et al., *Gene*, 10:157 (1980)) are commonly 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 number 44076 or PEP4-1 (Jones, *Genetics*, 85:12 (1977)). The presence of trpl damage then provides an effective environment for transformation by growth detection in the absence of tryptophan, as a genomic feature of the yeast host cell.

[0263] Treatment

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

[0265] The binding peptides disclosed herein can be used in a variety of applications. For example, in one embodiment, the subject-binding peptide can be used to reduce or eliminate cells carrying epitopes recognized by the binding domain of the binding peptide. In another embodiment, the subject-binding peptide effectively reduces or eliminates circulating soluble antigens. In yet another embodiment, the subject-binding peptide is effective as a T-cell entrainer. In one embodiment, the binding peptide can reduce tumor size, inhibit tumor growth, and / or prolong the survival time of tumor-bearing animals. Therefore, this disclosure also relates to methods for treating tumors in humans or other animals by administering an effective, non-toxic amount of a modified antibody to such humans or animals.

[0266] In one embodiment, the subject-binding peptide can be used to treat a disease or condition. For example, the subject-binding peptide can be used to treat antibody-related conditions or antibody-reactive conditions, illnesses, or diseases. As used herein, the terms “antibody-related condition” or “antibody-reactive condition” or “illness” refer to or describe a disease or condition that can be improved by administration of a pharmaceutical composition comprising an antibody or binding peptide of the present disclosure.

[0267] In one implementation, the subject-combined peptide can be used to treat cancer. As used herein, the term "cancer" or "carcinogenic" refers to or describes a physiological condition typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, germ cell tumor, sarcoma (including liposarcoma), neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of this type of cancer 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 cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney 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 topic-binding peptide can be used to treat other conditions, including but not limited to infectious diseases, autoimmune diseases, inflammatory diseases, lung diseases, neuronal or neurodegenerative diseases, liver diseases, spinal diseases, uterine diseases, depression, etc. Non-limiting examples of infectious diseases include those caused by RNA viruses (e.g., orthomyxoviruses (e.g., influenza), paramyxoviruses (e.g., respiratory syncytial virus, parainfluenza virus, metapneumovirus), rhabdoviruses (e.g., rabies virus), coronaviruses, alphaviruses (e.g., chikungunya virus), lentiviruses (e.g., HIV), 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 spp., Streptococcus spp., and 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 spirochetosis, leishmaniasis, anthrax, and meningitis. Exemplary autoimmune conditions 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). Therefore, this disclosure relates to a method for treating a variety of conditions that will benefit from the use of a subject-binding peptide having, for example, an increased half-life.

[0269] Through routine experiments, those skilled in the art will be able to determine that an effective, non-toxic amount of the modified binding peptide can be used for the treatment of malignant tumors. For example, the therapeutically active amount of the binding peptide disclosed herein can vary depending on 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 weight, as well as the ability of the modified antibody to elicit the desired response in the subject. Dosing regimens can be adjusted to provide optimal therapeutic response. For example, several separate doses may be administered daily, or the dose may be proportionally reduced as indicated by an emergency in the treatment situation.

[0270] Typically, the compositions provided in this disclosure can be used for the preventive or therapeutic treatment of any tumor containing an antigenic marker that allows the targeting of cancer cells by a modified antibody.

[0271] Pharmaceutical Compositions and Their Administration

[0272] Methods for preparing and administering the conjugated peptides of this disclosure to subjects are well known or readily determined by those skilled in the art. The conjugated peptides of this disclosure can be administered orally, parenterally, by inhalation, or topically. As used herein, parenterally includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all these forms of administration are clearly considered to be within the scope of this disclosure, the form of administration will be a solution for injection, particularly for intravenous or intra-arterial injection or infusion. Typically, suitable pharmaceutical compositions for injection may contain buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbate), optional stabilizers (e.g., human albumin), etc. In some embodiments, the conjugated peptide may be delivered directly to the site of the adverse cell population, thereby enhancing the exposure of the diseased tissue to the therapeutic agent.

[0273] Formulations intended 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 buffer media. In the compositions and methods disclosed herein, pharmaceutically acceptable carriers include, but are not limited to, 0.01–0.1 M, such as 0.05 M phosphate buffer or 0.8% saline. Other common parenteral media include sodium phosphate solutions, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextran), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (water-soluble) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In this case, the composition must be sterile and should be a fluid to the extent that it is easily injectable. It should be stable under manufacturing and storage conditions and generally protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, this can be achieved by using a coating such as lecithin, maintaining the desired particle size in the dispersed state, and maintaining appropriate flowability by using surfactants.

[0274] Antimicrobial activity can be achieved through a variety of antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, mercurochrome, etc.). In many cases, isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride, are included in the composition. Prolonged absorption of injectable compositions can be achieved by including delayed-absorption agents such as aluminum monostearate and gelatin.

[0275] In any case, a sterile injectable solution can be prepared by incorporating an active compound (e.g., a modified binding polypeptide itself or in combination with other active agents) in a desired amount into a suitable solvent, followed by filtration and sterilization. The solvent may, as needed, contain one or a combination of the components listed herein. Typically, a dispersion is prepared by incorporating the active compound into a sterile medium containing an alkaline dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, exemplary preparation methods include vacuum drying and freeze-drying, which produce a powder of the active ingredient and any additional desired components from a previously sterile filtered solution. The formulation for injection is processed, filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under sterile conditions according to methods known in the art. Furthermore, the formulation may be packaged and marketed as a kit. Such articles typically have labels or packaging inserts indicating that the relevant composition can be used to treat subjects with or susceptible to autoimmune or neoplastic conditions.

[0276] The effective dosage of the disclosed compositions for treating the above-mentioned conditions varies depending on many different factors, including the route of administration, target site, patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. The therapeutic dosage can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0277] The conjugating peptides of this disclosure can be administered multiple times. The interval between single doses can be weekly, monthly, or annually. The interval can also be irregular, as indicated by measuring the blood level of the modified conjugating peptide or antigen in the patient. In some methods, the dose is adjusted to achieve a plasma concentration of the modified conjugating peptide of about 1-1000 μg / ml, and in other methods, this concentration is about 25-300 μg / ml. Alternatively, the conjugating peptide can be administered as a sustained-release formulation, in which case less frequent administration is required. For antibodies, the dose and frequency vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit 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 prophylactic or therapeutic. In prophylactic use, a composition containing the antibody of the present invention or a mixture thereof 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 the "preventive effective dose." In this use, the precise amount also depends on the patient's health status and overall immunity, but is generally in the range of about 0.1 to about 25 mg per dose, particularly about 0.5 to about 2.5 mg per dose. Relatively low doses are administered for extended periods at relatively infrequent intervals. Some patients continue treatment for the rest of their lives. In therapeutic use, it is sometimes necessary to administer relatively high doses (e.g., about 1 to 400 mg / kg antibody per dose, with doses of about 5 to 25 mg more commonly used for radioimmunoconjugates and higher doses for cytotoxic-drug modified antibodies) at relatively short intervals until disease progression is reduced or terminated, or until the patient shows partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen can be administered to the patient.

[0279] The conjugated polypeptides of this disclosure may optionally be administered in combination with other pharmaceutical agents that are effective in treating conditions or illnesses requiring treatment (e.g., prophylactic or therapeutic). The present disclosure... 90 The effective single therapeutic dose (i.e., the effective therapeutic dose) of the Y-labeled modified antibody ranges from about 5 to about 75 mCi, such as from about 10 to about 40 mCi. 131 The effective single-dose non-myeloablation dose range for I-modified antibodies is between approximately 5 and approximately 70 mCi or between approximately 5 and approximately 40 mCi. 131 The effective single-treatment ablation dose of iodine-labeled antibodies (i.e., potentially requiring autologous bone marrow transplantation) ranges from approximately 30 to approximately 600 mCi, or from approximately 50 to less than approximately 500 mCi. Together with chimeric antibodies, due to the longer circulating half-life of mouse antibodies, the effective single-treatment non-bone marrow ablation dose of iodine-131-labeled chimeric antibodies ranges from approximately 5 to approximately 40 mCi, or less than approximately 30 mCi. For example, 111 Imaging standards for In-labeled markers are typically less than about 5 mCi.

[0280] While the conjugated peptide can be administered as described above, it must be emphasized that in other embodiments, the conjugated peptide can be administered as a first-line therapy to other healthy patients. In such embodiments, the conjugated peptide can be administered to patients with normal or average red bone marrow reserves and / or to patients who have not received treatment and are not currently receiving treatment. As used herein, the administration of a modified antibody or immunoadhesin together with or in combination with adjuvant therapy means the sequential, simultaneous, concurrent, parallel, accompanying, or simultaneous administration or application of the therapy and the disclosed antibody. Those skilled in the art will understand that various components of a combined therapeutic regimen can be administered or applied at regular intervals to enhance the overall effectiveness of the treatment.

[0281] As previously described, the conjugating peptides, immunoadhesins, or recombinants thereof disclosed herein can be administered in an effective amount for the in vivo treatment of mammalian diseases. In this regard, it should be understood that the disclosed conjugating peptides will be formulated to facilitate the administration of the active agent and enhance its stability.

[0282] The pharmaceutical compositions according to this disclosure may comprise a pharmaceutically acceptable, non-toxic, sterile carrier, such as physiological saline, non-toxic buffer solution, preservatives, etc. For the purposes of this application, the effective amount of the conjugated or unconjugated binding peptide, immunoadhesin, or recombinant thereof should be maintained, meaning an amount sufficient to achieve effective binding to the antigen and sufficient to obtain benefit (e.g., sufficient to improve symptoms of a disease or condition or to detect substances or cells). In the case of tumor cells, the modified binding peptide can interact with selected immunoreactive antigens on tumor cells or immune-responsive cells and provide an increase in the death of these cells. Of course, the pharmaceutical compositions of this disclosure can be administered in single or multiple doses to provide an effective amount of the modified binding peptide.

[0283] To be consistent with the scope of this disclosure, the conjugating peptides of this disclosure can be administered to humans or other animals in an amount sufficient to produce a therapeutic or prophylactic effect, as described in the treatment methods described above. The conjugating peptides of this disclosure can be administered to such humans or other animals in conventional dosage forms prepared by combining the antibody of this disclosure with a conventionally pharmaceutically acceptable carrier or diluent according to known techniques. Those skilled in the art will recognize that the form and characteristics of a pharmaceutically acceptable carrier or diluent depend on the amount of active ingredient combined with it, the route of administration, and other well-known variables. Those skilled in the art will further understand that mixtures comprising one or more conjugating peptides described in this disclosure can prove particularly effective.

[0284] The contents of any articles, patents, and patent applications mentioned or cited herein, as well as all other documents and electronically available information, are incorporated herein by reference in their entirety, to the extent that each individual publication is specifically and individually indicated as incorporated by reference. The applicant reserves the right to substantially incorporate any and all material and information from any such articles, patents, patent applications, or other physical and electronic documents into this application.

[0285] While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes and substitutions can be made without departing from the true spirit and scope of the invention. It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Furthermore, many modifications can be made to employ specific conditions, materials, compositions of matter, processes, process steps, or steps to achieve the objective spirit and scope of the invention. All such modifications are contemplated within the scope of the appended claims. Certain embodiments have now been described in detail and will become clearer from the following examples, which are for illustrative purposes only and are not intended to be limiting.

[0286] Example

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

[0288] Example 1: Materials and Methods

[0289] Protein reagents:

[0290] The following proteins were expressed and isolated: antigens with a C-terminal 8x histidine tag; rFcRn (UniProt: P1359, p51 subunit: residues 23-298; UniProt: P07151, β2-m: residues 21-119); biotinylated cynomolgus monkey FcRn (UniProt: Q8SPV9, p51 subunit: residues 24-297 with a C-terminal Avi- tag; UniProt: Q8SPW0, β2-m: residues 21-119); biotinylated hFcRn (UniProt: P55899, p51 subunit: residues 24-297 with a C-terminal Avi- tag; UniProt: P61769, β2-m: residues 21-119); human CD16a (UniProt: P08637, FcγRIIIa: residues 17-208, which have a C-terminal HPC4 tag and a valine residue at position 158 (V158)). H435A and H310A / H435Q heavy chain variants were obtained from HEK293 conditioned medium. The mAb2 variant was cloned via Evitria and purified from suspension CHO K1 conditioned medium using a mAbSelect SuRe affinity column (GE Healthcare), with the buffer exchanged for phosphate-buffered saline (PBS) at pH 7.4 for subsequent experiments.

[0291] Saturated library construction:

[0292] The heavy and light chains of the WT IgG1 mAb1 antibody, with leader DNA sequences, were incorporated into the mammalian expression plasmids pBH6414 and pBH6368, respectively, using NcoI and HindIII restriction enzyme sites. Saturated libraries were 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 number). Heavy chain DNA sequences of three control variants of the mAb1 backbone, AAA (T307A / E380A / N434A), LS (M428L / N434S), and YTE (M252Y / S254T / T256E), were constructed into the pBH6414 vector using LakePharma.

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

[0294] Recombinant antibody expression and purification:

[0295] For conditioned medium screening, DNA containing the mutant heavy chain of mAb1 and the wild-type light chain was transfected into 1 mL of Expi293 mammalian cells (Invitrogen) for expression according to the manufacturer's instructions. Cells were incubated at 37ºC, 5% CO2, and 80% humidity in 2 mL 96-well plates (Greiner Bio-One) with shaking at 900 rpm and sealed with an aerated membrane. Conditioned medium was collected five days after transfection and stored at -80ºC until use. Lead variants of the mAb1 and Ab3 backbones were expressed at a 30 mL scale in 125 mL flasks (Corning) with 0.2 μm discharge caps. The 125 mL flasks were shaken at 125 RPM throughout the expression duration. Conditioned medium was collected five days after transfection and filtered through a 0.22 μm, 50 mL conical filter (Corning) and stored at 4ºC until purification.

[0296] mAb1 and Ab3 were separated using a 1 mL mAbSelect SuRe HiTrap column (GE Healthcare). After a ten-column wash in PBS at pH 7.4, the antibody was eluted with five column volumes of 0.1 M citrate (Sigma) at pH 3.0 and neutralized with 0.5 mL of 1 M Tris base (Sigma) at pH 9.0. The eluted antibody was then buffer-exchanged in PBS at pH 7.4 and concentrated to >1 mg / mL using a 30 kDa MWCO Amicon concentrator (Millipore). -1 For subsequent research. The concentration of the purified antibodies was determined by their UV absorbance at 280 nm (UV). 280 The appropriate extinction coefficient is used to determine this.

[0297] Octet conditioned medium screening and analysis:

[0298] Screening for mAb1 variants was performed on Octet QK 384 (PALL Life Sciences) equipped with a Ni-NTA biosensor in conditioned media. His-labeled antigens were screened at 15 μg / mL in PBS, 0.1% bovine serum albumin (BSA, Sigma), and 0.01% Tween-20 (Sigma) (PBST-BSA 7.4) at pH 7.4. -1The antibody was captured for 300 sec, followed by washing with PBST-BSA at pH 7.4 for 20 sec. It was then captured for 200 sec in conditioned medium diluted 1:1 with PBST-BSA at pH 7.4. After a buffer wash in pH 6.0 buffer, association was performed with 200 nM rFcRn, and FcRn binding kinetics were obtained at pH 6.0 with dissociation times of 150 and 200 sec, respectively. Throughout all steps during Octet screening, the temperature was 30ºC and the shaking speed was 1000 RPM. The rFcRn binding kinetics were corrected for the onset 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 modified protocol with a direct FcRn immobilization or biotinylated CAPture kit (GE Healthcare) via a Biacore T200 instrument (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) was immobilized via amine coupling chemistry (GE Healthcare). -1 ) at 10 μL min -1 Immobilization in 10 mM sodium acetate (GE Healthcare) at pH 4.5 for 180 s resulted in approximately 20 RU bound to the C1 sensor chip surface. In the case of the Biotin CAPture kit, CAPture reagent was captured on the CAP chip surface until the bound RU > 2,000 RU, followed by 0.1 μg mL... -1 FcRn at 30 uL min -1 Capture for 24 s in the appropriate channel until the final binding RU is approximately 2 RU. The run buffer used for FcRn binding kinetics experiments was PBS with 0.05% surfactant P-20 (PBS-P+, GE Healthcare) at pH 6.0 or 7.4. For each variant (including a 0 nM control), a 4-fold serial dilution from a 1000 nM antibody concentration was performed in quadruplicate. [The text abruptly ends here, so the translation stops as well.] -1Kinetic measurements of association and dissociation times of 180 and 300 sec were obtained at flow rates of [missing information]. The C1 and CAP sensor chips were respectively treated with 10 mM sodium tetraborate and 1 M NaCl (pH 8.5) at 50 μL / min [missing information]. -1 Regenerate for 30 seconds, or use 6 M guanidine hydrochloride and 250 mM sodium hydroxide (GE Healthcare) at 50 μL / min. -1 Regeneration was performed for 120 seconds, followed by an additional 60–90 sec stabilization step in PBS-P+ at pH 6.0. Steady-state RU measurements of all variants at pH 7.4 were obtained in triplicate using the same C1 or CAP sensor chip and kinetic parameters as described above, with only a 10–20 fold increase in FcRn capture levels for both methods.

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

[0302] FcRn affinity chromatography:

[0303] In one experiment, the FcRn affinity column was produced using a protocol adapted from Schlothauer et al. 2013, mAbs 5: 576-586. 1 mL of streptavidin HP HiTrap column (GE Healthcare) was loaded with five column volumes of binding buffer (pH 7.4, 20 mM sodium phosphate (Sigma), 150 mM sodium chloride (NaCl; Sigma)) at a rate of 1 mL / min. -1 After equilibration, inject 4 mg of biotinylated cynoFcRn. Wash the column with binding buffer and store at 4ºC 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), followed by injection of 300 μg of each antibody. The antibody solution was then adjusted to pH 5.5 with low-pH buffer. After ten column volumes of washing with low-pH buffer, the column was flushed with more than 30 column volumes of high-pH buffer (20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (bistripropane; Sigma) pH 9.5; 150 mM NaCl) at a rate of 1 mL / min. -1 Antibodies were eluted from 1 mL fractions using a linear pH gradient, and UV was monitored. 280 Reequilibrate the FcRn affinity column with ten column volumes of low pH buffer for subsequent runs or with binding buffer for storage. All variants were performed in triplicate.

[0305] In Sigmaplot 11 (Systat Software, Inc.), Equation 1 is used to model the FcRn affinity column elution curves for each antibody as a single Gaussian distribution to determine the UV. 280 Elution volume at the maximum value.

[0306] (Equation 1)

[0307] Where x0 is UV 280 The elution volume at the peak value, y0 is the baseline UV. 280 The absorbance, and a and b, are related to the full width at half the maximum of the distribution. The pH of each fraction was measured using a Corning Pinnacle 540 pH meter and correlated with 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, in which biotinylated hFcRn was placed on a 1 mL Streptavidin HP HiTrap column (GE Healthcare). On the AKTA Pure system (AKTA), 300 μg of each antibody was injected into the column in a low pH buffer (pH 5.5 in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES; Sigma); 150 mM NaCl). A linear pH gradient was generated by using more than 30 column volumes of low and high pH buffer (pH 9.5 in 20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (bistripropane; Sigma); 150 mM NaCl) at 0.5 mL / min. -1Elute the antibody and monitor absorbance and pH. Reequilibrate the column with low pH buffer for subsequent runs. Perform all variations in triplicate. Fit the FcRn affinity column elution curves to a single Gaussian distribution in Sigmaplot 11 (Systat Software, Inc.) to determine UV. 280 Elution volume and pH at the maximum value.

[0309] Differential scanning fluorescence measurement:

[0310] Differential scanning fluorescence (DSF) assays were performed in 20 μL reactions using a BioRad CFX96 real-time thermal cycler. Antibody samples and 5000x stock solutions were diluted with Sypro Orange dye (Invitrogen) to 0.4 mg / mL in PBS at pH 7.4. -1 And 10x. The antibody and Sypro Orange were mixed 1:1 in a 96-well PCR plate and sealed with a 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 tested in triplicate. The thermal cycling procedure consisted of the following steps: a 2-minute equilibration step at 20ºC, followed by a constant heating rate of 0.5ºC / 5 sec to a final temperature of 100ºC. Fluorescence measurements for each well were obtained using a FAM excitation wavelength (485 nm) and a ROX emission (625 nm) detector suitable for Sypro Orange fluorescence (see, for example, Biggar et al. 2012, Biotechniques 53: 231-238). DSF fluorescence intensity curves and first derivatives were output from BioRad CFX Manager and analyzed in Sigmaplot 11. 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). Amine chemistry was employed to measure the binding kinetics in 50 μg mL of acetate at pH 4.5. -1 The anti-HPC4 antibody (Roche) was administered at 10 μl min. -1Surface coupling with the CM5 sensor chip for 600 sec achieved a final density > 20,000 RU. The run buffer used for FcγRIIIa binding kinetics experiments was a HEPES-buffered saline solution at pH 7.4 containing 0.05% surfactant P-20 (HBS-P+, GE Healthcare) and 2 mM calcium chloride (CaCl2, Fluka). The mixture was infused with 5 μl / min... -1 Capture 1.25 μg mL -1 HPC4-labeled FcγRIIIa-V158 was used to initialize each kinetic trace for 30 sec. Association and dissociation kinetics for each variant were measured at 300 nM, with each step for each variant measured in 5 μl / min. -1 Measure for 120–180 seconds. After completing the kinetic measurement, regenerate the CM5 chip with HBS-P+ buffer supplemented with 10 mM EDTA (Ambion). Before the next kinetic measurement, wash the CM5 chip with HBS-P+ containing CaCl2 for 120 seconds.

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

[0314] (Equation 2)

[0315] Where the offset is the baseline RU of the 0 nM antibody, R max This refers to the stable period RU at high antibody concentrations, where [antibody] is the antibody concentration, and K... D,app It is the apparent binding affinity between the variant and FcγRIIIa.

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

[0317] Isoelectric focusing

[0318] The isoelectric point (pI) of the leader variant was determined by capillary electrophoresis on Maurice C (Protein Simple). Each 200 μL sample contained 0.35% methylcellulose (Protein Simple), 4% amphoteric electrolyte (pharmalyte) 3-10 (GE Healthcare), 10 mM arginine (Protein Simple), and 0.2 mg / mL... -1 Antibodies and biomarkers (Protein Simple) at pIs of 4.05 and 9.99 were used. Samples were loaded into capillaries at 1500 V for 1 minute, followed by a separation phase at 3000 V for 6 minutes, monitored using tryptophan fluorescence. The pI of each variant was determined using Maurice C software and defined as the pH at which the fluorescence peak of the predominant species was observed.

[0319] Homogeneous bridging rheumatoid factor (RF) ELISA

[0320] According to the manufacturer's instructions, the antibodies were biotinylated and labeled with digitoxin using the EZ-Link Thio-NHS-LC-Biotin and Mix-n-Stain™ Digoxin Antibody Labeling Kit (Biotium). For each variant, a 4 μg mL solution was prepared. -1 Stock solutions of biotinylated and digitoxin-labeled antibodies were prepared and mixed with 300 U / mL RF (Abcam) at a 1:1 ratio. After incubation at room temperature for 20 hours, 100 μL of each antibody-RF mixture was added to a Streptawell plate (Sigma-Aldrich) and incubated at room temperature for 2 hours. The plate was washed three times with PBS at pH 7.4 with 0.05% Tween-20, and 100 μL of 1:2000 diluted HRP-conjugated anti-digitoxin secondary antibody (Abcam) was added to each well. After incubation at room temperature for 2 hours, the wells were washed and treated with 100 μL of TMB substrate (Abcam) at room temperature for 15 minutes. The reaction was terminated with 100 μL of stop solution (Abcam), and the absorbance was measured at 450 nm on a SpectraMax plate reader. Wells without antibody-RF mixtures were used as blanks, and the experiment was repeated three times. The p-value was determined using a Student's t-test.

[0321] In vivo pharmacokinetics

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

[0323] In hFcRn mice, the antibody variant was administered intravenously at a single dose of 2.5 mg / kg via the tail vein, 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 heparinized capillary. The collected blood samples were transferred to microtubes and centrifuged at 1500 g for 10 min at 4ºC. Plasma samples were collected, pooled for each time point (6 mice / sample), and stored at -80ºC prior to analysis.

[0324] All in vivo studies were conducted in accordance with Sanofi's institutional animal care policy. The cynomolgus monkey and mouse studies were approved by the French Ministry of Health and Welfare and the German Regierungspraesidium Darmstadt.

[0325] The concentrations of each mAb2 variant at each time point were determined by bottom-up LC-MS / MS assay. After plasma aliquot precipitation, the plasma precipitate was subjected to protein denaturation, reduction, alkylation, trypsin digestion, and solid-phase extraction, followed by analysis of the alternative peptides. mAb2 variants were analyzed at concentrations of 1.00, 2.00, 5.00, 10.0, 20.0, 50.0, 100, 200, and 400 µg / mL. -1 Calibration standards were prepared by incorporating them into plasma. A reverse-phase XBridge BEHC18 column (2.1 x 150 mm, 3.5 µM, 300 Å, Waters) was used on a Waters Acquity UPLC system with a flow rate of 300 µL / min. -1Peptide separation was performed using a stepwise gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For detection, a Sciex API 5500 mass spectrometer was used in cation mode with a source temperature of 700 ºC, an ion spray voltage of 5500 V, a curtain and nebulizing gas of 40, and a collision gas in the middle. The residence time was 20 ms, and the inlet potential for each conversion was 10 V. Multiple reaction monitoring of the two unique alternative peptides of the mAb2 backbone was used to determine the concentration relative to standards and controls using peak area determination with the MQIII integral algorithm from Analyst software. Clearance and serum half-life were obtained from a non-compartmental model of antibody concentration as a function of time using Phoenix software (Certara). All time points showing a sharp decrease in concentration were excluded from the mean plasma concentration due to (without being bound by any theory) assumed target-mediated drug disposal (TMDD) and / or anti-drug antibody (ADA) interference.

[0326] Example 2: Octet screening for saturation point mutations in conditioned media

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

[0328] To identify variants with slower FcRn dissociation rates than WT antibodies, 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 baseline variants that, compared to WT antibodies, exhibited either enhanced (AAA, LS, and YTE) or decreased (H435A, H310A / H435Q) affinity for FcRn at pH 6.0. The NiNTA biosensor captured the his-labeled antigen and subsequently captured each antibody variant at pH 7.4 to simulate the conditioned medium (…). Figure 2A For each of the six variants, the binding kinetics for rat FcRn (rFcRn) were measured at pH 6.0. Figure 2B The rat FcRn 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 dashes with interspersed single dots) and H310A / H435Q ( Figure 2BThe (long dashed line with two points) variant shows little or no FcRn binding kinetics (see also, for example, Shields et al. 2001, ibid.; Medesan et al. 1997, ibid.; and Raghavan et al. 1995, ibid.). Compared with WT ( Figure 2B Compared to (solid line), AAA ( Figure 2B (hyperline), LS ( Figure 2B (interspersed with short dashes with single dots) and YTE ( Figure 2B The (underlined) variants all exhibited slower dissociation kinetics, with FcRn dissociation rates reduced by 2–7.3 times. This suggests that the Octet screen is suitable for distinguishing variants with perturbed rFcRn dissociation kinetics.

[0329] IgG1 antibody mAb1 was used as a model system to generate a saturated mutagenic library for screening mutants with a reduced FcRn dissociation rate. Eleven sites in the Fc region of mAb1 were selected based on their proximity to or direct contribution to the FcRn interface. Figure 1 A and Figure 1 (B) (See, for example, Oganesyan et al. 2014, ibid.; and Shields et al. 2001, ibid.). All point mutations at these sites were constructed using site-directed mutagenesis and transfected into Expi293 cells for expression. Conditioned culture media were used to screen for saturated library mutants as described above. Normalized FcRn binding to Octet sensing for subsets of variants is illustrated in [image / image / description]. Figure 2C (long underline), and wild type ( Figure 2C (thick underline) and simulant negative control ( Figure 2C Normalized FcRn binding Octet sensing plot (dashed line). Simulated samples show a lack of observable FcRn binding. Several mutants significantly disrupt rFcRn binding, as almost no signal change is observed in the kinetic curves ( Figure 2C (Long dashed line, below the dashed line (simulant)). The cutoff value for variants with improved FcRn dissociation rates is defined as three standard deviations below the mean of the WT antibody. Figure 2C In the mutant subset shown, two ( Figure 2C (solid line) Compared with wild-type antibody, it has a significantly reduced dissociation rate ( Figure 2C (thick, long dashes), while the other variants have similar ( Figure 2C (interspersed with short dashes with single dots) or faster ( Figure 2C The dissociation rate of rFcRn (the long dashed line above the dashed line (simulation)).

[0330] The dissociation rate of rFcRn for all single-point mutations is at Figure 2D and Figure 14 This is shown through position and mutation. Figure 14 In this study, the data were classified into one of four categories based on the multiple change in rFcRn dissociation rate compared to the wild type, with the wild type represented by black squares.

[0331] exist Figure 14 In this study, the fold changes in the rFcRn dissociation rates at all eleven positions of the saturated library were normalized to the average value of the WT antibody and color-coded. All mutants fell into one of four categories: little or no binding (dark gray), faster rFcRn dissociation rate (gray), WT-like rFcRn dissociation rate (horizontal line), and slower rFcRn dissociation rate (grid). Several variants exhibited slower rFcRn dissociation rates (grid) than the WT antibody.

[0332] Figure 14 The mutant, colored dark gray, is compared to the analogue ( Figure 2C The dashed lines (indicating a similar pattern) show little or no binding to rFcRn and are located in the M252, I253, and S254 rings. The only mutations at I253 are methionine and valine, both of which significantly increase the rFcRn dissociation rate, further supporting the importance of I253 for FcRn interactions. An additional 120 variants (…) Figure 2D and Figure 14 (The light gray rectangle) makes the interaction with rFcRn unstable, with approximately 50% located in each C H 2 and C H Within the 3-domain structure, twenty-five mutants exhibited dissociation rates similar to WT. Figure 2D and Figure 14 (white rectangle), where eight of the eleven positions have at least one WT-like mutation ( Figure 14 (White rectangle). Compared to wild type, the following mutations have a significantly reduced rFcRn dissociation rate ( Figure 2D and Figure 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 have dissociation rates more than twice as slow as WT antibodies. Figure 2D These mutations were expressed and purified by protein A chromatography for further in vitro FcRn kinetic characterization.

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

[0334] In Biacore's FcRn binding kinetics assay, the AAA, LS, and YTE variants were used as positive controls for both human and rat FcRn at pH 6.0. The concentration-dependent binding of all variants to FcRn was observed, including wild-type, baseline, and standard. Figure 3 ) and precursor ( Figure 4A and Figure 4B Furthermore, the binding curves of a single injection of human and rat FcRn are shown in [figures to be inserted]. Figure 5A and Figure 5B The binding affinity of the wild-type antibody to human and rat FcRn was 2380 ± 470 nM and 207 ± 43 nM, respectively (Table 1).

[0335] Table 1: In vitro characterization parameters of the purified lead antibody for mAb1.

[0336]

[0337]

[0338] All the data shown in Table 1 were obtained using the experimental techniques shown at the top of each column.

[0339] As a comparison with the kinetic constants obtained from screening in conditioned media, the rFcRn dissociation rate obtained by Octet using purified protein was measured. Elution pH was determined in triplicate (n = 3) by FcRn affinity chromatography, and thermostability was probed in triplicate (n = 3) by DSF. FcRn binding kinetics for human and rat FcRn were obtained from Biacore in duplicate (n = 2) using a series of antibody concentrations and fitted independently. Steady-state binding response (RU) of each variant to human and rat FcRn at pH 7.4 was measured in triplicate (n = 3) using 1000 nM antibody in Biacore. Units for each measurement are as follows: Octet pH 6.0 rFcRn dissociation rate (x 10⁻¹⁰). -3 s -1 ); Elution pH (unitless); DSF T m (ºC); 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 reaction (RU).

[0340] exist Figure 5B Among them, the AAA (dashed line), LS (stretched line with two dots), and YTE (stretched line with one dot) variants showed 1.6 to 10.4 times enhanced binding affinity compared to WT. The characteristic of the baseline variant with the tightest FcRn affinity is species-specific, as LS has the tightest affinity for hFcRn, while rFcRn has a tighter affinity for YTE (Table 2A).

[0341] Table 2A: In vitro characterization parameters of the purified dual antibody for mAb1.

[0342]

[0343]

[0344] Most of the leader variants of human and rat FcRn ( Figure 5A and 5B The solid lines (various shaded areas) showed significantly slower (> 2-fold) association rates compared to WT or baseline variants (Table 1). The N434F and N434Y mutations were the only variants exhibiting increased association rates with both FcRn types. Without any theoretical constraint, the apparent binding affinity of the lead variants is generally weaker than WT due to the slower association kinetics with hFcRn, which is consistent with rFcRn (…). Figure 5C and Figure 5D (Table 1) is different. The affinity for rFcRn is weaker than that for YTE ( Figure 5D (Table 2A, facing the diagonal of the lower left corner). Without any theoretical constraints, these results indicate that a single mutation is insufficient to enhance affinity beyond the LS and YTE variants. Ranking the FcRn dissociation rates (due to the weak binding affinity of the variants to hFcRn) revealed subsets of reduced dissociation rates for human and rat FcRn: M252Y, N434F / P / Y, T256D / E, and T307A / E / F / Q / W (Table 2A). Further attention should be paid to combinations of these variants to further improve FcRn binding capacity in the Fc region beyond the baseline variants.

[0345] Table 2B shows the in vitro characterization parameters of the lead variant.

[0346] Table 2B: In vitro characterization parameters of the lead variant.

[0347]

[0348] In Table 2B, all data were obtained using the experimental techniques listed at the top of each column. FcRn affinity chromatography, DSF, and FcγRIIIa were performed in triplicate (n = 3). FcRn binding kinetics with human and rat FcRn were obtained in quadruplicate and fitted independently. Unit: DSF T m (ºC); 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 (x10 9 M); Biacore pH 6.0rFcRn K D,app (x10 9 M); Biacore pH 7.4 hFcRn and rFcRn steady-state RU (RU).

[0349] Example 4: Combinatorial variants further reduce the FcRn binding and dissociation rate

[0350] Multiple leader mutations located at a single location ( Figure 14 (Black rectangles), such as T307 and N434, from which six and three mutations, respectively, were identified, exhibiting slower FcRn dissociation kinetics. Only mutations with the slowest FcRn dissociation rate with hFcRn at these locations 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 duplex, triple, and quadruple variants. The combinatorial library consisted of a total of 54 variants, including seven single-lead variants, 18 duplex variants, 20 triple variants, 8 quadruple variants, and WT antibody. These variants were named as follows: wild-type background containing M252, T256, T307, and N434, and relabeled as MTTN. Thus, triple variants... Y T QY Contains M252 Y T307 Q and N434 Y The mutation, while maintaining WT threonine at position 256.

[0351] Similar to single mutations, the FcRn binding kinetics of Biacore at pH 6.0 were used to determine which combination variants had improved affinity. Figure 6A and Figure 6B The diagram shows representative FcRn binding kinetic traces for each of the single (long dashed line with two dots), double (long dashed line with one dot), triple (long dashed line), and quadruple (short dashed line) types, compared to WT (dashed line) and benchmark variants with the closest affinity for their respective species' FcRn (hFcRn: LS (long dashed line with two dots); rFcRn: YTE (solid line)). hFcRn association and dissociation rates ( Figure 6C The study revealed that two single, 15 dual, 18 triple, and eight quadruple variants exhibited enhanced binding affinity compared to the LS variant. Figure 6C (dashed line). Similarly, all combinations except one triple variant have a tighter affinity for rFcRn than YTE. Figure 6D (The diagonal facing the lower left). In the case of hFcRn, the additional mutations that enhance FcRn further increase binding affinity ( Figure 6C The five combinations with the closest affinity for hFcRn are all quadruple variants. Figure 6C (The one with squares) has a binding affinity approximately 500 times higher than the wild type. A similar phenomenon did not occur with rFcRn. Figure 6D ), because the variant with the highest affinity is the bivariate ( Figure 6D (Horizontal line). Triple ( Figure 6D (vertical line) and quadruple ( Figure 6D The (squared) variant typically shows only a slight decrease (less than 2-fold) in dissociation rate, but also exhibits a decreased association rate. Figure 6D Unbound by any theory, these results suggest there may be a lower limit (approximately 0.5 nM) on the apparent binding affinity of FcRn achieved with rFcRn, while this limit does not exist for hFcRn. Figure 6B In total, more than 40 combination variants exhibited tighter affinity than the baseline variants, and further characterization is needed to select combinations with favorable properties for in vivo studies.

[0352] Example 5: The combined variants maintained significant binding at physiological pH.

[0353] 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 directly measured the perturbation of pH dependence using a linear pH gradient. The variants H435A and H310A / H435Q, which exhibit weak FcRn binding, did not bind to the column regardless of pH. Figure 8AWT eluted near physiological pH (pH 7.37 ± 0.05), while AAA, LS, and YTE required higher pH (Table 2B). All combinatorial variants and the seven lead single variants required higher pH elution from the affinity column than WT. Figure 8A and Figure 8C The N434F / Y variants eluted at higher pH levels than LS (Table 2B), defying scientific theory and indicating that these variants (alone and in combination) disrupted pH dependence. Representative chromatograms show a significant shift in elution pH to higher levels with increasing number of mutations. Figure 9A and Figure 9B A strong correlation was found between elution pH and the dissociation rate of hFcRn (R² = 0.94). Figure 9C This indicates that the slower FcRn dissociation rate at pH 6.0 directly contributes to the increase in elution pH for FcRn variants.

[0354] 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 because some variants exhibit unreliable kinetics and show little or no binding at this pH. Figure 7A and Figure 7B The diagram shows representative dynamic traces of single (long dash with two points), double (long dash with one point), triple (long dash), and quadruple (short dash) variants, which are compared with LS ( Figure 7A (solid line) and YTE ( Figure 7B (solid line) Comparison. Both variants exhibited maximum residue binding to human and rat FcRn at pH 7.4. Most lead single variants showed slightly increased FcRn binding compared to WT (4.3 ± 1.0 RU), but this was less than that of AAA (13.1 ± 1.7 RU), LS (18.5 ± 2.6 RU), and YTE (13.1 ± 1.6 RU), except for the N434F / Y mutation (Tables 2A and 2B). The combined variant also showed significant residue binding to FcRn from both species at pH 7.4, even more so than N434F / Y (…). Figure 7A and Figure 7B Unbound by any theory, ideal candidates for in vivo studies are variants that exhibit increased FcRn binding at low pH but maintain low levels of binding in a manner similar to WT at elevated pH (such as AAA, LS, and YTE variants). Figure 7C and Figure 7D In the figure shown, these combinations will occupy the lower left quadrant, which is specified by the affinity of the LS and YTE variants for human and rat FcRn at each pH.

[0355] Example 6: FcRn affinity chromatography

[0356] The combined 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 Figure 7D Unbound by any theory, these results suggest that higher affinity at pH 6.0 generally translates to greater FcRn binding at pH 7.4. These variants can maintain FcRn binding in the bloodstream and have a short serum half-life and / or promote its clearance, similar to the high FcRn affinity abdeg mutation (see, for example, Swiercz et al. 2014, colleagues; and Vaccaro et al. 2005, ibid.). Since antibody-FcRn interactions are pH-dependent and occur only at low pH (< pH 6.5), saturation mutations can enhance the interaction through contributions from hydrophobicity or charge sources that may disrupt the deprotonation of key histidine residues. Figure 1 B (as shown in the figure) and this interaction is weakened at physiological pH.

[0357] FcRn affinity chromatography directly measures pH-dependent perturbations of FcRn interactions using a linear pH gradient (see, for example, Schlothauer et al. 2013, ibid.). FcRn affinity chromatography using AAA, LS, YTE, H435A, and H310A / H435Q variants reveals H435A (… Figure 8A (light gray solid line) and H310A / H435Q ( Figure 8A The wild-type antibody (AQ, dark gray solid line) did not bind to FcRn even at pH 5.5 and eluted in the effluent. Wild-type antibodies eluted near physiological pH (pH 7.37 ± 0.05), while AAA, LS, and YTE (via Octet...) eluted... Figures 2A-2D ) and Biacore ( Figure 3 (With a slower dissociation rate and tighter FcRn binding affinity than the wild type) required considerably higher pH for dissociation from the column (AAA: 7.94 ± 0.06; LS: 8.29 ± 0.03; YTE: 8.14 ± 0.03). Elution profiles revealed that all variants in the combinatorial library required higher pH than the wild type for elution from the affinity column. Figure 9AThe diagram shows representative chromatograms at average elution pH for single (long streaks with two dots), double (long streaks with one dot), triple (long streaks), and quadruple (short streaks) variants. Compared to WT, the seven lead single variants required higher pH for dissociation from the column. Figure 10A (Table 3), while those variants (K288D / N, Y436H / H / W) with similar wild-type kinetics for hFcRn all eluted at pH levels similar to the wild type.

[0358] Table 3: In vitro characterization parameters of the lead antibody variant

[0359]

[0360] All data were obtained using the experimental techniques listed at the top of each column. Elution pH was determined in triplicate (n = 3) by FcRn affinity chromatography, and thermostability was probed in triplicate (n = 3) by DSF. FcRn binding kinetics for human and rat FcRn were obtained from Biacore using a series of antibody concentrations (n ​​= 4) and fitted independently. Units for each measurement are as follows: elution pH (unitless); DSF Tm (ºC); Biacore pH 6.0 h FcRn association rate (x10⁻¹⁰). 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).

[0361] Both N434F / Y variants eluted at higher pH levels than the LS variant (N434F: 8.30 ± 0.05; N434Y: 8.46 ± 0.02) and exhibited considerable FcRn binding at pH 7.4 (Table 4). These results indicate that these variants alone can disrupt pH dependence. Generally, the average elution pH increases with increasing number of mutants enhancing FcRn binding. Figure 9B Unbound by any theory, it shows a strong correlation with elution pH compared to the dissociation rate of hFcRn (R). 2 = 0.94)( Figure 9CThis indicates that disruption of the pH-dependent nature of the interaction directly contributes to the slower FcRn dissociation rate observed in the combinatorial library at pH 6.0.

[0362] Example 7: Thermal Stability

[0363] Most proteins (including antibodies) with low thermodynamic stability exhibit increased tendency to misfold and aggregate, which will limit or hinder their activity, efficacy, and potential as novel therapeutic agents. The thermal stability of each variant was determined using DSF, and the melting temperature (T0) was reported. m () is defined as the midpoint of the first transition in the Sypro Orange fluorescence intensity curve. (T) m Compared to WT (69.0 ± 0.2ºC), the LS variant is similar to WT (68.5 ± 0.3ºC), and AAA and YTE are thermally unstable, differing by approximately 8ºC (AAA: 61.3 ± 0.6ºC; YTE: 61.2 ± 0.3ºC). Figure 8B , Figure 9B and Figure 10B ; and Tables 2B, 3, and 4). (with T) m Compared to WT and LS, which have a thermal stability of 69.0 ± 0.2ºC, the DSF, AAA, and YTE variants exhibit lower thermal stability, with a difference of approximately 8ºC.

[0364] Table 4: In vitro characterization parameters of the baseline and lead combination

[0365]

[0366] Mutations introduced into the wild-type backbone are indicated in bold and underline. All data were obtained using the experimental techniques shown at the top of each column. Elution pH and T were determined in triplicate (n = 3). m FcRn binding kinetics (n = 4) to human and rat FcRn at pH 6.0 were obtained from Biacore and independently fitted. Steady-state FcRn binding responses at pH 7.4 were measured in triplicate at a single antibody concentration using Biacore. FcγRIIIa binding affinity was determined in duplicate from a series of antibody concentrations using Biacore. Units for each measurement are as follows: elution pH (unitless); DSF Tm (ºC); Biacore pH 6.0 h FcRn association rate (x10⁻¹). 5 M -1 s -1 ), dissociation rate (x10) -2 s -1 ) and K D,app (x10 9 M); Biacore pH 6.0rFcRn 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).

[0367] Compared to the wild type, 12 of the 18 lead saturated variants had reduced T... m Furthermore, several T307 mutants (T307E / F / M / Q) showed slight stabilizing effects (Table 4). Seven single variants were used for combination ( Figure 10B None of the results in Table 4 are significantly unstable relative to YTE. Figure 8B (and Table 5). With a single variant ( Figure 9D Compared to the white circle, add a double ( Figure 9D (horizontal line), triple ( Figure 9D (vertical line) and quadruple ( Figure 9D The (squared) variants further reduced overall thermal stability. Several variants showed TA values ​​below AAA or YTE (61.2 ± 0.ºC). m Of these variants, >60% contain T307W. Tetrapolyvariates ( Figure 9D The gridded structures show a distinct bimodal distribution of melting temperature, with the combination containing T307Q exhibiting approximately 6ºC higher thermal stability than those containing T307W. Figure 9D ).

[0368] Example 8: Binding Interaction of Fc Variant Alterations with FcγRIIIa

[0369] In addition to its interaction with FcRn, the Fc region hinge and C H The 2 domain is responsible for interactions with other Fc receptors, including FcγRIIIa. Since five of the seven single variants used to construct the combinatorial saturated library are located in the C domain... H In the 2-domain structure, although their location is far from the interaction interface, the ability to interact with these receptors may be impaired compared to the wild type. Using Biacore to measure FcγRIIIa binding in a manner similar to FcRn binding at pH 7.4 revealed that YTE ( Figure 11A The dark gray variant shows similarities to the wild type ( Figure 11ACompared to the black color, the binding reaction is reduced by about 50%. Unbound by any theory, the reduced FcγRIIIa binding of YTE is due to the M252Y mutation (…). Figure 11B The result (the lowest white circle) is due to the fact that this variant alone has a significantly reduced affinity for the receptor. Other single mutations do not have this reduced affinity. Figure 11B (white circle), and the N434F / Y variant alone enhances binding by 16%–40%. These effects transfer to most, but not all, of their respective combinations. For example, combinations containing M252Y show reductions of 17% to 72% in FcγRIIIa binding (Table 5).

[0370] Table 5: Concentration of saturated library variants in conditioned medium

[0371]

[0372] A variant M DQF ( Figure 11B The highest among the triple variants showed a significant increase of 140% in FcγRIIIa binding. Therefore, the combined saturated library provides variants with multiple Fc receptor functions, which can be used to tailor therapeutic antibodies with specific effector functions.

[0373] Figure 11C Box plots of FcγRIIIa binding responses for seven lead monovariates are shown compared to the WT and YTE variants.

[0374] Example 9: pH dependence of balanced FcRn interactions in seven lead combinations

[0375] Unbound by any theory, candidate variants for further in vivo studies account for [a significant portion]. Figure 7C and Figure 7D The lower left quadrant of the diagram shown. Seven variants satisfy these criteria for hFcRn and include five double and two triple combinations (M DQ N, M DW N、 YD TN YE TN Y T W N、 YDQ N and YEQ N) and does not contain the mutation at the N434 position (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 YDQ N was eluted at a maximum pH of 8.51 ± 0.14. Figure 12A(Table 5), indicating only a slight perturbation to pH dependence and greater residue binding at pH 7.4 (Table 2A). One variant (M) DQ N) exhibits similar thermal stability to the wild type, and the six variants have similar or decreased T values ​​compared to the YTE variant. m ( Figure 12B (Table 4). In FcγRIIIa binding assays, five combinatorial variants showed reductions similar to YTE (Table 4). Further studies of single mutations revealed that M252Y significantly affects FcγRIIIa binding, and this effect is not bound by any theory, but translates to combinations with this mutation. The remaining six single mutations were similar to WT or showed slightly improved binding to the receptor.

[0376] Based on their FcRn binding properties, thermal stability, and FcγRIIIa binding, three combined variants were selected for further investigation. DQ (T256D / T307Q), DW (T256D / T307W), and YD (M252Y / T256D) each provided the best FcRn binding properties (Table 2B) as LS variants. Figure 12E Each variant offers a different range of thermal stability and FcγRIIIa binding properties. Figure 12F and Figure 12G (Table 2B) provides a range of functions. Figure 12H This is a diagram of a homogeneous bridged RF.

[0377] respectively with LS ( Figure 13A (thick long dash) and YTE variant ( Figure 13B Compared to (thick underline), the enhanced apparent binding affinity for human and rat FcRn at pH 6.0 represents a trade-off between association and dissociation rates. Figure 13A and Figure 13B (Table 4). Generally, combinations with faster dissociation rates also have faster association rates, and vice versa. This observation was maintained between human and rat FcRn (Table 4). Furthermore, all these variants showed better affinity for hFcRn at pH 7.4 than the LS variant ( Figure 13C (Thick underline) Lower steady-state reaction. These results are inconsistent with rFcRn because, compared to YTE, the five M252Y-containing variants showed lower steady-state reactions at pH 7.4. YD TN YE TN Y T W N、 YDQ N and YEQ N exhibits increased FcRn binding (Figure 13, Table 5). M DQ N and M DWThe N variant is the only combination of human and rat FcRn variants that exhibit cross-reactivity. Furthermore, neither of these variants disrupts the interaction with FcγRIIIa to a similar degree as the variant containing M252Y. Figure 12C and Figure 12D And Table 5; M DQ N: 600 ± 4 nM; MDWN: 512 ± 30 nM; WT: 467 ± 99 nM). Therefore, saturation and combinatorial mutagenesis at key FcRn interaction sites have led to the identification of lead variants that balance the pH dependence of the interaction, maintain Fc receptor function, enhance FcRn function in vivo, and prolong the serum half-life of therapeutic antibodies.

[0378] Example 10: Rheumatoid factor binding characteristics of the lead combination variant

[0379] The isoelectric point and RF binding of the lead variants were investigated, as these mutations can alter antibody surface charge and immunogenicity. More acidic antibodies have been thought to prolong antibody pharmacokinetics. Compared to WT and LS controls, all three leads resulted in a pI decrease of approximately 0.2 pH units due to T256D substitution. Mutations enhancing FcRn can simultaneously alter binding to host antibodies, such as rheumatoid factor (RF), due to overlapping interaction interfaces. Homogeneous bridging ELISA is suitable for measuring changes in RF binding of lead variants. Interestingly, LS and YTE showed completely opposite changes in RF binding compared to WT. Figure 12H LS significantly increased RF binding, while YTE showed a significant decrease (p < 0.001). YD (p < 0.001) and DW (p < 0.01) also significantly decreased RF binding, while DQ produced a similar response to WT. Unbound from any theoretical constraints, these results suggest that DQ, DW, and YD offer immunogenic advantages compared to LS. The YD, DW, and DQ variants represent a range of key antibody characteristics that can be combined with improved FcRn binding properties relative to the baseline YTE and LS variants.

[0380] Example 11: Lead combination variants can be transferred to other antibodies

[0381] A novel binding assay was developed using the CM5 sensor chip, such as... Figure 15A As shown. The binding assay includes the step of immobilizing streptavidin on a CM5 sensor chip to capture biotinylated FcRn to approximately 30 RU (supplement as needed). Antibody binding kinetics were measured at pH 6.0 and 7.4, and regeneration was measured at pH 8.5. Figure 15B and Figure 15CThe use of novel binding assays to perform direct immobilization of FcRn and streptavidin capture of biotinylated FcRn are illustrated.

[0382] Antibody 2 binding to FcRn at pH 6.0: In the case of mouse FcRn, the lead antibody 2 variant showed a slower dissociation rate than the LS variant (stretched) and wild-type (black). Figure 16A For human FcRn, all leader variants exhibited faster association rates but similar dissociation rates to LS (stretched). Figure 16B ).

[0383] FcRn binding of antibody 2 at pH 7.4: Compared to LS (strikethrough), all lead variants showed reduced human FcRn binding at pH 7.4. Figure 17A Similar to the background of antibody 1, DW (M) was observed at pH 7.4. DW N) and DQ (M) DQ The N) variant also showed binding to lower residues of mouse (rat) FcRn. Figure 17B ).

[0384] Compared to LS, the lead variant maintained higher binding affinity at pH 6.0 and lower residue binding at pH 7.4. Figure 18 Importantly, it was found that the variant could transfer between different IgG1 backgrounds with minimal effect on FcRn binding. Figure 19 As shown, LS exhibits similar elution pH regardless of the background. WT, DQ, and DW in the antibody 2 background show a higher elution pH than in the antibody 1 background, which is likely a result of tighter binding at pH 6.0 in the antibody 2 background.

[0385] All background variants of antibody 2 showed slightly increased thermal stability, such as Figure 20 As shown.

[0386] like Figure 21 As shown, similar to the background of antibody 1, YD ( YD TN shows a decrease in FcγRIIIa binding reaction (left) and affinity (right). DQ (light gray) and DW (dark gray) show FcγRIIIa binding properties similar to WT (black) against the background of antibody 2. The effect on FcγRIIIa binding of LS is consistent between antibody 1 and antibody 2.

[0387] Therefore, compared with the same lead variant in the antibody 1 background, the lead variant in the antibody 2 background does not significantly affect FcRn binding, pH dependence, thermal stability, or FcγRIIIa binding.

[0388] In one implementation, DQ (T256D / T307Q), DW (T256D / T307W), and YD (M252Y / T256D) variants are incorporated into additional IgG1 antibodies and recombinant Fc fragments: mAb2 recognizes a different antigen from mAb1, and Ab3 is an Fc fragment. In each case, in addition to elution pH, thermal stability, and FcγRIIIa binding affinity, pH-dependent FcRn binding kinetics (…) Figure 22 The results are also highly similar (Tables 2B and 6). Unbound by any theory, these results suggest that the DQ, DW, and YD variants confer improved FcRn binding properties to proteins composed of Fc domains.

[0389] Table 6: Concentration of saturated library variants in conditioned medium

[0390]

[0391] Example 12: Leader variant prolongs the elimination half-life of plasma antibodies in vivo

[0392] The effects of pharmacokinetic (PK) analysis of DQ, DW, and YD variants compared to WT and LS controls on the antibody circulating half-life in cynomolgus monkeys and hFcRn transgenic mice (strain Tg32) were investigated (see, for example, Avery et al., Mabs (2016) 8: 1064-1078). FcRn binding studies with cynomolgus monkey FcRn revealed similar binding affinity to hFcRn. Figures 23A to 23B Table 6). Each animal was intravenously injected with WT, LS, DQ, DW, or YD variants, and antibody concentrations were quantified by mass spectrometry to determine the monkey ( Figure 24A ) and hFcRn transgenic mice ( Figure 24B The clearance rate and serum half-life of the antibody were determined. Clearance rate and serum half-life were obtained 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 showed significantly reduced clearance rates compared to WT (p < 0.001). The plasma half-lives of the WT antibody were 9.9 ± 0.5 days and 11.7 days in monkeys and mice, respectively. Furthermore, the identified LS baseline and variants showed a significant increase in elimination half-life compared to the wild type in both species (2.5-fold and 1.7-fold increases in monkeys and mice, respectively) (Table 7). DQ, DW, and YD showed similarly prolonged half-lives compared to the LS baseline (Table 7). The DQ, DW, and YD mutations identified by saturation mutagenesis demonstrate that plasma half-lives are significantly prolonged compared to their WT counterparts in mouse and non-human primate models.

[0393] Table 7: Clearance and serum half-life of baseline and lead variants

[0394]

[0395] In Table 7, clearance and plasma half-life were determined using mAb2. Each clearance and half-life is a mean for n = 3 cynomolgus monkeys and a single assessment from a n = 6 hFcRn transgenic mouse library. Folds relative to WT and folds 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

[0396] Example 13: Combinatorial variants with enhanced FcRn binding at pH 6.0 and pH 7.4

[0397] Based on the Octet screening (BLI-based screening) as described in Example 2, a variety of single, dual, triple, and quadruple variants were generated, and their binding to FcRn at pH 6.0 and pH 7.4 was evaluated (Table 8).

[0398] Table 8: Binding affinity (pH 6.0) and steady-state binding (pH 7.4) of variants

[0399]

[0400]

[0401] Table 8 shows the binding affinity of various single, dual, triple, and quadruple mutants, as well as the baseline variants (AAA, LS, YTE), to FcRn at pH 6.0 and the steady-state binding to FcRn at pH 7.4.

[0402] Plot these values ​​on Figure 25 middle. Figure 25 The binding affinity at pH 6.0 and RU at pH 7.4 are shown. As shown in the figure, the baseline variant LS has the tightest binding affinity at pH 6.0, and the tested baseline variants (AAA, LS, YTE) have the largest residue binding at pH 7.4.

[0403] Sure Figure 25Several of the combined variants shown exhibited enhanced FcRn binding affinity at pH 6.0 and pH 7.4. To investigate whether any of the combined variants showed tighter binding at pH 6.0 and pH 7.4 than the MST-HN variant (referred to herein as the “YTEKF baseline,” which contains mutations from Met252, Ser254, Thr256, His433, and Asn434 to Tyr252, Thr254, Glu256, Lys433, and Phe434), the following methods were performed. Biotinylated human, cynomolgus monkey, and mouse FcRn were captured via the biotin CAPture method (see [link to biotin CAPture method]). Figure 26 (Schematic diagram). For pH 6.0, a series of concentrations from 1000 nM (5 pts) were performed in duplicate. For pH 7.4, a single concentration (1000 nM) was injected in triplicate (the capture level of each FcRn was increased 10-fold to observe binding at that pH). Association: 180 sec; Dissociation: 300 sec.

[0404] Figure 27 Human FcRn binding kinetics at pH 6.0, based on the YTEKF standard and with various combined variants, are shown. Figure 27 As shown, all the tested variants exhibited a two-order-of-magnitude closer affinity for human FcRn compared to the wild-type (WT).

[0405] Figure 28A and Figure 28B It was shown at pH 6.0 ( Figure 28A ) and pH 7.4 ( Figure 28B FcRn binding kinetics of the combined variant compared to the YTEKF benchmark. Figure 28A In this study, most variants exhibited slower dissociation rates than the YTEKF benchmark, and similarly or even slower association rates. Figure 28B In this study, YTEKF showed significant binding at pH 7.4, and the four variants showed even higher residue binding.

[0406] Table 9: Binding affinity (pH 6.0) and steady-state binding (pH 7.4) of selected variants

[0407]

[0408] Table 9 shows the binding affinity of the selected combination variants, as well as the binding affinity of the YTEKF baseline and WT to FcRn at pH 6.0 and the steady-state binding affinity to FcRn at pH 7.4.

[0409] Figure 29The binding affinity of the selected combination variants shown in Table 9 is compared with that of RU at pH 6.0 and at pH 7.4. (See Table 9 and...) Figure 29 As shown, the four tetrad variants exhibited higher affinity for FcRn at pH 6.0 and pH 7.4 compared to the YTEKF baseline. The four tetrad variants favored the T256D, T307Q, and N434Y mutations. These tetrad variants showed approximately 500-fold and 3-fold increases in affinity (at pH 6.0) compared to WT and YTEKF, respectively.

[0410] Other characterization parameters, such as thermal stability, binding with FcγRIIIa, and elution pH, were determined and are shown in Table 10.

[0411] Table 10: Other characterization parameters of the lead quaternion variant

[0412]

[0413] *Unbound by theory, due to the presence of “YTE” in YTEKF, thermal stability and FcγRIIIa binding are expected to be similar to the lead quaternary variant.

[0414] As shown in Table 10, all the lead quartet variants were found to be thermally unstable and exhibited reduced FcγRIIIa binding capacity.

[0415] This disclosure relates to the following implementation plan:

[0416] 1. A separated binding polypeptide comprising a modified Fc domain, the modified Fc domain comprising:

[0417] 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:

[0418] The amino acid at position 434 contains either phenylalanine (F) or tyrosine (Y); or

[0419] Tyrosine (Y) at amino acid position 252,

[0420] The amino acid positions are based on EU designations.

[0421] 2. A separated binding polypeptide comprising a modified Fc domain, the modified Fc domain comprising a combination of amino acid substitutions selected from the following positions:

[0422] a) Tyrosine (Y) at amino acid position 252 and aspartic acid (D) at amino acid position 256;

[0423] b) Aspartic acid (D) at amino acid position 256 and phenylalanine (F) at amino acid position 434;

[0424] c) Aspartic acid (D) at amino acid position 256 and tyrosine (Y) at amino acid position 434;

[0425] d) Tryptophan (W) at amino acid position 307 and phenylalanine (F) at amino acid position 434;

[0426] e) Tyrosine (Y) at amino acid position 252 and tryptophan (W) at amino acid position 307, where tyrosine (Y) is not at amino acid position 434;

[0427] f) Aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307, where tyrosine (Y) is not at amino acid position 434;

[0428] g) Aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307, where tyrosine (Y) is not at amino acid position 434;

[0429] 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

[0430] 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.

[0431] The amino acid substitutions are based on EU codes.

[0432] 3. A separated binding polypeptide comprising a modified Fc domain, the modified Fc domain comprising:

[0433] a) Double amino acid substitutions selected from M252Y / T256D, M252Y / T256E, M252Y / T307Q, M252Y / T307W, T256D / T307Q, T256D / T307W, T256E / T307Q, and 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; or

[0434] b) Triple amino acid substitutions 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;

[0435] The amino acid substitutions are based on EU codes.

[0436] 4. The isolated binding polypeptide according to any one of claims 1-3, wherein the modified Fc domain is a modified human Fc domain.

[0437] 5. The isolated binding polypeptide according to any one of claims 1-4, wherein the modified Fc domain is a modified IgG1 Fc domain.

[0438] 6. The isolated binding polypeptide according to any one of claims 1-5, wherein the binding polypeptide has human FcRn binding affinity.

[0439] 7. The isolated binding polypeptide according to any one of items 1-5, wherein the binding polypeptide has rat FcRn binding affinity.

[0440] 8. The isolated binding polypeptide according to any one of items 1-7, wherein the binding polypeptide has binding affinity for human and rat FcRn.

[0441] 9. The isolated binding polypeptide according to any one of claims 2-8, wherein the isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide containing a wild-type Fc domain.

[0442] 10. The isolated binding polypeptide according to claim 9, wherein the isolated binding polypeptide has an increased serum half-life compared with a binding polypeptide containing a wild-type Fc domain.

[0443] 11. The isolated binding polypeptide according to any one of claims 1-8, wherein the isolated binding polypeptide has a modified FcRn binding affinity compared to a binding polypeptide containing a wild-type Fc domain.

[0444] 12. The isolated binding polypeptide according to claim 11, wherein the isolated binding polypeptide has enhanced FcRn binding affinity compared with a binding polypeptide containing a wild-type Fc domain.

[0445] 13. The isolated binding polypeptide according to any one of claims 1-12, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared with a binding polypeptide containing a wild-type Fc domain.

[0446] 14. The isolated binding polypeptide according to any one of claims 1-13, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared to the FcRn binding affinity of the binding polypeptide at elevated non-acidic pH.

[0447] 15. The isolated binding polypeptide according to any one of claims 12-14, wherein the enhanced FcRn binding affinity comprises a reduced FcRn binding dissociation rate.

[0448] 16. The isolated bound polypeptide according to any one of claims 13-15, wherein the acidic pH is about 6.0.

[0449] 17. The isolated binding polypeptide according to any one of claims 13-16, wherein the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0450] 18. The isolated binding polypeptide according to any one of claims 1-17, wherein the isolated binding polypeptide has a modified FcγRIIIa binding affinity compared to a binding polypeptide containing a wild-type Fc domain. 19. The isolated binding polypeptide according to any one of claims 1-18, wherein the isolated binding polypeptide has a reduced FcγRIIIa binding affinity compared to a binding polypeptide containing a wild-type Fc domain.

[0451] 20. The isolated binding polypeptide according to any one of claims 1-18, wherein the isolated binding polypeptide has enhanced FcγRIIIa binding affinity compared with a binding polypeptide containing a wild-type Fc domain.

[0452] 21. The isolated binding polypeptide according to any one of claims 1-18, wherein the isolated binding polypeptide has substantially the same FcγRIIIa binding affinity as the binding polypeptide containing the wild-type Fc domain.

[0453] 22. The isolated binding polypeptide according to any one of claims 1-21, wherein the isolated binding polypeptide has substantially the same thermal stability as a binding polypeptide containing a wild-type Fc domain.

[0454] 23. The isolated binding polypeptide according to any one of claims 1-21, wherein the isolated binding polypeptide has substantially the same thermal stability as a binding polypeptide comprising a modified Fc domain having triple amino acid substitutions M252Y / S254T / T256E according to EU designations.

[0455] 24. The isolated binding polypeptide according to any one of items 1-23, wherein the isolated binding polypeptide is an antibody.

[0456] 25. The isolated binding polypeptide according to any one of items 1-24, wherein the isolated binding polypeptide is a monoclonal antibody.

[0457] 26. The isolated binding polypeptide according to any one of claims 24-25, wherein the isolated antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0458] 27. The isolated binding polypeptide according to any one of items 24-26, wherein the isolated antibody is a full-length antibody.

[0459] 28. The isolated binding polypeptide according to any one of claims 1-27, wherein the isolated binding polypeptide specifically binds to one or more human targets.

[0460] 29. An isolated nucleic acid molecule comprising a nucleic acid encoding an isolated polypeptide according to any one of claims 1-28.

[0461] 30. A vector comprising the isolated nucleic acid molecule as described in claim 29.

[0462] 31. The carrier according to claim 30, wherein the carrier is an expression carrier.

[0463] 32. A host cell comprising a vector according to any one of claims 30-31.

[0464] 33. The host cell according to item 32, wherein the host cell is of eukaryotic or prokaryotic origin.

[0465] 34. The host cell according to any one of items 32-33, wherein the host cell is of mammalian origin.

[0466] 35. The host cell according to any one of items 32-33, wherein the host cell is of bacterial origin.

[0467] 36. A pharmaceutical composition comprising the isolated binding polypeptide according to any one of claims 1-28.

[0468] 37. A pharmaceutical composition comprising the isolated antibody according to any one of claims 24-27.

[0469] 38. A separated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises aspartic acid (D) at amino acid position 256 according to EU number and glutamine (Q) at amino acid position 307.

[0470] 39. A separated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307 according to EU designation.

[0471] 40. A separated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises tyrosine (Y) at amino acid position 252 and aspartic acid (D) at amino acid position 256 according to EU designation.

[0472] 41. The isolated binding polypeptide according to any one of claims 38-40, wherein the modified Fc domain is a modified human Fc domain.

[0473] 42. The isolated binding polypeptide according to any one of claims 38-41, wherein the modified Fc domain is a modified IgG1 Fc domain.

[0474] 43. The isolated binding polypeptide according to any one of claims 38-42, wherein the binding polypeptide has human FcRn binding affinity.

[0475] 44. The isolated binding polypeptide according to any one of claims 38-42, wherein the binding polypeptide has rat FcRn binding affinity.

[0476] 45. The isolated binding polypeptide according to any one of claims 38-44, wherein the isolated binding polypeptide has an increased serum half-life compared with a binding polypeptide containing a wild-type Fc domain.

[0477] 46. ​​The isolated binding polypeptide according to any one of claims 38-44, wherein the isolated binding polypeptide has enhanced FcRn binding affinity compared with a binding polypeptide containing a wild-type Fc domain.

[0478] 47. The isolated binding polypeptide according to any one of claims 38-44, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared with a binding polypeptide containing a wild-type Fc domain.

[0479] 48. The isolated binding polypeptide according to any one of claims 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 elevated non-acidic pH.

[0480] 49. The isolated binding polypeptide according to any one of claims 46-48, wherein the enhanced FcRn binding affinity comprises a reduced FcRn binding dissociation rate.

[0481] 50. The isolated bound polypeptide according to item 47 or 48, wherein the acidic pH is about 6.0.

[0482] 51. The isolated binding polypeptide according to claim 47, wherein the acidic pH is about 6.0 and the non-acidic pH is about 7.4.

[0483] 52. The isolated bound polypeptide according to claim 48, wherein the acidic pH is about 6.0.

[0484] 53. The isolated binding polypeptide according to any one of claims 38-52, wherein the isolated binding polypeptide has a modified FcγRIIIa binding affinity compared to a binding polypeptide containing a wild-type Fc domain.

[0485] 54. The isolated binding polypeptide according to any one of claims 38-53, wherein the isolated binding polypeptide is a monoclonal antibody.

[0486] 55. The isolated binding polypeptide according to any one of claims 38-54, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0487] 56. The isolated binding polypeptide according to any one of claims 38-55, wherein the isolated binding polypeptide specifically binds to one or more human targets.

[0488] 57. An isolated nucleic acid molecule comprising a nucleic acid encoding an isolated polypeptide according to any one of claims 38-56.

[0489] 58. An expression vector comprising the isolated nucleic acid molecule as described in claim 57.

[0490] 59. A host cell comprising the expression vector according to claim 58.

[0491] 60. A pharmaceutical composition comprising the isolated binding polypeptide according to any one of claims 38-58.

[0492] 61. A separated binding polypeptide comprising a modified Fc domain, wherein the modified Fc domain comprises a combination of at least four amino acid substitutions, the combination comprising:

[0493] The amino acid at position 256 is either aspartic acid (D) or glutamic acid (E) and amino acid at position 307 is either tryptophan (W) or glutamine (Q), wherein amino acid at position 254 is not threonine (T), and further comprises:

[0494] The amino acid at position 434, either phenylalanine (F) or tyrosine (Y); and

[0495] Tyrosine (Y) at amino acid position 252,

[0496] The amino acid positions are based on EU designations.

[0497] 62. A separated binding polypeptide comprising a modified Fc domain having a combination of amino acid substitutions selected from the following positions:

[0498] 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;

[0499] 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;

[0500] 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;

[0501] 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

[0502] 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.

[0503] The amino acid substitutions therein are in accordance with EU designations.

[0504] 63. A separated binding polypeptide comprising a modified Fc domain, the modified Fc domain comprising:

[0505] The four amino acid substitutions are selected from M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307W / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307Q / N434F, and M252Y / T256D / T307W / N434Y.

[0506] The amino acid substitutions therein are in accordance with EU designations.

[0507] 64. The isolated binding polypeptide according to any one of claims 61-63, wherein the modified Fc domain is a modified human Fc domain.

[0508] 65. The isolated binding polypeptide according to any one of claims 61-64, wherein the modified Fc domain is a modified IgG1 Fc domain.

[0509] 66. The isolated binding polypeptide according to any one of claims 61-65, wherein the binding polypeptide has human FcRn binding affinity.

[0510] 67. The isolated binding polypeptide according to any one of claims 61-65, wherein the binding polypeptide has rat FcRn binding affinity.

[0511] 68. The isolated binding polypeptide according to any one of claims 61-67, wherein the binding polypeptide has binding affinity for human and rat FcRn.

[0512] 69. The isolated binding polypeptide according to any one of claims 61-68, wherein the isolated binding polypeptide has a modified FcRn binding affinity compared to a binding polypeptide containing a wild-type Fc domain.

[0513] 70. The isolated binding polypeptide according to items 61-69, wherein the isolated binding polypeptide has enhanced FcRn binding affinity compared with binding polypeptides containing wild-type Fc domains.

[0514] 71. The isolated binding polypeptide according to any one of claims 61-70, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at acidic pH compared with a binding polypeptide containing a wild-type Fc domain.

[0515] 72. The isolated binding polypeptide according to any one of claims 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.

[0516] 73. The isolated binding polypeptide according to any one of claims 61-72, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at a non-acidic pH compared to a binding polypeptide containing a wild-type Fc domain.

[0517] 74. The isolated binding polypeptide according to any one of claims 61-73, wherein the isolated binding polypeptide has enhanced FcRn binding affinity at a non-acidic pH compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0518] 75. The isolated binding polypeptide according to any one of claims 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 containing a wild-type Fc domain.

[0519] 76. The isolated binding polypeptide according to any one of claims 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.

[0520] 77. The isolated bound polypeptide according to any one of claims 61-76, wherein the acidic pH is about 6.0.

[0521] 78. The isolated bound polypeptide according to any one of claims 61-77, wherein the non-acidic pH is about 7.4.

[0522] 79. The isolated binding polypeptide according to any one of claims 61-78, wherein the isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide containing a wild-type Fc domain.

[0523] 80. The isolated binding polypeptide according to any one of claims 61-79, wherein the isolated binding polypeptide has a reduced serum half-life compared with a binding polypeptide containing a wild-type Fc domain.

[0524] 81. The isolated binding polypeptide according to any one of claims 61-80, wherein the isolated binding polypeptide has a reduced serum half-life compared with a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0525] 82. The isolated binding polypeptide according to any one of claims 61-81, wherein the isolated binding polypeptide has a modified FcγRIIIa binding affinity compared to a binding polypeptide containing a wild-type Fc domain.

[0526] 83. The isolated binding polypeptide according to any one of claims 61-82, wherein the isolated binding polypeptide has reduced FcγRIIIa binding affinity compared with a binding polypeptide containing a wild-type Fc domain.

[0527] 84. The isolated binding polypeptide according to any one of claims 61-83, wherein the isolated binding polypeptide has reduced FcγRIIIa binding affinity compared with a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0528] 85. The isolated binding polypeptide according to any one of claims 61-84, wherein the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide containing a wild-type Fc domain.

[0529] 86. The isolated binding polypeptide according to any one of claims 61-85, wherein the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0530] 87. The isolated binding polypeptide according to any one of claims 61-86, wherein the isolated binding polypeptide is an antibody.

[0531] 88. The isolated binding polypeptide according to any one of claims 61-87, wherein the isolated binding polypeptide is a monoclonal antibody.

[0532] 89. The isolated binding polypeptide according to any one of claims 61-88, wherein the isolated antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0533] 90. The isolated binding polypeptide according to any one of claims 61-89, wherein the isolated antibody is a full-length antibody.

[0534] 91. The isolated binding polypeptide according to any one of claims 61-90, wherein the isolated binding polypeptide specifically binds to one or more targets.

[0535] 92. An isolated nucleic acid molecule comprising a nucleic acid encoding an isolated polypeptide according to any one of claims 61-91.

[0536] 93. A carrier comprising the isolated nucleic acid molecule as described in claim 92.

[0537] 94. The carrier according to claim 93, wherein the carrier is an expression carrier.

[0538] 95. A host cell comprising a vector according to any one of claims 93-94.

[0539] 96. The host cell according to claim 95, wherein the host cell is of eukaryotic or prokaryotic origin.

[0540] 97. The host cell according to any one of items 95-96, wherein the host cell is of mammalian origin.

[0541] 98. The host cell according to any one of items 95-96, wherein the host cell is of bacterial origin.

[0542] 99. A pharmaceutical composition comprising the isolated binding polypeptide according to any one of claims 61-92.

[0543] 100. A pharmaceutical composition comprising the isolated antibody according to any one of claims 87-90.

[0544] 101. A separated 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 tyrosine (Y) at amino acid position 434, according to EU designation.

[0545] 102. A separated binding polypeptide comprising a modified Fc domain comprising 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 according to EU numbers.

[0546] 103. A separated binding polypeptide comprising a modified Fc domain comprising 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, according to EU designation.

[0547] 104. A separated 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 designation.

[0548] 105. A separated binding polypeptide comprising a modified Fc domain comprising 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, according to EU designation.

[0549] 106. The isolated binding polypeptide according to any one of claims 101-105, wherein the modified Fc domain is a modified human Fc domain.

[0550] 107. The isolated binding polypeptide according to any one of claims 101-106, wherein the modified Fc domain is a modified IgG1 Fc domain.

[0551] 108. The isolated binding polypeptide according to any one of claims 101-107, wherein the binding polypeptide has human FcRn binding affinity.

[0552] 109. The isolated binding polypeptide according to any one of claims 101-108, wherein the isolated binding polypeptide has a reduced serum half-life compared with a binding polypeptide containing a wild-type Fc domain.

[0553] 110. The isolated binding polypeptide according to any one of claims 101-109, wherein the isolated binding polypeptide has a reduced serum half-life compared with a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0554] 111. The isolated binding polypeptide according to any one of claims 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 containing a wild-type Fc domain.

[0555] 112. The isolated binding polypeptide according to any one of claims 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.

[0556] 113. The 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.

[0557] 114. The isolated binding polypeptide according to any one of claims 101-113, wherein the isolated binding polypeptide has reduced FcγRIIIa binding affinity compared with a binding polypeptide containing a wild-type Fc domain.

[0558] 115. The isolated binding polypeptide according to any one of claims 101-114, wherein the isolated binding polypeptide has reduced FcγRIIIa binding affinity compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0559] 116. The isolated binding polypeptide according to any one of claims 101-115, wherein the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide containing a wild-type Fc domain.

[0560] 117. The isolated binding polypeptide according to any one of claims 101-116, wherein the isolated binding polypeptide has reduced thermal stability compared to a binding polypeptide comprising M252Y / S254T / T256E / H433K / N434F.

[0561] 118. The isolated binding polypeptide according to any one of claims 101-117, wherein the isolated binding polypeptide is a monoclonal antibody.

[0562] 119. The isolated binding polypeptide according to claim 118, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0563] 120. The isolated binding polypeptide according to any one of claims 101-119, wherein the isolated binding polypeptide specifically binds to one or more targets.

[0564] 121. An isolated nucleic acid molecule comprising a nucleic acid encoding an isolated polypeptide according to any one of claims 101-120.

[0565] 122. An expression vector comprising the isolated nucleic acid molecule as described in claim 121.

[0566] 123. A host cell comprising the expression vector according to item 122.

[0567] 124. A pharmaceutical composition comprising the isolated binding polypeptide according to any one of claims 101-120.

[0568] 125. A method of treating a disease or condition of a subject in need, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide according to any one of claims 1-28, 38-58, 61-91 and 101-120, or administering to the subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 36-37, 60, 99-100 and 124.

[0569] 126. The method described in item 125, wherein the disease or condition is cancer.

[0570] 127. The method according to item 126, wherein the cancer is a tumor.

[0571] 128. The method according to item 125, wherein the disease or condition is an autoimmune disease.

[0572] 129. A method of treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide according to any one of claims 1-28 and 38-58, or administering to the subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 36, 37 and 60.

[0573] 130. A method of treating an autoimmune disease in a subject with such need, comprising administering to the subject a therapeutically effective amount of the isolated binding polypeptide according to any one of claims 61-91 and 101-120, or administering to the subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 99, 100 and 124.

Claims

1. A separated 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: The amino acid at position 434 contains either phenylalanine (F) or tyrosine (Y); or Tyrosine (Y) at amino acid position 252, The amino acid positions are based on EU designations.

2. A separated binding polypeptide comprising a modified Fc domain, the modified Fc domain comprising a combination of amino acid substitutions selected from the following positions: 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) Tyrosine (Y) at amino acid position 252 and tryptophan (W) at amino acid position 307, where 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, where 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, where 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 based on EU codes.

3. A separated binding polypeptide comprising a modified Fc domain, the modified Fc domain comprising: a) Double amino acid substitutions selected from M252Y / T256D, M252Y / T256E, M252Y / T307Q, M252Y / T307W, T256D / T307Q, T256D / T307W, T256E / T307Q, and 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; or b) Triple amino acid substitutions 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; The amino acid substitutions are based on EU codes.

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 according to any one of claims 1-5, wherein the binding polypeptide has rat FcRn binding affinity.

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

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

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

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