RU2795592C2 - Fc OPTIONS WITH INCREASED FcRn BINDING AND PROLONGED HALF-LIFE - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: new binding polypeptides applied in medicine. The invention discloses polypeptides with a modified Fc domain in which point mutations have been introduced to increase its ability to bind to FcRn and FcγRIIIa.

EFFECT: polypeptides containing such a modified Fc domain have a prolonged half-life of serum compared to a binding polypeptide containing a wild-type Fc domain.

34 cl, 29 dwg, 10 tbl, 13 ex

Description

RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application No. 62/622,468, filed January 26, 2018, the full disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The interaction of antibodies with the neonatal Fc receptor (FcRn) is a critical factor in maintaining and prolonging the serum half-life of antibodies and other Fc-derived therapeutics. FcRn is a heterodimer of the α-domain and β2-macroglobulin (β2-m) subunit in MHC class I, which recognizes antibody Fc heavy chain regions distinct from other Fcγ receptors (FcγR). Although FcRn is expressed in various tissues, it is thought to act primarily on the vascular endothelium, kidney, and blood-brain barrier to prevent IgG degradation, excretion, and inflammatory reactions, respectively.

Antibody binding to FcRn is highly pH dependent, and interaction occurs with high affinity (high nanomolar to low micromolar) only at low pH (pH<6.5) but not at physiological pH (pH about 7.4). Acidification of the endosome to pH less than 6.5 creates favorable conditions for the interaction between IgG and FcRn, which is directly responsible for inhibiting degradation and stimulating the recycling of FcRn-bound antibodies to the cell surface. An increase in pH weakens the interaction and promotes the release of antibodies into the bloodstream.

Fc engineering using high throughput mutagenesis has been extensively used to identify variants that increase FcRn binding capacity, as increased binding is expected to result in increased efficacy and reduced dosing frequency of therapeutic antibodies due to extended serum half-life compared to wild-type IgG antibody. . However, variants that increase the ability to bind to FcRn can have unpredictable results. For example, some IgG variants, such as N434W or P257I/Q311I among others, which in transgenic mice, Cynomolgus monkeys, and human FcRn (hFcRn) studies show a significant increase in affinity for FcRn at pH 6.0, have serum half-lives , as for wild-type molecules, or they become extremely short (see, for example, Kuo et al. 2011 supra; Datta-Mannan et al. 2007, J. Biol. Chem. 282: 1709-1717; and Datta-Mannan et al 2007, Metab Dispos 35 86-94). The T250Q/M428L (QL) variant has shown IgG framework-specific results in animal models (see, e.g., Datta-Mannan et al. 2007, J. Biol. Chem. 282: 1709-1717; and Hinton et al. 2006, J Immunol 176: 346-356). The M252Y/S254T/T256E variant (YTE, EU numbering) showed a 10-fold increase in vitro but exhibited reduced antibody-dependent cell-mediated cytotoxicity (ADCC) in vivo due to a 2-fold decrease in affinity for the FcγRIIIa receptor (see, e.g., Dall'Acqua et al 2002, supra).

Thus, there continues to be a need for Fc alternatives that have improved FcRn binding and a prolonged circulatory half-life.

SHORT DESCRIPTION

The present invention is based on the discovery of new IgG antibodies having one or more of the following characteristics: longer serum half-life, increased FcRn binding, increased FcRn binding at acidic pH, increased FcγRIIIa binding compared to antibody IgG wild-type and similar thermal stability.

Accordingly, in some aspects, an isolated binding polypeptide comprising a modified Fc domain containing 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 a threonine (T), and additionally contains phenylalanine (F) or tyrosine (Y) at amino acid position 434, or tyrosine (Y) at amino acid position 252, where the amino acid positions correspond to EC numbering.

In some exemplary embodiments, the altered Fc domain is an altered human Fc domain. In some exemplary embodiments, the altered Fc domain is an altered IgG1 Fc domain.

In some exemplary embodiments, the binding polypeptide has human FcRn binding ability, rat FcRn binding ability, or both human and rat FcRn binding ability.

In some exemplary embodiments, an isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, an isolated binding polypeptide has a longer serum half-life compared to a binding polypeptide containing a wild-type Fc domain.

In some illustrative embodiments, the isolated binding polypeptide has an altered ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to the ability to bind to an FcRn binding polypeptide at an elevated, non-acidic pH. In some exemplary embodiments, increased FcRn binding capacity includes a reduced rate of dissociation from FcRn.

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.

In some exemplary embodiments, the isolated binding polypeptide has an altered ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has a reduced ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has an increased ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain.

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

In some exemplary embodiments, the isolated binding polypeptide has about the same thermal stability as a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has approximately the same thermal stability as a binding polypeptide containing an altered Fc domain containing the triple amino acid substitution M252Y/S254T/T256E, according to EC numbering.

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, humanized, or human antibody. In some exemplary embodiments, the isolated antibody is a full length antibody.

In some exemplary embodiments, an isolated binding polypeptide specifically binds one or more targets in a human.

In other aspects, an isolated binding polypeptide is provided comprising an altered Fc domain comprising a combination of amino acid substitutions at positions selected from the group consisting of 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) 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, where 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, where 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, where the amino acid substitutions are represented by EC numbering.

In some exemplary embodiments, the altered Fc domain is an altered human Fc domain. In some exemplary embodiments, the altered Fc domain is an altered IgG1 Fc domain.

In some exemplary embodiments, the binding polypeptide has human FcRn binding ability, rat FcRn binding ability, or both human and rat FcRn binding ability.

In some exemplary embodiments, an isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, an isolated binding polypeptide has a longer serum half-life compared to a binding polypeptide containing a wild-type Fc domain.

In some illustrative embodiments, the isolated binding polypeptide has an altered ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to the ability to bind to an FcRn binding polypeptide at an elevated, non-acidic pH. In some exemplary embodiments, increased FcRn binding capacity includes a reduced rate of dissociation from FcRn. In some illustrative embodiments, the isolated binding polypeptide has a weaker ability to bind to FcRn at non-acidic pH than a binding polypeptide containing an altered Fc domain containing the double amino acid substitution M428L/N434S, according to EC numbering.

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.

In some exemplary embodiments, the isolated binding polypeptide has an altered ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has a reduced ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has an increased ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain.

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

In some exemplary embodiments, the isolated binding polypeptide has about the same thermal stability as a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has approximately the same thermal stability as a binding polypeptide containing an altered Fc domain containing the triple amino acid substitution M252Y/S254T/T256E, according to EC numbering.

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, humanized, or human antibody. In some exemplary embodiments, the isolated antibody is a full length antibody.

In some exemplary embodiments, an isolated binding polypeptide specifically binds one or more targets in a human.

In other aspects, an isolated binding polypeptide comprising an altered Fc domain comprising a) a double amino acid substitution selected from the group consisting of M252Y/T256D, M252Y/T256E, M252Y/T307Q, M252Y/T307W, T256D/T307Q, T256D/T307W, T256E/T307Q and T256E/T307W, where 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) a triple amino acid substitution of your choice from the group consisting of M252Y/T256D/T307Q, M252Y/T256D/T307W, M252Y/T256E/T307Q and M252Y/T256E/T307W, where threonine (T) is not the amino acid at position 254 histidine (H) is not at the amino acid position 311 , and tyrosine (Y) is not at amino acid position 434, where amino acid substitutions correspond to EC numbering.

In some exemplary embodiments, the altered Fc domain is an altered human Fc domain. In some exemplary embodiments, the altered Fc domain is an altered IgG1 Fc domain.

In some exemplary embodiments, the binding polypeptide has human FcRn binding ability, rat FcRn binding ability, or both human and rat FcRn binding ability.

In some exemplary embodiments, an isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, an isolated binding polypeptide has a longer serum half-life compared to a binding polypeptide containing a wild-type Fc domain.

In some illustrative embodiments, the isolated binding polypeptide has an altered ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to the ability to bind to an FcRn binding polypeptide at an elevated, non-acidic pH. In some exemplary embodiments, increased FcRn binding capacity includes a reduced rate of dissociation from FcRn. In some illustrative embodiments, the isolated binding polypeptide has a weaker ability to bind to FcRn at non-acidic pH than a binding polypeptide containing an altered Fc domain containing the double amino acid substitution M428L/N434S, according to EC numbering.

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.

In some exemplary embodiments, the isolated binding polypeptide has an altered ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has a reduced ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has an increased ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain.

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

In some exemplary embodiments, the isolated binding polypeptide has about the same thermal stability as a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has approximately the same thermal stability as a binding polypeptide containing an altered Fc domain containing the triple amino acid substitution M252Y/S254T/T256E, according to EC numbering.

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, humanized, or human antibody. In some exemplary embodiments, the isolated antibody is a full length antibody.

In some exemplary embodiments, an isolated binding polypeptide specifically binds one or more targets in a human.

In certain aspects, an isolated binding polypeptide is provided comprising an altered Fc domain, wherein the altered Fc domain contains aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307, according to EC numbering.

In some exemplary embodiments, the altered Fc domain is an altered human Fc domain. In some exemplary embodiments, the altered Fc domain is an altered IgG1 Fc domain.

In some illustrative embodiments, the binding polypeptide has the ability to bind to human FcRn, or the ability to bind to rat FcRn, or the ability to bind to both human and rat FcRn.

In some exemplary embodiments, an isolated binding polypeptide has a longer serum half-life compared to a binding polypeptide containing a wild-type Fc domain.

In some exemplary embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to the ability to bind to an FcRn binding polypeptide at an elevated, non-acidic pH. In some exemplary embodiments, increased FcRn binding capacity includes a reduced rate of dissociation from FcRn.

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.

In some exemplary embodiments, the isolated binding polypeptide has an altered ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain.

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

In some exemplary embodiments, an isolated binding polypeptide specifically binds one or more targets in a human.

In some aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding an isolated polypeptide.

In some aspects, a vector is provided that contains an isolated nucleic acid molecule. In some exemplary embodiments, the vector is an expression vector. In some aspects, an expression vector is provided that contains an isolated nucleic acid molecule.

In some aspects, a host cell containing a vector is provided. In some aspects, a host cell is provided that contains an expression vector.

In some exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In some illustrative embodiments, the implementation of the host cell is from a mammal. In some exemplary embodiments, the host cell is of bacterial origin.

In some aspects, a pharmaceutical formulation is provided comprising an isolated binding polypeptide.

In some aspects, a pharmaceutical formulation is provided comprising an isolated antibody.

In some aspects, an isolated binding polypeptide is provided comprising an altered Fc domain, wherein the altered Fc domain contains aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307, according to EC numbering.

In some exemplary embodiments, the altered Fc domain is an altered human Fc domain. In some exemplary embodiments, the altered Fc domain is an altered IgG1 Fc domain.

In some illustrative embodiments, the binding polypeptide has the ability to bind to human FcRn, or the ability to bind to rat FcRn, or the ability to bind to both human and rat FcRn.

In some exemplary embodiments, an isolated binding polypeptide has a longer serum half-life compared to a binding polypeptide containing a wild-type Fc domain.

In some exemplary embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to the ability to bind to an FcRn binding polypeptide at an elevated, non-acidic pH. In some exemplary embodiments, increased FcRn binding capacity includes a reduced rate of dissociation from FcRn.

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.

In some exemplary embodiments, the isolated binding polypeptide has an altered ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain.

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

In some exemplary embodiments, an isolated binding polypeptide specifically binds one or more targets in a human.

In some aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding an isolated polypeptide.

In some aspects, a vector is provided that contains an isolated nucleic acid molecule. In some exemplary embodiments, the vector is an expression vector. In some aspects, an expression vector is provided that contains an isolated nucleic acid molecule.

In some aspects, a host cell containing a vector is provided. In some aspects, a host cell is provided that contains an expression vector.

In some exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In some illustrative embodiments, the implementation of the host cell is from a mammal. In some exemplary embodiments, the host cell is of bacterial origin.

In some aspects, a pharmaceutical formulation is provided comprising an isolated binding polypeptide.

In some aspects, a pharmaceutical formulation is provided comprising an isolated antibody.

In some aspects, an isolated binding polypeptide is provided comprising an altered Fc domain, wherein the altered Fc domain contains tyrosine (Y) at amino acid position 252 and aspartic acid (D) at amino acid position 256, according to EC numbering.

In some exemplary embodiments, the altered Fc domain is an altered human Fc domain. In some exemplary embodiments, the altered Fc domain is an altered IgG1 Fc domain.

In some illustrative embodiments, the binding polypeptide has the ability to bind to human FcRn, or the ability to bind to rat FcRn, or the ability to bind to both human and rat FcRn.

In some exemplary embodiments, an isolated binding polypeptide has a longer serum half-life compared to a binding polypeptide containing a wild-type Fc domain.

In some exemplary embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to the ability to bind to an FcRn binding polypeptide at an elevated, non-acidic pH. In some exemplary embodiments, increased FcRn binding capacity includes a reduced rate of dissociation from FcRn.

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.

In some exemplary embodiments, the isolated binding polypeptide has an altered ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain.

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

In some exemplary embodiments, an isolated binding polypeptide specifically binds one or more targets in a human.

In some aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding an isolated polypeptide.

In some aspects, a vector is provided that contains an isolated nucleic acid molecule. In some exemplary embodiments, the vector is an expression vector. In some aspects, an expression vector is provided that contains an isolated nucleic acid molecule.

In some aspects, a host cell containing a vector is provided. In some aspects, a host cell is provided that contains an expression vector.

In some exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In some illustrative embodiments, the implementation of the host cell is from a mammal. In some exemplary embodiments, the host cell is of bacterial origin.

In some aspects, a pharmaceutical formulation is provided comprising an isolated binding polypeptide.

In some aspects, a pharmaceutical formulation is provided comprising an isolated antibody.

In some aspects, an isolated binding polypeptide comprising an altered Fc domain, wherein the altered 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, where amino acid position 254 is not threonine (T), and additionally contains phenylalanine (F) or tyrosine (Y) at amino acid position 434; and tyrosine (Y) at amino acid position 252, where the amino acid positions correspond to the EC numbering.

In some aspects, an isolated binding polypeptide is provided comprising an altered Fc domain comprising a combination of amino acid substitutions at positions selected from the group consisting of: a) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256 , glutamine (Q) at amino acid position 307 and tyrosine (Y) at amino acid position 434; b) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307 and tyrosine (Y) at amino acid position 434; c) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, 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, where the amino acid substitutions correspond to EC numbering.

In some aspects, an isolated binding polypeptide is provided that contains an altered Fc domain, comprising: a four amino acid substitution selected from the group consisting of M252Y/T256D/T307Q/N434Y, M252Y/T256E/T307W/N434Y, M252Y/T256E/T307Q/N434Y, M252Y/T256D/T307Q/N434F and M252Y/T256D/T307W/N434Y, in which amino acid substitutions are presented according to EU numbering.

In some exemplary embodiments, the altered Fc domain is an altered human Fc domain. In some exemplary embodiments, the altered Fc domain is an altered IgG1 Fc domain.

In some exemplary embodiments, the binding polypeptide has the ability to bind to human FcRn. In some exemplary embodiments, the binding polypeptide has the ability to bind to rat FcRn. In some exemplary embodiments, the binding polypeptide is capable of binding to both human and rat FcRn.

In some illustrative embodiments, the isolated binding polypeptide has an altered ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain.

In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to a binding polypeptide containing M252Y/S254T/T256E/H433K/N434F.

In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at a non-acidic pH compared to a binding polypeptide containing a wild-type Fc domain. In some illustrative embodiments, the isolated binding polypeptide has an increased ability to bind to FcRn at non-acidic pH compared to a binding polypeptide containing M252Y/S254T/T256E/H433K/N434F.

In some illustrative embodiments, the isolated binding polypeptide has increased FcRn binding capacity at acidic pH and increased FcRn binding capacity at non-acidic pH compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has increased FcRn binding capacity at acidic pH and increased FcRn binding capacity at non-acidic pH compared to a binding polypeptide containing M252Y/S254T/T256E/H433K/N434F.

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

In some exemplary embodiments, an isolated binding polypeptide has an altered serum half-life compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, an isolated binding polypeptide has a shorter serum half-life compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has a shorter serum half-life compared to a binding polypeptide containing M252Y/S254T/T256E/H433K/N434F.

In some exemplary embodiments, the isolated binding polypeptide has an altered ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has a reduced ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has a reduced ability to bind to FcγRIIIa compared to a binding polypeptide containing M252Y/S254T/T256E/H433K/N434F.

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

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, humanized, or human antibody. In some exemplary embodiments, the isolated antibody is a full length antibody.

In some exemplary embodiments, an isolated binding polypeptide specifically binds one or more targets.

In some aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding an isolated polypeptide.

In some aspects, a vector is provided that contains an isolated nucleic acid molecule.

In some exemplary embodiments, the vector is an expression vector.

In some aspects, a host cell containing a vector is provided.

In some exemplary embodiments, the host cell is of eukaryotic or prokaryotic origin. In some illustrative embodiments, the implementation of the host cell is from a mammal. In some exemplary embodiments, the host cell is of bacterial origin.

In some aspects, a pharmaceutical formulation is provided comprising an isolated binding polypeptide.

In some aspects, a pharmaceutical formulation is provided comprising an isolated antibody.

In some aspects, an isolated binding polypeptide is provided comprising an altered Fc domain containing 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 the EU numbering.

In some aspects, an isolated binding polypeptide is provided comprising an altered Fc domain containing 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 the EU numbering.

In some aspects, an isolated binding polypeptide is provided comprising an altered Fc domain containing 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 the EU numbering.

In some aspects, an isolated binding polypeptide is provided comprising an altered Fc domain containing 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 the EU numbering.

In some aspects, an isolated binding polypeptide is provided comprising an altered Fc domain containing 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 the EU numbering.

In some exemplary embodiments, the altered Fc domain is an altered human Fc domain. In some exemplary embodiments, the altered Fc domain is an altered IgG1 Fc domain.

In some exemplary embodiments, the binding polypeptide has the ability to bind to human FcRn.

In some exemplary embodiments, an isolated binding polypeptide has a shorter serum half-life compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has a shorter serum half-life compared to a binding polypeptide containing M252Y/S254T/T256E/H433K/N434F.

In some illustrative embodiments, the isolated binding polypeptide has increased FcRn binding capacity at acidic pH and increased FcRn binding capacity at non-acidic pH compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has increased FcRn binding capacity at acidic pH and increased FcRn binding capacity at non-acidic pH compared to a binding polypeptide containing M252Y/S254T/T256E/H433K/N434F.

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

In some exemplary embodiments, the isolated binding polypeptide has a reduced ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some exemplary embodiments, the isolated binding polypeptide has a reduced ability to bind to FcγRIIIa compared to a binding polypeptide containing M252Y/S254T/T256E/H433K/N434F.

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

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

In some exemplary embodiments, an isolated binding polypeptide specifically binds one or more targets.

In some aspects, an isolated nucleic acid molecule is provided, comprising a nucleic acid encoding an isolated polypeptide.

In some aspects, an expression vector is provided that contains an isolated nucleic acid molecule.

In some aspects, a host cell is provided that contains an expression vector.

In some aspects, a pharmaceutical formulation is provided comprising an isolated binding polypeptide.

In some aspects, a method is provided for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated binding polypeptide or administering to the subject a therapeutically effective amount of a pharmaceutical composition.

In some exemplary embodiments, the disease or disorder is a cancer. In some exemplary embodiments, the cancer is a tumor.

In some exemplary embodiments, the disease or disorder is an autoimmune disorder.

In some aspects, there is provided a method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated binding polypeptide or administering to the subject a therapeutically effective amount of a pharmaceutical formulation.

In some aspects, a method of treating an autoimmune disorder in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of an isolated binding polypeptide or administering to the subject a therapeutically effective amount of a pharmaceutical composition.

BRIEF DESCRIPTION OF GRAPHICS

The foregoing and other features and advantages of the present invention will be better understood from the following detailed description of illustrative embodiments in conjunction with the accompanying drawings.

Fig. 1A - Fig. 1B shows the structure of FcRn interacting with the Fc region of IgG1. On FIG. 1A depicts the interaction between hFcRn and IgG1 Fc (pdb: 4n0u) showing a single Fc monomer (dark gray ribbon) including glycosylation shown as rods labeled "glycan" in complex with the α domain (grey) and subunits β2-m (light gray) hFcRn. Most of the antibody residues involved in interaction with FcRn are located in loops immediately adjacent to the C _H 2- _CH 3 contact area (dashed line) and opposite the glycosylation site. On FIG. 1B shows a surface image of the crystal structure of Fc IgG1 (pdb: 5d4q) rotated 75º with respect to FIG. 1A. The FcRn binding site consists of residues in the _CH 2 and _CH 3 domains. The saturation library was built at eleven positions, shown as rods, as indicated: M252; I253; S254; T256; K288; T307; K322; E380; L432; N434 and Y436. All of these residues are in close proximity or in direct contact with FcRn. The surfaces of the critical histidine residues responsible for the pH-dependence cluster (H310, H433, H435) are near the positions of interest and are as indicated.

Fig. 2A - Fig. 2D depicts the Octet screening assay and its results. On FIG. 2A is a schematic representation of an Octet screening assay. NiNTA biosensors capture histidine-labeled antigen and then antibody variants to study the kinetics of binding to rat FcRn (rFcRn). On FIG. 2B shows the kinetic profiles of binding to c rFcRn at pH 6.0 of wild-type variant (solid line), T307A/E380A/N434A (AAA) variant (short dashes), LS (short dotted dashes), YTE (long dashes), antibodies to H435A (long dashes with dots) and H310A/H435Q antibodies (long dashes with two dots) aligned to the beginning of the rFcRn association phase. The H435A and H310A/H435Q variants hardly bind to FcRn. The YTE variant has the lowest FcRn dissociation rate tested in the Octet rFcRn binding assay. On FIG. 2C is a graphical representation of the normalization of FcRn binding kinetics at pH 6.0 of the reduced number of mutants obtained from the Octet screen. Most of the mutants retained a significant degree of binding to rFcRn, but some results were similar to the test control (dashed line), indicating the loss of all binding to rFcRn (long dashes below the dashed line (test)). Two variants (solid lines) had a slower rate of dissociation from rFcRn than wild-type antibodies (bold long dashes). On FIG. 2D depicts an analysis of the rFcRn dissociation rate plot for all point mutations with observed rFcRn binding kinetics separated by residue position. Saturation variants fell into one of the following four dissociation rate modes from rFcRn: no binding (not shown), faster binding (black), wild-type binding (white), slow binding (grey). Eighteen mutants showed a significantly slower rate of dissociation from rFcRn than wild-type antibodies (black dotted lines).

On FIG. 3 graphically depicts Biacore kinetics of reference samples and wild-type variants with human and rat FcRns at pH 6.0 and pH 7.4. All FcRn binding curves for the dilution series of wild type variants (upper left), AAA variant (upper right), M428/N434S (LS) variant (lower left), and M252Y/S254T/T256E (YTE) variant (lower right) are shown for Human (first and third columns) and rat (second and fourth columns) FcRn at pH 6.0 (first and third rows) and pH 7.4 (second and fourth rows). Variants AAA, LS and YTE showed slower dissociation from FcRn than wild-type antibodies. In general, the antibodies bind rFcRn with an approximately 10-fold increased capacity compared to the wild-type antibody. The LS variant had the strongest affinity for hFcRn at pH 7.4 and the highest residual binding at pH 7.4, while the rFcRn binding of the YTE variant was the closest.

On FIG. 4A graphically depicts Biacore kinetics of saturation leading variants with human and rat FcRn at pH 6.0. Traces of kinetic binding of FcRn for a series of dilutions are shown for the 18 leading saturation options. Dissociation rates of M252Y, T256D, T256E, N434F, N434P, N434Y, T307A, T307E, T307F, T307Q, and T307W with human and rat FcRn were slower. The remaining variants were specific for rat FcRn only.

On FIG. 4B is a graphical representation of FcRn binding kinetics for WT, comparison variant, and leading single saturation variants with human FcRn at pH 6.0. Sensorgrams of FcRn binding with serial dilutions of WT, LS, YTE and 18 saturation variants with human FcRn at pH 6.0. Single saturation options used for the combination library are underlined and in bold.

On FIG. 5A - Fig. 5D depicts data showing that several variants have slower dissociation rates from human and rat FcRn at pH 6.0. On FIG. 5A and 5B show Biacore sensorgrams of various variants. On fig. 5A shows dissociation rates from human FcRn at pH 6.0 for the YTE variant (long dashes with dots), LS variant (long dashes with two dots), wild-type (WT; dotted line) and saturation leading variants (leading; solid lines). different shades). On FIG. 5A shows normalized sensorgrams showing improved dissociation rates with hFcRn compared to WT. On FIG. 5B shows dissociation rates from rat FcRn at pH 6.0 for AAA variant (dashed line), LS variant (two-dot dashes), YTE variant (single-dot dashes), wild-type variant (solid line), and saturation leading variants (dashed lines). lines of different frequency and thickness). A representative injection of each of the top eleven antibodies is shown for clarity. These leading single variants showed improved dissociation kinetics from human and rat FcRn compared to wild type. On FIG. 5C and 5D show plots of binding capacity for leading antibody saturation (white circles) and wild-type antibodies (black circle) to human (FIG. 5C) and rat (FIG. 5D) FcRn using binding and dissociation rates derived from kinetic measurements. Biacore. Comparisons shown are AAA (diagonal lines pointing down to the right), LS (dotted) and YTE (diagonal lines pointing down to the left). Despite the improvement in FcRn dissociation rate, most variants did not have stronger affinity for human or rat FcRn due to slower association kinetics. Eleven variants had slower dissociation rates from both FcRn species.

On FIG. 6A - Fig. 6D depicts data showing that combinations of lead saturation mutations further improve FcRn dissociation rates and binding abilities. On FIG. 6A and 6B are typical Biacore sensorgrams showing dissociation from FcRn for human and rat FcRn, respectively. On FIG. 6A shows normalized sensorgrams for human FcRn typical variants with single (dashed line), double (solid light gray), triple (solid gray line) and quadruple (solid black line) combinations compared to the wild-type variant (dashed line) and the variant LS (long strokes with two dots). On FIG. 6B shows normalized sensorgrams for rat FcRn typical variants with single (long dashes with two dots), double (long dashes with one dot), triple (long dashes), and quadruple (short dashes) combinations compared to the wild-type variant (dashed line) and the YTE variant (solid line). Inclusion of multiple mutations decreased the dissociation rate and increased the FcRn binding capacity to a greater extent than comparison variants. On FIG. 6C and FIG. 6D is a plot of the combined saturation variants showing the rate of interaction as a function of the rate of dissociation for either human (FIG. 6C) or rat (FIG. 6D) FcRn, showing that most variants had increased binding to FcRn at pH 6.0 compared to comparison options. The variants with the closest binding to human and rat FcRn were quadruple and double combinations, respectively.

(Уравнение 2)) всех вариантов насыщения с FcRn человека (Фиг. 7C) или крысы (Фиг. 7D) при pH 7,4 как функции способности связывания при рН 6,0. На Фиг. 7С показано сравнение остаточного связывания с FcRn при рН 7,4 со способностью связывания с FcRn при рН 6,0. Лидирующие комбинации с улучшенными свойствами связывания с FcRn занимают нижнюю левую четверть изображения относительно варианта сравнения LS (ромб). На рис. 7D варианты LS (ромб) и YTE (треугольник) служат граничными условиями для подтверждения лидирования, соответственно. Эти два варианта имели способность к наиболее тесному связыванию при рН 6,0 и наибольшее остаточное связывание при рН 7,4 для FcRn человека и крысы, соответственно. На обеих Фиг. 7C и 7D показаны одинарный (белые кружки), двойной (светло-серые кружки), тройной (темно-серые кружки) и четверной (черные кружки) варианты, а также вариант YTE (треугольник). On figa - fig.7D presents data showing that increased binding to FcRn at pH 6.0 breaks the dependence of the interaction on the pH. On FIG. 7A and FIG. 7B shows representative Biacore sensorgrams of FcRn binding kinetics at pH 7.4 of variants with single (long dashes with two dots), double (long dashes with one dot), triple (long dashes), and quadruple (short dashes) combinations compared to the variant. wild-type (dashed line) and LS variant (Fig. 7A, solid line) and YTE variant (Fig. 7B, solid line). Increasing FcRn binding mutations resulted in more residual binding at physiological pH, with most double, triple and quadruple combination variants showing stable binding to both FcRn species. On FIG. 7C and FIG. 7D is a graphical representation of the steady state of RU (

(Equation 2)) of all saturation options with human (FIG. 7C) or rat (FIG. 7D) FcRn at pH 7.4 as a function of binding capacity at pH 6.0. On FIG. 7C shows a comparison of residual FcRn binding at pH 7.4 with FcRn binding capacity at pH 6.0. Leading combinations with improved FcRn binding properties occupy the lower left quarter of the image relative to the LS comparison (diamond). On fig. The 7D options LS (diamond) and YTE (triangle) serve as boundary conditions for leading confirmation, respectively. These two variants had the highest binding capacity at pH 6.0 and the highest residual binding at pH 7.4 for human and rat FcRn, respectively. On both Figs. 7C and 7D show the single (white circles), double (light gray circles), triple (dark gray circles), and quadruple (black circles) options, as well as the YTE (triangle) option.

8A - FIG. 8C shows data obtained from FcRn affinity chromatography and differential scanning fluorometry (DSF) comparisons. On fig. 8A shows normalized elution profiles for WT (solid black line), AAA (dashed line), LS (long dashes with two dots), YTE (long dashes with one dot), H435A (solid light gray line) and H310A/H435Q (AQ; solid dark gray line). The pH is indicated at the top of the graph. Null FcRn binding variants (H435A, H310A/H435Q) do not bind on the column and elute with flow (<10 ml). The AAA, LS and YTE variants elute at a higher pH than the WT antibody. On FIG. 8B shows the DSF profiles of the WT (black), LS (grey), and YTE (dark grey) variants. YTE was destabilized compared to WT and LS. On FIG. 8C shows the FcRn affinity column elution profiles for the top seven single variants used for the combined variants compared to the WT and LS variants (vertical dotted line). Two variants (N434F/Y) eluted at a higher pH than LS, indicating a reduced pH dependence of interaction with FcRn in variants containing these mutations.

On figa - Fig.9D shows data showing that the combined options significantly violate the dependence on pH and thermal stability. On FIG. 9A shows typical FcRn affinity chromatograms with single (long dashes with two dots), doubles (long dashes with one dot), triple (long dashes), and quadruple (short dashes) variants. An increase in the number of mutations that enhance binding to FcRn shifted the elution towards higher pH values; LS variant (small dotted vertical line). On FIG. 9B is a boxplot of elution pH for the leading saturation and combination variants, including single (white circles), double (horizontal lines), triple (vertical lines), and quadruple (checkered) mutants, indicating a tendency for pH to increase with increasing number mutants that enhance binding to FcRn. On FIG. 9C shows that the high correlation (R ² =0.94) between the pH of the FcRn affinity eluting and the rate of dissociation from hFcRn using Biacore revealed a loss of pH dependence of the antibody-FcRn interaction while improving the kinetics of dissociation from FcRn. Options AAA (diagonal lines pointing right down), LS (dashed lines) and YTE (diagonal lines pointing left down) had similar dissociation rates from hFcRn and elution pH to the dual options. On Fig.9D presents a box plot for T _pl. , derived from DSF for combined saturation variants, from which it is seen that additional mutations that enhance binding to FcRn destabilize the antibody compared to WT variants, single variants or comparison variants.

On FIG. 10A - Fig. 10B shows data obtained from FcRn and DSF affinity chromatography for the seven leading variants. Figure 10A shows FcRn affinity chromatography data for variants M252Y (solid line), T256D (short dashes with one dot), T256E (long dashes), T307Q (long dashes with one dot), T307W (long dashes with two dots), N434F (dashed) and N434Y (short dashes). Chromatograms show the pH shift during elution compared to wild-type and LS antibodies (vertical dotted lines). N434F and N434Y had a higher eluting pH (pH about 8.3) than the LS variant (vertical dotted line). The pH value at certain elution volumes is indicated above the chromatograms for reference. On FIG. 10B depicts the DSF profiles of the seven leading variants, which showed that none of the seven leading variants destabilized antibodies to the same extent as the YTE variant (vertical dotted line). All variants except T307Q (long dashes with one dot) were destabilized compared to WT (vertical dotted line).

On FIG. 11A - FIG. 11C shows data showing that FcγRIIIa binding was reduced in M252Y-containing combination variants. FIG. 11A shows the FcγRIIIa binding sensorgrams of the WT (black), LS (grey) and YTE (dark gray) variants showing the reduced binding response of the YTE variant. On FIG. 11B shows a box plot of FcγRIIIa binding interaction of the comparison, single and combined variants as indicated. Variants with M252Y mutations contain a reduced response to FcγRIIIa binding, including all quad variants. Combinations with N434F/Y usually show increased interaction with FcγRIIIa. On FIG. 11C shows FcγRIIIa binding responses for the top seven single variants compared to WT and YTE variants (dashed horizontal line). The M252Y mutation shows reduced FcγRIIIa binding compared to WT, while six show WT-like or increased binding to this receptor.

On FIG. 12A - FIG. 12D shows FcRn, DSF affinity chromatography and FcγRIIIa binding data for the top seven combination variants. FIG. 12A shows the FcRn affinity chromatograms for the top seven combination variants compared to the wild-type antibody and the LS variant (vertical dotted line and solid vertical line, respectively). Each leading variant had an eluting pH close to that of the LS variant. FIG. 12B shows the DSF profiles of the leading combined variants compared to the YTE and wild type variants (vertical dotted lines as indicated). Six of the seven leading options had T _pl. , which was similar to or more destabilized than the YTE variant: MDWN (long dashes with two dots); YTWN (long strokes); YDTN (solid line); YETN (long strokes with one dot); YDQN (dotted line); YEQN (short strokes with one dot). Option M DQ N had T _pl. similar to the wild-type antibody (short dashes). On FIG. 12C shows Biacore sensorgrams of FcγRIIIa binding kinetics for the top seven variants compared to wild type (large dotted line) and YTE variant (thick long dashes). Variants containing M252Y, YD TN (solid line), YDQ N (short dashes with one dot), Y T W N (long dashes), YE TN (long dashes with one dot) and YE QN (smaller dotted line), each had a reduced steady state RU in the same way as YTE. (D) shows the steady state of the RUs of the seven leading variants, the wild type and the YTE variant. Only the M DW N and M DQ N variants had similar affinity for FcγRIIIa as the wild-type antibody.

On FIG. 12E - FIG. 12H shows data showing that the top three variants exhibit a number of key antibody properties. On FIG. 12E shows the FcRn affinity chromatography elution profiles for DQ (solid), DW (dashed), and YD (dashed) variants compared to WT and LS (vertical dashed lines). Each duplicate showed an elution pH that was between WT and LS. On FIG. 12F shows DSF fluorescence profiles for three variants compared to YTE and WT (vertical dotted line), showing that YD (dashed line) and DW (dashed line) were slightly destabilized compared to YTE, but DQ (solid) was similar to WT . On FIG. 12G shows FcγRIIIa binding sensorgrams compared to WT and YTE (horizontal dotted line). YD (dashed) showed a similar binding response as YTE, while DQ (solid) and DW (dashed) showed a slight decrease compared to WT. On FIG. 12H presents data showing that homogeneous bridged RF-ELISA revealed three leading variants, and YTE showed significantly reduced or WT-like RF binding, in contrast to LS. **p<0.001, *p<0.01.

On FIG. 13A - Fig. 13D is data showing a comparison of the FcRn binding kinetics of the leading combination variants at pH 6.0 and pH 7.4. On FIG. 13A and FIG. 13B shows Biacore sensograms of FcRn binding of the leading combined variants for human FcRn (FIG. 13A) or rat FcRn (FIG. 13B) compared to the wild-type (dashed line) and either LS (hFcRn, FIG. 13A, bold long dashes) variant. , or YTE (rFcRn, Fig. 13B, bold long strokes) at pH 6.0. Each combination variant had an overall ability to bind more closely to the respective FcRn despite altered rates of interaction and dissociation. On FIG. 13C and FIG. 13D shows Biacore FcRn sensograms at pH 7.4. Each leading hFcRn variant had a similar or reduced response to FcRn binding at steady state compared to the LS variant. Only the M DQ N and M DW N variants showed less binding to rFcRn at pH 7.4 than the YTE variant.

On FIG. 14 is a table depicting the dissociation rates of rFcRn binding by the Octet technology of a saturation library in accordance with some embodiments. Wild-type (WT) and wild-type (WT-like) samples are indicated by white boxes; WT samples are as specified. Variants with little or no binding to rFcRn compared to wild type are indicated by dark gray boxes. Variants with a higher rate of dissociation from rFcRn compared to wild type are indicated by light gray boxes, and variants with a lower rate of dissociation from rFcRn than wild type are indicated by black boxes.

On FIG. 15A - Fig. 15C shows a new binding assay developed using the CM5 sensor chip. On FIG. 15A is a diagram of the analysis. On FIG. 15B shows direct immobilization of FcRn. On FIG. 15C shows streptavidin capture of biotinylated FcRn.

On FIG. 16A - Fig. 16B depicts antibody-2 FcRn binding at pH 6.0. Figure 16A depicts a human FcRn. On FIG. 16B depicts mouse FcRn.

On FIG. 17A - Fig. 17B depicts antibody-2 FcRn binding at pH 7.4. Figure 17A depicts a human FcRn. On FIG. 17B depicts mouse FcRn.

On FIG. 18 is a graphical representation of the pH-dependence for various antibody-2 variants. The leading variants retained higher binding capacity at pH 6 and lower residual binding at pH 7.4 than LS.

On FIG. 19 shows a comparison of the pH dependence of binding to FcRn using antibody-1 and antibody-2 scaffolds.

On FIG. 20 shows a comparison of thermal stability using antibody-1 and antibody-2 scaffolds.

On FIG. 21 shows a comparison of binding to FcγRIIIa using antibody-1 and antibody-2 scaffolds.

On FIG. 22A - FIG. 22I depicts multiple graphs showing that DQ, DW, and YD variants can be transferred between IgG1 scaffolds. Plots a-c depict normalized sensograms of FcRn binding at pH 6.0 in three IgG1 scaffolds with WT (light grey), LS (dark grey), DQ (solid black), DW (dotted) and YD (dashed) variants showing similar kinetics at low pH. These three variants, DQ, DW, and YD, had slightly higher binding and dissociation rates than the LS variant, but retained the ability to bind more closely to FcRn. Plots d-f depict FcRn binding sensorgrams at pH 7.4; comparison variant LS (solid black). Plots g-i depict FcRn binding response at pH 7.4 versus binding capacity at pH 6.0 for each antibody scaffold with options WT (grey), LS (dark grey), DQ (solid black), DW (open ) and YD (empty square). DQ, DW and YD show improved FcRn performance with increased binding at pH 6.0 and minimal binding at pH 7.4.

On FIG. 23A - FIG. 23C shows that the top three variants in the mAb2 framework similarly improve binding to cynomolgus FcRn. FIG. 23A shows normalized cFcRn binding sensograms at pH 6.0 for WT (grey), LS (dark grey), DQ (solid black), DW (dashed) and YD (dashed), demonstrating similar binding kinetics and capacity. hFcRn. On FIG. 23B shows that the cFcRn binding response for the three variants was drastically reduced at physiological pH; however, LS (dark grey) showed more binding than WT (grey), similar to hFcRn. On FIG. 23C shows a comparison of the residual binding reaction of cFcRn at pH 7.4 with the binding capacity of cFcRn at pH 6.0 WT (grey), LS (dark grey), DQ (solid black), DW (open) and YD (open box), which shows that all three variants retained the improved FcRn binding properties observed with hFcRn.

From FIG. 24A-24B show that the leading variants prolong the serum half-life of antibodies. Pharmacokinetic profiles of plasma antibody concentration as a function of time in cynomolgus monkeys (Fig. 24A) and hFcRn transgenic mice (Fig. 24B) with WT (wild type) antibodies (black circles with solid black line), LS (white circles with broken black line). line), DQ (light gray circles with a solid light gray line), DW (dark gray circles with a solid dark gray line), and YD (black circles with a dotted black line). All three leading options prolong the half-life of antibodies compared to WT.

Figure 25 is a graph of the steady state of RU of all saturation variants with human FcRn at pH 7.4 as a function of binding capacity at pH 6.0. Comparison of residual FcRn binding at pH 7.4 with FcRn binding capacity at pH 6.0 is shown. Quadruple combinations with improved FcRn binding properties at both pH 6.0 and pH 7.4 are shown in the box in the top right quarter of the image. Single (white circles), double (light gray circles), triple (dark gray circles), and quadruple (black circles) options are shown, as well as AAA, LS, and YTE comparative options (as indicated).

On FIG. 26 is a schematic of the Biotin CAPture method used to capture biotinylated FcRn.

On FIG. 27 depicts human FcRn binding kinetics at pH 6.0 for the YTEKF comparison variant and combined variants as indicated.

On FIG. 28A - FIG. 28B shows the FcRn binding kinetics of the combined variants compared to the YTEKF comparison variant at pH 6.0 (FIG. 28A) and at pH 7.4 (FIG. 28B). The wild type is indicated by the solid black line (WT) and the YTEKF comparison variant by the dotted line.

On FIG. 29 is a plot of RU steady state of selected human FcRn variants at pH 7.4 versus binding capacity at pH 6.0 compared to the YTEKF comparison. Several variants (leading quad variants) exhibited increased binding to human FcRn at pH 6.0 and pH 7.4 compared to the YTEKF reference variant.

DETAILED DESCRIPTION

The present invention relates to binding polypeptides (eg, antibodies) having an altered ability to bind to the neonatal Fc receptor (FcRn). In some embodiments, the binding polypeptides contain an altered Fc domain that enhances FcRn binding ability compared to a binding polypeptide that contains a wild-type (eg, unmodified) Fc domain. The present invention also provides nucleic acids encoding binding polypeptides, recombinant expression vectors and host cells for producing binding polypeptides, and pharmaceutical compositions containing binding polypeptides disclosed herein. Also provided are methods for using the binding polypeptides of the present invention to treat diseases.

Fc domains of immunoglobulins are involved in non-antigen binding processes and have several effector functions that are performed by binding to effector molecules, such as binding to FcRn. As shown in FIG. 1A, Fc domains consist of a CH2 domain and a CH3 domain. Most of the residues involved in the interaction with FcRn are located in the loops immediately adjacent to the C _H 2- _CH 3 contact area (Figure 1A, dotted line) and opposite the glycosylation site. Figure 1B shows an image of the surface of the crystal structure of Fc IgG1 (pdb: 5d4q) and shows the residues in the CH2 and CH3 domains that constitute the contact area when binding to FcRn. The present invention relates to binding polypeptides containing a modified Fc domain. Binding polypeptides containing a modified Fc domain may be antibodies or immunoadhesins or Fc fusion proteins.

In some embodiments, the binding polypeptide may comprise a modified Fc domain containing an amino acid substitution that alters the antigen-independent effector functions of the antibody, in particular the circulating half-life (e.g., serum half-life) of the binding polypeptide. In some embodiments, the binding polypeptide may comprise a modified Fc domain containing an amino acid substitution that alters the serum half-life of the binding polypeptide compared to a binding polypeptide containing a wild-type (i.e., unmodified) Fc domain. In some embodiments, the binding polypeptide may comprise a modified Fc domain containing an amino acid substitution that prolongs the serum half-life of the binding polypeptide compared to a binding polypeptide containing a wild-type (i.e., unmodified) Fc domain. In some embodiments, the binding polypeptide may comprise a modified Fc domain containing an amino acid substitution that results in a shorter serum half-life of the binding polypeptide compared to a binding polypeptide containing a wild-type (i.e., unmodified) Fc domain.

In some embodiments, a binding polypeptide comprising a modified Fc domain that alters (i.e., prolongs or shortens) the circulating half-life (e.g., serum half-life) further comprises one or more mutations in addition to the mutation (pits), which change the half-life from the bloodstream. In some embodiments, one or more mutations, in addition to mutations that alter the circulating half-life, provide one or more desired biochemical characteristics, such as, for example, one or more reduced or enhanced effector functions, the ability to non-covalently dimerize, increased ability to localize at the tumor site, a shorter serum half-life, a longer serum half-life compared to a whole unchanged antibody of approximately the same immunogenicity, and the like.

The binding polypeptides described herein may exhibit either increased or decreased binding to the neonatal Fc receptor (FcRn) compared to binding polypeptides lacking these substitutions and therefore have a longer or shorter serum half-life, respectively. Fc domains with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules may be usefully used in methods of treating mammals where a long half-life of the administered antibody is required, such as for the treatment of chronic diseases or disorders. In contrast, Fc domains with reduced FcRn binding capacity are expected to have shorter half-lives, and such molecules are also useful, for example, for administration to a mammal where reduced circulation time may be preferred, for example, for in vivo diagnostic imaging. or in situations where the parent antibody has toxic side effects if present in the bloodstream for extended periods of time. Fc domains with reduced FcRn binding ability are also less likely to cross the placental barrier and are thus also useful in the treatment of diseases or disorders in pregnant women. In addition, other applications that may require a reduction in the ability to bind to FcRn include applications localized in the brain, kidney and/or liver.

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

In addition, the experiments described here, unless otherwise indicated, use conventional molecular and cellular biological and immunological methods known to those skilled in the art. Such methods are well known to the skilled person and are fully explained in the literature. See, for example, Ausubel, et al. , ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, New York (1987-2008), including all applications, Molecular Cloning: A Laboratory Manual (Fourth Edition) by 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).

Unless otherwise indicated, scientific and technical terms used in this document have meanings that are commonly understood by those skilled in the art. In the event of any latent ambiguity, the definitions given in this document take precedence over any definition given in the dictionary or used outside of this document. Unless the context otherwise requires, singular terms will include the plural forms, and plural terms will include the singular form. The use of "or" means "and/or" unless otherwise noted. The use of the term "including", as well as other forms such as "include", "includes" and "included", is not limiting.

Generally, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and chemistry, and hybridization of proteins and nucleic acids, as well as the corresponding techniques described herein, are well known and widely used in the art. The methods and techniques provided herein are generally performed, unless otherwise indicated, in accordance with conventional methods well known in the art and as described in the various general and more specific references cited and discussed herein. Enzymatic reactions and purification procedures are performed according to the manufacturer's descriptions, as is commonly done in the art or as described herein. The terminology, as well as laboratory procedures and their techniques used in the context of analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry and described in this document, are well known and widely used in this field of technology. Standard techniques are used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and in the treatment of patients.

To make the invention more understandable, selected terms are defined below.

The term "polypeptide" refers to any polymeric chain of amino acids and includes natural or artificial proteins, polypeptide analogs or variants of the protein sequence or fragments thereof, unless the context is contrary to this. The polypeptide may be monomeric or polymeric. The polypeptide fragment contains, for example, at least about 5 adjacent amino acids, at least about 10 adjacent amino acids, at least about 15 adjacent amino acids, or at least about 20 adjacent amino acids. .

The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, by virtue of its origin or source of preparation, is not associated with its naturally associated components that accompany it in its natural state, essentially does not contain other proteins from the same species, are expressed by a cell from another species, or are not found in nature. Thus, a protein or polypeptide synthesized chemically or synthesized in a cellular system other than the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein or polypeptide can also be made substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

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

The term "ligand" refers to any substance that can bind to or be associated with another substance. Likewise, the term "antigen" refers to any substance against which an antibody can be made. Although "antigen" is commonly used when referring to an antibody-binding substrate and "ligand" is often used when referring to receptor-binding substrates, the terms are indistinguishable and cover a wide range of overlapping chemical entities. To avoid confusion, the terms antigen and ligand are used interchangeably herein. Antigens/ligands can be a peptide, polypeptide, protein, aptamer, polysaccharide, sugar molecule, carbohydrate, lipid, oligonucleotide, polynucleotide, synthetic molecule, inorganic molecule, organic molecule, and any other combination thereof.

As used herein, the term "specifically binds" refers to the ability of an antibody or immunoadhesin to bind to an antigen with a dissociation constant (Kd) of at most about 1 x 10 ^-6 M, about 1 x 10 ^-7 M, about 1 x 10 ^-8 M, about 1×10 ^-9 M, about 1×10 ^-10 M, about 1×10 ^-11 M, about 1×10 ^-12 M or less, and/or for antigen binding with an affinity that is at least approximately twice its affinity for a nonspecific antigen.

As used herein, the term "antibody" refers to such aggregations (eg, intact antibody molecules, immunoadhesins, or variants thereof) that have significant known specific immunoreactive activity for an antigen of interest (eg, a tumor-associated antigen). Antibodies and immunoglobulins contain light and heavy chains with or without an interchain covalent bond between them. The basic structures of immunoglobulins in vertebrate organisms are relatively well understood.

As will be discussed in more detail below, the general term "antibody" includes five different classes of antibodies that can be distinguished biochemically. While all five classes of antibodies are clearly within the scope of the present disclosure, the discussion that follows will primarily be directed to IgG class immunoglobulin molecules. With regard to IgG, immunoglobulins include 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 connected by disulfide bonds in a "Y" configuration, where the connection of light chains to heavy chains begins at the mouth of the "Y" and continues through the variable region.

Immunoglobulin light chains are classified as kappa (κ) or lambda (λ). Each class of heavy chains can be linked to a kappa or lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds in the creation of immunoglobulins, either by hybridomas, B cells, or genetically modified host cells. In the heavy chain, the amino acid sequences run from the N-terminus at the branched ends of the Y-configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) with some subclasses among them (eg, γl-γ4). The nature of this chain defines the "class" of the antibody, namely IgG, IgM, IgA IgG or IgE, respectively. Subclasses of immunoglobulin isotypes (eg, IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to confer functional specialization. The person skilled in the art will easily recognize modified versions of each of these classes and isotypes in light of the present invention and, accordingly, they are within the scope of the present invention.

Both light and heavy chains are divided into regions of structural and functional homology. The term "region" refers to a portion or segment of an immunoglobulin or antibody chain and includes the constant region or variable regions, as well as more disparate portions or segments of these regions. For example, light chain variable regions include "complementarity determining regions" or "CDRs" located between "framework regions" or "FRs" as defined herein.

Regions of the heavy or light chain of an immunoglobulin may be defined as a "constant" (C) region or "variable" (V) regions based on the relative absence of change in the sequence regions of different members of the class in the case of a "constant region", or on the basis of a significant change in sites in different members of the class in the case of "variable sites". The terms "constant region" and "variable region" can also be used functionally. In this regard, it should be understood that the variable regions of the immunoglobulin or antibody determine the recognition and specificity of the antigen. Conversely, the constant regions of an immunoglobulin or antibody confer important effector functions such as secretion, transplacental motility, Fc receptor binding, complement binding, and the like. The subunit structures and the three-dimensional configurations of the constant regions of various classes of immunoglobulins are well known.

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 a heavy or light chain containing peptide loops (eg, containing 3 to 4 peptide loops) stabilized, for example, by a β-sheet conformation and/or an intrachain disulfide bond. Constant region domains on an immunoglobulin light chain are interchangeably referred to as "light chain constant region domains", "CL regions", or "CL domains". Heavy chain constant domains (eg, hinge, CH1, CH2 or CH3 domains) are interchangeably referred to as "heavy chain constant region domains", "CH region domains", or "CH domains". Light chain variable domains are interchangeably referred to as "light chain variable region domains", "VL region domains", or "VL domains". Heavy chain variable domains are interchangeably referred to as "heavy chain variable region domains", "VH region domains", or "VH domains".

By convention, the amino acid numbering of the variable constant region domains increases as they move away from the antigen-binding site or amine terminus of an immunoglobulin or antibody. The N-terminus of each immunoglobulin heavy and light chain is a variable region, and the C-terminus is a constant region. The CH3 and CL domains contain the carboxy terminus of the heavy and light chains, respectively. Accordingly, the light chain immunoglobulin domains are oriented as VL-CL, while the heavy chain domains are oriented as VH-CH1-hinge-CH2-CH3.

The amino acid distribution for each variable region domain is as defined by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Bethesda, MD, 1987 and 1991). Kabat also provides a commonly used numbering convention (Kabat numbering) in which the corresponding residues between different heavy chain variable regions or between different light chain variable regions are assigned the same number. VL domain CDRs 1, 2, and 3 are also referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively. VH domain CDRs 1, 2, and 3 are also referred to herein as CDR-H1, CDR-H2, and CDR-H3, respectively. If indicated, CDR allocation can be made according to IMGT® (Lefranc et al., Developmental & Comparative Immunology 27:55-77; 2003) instead of Kabat. The numbering of the heavy chain constant region is performed using the EC index as described in Kabat (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991).

As used herein, the term "VH domain" includes the variable domain of the amine end of an immunoglobulin heavy chain, and the term "VL domain" includes the variable domain of the amine end of an immunoglobulin light chain.

The term "CH1 domain" as used herein includes the first (amino terminus closest to the end) domain of an immunoglobulin heavy chain constant region, which extends from, for example, approximately positions 114-223 according to the Kabat numbering system (positions 118-215 according to EU numbering) . The CH1 domain is adjacent to the VH domain and the amino terminus of the hinge region of the immunoglobulin heavy chain molecule and does not form part of the Fc region of the immunoglobulin heavy chain.

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

The term "CH2 domain" as used herein includes the heavy chain portion of an immunoglobulin molecule that extends from, for example, approximately positions 244-360 according to the Kabat numbering system (positions 231-340 according to EU numbering). The CH2 domain is unique in that it is not closely related to another domain. More precisely, two N-linked branched carbohydrate chains are located between two CH2 domains of an intact native IgG molecule. In one embodiment, the binding polypeptide of the present invention comprises a CH2 domain derived from an IgG1 molecule (eg, a human IgG1 molecule).

The term "CH3 domain" as used herein includes the heavy chain portion of an immunoglobulin molecule that is approximately 110 residues from the N-terminus of the CH2 domain and extends, for example, from approximately positions 361-476 according to the Kabat numbering system (positions 341-445 by numbering EU). The CH3 domain usually 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 (eg, the CH4 domain in IgM μ chain and IgE e chain). In one embodiment, the binding polypeptide of the present invention comprises a CH3 domain derived from an IgG1 molecule (eg, a human IgG1 molecule).

The term "CL domain" as used herein includes an immunoglobulin light chain constant region domain that extends, for example, from Kabat position 107A to Kabat position 216. The CL domain borders directly on the VL domain. In one embodiment, the binding polypeptide of the present invention comprises a CL domain derived from a kappa light chain (eg, a human kappa light chain).

The term "Fc region" as used herein is defined as the part of the heavy chain constant region starting at the hinge region immediately before the papain cleavage site (i.e. residue 216 in IgG, taking the position of the first residue of the heavy chain constant region as 114) and ending at C the end of the antibody. Accordingly, a complete Fc region contains at least a hinge domain, a CH2 domain, and a CH3 domain.

As used herein, the terms "natural Fc", "native Fc", and "wild-type Fc" refer to a molecule, in both monomeric and multimeric form, containing a sequence that is not part of an antigen-binding fragment, which is obtained by cleavage of an antibody or by other means. , and at the same time it may contain a hinge section. The original source of immunoglobulin for natural Fc is usually of human origin and can be any of the immunoglobulins, such as IgG1 and IgG2. Native Fc molecules are composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (ie disulfide bonds) and non-covalent bonds. The number of intermolecular disulfide bonds between the monomeric subunits of natural Fc molecules varies from 1 to 4, depending on the class (eg IgG, IgA and IgE) or subclass (eg IgG1, IgG2, IgG3, IgA1 and IgGA2). One example of a naturally occurring Fc is a disulfide dimer resulting from papain cleavage of IgG. The term "natural Fc" or "native Fc" as used herein is generic to monomeric, dimeric, and multimeric forms.

The term "Fc variant" or "modified Fc" as used herein refers to a molecule or sequence that is obtained by modifying a natural wild-type Fc/Fc but still contains an FcRn binding site. Thus, the term "Fc variant" may include a molecule or sequence derived from the humanization of a native non-human Fc. In addition, native Fc contains regions that can be removed because they provide structural features or biological effects that are not required for the antibody-like binding polypeptides described herein. Thus, the term "Fc variant" refers to a molecule or sequence that lacks one or more native Fc sites or residues or that has been modified with one or more Fc sites or residues that affect or are involved in: (1) formation of a disulfide bond, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity when expressed in the selected host cell, (4) glycosylation, (5) interaction with the complement system, (6) binding to an Fc receptor other than the receptor recycling, or (7) antibody-dependent cellular cytotoxicity (ADCC).

In some exemplary embodiments, the Fc variant provided herein has one or more of the following characteristics: longer serum half-life, increased FcRn binding, increased FcRn binding at acidic pH, increased FcγRIIIa binding, and /or similar thermal stability compared to an IgG antibody containing wild-type Fc.

The term "Fc-domain" in the context of this document, covers natural wild-type Fc/Fc, as well as Fc variants and sequences as defined above. With regard to Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether formed by cleavage of the whole antibody or obtained by other means.

As noted above, the variable regions of an antibody allow it to selectively recognize and specifically bind to epitopes on antigens. That is, the VL domain and the VH domain of an antibody combine to form a variable region (Fv) that defines a three-dimensional antigen binding site. This antibody quaternary structure forms an antigen-binding site present at the end of each Y arm. More specifically, the antigen-binding site is defined by three complementary defining regions (CDRs) in each of the heavy and light chain variable regions. The term "antigen binding site" as used herein includes a site that specifically binds (immunoreacts) to an antigen (eg, cell surface antigen or soluble antigen). The antigen binding site includes the variable region of the heavy chain and light chain of an immunoglobulin; the binding site formed by these variable regions determines the specificity of the antibody. The antigen-binding site is formed by variable regions that differ depending on the antibody. The modified antibodies of the present invention contain at least one antigen-binding site.

In some embodiments, the binding polypeptides of the present invention comprise at least two antigen-binding domains that allow the binding polypeptide to interact with the selected antigen. Antigen binding domains do not have to be derived from the same immunoglobulin molecule. In this regard, the variable region may be from any type of animal, or may be derived from any type of animal in which a humoral response can be induced and immunoglobulins against the desired antigen can be generated. As such, the variable region of the binding polypeptide may, for example, be from a mammal, for example, may be from a human, mouse, rat, goat, sheep, non-human primate (such as cynomolgus monkeys, macaques, etc.), the family wolf or camelid (for example, from camels, llamas and related species).

In naturally occurring antibodies, the six CDRs present in each monomeric antibody are short, non-bordering amino acid sequences that are specifically arranged to form an antigen-binding site when the antibody assumes a three-dimensional configuration in an aqueous environment. The remaining heavy and light variable domains have less intermolecular variability in amino acid sequence and are called framework regions. The framework regions generally adopt the β-sheet conformation, and the CDRs form loops that connect, and in some cases are part of, the β-sheet structure. Thus, under the influence of these framework regions, a framework structure is formed, which ensures the positioning of six CDRs in the correct orientation due to inter-chain non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to the immunoreactive antigen epitope.

Exemplary binding polypeptides include antibody variants. The term "antibody variant" as used herein includes synthetic and engineered forms of antibodies that are modified so that they do not occur naturally, for example, antibodies that contain at least two heavy chain portions but not two complete heavy chains (such as antibodies deleted domain or mini-antibody); multispecific antibody forms (eg, bispecific, trispecific, etc.) modified to bind to two or more different antigens or to different epitopes on the same antigen); heavy chain molecules linked to scFv molecules; and the like. In addition, the term "antibody variant" includes multivalent forms of antibodies (eg, trivalent, tetravalent, etc. antibodies that bind to three, four or more copies of the same antigen).

The term "valence" as used herein refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds one target molecule or a specific site on the 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 (eg, may bind to different ligands or different antigens or different epitopes on the same antigen). A subject's binding polypeptides typically have at least one binding site specific for a human antigen molecule.

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

The term "antigen" or "target antigen" in the context of the present description refers to a molecule or part of a molecule to which the binding site of a binding polypeptide is able to bind. The target antigen may have one or more epitopes.

The term "about" or "approximately" means within 20%, for example, within 10%, within 5%, or within 1% or less of a given value or range.

As used herein, the term "administer" or "administration" refers to performing an injection or otherwise performed physical delivery of a substance as it exists outside the body (e.g., the isolated binding polypeptide provided herein) to a patient, for example, but not limited to pulmonary (eg, inhalation), mucosal (eg, intranasal), intradermal, intravenous, intramuscular delivery, and/or any other physical delivery method described herein or known in the art. In the management or treatment of a disease or symptom thereof, the administration of a substance generally occurs after the disease or its symptoms have been identified. In the prevention of a disease or its symptoms, the administration of a substance usually occurs before the onset of the disease or the onset of its symptoms and may be continued continuously to delay or reduce the onset or severity of the symptoms associated with the disease.

As used herein, the term "composition" is intended to encompass a product containing the indicated ingredients (e.g., the isolated binding polypeptide provided herein), optionally in the amounts indicated, as well as any product that is directly or indirectly obtained by combining these ingredients, if necessary, in the indicated quantities.

"Effective amount" means an amount of an active pharmaceutical ingredient (eg, an isolated binding polypeptide of the present invention) sufficient to achieve the desired physiological result in a person in need of this active substance. The effective amount may vary among individuals depending on the health and physical condition of the person in need of treatment, the taxonomic group of people in need of treatment, composition of the composition, assessment of the health status of the person, and other relevant factors.

As used herein, the terms "subject" and "patient" are used interchangeably. The subject in the context of this document may be a non-primate mammal (eg, cows, pigs, horses, cats, dogs, rats, etc.) or a primate (eg, monkey and human). In certain embodiments, the term "subject" as used herein refers to a vertebrate animal, such as a mammal. Mammals include, without limitation, humans, non-human primates, wild animals, feral animals, farm animals, sport animals, and pets.

As used herein, the term "therapy" refers to any protocol, method, and/or agent that can be used to prevent, manage, treat, and/or ameliorate a disease or associated symptom. In some embodiments, the term “therapy” refers to any protocol, method, and/or agent that can be used to modulate a subject's immune response to an infection or associated symptom. In some embodiments, the terms “therapies” and “therapies” refer to biological therapies, supportive therapies, and/or other therapies useful in the prevention, control, treatment, and/or amelioration of a disease or associated symptom known to those skilled in the art. area, such as medical personnel. In other embodiments, the terms "therapies" and "therapies" refer to biological therapies, maintenance therapies, and/or other therapies useful for modulating a subject's immune response to an infection or associated symptom known to one of skill in the art, such as medical staff.

As used herein, the terms "treat", "treatment", and "treating" refer to the reduction or amelioration of the progression, severity and/or duration of a disease or associated symptom, as a result of the administration of one or more therapies (including, but not limited to , administration of one or more prophylactic or therapeutic agents, such as the isolated binding polypeptide provided herein). As used herein, the term "treatment" may also refer to a change in the course of a disease in a subject receiving treatment. The therapeutic effect of treatment includes, without limitation, preventing the onset or recurrence of the disease, ameliorating the symptom(s), reducing the direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or mitigating the severity of the disease, and remission or improved prognosis.

Binding polypeptides

In one aspect of the present disclosure, the binding of polypeptides (eg, antibodies, immunoadhesins, antibody variants, and fusion proteins) containing a modified Fc domain is provided. Binding polypeptides disclosed herein include any binding polypeptide that contains a modified Fc domain. In certain embodiments, the binding polypeptide is an antibody, or an immunoadhesin, or a derivative thereof. Any antibody from any source or species can be used in the binding polypeptides disclosed here. Suitable antibodies include, without limitation, human antibodies, humanized antibodies, or chimeric antibodies. Suitable antibodies include, without limitation, monoclonal antibodies, polyclonal antibodies, full length antibodies, or single chain antibodies.

Fc domains from any class of immunoglobulins (eg, IgM, IgG, IgD, IgA and IgE) and species can be used in the binding polypeptides disclosed here. It is also possible to use chimeric Fc domains containing portions of Fc domains from different Ig species or classes. 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 embodiments, the Fc domain is a human IgG1 or IgG4 Fc domain. In some embodiments, the Fc domain is a human IgG1 Fc domain. In the case of Fc domains of other Ig species and/or classes or isotypes, one skilled in the art will appreciate that any of the amino acid substitutions described herein may be adapted accordingly. In some embodiments, the modified Fc domain may contain an amino acid substitution selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, or Y436, and any combination thereof, according to EU numbering. In some embodiments, the modified Fc domain may contain a double amino acid substitution at any two amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436, according to EU numbering. In some embodiments, the modified Fc domain may contain a triple amino acid substitution at any three amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436, according to EU numbering. In some embodiments, the modified Fc domain may contain a quaternary amino acid substitution at any four amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436, according to EU numbering. In some embodiments, it may be desirable for the modified Fc domain to contain an amino acid substitution at any of the amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, or Y436 and any combinations thereof, where the position amino acid N434 is not substituted (ie the amino acid position of wild-type N434), according to EC numbering.

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

In certain embodiments, the modified Fc domain may contain an amino acid substitution selected from M252, T256, T307, or N434 and any combination thereof, according to EC numbering. In certain embodiments, the modified Fc domain may contain a double amino acid substitution at any two amino acid positions selected from M252, T256, T307, and N434, according to EC numbering. In certain embodiments, the modified Fc domain may contain a triple amino acid substitution at any three amino acid positions selected from M252, T256, T307, and N434, according to EC numbering. In certain embodiments, the modified Fc domain may contain a quaternary amino acid substitution at amino acid positions M252, T256, T307, and N434, according to EC numbering. In some embodiments, it may be desirable for the modified Fc domain to contain an amino acid substitution selected from M252, T256, or T307 and any combinations thereof, where the N434 amino acid position is not substituted (i.e., wild-type N434 amino acid position), according to EC numbering.

In exemplary embodiments, the modified Fc domain may comprise an amino acid substitution selected from M252, where the substitution is M252Y; T256 where the replacement is T256D or T256E; T307 where the replacement is T307Q or T307W; or N434, where the replacement is N434F or N434Y, and any combination thereof, in accordance with EC numbering. In exemplary embodiments, the modified Fc domain may contain a double amino acid substitution at any two amino acid positions selected from M252, where the substitution is M252Y; T256 where the replacement is T256D or T256E; T307 where the replacement is T307Q or T307W; or N434 where the replacement is N434F or N434Y, according to EC numbering. In exemplary embodiments, the modified Fc domain may contain a triple amino acid substitution at any three amino acid positions selected from M252, where the substitution is M252Y; T256 where the replacement is T256D or T256E; T307 where the replacement is T307Q or T307W; or N434 where the replacement is N434F or N434Y, according to EC numbering. In illustrative embodiments, the modified Fc domain may contain a quaternary amino acid substitution at amino acid positions selected from M252, where the substitution is M252Y; T256 where the replacement is T256D or T256E; T307 where the replacement is T307Q or T307W; or N434 where the replacement is N434F or N434Y, according to EC numbering. In some embodiments, it may be desirable that the modified Fc domain contains an amino acid substitution selected from M252Y, T256D, T256E, T307Q, or T307W, and any combinations thereof, wherein the N434 amino acid position is not substituted by phenylalanine (F) or tyrosine (Y), according to EU numbering. In some embodiments, it may be desirable for the modified Fc domain to contain an amino acid substitution selected from M252Y, T256D, T256E, T307Q, or T307W, and any combinations thereof, wherein the N434 amino acid position is not substituted with tyrosine (Y), according to EU numbering. In some embodiments, it may be desirable for the modified Fc domain to contain an amino acid substitution selected from M252Y, T256D, T256E, T307Q, or T307W, and any combinations thereof, wherein the N434 amino acid position is not substituted (i.e., wild-type N434 amino acid position), according to EU numbering.

In some embodiments, the modified Fc domain may comprise an amino acid substitution selected from T256D or T256E and/or T307W or T307Q, and further comprises an amino acid substitution selected from N434F or N434Y or M252Y, according to EU numbering. In some embodiments, it may be desirable that the modified Fc domain contains an amino acid substitution selected from T256D or T256E and/or T307W or T307Q and further comprises an M252Y amino acid substitution wherein the N434 amino acid position is not substituted with phenylalanine (F) or tyrosine (Y), according to the EU numbering. In some embodiments, it may be desirable that the modified Fc domain contains an amino acid substitution selected from T256D or T256E and/or T307W or T307Q and further comprises an M252Y amino acid substitution wherein the N434 amino acid position is not substituted with tyrosine (Y), according to EU numbering. In some embodiments, it may be desirable that the modified Fc domain contains an amino acid substitution selected from T256D or T256E and/or T307W or T307Q and further comprises an M252Y amino acid substitution where the N434 amino acid position contains no substitution (i.e. . is the amino acid position of wild-type N434), according to EC numbering.

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/N434F, T25 6D/ N434Y, T256E/T307Q, T256E/T307W, T256E/N434F, T256E/N434Y, T307Q/N434F, T307Q/N434Y, T307W/N434F and T307W/N434Y, according to EU numbering. In some embodiments, the modified Fc domain may contain a triple amino acid substitution selected from M252Y/T256D/T307Q, M252Y/T256D/T307W, M252Y/T256D/N434F, M252Y/T256D/N434Y, M252Y/T256E/T307Q, M252Y/T256 E/ M252Y/T307Q/N434Y, M252Y/T307W/N434F, M252T/T307W/N434Y, T256D/30 7Q/N434F, T256D/307W/N434F, T256D/307Q/N434Y, T256D/307W/N434Y, T256E/307Q/N434F, T256E/307W/N434F, T256E/307Q/N434Y and T256E/307W/N434Y, according to EU numbering.

In some embodiments, the modified Fc domain may contain a quad amino acid substitution selected from M252Y/T256D/T307Q/N434F, M252Y/T256E/T307Q/N434F, M252Y/T256D/T307W/N434F, M252Y/T256E/T307W/N434F, M252 Y/ T256D/T307Q/N434Y, M252Y/T256E/T307Q/N434Y, M252Y/T256D/T307W/N434Y and M252Y/T256E/T307W/N434Y, according to EU numbering.

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 EC numbering. In some embodiments, it may be desirable that the Fc domain does not contain phenylalanine (F) or tyrosine (Y) at amino acid position N434, according to EC numbering. In some embodiments, it may be desirable that the Fc domain does not contain a tyrosine (Y) at amino acid position N434, according to EC numbering. 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 with EU numbering. 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 numbering.

In one embodiment, the FcRn-binding binding polypeptide comprises an Fc domain having one or more amino acid substitutions as disclosed herein. In one embodiment, a binding polypeptide with enhanced FcRn binding capacity comprises an Fc domain having one or more amino acid substitutions as disclosed herein. In one embodiment, a binding polypeptide with enhanced FcRn binding capacity comprises an Fc domain having two or more amino acid substitutions as disclosed herein. In one embodiment, a binding polypeptide with enhanced FcRn binding capacity comprises an Fc domain having three or more amino acid substitutions as disclosed herein.

In some embodiments, a binding polypeptide may exhibit a species-specific FcRn binding ability. In one embodiment, the binding polypeptide may exhibit the ability to bind to human FcRn. In one embodiment, the binding polypeptide may exhibit the ability to bind to rat FcRn. In some embodiments, a binding polypeptide may exhibit cross-species FcRn binding ability. Such binding polypeptides are said to be cross-reactive with one or more different species. In one embodiment, the binding polypeptide may exhibit the ability to bind to both human and rat FcRn.

The neonatal Fc receptor (FcRn) interacts with the Fc region of antibodies to promote recycling by restoring normal lysosomal degradation. This process is a pH-dependent process that occurs in endosomes at an acidic pH (eg, pH less than 6.5), but not under physiological bloodstream pH conditions (eg, non-acidic pH). In some embodiments, a binding polypeptide of the present invention containing an altered Fc domain has an increased ability to bind to FcRn at an acidic pH compared to a binding polypeptide containing a wild-type Fc domain. In some embodiments, a binding polypeptide comprising an altered Fc domain has enhanced FcRn binding at a pH below 7, e.g., at a pH of about 6.5, at a pH of about 6.0, at a pH of about 5.5, at a pH of about 5.0 compared to a binding polypeptide containing a wild-type Fc domain. In some embodiments, a binding polypeptide comprising an altered Fc domain has increased FcRn binding ability at a pH below 7, e.g., at a pH of about 6.5, at a pH of about 6.0, at a pH of about 5.5, at a pH of about 5.0 compared to the FcRn binding capacity of the binding polypeptide at elevated non-acidic pH. An elevated non-acidic pH can be, for example, a pH greater than 7, a pH of about 7, a pH of about 7.4, a pH of about 7.6, a pH of about 7.8, a pH of about 8.0, a pH of about 8.5, a pH of about 9, 0.

In some embodiments, it may be desirable for a binding polypeptide containing an altered Fc domain to exhibit approximately the same ability to bind to FcRn at a non-acidic pH as a binding polypeptide containing a wild-type Fc domain. In some embodiments, it may be desirable for a binding polypeptide containing an altered Fc domain to exhibit a weaker ability to bind to FcRn at non-acidic pH values than a binding polypeptide containing an altered Fc domain containing the double amino acid substitution M428L/N434S, as numbered EU. Accordingly, it may be desirable for a binding polypeptide containing an altered Fc domain to exhibit minimal disruption of pH-dependent FcRn binding.

In some embodiments, a binding polypeptide containing an altered Fc domain having an increased ability to bind to FcRn at an acidic pH had a reduced (i.e., slower) rate of dissociation from FcRn compared to a binding polypeptide containing a wild-type Fc domain. . In some embodiments, a binding polypeptide comprising an altered Fc domain having an increased ability to bind to FcRn at an acidic pH compared to the ability to bind to an FcRn of a binding polypeptide at an increased non-acidic pH has a slower rate of dissociation from FcRn at an acidic pH by compared to the rate of dissociation from the FcRn binding polypeptide at elevated non-acidic pH.

In some embodiments, a binding polypeptide comprising an altered Fc domain is provided that exhibits a stronger FcRn binding ability at non-acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising an altered Fc domain is provided that exhibits a stronger FcRn binding ability at acidic pH compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, a binding polypeptide comprising an altered Fc domain is provided that exhibits stronger binding to FcRn at non-acidic pHs compared to a binding polypeptide comprising a wild-type Fc domain, exhibits stronger FcRn binding at acidic pHs compared to with a binding polypeptide containing a wild-type Fc domain. Accordingly, in certain embodiments, a binding polypeptide is provided that contains an altered Fc domain that exhibits loss of pH-dependent binding to FcRn.

Certain embodiments include antibodies that, in addition to the Fc mutations described herein that alter FcRn binding ability, 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 domains. sites that have been removed or otherwise altered to provide desired biochemical characteristics, such as, for example, decreased or enhanced effector functions, ability to dimerize non-covalently, increased ability to localize at the tumor site, shorter serum half-life, longer half-life from blood serum compared to a whole unchanged antibody of approximately the same immunogenicity, etc.

In some other embodiments, the binding polypeptides comprise constant regions derived from different antibody isotypes (eg, constant regions from two or more human IgG1, IgG2, IgG3, or IgG4). In other embodiments, anti-PAI-1 antibodies comprise a chimeric hinge (i.e., a hinge containing portions of the hinge derived from hinge domains of different antibody isotypes, eg, an upper hinge from an IgG4 molecule and a middle hinge from IgG1).

In certain embodiments, the Fc domain may be mutated to increase or decrease effector function using techniques known in the art. In some embodiments, a binding polypeptide of the present invention containing an altered Fc domain has an altered ability to bind to an Fc receptor. There are several different types of Fc receptors, which are classified according to the type of antibody they recognize. For example, Fc gamma receptors (FcγR) bind to IgG class antibodies, Fc alpha receptors (FcαR) bind to IgA class antibodies, and Fc epsilon receptors (FcεR) bind to IgE class antibodies. FcγRs belong to a family that includes members such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb. In some embodiments, a binding polypeptide containing an altered Fc domain has an altered ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some embodiments, a binding polypeptide containing an altered Fc domain has a reduced ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some embodiments, a binding polypeptide containing an altered Fc domain has an increased ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain. In some embodiments, a binding polypeptide comprising an altered Fc domain has approximately the same FcγRIIIa binding capacity as a binding polypeptide comprising a wild-type Fc domain.

In other embodiments, binding polypeptides for use in the diagnostic and therapeutic methods described herein have a constant region, eg, an IgG1 heavy chain constant region, modified to reduce or eliminate glycosylation. For example, binding polypeptides (eg, antibodies or immunoadhesins) containing an altered Fc domain may further comprise an amino acid substitution that alters the Fc glycosylation of the antibody. For example, said altered Fc domain may be characterized by reduced glycosylation (eg, N- or O-linked glycosylation).

Exemplary amino acid substitutions conferring reduced or altered glycosylation are disclosed in PCT International Publication No. WO05/018572, incorporated herein by reference in its entirety. In some embodiments, the binding polypeptides are modified to eliminate glycosylation. Such binding polypeptides may be referred to as "agly" binding polypeptides (eg, "agly" antibodies). Without being bound by any theory, it is believed that "agly" binding polypeptides may have an improved in vivo safety and stability profile. Agly-binding polypeptides may be of any isotype or subclass thereof, such as IgG1, IgG2, IgG3 or IgG4. To obtain "agly"-antibodies or antibodies with altered glycans, many methods recognized in this field are used. For example, genetically engineered host cells (eg, modified yeasts, eg, Picchia cells or CHO cells) with modified glycosylation signaling pathways (eg, deletion of glycosyltransferase) can be used to generate such antibodies.

In some embodiments, binding polypeptides may comprise an antibody constant region (eg, an IgG constant region, eg, a human IgG constant region, eg, a human IgG1 constant region) that is involved in one or more effector functions. For example, binding of the C1 complex of the system to the constant region of an antibody can activate the complement system. Activation of the complement system is important for opsonization and lysis of cellular pathogens. Activation of the complement system also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. In addition, antibodies bind to receptors on various cells via the Fc domain (Fc receptor binding sites in the Fc region of an antibody bind to an Fc receptor (FcR) on the cell surface). There are several Fc receptors specific to different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors), and IgM (mu receptors). The binding of an antibody to Fc receptors on the cell surface triggers several important and varied biological reactions, including the uptake and destruction of antibody-coated particles, the clearance of immune complexes, the lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, transplacental transfer and regulation of immunoglobulin production. In some embodiments, binding polypeptides (eg, antibodies or immunoadhesins) bind to the Fc-gamma receptor. In alternative embodiments, the binding polypeptides may comprise a constant region lacking one or more effector functions (eg, ADCC activity) and/or Fcγ receptor binding ability.

Proteins, including antibodies, with low thermodynamic stability have an increased tendency to misfold and aggregate and may limit or interfere with the protein's activity, efficacy, and efficacy as a useful therapeutic agent. In some embodiments, a binding polypeptide containing an altered Fc domain has approximately the same thermal stability as a binding polypeptide containing a wild-type Fc domain. In some embodiments, a binding polypeptide comprising an altered Fc domain has approximately the same thermal stability as a binding polypeptide comprising an altered Fc domain having the M252Y/S254T/T256E (YTE) triple amino acid substitution.

The resulting physiological profile, bioavailability, and other biochemical effects of these changes, such as tumor localization, biodistribution, and serum half-life, can be easily measured and quantified using well known immunological techniques without undue experimentation.

In some embodiments, a binding polypeptide of the present invention 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 an antigen or competes with an intact antibody (i.e., the intact antibody from which it was derived) for antigen binding (i.e., specific binding). Antigen binding fragments can be obtained by recombinant or biochemical methods, which are well known in this field. Examples of antigen binding fragments include Fv, Fab, Fab'and (Fab')2. In exemplary embodiments, a binding polypeptide of the present invention comprises an antigen-binding fragment and an altered Fc domain.

In some embodiments, the binding polypeptide comprises a single chain variable region (ScFv) sequence. A single chain variable region sequence comprises a single polypeptide having one or more antigen binding sites, such as a VL domain connected by a flexible linker to a VH domain. ScFv molecules can be designed in the VH-linker-VL orientation or the VL-linker-VH orientation. The flexible hinge that connects the VL and VH domains that form the antigen binding site comprises from about 10 to about 50 amino acid residues. Binding peptides are known in the art. Binding polypeptides may contain at least one scFv and/or at least one constant region. In one embodiment, the binding polypeptide of the present invention may comprise at least one scFv attached to or fused to an altered Fc domain.

In some embodiments, the binding polypeptide of the present invention is a multivalent (eg, tetravalent) antibody, which is obtained by fusing the DNA sequence encoding the antibody to a ScFv molecule (eg, an altered ScFv molecule). For example, in one embodiment, these sequences are combined such that the ScFv molecule (eg, an altered ScFv molecule) is linked at its N-terminus or C-terminus to the antibody Fc fragment via a flexible linker (eg, gly/ser linker). In another embodiment, a tetravalent antibody of the present invention can be made by fusing a ScFv molecule to a junction peptide that is linked to an altered Fc domain to construct a tetravalent ScFv-Fab molecule.

In another embodiment, the binding polypeptide of the present invention is a modified mini-antibody. The altered mini-antibody of the present disclosure is a dimeric molecule consisting of two polypeptide chains, each containing a ScFv molecule that is linked to the altered Fc domain via a link peptide. Minibodies can be obtained by constructing the ScFv component and connecting the peptide components using the methods described in this field (see, for example, US patent 5837821 or WO 94/09817A1). In another embodiment, a tetravalent mini-antibody can be engineered. Quadrivalent mini-antibodies can be constructed in the same way as mini-antibodies, except that two ScFv molecules are connected by a flexible linker. The associated scFv-scFv construct is then attached to the modified Fc domain.

In another embodiment, the binding polypeptide of the present invention contains a dimeric antibody. Dimeric antibodies are dimeric, tetravalent molecules, each having a polypeptide similar to scFv molecules, but typically containing a short linker of (less than 10, e.g., about 1 to about 5) amino acid residues connecting both variable domains, such that VL and VH domains on the same polypeptide chain cannot interact with each other. Instead, the VL and VH domain of one polypeptide chain interact with the VH and VL domain (respectively) on the second polypeptide chain (see, for example, WO 02/02781). The dimeric antibodies of the present invention comprise an scFv-like molecule linked to an altered Fc domain.

In other embodiments, the binding polypeptides comprise multispecific or multivalent antibodies comprising one or more variable domains sequentially on the same polypeptide chain, such as tandem variable domain (TVD) polypeptides. Exemplary TVD polypeptides include the "dual head" or "Dual-Fv" configuration described in US Pat. No. 5,989,830. In the Dual-Fv configuration, the variable domains of two different antibodies are expressed in tandem orientation on two separate chains (one heavy chain and chain), where one polypeptide chain has two consecutive VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains connected in series by a peptide linker (VL1-linker-VL2). In a crossed double head configuration, the variable domains of two different antibodies are expressed in tandem orientation on two separate polypeptide chains (one heavy chain and one light chain), where one polypeptide chain has two consecutive VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains connected in series by a peptide linker in opposite orientation (VL2-linker-VL1). Additional variants of antibodies in the "Dual-Fv" format include bispecific antibody with two IgG variable domains (DVD-IgG) (see US patent No. 7612181) and TBTI format (see US 2010/0226923 A1). In some embodiments, the binding polypeptides comprise multispecific or multivalent antibodies comprising one or more variable domains sequentially on the same polypeptide chain linked to an altered Fc domain.

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

In another exemplary embodiment, the binding polypeptide is an immunoadhesin. As used herein, the term "immunoadhesin" refers to a binding polypeptide containing one or more binding domains (e.g., from a receptor, ligand, or cell adhesion molecule) associated with an immunoglobulin constant domain (i.e., Fc region) (see, e.g., Ashkenazi et al 1995, Methods 8 (2): 104-115, and Isaacs (1997) Brit J Rheum 36:305, which are incorporated herein by reference in their entirety). Immunoadhesins are designated by the suffix "-cept" in their International Nonproprietary Names (INN). Like antibodies, immunoadhesins have a long half-life, are easy to purify using affinity-based methods, and have avidity advantages due to their bivalent nature. Examples of commercially available therapeutic immunoadhesins include etanercept (ENBREL®), abatacept (ORENCIA®), rilonacept (ARCALYST®), aflibercept (ZALTRAP®/EYLEA®), and belatacept (NULOJIX®).

In certain embodiments, the binding polypeptide includes immunoglobulin-like domains. Suitable immunoglobulin-like domains include, without limitation, fibronectin domains (see, e.g., Koide et al. (2007), Methods Mol. Biol. 352:95-109, which is incorporated herein by reference in its entirety), DARPin (see, e.g., , Stumpp et al (2008) Drug Discov Today 13 (15-16): 695-701, which is hereby incorporated by reference in its entirety), Protein A Z-domains (see Nygren et al. (2008) FEBS J. 275 (11): 2668-76, which is incorporated herein by reference in its entirety), lipocalins (see, e.g., Skerra et al. (2008) FEBS J. 275 (11): 2677-83, which is incorporated in its entirety in this description by reference), affilins (see e.g. Ebersbach et al. (2007) J. Mol. Biol. 372 (1): 172-85, which is incorporated herein by reference in its entirety), affinins (see e.g. Krehenbrink et al (2008) J Mol Biol 383 (5): 1058-68 (which is incorporated herein by reference in its entirety), avimers (see e.g. Silverman et al (2005) Nat. Biotechnol 23 (12): 1556-61 (which is incorporated herein by reference in its entirety), finomers (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, e.g., Nixon et al. (2006) Curr Opin Drug Discov Devel 9 (2 ): 261-8, which is incorporated herein by reference in its entirety).

For the binding polypeptides and immunoadhesins of the present invention, the binding polypeptides can be targeted to virtually any antigen, including, but not limited to, proteins, subunits, domains, motifs, and/or epitopes of target antigens that include both soluble factors such as cytokines and associated with membrane factors and transmembrane receptors.

A binding polypeptide of the present invention containing an altered Fc domain as described herein may include CDR sequences or variable domain sequences of a known "parent" antibody. In some embodiments, the implementation of the parent antibody and the antibody of the present invention may have similar or identical sequences, with the exception of the altered Fc domain, as described herein.

Nucleic acids and expression vectors

In one aspect, the invention provides polynucleotides encoding the binding polypeptides disclosed herein. Methods for obtaining a binding polypeptide are also provided, including the expression of these polynucleotides.

The polynucleotides encoding the binding polypeptides disclosed herein are typically inserted into an expression vector for introduction into host cells, which can be used to produce the desired amount of claimed antibodies or immunoadhesins. Accordingly, in certain aspects of the invention, expression vectors containing the polynucleotides disclosed herein and host cells containing these vectors and polynucleotides are provided.

The term "vector" or "expression vector" is used throughout this specification and claims to refer to vectors used to introduce and express a desired gene in a cell. As known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses and retroviruses. Typically, the vector will contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.

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

In other embodiments, a binding polypeptide as described herein may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as antibody heavy and light chains, can be generated from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in US Pat. No. 6,193,980, incorporated herein by reference. Those skilled in the art will appreciate that such expression systems can be used to efficiently produce the full range of polypeptides disclosed in this application.

More generally, once a vector or DNA sequence encoding a binding polypeptide of the present invention has been obtained, the expression vector can be introduced into an appropriate host cell. In other words, the host cell can be transformed. Introduction of a plasmid into a host cell can be accomplished using a variety of techniques well known to those skilled in the art. These include, without limitation, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, DNA-enveloped cell fusion, microinjection, and infection with intact virus. See Ridgway, A. A. G. "Mammalian Expression Vectors" Chapter 24.2, pp. 470-472, Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, MA 1988). Transformed cells are grown under conditions suitable for the production of light chains and heavy chains and analyzed for heavy and/or light chain protein synthesis. Exemplary assay technologies include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence-activated cell sorter (FACS), immunohistochemistry, and the like.

As used herein, the term "transformation" should be used in a broad sense to refer to the introduction of DNA into a recipient host cell that alters the genotype and thereby results in a change in the recipient cell.

In the same way, "host cells" refer to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of methods for isolating polypeptides from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to refer to the source of the antibody, unless otherwise clearly defined. In other words, isolation of a polypeptide from "cells" may mean isolation from whole cells pelleted by centrifugation or from a cell culture containing both medium and suspended cells.

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 for expression of the binding polypeptide is mammalian; those skilled in the art can identify specific host cell lines that are best suited to express the desired gene product. Exemplary host cell lines include, without limitation, DG44 and DUXB11 (Chinese Hamster Ovary cell lines, DHFR-negative), HELA (human cervical carcinoma cells), CVI (monkey kidney cell line), COS (CVI derivative with T antigen SV40), R1610 (Chinese hamster fibroblasts), BALBC/3T3 (mouse fibroblasts), HAK (hamster kidney cell line), SP2/O (mouse myeloma cells), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney cells). In one embodiment, the cell line provides for altered glycosylation, e.g., afucosylation, of an antibody expressed therein (e.g., PER.C6.RTM. cell lines (Crucell) or FUT8 knockout CHO cell lines (POTELLIGENT ^T cells) (Biowa, Princeton, New Jersey)). In one embodiment, NS0 cells can be used. Host cell lines are commonly available from commercial services, the American Type Culture Collection, or from published literature.

In vitro production allows scaling up to produce large amounts of desired binding polypeptides. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include culturing in a homogeneous suspension, such as in an airlift or continuously stirred reactor, or culturing immobilized or trapped cells, such as in hollow fibers, microcapsules, agarose microbeads, or in ceramic cartridges. If necessary and/or desired, polypeptide solutions can be purified using conventional chromatographic methods, for example gel filtration, ion exchange chromatography, DEAE-cellulose chromatography and/or (immuno-)affinity chromatography.

One or more genes encoding binding polypeptides can also be expressed in cells other than mammalian cells, such as bacteria, yeast, or plant cells. In this regard, it will be understood that various unicellular microorganisms other than mammals, such as bacteria, i. capable of growing in crops or fermentation can also be transformed. Bacteria subject to transformation include members of the Enterobacteriaceae such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; pneumococcus; Streptococcus and Haemophilus influenzae. Additionally, it will be appreciated that when expressed in bacteria, the polypeptides may become part of inclusion bodies. Polypeptides must be isolated, purified and then assembled into functional molecules.

In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most widely used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, for example, the YRp7 plasmid is used (Stinchcomb et al ., Nature, 282:39 (1979); Kingsman et al ., Gene, 7:141 (1979); Tschemper et al ., Gene, 10:157 (1980)). This plasmid already contains the TRP1 gene, which is a selection marker for a mutant yeast strain that lacks the ability to grow on tryptophan, such as ATCC #44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of TRP1 damage as a characteristic of the yeast host cell genome in this case provides an efficient environment for detecting transformation by growth in the absence of tryptophan.

Methods of treatment

In one aspect, the invention relates to methods for treating or diagnosing a patient in need thereof, comprising administering an effective amount of a binding polypeptide disclosed herein. In certain embodiments, the present disclosure provides kits and methods for diagnosing and/or treating disorders, such as tumors, in a mammal in need of such treatment. In some exemplary embodiments, the subject is a human.

The binding polypeptides of the present invention are useful in a number of different applications. For example, in one embodiment, the binding polypeptides of the present invention are useful for reducing or eliminating cells bearing an epitope recognized by the binding domain of the binding polypeptide. In another embodiment, said binding polypeptides are effective in reducing or removing soluble antigen from the bloodstream. In another embodiment, the binding polypeptides of this invention are effective as T cell activators. In one embodiment, binding polypeptides can reduce tumor size, suppress tumor growth, and/or prolong the survival time of animals harboring the tumor. Accordingly, this invention also relates to a method of treating tumors in a human or other animal by administering to such human or animal an effective, non-toxic amount of the modified antibody.

In one embodiment, the binding polypeptides of the present invention are useful in the treatment of a disease or disorder. For example, the binding polypeptides of the present invention are useful in the treatment of an antibody-associated disorder or an antibody-responsive disorder, condition, or disease. As used herein, the terms "antibody related disorder" or "antibody responsive disorder" or "condition" or "disease" refer to or describe a disease or disorder that can be ameliorated by administration of a pharmaceutical composition comprising an antibody or binding polypeptide of the present inventions.

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

In another embodiment, the binding polypeptides are useful in the treatment of other disorders, including, without limitation, infectious diseases, autoimmune disorders, inflammatory disorders, lung diseases, neuronal or neurodegenerative diseases, liver diseases, spinal diseases, uterine diseases, depressive disorders, and the like. . Non-limiting examples of infectious diseases include diseases caused by RNA viruses (eg, orthomyxoviruses (eg, influenza virus), paramyxoviruses (eg, respiratory syncytial virus, parainfluenza virus, metapneumovirus), rhabdoviruses (eg, rabies virus), coronaviruses, alpha viruses (eg, chikungunya virus), lentiviruses (eg, HIV), etc.) or DNA viruses. Examples of infectious diseases also include, without limitation, bacterial infectious diseases caused by, for example, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus, Streptococcus, Escherichia coli, and other infectious diseases, including, for example, those caused by Candida albicans. Other infectious diseases include, without limitation, malaria, SARS, yellow fever, Lyme borreliosis, leishmaniasis, anthrax, and meningitis. Typical autoimmune disorders include, but are not limited to, psoriasis, rheumatoid arthritis, Sjögren's syndrome, transplant rejection, Graves' disease, myasthenia gravis, and lupus (eg, systemic lupus erythematosus). Accordingly, this invention relates to a method of treating various conditions that would benefit from the use of a binding polypeptide of the present invention, such as an increase in half-life.

One of ordinary skill in the art will be able to determine by routine experimentation what effective, non-toxic amount of the modified binding polypeptide can be used to treat cancer. For example, the therapeutically active amount of a binding polypeptide of the present invention may vary depending on such factors as disease stage (e.g., stage I versus stage IV), age, sex, medical complications (e.g., immunosuppressive conditions or diseases), and body weight of the subject. , as well as the ability of the antibody to elicit the desired response in the subject. The dosage regimen can be adjusted to obtain the optimal therapeutic effect. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as dictated by the requirements of the therapeutic situation.

In general, the compositions of the present invention can be used for the prophylactic or therapeutic treatment of any neoplasm containing an antigenic marker with which the modified antibody can be targeted to cancer cells.

Pharmaceutical formulations and their uses

Methods for preparing and administering the binding polypeptides of the present invention to a subject are well known or can be easily determined by those skilled in the art. The route of administration of the binding polypeptides of the present invention may be oral, parenteral, inhalation, or topical. As used herein, the term "parenteral" includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all of these forms of administration are clearly within the scope of the present invention, the form of administration will be a solution for injection, in particular for intravenous or intra-arterial injection or drip infusion. Generally, a suitable pharmaceutical composition for injection may contain a buffer (eg, an acetate, phosphate or citrate buffer), a surfactant (eg, polysorbate), optionally a stabilizing agent (eg, human albumin), and the like. In some embodiments, binding polypeptides can be delivered directly to the site of an unfavorable cell population, thereby increasing exposure of the affected tissue to the therapeutic agent.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffer media. In the compositions and methods of the present invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, for example, 0.05 M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, dextrose Ringer's solution, dextrose and sodium chloride solution, Ringer's lactate solution, or fixed oils. Carrier media for intravenous administration include fluid and nutrient supplemented solutions, electrolyte supplemented solutions such as those based on dextrose Ringer's solution, and the like. Preservatives and other additives such as, for example, antimicrobials may also be present. agents, antioxidants, chelating agents and inert gases, and the like. More specifically, pharmaceutical formulations suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions for immediate administration. In such cases, the composition must be sterile and must be fluid to the extent that it is easy to administer via syringe. It must be stable under the conditions of manufacture and storage, and preferably protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier medium may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by applying a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants.

The undesirable action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride will be included in the composition. Sustained absorption of injectable compositions can be achieved by including an absorption delaying agent such as aluminum monostearate and gelatin in the composition.

In any case, sterile injectable solutions can be obtained by incorporating the active substance (for example, the modified binding polypeptide alone or in combination with other active principles) in the required amount in an appropriate solvent along with one ingredient or combination thereof listed herein, in the case of necessary, followed by sterilizing filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile inert material which contains the basic dispersion medium and the other necessary ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, illustrative methods of preparation include vacuum drying and freeze drying, which result in a powder form of the active ingredient with any additional required ingredient from its previously sterile filtered solution. Injectable preparations are processed and filled into containers such as ampoules, sachets, flasks, syringes or vials, which are then hermetically sealed under aseptic conditions according to methods known in the art. In addition, preparations may be packaged and marketed in kit form. Such articles will typically have labels or package inserts stating that the respective compositions are useful for treating a subject suffering from or predisposed to autoimmune or neoplastic disorders.

Effective doses of the compositions of the present invention for the treatment of the conditions described above will vary depending on many different factors, including the route of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is preventive or therapeutic. Usually the patient is a human, however, non-human mammals, including transgenic mammals, can also be treated. Therapeutic doses can be adjusted using standard methods known to those skilled in the art to optimize safety and efficacy.

The binding polypeptides of the present invention can be administered multiple times. Intervals between single doses may be weekly, monthly or yearly. The intervals may also be irregular and determined by measuring levels of the modified binding polypeptide or antigen in the patient's blood. In some methods, the dosage is adjusted to achieve a plasma concentration of the modified binding polypeptide of about 1-1000 µg/ml, and in some methods, about 25-300 µg/ml. Alternatively, the binding polypeptides can be administered as a sustained release formulation, in which case less frequent administration is required. In the case of antibodies, the dose and frequency will vary depending on the half-life of the antibody in the patient. Typically, humanized antibodies have the longest half-life, followed by chimeric and non-human antibodies.

The dose and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, formulations containing antibodies of the present invention, or a cocktail thereof, are administered to a patient who is not yet in a disease state to increase the patient's resistance. Such an amount is defined as a "prophylactic effective dose". When used in this manner, the precise amounts again depend on the health and general immunity of the patient, but are typically in the range of about 0.1 to about 25 mg per dose, especially about 0.5 to about 2.5 mg per dose. A relatively low dose is administered at relatively long intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer a relatively high dose (e.g., about 1 to 400 mg/kg of antibody per dose, with doses of about 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxic-conjugated molecules). drugs) at relatively short intervals until the progression of the disease is reduced or stopped and, in specific embodiments, until the patient experiences partial or complete relief of the symptoms of the disease. After that, the patient can be administered prophylactically.

The binding polypeptides of the present invention may optionally be administered in combination with other agents effective in treating a disorder or condition requiring treatment (eg, prophylactic or therapeutic). Effective single therapeutic doses (ie, therapeutically effective amounts) of the modified ⁹⁰ Y labeled antibodies of the present invention range from about 5 to about 75 mCi, eg, from about 10 to about 40 mCi. Effective single therapeutic doses of non-marrow-damaging ¹³¹ I-modified antibodies range from about 5 to about 70 mCi and from about 5 to about 40 mCi. Effective single therapeutic disruptive doses (ie, which may require autologous bone marrow transplantation) of ¹³¹ I-labeled antibodies range from about 30 to about 600 mCi, eg, from about 50 to less than about 500 mCi. In the case of a chimeric antibody, due to the longer blood half-life compared to mouse antibodies, effective single doses of non-marrow-damaging chimeric antibodies labeled with iodine-131 are in the range of about 5 to about 40 mCi, as , for example, less than about 30 mCi. The imaging criteria for, for example, ^{the 111} In label is typically less than about 5 mCi.

While the binding polypeptides may be administered as described above, it should be emphasized that in other embodiments, the binding polypeptide may be administered to otherwise healthy patients as first line therapy. In such embodiments, binding polypeptides can be administered to patients with normal or moderate red bone marrow reserve and/or to patients who have not yet and are not currently undergoing treatment. As used herein, administration of modified antibodies or immunoadhesins together with or in combination with additional therapy means sequential, simultaneous, concurrent, accompanying or concurrent administration or use of therapy and the disclosed antibodies. Those skilled in the art will appreciate that the administration or use of the various components of a combination therapy regimen can be timed to improve the overall effectiveness of the treatment.

As discussed previously, the binding polypeptides of the present invention, immunoadhesins, or recombinant molecules thereof, may be administered in a pharmaceutically effective amount to treat diseases in a mammal in vivo . In this regard, it will be appreciated that the disclosed binding polypeptides will be formulated to facilitate administration and ensure stability of the active principle.

The pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable, non-toxic, sterile carrier such as saline, non-toxic buffers, preservatives, and the like. For the purposes of the present invention, a pharmaceutically effective amount of a binding polypeptide, immunoadhesin, or recombinant molecule thereof, conjugated or unconjugated to a therapeutic agent, means an amount sufficient to achieve effective binding to a target and to achieve a beneficial effect, for example, to reduce the severity of symptoms of a disease or disorder, or to detection of a substance or cell. In the case of tumor cells, the modified binding polypeptide can interact with selected immunoreactive antigens on neoplastic or immunoreactive cells and lead to increased cell death. Of course, the pharmaceutical compositions of the present invention may be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

Within the scope of the present invention, the binding polypeptides of the present invention may be administered to a human or other animal in accordance with the aforementioned treatments in an amount sufficient to produce a therapeutic or prophylactic effect. The binding polypeptides of the present invention may be administered to such a human or other animal in a conventional dosage form prepared by combining an antibody of the present invention with a conventional pharmaceutically acceptable carrier or diluent according to known procedures. One skilled in the art will appreciate that the form and type of pharmaceutically acceptable carrier or diluent is determined by the amount of active ingredient with which it is to be combined, by administration, and other well known variables. Those skilled in the art will also appreciate that a cocktail containing one or more of the binding polypeptides described herein may be particularly effective.

The contents of articles, patents and patent applications, and all other documents and information available electronically, mentioned or cited in this document, are incorporated by reference in their entirety into this document to the same extent as if each individual publication were specifically and individually specified for inclusion by reference. Applicants reserve the right to physically incorporate into this application any materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

While the present invention has been described with reference to specific embodiments thereof, those skilled in the art will appreciate that various changes and equivalent substitutions may be made without departing from the true spirit and scope of the invention. Those skilled in the art will appreciate that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many changes can be made to adapt a particular situation, material, composition of matter, process, process step or steps to the object, spirit and scope of the present invention. All such variations are intended to be within the scope of the appended claims. Now, after a detailed description of some embodiments, they will be better understood by considering the following examples, which are included for purposes of illustration only and are not intended to be limiting.

EXAMPLES

The present invention is further illustrated by the following examples, which should not be construed as a further limitation.

Example 1: Materials and Methods

Protein reagents:

The following proteins have been expressed and isolated: 8xHistidine C-terminal antigen; rFcRn (UniProt: P1359, p51 subunit: residues 23-298; UniProt: P07151, β2-m: residues 21-119); biotinylated FcRn from cynomolgus (UniProt: Q8SPV9, p51 subunit: residues 24-297 with C-terminal tag Avi; UniProt: Q8SPW0, β2-m: residues 21-119); biotinylated hFcRn (UniProt: P55899, p51 subunit: residues 24-297 with C-terminal tag Avi; UniProt: P61769, β2-m: residues 21-119); human CD16a (UniProt: P08637, FcγRIIIa: HPC4 C-terminal residues 17-208 and valine at position 158 (V158)). Heavy chain variants H435A and H310A/H435Q were prepared from HEK293 conditioned medium. mAb2 variants were cloned with Evitria and purified from suspension conditioned CHO K1 medium using mAbSelect SuRe affinity columns (GE Healthcare) and buffer exchanged in phosphate buffered saline (PBS) pH 7.4 for subsequent experiments.

Building the saturation library:

The heavy and light chains of the WT IgG1 mAb1 DNA leader were incorporated into the mammalian expression plasmids pBH6414 and pBH6368, respectively, using the NcoI and HindIII restriction sites. The saturation library was created using Lightning Site Directed Mutagenesis Kit (Agilent) and NNK (N= A/C/G/T, K=G/T) and WWC primers (W=A/T) (IDT Technologies) to insert all possible amino acids at the following positions: M252, I253, S254, T256, K288, T307, K322, E380, L432, N434 and Y436 (EC numbering). The heavy chain DNA sequences of the three control variants in the mAb1 framework, AAA (T307A/E380A/N434A), LS (M428L/N434S), and YTE (M252Y/S254T/T256E), were inserted into the pBH6414 vector, manufactured by LakePharma.

A combinatorial saturation library was generated by site-directed mAb1 heavy chain mutagenesis using the Q5 mutagenesis kit (Q5 Mutagenesis Kit, NEBiolabs) and primers T256D, T256E, T307Q, T307W, N434F, and N434Y with WT and M252Y templates in a PCR reaction. Insertion of mutations into the Ab3 scaffold was performed using the Q5 Mutagenesis Kit (NEBiolabs) with primers M252Y, T256D, T307Q, and T307W. Creation of all Fc variants was validated by Sanger Sequencing (Genewiz, Inc.).

Expression and purification of recombinant antibodies:

For screening in conditioned media, DNA containing the mAb1 wild-type mutant heavy chain and light chain was transfected into 1 ml Expi293 mammalian cells (Invitrogen) for expression according to the manufacturer's instructions. Cells were incubated at 37°C, 5% carbon dioxide and 80% humidity with shaking at 900 revolutions per minute (rpm) in a 2 ml 96-well plate (Greiner Bio-One) and sealed with an aerated membrane. Conditioned media were collected five days after transfection and stored at -80°C until use. The leading variants in the mAb1 and Ab3 frameworks were expressed at a 30 ml scale in 125 ml 0.2 μm vented flasks (Corning). 125 ml culture flasks were shaken at 125 rpm for the duration of expression. Conditioned medium was collected five days after transfection, filtered through 0.22 μm 50 ml conical filters (Corning) and stored at 4° C. until purified.

Isolation of mAb1 and Ab3 was performed using 1 ml mAbSelect SuRe HiTrap columns (GE Healthcare). After a washing step using ten column volumes of PBS pH 7.4, antibodies were eluted with five column volumes of 0.1 M citric acid pH 3.0 (Sigma) and neutralized with 0.5 ml of 1 M tris base pH 9.0 (Sigma ). The eluted antibodies were buffer exchanged with PBS pH 7.4 and concentrated to >1 mg ml ^-1 using 30 kDa MWCO Amicon Concentrators (Millipore) for further analysis. The concentration of purified antibodies was determined by their UV absorption at 280 nm (UV ₂₈₀ ) with the corresponding extinction coefficient.

Sampling and analysis of Octet conditioned media:

Screening of conditioned media containing mAb1 variants was performed on an Octet QK 384 with Ni-NTA biosensors (PALL Life Sciences). His-tagged antigen was maintained at a concentration of 15 μg/ml for 300 sec in PBS, 0.1% bovine serum albumin (BSA, Sigma) and 0.01% Tween-20 (Sigma), pH 7.4 (PBST-BSA 7.4) and then washed for 20 sec in PBST-BSA pH 7.4. Antibodies were kept for 200 sec in conditioned medium diluted 1:1 with PBST-BSA, pH 7.4. After washing steps with pH 6.0 buffer, FcRn binding kinetics were obtained using 200 nM rFcRn for association and dissociation times of 150 and 200 sec, respectively, at pH 6.0. At all stages during Octet screening, the temperature was 30ºC with a shaking speed of 1000 rpm. rFcRn binding kinetic profiles were adjusted prior to the start of the FcRn association phase and modeled according to a 1:1 binding model using Octet Analysis Software 7.1 analysis software.

FcRn binding kinetics:

FcRn binding kinetics at pH 6.0 and pH 7.4 were measured on a Biacore T200 instrument (GE Healthcare) using modified protocols with either direct FcRn immobilization or the CAPture biotin capture kit (GE Healthcare) (see e.g. Abdiche et al., MAbs (2015) 7: 331-343; Karlsson et al., Anal Biochem. (2016) 502: 53-63). For direct immobilization, biotinylated FcRn at a concentration of 20 μg/ml was immobilized for 180 sec at 10 μl/min in 10 mM sodium acetate pH 4.5 (GE Healthcare) to ~20 RU on the C1 sensor chip surface using an amino group coupling reaction ( GE Healthcare). When using the CAPture biotin kit, the CAPture reagent was captured on the surface of the CAP chip to RU binding >2000 RU, followed by the addition of 0.1 µg/mL FcRn in the appropriate channels for 24 sec at 30 µL/min until final RU binding at ~ 2RU. The working buffer for FcRn binding kinetics experiments was PBS with 0.05% P-20 surfactant (PBS-P+, GE Healthcare) at pH 6.0 or 7.4. A series of 4-fold serial dilution concentrations, starting from 1000 nM antibody, were run in quadruplicate for each variant, including a 0 nM control. Kinetic measurements were made for association and dissociation times of 180 and 300 sec, respectively, at a flow rate of 10 μl/min. C1 and CAP sensor chips were regenerated with 10 mM sodium tetraborate, 1 M NaCl, pH 8.5 (GE Healthcare) for 30 sec at 50 μl/min or 6 M guanidine hydrochloride, 250 mM sodium hydroxide (GE Healthcare) for 120 sec at 50 µl/min, respectively, followed by an additional stabilization step for 60-90 sec in PBS-P+ pH 6.0. Steady state RU measurements at pH 7.4 were obtained for all variants at 1000 nM in triplicate using the same C1 or CAP sensor chip and kinetic parameters as described above, except that the FcRn uptake level was increased 10- 20 times for both methods.

The kinetic parameters for the concentration series at pH 6.0 were matched to the bivalent model using the Biacore T200 evaluation software due to the avidity effect. See, for example, Suzuki et al., J. Immunol. (2010) 184: 1968-1976. Each series of dilutions was selected independently to obtain the average values of the rates of binding and dissociation and the ability to bind. The observed binding capacity was calculated from the first binding and dissociation rates obtained from the bivalent model. Residual binding at pH 7.4 was measured using 1000 nM of each antibody in triplicate at the same time to compare response. The steady state response in each replicate was averaged to obtain the mean and standard deviation.

Affinity Chromatography FcRn:

In one experiment, an FcRn affinity column was performed according to protocols adapted from Schlothauer et al. 2013, mAbs 5:576-586. A 1 ml Streptavidin HP HiTrap column (GE Healthcare) was equilibrated with binding buffer (20 mM sodium phosphate (Sigma) pH 7.4, 150 mM sodium chloride (NaCl; Sigma)) at 1 ml/min at a rate of five column volumes followed by injection of 4 milligrams of biotinylated cynoFcRn. The column was washed with binding buffer and stored at 4°C until use.

The FcRn affinity column was equilibrated with low pH buffer (20 mM 2-(N-morpholino)ethanesulfonic acid (MES; Sigma), pH 5.5; 150 mM NaCl) at five column volumes prior to injecting 300 μg of each antibody. The pH of the antibody solutions was adjusted to 5.5 with a low pH buffer. After washing ten column volumes with low pH buffer, antibodies were eluted with a linear pH gradient using high pH buffer (20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (bis-trispropane; Sigma) pH 9.5; 150 mM NaCl) in 30 column volumes at 1 ml/min in 1 ml fractions, observing with UV ₂₈₀ . The FcRn affinity column was re-equilibrated with ten column volumes of low pH buffer for subsequent runs or with binding buffer in case of preparation for storage. All variants were performed in triplicate.

The elution profile of the FcRn affinity column for each variant was modeled from a single Gaussian distribution using Equation 1 in Sigmaplot 11 (Systat Software, Inc.) to determine the elution volume at UV ₂₈₀ maximum.

(Уравнение 1)

(Equation 1)

where x ₀ is the elution volume at the maximum of the UV ₂₈₀ peak, y ₀ is the initial level of UV ₂₈₀ absorbance, a and b refer to the full width at half maximum of the distribution. The pH of each fraction was measured with a Corning Pinnacle 540 pH meter and correlated with elution volume using linear regression.

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

Differential Scanning Fluorimetry:

Differential scanning fluorometry (DSF) experiments were performed on a BioRad CFX96 real-time system thermal cycler (BioRad) for 20 µl reactions. Antibody samples and a 5000-fold supply of Sypro Orange dye (Invitrogen) were diluted to 0.4 mg/ml and 10x, respectively, in PBS pH 7.4. Antibodies and Sypro Orange were mixed 1:1 in 96-well PCR plates and sealed with sticky microseal (BioRad) to a final concentration of 0.2 mg/ml of each antibody and 5x Sypro Orange dye. All antibody variants were performed in triplicate. The thermal cycling program consisted of a 2 minute equilibration step at 20ºC followed by a constant temperature change rate of 0.5ºC/5 sec to a final temperature of 100ºC. Fluorescence measurements of each well were obtained using FAM excitation (485 nm) and ROX emission (625 nm) wavelength detectors suitable for Sypro Orange fluorescence (see e.g. Biggar et al. 2012, Biotechniques 53: 231-238) . The DSF fluorescence intensity profile and the first derivative were taken from the BioRad CFX Manager and analyzed in Sigmaplot 11. T _pl. was defined as the middle of the first transition in the fluorescence intensity profile.

Kinetics of binding to FcγRIIIa:

The kinetics and binding capacity were measured on a Biacore T200 instrument (GE Healthcare) (Zhou et al., 2008 Biotechnol. Bioeng. 99. 652-665). An anti-HPC4 antibody (Roche), at 50 μg/ml in acetate, pH 4.5, was bound to the surface of the CM5 sensor chip for 600 sec at 10 μl/min by amino group reaction to a final density of >20,000 RU. The working buffer for FcγRIIIa binding kinetics experiments was HEPES buffered saline with 0.05% P-20 surfactant (HBS-P+, GE Healthcare) and 2 mM calcium chloride (CaCl ₂ , Fluka) at pH 7.4. Each kinetic trace was initialized by capturing 1.25 μg/ml HPC4-tagged FcγRIIIa-V158 for 30 sec at 5 μl/min. The kinetics of association and dissociation at 300 nM of each variant was measured for each variant for 120-180 sec for each step at 5 μl/min. After completion of kinetic measurements, the CM5 chip was regenerated with HBS-P+ buffer supplemented with 10 mM EDTA (Ambion). Before the next kinetic measurements, the CM5 chip was washed for 120 sec using HBS-P+ with CaCl ₂ .

In one experiment, FcγRIIIa kinetic experiments were analyzed in the same manner as described for binding to FcRn at pH 7.4. For WT, comparative, leading single and combined variants, kinetics were obtained in a series of 3-fold serial dilution from 1000 nm to determine the ability to bind to FcγRIIIa. Steady state RU at each concentration and replication was determined, plotted as a function of antibody concentration, and adjusted to the steady state model as shown in Equation 2.

(Уравнение 2)

(Equation 2)

where offset is the baseline RU level at 0 nM antibody, R _max is the RU plateau at high antibody concentrations, [Antibody] is the antibody concentration _{, and K D obs.} - observed binding capacity in the interaction between variants and FcγRIIIa.

In another experiment, FcγRIIIa kinetic experiments were analyzed in the same manner as described for FcRn binding at pH 7.4 using the average steady state binding response. For all variants, the steady state of the RU 300 nM antibody was determined in triplicate and averaged. The fold change in response change relative to WT (fold change in response) was determined to compare options within each framework.

Isoelectric focusing

The isoelectric point (pI) of the leading variants was determined by capillary electrophoresis on a Maurice C instrument (Protein Simple). Each 200 µl sample contained 0.35% methylcellulose (Protein Simple), 4% farmalite 3-10 (GE Healthcare), 10 mM arginine (Protein Simple), 0.2 mg/ml antibody, and 4.05 and 9.99 pI markers (Protein Simple). The sample was loaded into a capillary for 1 min at 1500 V followed by a separation phase for 6 min at 3000 V and monitored for tryptophan fluorescence. The pI for each variant was determined using Maurice C software and was defined as the pH at maximum fluorescence for the major species.

ELISA with homogeneous bridging rheumatoid factor (RF)

Antibodies were biotinylated and labeled with digoxigen using EZ-Link Sulfo-NHS-LC-Biotin and Mix-n-Stain™ Digoxigenin Antibody Labeling Kits (Biotium) according to the manufacturer's instructions. A stock solution containing 4 μg/ml biotinylated and digoxigenin-labeled antibodies was prepared for each variant and mixed 1:1 with 300 U/ml RF (Abcam). After incubation at room temperature for 20 hours, 100 μl of each antibody-RF mixture was added to Streptawell plates (Sigma-Aldrich) and incubated at room temperature for 2 hours. The plate was washed three times with PBS, pH 7.4, with 0.05% Tween-20 and 100 μl diluted 1:2000 HRP-conjugated anti-digoxigenin secondary antibody (Abcam) was added to each well. After a 2 hour incubation at room temperature, the wells were washed and treated with 100 μl of TMB substrate (Abcam) for 15 minutes at room temperature. The reaction was stopped with 100 μl stop solution (Abcam) and the absorbance was measured at 450 nm on a SpectraMax plate reader. The well containing no antibody-RF mixture allowed blank subtractions and the experiment was repeated three times. P-values were determined using Student's t-test.

Pharmacokinetics in vivo

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

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

All in vivo studies were conducted in accordance with Sanofi's animal welfare policy. Studies in monkeys and mice were approved by the French "Ministère de l'Enseignement Superieur et de la Recherche" and the German "Regierungspraesidium Darmstadt".

The concentration of each mAb2 variant at each time point was determined by bottom-up LC-MS/MS analysis. After pelleting an aliquot of plasma, the plasma pellet was subjected to denaturation, reduction, alkylation, trypsin digestion, and protein solid phase extraction prior to analysis of surrogate peptides. Calibration standards were prepared by infusing the mAb2 variant into plasma at 1.00, 2.00, 5.00, 10.0, 20.0, 50.0, 100, 200 and 400 µg/mL. Peptide separation was performed on a Waters Acquity UPLC system with an XBridge BEH C18 reverse phase column (2.1×150 mm, 3.5 μM, 300Å, Waters) at a flow rate of 300 μl/min with a step gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For detection, a Sciex API5500 mass spectrometer was used in positive ion mode with a source temperature of 700°C, ion sputter voltage of 5500 V, curtain and nebulizer gases at 40°C, and collision gas in the middle. The dwell time was 20 ms and the input potential was 10 V for each transition. To determine concentration relative to standards and controls, multiple reaction monitoring transformations were used for two unique mAb2 framework surrogate peptides using the peak area from the Analyst software MQIII integration algorithm. The clearance rate 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 when a sharp decrease in concentration was observed were excluded from the mean plasma concentration due to an assumption, not associated with any theory, about the distribution of the drug, due to the target (target-mediated drug disposition, TMDD), and / or interaction of antibodies with a drug (anti-drug antibody, ADA).

Example 2 Octet Screening for Saturating Point Mutations in Conditioned Media

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

To identify variants with slower rates of dissociation from FcRn than WT antibody, a bilayer interferometry (BLI) assay was developed to screen for antibody variants in high throughput conditioned media (Figure 2A). This assay was designed using several comparators that either increase (AAA, LS and YTE) or decrease (H435A, H310A/H435Q) affinity for FcRn at pH 6.0 compared to the WT antibody. NiNTA biosensors captured the his-tagged antigen and then simulated conditioned media at pH 7.4 for each antibody variant (Figure 2A). Binding kinetics to rat FcRn (rFcRn) at pH 6.0, which has a ~25-fold slower dissociation rate than human IgG1 and is easier to evaluate by Octet than human FcRn (hFcRn), was measured for each of the six variants ( Figure 2B). Variants H435A (Figure 2B, long dashes with one dot interspersed) and H310A/H435Q (Figure 2B, long dashes with two dots) show almost no FcRn binding kinetics (see also, e.g., Shields et al. 2001 , supra; Medesan et al., 1997, supra, and Raghavan et al., 1995, supra). Options AAA (Figure 2B, short dashes), LS (Figure 2B, short dashes with one dot) and YTE (Figure 2B, long dashes) show slower dissociation kinetics compared to WT (Figure 2B, solid line) with a decrease in dissociation rate with FcRn by 2-7.3 times. This demonstrated that Octet screening is suitable for variant recognition using aberrant dissociation kinetics from rFcRn.

The IgG1 antibody, mAb1, served as a model system for creating a saturation mutagenesis library to screen for mutants with a reduced dissociation rate from FcRn. Eleven positions in the Fc region of mAb1 were selected based on their proximity or direct involvement in the area of contact with FcRn (Figures 1A and 1B) (see, e.g., Oganesyan et al. 2014, supra; and Shields et al. 2001, supra) . All point mutations at these positions were engineered using site-directed mutagenesis and transfected into Expi293 cells for expression. The conditioned media were screened for the saturation mutagenesis library as described above. Normalized FcRn-binding Octet sensograms for a subset of variants are shown in Figure 2C (long dashes) with wild type (Figure 2C, thick long dashes) and sham negative control (Figure 2C, dotted line). Simulation control showed no noticeable binding to FcRn. Several mutants clearly disrupted binding to rFcRn as little to no signal change was observed in the kinetic profiles (Figure 2C, long dashes below the dotted line (sham control)). The cutoff for variants with improved FcRn dissociation rate was defined as three standard deviations below the WT antibody mean. In the subset of mutations shown in Figure 2C, two (Figure 2C, solid lines) had significantly reduced dissociation rates compared to wild-type antibodies (Figure 2C, thick long dashes), while the remaining variants had similar values (Figure 2C, short dashes with one dot) or faster (Figure 2C, long dashes above the dotted line (simulation control)) dissociation rate from rFcRn.

Dissociation rates from rFcRn for all single point mutations are shown in Figure 2D and Figure 14 by position and mutation. In Figure 14, the data are sorted into four categories based on the fold change in dissociation rate from rFcRn compared to wild type, and wild types are indicated by black squares.

In Figure 14, the fold change in dissociation rate from rFcRn for all possible substitutions at eleven positions in the saturation library was normalized to the WT antibody mean and color coded. All mutants fall into one of four categories: virtually non-binding (dark gray), faster dissociation rate with rFcRn (grey), WT-like dissociation rate with rFcRn (horizontal lines), and slower dissociation rate with rFcRn (network). Multiple variants had slower dissociation rates from rFcRn than WT antibodies (network).

The mutants shown in dark gray in Figure 14 did not bind to rFcRn substantially as did the sham control (Figure 2C, dotted line) and were located in the M252, I253 and S254 loop. The only mutations in I253 were methionine and valine, and both significantly increased the rate of dissociation from rFcRn, further confirming the importance of I253 for interaction with FcRn. An additional 120 variants (Figure 2D and Figure 14, light gray boxes) destabilize the interaction with rFcRn, with approximately 50% of them located in each of the C _H 2 and C _H 3 domains. Twenty-five mutants have a dissociation rate similar to WT (Figure 2D and Figure 14, white boxes), with eight of the 11 positions having at least one WT-like mutation (Figure 14, white boxes). The following mutations had a significantly reduced rate of dissociation from rFcRn compared to wild type (Figure 2D and Figure 14, black boxes): M252Y, T256D/E, K288D/N, T307A/E/F/M/Q/W, E380C, N434F /P/Y and Y436H/N/W. Mutations M252Y, N434F and N434Y had a dissociation rate more than two times slower than that of the WT antibody (Figure 2D). These mutations were expressed and purified by protein A chromatography for further kinetic study with FcRn in vitro .

Example 3: Kinetics of Biacore Binding to FcRn at pH 6.0

Variants AAA, LS and YTE served as positive controls in FcRn binding kinetics measurements using Biacore with both human and rat FcRn at pH 6.0. Concentration-dependent binding to FcRn was observed for all variants, including wild type, reference (Figure 3), and leaders (Figures 4A and 4B), and the binding profile of a single injection to human and rat FcRn is shown in Figures 5A and 5B, respectively. The wild-type antibody had human and rat FcRn binding capacities of 2380 ± 470 nM and 207 ± 43 nM, respectively (Table 1).

Table 1 Characterization parameters obtained in vitro for purified anti-mAb1 leader antibodies.

Octet
pH 6.0 Affinity column with FcRn DSF Biacore pH 6.0 Biacore pH 7.4 hFcRn rFcRn hFcRn rFcRn Mutant rFcRn
Dissociation rate Elution
pH value T _pl. (ºC) Binding speed Dissociation rate K _{D, obs.} Binding speed Dissociation rate K _{D, obs.} Steady state RU Steady state RU E380C 2.08 ± 0.18 7.18 ± 0.11 64.7±0.5 1.73 ± 0.39 4.57 ± 1.50 >10,000 1.71±0.25 106±1 6310±880 5.7±0.1 27.7 ± 4.0 K288D 3.79±0.06 7.33±0.10 65.8±0.1 3.31 ± 1.10 5.13±0.75 >10,000 6.61 ± 3.01 8.43±0.60 149 ± 63 3.8±0.2 21.9 ± 3.1 K288N 4.11 ± 0.08 7.39±0.02 66.7±0.3 4.12 ± 1.42 4.54±0.34 >10,000 6.42 ± 2.80 10.7±0.9 190 ± 73 3.9±0.2 20.4 ± 2.7 M252Y 0.95±0.03 7.88±0.03 64.4±0.2 5.50 ± 1.83 1.43 ± 0.23 3100±1500 10.6 ± 3.1 2.64±0.62 25±3 8.6±0.9 57.7 ± 10.7 N434F 1.18±0.05 8.30±0.05 67.8±0.2 35.4 ± 15.3 0.50 ± 0.08 165 ± 73 12.6 ± 1.4 3.36 ± 1.79 26 ± 13 20.1 ± 3.4 54.9 ± 9.1 N434P 3.80±0.08 7.56±0.02 63.6±0.5 2.42 ± 0.54 3.35±1.10 >10,000 3.44 ± 0.07 6.67 ± 0.18 194 ± 9 3.5±0.4 4.3±0.6 N434Y 1.33±0.04 8.46±0.02 67.3±0.5 35.9 ± 9.6 0.52 ± 0.10 137 ± 33 14.5 ± 1.82 3.46 ± 1.86 23 ± 12 22.6 ± 4.2 47.0±6.3 T256D 2.24±0.03 7.82±0.07 64.7±0.2 4.41 ± 1.72 2.51±0.65 6700±3540 6.09 ± 1.84 4.84±0.08 86 ± 27 5.8±0.6 30.4 ± 3.4 T256E 3.26±0.04 7.63±0.06 66.3±0.6 3.90±2.37 3.38±0.28 >10,000 6.29 ± 1.59 6.94±0.97 113±15 4.8±0.5 23.0±2.7 T307A 2.98±0.06 7.61±0.03 68.0±0.4 2.85±0.72 2.91±0.46 >10,000 6.09±2.72 7.08±0.67 132 ± 48 5.2±0.6 23.1 ± 2.8 T307E 3.29±0.08 7.58±0.03 70.2±0.5 4.37 ± 1.63 2.98±0.37 8130±5070 5.55 ± 2.82 6.03±0.20 141 ± 65 5.7±0.7 21.5 ± 2.6 T307F 2.80±0.07 7.61±0.03 70.2±0.3 2.70±0.83 2.92±0.13 >10,000 5.69±2.83 6.15±0.14 131 ± 63 4.9±0.7 21.9 ± 2.7 T307M 3.47 ± 0.13 7.40±0.08 70.0±0.4 4.08 ± 0.83 3.87 ± 0.28 >10,000 6.81 ± 2.34 17.1 ± 4.0 279 ± 140 4.4±0.7 15.3 ± 1.9 T307Q 1.84±0.05 7.86±0.06 70.3±0.6 3.96 ± 1.10 2.15±0.22 5720±1530 7.35±2.15 4.04±0.28 58±16 6.4±0.8 24.3 ± 3.0 T307W 2.42±0.08 7.75±0.07 63.0±0.5 3.33±0.83 2.77 ± 0.29 8740 ± 2440 7.11 ± 2.29 7.37 ± 0.41 111 ± 32 5.8±0.7 19.2 ± 3.3 Y436H 3.22±0.08 7.33±0.05 68.7±0.3 2.59±0.78 6.06 ± 1.00 >10,000 5.06±0.13 7.30±0.88 131 ± 9 3.6±0.5 16.3 ± 1.8 Y436N 5.25±0.22 7.22±0.05 65.8±0.5 4.60±2.36 7.37 ± 3.34 >10,000 10.1 ± 4.9 20.3 ± 3.1 233 ± 86 3.6±0.4 17.4±2.0 Y436W 5.18±0.18 7.39±0.03 68.6±0.7 2.84 ± 1.90 4.62±0.92 >10,000 3.44 ± 2.18 23.7 ± 7.7 1140±950 3.7±0.4 10.3±1.2 wt 5.01±0.45 7.37±0.05 69.0±0.2 16.2 ± 2.9 3.86±0.38 2380±470 7.26 ± 1.01 15.2±0.23 207 ± 43 4.3±1.0 12.2±0.5 AAA 3.77 ± 1.03 7.94±0.06 61.3±0.6 8.37 ± 1.82 1.44 ± 0.04 1780±380 15.7 ± 3.3 11.7 ± 1.1 77 ± 18 13.9±3.1 23.6 ± 4.9 LS 3.38 ± 0.23 8.29±0.03 68.5±0.3 19.3±3.5 0.52 ± 0.03 272 ± 40 9.08 ± 1.58 6.58±0.39 74 ± 9 18.3 ± 4.6 24.8 ± 4.8 YTE 0.66 ± 0.17 8.14 ± 0.03 61.2±0.3 14.3 ± 4.4 0.45 ± 0.07 342 ± 117 6.52±0.46 1.21 ± 0.21 18±2 13.2 ± 3.5 53.9 ± 1.2

All data shown in Table 1 was obtained by the experimental methods indicated at the top of each column.

The rate of dissociation from rFcRn by Octet using purified proteins was measured as a comparison with the kinetic constants obtained from screening in conditioned media. The elution pH was determined by FcRn affinity chromatography in triplicate (n=3) and thermal stability was examined in triplicate by DSF (n=3). FcRn binding kinetics for human and rat FcRn were obtained from Biacore with duplicate antibody serial dilutions (n=2) and adjusted independently. The steady state binding response (RU) of each variant to human and rat FcRn at pH 7.4 was measured with 1000 nM antibody in triplicate (n=3) using Biacore. Units for each measurement: Octet, pH 6.0, dissociation rate with rFcRn (x10 ^-3 sec ^-1 ); elution pH (dimensionless value); DSF T _pl. (°C); Biacore, pH 6.0, hFcRn binding rate (x10 ⁴ M ^-1 sec ^-1 ), dissociation rate (x10 ^-1 sec ^-1 ) and K _{D obs.} (x10 ⁹ M); Biacore, pH 6.0, rFcRn binding rate (x10 ⁴ M ^-1 sec ^-1 ), dissociation rate (x10 ^-3 sec ^-1 ) and K _{D obs.} (x10 ⁹ M); Biacore, pH 7.4, steady state binding response (RU).

On FIG. 5B variants AAA (dashed), LS (dashed with two dots) and YTE (dashed with one dot) had a 1.6-10.4-fold improved binding capacity compared to WT. The nature of the comparative variant with the strongest affinity for FcRn was species-specific, as LS had the strongest affinity for hFcRn, while rFcRn had a stronger affinity for YTE (Table 2A).

Table 2A. Characteristic parameters obtained in vitro for purified mAb1 dual combination antibodies.

Affinity column with FcRn DSF Biacore pH 6.0 Biacore pH 7.4 hFcRn rFcRn hFcRn rFcRn Option elution pH T _pl. (ºC) Binding speed Dissociation rate K _{D, obs.} Binding speed Dissociation rate K _{D, obs.} Steady state RU Steady state RU M DQ N 7.92±0.06 67.9±0.4 3.76±0.38 8.72 ± 0.10 232 ± 24 1.33±0.22 1.27 ± 0.07 9.54 ± 1.66 10.7±1.0 39.1 ± 4.5 M D T F 8.41 ± 0.07 62.3±0.2 9.68±0.64 2.90±0.04 29.9±2.0 2.18±0.20 0.39±0.01 1.78±0.17 29.2 ± 4.0 63.0±6.0 M D T Y 8.45±0.04 61.5±0.2 18.3±0.6 2.85±0.01 15.6±0.5 4.13±0.15 0.25±0.35 0.60±0.85 37.1±5.0 71.6 ± 6.6 M DW N 7.92±0.04 57.8±0.4 5.29±0.14 8.92±0.32 169±8 1.57 ± 0.12 1.29±0.01 8.24±0.85 12.2 ± 1.3 42.1 ± 4.7 M EQ N 7.84±0.06 68.0±0.5 2.87±0.01 14.3±0.2 499±6 1.46±0.25 1.56±0.94 10.7 ± 6.7 5.8±0.6 23.4 ± 3.1 M E T F 8.23±0.03 64.1±0.7 5.36 ± 1.41 4.19±0.03 78.2±20.5 1.08 ± 0.11 0.81 ± 0.07 7.53 ± 1.03 23.3 ± 3.2 59.0±5.6 M E T Y 8.38±0.04 63.5±0.6 6.28 ± 1.62 3.93±0.02 62.6 ± 16.2 1.27 ± 0.11 0.98 ± 0.07 7.71±0.84 26.8 ± 3.7 62.2 ± 6.3 M EW N 7.78 ± 0.04 58.2±0.4 4.32±0.36 14.0±0.1 323 ± 27 1.81±0.01 1.92±0.05 10.6±0.26 7.7±0.9 33.0±4.2 MT QF 8.56 ± 0.14 69.3±0.2 5.74 ± 1.05 2.46±0.05 42.9 ± 7.9 1.04 ± 0.07 0.40±0.01 3.89±0.26 34.2 ± 4.6 62.5 ± 7.6 MT QY 8.68±0.15 69.2±0.2 6.22 ± 1.38 2.02±0.06 32.4 ± 7.3 1.16±0.06 0.48 ± 0.01 4.11 ± 0.23 38.4±5.2 63.0 ± 7.7 MTWF 8.61±0.06 60.9±0.2 7.87 ± 0.31 3.01±0.08 38.2 ± 1.8 2.11 ± 0.06 0.57 ± 0.01 2.70±0.08 30.5 ± 4.2 65.1 ± 6.4 MTWY 8.62±0.14 62.1±0.5 14.8±0.8 3.17±0.03 21.5 ± 1.2 4.80±0.07 0.30±0.04 0.62 ± 0.08 37.2 ± 4.9 69.6 ± 7.0 YD TN 8.29±0.06 59.6±0.9 6.33 ± 1.23 5.93±0.20 93.6 ± 18.4 2.85±0.11 1.67 ± 0.16 5.86±0.61 9.7 ± 1.8 56.0±5.6 YE TN 7.83±0.06 60.7±0.7 5.92±0.05 7.57±0.30 128±5 3.24 ± 0.07 2.73±0.02 8.43 ± 0.20 9.8±1.2 55.1 ± 6.1 Y T Q N 7.87±0.06 63.1±0.1 3.45 ± 0.29 9.60±0.01 278 ± 23 2.55±0.24 1.05±0.63 4.11 ± 2.51 10.6 ± 1.2 49.2 ± 5.6 Y TT F 8.56 ± 0.09 62.2±0.1 12.7±0.7 2.30±0.01 18.0±1.0 4.03±0.06 0.12±0.02 0.29±0.04 43.8±5.8 79.3 ± 9.9 Y TT Y 8.95±0.02 62.0±0.1 20.6±0.6 1.71 ± 0.02 8.32±0.2 5.40±0.07 0.14 ± 0.04 0.26 ± 0.08 54.1 ± 7.1 67.9 ± 7.9 Y T W N 8.14 ± 0.02 59.3±0.2 4.83 ± 0.20 5.70±0.03 118±5 2.08 ± 0.07 1.09±0.03 5.21±0.22 15.9±1.7 66.1 ± 7.1

The vast majority of the leading options for both human and rat FcRn (Figures 5A and 5B, solid lines in different shades) had significantly lower binding rates than WT or comparison options (>2 times) (table 1). The N434F and N434Y mutations were the only variants that showed increased binding rates in both FcRn species. Without being bound by any theory, as a result of the slower association kinetics with hFcRn, the observed binding abilities of the leading variants were generally weaker than those of WT, in contrast to rFcRn (Figures 5C and 5D, Table 1). The affinity for rFcRn was weaker than that of YTE (Figure 5D, diagonal lines facing bottom left, Table 2A). Without being bound by any theory, these results indicated that a single mutation was not sufficient to increase affinity that would be better than that of the LS and YTE variants. A ranking of FcRn dissociation rates (due to the poor binding ability of variants to hFcRn) revealed a subset with reduced rates of dissociation from both human and rat FcRn: M252Y, N434F/P/Y, T256D/E, and T307A/E/F/Q/ W (Table 2A). These variants in combination are of additional interest to further improve the FcRn binding capacity of the Fc region to outperform the comparison variants.

The in vitro characterization parameters for the leading variants are shown in Table 2B.

Table 2B. In vitro characteristics of the leading variants.

mAb1 Affinity column with FcRn DSF Biacore Biacore pH 6.0 Biacore pH 7.4 FcγRIIIa
V158 hFcRn rFcRn hFcRn rFcRn Option pH value T _pl. (ºC) The fold change in affinity Binding speed Dissociation rate K _{D, obs.} K _{D, obs.} Steady state RU Steady state RU wt 7.37 69.0±0.2 1.00 16.2 ± 2.9 3.9±0.4 2380±470 207 ± 43 4.2±0.9 13.0±3.2 LS 8.29 68.5±0.3 1.04±0.04 1.9±0.4 5.0±0.1 272 ± 40 74 ± 9 18.3 ± 4.6 24.8 ± 4.8 YTE 8.14 61.2±0.3 0.52 ± 0.03 1.4±0.4 4.7±0.1 342 ± 117 18±2 13.2 ± 3.5 53.9 ± 1.2 M252Y 7.88 64.4±0.2 0.46 ± 0.03 5.5±1.8 1.4±0.2 >3000 25±3 8.6±1.0 50.5±5.6 N434F 8.30 67.8±0.2 1.16 ± 0.08 35±15 0.5±0.1 165 ± 73 26 ± 13 22.2 ± 2.6 61.5 ± 6.5 N434Y 8.46 67.3±0.5 1.41±0.06 36±10 0.5±0.1 137 ± 33 23 ± 12 25.5 ± 2.9 66.0 ± 7.1 T256D 7.82 64.7±0.2 0.92 ± 0.04 4.4 ± 1.7 2.5±0.7 >3000 86 ± 27 5.8±0.5 23.2 ± 2.8 T256E 7.63 66.3±0.6 0.89±0.04 3.9±2.4 3.4±0.2 >3000 113±15 4.5±0.4 17.0±2.1 T307Q 7.86 70.3±0.6 1.00±0.04 4.0±1.1 2.2±0.2 >3000 58±16 6.6±0.7 31.3 ± 3.7 T307W 7.75 63.0±0.5 0.97 ± 0.04 3.3±0.8 2.8±0.3 >3000 111 ± 32 5.6±0.7 21.9 ± 2.8 DQ 7.92 67.9±0.4 0.73 ± 0.03 3.8±0.4 8.7±0.1 232 ± 24 9.5±1.7 10.7±1.0 39.1 ± 4.5 DW 7.92 57.8±0.4 0.89±0.04 5.3±0.1 8.9±0.3 169±8 8.2±0.9 12.2 ± 1.3 42.1 ± 4.7 YD 8.29 59.6±0.9 0.48 ± 0.02 6.3 ± 1.2 5.9±0.20 94±18 5.9±0.6 9.7 ± 1.8 56.0±5.6

All data shown in Table 2B was obtained using the experimental procedures indicated at the top of each column. Affinity chromatography with FcRn, DSF and binding with FcγRIIIa was performed three times (n=3). FcRn binding kinetics to human and rat FcRn were obtained in quadruplicate and adjusted independently. Units: DSF T _pl. (°C); binding to FcγRIIIa (fold change relative to WT), Biacore, pH 6.0, binding rate to hFcRn (x10 ⁴ M ^-1 sec ^-1 ), dissociation rate (x10 ^-1 sec ^-1 ) and K _{D obs.} (x10 ⁹ M); Biacore, pH 6.0, rFcRn K _{D binding rate, obs.} (x10 ⁹ M); Biacore, pH 7.4, hFcRn and rFcRn at steady state RU (RU).

Example 4 Combined Variants Further Reduce FcRn Binding Dissociation Rate

Multiple leader mutations were localized to a single position (Figure 14, black boxes) such as T307 and N434 where six and three mutations, respectively, were identified that showed slower dissociation kinetics from FcRn. Only the mutations with the slowest dissociation rates from FcRn to hFcRn at these positions were used to generate combined variants. In this case, T307Q, T307W, N434F, and N434Y were mixed with M252Y, T256D, and T256E to generate double, triple, and quadruple variants using mixed primer PCR and site-directed mutagenesis. In total, the combinatorial library consisted of 54 variants, including seven leading single, 18 double, 20 triple, 8 quadruple variants and the WT antibody. The nomenclature of these variants is as follows: the wild-type background contains M252, T256, T307 and N434 and is designated MTTN. Thus, the triple variant - Y T QY - contains the mutations M252 Y , T307 Q and N434 Y , while retaining the WT threonine at position 256.

As with single mutations, FcRn binding kinetics at pH 6.0 using Biacore was used to determine which combination variants had improved affinity. A typical FcRn binding kinetic signature of each of single (long dashes with two dots), double (long dashes with one dot), triple (long dashes) and quadruple (short dashes) is shown in Figures 6A and 6B compared to WT (dashed line). ) and the comparison variant with the strongest affinity for their respective FcRn species (hFcRn: LS (long dashes with two dots); rFcRn: YTE (solid line)). From the rates of binding to and dissociation from hFcRn (Figure 6C) it can be seen that two single, 15 double, 18 triple and eight quadruple variants had increased binding capacity than the LS variant (Figure 6C, dotted line). In the same way, all combinations except one triple variant had a stronger affinity for rFcRn than YTE (Figure 6D, diagonal lines pointing down to the left). In the case of hFcRn, additional FcRn-enhancing mutations further increased binding capacity (Figure 6C). All five combinations with the strongest affinity for hFcRn were quadruple variants (Figure 6C, shaded) with about 500 times the wild-type binding capacity. A similar phenomenon did not occur with rFcRn (Figure 6D), as the highest affinity variants were double variants (Figure 6D, horizontal lines). The triple (Figure 6D, vertical lines) and quadruple (Figure 6D, shaded) variants generally showed only a slight decrease in dissociation rate (less than 2-fold) and also showed reduced association rates (Figure 6D). Without being bound by any theory, these results suggest that there may be a lower limit in terms of observed FcRn binding capacity (about 0.5 nM) that was achieved with rFcRn but not with hFcRn (Figure 6B). Overall, more than 40 combination variants had a stronger affinity than the comparison variants, and more research is required to select combinations with favorable properties for in vivo studies.

Example 5 Combined Variants Retain Significant Binding at Physiological pH

As a result of the significantly improved affinity for FcRn at pH 6.0, the effect on pH dependence was investigated using FcRn affinity chromatography and Biacore steady state measurements at pH 7.4. Affinity chromatography for FcRn uses a linear pH gradient to directly measure the disruption of pH dependence due to mutations. H435A and H310A/H435Q, weak FcRn binding variants, did not bind to the column regardless of pH (Figure 8A). WT elution occurs at approximately physiological pH (pH 7.37 ± 0.05), while AAA, LS and YTE require higher pH (Table 2B). All combination options and the top seven options required a higher pH to elute from the affinity column than WT (Figures 8A and 8C). The N434F/Y variants eluted at a higher pH than LS (Table 2B), which, without regard to scientific theory, indicates that these variants, either alone or in combination, disrupted the pH dependence. Representative chromatograms showed a distinct shift towards higher pH elution with increasing number of mutations (Figures 9A and 9B). The strong correlation (R2=0.94) between elution pH and hFcRn dissociation rates (Figure 9C) indicates that the slower FcRn dissociation rate at pH 6.0 directly contributed to the increased elution pH for the FcRn variants.

FcRn binding kinetic experiments were performed at pH 7.4 using Biacore to measure residual binding activity under physiological conditions. Steady state RU was used as a measure of residual FcRn binding capacity, as some variants exhibited unreliable kinetics and hardly bound at this pH. Characteristic kinetic traces of single (long dashes with two dots), double (long dashes with one dot), triple (long dashes), and quadruple (short dashes) options are shown in Figures 7A and 7B compared to LS (Figure 7A, solid) and YTE (Figure 7B, solid). These two variants showed the highest residual binding to human and rat FcRn at pH 7.4, respectively. Most of the leading single variants had slightly increased binding to FcRn compared to WT (4.3 ± 1.0 RU), but less than AAA (13.1 ± 1.7 RU), LS (18.5 ± 2.6 RU) and YTE (13.1 ± 1.6 RU), except for the N434F/Y mutations (Tables 2A and 2B). The combined variants also had significant residual binding to both FcRn species at pH 7.4 (Figures 7A and 7B) to an even greater extent than N434F/Y. Without wishing to be bound by theory, ideal candidates for in vivo studies are variants with increased FcRn binding at low pH (such as AAA, LS and YTE variants) but maintaining low binding at elevated pH, similar to WT. In the graph shown in Figures 7C and 7D, these combinations will occupy the lower left quarter of the image, indicated by the affinity of the LS and YTE variants at each pH for human and rat FcRn, respectively.

Example 6: FcRn Affinity Chromatography:

The combined variants showed a moderate positive correlation (hFcRn: R ² =0.69, rFcRn: R ² =0.71) between the observed binding capacity at pH 6.0 and the steady state of RU at pH 7.4 (Figures 7C and 7D). Without being bound by any theory, these results indicate that higher affinity at pH 6.0 generally results in more residual binding to FcRn at pH 7.4. These variants may remain associated with FcRn in the circulation and have short serum half-lives and/or facilitate their clearance, similar to high affinity abdeg mutations for FcRn (see e.g. Swiercz et al. 2014, supra; and Vaccaro et al. 2005, supra). Because the antibody-FcRn interaction is pH dependent and only occurs at low pH (<pH 6.5), saturation mutations can enhance the interaction between hydrophobic or charge contributors, which can interfere with the deprotonation of particularly important histidine residues (Figure 1B, as indicated ) and weaken this interaction at physiological pH values.

FcRn affinity chromatography uses a linear pH gradient to directly measure the disruption of the pH-dependency of interaction with FcRn (see, for example, Schlothauer et al. 2013, supra). FcRn affinity chromatography with variants AAA, LS, YTE, H435A and H310A/H435Q showed that H435A (Figure 8A, solid light gray line) and H310A/H435Q (Figure 8A, AQ, solid dark gray line) did not bind to FcRn even at pH 5.5 and eluted with flow. The wild-type antibody eluted at physiological pH (pH 7.37±0.05), while AAA, LS and YTE, which have a slower dissociation rate and the ability to bind more closely to FcRn than wild-type, according to Octet (Figure 2) and Biacore (Figure 3), a significantly higher pH is required to dissociate from the column (AAA: 7.94 ± 0.06; LS: 8.29 ± 0.03; YTE: 8.14 ± 0.03 ). It can be seen from the elution profiles that all combinatorial library variants require a higher pH to elute from the affinity column than the wild type variant. Representative chromatograms at medium pH elution for single (long dashes with two dots), double (long dashes with one dot), triple (long dashes) and quadruple (short dashes) options are shown in Figure 9A. The leading seven variants required a higher pH to dissociate from the column compared to WT (Figure 10A, Table 3), while variants having hFcRn kinetics like the wild-type variant (K288D/N, Y436H/H/ W) eluted at a pH similar to that of the wild type variant.

Table 3. Characteristic parameters of the leading antibody variants obtained in vitro

Affinity column with FcRn DSF Biacore pH 6.0 hFcRn rFcRn Option pH value T _pl. (ºC) Binding speed
(x10 ⁴ M ^-1 sec ^-1 ) Dissociation rate
(x10 ^-1 sec ^-1 ) K _{D, obs.}
( ^x109M ) Binding speed (x10 ⁴ M ^-1 sec ^-1 ) Dissociation rate
(x10 ^-3 sec ^-1 ) K _{D, obs. (x109M)} wt 7.37 69.0±0.2 16.2 ± 2.9 3.9±0.4 2380±470 7.3±1.0 15.2±0.2 207 ± 43 E380C 7.18 64.7±0.5 1.7±0.4 4.6±1.5 >10,000 1.7±0.25 106±1 6310±880 K288D 7.33 65.8±0.1 3.3 ± 1.1 5.1±0.8 >10,000 6.6±3.0 8.4±0.6 149 ± 63 K288N 7.39 66.7±0.3 4.1 ± 1.4 4.5±0.3 >10,000 6.4±2.8 10.7±0.9 190 ± 73 M252Y 7.88 64.4±0.2 5.5±1.8 1.4±0.2 3100±1500 10.6 ± 3.1 2.6±0.6 25±3 N434F 8.30 67.8±0.2 35±15 0.5±0.1 165 ± 73 12.6 ± 1.4 3.4±1.8 26 ± 13 N434P 7.56 63.6±0.5 2.4±0.5 3.4 ± 1.1 >10,000 3.4±0.1 6.7±0.2 194 ± 9 N434Y 8.46 67.3±0.5 36±10 0.5±0.1 137 ± 33 14.5 ± 1.8 3.5 ± 1.9 23 ± 12 T256D 7.82 64.7±0.2 4.4 ± 1.7 2.5±0.7 6700±3540 6.1 ± 1.8 4.8±0.1 86 ± 27 T256E 7.63 66.3±0.6 3.9±2.4 3.4±0.2 >10,000 6.3 ± 1.6 6.9±1.0 113±15 T307A 7.61 68.0±0.4 2.9±0.7 2.9±0.5 >10,000 6.1 ± 2.7 7.1±0.7 132 ± 48 T307E 7.58 70.2±0.5 4.4 ± 1.6 3.0±0.4 8130±5070 5.6±2.8 6.0±0.2 141 ± 65 T307F 7.61 70.2±0.3 2.7±0.8 2.9±0.1 >10,000 5.7 ± 2.8 6.2±0.1 131 ± 63 T307M 7.40 70.0±0.4 4.1±0.8 3.9±0.3 >10,000 6.8±2.3 17.1 ± 4.0 279 ± 140 T307Q 7.86 70.3±0.6 4.0±1.1 2.2±0.2 5720±1530 7.4±2.2 4.0±0.3 58±16 T307W 7.75 63.0±0.5 3.3±0.8 2.8±0.3 8740 ± 2440 7.1 ± 2.3 7.4±0.4 111 ± 32 Y436H 7.33 68.7±0.3 2.6±0.8 6.1 ± 1.0 >10,000 5.1±0.1 7.3±0.9 131 ± 9 Y436N 7.22 65.8±0.5 4.6±2.4 7.4 ± 3.3 >10,000 10.1 ± 4.9 20.3 ± 3.1 233 ± 86 Y436W 7.39 68.6±0.7 2.8 ± 1.9 4.6±0.9 >10,000 3.4 ± 2.2 23.7 ± 7.7 1140±950

All data were generated using the experimental procedures indicated at the top of each column. The elution pH was determined by FcRn affinity chromatography in triplicate (n=3) and thermal stability was examined in triplicate by DSF (n=3). FcRn binding kinetics for human and rat FcRn were obtained from Biacore with antibody serial dilutions (n=4) and adjusted independently. Units for each measurement: elution pH (dimensionless value); DSF T _pl. (°C); Biacore, pH 6.0, hFcRn binding rate (x10 ⁴ M ^-1 sec ^-1 ), dissociation rate (x10 ^-1 sec ^-1 ) and K _{D obs.} (x10 ⁹ M); Biacore, pH 6.0, rFcRn binding rate (x10 ⁴ M ^-1 sec ^-1 ), dissociation rate (x10 ^-3 sec ^-1 ) and K _{D obs.} (x10 ⁹ M).

Both N434F/Y variants eluted at a higher pH than the LS variant (N434F: 8.30 ± 0.05; N434Y: 8.46 ± 0.02) and showed significant binding to FcRn at pH 7.4 (Table 4) . These results indicate that only these variants can break the pH dependence. In general, the average elution pH increased with the increase in FcRn binding-enhancing mutations (Figure 9B). There was a strong correlation (R ² =0.94) with elution pH versus hFcRn dissociation rates (Figure 9C); without being bound by any theory, this indicates that the disruption of the pH dependence of the interaction directly contributes to the slower FcRn dissociation rates observed for the combinatorial library at pH 6.0.

Example 7: Thermal Stability

Most proteins, including antibodies, with low thermodynamic stability have an increased tendency to misfold and aggregate and may limit or interfere with their potency, efficacy, and efficacy as novel therapeutic agents. The thermal stability of each variant was determined using DSF and the reported melting point ( _Tm ) was determined as the middle of the first transition in the fluorescence intensity profile of the Sypro Orange dye. Compared with WT, T _pl. which is 69.0 ± 0.2°C, the LS variant is WT-like (68.5 ± 0.3°C), while AAA and YTE are thermally destabilized by ~8°C (AAA: 61.3 ± 0, 6°C; YTE: 61.2 ± 0.3°C) (Figures 8B, 9B and 10B; and tables 2B, 3 and 4). Compared with WT and LS, T _pl. which is 69.0 ± 0.2°C, variants AAA and YTE had thermal stability lower by ~8°C according to DSF data.

Table 4 Characteristic parameters of comparative and leading combinations obtained in vitro

Affinity column with FcRn DSF Biacore pH 6.0 Biacore pH 7.4 hFcRn rFcRn hFcRn rFcRn FcγRIIIa
V158 Option pH value T _pl.
(ºC) Binding speed
(x10 ⁴
M ^-1 sec ^-1 ) Dissociation rate
(x10 ^-1
sec ^-1 ) K _{D, obs.}
( ^x109M ) Binding speed (x10 ⁴
M ^-1 sec ^-1 ) Dissociation rate
(x10 ^-3
sec ^-1 ) K _{D, obs. (x109M)} Steady state RU Steady state RU K _{D, obs. (} ^x109M ₎ wt 7.37 69.0±0.2 16.2 ± 2.9 3.9±0.4 2380±470 7.3±1.0 15.2±0.2 207 ± 43 4.2±0.9 13.0±3.2 467 ± 99 AAA 7.94 61.3±0.6 8.4 ± 1.8 1.4±0.1 1780±380 15.7 ± 3.3 11.7 ± 1.1 77 ± 18 13.9±3.1 23.6 ± 4.9 450±19 LS 8.29 68.5±0.3 19.3±3.5 0.5±0.1 272 ± 40 9.1 ± 1.6 6.6±0.4 74 ± 9 18.3 ± 4.6 24.8 ± 4.8 369 ± 19 YTE 8.14 61.2±0.3 14.3 ± 4.4 0.5±0.1 342 ± 117 6.5±0.5 1.2±0.2 18±2 13.2 ± 3.5 53.9 ± 1.2 1040±160 M DQ N 7.92 67.9±0.4 3.8±0.4 8.7±0.1 232 ± 24 1.3±0.2 1.3±0.1 9.5±1.7 10.7±1.0 39.1 ± 4.5 600±4 M DW N 7.92 57.8±0.4 5.3±0.1 8.9±0.3 169±8 1.6±0.1 1.3±0.1 8.2±0.9 12.2 ± 1.3 42.1 ± 4.7 512±30 YD TN 8.29 59.6±0.9 6.3 ± 1.2 5.9±0.20 94±18 2.9±0.1 1.7±0.2 5.9±0.6 9.7 ± 1.8 56.0±5.6 1060±60 YE TN 7.83 60.7±0.7 5.9±0.1 7.6±0.3 128±5 3.2±0.1 2.7±0.1 8.4±0.2 9.8±1.2 55.1 ± 6.1 878 ± 101 Y T W N 8.14 59.3±0.2 4.8±0.2 5.7±0.1 118±5 2.1±0.1 1.1±0.1 5.2±0.2 15.9±1.7 66.1 ± 7.1 896±53 YDQ N 8.51 60.5±0.1 2.5±0.2 2.8±0.1 115±7 0.5±0.1 0.3 ± 0.1 5.1±0.3 19.8 ± 2.7 65.2 ± 6.6 1060±50 YEQ N 8.12 61.9±0.8 1.9±0.1 4.1±0.1 218±6 0.5±0.1 0.4 ± 0.1 7.8±0.4 15.2±2.1 61.8 ± 6.4 1620±210

Mutations introduced into the wild-type framework are in bold and underlined. All data were generated using the experimental procedures indicated at the top of each column. elution pH and T _pl. determined in triplicate (n=3). FcRn binding kinetics at pH 6.0 to human and rat FcRn were obtained using Biacore (n=4) and adjusted independently. Steady state c FcRn binding response at pH 7.4 was measured using Biacore at one antibody concentration in triplicate. The ability to bind to FcγRIIIa was determined by a duplicate antibody dilution series using Biacore. Units for each measurement: elution pH (dimensionless value); DSF T _pl. (°C); Biacore, pH 6.0, hFcRn binding rate (x10 ⁵ M ^-1 sec ^-1 ), dissociation rate (x10 ^-2 sec ^-1 ) and K _{D obs.} (x10 ⁹ M); Biacore, pH 6.0, rFcRn binding rate (x10 ⁵ M ^-1 sec ^-1 ), dissociation rate (x10 ^-3 sec ^-1 ) and K _{D obs.} (x10 ⁹ M); Biacore, pH 7.4, steady state binding response (RU) and FcγRIIIa K _{D, obs.} (x10 ⁹ M).

Twelve of the 18 leading saturation options had a reduced T _pl. compared to wild type, and some of the T307 mutants (T307E/F/M/Q) showed little stabilization (Table 4). None of the seven single variants used for the combinations (Figure 10B and Table 4) were significantly destabilized compared to YTE (Figure 8B and Table 5). The addition of double (Figure 9D, horizontal lines), triple (Figure 9D, vertical lines) and quadruple (Figure 9D, shaded in a box) options led to a further decrease in overall thermal stability compared to single options (Figure 9D, open circles). In multiple options T _pl. was lower than AAA or YTE (61.2 ± 0ºC), with >60% of these variants containing T307W. Quadruple variants (Figure 9D, shaded in a box) showed a clear bimodal distribution of melting temperatures, while combinations containing T307Q had about 6ºC higher thermal stability than variants bearing T307W (Figure 9D).

Example 8 Fc Variants Change FcγRIIIa Binding Interaction

In addition to interacting with FcRn, Fc hinge regions and _CH 2 domains are responsible for interaction with other Fc receptors, including FcγRIIIa. Because five of the seven individual variants used to create the combinatorial saturation library are located in the _CH 2 domain, the ability to interact with these receptors may be impaired compared to wild type, despite being located far from the interaction site. Using Biacore to measure binding to FcγRIIIa in the same manner as binding to FcRn at pH 7.4 revealed that the YTE variant (Figure 11A, dark grey) showed an approximately 50% reduction in binding response compared to wild type. (Figure 11A, black). Without wishing to be bound by any theory, the reduced binding of FcγRIIIa to YTE is the result of the M252Y mutation (Figure 11B, lowermost white circle), since this variant alone significantly reduces affinity for this receptor. Other single mutations did not have this reduced affinity (Figure 11B, white circles), and only the N434F/Y variants increased binding by 16-40%. These effects were transferred to most, but not all, of their respective combinations. For example, combinations containing M252Y had a 17-72% reduction in FcγRIIIa binding (Table 5).

Table 5. Concentrations of saturation library variants in conditioned media

Position M252 I253 S254 T256 K288 T307 K322 E380 L432 N434 Y436 Mutant Concentration (µg/ml) A 89.4 3.4 99.5 4.5 106 < 0.1 110 206 173 108 22.0 C < 0.1 0.2 108 < 0.1 < 0.1 < 0.1 323 < 0.1 138 31.8 < 0.1 D 22.7 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 169 127 133 < 0.1 2.2 E < 0.1 136 93.0 163 < 0.1 23.5 167 wt 2.7 65.9 8.9 F < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 154 127 118 < 0.1 < 0.1 G 46.7 < 0.1 172 106 < 0.1 4.9 124 146 107 < 0.1 < 0.1 H < 0.1 < 0.1 < 0.1 207 < 0.1 7.0 229 103 124 < 0.1 173 I < 0.1 wt < 0.1 193 53.9 < 0.1 63.0 60.2 182 16.3 < 0.1 K 1.2 4.1 < 0.1 3.2 wt 88.8 wt 69.2 25.0 < 0.1 67.0 L < 0.1 < 0.1 103 124 < 0.1 < 0.1 181 169 wt 77.7 < 0.1 M wt 80.0 1.5 122 < 0.1 < 0.1 112 76.8 97.3 30.5 90.1 N < 0.1 < 0.1 89.6 46.8 < 0.1 126 231 190 235 wt 18.9 P 0.7 < 0.1 18.5 2.7 92.6 < 0.1 1.3 175 < 0.1 120 < 0.1 Q 176 35.7 20.9 123 1.0 14.0 216 197 188 55.4 96.9 R 0.8 < 0.1 < 0.1 189 < 0.1 1.5 209 9.1 142 102 25.0 S < 0.1 71.1 wt 141 < 0.1 88.8 99.3 153 106 < 0.1 3.0 T 19.7 12.5 < 0.1 wt 83.8 wt 218 176 114 75.2 34.1 V 63.6 89.9 67.1 150 < 0.1 < 0.1 88.9 239 114 68.0 18.0 W 2.8 < 0.1 < 0.1 2.9 4.9 < 0.1 66.8 171 < 0.1 21.6 128 Y 0.7 < 0.1 150 117 < 0.1 1.1 143 11.3 26.1 < 0.1 wt

One variant, M DQF (Figure 11B, highest in the triple variant category), showed a significant increase in FcγRIIIa binding by 140%. Thus, the combinatorial saturation library offers options with a wide range of Fc receptor functionality that can be used to tailor therapeutic antibodies with specific effector functions.

Figure 11C shows a boxplot of FcγRIIIa binding responses for the top seven variants versus WT and YTE variants.

Example 9 Leading Seven Combinations Balance pH-Dependent Interaction with FcRn

Without being bound by any theory, candidate variants for further in vivo study occupied the bottom left of the graphs shown in Figures 7C and 7D. Seven variants met these criteria for hFcRn and included five double and two triple combinations (M DQ N, M DW N, YD TN, YE TN, Y T W N, YDQ N, and YEQ N) and did not contain a mutation at position N434 (table 3). Each of these combinations was eluted from the FcRn affinity column between AAA (pH 7.94 ± 0.06) and LS (pH 8.29 ± 0.03), with YDQ N eluting at the highest pH of 8.51 ± 0. 14 (Figure 12A, Table 5), indicating only slight pH derangement and more residual binding at pH 7.4 (Table 2A). One of the variants (M DQ N) had a thermal stability similar to the wild type, and six had a similar or reduced T _pl. compared to the YTE variant (Figure 12B, table 4). In the FcγRIIIa binding assay, the five combined variants showed the same reduction as YTE (Table 4). Further study with individual mutations showed that M252Y significantly affects FcγRIIIa binding and, without being bound by any theory, translates this effect in combination with this mutation. The remaining six single mutations were WT-like or had slightly improved binding to this receptor.

Three combination variants were selected for further studies based on their FcRn binding properties, thermal stability, and FcγRIIIa binding. DQ (T256D/T307Q), DW (T256D/T307W) and YD (M252Y/T256D) provided optimal FcRn binding properties (Table 2B) as the LS variant (Figure 12E). Each variant has a varied range of thermal stability and FcγRIIIa binding properties (Figures 12F and 12G, Table 2B), providing a range of functionality. Figure 12H is a diagram of a homogeneous RF bridge.

The increase in observed binding capacity for both human and rat FcRn at pH 6.0 compared to LS variants (Figure 13A, thick long dashes) and YTE variants (Figure 13B, thick long dashes), respectively, was a compromise between binding and dissociation rates. (Figures 13A and 13B, table 4). As a rule, combinations with faster dissociation rates also have faster binding rates and vice versa. This was observed for both human and rat FcRn (Table 4). In addition, all of these variants had a weaker steady state response than the LS variant (Figure 13C, thick long dashes) to hFcRn at pH 7.4. These results were not consistent with rFcRn as the five variants containing M252Y, YD TN, YE TN, Y T W N, YDQ N and YEQ N had increased binding to FcRn at pH 7.4 (Figure 13, Table 5) compared to YTE. The M DQ N and M DW N variants were the only combinations that were cross-reactive with human and rat FcRn. In addition, these two variants did not interfere with FcγRIIIa interaction to the same extent as M252Y-containing variants (Figures 12C and 12D and Table 5; M DQ N: 600 ± 4 nM; MDWN: 512 ± 30 nM; WT: 467 ± (99 nM) Thus, saturation and combination mutagenesis at key positions of the interaction with FcRn led to the identification of leading variants that balanced the pH-dependence of the interaction, retained functionality with the Fc receptor, could enhance the functionality of FcRn in vivo , and could increase the half-life from blood serum of therapeutic antibodies.

Example 10: Rheumatoid Factor Binding Characteristics of Leading Combination Variants

The isoelectric point and RF binding of the leading variants have been investigated as these mutations can alter antibody surface charge and immunogenicity. It is believed that more acidic antibodies prolong the pharmacokinetics of antibodies. Compared to control WT and LS, all three leaders resulted in a decrease in pH by ~0.2 units in pI as a result of T256D substitution. Mutations that enhance FcRn can simultaneously alter binding to host antibodies such as rheumatoid factor (RF) due to overlapping contact areas. Homogeneous bridging ELISA was adapted to measure the change in RF binding in the leading variants. Interestingly, LS and YTE showed completely opposite shifts in RF binding compared to WT (Figure 12H). LS significantly increased RF binding, while YTE showed a significant decrease (p < 0.001). YD (p<0.001) and DW (p<0.01) also significantly reduced RF binding, while DQ elicited a similar response to WT. Without being bound by any theory, these results indicate that DQ, DW, and YD may provide an immunogenic advantage over LS. The YD, DW and DQ variants have a number of key antibody characteristics that can be used in combination with improved FcRn binding properties compared to the YTE and LS comparison variants.

Example 11 Leading combination variants can be transferred to other antibodies

A new binding assay was designed using the CM5 sensor chip as shown in Figure 15A. The binding assay includes the step of immobilizing streptavidin on a CM5 sensor chip to capture biotinylated FcRn up to about 30 RU, which can be replenished as needed. Antibody binding kinetics were measured at pH 6.0 and 7.4 and pH 8.5 for regeneration. Figures 15B and 15C show direct FcRn immobilization and streptavidin capture of biotinylated FcRn, respectively, using the new binding assay.

Antibody-2 FcRn Binding at pH 6.0: In the case of mouse FcRn, the leading antibody-2 variants show slower dissociation rates than the LS variant (dashed) and wild-type (black) (Figure 16A). In the case of human FcRn, all leading variants have higher binding rates but the same dissociation rates as LS (dashes) (Figure 16B).

Antibody-2 FcRn Binding at pH 7.4: All leading variants showed reduced binding to human FcRn at pH 7.4 compared to LS (dashes) (Figure 17A). As with antibody-1, variants DW (M DW N) and DQ (M DQ N) also showed lower residual binding to mouse (rat) FcRn at pH 7.4 (Figure 17B).

The leading variants maintained higher binding capacity at pH 6.0 and lower residual binding at pH 7.4 compared to LS (Figure 18). An important observation was made that variants can be transferred between different IgG1 baseline without any significant effect on FcRn binding. As shown in Figure 19, LS had the same elution pH regardless of base. Antibody-2 based WT, DQ and DW showed a higher elution pH than antibody-1 based, possibly as a result of tighter binding at pH 6.0 to antibody-2 base.

All antibody-2-based variants showed slightly increased thermal stability, as shown in Figure 20.

As shown in Figure 21, similar to the antibody-1 base, YD ( YD TN) showed a decrease in FcγRIIIa binding response (left) and affinity (right). DQ (light gray) and DW (dark gray) showed FcγRIIIa binding properties similar to WT (black) based on antibody-2. The effect on FcγRIIIa binding for LS is consistent between antibody-1 and antibody-2.

Thus, FcRn binding, pH dependence, thermal stability, or FcγRIIIa binding of antibody-2-based leading variants do not differ significantly from those of the same antibody-1-based leading variants.

In one embodiment, DQ (T256D/T307Q), DW (T256D/T307W) and YD (M252Y/T256D) variants were included in an additional IgG1 antibody and a recombinant Fc fragment: mAb2 recognizes an antigen other than mAb1 and Ab3 is an Fc fragment . In each case, the pH dependent FcRn binding kinetics (Figure 22) were very similar along with elution pH, thermal stability and FcγRIIIa binding capacity (Table 2B and Table 6). Without wishing to be bound by theory, these results indicate that the DQ, DW, and YD variants confer their improved FcRn binding properties on Fc domain-consisting proteins.

Table 6. Concentrations of saturation library variants in conditioned media

Affinity column with FcRn DSF Biacore Biacore pH 6.0 Biacore pH 7.4 FcγRIIIa
V158 hFcRn cFcRn mFcRn hFcRn cFcRn mFcRn Ab Option pH value T _pl. The fold change in affinity *K _{D, obs.} *K _{D, obs.} *K _{D, obs.} Steady state RU Steady state RU Steady state RU 2 wt 7.61 69.3±0.1 1.0 678±
97 1440±
360 107 ± 9 1.3±0.2 1.3±0.3 92±8 2 LS 8.32 69.0±0.1 1.23±0.02 113 ± 22 210 ± 43 20 ± 9 44 ± 4 38±6 285±8 2 DQ 8.06 69.3±0.1 1.05±0.01 97 ± 33 110±42 24 ± 11 14±1 11±1 248±5 2 DW 8.11 58.1±0.1 1.15±0.01 69 ± 25 99 ± 9 13±5 18±2 15±1 257±8 2 YD 8.25 60.5±0.1 0.58±0.01 99 ± 49 120±6 10±3 27 ± 2 22±3 394 ± 11 3 wt 7.62 67.5±0.2 1.0 717 ± 23 61±1 2.2±0.3 72±2 3 LS 8.32 66.6±0.2 1.11±0.03 51±2 16±2 32±2 103±1 3 DQ 8.07 63.8±0.2 0.87±0.02 51±1 24±1 19±2 89±1 3 DW 8.12 57.1 ± 0.2 0.93±0.02 39±6 23±1 22±2 99±1 3 YD 8.23 59.5±0.1 0.73±0.02 54±1 0.5±0.2 28±3 125±2

Example 12 Leading Variants Extend the Half-Life of Antibodies in Plasma in Vivo

The pharmacokinetics (PK) of the DQ, DW, and YD variants were studied in terms of the effect on the half-life of antibodies in cynomolgus monkeys and hFcRn (Tg32 strain) transgenic mice (see, e.g., Avery et al. Mabs (2016) 8: 1064-1078) by compared with control WT and LS. FcRn binding studies on Cynomolgus FcRn revealed binding abilities similar to hFcRn (Figures 23A-23B; Table 6). WT, LS, DQ, DW, or YD variants were intravenously injected with WT, LS, DQ, DW, or YD variants and antibody concentration was quantified using a mass spectrometry approach to determine clearance rate and serum half-life in monkeys (Figure 24A) and hFcRn transgenic mice (Figure 24B) . The clearance rates and serum half-life were derived from a non-compartmental model of antibody concentration as a function of time. All three leading variants and LS showed a significantly reduced clearance rate compared to WT in both monkeys and mice (p<0.001). The plasma half-life of the WT antibody in monkeys and mice was 9.9 ± 0.5 and 11.7 days, respectively. In addition, comparative LS and identified variants showed a significant increase in half-life compared to wild type in both species (2.5 and 1.7-fold increase, respectively, in monkeys and mice) (Table 7). DQ, DW and YD showed a similar prolongation of the half-life compared to comparative LS (table 7). The DQ, DW, and YD mutations identified here by saturation mutagenesis demonstrated significantly longer plasma half-lives than their WT counterparts in non-human primate and mouse animal models.

Table 7. Clearance rate and serum half-life of comparative and leading variants

Monkey Cynomolgus (n=3) Mouse hFcRn Tg32 (n=6) mAb2 Clearance
(ml/
day/
kg) t _1/2 (days) Clearance
(ml/
day/
kg) t _1/2 (days) Option Mean ± SD Mean ± SD Multiplicity in relation to WT Multiplicity with respect to LS Average Average Multiplicity in relation to WT Multiplicity with respect to LS wt 5.6±0.5 9.9±0.5 1.0 0.4 7.4 11.7 1.0 0.6 LS 2.1** 22.5 ± 2.4 2.3 1.0 4.6 19.5 1.7 1.0 DQ 3.4* 20.8 2.1 0.9 3.2 24.5 2.1 1.3 DW 2.5±0.2 20.4±0.9 2.1 0.9 3.5 20.1 1.7 1.0 YD 2.4±0.3 23.5 ± 2.1 2.4 1.0 4.5 17.5 1.5 0.9

In Table 7, the clearance rate and plasma half-life were determined using mAb2. Each clearance rate and half-life was the mean n=3 for the cynomolgus monkey and a single score from a pool of n=6 hFcRn transgenic mice. The fold versus WT and the fold versus LS indicate the relative improvement in serum half-life compared to WT and LS, respectively. *n=2 due to antibody-drug interaction, ** n=2 due to partial subcutaneous route of administration

Example 13 Combined Variants with Increased FcRn Binding at pH 6.0 and pH 7.4

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

Table 8. Binding capacity (pH 6.0) and binding at steady state (pH 7.4) of variants

Option Type Binding capacity (M ^-1 ) Binding ability error (M ^-1 ) Binding at steady state, pH 7.4 (RU) Binding error at steady state, pH 7.4 (RU) Ratio (pH 6.0/pH 7.4) Ratio (pH 6.0/pH 7.4) WT (MTTN) Comparative 420000 83000 4.2 0.9 100000 1.00E-05 AAA Comparative 562000 120000 13.9 3.1 40432 2.47E-05 LS Comparative 3680000 541000 18.3 4.6 201093 4.97E-06 YTE Comparative 2920000 1000000 13.2 3.5 221212 4.52E-06 MDQN Double 4310000 446000 10.7 1 402804 2.48E-06 MDTF Double 33400000 2240000 29.2 4 1143836 8.74E-07 MDTY Double 64100000 2050000 37.1 5 1727763 5.79E-07 MDWN Double 5920000 280000 12.2 1.3 485246 2.06E-06 MEQN Double 2000000 24100 5.8 0.6 344828 2.90E-06 METF Double 12800000 3350000 23.3 3.2 549356 1.82E-06 METY Double 16000000 4130000 26.8 3.7 597015 1.68E-06 MEWN Double 3100000 259000 7.7 0.9 402597 2.48E-06 MTQF Double 23300000 4290000 34.2 4.6 681287 1.47E-06 MTQY Double 30900000 6950000 38.4 5.2 804688 1.24E-06 MTWF Double 26200000 1230000 30.5 4.2 859016 1.16E-06 MTWY Double 46500000 2600000 37.2 4.9 1250000 8.00E-07 YDTN Double 10700000 2100000 9.7 1.8 1103093 9.07E-07 YETN Double 7810000 305000 9.8 1.2 796939 1.25E-06 YTQN Double 3600000 298000 10.6 1.2 339623 2.94E-06 YTTF Double 55600000 3090000 43.8 5.8 1269406 7.88E-07 YTTY Double 120000000 2890000 54.1 7.1 2218115 4.51E-07 YTWN Double 8470000 359000 15.9 1.7 532704 1.88E-06 MDQF Triple 6620000 1840000 55.3 11.2 119711 8.35E-06 MDQY Triple 36900000 4360000 49.4 6.7 746964 1.34E-06 MDWF Triple 28100000 2840000 47.1 6.4 596603 1.68E-06 MDWY Triple 84000000 9890000 59 7.9 1423729 7.02E-07 MEQF Triple 142000 6490 8.6 0.8 16512 6.06E-05 MEQY Triple 23800000 2660000 38.6 5.2 616580 1.62E-06 MEWF Triple 56200000 8520000 41.9 5.6 1341289 7.46E-07 MEWY Triple 70400000 7440000 46.6 6.3 1510730 6.62E-07 YDQN Triple 8700000 560000 19.8 2.7 439394 2.28E-06 YDTF Triple 29600000 2540000 57.7 7.7 512998 1.95E-06 YDTY Triple 90100000 812000 65.4 8.9 1377676 7.26E-07 YDWN Triple 10100000 1540000 25.9 3.6 389961 2.56E-06 YEQN Triple 4590000 126000 15.2 2.1 301974 3.31E-06 YETY Triple 33400000 3580000 22.6 2.9 1477876 6.77E-07 YEWN Triple 6410000 904000 69.6 9.3 92098 1.09E-05 YTQF Triple 56500000 1280000 59.9 8 943239 1.06E-06 YTQY Triple 63300000 4010000 71.5 9.6 885315 1.13E-06 YTWF Triple 65400000 2990000 62.6 8.2 1044728 9.57E-07 YTWY Triple 106000000 10600000 75.1 9.8 1411451 7.08E-07 YDQF quadruple 111000000 4320000 68.6 10 1618076 6.18E-07 YDQY quadruple 235000000 6060000 80.2 12 2930175 3.41E-07 YDWF quadruple 166000000 3050000 71.8 9.9 2311978 4.33E-07 YDWY quadruple 266000000 19100000 88.5 11.7 3005650 3.33E-07

Table 8 shows FcRn binding capacity at pH 6.0 and steady state FcRn binding at pH 7.4 for various single, double, triple and quadruple mutants and comparative variants (AAA, LS, YTE).

These values are shown in Figure 25. Figure 25 shows a comparison of binding capacity at pH 6.0 and RU at pH 7.4. As can be seen, the LS comparison variant has the highest binding capacity at pH 6.0 and the highest residual binding at pH 7.4 of the comparatives tested (AAA, LS, YTE).

Several combination variants shown in Figure 25 were found to exhibit increased FcRn binding capacity at pH 6.0 and at pH 7.4. To investigate if any of the combined variants showed tighter binding than the MST-HN variant (referred to here as the "YTEKF comparative variant") containing mutations in Met252, Ser254, Thr256, His433 and Asn434 in Tyr252, Thr254, Glu256, Lys433 and Phe434) at pH 6.0 and pH 7.4, a study was performed according to the following methodology. Capture of biotinylated human, Cynomolgus and mouse FcRn was performed using the Biotin CAPture method (see diagram in Figure 26). In the case of pH 6.0, a series of dilutions (5 units) from 1000 nM was performed in duplicate. In the case of pH 7.4, a single concentration injection (1000 nM) was performed in triplicate (the capture level of each FcRn was increased 10-fold to observe binding at this pH). Association: 180 sec; Dissociation: 300 sec.

The binding kinetics to human FcRn at pH 6.0 of the YTEKF comparative variant and various combination variants are shown in Figure 27. As can be seen from Figure 27, all tested variants showed two orders of magnitude closer affinity for human FcRn compared to wild type (WT).

Figures 28A and 28B show the FcRn binding kinetics of the combined variants compared to the comparative YTEKF variant at pH 6.0 (Figure 28A) and at pH 7.4 (Figure 28B). From Figure 28A it can be seen that most variants had slower dissociation rates than the comparative YTEKF variant and had similar or slower binding rates. Figure 28B shows significant binding to YTEKF at pH 7.4, and four variants show higher residual binding.

Table 9. Binding capacity (pH 6.0) and binding at steady state (pH 7.4) of selected variants

Human FcRn pH 6.0 pH 7.4 Option KD (nM) SD EN SD YDQY 2.7 0.1 103.1 0.6 YEWY 8.3 0.5 98.2 0.9 YEQY 4.8 0.2 93.6 0.8 YDQF 4.7 0.1 91.7 0.6 YDWY 5.2 0.2 91.2 0.5 YTEKF 14.8 0.3 89.9 1.3 YWY 10.0 0.4 77.1 0.3 YDWF 11.0 0.4 75.7 0.5 YDY 15.1 0.8 69.7 0.4 DWY 18.1 1.1 59.8 0.8 YY 22.9 0.9 54.4 0.1 wt 1288 201 0.4 0.1

Table 9 shows the FcRn binding capacity at pH 6.0 and steady state FcRn binding at pH 7.4 for selected combination variants and a comparison of YTEKF and WT.

Figure 29 shows a comparison of the binding capacity at pH 6.0 and RU at pH 7.4 for the selected combination variants as indicated in Table 9. As shown in Table 9 and Figure 29, the four quad variants were found to have higher affinity at pH 6.0 and pH 7.4 for FcRn compared to a comparative YTEKF variant. Mutations T256D, T307Q and N434Y in four quadruple variants had a beneficial effect. These quad variants show approximately 500-fold and 3-fold improvement in affinity (at pH 6.0) compared to WT and YTEKF, respectively.

Other characterization parameters such as thermal stability, FcγRIIIa binding and elution pH were determined and are listed in Table 10.

Table 10. Other characteristic parameters of the quadruple leading options

Option Affinity, pH 6.0 (nM) Steady state RU, pH 7.4 T _pl (°С) Binding to FcγRIIIa (RU) elution pH YDQY 2.7 103.1 59.0 119 9.2 YEWY 8.3 98.2 52.7 92 9.3 YEQY 4.8 93.6 59.5 108 9.1 YDQF 4.7 91.7 59.1 93.2 8.8 YDWY 5.2 91.2 52.5 104 9.5 wt ~1500 <1 69.0 142 7.37 YTEKF 14.8 89.9 N.P.* N.P.* N.P.

* Without intending to be bound by theory, due to the presence of "YTE" in YTEKF, thermal stability and binding to FcγRIIIa is expected to be similar to the quad leader variants

As shown in Table 10, all four leading variants were found to be thermally destabilized and exhibit reduced FcγRIIIa binding abilities.

Claims (84)

1. An isolated binding polypeptide containing an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, wherein modifications to the human IgG Fc domain include: aspartic acid (D) or glutamic acid (E) at amino acid position 256 and/or tryptophan (W) or glutamine (Q) at amino acid position 307, where amino acid position 254 is not threonine (T), and additionally: phenylalanine (F) or tyrosine (Y) at amino acid position 434; or tyrosine (Y) at amino acid position 252, where the positions of the amino acids correspond to the EC numbering. 2. An isolated binding polypeptide containing an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, wherein the modifications to the human IgG Fc domain (I) include a combination of amino acid residues selected from the group consisting of: 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) tyrosine (Y) at amino acid position 252, 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; And h) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256 and glutamine (Q) at amino acid position 307, where threonine (T) is not at amino acid position 254, histidine (H) is not at position amino acid 311 and tyrosine (Y) is not at amino acid position 434; or (II) consists of 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; where the amino acid substitutions correspond to the EC numbering. 3. An isolated binding polypeptide containing a modified human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, where the modifications to the human IgG Fc domain are: a) include a double amino acid substitution selected from the group consisting of M252Y/T256D, M252Y/T256E, M252Y/T307Q, M252Y/T307W, T256D/T307W, T256E/T307Q and T256E/T307W, where the threonine (T) is not in position amino acid 254, histidine (H) is not at amino acid position 311, and tyrosine (Y) is not at amino acid position 434; b) consist of a double amino acid substitution T256D/T307Q; or c) include a triple amino acid substitution selected from the group consisting of M252Y/T256D/T307Q, M252Y/T256D/T307W, M252Y/T256E/T307Q and M252Y/T256E/T307W, where 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; where the amino acid substitutions correspond to the EC numbering. 4. An isolated binding polypeptide containing an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, wherein the human IgG Fc domain modifications consist of aspartic acid (D) at the amino acid position 256 and glutamine (Q) at amino acid position 307, according to the EC numbering. 5. An isolated binding polypeptide containing an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, wherein the modifications to the human IgG Fc domain include aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307, according to the EC numbering. 6. An isolated binding polypeptide comprising an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide comprising a wild-type Fc domain, wherein the modifications to the human IgG Fc domain include tyrosine (Y) at amino acid position 252 and aspartic acid (D) at amino acid position 256, according to the EC numbering. 7. Selected binding polypeptide according to any one of paragraphs. 1-6, wherein the isolated binding polypeptide has a longer serum half-life compared to the binding polypeptide containing the wild-type Fc domain. 8. Selected binding polypeptide according to any one of paragraphs. 1-7, wherein the isolated binding polypeptide has an increased ability to bind to FcRn at an acidic pH compared to a binding polypeptide containing a wild-type Fc domain, optionally having an acidic pH of about 6.0. 9. Selected binding polypeptide according to any one of paragraphs. 1-8, wherein the isolated binding polypeptide has a higher FcRn binding capacity at an acidic pH compared to the binding polypeptide's FcRn binding capacity at a non-acidic pH, where the optionally acidic pH is about 6.0, and where the optional non-acidic pH is is approximately 7.4. 10. Selected binding polypeptide according to any one of paragraphs. 1-9, wherein the isolated binding polypeptide has an altered ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain, where optionally FcγRIIIa is human FcγRIIIa. 11. An isolated binding polypeptide containing an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, wherein modifications to the human IgG Fc domain include: tyrosine (Y) at amino acid position 252, aspartic acid (D) or glutamic acid (E) at amino acid position 256, tryptophan (W) or glutamine (Q) at amino acid position 307, and phenylalanine (F) or tyrosine (Y) at amino acid position 434, where the positions of the amino acids correspond to the EC numbering. 12. An isolated binding polypeptide comprising an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide comprising a wild-type Fc domain, wherein the modifications to the human IgG Fc domain comprise a combination of amino acid residues selected from the group consisting of from: 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; 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; And f) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307 and phenylalanine (F) at amino acid position 434, where the amino acid substitutions correspond to the EC numbering. 13. An isolated binding polypeptide comprising an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide comprising a wild-type Fc domain, wherein the modifications to the human IgG Fc domain comprise a quaternary amino acid substitution selected from the group consisting of from M252Y/T256D/T307Q/N434Y, M252Y/T256E/T307W/N434Y, M252Y/T256E/T307Q/N434Y, M252Y/T256D/T307Q/N434F, M252Y/T256D/T307W/N434Y and M252Y/T256D/T307W/N434F, where the amino acid substitutions correspond to the EC numbering. 14. An isolated binding polypeptide containing an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, wherein the modifications to the human IgG Fc domain include 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 EC numbering. 15. An isolated binding polypeptide containing an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, wherein the modifications to the human IgG Fc domain include 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 EC numbering. 16. An isolated binding polypeptide containing an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, wherein the modifications to the human IgG Fc domain include 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 EC numbering. 17. An isolated binding polypeptide containing an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, wherein the modifications to the human IgG Fc domain include 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 EC numbering. 18. An isolated binding polypeptide containing an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, wherein the modifications to the human IgG Fc domain include 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 the EC numbering. 19. An isolated binding polypeptide containing an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide containing a wild-type Fc domain, wherein modifications to the human IgG Fc domain include: a) tyrosine (Y) at amino acid position 252 and tyrosine (Y) at amino acid position 434, b) tyrosine (Y) at amino acid position 252, tryptophan (W) at amino acid position 307 and tyrosine (Y) at amino acid position 434, c) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256 and tyrosine (Y) at amino acid position 434, or d) aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307 and tyrosine (Y) at amino acid position 434, where the positions of the amino acids correspond to the EC numbering. 20. An isolated binding polypeptide comprising an altered human IgG Fc domain having an increased ability to bind to FcRn compared to a binding polypeptide comprising a wild-type Fc domain, wherein the modifications to the human IgG Fc domain comprise a combination of amino acid substitutions selected from the group consisting of from M252Y/N434Y, M252Y/T307W/N434Y, M252Y/T256D/N434Y and T256D/307W/N434Y, where the amino acid substitutions correspond to the EC numbering. 21. Selected binding polypeptide according to any one of paragraphs. 11-20, wherein the altered human IgG Fc domain has increased FcRn binding capacity at acidic pH and increased FcRn binding capacity at non-acidic pH compared to wild-type Fc domain, where optionally acidic pH is about 6.0 , and where optionally the non-acidic pH is about 7.4, where further optionally, the FcRn is a human FcRn. 22. Selected binding polypeptide according to any one of paragraphs. 11-21, where the altered human IgG Fc domain has increased FcRn binding capacity at acidic pH and increased FcRn binding capacity at non-acidic pH compared to an Fc domain containing five amino acid substitutions M252Y/S254T/T256E/H433K/ N434F, where the optionally acidic pH is about 6.0, and where the optional non-acidic pH is about 7.4, where the optional FcRn is a human FcRn. 23. Selected binding polypeptide according to any one of paragraphs. 11-22, wherein the altered human IgG Fc domain has a reduced ability to bind to FcγRIIIa compared to a binding polypeptide containing a wild-type Fc domain, where the FcγRIIIa is further optionally human FcγRIIIa. 24. Selected binding polypeptide according to any one of paragraphs. 11-23, where the altered human IgG Fc domain has a reduced ability to bind to FcγRIIIa compared to an Fc domain containing five amino acid substitutions M252Y/S254T/T256E/H433K/N434F, where additionally optionally FcγRIIIa is human FcγRIIIa. 25. Selected binding polypeptide according to any one of paragraphs. 1-24, wherein the altered human IgG Fc domain is an altered human IgG1 Fc domain. 26. Selected binding polypeptide according to any one of paragraphs. 1-25, where the binding polypeptide has the ability to bind to human FcRn, and optionally, the ability to bind to rat FcRn. 27. Selected binding polypeptide according to any one of paragraphs. 1-26, wherein the isolated binding polypeptide specifically binds one or more human targets. 28. Selected binding polypeptide according to any one of paragraphs. 1-27, wherein the isolated binding polypeptide is a monoclonal antibody. 29. The isolated binding polypeptide of claim 28, wherein the antibody is a chimeric, humanized, or human antibody. 30. An isolated nucleic acid molecule for encoding an isolated binding polypeptide according to any one of paragraphs. 1-29, containing a nucleotide sequence encoding an isolated binding polypeptide according to any one of paragraphs. 1-29. 31. An expression vector containing the isolated nucleic acid molecule according to claim 30. 32. A host cell to obtain an isolated binding polypeptide according to any one of paragraphs. 1-29, containing the expression vector according to item 31. 33. The host cell of claim 32, wherein the host cell is a mammalian cell. 34. A method for producing an isolated binding polypeptide, comprising culturing the host cell according to claim 33 under conditions that allow expression of the binding polypeptide, and isolation of the binding polypeptide from the culture.

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