Fc domain variants with enhanced fe receptor binding

Abstract

The present disclosure provides Fc domain variant polypeptides comprising Fc domain variants with effector capacity. The present disclosure also provides Fc domain variant polypeptides comprising at least one substitution or at least two substitutions at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373, and 376, according to EU numbering, as compared to a Fc domain parent polypeptide. The present disclosure also provides nucleic acids encoding Fc domain variant polypeptides and host cells for making Fc domain variant polypeptides. Methods for increasing the yield of Fc domain variant polypeptides are also provided, as are methods of using Fc domain variant polypeptides to treat disease.

Description

priority

[0001] This application claims the benefit of U.S. Serial No. 63 / 638,212, filed April 24, 2024, and U.S. Serial No. 63 / 593,011, filed October 25, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0002] The specific binding between the crystallizable (Fc) region of an antibody and the Fcγ receptor (FcγR) is the initial step in effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC). In humans, activated FcγRIIIA is expressed on the surface of natural killer cells. FcγRIIIA is a low-affinity receptor, and cell activation is caused by cross-linking of these surface receptors after binding to the Fc region of the antibody-antigen immune complex. The Fc region also interacts with the neonatal Fc receptor (FcRn). This interaction has been shown to prolong the half-life of IgG by reducing lysosomal degradation in endothelial cells.

[0003] Attempts have been made to enhance ADCC efficacy through Fc engineering, thereby identifying Fc domain variants that enhance affinity for Fc receptors and thus improve ADCC activity and / or serum half-life. Despite these efforts, there remains a need to create novel Fc variant domain peptides with enhanced efficacy and improved manufacturability for the treatment of various diseases. Summary of the Invention

[0004] Compared to the parental Fc domain peptide, the Fc domain variant peptides disclosed herein, according to EU designation, contain at least one or at least two substitutions at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373, and 376. Compared to the parental Fc domain peptide, the Fc domain variant peptides exhibit increased binding to the Fc receptor and possess higher protein expression and purity profiles.

[0005] In one aspect, the Fc domain variant polypeptide, according to EU designation, contains at least two substitutions at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373, and 376, compared to the parent Fc domain polypeptide. In another aspect, the Fc domain variant polypeptide exhibits increased binding to the Fc receptor relative to the parent Fc domain polypeptide. In the other case, at least two amino acid substitutions are selected from: (i) alanine (A), histidine (H), isoleucine (I), phenylalanine (F), glutamine (Q), or tryptophan (W) at amino acid position 251; (ii) alanine (A) or aspartic acid (D) at amino acid position 267; (iii) aspartic acid (D) or glutamic acid (E) at amino acid position 268; (iv) alanine (A) at amino acid position 298; (v) glycine (G), lysine (K), asparagine (N), methionine (M), serine (S), threonine (T), valine (V), glutamic acid (E), or tryptophan (W) at amino acid position 314; (vi) phenylalanine (F), methionine (M), or tyrosine (Y) at amino acid position 330; (vii) threonine (T) at amino acid position 339; (viii) tryptophan (W) at amino acid position 373; or (ix) The amino acid position 376 contains valine (V), and amino acid positions are numbered according to EU. In one aspect, the Fc domain parent polypeptide is the wild-type Fc domain.

[0006] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, tryptophan (W) at amino acid position 373, and alanine (A) at amino acid position 298.

[0007] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and alanine (A) at amino acid position 298.

[0008] In one aspect, according to EU designation, the Fc domain variants include aspartic acid (D) at amino acid position 268, isoleucine (I) at amino acid position 251, phenylalanine (F) at amino acid position 330, and alanine (A) at amino acid position 298.

[0009] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, threonine (T) at amino acid position 339, and alanine (A) at amino acid position 298.

[0010] In one aspect, according to EU designations, the Fc domain variants include glutamic acid at amino acid position 268 (E), valine at amino acid position 376 (V), alanine at amino acid position 251 (A), and methionine at amino acid position 330 (M).

[0011] In one aspect, according to EU designation, the Fc domain variants include aspartic acid (D) at amino acid position 268, aspartic acid (D) at amino acid position 267, isoleucine (I) at amino acid position 251, and threonine (T) at amino acid position 339.

[0012] In one aspect, according to EU designations, the Fc domain variants contain aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, threonine (T) at amino acid position 339, and tryptophan (W) at amino acid position 373.

[0013] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and tryptophan (W) at amino acid position 373.

[0014] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and threonine (T) at amino acid position 314.

[0015] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and valine (V) at amino acid position 314.

[0016] In one aspect, according to EU designations, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and valine (V) at amino acid position 376.

[0017] In one aspect, according to EU designation, the Fc domain variant polypeptide contains alanine (A) at amino acid position 298, tyrosine (Y) at amino acid position 330, threonine (T) at amino acid position 339, and tryptophan (W) at amino acid position 373.

[0018] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and alanine (A) at amino acid position 251.

[0019] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 267, tyrosine (Y) at amino acid position 330, and threonine (T) at amino acid position 339.

[0020] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 267, tyrosine (Y) at amino acid position 330, and tryptophan (W) at amino acid position 373.

[0021] In one aspect, according to EU designation, the Fc domain variant contains glutamic acid (E) at amino acid position 268, histidine (H) at amino acid position 251, and phenylalanine (F) at amino acid position 330.

[0022] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 373, and alanine (A) at amino acid position 298.

[0023] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and alanine (A) at amino acid position 298.

[0024] In one aspect, according to EU designations, the Fc domain variants include aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 330, and alanine (A) at amino acid position 298.

[0025] In one aspect, according to EU designations, the Fc domain variants include glutamic acid at amino acid position 268 (E), valine at amino acid position 314 (V), and methionine at amino acid position 330 (M).

[0026] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, aspartic acid (D) at amino acid position 267, and threonine (T) at amino acid position 339.

[0027] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and tryptophan (W) at amino acid position 373.

[0028] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and valine (V) at amino acid position 314.

[0029] In one aspect, according to EU designation, the Fc domain variant contains glutamic acid (E) at amino acid position 268 and phenylalanine (F) at amino acid position 330.

[0030] In one aspect, according to EU designations, the Fc domain variants include aspartic acid at amino acid position 268 (D), glutamine at amino acid position 251 (Q), and glutamic acid at amino acid position 314 (E).

[0031] In one aspect, according to EU designations, the Fc domain variants include aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

[0032] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, and serine (S) at amino acid position 314.

[0033] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, and asparagine (N) at amino acid position 314.

[0034] In one aspect, according to EU designations, the Fc domain variants include aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

[0035] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 314, tryptophan (W) at amino acid position 373, and methionine (M) at amino acid position 330.

[0036] In one aspect, according to EU designation, the Fc domain variant contains glutamic acid at amino acid position 268 (E), isoleucine at amino acid position 251 (I), threonine at amino acid position 314 (T), and valine at amino acid position 376 (V).

[0037] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and glycine (G) at amino acid position 314.

[0038] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, glycine (G) at amino acid position 314, and alanine (A) at amino acid position 298.

[0039] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and lysine (K) at amino acid position 314.

[0040] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, threonine (T) at amino acid position 314, and alanine (A) at amino acid position 298.

[0041] In one aspect, according to EU designations, the Fc domain variants include glutamic acid at amino acid position 268 (E), valine at amino acid position 314 (V), alanine at amino acid position 251 (A), and methionine at amino acid position 330 (M).

[0042] In one aspect, according to EU designation, the Fc domain variant contains alanine (A) at amino acid position 298, tyrosine (Y) at amino acid position 330, and threonine (T) at amino acid position 339.

[0043] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and lysine (K) at amino acid position 314.

[0044] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and glycine (G) at amino acid position 314.

[0045] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, tryptophan (W) at amino acid position 251, and valine (V) at amino acid position 376.

[0046] In one aspect, according to EU designations, the Fc domain variants include tyrosine (Y) at amino acid position 330, alanine (A) at amino acid position 267, valine (V) at amino acid position 376, and alanine (A) at amino acid position 298.

[0047] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and threonine (T) at amino acid position 314.

[0048] In one aspect, according to EU designations, the Fc domain variants include aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

[0049] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and tryptophan (W) at amino acid position 373.

[0050] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and glutamic acid (E) at amino acid position 314.

[0051] In one aspect, according to EU designations, the Fc domain variants include aspartic acid (D) at amino acid position 268, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

[0052] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 373, and alanine (A) at amino acid position 298.

[0053] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and serine (S) at amino acid position 314.

[0054] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and asparagine (N) at amino acid position 314.

[0055] In one aspect, according to EU designations, the Fc domain variants include glutamic acid (E) at amino acid position 268, threonine (T) at amino acid position 314, and valine (V) at amino acid position 376.

[0056] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and glycine (G) at amino acid position 314.

[0057] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, threonine (T) at amino acid position 314, and alanine (A) at amino acid position 298.

[0058] In one aspect, according to EU designations, the Fc domain variants include glutamic acid at amino acid position 268 (E), valine at amino acid position 314 (V), and methionine at amino acid position 330 (M).

[0059] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and lysine (K) at amino acid position 314.

[0060] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and valine (V) at amino acid position 376.

[0061] In one aspect, according to EU designations, the Fc domain variants include aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

[0062] In one aspect, according to EU designations, the Fc domain variants include aspartic acid (D) at amino acid position 268, aspartic acid (D) at amino acid position 267, and threonine (T) at amino acid position 339.

[0063] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and threonine (T) at position 314.

[0064] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and glutamic acid (E) at position 314.

[0065] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and glycine (G) at position 314.

[0066] In one aspect, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268 and serine (S) at position 314.

[0067] In one aspect, according to EU designations, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and valine (V) at position 376.

[0068] In one aspect, according to EU designations, the Fc domain variants contain glutamic acid at amino acid position 268 (E), threonine at amino acid position 314 (T), and valine at position 376 (V).

[0069] In one aspect, according to EU designations, the Fc domain variants contain aspartic acid (E) at amino acid position 268, valine (V) at amino acid position 314, and methionine (M) at amino acid position 330.

[0070] In one aspect, the Fc domain variants disclosed herein include mammalian-derived Fc domain variants. In another aspect, the Fc domain variants are of human origin.

[0071] In another aspect, Fc domain variants are derived from immunoglobulin classes selected from the group consisting of: IgM, IgG, IgD, IgA, and IgE. In yet another aspect, Fc domain variants are derived from the IgG Fc domain. In yet another aspect, Fc domain variants are derived from either the IgG1 Fc domain or the IgG4 Fc domain.

[0072] In one aspect, the Fc domain variant of this disclosure further includes aspartic acid (D) at amino acid position 239.

[0073] In one aspect, the Fc domain variant of this disclosure further includes glutamic acid (E) at amino acid position 332.

[0074] In one aspect, the Fc domain variant of this disclosure further includes aspartic acid (D) at amino acid position 239 and glutamic acid (E) at amino acid position 332.

[0075] In one aspect, the Fc domain variants of this disclosure are about 20%, 30%, 40%, 50%, 60%, 70%, 90% or more unfucosylated.

[0076] In one aspect, the Fc domain variant of this disclosure further includes cysteine ​​(C) at amino acid position 292 and cysteine ​​(C) at amino acid position 302.

[0077] In one aspect, the Fc domain variant of this disclosure further comprises at least one N-glycan. In another aspect, the N-glycan comprises mannose and / or GlcNAc. In another aspect, the N-glycan is oligomannose-type. In another aspect, the oligomannose-type N-glycan comprises oligosaccharides selected from the group consisting of: Man9(GlcNAc)2, Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2, and Man5(GlcNAc)2. In another aspect, the Fc domain variant polypeptide comprises 20%, 30%, 40%, 50%, 60%, 70%, 90%, or more Man9(GlcNAc) in a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 glycan. In another aspect, the Fc domain variant peptide contains greater than 70%, 75%, 80%, 85%, 90%, or 95% Man, in a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 N-glycan. In another aspect, the Fc domain variant peptide contains Man8 and Man9 together as Man... 5-9 (GlcNAc)2 is the main type of N-glycan. On the other hand, Fc domain variant peptides contain at least 97% Mann based on a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 N-glycan. In another aspect, the Fc domain variant peptides contain less than 30%, 20%, 10%, 5%, and 1% Man, respectively, in molar ratios relative to all N-glycans. 5-9 (GlcNAc)2 polysaccharide, or essentially no man in molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 polysaccharide.

[0078] In one aspect, the Fc domain variant of this disclosure further includes cysteine ​​(C) at amino acid position 292 and cysteine ​​(C) at amino acid position 302.

[0079] In one aspect, the Fc domain variant of this disclosure is generated by culturing cells expressing the Fc domain variant peptide in the presence of a mannosidase inhibitor. In another aspect, the mannosidase inhibitor is kifunensine. In yet another aspect, the concentration of kifunensine is from about 60 ng / mL to about 2500 ng / mL. In yet another aspect, the concentration of kifunensine is about 2000 ng / mL.

[0080] In one aspect, the Fc domain variant of this disclosure includes at least one binding site for selectively binding an antigen of interest and an Fc domain variant polypeptide. In another aspect, the binding polypeptide is a multispecific binding polypeptide. In another aspect, the binding polypeptide is an antibody or an antigen fragment thereof. In another aspect, the binding polypeptide is a multispecific antibody. In another aspect, the binding polypeptide includes VHH. In another aspect, the binding polypeptide includes at least one antigen-binding fragment selected from the group consisting of: variable fragments (Fv), Fab, Fab', F(ab')2, microantibodies, biantibodies, triantibodies, tetraantibodies, tandem di-scFv, tandem tri-scFv, and immunoglobulin single variable domain (ISV).

[0081] In one aspect, the Fc domain variants of this disclosure comprise human Fc receptors. In another aspect, the Fc receptor is an Fcγ receptor (FcγR). In yet another aspect, the FcγR is FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB. In yet another aspect, according to EU designations, FcγRIIIa is hFcγRIIIa (FcγRIIIa) containing a valine residue at amino acid position 158. V158 On the other hand, according to EU designation, FcγRIIIa is hFcγRIIIa (FcγRIIIa) with phenylalanine at amino acid position 158. F158 On the other hand, the Fc receptor is the neonatal Fc receptor (FcRn).

[0082] In one aspect, the Fc domain variant of this disclosure comprises an Fc domain parent polypeptide, which is a wild-type Fc domain. In another aspect, the Fc domain parent polypeptide comprises a modified Fc domain. In yet another aspect, the Fc domain parent polypeptide comprises at least one modified glycan. In yet another aspect, the modified glycan is bis-mannose-6-phosphate (bisM6P), disaccharide-mannose-6-phosphate, or mannose-6-phosphate monosaccharide. In yet another aspect, the modified glycan is an oligomannose-type N-glycan comprising an oligosaccharide selected from the group consisting of: Man9(GlcNAc)2, Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2, or Man5(GlcNAc)2.

[0083] In one respect, the Fc domain variants of this disclosure exhibit approximately 1.5 to 20 times increased binding affinity to the Fc receptor compared to the Fc domain parent polypeptide.

[0084] In one aspect, the Fc domain variants disclosed herein comprise Fc domain variants having a production titration ranging from about 5 mg / L to 500 mg / L.

[0085] In one aspect, when measured by size exclusion chromatography (SEC), the Fc domain variants of this disclosure containing Fc domain variants have a purity of at least 60%, 70%, 80%, 90%, or 100%.

[0086] In one aspect, the pharmaceutical composition comprises an Fc domain variant peptide and a pharmaceutically acceptable carrier or diluent.

[0087] In one aspect, the nucleic acid molecule encodes a disclosed Fc domain variant polypeptide. In another aspect, the vector contains a nucleic acid molecule encoding an Fc domain variant polypeptide. In yet another aspect, a cell-expressing nucleic acid molecule is produced.

[0088] To provide a clear understanding of the specification and claims, the following definitions are provided. Attached Figure Description

[0089] The foregoing and other features and advantages of this application will be more fully understood from the following detailed description of illustrative aspects, taken in conjunction with the accompanying drawings.

[0090] Figure 1 It is a bar graph showing the protein yield of Fc domain variant peptides in milligrams (mg).

[0091] Figure 2 This is a bar chart showing the percentage of protein purity of Fc domain variant peptides as measured by size exclusion chromatography (SEC).

[0092] Figure 3 The sensor data were collected from four Fc domain peptides used as controls. The four controls were: (1) a commercially available antibody with a wild-type IgG domain (adalimumab; top left); (2) the same antibody as (1) but with a glycoengineered Fc domain mutation that eliminated effector functions, including hFcγRIIIA binding (antibody with an S298N / T299A / Y300SFc domain mutation; top right); (3) an antibody with an Fc domain mutation that enhanced hFcγRIIIA binding (bottom left); and (4) the antibody in (3) which was glycoengineered to further enhance hFcγRIIIA binding.

[0093] Figure 4 It is a sensor map data measuring the binding affinity of hFcγRIIIA from 80 Fc domain variant peptides and corresponding controls.

[0094] Figure 5It is a sensor map data measuring the binding affinity of hFcγRIIIA from 45 Fc domain variant peptides and corresponding controls.

[0095] Figure 6 This is an affinity plot of 125 Fc domain variant peptides relative to the control (Ka, 1 / Ms, y-axis; Kd, 1 / s, x-axis).

[0096] Figure 7 This is a table showing the binding affinity of 125 Fc domain variant peptides relative to the binding affinity of wild-type IgG Fc domain (adalimumab).

[0097] Figure 8A -B: Figure 8A A table of 16 Fc domain variant peptide candidates with favorable protein expression, protein purity, and hFcγRIIIa binding kinetics is shown. Figure 8B It is applied to Figure 8A The standard legend.

[0098] Figure 9 This is a table mapping the unique IMGT number, IMGT exon number, EU, and Kabat number for the C domain (C-DOMAIN). These tables are for the CH2 and CH3 domains and are taken from https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html, which is incorporated herein by reference in its entirety. Detailed Implementation

[0099] This disclosure provides novel Fc domain variants (e.g., novel binding peptides comprising Fc domain variants) that have improved binding to Fc receptors. This disclosure further provides novel Fc domain variants (e.g., binding peptides comprising Fc domain variants) that, according to EU designations, contain at least one or at least two substitutions at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373, and 376, compared to the parental Fc domain peptide.

[0100] This disclosure also provides nucleic acids encoding Fc domain variants (e.g., novel binding peptides containing Fc domain variants), recombinant expression vectors and host cells for preparing Fc domain variants (e.g., novel binding peptides containing Fc domain variants), and pharmaceutical compositions comprising isolated Fc domain variants (e.g., novel binding peptides containing Fc domain variants). Methods for treating one or more diseases or conditions using the Fc domain variants (e.g., novel binding peptides containing Fc domain variants) of this disclosure are also provided. definition

[0101] It should be understood that the methods described in this disclosure are not limited to specific methods and experimental conditions, and that methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0102] Unless otherwise stated, the experiments described herein utilize conventional molecular and cell biology and immunological techniques within the scope of the art. These techniques are well known to those skilled in the art and are well explained in the literature. See, for example, Ausubel, et al., eds., Current Protocols in Molecular Biology, JohnWiley & Sons, Inc., NY, NY (1987–2008), including all supplementary materials, Molecular Cloning: A Laboratory Manual, written 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 defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Furthermore, unless the context requires otherwise, singular terms will include plural and plural terms will include singular. Unless otherwise stated, “or” is used to mean “and / or”. The use of the term "including" and other forms such as "includes" and "included" is non-limiting. The embodiments described illustratively herein may be implemented where no one or more elements or limitations specifically disclosed or not specifically disclosed herein are present. Therefore, for example, in each case herein, any one of the terms "comprising," "consisting substantially of," and "consisting of" may be replaced by any of the other two terms while retaining their ordinary meaning. Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein may be specifically excluded from the claim.

[0103] Generally, the nomenclature used in conjunction with the cell culture, molecular biology, immunology, microbiology, genetics, protein biology, and chemistry described herein is well-known and commonly used in the field. The nomenclature, laboratory procedures, and techniques used in conjunction with the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are also well-known and commonly used in the field.

[0104] To make this disclosure easier to understand, selected terms are defined below. Peptides and isolated peptides

[0105] The term "peptide" refers to any polymeric amino acid chain and encompasses natural or artificial proteins, peptide analogs or variants of protein sequences, or fragments thereof, unless otherwise contradicted by the context. Peptides can be monomeric or polymeric. For peptides (e.g., variant peptides containing at least one or at least two substituted Fc domains at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373, and 376, according to EU designations), fragments of the peptide optionally contain at least one continuous or non-linear epitope of the peptide. The length of the fragmented peptide can be about 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, or more amino acids while retaining the ability to bind to Fc receptors. The precise boundaries of at least one epitope fragment can be verified by those skilled in the art. The polypeptide fragment contains, for example, at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, or at least about 20 consecutive amino acids, at least about 50 consecutive amino acids, at least about 100 consecutive amino acids, at least about 150 consecutive amino acids, at least about 200 consecutive amino acids, at least about 250 consecutive amino acids, at least about 300 consecutive amino acids, or at least about 400 consecutive amino acids.

[0106] In some respects, Fc domain variant peptides or Fc domain parent peptides are “isolated peptides.” The term “isolated peptide” refers to a protein or peptide that, due to its source or derivative, is not associated with its naturally occurring associated components in its native state; and is substantially free of other proteins from the same species. Isolated recombinant peptides can be expressed by cells that do not naturally express recombinant peptides. In some respects, isolated peptides are not naturally occurring. Chemically synthesized or synthesized in cellular systems, proteins or peptides may differ from the cells of their natural origin and thus be “isolated” from their naturally occurring associated components. Proteins or peptides can also be isolated to be substantially free of their naturally occurring associated components using protein purification techniques. Natural (wild-type) residues compared to variant peptides

[0107] As used herein, the terms “natural residue,” “wild-type residue,” and “parental residue” refer to amino acid residues that are naturally present at a specific amino acid position (e.g., the wild-type IgG1 or IgG4 Fc domain) of a binding polypeptide and that have not been artificially modified, introduced, or altered. Therefore, the “parental polypeptide” can refer to the wild-type amino acid sequence (e.g., the wild-type IgG1 or IgG4 Fc domain) encoding the binding polypeptide.

[0108] The term "parental polypeptide" can refer to an amino acid sequence that has been modified but still serves as a reference binding polypeptide when compared to a variant-binding polypeptide. For example, an Fc domain parental polypeptide could be a humanized IgG Fc domain. In another instance, the parental polypeptide sequence is altered to remove a C'-terminal amino acid residue. In yet another instance, the parental polypeptide sequence has amino acid substitutions; for example, the parental polypeptide sequence is:

[0109] As used herein, the terms “altered binding protein,” “altered binding peptide,” “modified binding protein,” “modified binding peptide,” “variant peptide,” “mutant peptide,” or “engineered peptide” refer to a binding peptide and / or binding protein (e.g., an antibody or fragment thereof) that contains at least one amino acid substitution, deletion, and / or addition relative to the native (i.e. wild-type) amino acid sequence, and / or a mutant binding peptide and / or binding protein (e.g., an antibody or fragment thereof) that produces altered glycosylation (e.g., hyperglycosylation, hypoglycosylation, and / or deglycosylation) at one or more amino acid positions relative to the native, parental (i.e. wild-type) amino acid sequence.

[0110] In one aspect, the Fc domain parent peptide contains the wild-type human IgG1 Fc domain.

[0111] In one aspect, the Fc domain parent polypeptide is a wild-type Fc domain containing the human IgG1 constant domain (CH1-hinge-CH2-CH3) as shown in the following amino acid sequence: .

[0112] In one aspect, the Fc domain parent polypeptide is a wild-type Fc domain containing the human IgG1 constant domain (CH1-hinge-CH2-CH3) as shown in the amino acid sequence above, and lacking the C-terminal lysine (K) as shown in the following amino acid sequence: .

[0113] In one aspect, the Fc domain parent polypeptide is an Fc domain containing the human IgG1 Fc (hinge-CH2-CH3) as shown in the following amino acid sequence: .

[0114] In one aspect, the Fc domain parent polypeptide is an Fc domain containing the human IgG1 Fc (hinge-CH2-CH3) as shown in the amino acid sequence above, but without the C-terminal lysine (K) as shown in the following amino acid sequence:

[0115] .

[0116] In one aspect, the Fc domain parent polypeptide is an Fc domain comprising human IgG1 Fc, which includes amino acid residue substitutions from cysteine ​​(C) to serine (S) and does not include a C-terminal lysine (K) resulting in the amino acid sequence shown below:

[0117] In one aspect, the Fc domain parental polypeptide contains the wild-type human IgG4 Fc domain.

[0118] In one aspect, the Fc domain parent polypeptide is the Fc domain, which contains the human IgG4P constant domain (CH1-hinge-CH2-CH3) as shown in the following amino acid sequence: .

[0119] In one aspect, the Fc domain parent polypeptide is an Fc domain containing the human IgG4P constant domain (CH1-hinge-CH2-CH3) as shown in the amino acid sequence above, but without the C-terminal lysine (K) as shown in the following amino acid sequence: .

[0120] In one aspect, the Fc domain parent polypeptide is an Fc domain containing the human IgG4PFc (hinge-CH2-CH3) constant domain as shown in the following amino acid sequence: .

[0121] In one aspect, the Fc domain parent polypeptide is an Fc domain containing the human IgG4PFc (hinge-CH2CH3) constant domain but lacking the C-terminal lysine (K) shown in the amino acid sequence below: .

[0122] In one aspect, the Fc domain parent polypeptide is encoded by an amino acid sequence having at least 80%, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequences shown in SEQ ID Nos. 50 to 58.

[0123] In one aspect, the Fc domain parent polypeptide is encoded by an amino acid sequence having at least 80%, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 50.

[0124] In one aspect, the Fc domain parent polypeptide is encoded by an amino acid sequence having at least 80%, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 51.

[0125] In one aspect, the Fc domain parent polypeptide is encoded by an amino acid sequence having at least 80%, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 52.

[0126] In one aspect, the Fc domain parent polypeptide is encoded by an amino acid sequence having at least 80%, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 53.

[0127] In one aspect, the Fc domain parent polypeptide is encoded by an amino acid sequence having at least 80%, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 54.

[0128] In one aspect, the Fc domain parent polypeptide is encoded by an amino acid sequence having at least 80%, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 55.

[0129] In one aspect, the Fc domain parent polypeptide is encoded by an amino acid sequence having at least 80%, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 56.

[0130] In one aspect, the Fc domain parent polypeptide is encoded by an amino acid sequence having at least 80%, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 57.

[0131] In one aspect, the Fc domain parent polypeptide is encoded by an amino acid sequence having at least 80%, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID No: 58. Binding proteins or binding peptides

[0132] As used herein, the terms "binding protein," "binding polypeptide," or "multispecific binding polypeptide or protein" refer to a protein or polypeptide (e.g., an antibody or antigen-binding fragment thereof) containing at least one binding site responsible for selectively binding to a protein of interest (e.g., a human antigen) and an Fc domain (e.g., the wild-type Fc domain or an Fc domain variant described herein). Exemplary binding sites include antibody variable domains, ligand-binding sites of receptors, or receptor-binding sites of ligands. In some aspects, the binding protein or binding polypeptide contains multiple (e.g., two, three, four, or more) binding sites. In one aspect, the binding polypeptide may have one or more antigen-binding sites and one or more Fc receptor-binding sites. In another aspect, the binding protein or binding polypeptide is not a therapeutic enzyme. Ligands and antigens

[0133] The term "ligand" refers to any substance that can bind to or be bound by another substance. As used herein, the term "antigen" or "target antigen" refers to a molecule or part of a molecule that can be bound to a binding site of a polypeptide, for example, any substance that can generate an antibody. A target antigen may have one or more epitopes.

[0134] Although the term "antigen" is commonly used when referring to antibody-binding substrates and "ligand" is often used when referring to receptor-binding substrates, these terms are interchangeable and cover a wide range of overlapping chemical entities. To avoid confusion, antigen and ligand are used interchangeably throughout this article.

[0135] Examples of antigens / ligands can be peptides, polypeptides, proteins, aptamers, polysaccharides, sugar molecules, carbohydrates, lipids, oligonucleotides, polynucleotides, synthetic molecules, inorganic molecules, organic molecules, and any combination thereof. Immunoglobulin domain

[0136] As used herein, the term immunoglobulin domain can refer to immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), immunoglobulin G (IgG), or immunoglobulin M (IgM). An immunoglobulin domain can be a region of the immunoglobulin heavy chain or a fragment thereof. In some cases, the immunoglobulin domain originates from an antibody (e.g., mammalian antibody, recombinant antibody, chimeric antibody, engineered antibody, human antibody, humanized antibody) or an antigen-binding fragment thereof.

[0137] In one aspect, Fc domain variant peptides or Fc domain parent peptides are derived from immunoglobulin classes selected from the group consisting of: IgM, IgG, IgD, IgA, and IgE.

[0138] In one respect, the Fc domain variant peptide or the Fc domain parent peptide is derived from the IgG Fc domain. In the other respect, the Fc domain variant peptide or the Fc domain parent peptide is derived from the IgG1 Fc domain or the IgG4 Fc domain. Fc domain peptides

[0139] In one aspect, Fc domain peptides are provided, for example, Fc domain variant peptides. As used herein, the term "Fc region" or "Fc domain" refers to a portion of the heavy chain constant region that begins in the hinge region, just upstream of the papain cleavage site (i.e., residue 216 in IgG, taking the first residue of the heavy chain constant region as 114), and terminates at the C-terminus of the antibody. Thus, a complete Fc region contains at least a hinge domain, a CH2 domain, and a CH3 domain.

[0140] The Fc domain of an antibody participates in non-antigen binding and can mediate effector function by binding to Fc receptors. There are many different types of Fc receptors, classified according to the types of antibodies they recognize. For example, the Fc-γ receptor (FcγR) binds to IgG antibodies, the Fc-α receptor (FcαR) binds to IgA antibodies, and the Fc-ε receptor (FcεR) binds to IgE antibodies. Neonatal Fc receptors (FcRn) interact with the Fc region of antibodies, promoting antibody recycling by rescuing normal lysosomal degradation. FcγR belongs to a family that includes several members, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb.

[0141] In one aspect, the Fc domain variant peptide is of human origin. In another aspect, the Fc domain variant peptide is derived from an immunoglobulin class selected from the group consisting of IgM, IgG, IgD, IgA, and IgE. In another aspect, the Fc domain variant peptide is derived from the IgG Fc domain. In yet another aspect, the Fc domain peptide is derived from either the IgG1 Fc domain or the IgG4 Fc domain.

[0142] In one aspect, the human IgG1 Fc domain contains: .

[0143] In one aspect, the fifth amino acid is serine (S).

[0144] In one aspect, the human IgG1 Fc domain contains: In one aspect, the C-terminal lysine (K) is missing.

[0145] Note that in some respects, the positions of amino acid residues are indicated by EU numbers, such as... Figure 9 As shown. For example, according to EU designation, in SEQ ID NO: 50, the M amino acid at position 220 of SEQ ID NO: 50 is amino acid 428. FcγRIIIa

[0146] FcγRIIIa V158, or human CD16a-V receptor, or CD16aV, refers to a polypeptide construct containing a fragment of the human CD16 receptor that binds to the Fc region of a natural antibody. It mediates antibody-dependent cell cytotoxicity and has a valine (V) residue at position 158. It has also been reported in the literature as an allotype CD16a V158.

[0147] FcγRIIIa F158, or human CD16a-F receptor, or CD16aF, refers to a polypeptide construct containing a fragment of the human CD16 receptor that binds to the Fc region of a natural antibody. It mediates antibody-dependent cell cytotoxicity and contains a phenylalanine (F) at position 158. It is also known as allotype CD16a F158.

[0148] In one aspect, compared to the parental Fc domain polypeptide, the Fc domain variant polypeptide, according to EU designations, contains at least two substitutions at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373, and 376. In another aspect, the Fc domain variant polypeptide exhibits increased binding to the Fc receptor compared to the parental Fc domain polypeptide. In another aspect, the Fc receptor is the human Fc receptor. In another aspect, the Fc receptor is the Fcγ receptor (FcγR). In another aspect, the FcγR is FcγRIIIa or FcγRIIIb. In another aspect, according to EU designations, FcγRIIIa is hFcγRIIIa (FcγRIIIa) containing valine at amino acid position 158. V158 On the other hand, according to EU designation, FcγRIIIA is hFcγRIIIa (FcγRIIIa) containing phenylalanine at amino acid position 158. F158 ). Fc domain parent peptide

[0149] As used herein, the terms “natural Fc” or “wild-type Fc” or “Fc domain parent polypeptide” refer to a molecule, whether in monomeric or multimeric form, that corresponds to a sequence of a non-antigen-binding fragment produced by antibody digestion or otherwise, and may contain a hinge region. The original immunoglobulin source of natural Fc is typically human and can be any immunoglobulin, such as IgG1 and IgG2. Natural Fc can also be of non-human species origin but has been modified, for example, to resemble, humanized mouse immunoglobulins.

[0150] Natural Fc molecules are composed of monomeric polypeptides that can be linked together covalently (i.e., by disulfide bonds) and non-covalently to form dimers or polymers. Depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2), the number of intermolecular disulfide bonds between the monomeric subunits of a natural Fc molecule ranges from 1 to 4. One example of natural Fc is a disulfide-bonded dimer produced by the papain digestion of IgG. As used herein, the terms “natural Fc” or “wild-type Fc” are general terms for monomeric, dimer, and polymeric forms.

[0151] In one instance, the Fc domain parent peptide is a wild-type Fc domain. In another instance, the Fc domain parent peptide is a wild-type Fc IgG domain. In one instance, the Fc domain parent peptide is derived from a commercially available antibody. In yet another instance, the Fc domain parent peptide is derived from a modified Fc domain.

[0152] In one aspect, the Fc domain parent polypeptide comprises at least one modified glycan. In another aspect, the Fc domain parent polypeptide comprises at least one N-glycan. In yet another aspect, the Fc domain parent polypeptide comprises bisM6P, disaccharide mannose-6-phosphate, or mannose-6-phosphate monosaccharide. In yet another aspect, the Fc domain parent polypeptide comprises an oligomannose-type N-glycan comprising an oligosaccharide selected from the group consisting of: Man9(GlcNAc)2, Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2, or Man5(GlcNAc)2. In yet another aspect, the Fc domain parent polypeptide is about 20%, 30%, 40%, 50%, 60%, 70%, 90%, or more unfucosylated.

[0153] In one respect, compared with the Fc domain parent polypeptide, the Fc domain variant polypeptide, according to EU designation, contains at least one or at least two substitutions at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373 and 376. Fc domain variant peptides and Fc domain

[0154] As used herein, the terms “Fc domain variant,” “Fc variant,” “modified Fc,” or “Fc domain variant peptide” refer to a molecule or sequence modified from natural / wild-type Fc but still containing a binding site for the Fc receptor. Thus, the term “Fc variant” can encompass molecules or sequences humanized from non-human natural Fc. Furthermore, natural Fc contains regions that can be removed because they provide structural features or biological activities not required by the antibody-like binding peptides described herein. Therefore, the term “Fc domain variant peptide” encompasses molecules or sequences lacking one or more natural Fc sites or residues, or where one or more Fc sites or residues have been modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity after expression in a selected host cell, (4) glycosylation, (5) complement interaction, (6) binding to an Fc receptor other than a rescue receptor (e.g., FcγRIIIa), or (7) antibody-dependent cytotoxicity (ADCC).

[0155] As used herein, “effecton-capable Fc domain variant” or “effecton-capable polypeptide” refers to an Fc domain variant polypeptide having one or more Fc effector functions as further described herein.

[0156] In one respect, compared with the Fc domain parent peptide, the Fc domain variant peptides characterized in this paper have one or more of the following: increased serum half-life, enhanced FcRn binding affinity, enhanced FcRn binding affinity at acidic pH, enhanced FcγRIIIa binding affinity, and / or similar thermal stability.

[0157] As used herein, the term "Fc domain" encompasses both parental Fc domain peptides (e.g., natural / wild-type Fc domains) and Fc domain variant peptides and sequences as defined herein. Similar to Fc domain variant peptides and parental Fc domain peptides (e.g., natural Fc domain molecules), the term "Fc domain" includes molecules in monomeric or multimeric form, whether produced from whole antibody digestion or by other means.

[0158] In one respect, the Fc domain variant peptides described herein are thermally stable. Exemplary Fc domain variant peptides that may enhance thermal stability are described in U.S. Provisional Patent Application No. 63 / 193,655, which is incorporated herein by reference in its entirety.

[0159] In one aspect, the Fc domain described herein comprises at least one N-glycan. In one aspect, the Fc domain described herein is glycosylated (e.g., via N-linked glycosylation). In one aspect, the Fc domain comprises N-linked glycosylation, for example, located at an N-linked glycosylation motif containing the amino acid sequence NXT or NXTS (where X is any amino acid residue other than proline). In some exemplary embodiments, according to EU numbering, the Fc domain is glycosylated at amino acid position 297.

[0160] In one respect, the Fc structural domain described in this paper has effector capability.

[0161] In one respect, the Fc domains described in this paper are any combination of thermally stable, glycosylated, and effective quantum capabilities. Fc domain variants of enhancement effectors

[0162] In one aspect, this disclosure provides Fc domain variant peptides comprising effector-enhancing amino acid substitutions.

[0163] In one respect, Fc domain variant peptides exhibit altered binding affinity to Fc receptors. There are many different types of Fc receptors, classified according to the types of antibodies they recognize. For example, the Fcγ receptor (FcγR) binds to IgG antibodies, the Fcα receptor (FcαR) binds to IgA antibodies, and the Fcε receptor (FcεR) binds to IgE antibodies. FcγR belongs to a family comprising several members, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb.

[0164] In one respect, the Fc domain variant peptide exhibits altered FcγRIIIa binding affinity compared to the Fc domain parent peptide. In another respect, the Fc domain variant has decreased FcγRIIIa binding affinity compared to the wild-type Fc domain peptide. In yet another respect, the Fc domain variant has enhanced FcγRIIIa binding affinity compared to the wild-type IgG Fc domain peptide. In yet another respect, the Fc domain modified in the Fc domain variant peptide has approximately the same FcγRIIIa binding affinity compared to the Fc domain parent peptide.

[0165] In another aspect, this document discloses Fc domain variant peptides with altered FcγRIIIa binding affinity, comprising one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, or more). In another aspect, Fc domain variant peptides with enhanced FcγRIIIa binding affinity as disclosed herein have one or more amino acid substitutions. In another aspect, Fc domain variant peptides with enhanced FcγRIIIa binding affinity as disclosed herein comprise two or more amino acid substitutions. In another aspect, Fc domain variant peptides with enhanced FcγRIIIa binding affinity as disclosed herein comprise three or more amino acid substitutions. In another aspect, Fc domain variant peptides with enhanced FcγRIIIa binding affinity as disclosed herein comprise four or more amino acid substitutions.

[0166] In one aspect, the Fc domain variant polypeptide with modified FcRn binding comprises an Fc domain having one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5 or more) as disclosed herein. In another aspect, the Fc domain variant polypeptide with enhanced FcRn binding affinity as disclosed herein comprises an Fc domain having one or more amino acid substitutions. In another aspect, the Fc domain variant polypeptide with enhanced FcRn binding affinity as disclosed herein comprises an Fc domain having two or more amino acid substitutions. In another aspect, the Fc domain variant polypeptide with enhanced FcRn binding affinity as disclosed herein comprises an Fc domain having three or more amino acid substitutions. In another aspect, the Fc domain variant polypeptide with enhanced FcRn binding affinity as disclosed herein comprises four or more amino acid substitutions.

[0167] In one respect, Fc domain variant peptides can exhibit species-specific FcRn binding affinity. In another respect, Fc domain variant peptides can exhibit human FcRn binding affinity. In another respect, Fc domain variant peptides can exhibit cynomolgus monkey FcRn binding affinity. In yet another respect, Fc domain variant peptides can exhibit cross-species FcRn binding affinity. It is said that such Fc domain variant peptides exhibit cross-reactivity across one or more different species. In yet another respect, Fc domain variants can exhibit both human and cynomolgus monkey FcRn binding affinity.

[0168] In some embodiments, the Fc domain variant peptide includes an antibody constant region (e.g., an IgG constant region, such as a human IgG constant region, such as a human IgG1 constant region) that mediates one or more effector functions. For example, binding of the C1 complex to the antibody constant region can activate the complement system. Activation of the complement system is crucial for opsonization and lysis of cellular pathogens. Activation of the complement system can also stimulate inflammatory responses and may be involved in autoimmune hypersensitivity reactions.

[0169] Furthermore, antibodies and antibodies containing Fc domain variant peptides can bind to various receptors on cells via the Fc domain (the Fc receptor binding site on the antibody's Fc region binds to the cell's Fc receptor (FcR)). Many Fc receptors are specific to different classes of antibodies, including IgG (γ receptor), IgE (ε receptor), IgA (α receptor), and IgM (μ receptor). Binding of antibodies and / or Fc domain variant peptides to Fc receptors on cell surfaces triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by cytotoxic cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In one aspect, Fc domain variant peptides, such as binding peptides (e.g., antibodies or immunoadhesins), bind to the Fc-γ (Fcγ) receptor. In the other case, Fc domain variant peptides contain a constant region that lacks one or more effector functions (e.g., ADCC activity) and / or cannot bind to the Fcγ receptor. Amino acid substitution

[0170] In one aspect, compared to the Fc domain parent polypeptide, the Fc domain variant polypeptide, according to EU designations, contains at least one substitution (e.g., 1, 2, 3, 4, 5 or more) at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373, and 376. In another aspect, compared to the Fc domain parent polypeptide, the Fc domain variant polypeptide, according to EU designations, contains at least one or two substitutions at amino acid positions 251 or 376. In yet another aspect, compared to the Fc domain parent polypeptide, the Fc domain variant polypeptide, according to EU designations, contains at least two substitutions at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373, and 376.

[0171] In one aspect, the Fc domain variant polypeptide contains at least two amino acid substitutions selected from the following: (i) alanine (A), histidine (H), isoleucine (I), phenylalanine (F), glutamine (Q), or tryptophan (W) at amino acid position 251; (ii) alanine (A) or aspartic acid (D) at amino acid position 267; (iii) aspartic acid (D) or glutamic acid (E) at amino acid position 268; (iv) alanine (A) at amino acid position 298; (v) glycine (G), lysine (K), asparagine (N), methionine (M), serine (S), threonine (T), valine (V), glutamic acid (E), or tryptophan (W) at amino acid position 314; (vi) phenylalanine (F), methionine (M), or tyrosine (Y) at amino acid position 330; (vii) threonine (T) at amino acid position 339; (viii) ... Tryptophan (W) at amino acid position 373, or valine (V) at amino acid position 376, amino acid positions according to EU numbering.

[0172] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, tryptophan (W) at amino acid position 373, and alanine (A) at amino acid position 298.

[0173] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and alanine (A) at amino acid position 298.

[0174] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, isoleucine (I) at amino acid position 251, phenylalanine (F) at amino acid position 330, and alanine (A) at amino acid position 298.

[0175] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, threonine (T) at amino acid position 339, and alanine (A) at amino acid position 298.

[0176] In one aspect, according to EU designation, the Fc domain variant polypeptide contains glutamic acid (E) at amino acid position 268, valine (V) at amino acid position 376, alanine (A) at amino acid position 251, and methionine (M) at amino acid position 330.

[0177] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, aspartic acid (D) at amino acid position 267, isoleucine (I) at amino acid position 251, and threonine (T) at amino acid position 339.

[0178] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, threonine (T) at amino acid position 339, and tryptophan (W) at amino acid position 373.

[0179] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and tryptophan (W) at amino acid position 373.

[0180] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and threonine (T) at amino acid position 314.

[0181] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and valine (V) at amino acid position 314.

[0182] In one aspect, according to EU designations, the Fc domain variant contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and valine (V) at amino acid position 376.

[0183] In one aspect, according to EU designation, the Fc domain variant polypeptide contains alanine (A) at amino acid position 298, tyrosine (Y) at amino acid position 330, threonine (T) at amino acid position 339, and tryptophan (W) at amino acid position 373.

[0184] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and alanine (A) at amino acid position 251.

[0185] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 267, tyrosine (Y) at amino acid position 330, and threonine (T) at amino acid position 339.

[0186] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 267, tyrosine (Y) at amino acid position 330, and tryptophan (W) at amino acid position 373.

[0187] In one aspect, according to EU designation, the Fc domain variant polypeptide contains glutamic acid (E) at amino acid position 268, histidine (H) at amino acid position 251, and phenylalanine (F) at amino acid position 330.

[0188] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 373, and alanine (A) at amino acid position 298.

[0189] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and alanine (A) at amino acid position 298.

[0190] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 330, and alanine (A) at amino acid position 298.

[0191] In one aspect, according to EU designation, the Fc domain variant polypeptide contains glutamic acid at amino acid position 268 (E), valine at amino acid position 314 (V), and methionine at amino acid position 330 (M).

[0192] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, aspartic acid (D) at amino acid position 267, and threonine (T) at amino acid position 339.

[0193] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and tryptophan (W) at amino acid position 373.

[0194] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and valine (V) at amino acid position 314.

[0195] In one aspect, according to EU designation, the Fc domain variant polypeptide contains glutamic acid (E) at amino acid position 268 and phenylalanine (F) at amino acid position 330.

[0196] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and glutamic acid (E) at amino acid position 314.

[0197] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

[0198] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, tryptophan (W) at amino acid position 373, and alanine (A) at amino acid position 298.

[0199] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, and serine (S) at amino acid position 314.

[0200] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and alanine (A) at amino acid position 298.

[0201] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, isoleucine (I) at amino acid position 251, phenylalanine (F) at amino acid position 330, and alanine (A) at amino acid position 298.

[0202] In one aspect, according to EU designations, the Fc domain variants include aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

[0203] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, and asparagine (N) at amino acid position 314.

[0204] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

[0205] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 314, tryptophan (W) at amino acid position 373, and methionine (M) at amino acid position 330.

[0206] In one aspect, according to EU designation, the Fc domain variant polypeptide contains glutamic acid at amino acid position 268 (E), isoleucine at amino acid position 251 (I), threonine at amino acid position 314 (T), and valine at amino acid position 376 (V).

[0207] In one aspect, the Fc domain variant polypeptide, wherein, according to EU designation, the Fc domain variant contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and glycine (G) at amino acid position 314.

[0208] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, glycine (G) at amino acid position 314, and alanine (A) at amino acid position 298.

[0209] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and lysine (K) at amino acid position 314.

[0210] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, threonine (T) at amino acid position 314, and alanine (A) at amino acid position 298.

[0211] In one aspect, according to EU designation, the Fc domain variant polypeptide contains glutamic acid (E) at amino acid position 268, valine (V) at amino acid position 314, alanine (A) at amino acid position 251, and methionine (M) at amino acid position 330.

[0212] In one aspect, according to EU designation, the Fc domain variant polypeptide contains alanine (A) at amino acid position 298, tyrosine (Y) at amino acid position 330, and threonine (T) at amino acid position 339.

[0213] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and lysine (K) at amino acid position 314.

[0214] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and glycine (G) at amino acid position 314.

[0215] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, tryptophan (W) at amino acid position 251, and valine (V) at amino acid position 376.

[0216] In one aspect, according to EU designation, the Fc domain variant polypeptide contains tyrosine (Y) at amino acid position 330, alanine (A) at amino acid position 267, valine (V) at amino acid position 376, and alanine (A) at amino acid position 298.

[0217] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and threonine (T) at amino acid position 314.

[0218] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

[0219] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and tryptophan (W) at amino acid position 373.

[0220] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and glutamic acid (E) at amino acid position 314.

[0221] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

[0222] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 373, and alanine (A) at amino acid position 298.

[0223] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and serine (S) at amino acid position 314.

[0224] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and asparagine (N) at amino acid position 314.

[0225] In one aspect, according to EU designation, the Fc domain variant polypeptide contains glutamic acid (E) at amino acid position 268, threonine (T) at amino acid position 314, and valine (V) at amino acid position 376.

[0226] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and glycine (G) at amino acid position 314.

[0227] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, threonine (T) at amino acid position 314, and alanine (A) at amino acid position 298.

[0228] In one aspect, according to EU designation, the Fc domain variant polypeptide contains glutamic acid (E) at amino acid position 268, valine (V) at amino acid position 314, and methionine (M) at amino acid position 330.

[0229] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and lysine (K) at amino acid position 314.

[0230] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and valine (V) at amino acid position 376.

[0231] In one aspect, the effector-enhancing Fc domain variant peptide has one or more amino acid substitutions selected from the group consisting of: aspartic acid at amino acid position 221 (D); cysteine ​​at amino acid position 222 (C); tyrosine at amino acid position 234 (Y); alanine at amino acid position 236 (A); tryptophan at amino acid position 236 (W); aspartic acid at amino acid position 239 (D); leucine at amino acid position 243 (L); glutamic acid at amino acid position 267 (E); phenylalanine at amino acid position 268 (F); proline at amino acid position 292 (P); alanine at amino acid position 298 (A); and amino acid position 221 (D); cysteine ​​at amino acid position 222 (C); tyrosine at amino acid position 234 (Y); alanine at amino acid position 236 (A); glutamic acid at amino acid position 267 (E); phenylalanine at amino acid position 268 (F); proline at amino acid position 292 (P); glutamic acid at amino acid position 298 (A); glutamic acid at amino acid position 234 (Y); glutamic acid at amino acid position 236 (A); glutamic acid at amino acid position 236 (D); glutamic acid at amino acid position 236 (Y); glutamic acid at amino acid position 236 (F); glutamic acid at amino acid position 236 (W); glutamic acid at amino acid position 239 (D); glutamic acid at amino acid position 236 (Y ... Leucine (L) at amino acid position 300; isoleucine (I) at amino acid position 305; threonine (T) at amino acid position 324; tryptophan (W) at amino acid position 326; alanine (A) at amino acid position 326; leucine (L) at amino acid position 330; glutamic acid (E) at amino acid position 332; alanine (A) at amino acid position 333; serine (S) at amino acid position 333; alanine (A) at amino acid position 334; alanine (A) at amino acid position 336; arginine (R) at amino acid position 345; and leucine (L) at amino acid position 396, according to EU numbers. See Saunders, 2009, Front. Immunol. doi: 10.3389 / fimmu.2019.01296 for reference.

[0232] In one aspect, according to EU designations, the Fc domain variant polypeptide may contain amino acid substitutions at positions selected from amino acid positions 236, 239, 330, and 332. In another aspect, according to EU designations, the substitutions may include alanine (A) at amino acid position 236, aspartic acid (D) at amino acid position 239, leucine (L) at amino acid position 330, and glutamic acid (E) at amino acid position 332. In another aspect, the Fc domain variant polypeptide may contain diamino acid substitutions at any two amino acid positions, the diamino acid substitutions being selected from alanine (A) at amino acid position 236, aspartic acid (D) at amino acid position 239, leucine (L) at amino acid position 330, and glutamic acid (E) at amino acid position 332. In one aspect, the Fc domain variant peptide may contain a triamino acid substitution at any three amino acid positions, the triamino acid substitution being selected from alanine (A) at amino acid position 236, aspartic acid (D) at amino acid position 239, leucine (L) at amino acid position 330, and glutamic acid (E) at amino acid position 332. In another aspect, the Fc domain variant peptide may contain a tetraamino acid substitution at any four amino acid positions, the tetraamino acid substitution being selected from alanine (A) at amino acid position 236, aspartic acid (D) at amino acid position 239, leucine (L) at amino acid position 330, and glutamic acid (E) at amino acid position 332. In another aspect, the Fc domain variant peptide may contain a combination of amino acid substitutions including aspartic acid (D) at amino acid position 239 and glutamic acid (E) at amino acid position 332. In yet another aspect, the Fc domain variant peptide may contain a combination of amino acid substitutions including alanine (A) at amino acid position 236, aspartic acid (D) at amino acid position 236, and glutamic acid at position 332.

[0233] In one aspect, according to EU designations, the Fc domain variant polypeptide may further include amino acid substitutions at amino acid positions 256 and / or 307. In another aspect, the Fc domain variant polypeptide may include combinations of amino acid substitutions comprising aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307. See Mackness et al., 2019 MAbs 11:1276-88 and WO 2019147973A1, which are incorporated herein by reference in their entirety.

[0234] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

[0235] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, aspartic acid (D) at amino acid position 267, and threonine (T) at amino acid position 339.

[0236] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and threonine (T) at position 314.

[0237] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and glutamic acid (E) at position 314.

[0238] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and glycine (G) at position 314.

[0239] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268 and serine (S) at position 314.

[0240] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and valine (V) at position 376.

[0241] In one aspect, according to EU designation, the Fc domain variant polypeptide contains glutamic acid (E) at amino acid position 268, threonine (T) at amino acid position 314, and valine (V) at position 376.

[0242] In one aspect, according to EU designation, the Fc domain variant polypeptide contains aspartic acid (E) at amino acid position 268, valine (V) at amino acid position 314, and methionine (M) at amino acid position 330. pH-dependent Fc domain variant peptides

[0243] Neonatal Fc receptors (FcRn) interact with the Fc region of antibodies, promoting recycling by rescuing normal lysosomal degradation. This process is pH-dependent and occurs in endosomes at acidic pH levels (e.g., pH less than 6.5) but not at physiological pH conditions in the bloodstream (e.g., non-acidic pH).

[0244] In one aspect, the Fc domain variant peptide exhibits enhanced FcRn binding affinity at acidic pH compared to the Fc domain parent peptide. In another aspect, the Fc domain variant peptide exhibits enhanced FcRn binding affinity at pH less than 7 (e.g., at about pH 6.5, about pH 6.0, about pH 5.5, and about pH 5.0) compared to the Fc domain parent peptide at elevated non-acidic pH. Elevated non-acidic pH values ​​can be, for example, pH greater than 7, approximately pH 7, approximately pH 7.4, approximately pH 7.6, approximately pH 7.8, approximately pH 8.0, approximately pH 8.5, or approximately pH 9.0.

[0245] On the one hand, Fc domain variant peptides are expected to exhibit approximately the same FcRn binding affinity as the Fc domain parent peptide at non-acidic pH. On the other hand, Fc domain variants are expected to exhibit lower FcRn binding affinity at non-acidic pH than binding peptides containing Fc domains modified with diamino acid substitutions M428L / N434S (according to EU number (see USPN 8,088,376)). Therefore, Fc domain variant peptides are expected to exhibit minimal perturbation to pH-dependent FcRn binding.

[0246] In one respect, compared to the parent peptide with the Fc domain, the Fc domain variant peptide exhibiting enhanced FcRn binding affinity at acidic pH has a decreased (i.e., slower) FcRn dissociation rate. In another respect, compared to the binding peptide's FcRn binding affinity at elevated non-acidic pH, the Fc domain variant peptide exhibiting enhanced FcRn binding affinity at acidic pH has a slower FcRn dissociation rate at acidic pH compared to the Fc domain parent peptide's FcRn dissociation rate at elevated non-acidic pH. Other Fc domain variant peptides

[0247] Some aspects include Fc domain variant peptides, wherein at least one amino acid in one or more constant domains has been deleted or otherwise altered to provide desired biochemical characteristics, such as reduced or enhanced effector function, nonvalent dimerization ability, enhanced protein expression, increased protein purity, increased ability to localize to tumor sites, shortened serum half-life, or prolonged serum half-life when compared with intact, unaltered antibodies of substantially the same immunogenicity or when compared with Fc domain parent peptides.

[0248] In one aspect, the Fc domain variant peptide includes constant regions derived from different antibody isotypes (e.g., constant regions from two or more of human IgG1, IgG2, IgG3, or IgG4). In another aspect, the Fc domain variant peptide includes a chimeric hinge (i.e., a hinge comprising hinge portions of hinge domains derived from different antibody isotypes, e.g., an upper hinge domain from the IgG4 molecule and an intermediate hinge domain from IgG1). In yet another aspect, techniques known in the art can be used to mutate the Fc domain to increase or decrease effector function relative to the Fc domain parent peptide. Antibody

[0249] As used herein, the term "antibody" refers to assemblies (e.g., complete antibody molecules, antibody fragments, or variants thereof) that exhibit significant, known specific immunoreactivity against a target antigen (e.g., CD25-associated antigens or protein-associated antigens of interest). Antibodies and immunoglobulins comprise light and heavy chains, with or without interchain covalent linkages.

[0250] As will be discussed in more detail below, the general term "antibody" includes five distinct classes of antibodies that can be distinguished biochemically. While all five classes of antibodies are clearly within the scope of this disclosure, the following discussion will generally refer to the IgG class of immunoglobulin molecules. Regarding IgG, immunoglobulins consist of two identical light chains with a molecular weight of approximately 23,000 Daltons and two identical heavy chains with molecular weights ranging from 53,000 to 7,000 Daltons. The four chains are linked by disulfide bonds in a "Y" configuration, where the light chain begins at the opening of the "Y" and continues to surround the heavy chain throughout the variable region.

[0251] Immunoglobulin light chains are classified as κ or λ (κ, λ). Each heavy chain class can bind to either a κ or λ light chain. Typically, light and heavy chains are covalently bonded to each other, and when immunoglobulins are generated from fusion tumors, B cells, or genetically engineered host cells, the “tail” portions of the two heavy chains are bonded to each other via covalent disulfide linkages or non-covalent linkages. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration to the C-terminus at the bottom of each chain. Heavy chains are classified as γ, μ, α, δ, or ε (γ, μ, α, δ, ε), with several subclasses (e.g., γ1–γ4). The properties of this chain determine the “class” of the antibody, namely IgG, IgM, IgA, IgD, or IgE. Immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) confer functional specialization. The modifications of each of these categories and of the same type are readily discernible to a person skilled in the art in consideration of this disclosure, and are therefore within the scope of this disclosure.

[0252] Both light and heavy chains are categorized into regions with structural and functional homology. The term "region" refers to a part or portion of an immunoglobulin or antibody chain and includes constant or variable regions, as well as more discrete portions of said regions. For example, variable regions of light chains include "complementarity-determining regions" or "CDRs" scattered within "frame regions" or "FRs" as defined herein.

[0253] In one aspect, the antibody comprises an Fc domain variant polypeptide as described herein. In another aspect, compared to the parental Fc domain polypeptide, the antibody, according to EU designations, comprises an Fc domain variant polypeptide containing at least one or at least two substitutions (e.g., 1, 2, 3, 4, 5, or more) at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373, and 376. In another aspect, compared to the parental Fc domain polypeptide, the antibody, according to EU designations, comprises an Fc domain variant polypeptide containing at least two substitutions at amino acid positions 251 or 376. In another aspect, compared to the parental Fc domain polypeptide, the antibody, according to EU designations, comprises an Fc domain variant polypeptide containing at least two substitutions at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373, and 376.

[0254] In another embodiment, the antibody comprises an Fc domain variant polypeptide containing at least two amino acid substitutions selected from the following: (i) alanine (A), histidine (H), isoleucine (I), phenylalanine (F), glutamine (Q), or tryptophan (W) at amino acid position 251; (ii) alanine (A) or aspartic acid (D) at amino acid position 267; (iii) aspartic acid (D) or glutamic acid (E) at amino acid position 268; (iv) alanine (A) at amino acid position 298; (v) glycine (G), lysine (K), asparagine (N), methionine (M), serine (S), threonine (T), valine (V), glutamic acid (E), or tryptophan (W) at amino acid position 314; (vi) phenylalanine (F), methionine (M), or tyrosine (Y) at amino acid position 330; (vii) threonine (T) at amino acid position 339; (viii) ... Tryptophan (W) at amino acid position 373, or valine (V) at amino acid position 376, amino acid positions according to EU numbering. Constant and variable structural domains

[0255] Immunoglobulin heavy or light chain regions can be defined as "constant" (C) regions or "variable" (V) regions, based on whether, in the case of a "constant region," there is a relative lack of sequence variation within the region of each member, or in the case of a "variable region," there is significant variation within the region of each member. The terms "constant region" and "variable region" can also be used functionally. In this regard, it should be understood that the variable regions of immunoglobulins or antibodies determine antigen recognition and specificity. Conversely, the constant regions of immunoglobulins or antibodies confer important effector functions, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. The subunit structures and three-dimensional conformations of the constant regions of various immunoglobulin classes are known.

[0256] The constant and variable regions of the immunoglobulin heavy and light chains fold into domains. The term "domain" refers to a globular region of a heavy or light chain containing a peptide ring (e.g., containing 3 to 4 peptide rings), which is stabilized, for example, by β-folds and / or intrachain disulfide bonds. Constant region domains on the immunoglobulin light chain are interchangeably referred to as "light chain constant region domains," "CL regions," or "CL domains." Constant domains on the heavy chain (e.g., hinge, CH1, CH2, or CH3 domains) are interchangeably referred to as "heavy chain constant region domains," "CH region" domains, or "CH domains." Variable domains on the light chain are interchangeably referred to as "light chain variable region domains," "VL region domains," or "VL domains." "Variable domains" on the heavy chain are interchangeably referred to as "heavy chain variable region domains," "VH region domains," or "VH domains."

[0257] By convention, the variable constant region domains are numbered as they move further away from the antigen-binding site or N-terminus of the immunoglobulin or antibody. The N-terminus of each immunoglobulin heavy and light chain is the variable region, and the C-terminus is the constant region; the CH3 and CL domains actually contain the C-termini of the heavy and light chains, respectively. Therefore, the domains of light chain immunoglobulins are aligned in a VL-CL orientation, while the domains of heavy chains are aligned in a VH-CH1-hinge-CH2-CH3 orientation.

[0258] The amino acid assignments for each variable region domain are based on the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chain variable regions or between different light chain variable regions are assigned the same number. The CDRs 1, 2, and 3 of the VL domain are also referred to as CDR-L1, CDR-L2, and CDR-L3, respectively, in this paper. The CDRs 1, 2, and 3 of the VH domain are also referred to as CDR-H1, CDR-H2, and CDR-H3, respectively, in this paper. Where so, the CDR assignments can be based on IMGT® (Lefran C et al., Developmental & Comparison Immunology 27:55-77; 2003) instead of Kabat. The heavy chain constant regions were numbered using the EU index as described in Kabat (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991).

[0259] In one aspect, the binding portion of the target protein comprises an antibody or an antigen-binding fragment thereof. In another aspect, the target protein-specific antibody or its antigen-binding fragment comprises a variable domain. In yet another aspect, the target protein-specific variable domain is operatively linked to an Fc domain variant polypeptide of this disclosure. VH and VL domains

[0260] As used herein, the term "VH domain" includes the N-terminal variable domain of the immunoglobulin heavy chain, and the term "VL domain" includes the N-terminal variable domain of the immunoglobulin light chain. In one aspect, the Fc domain variant polypeptide comprises the VH domain. In another aspect, the Fc domain variant polypeptide comprises the VL domain.

[0261] As used herein, the term "CH1 domain" includes, for example, the first (amino-terminal) constant region domain of the immunoglobulin heavy chain extending from approximately positions 114-223 (EU positions 118-215) in the Kabat numbering system. The CH1 domain is adjacent to the amino terminus of the VH domain and hinge region of the immunoglobulin heavy chain molecule and does not form part of the Fc region of the immunoglobulin heavy chain. In one aspect, Fc domain variant polypeptides contain the CH1 domain.

[0262] As used herein, the term "hinge region" refers to the portion of a heavy-chain molecule that connects the CH1 and CH2 domains. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol. 1998, 161:4083). In one aspect, Fc domain variant peptides contain the hinge domain. CH2 domain

[0263] As used herein, the term "CH2 domain" includes, for example, a portion of a heavy chain immunoglobulin molecule extending from approximately positions 244-360 (EU positions 231-340) in the Kabat numbering system. The unique feature of the CH2 domain is that it does not pair tightly with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. In some respects, Fc domain variant peptides contain the CH2 domain (e.g., the human IgG1 molecule or a variant thereof). CH3 domain

[0264] As used herein, the term "CH3 domain" encompasses a portion of a heavy chain immunoglobulin molecule extending approximately 110 residues from the N-terminus of the CH2 domain (e.g., approximate positions 361-476 (EU position 341445) in the Kabat numbering system). The CH3 domain typically forms the C-terminal portion of an antibody. However, in some immunoglobulins, additional domains may extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ chain of IgM and the e chain of IgE). In some respects, Fc domain variant peptides contain a CH3 domain (e.g., the human IgG1 molecule or a variant thereof). CL domain

[0265] As used herein, the term "CL domain" includes a constant region domain of an immunoglobulin light chain that extends, for example, from approximately Kabat position 107A-216. The CL domain is adjacent to the VL domain. In some aspects, the Fc domain variant polypeptides of this disclosure contain a CL domain derived from the κ light chain (e.g., the human κ light chain).

[0266] As described above, the variable regions of an antibody enable it to selectively recognize and specifically bind to epitopes on antigens. That is, the VL and VH domains of the antibody combine to form a variable region (Fv) defining a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites at the ends of each arm of the Y-axis. More specifically, the antigen-binding site is defined by three complementarity-determining regions (CDRs) on each of the heavy chain and light chain variable regions. As used herein, the term "antigen-binding site" includes a site that specifically binds to (in conjunction with) an antigen (e.g., a cell surface or soluble antigen). Antigen-binding sites include immunoglobulin heavy and light chain variable regions, and the binding sites formed by these variable regions determine the specificity of the antibody. Antigen-binding sites are formed by variable regions that differ between one antibody and another. In some aspects, antibodies comprising Fc domain variant peptides of this disclosure contain at least one antigen-binding site. In some aspects, antibodies comprising Fc domain variant peptides of this disclosure contain at least two antigen-binding sites.

[0267] In some respects, the antigen-binding domain is not derived from the same immunoglobulin molecule. In this respect, the variable region can be derived from any type of animal that can be induced to produce a humoral response and generate immunoglobulins against the desired antigen. Therefore, the variable region of the binding protein can be of mammalian origin, for example, from humans, mice, rats, goats, sheep, non-human primates (e.g., cynomolgus monkeys, macaques, etc.), wolves, or camelids (e.g., from camels, llamas, and related species).

[0268] In naturally occurring antibodies, each monomeric antibody possesses six short, discontinuous amino acid sequences (CDRs) that are specifically localized to form antigen-binding sites when the antibody assumes its three-dimensional conformation in an aqueous environment. The remainder of the heavy and light chain variable domains exhibits less intermolecular variability in the amino acid sequence and is referred to as the framework region. The framework region primarily adopts a β-sheet conformation, and the CDRs form loops connecting the β-sheet structures, and in some cases, form part of the β-sheet structure. Thus, these framework regions act as a scaffold, positioning the six CDRs in the correct orientation through non-covalent interchain interactions. The antigen-binding domain formed by the localized CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to the immunoreactive antigen epitope. CDR and FR

[0269] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid sequence within the antibody variable region that confers antigen specificity and binding affinity. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The "frame region" or "FR" refers to the non-CDR portion of both the heavy and light chain variable regions. Typically, there are four FRs (FR-H1, FR-H2, FRH3, and FR-H4) in each heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each light chain variable region.

[0270] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of many well-known schemes, including the scheme described by Kabat et al., (1991) “Sequences of Proteins of Immunological Interest” 5th ed., PubliC Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact Analysis And binding site topography” J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme); LefranCM P et al., “IMGT unique numbering for immunoglobulin And T cell receptor variable domains And Igsuperfamily V-like domains”, Dev Comp Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); and Honegger A and Pluckthun A, “Yet Another numbering scheme for immunoglobulin variable domains: An Automatic C modeling And Analysis tool”, JMol Biol, June 8, 2001; 309(3):657-70 (AHo numbering scheme).

[0271] The boundaries of a given CDR or FR can vary depending on the protocol used for identification. For example, the Kabat protocol is based on structure alignment, while the Chothia protocol is based on structural information. Both the Kabat and Chothia protocols number antibodies based on the length of the most common antibody region sequence, using insertions (e.g., "30a"), and some antibodies show deletions. These two protocols place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact protocol is based on the analysis of complex crystal structures and is similar to the Chothia numbering protocol in many ways.

[0272] A CDR or its complement-determining region, or individually designated CDRs (e.g., “HCDR1”, “HCDR2”, “HCDR3”), for a given antibody or its region (such as its variable region) should be understood to encompass a complement-determining region as defined (or specific) by any known protocol. Similarly, a “FR” or “frame region”, or individually designated FRs (e.g., “FR-H1”, “FR-H2”), for a given antibody or its region (such as its variable region) should be understood to encompass a frame region as defined (or specific) by any known protocol. In some cases, a protocol for identifying a particular CDR or FR is specified, such as a CDR defined by the IMGT, Kabat, Chothia, AbM, or Contact method. In other cases, a specific amino acid sequence of the CDR or FR is given. Unless otherwise specified, all specific CDR amino acid sequences mentioned in this disclosure are IMGT CDRs. However, this disclosure also covers alternative CDRs defined by other schemes, such as those determined by abysis Key Annotation (website: abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi). Antibody variants

[0273] Exemplary antibody variants and / or specific antibody-binding fragment variants may comprise Fc domain variant peptides of this disclosure. As used herein, the terms “antibody variant” and “antibody-specific binding fragment variant” include synthetic and engineered forms of antibodies that are modified such that they are not naturally occurring, such as antibodies containing at least two heavy chain moieties but not two complete heavy chains (e.g., domain-deficient antibodies or microantibodies) and multispecific forms of antibodies modified to bind to two or more different antigens or to different epitopes on a single antigen; heavy chain molecules linked to scFv molecules, etc. antigen-binding fragments

[0274] Unless otherwise specified, the term "antibody" as used herein should be understood to encompass antibody molecules (i.e., complete antibody molecules) containing two immunoglobulin heavy chains and two immunoglobulin light chains, as well as their antigen-binding fragments. In one aspect, the Fc domain variant polypeptides of this disclosure comprise antibodies or are complexed with antibodies (e.g., fused). In another aspect, the binding polypeptides of this disclosure comprise antibodies and Fc domain variant polypeptides.

[0275] Other engineered molecules such as domain-specific binding proteins, single-domain binding proteins, domain-deficient binding proteins, chimeric binding proteins, CDR transplantation binding proteins, biantibodies, triantibodies, tetraantibodies, microantibodies, immunoglobulin single variable domain (ISV) (e.g., monovalent ISV, bivalent ISV, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also included in the term "antigen-binding fragment" as used herein. In one aspect, the Fc domain variant peptides of this disclosure comprise or are complexed with an antigen-binding fragment (e.g., fused). In another aspect, the binding peptides of this disclosure comprise an antigen-binding fragment and an Fc domain variant peptide.

[0276] The term "multispecific antibody" indicates that its binding fragment or derivative binds the antigen-binding sites of two or more antibodies in a single molecule. As used herein, the terms "antigen-binding moiety," "antigen-binding fragment," "binding protein," or "binding portion," etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to at least one target antigen to form a complex. In one aspect, the Fc domain variant polypeptide of this disclosure comprises or is complexed with a multispecific antibody (e.g., fused). In another aspect, the binding polypeptide of this disclosure comprises a multispecific antibody and an Fc domain variant polypeptide.

[0277] In some respects, the binding portion can refer to one or more segments within an antibody-binding fragment that retain the ability to specifically bind to a target protein. In some respects, the term "antigen-binding fragment" refers to a polypeptide segment of a binding protein. In one respect, an Fc domain variant polypeptide may contain or be linked to an antigen-binding fragment that specifically binds to a target protein.

[0278] The binding fragment or its derivatives can be derived using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and (optionally) constant domains of a multispecific binding protein or its binding fragment or derivatives, for example, derived from an intact multispecific binding protein molecule. This DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable conformation or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc. Multispecific

[0279] As used herein, a "multispecific" binding protein is a binding protein that specifically binds to two or more types of antigens or epitopes. A multispecific binding protein that binds to two antigens and / or two different epitopes of different antigens is also referred to herein as a "bispecific" binding protein. A multispecific binding protein that binds to three antigens and / or three different epitopes is also referred to herein as a "trispecific" binding protein. Therefore, a multispecific binding protein can bind to two or more different targets simultaneously. A multispecific binding protein may contain two different antigen-binding sites and an Fc domain variant polypeptide that can bind to an Fc receptor. Genetic engineering can be used to design, modify, and generate multispecific binding proteins, or their binding fragments or derivatives, with a desired set of binding properties and effector functions.

[0280] Multispecific binding proteins may include antibodies or their antigen-binding fragments (e.g., scFv, Fab, Fab', Fv, F(ab')2), microantibodies, biantibodies, triantibodies, tetraantibodies, tandem di-scFv, tandem tri-scFv, immunoglobulin single variable domain (ISV), such as VHH (including humanized VHH), camelified VH, single domain antibody, domain antibody, or dAb).

[0281] In one aspect, the Fc domain variant peptide of this disclosure comprises or is complexed with a multispecific binding protein (e.g., fused with a multispecific binding protein). In another aspect, the binding peptide of this disclosure comprises a multispecific binding protein and an Fc domain variant peptide. Fc-containing binding peptides

[0282] In one aspect, this disclosure provides an isolated Fc domain variant polypeptide comprising or complexed with (e.g., fused with) at least one binding domain. In another aspect, the binding domain comprises one or more antigen-binding domains. The antigen-binding domain need not be derived from the same molecule as the Fc domain parent polypeptide. In another aspect, the Fc domain variant is present in an antibody. In one aspect, the binding polypeptide of this disclosure comprises a binding domain and an Fc domain variant polypeptide.

[0283] In one aspect, the Fc domain variant peptide is present in or complexed with an antibody. Any antibody from any source or species may be used with the Fc domain variant peptide disclosed herein. Suitable antibodies include, but are not limited to, chimeric antibodies, humanized antibodies, or human antibodies. Suitable antibodies include, but are not limited to, full-length antibodies, monoclonal antibodies, polyclonal antibodies, or single-domain antibodies, such as VHH antibodies. In one aspect, the binding peptide disclosed herein comprises an antibody and an Fc domain variant peptide.

[0284] In one aspect, the Fc domain variant peptides disclosed herein can bind to or complex with an antigen-binding fragment of an antibody. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes for antigen binding with the intact antibody (i.e., with the intact antibody from which they are derived) (i.e., specific binding). Antigen-binding fragments can be generated by recombinant or biochemical methods well known in the art. Exemplary antigen-binding fragments include variable fragments (Fv), Fab, Fab', and F(ab)2, microantibodies, biantibodies, triantibodies, tandem di-scFv, tandem tri-scFv, and immunoglobulin single variable domain (ISV). In one aspect, the binding peptides of this disclosure comprise at least one antigen-binding fragment and an Fc domain variant peptide.

[0285] The term "Fab" refers to a binding protein or its binding fragment having an antigen-binding molecular weight of about 50,000 DA and possessing antigen-binding activity, wherein in a fragment obtained by treating IgG with papain, approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds. In one aspect, the Fc domain variant peptides disclosed herein can bind to or complex with Fab. In another aspect, the binding peptides disclosed herein comprise at least one Fab and an Fc domain variant peptide.

[0286] The term F(ab')2 refers to a binding protein or its binding fragment with a molecular weight of about 100,000 DA and possessing antigen-binding activity, which, in the case of a fragment obtained by treating IgG with pepsin, is slightly larger than Fab bound via disulfide bonds in the hinge region. In one aspect, the Fc domain variant peptides disclosed herein can bind to or complex with F(ab')2. In another aspect, the binding peptides disclosed herein comprise at least one F(ab')2 and an Fc domain variant peptide.

[0287] The term Fab' refers to a binding protein or its binding fragment with a molecular weight of about 50,000 DA and possessing antigen-binding activity, obtained by cleaving the disulfide bonds in the F(ab')2 hinge region. In one aspect, the Fc domain variant peptides disclosed herein can bind to or complex with Fab'. In another aspect, the binding peptides disclosed herein comprise at least one Fab' and an Fc domain variant peptide.

[0288] In one aspect, the Fc domain variant polypeptide of this disclosure comprises a single-chain variable region sequence (ScFv). The single-chain variable region sequence comprises a single polypeptide having one or more antigen-binding sites, for example, a VL domain linked to a VH domain via a flexible linker. The ScFv molecule can be constructed with a VH-linker-VL orientation or a VL-linker-VH orientation. The flexible hinge connecting the VL and VH domains constituting the antigen-binding sites comprises about 10 to about 50 amino acid residues. Tandem scFvs (e.g., tandem di-scFvs or tandem tri-scFvs) are two or more ScFvs linked by peptide linkers. Linking peptides are known in the art. The binding polypeptide may comprise at least one scFv and / or at least one constant region. In one aspect, the Fc domain variant polypeptide of this disclosure may comprise at least one scFv linked to or fused with an Fc domain variant. In one aspect, the Fc domain variant polypeptide of this disclosure may comprise a tandem di-scFv or a tandem tri-scFv. In one aspect, the binding polypeptide disclosed herein comprises one or more ScFv and Fc domain variant polypeptides.

[0289] In one aspect, the Fc domain variant peptide of this disclosure is a multispecific antibody, for example, a multivalent or tetravalent antibody generated by fusing a DNA sequence encoding an antibody with an ScFv molecule (e.g., a modified ScFv molecule). For example, in one aspect, the Fc domain variant peptide sequence is combined such that the ScFv molecule (e.g., a modified ScFv molecule) is linked to the Fc domain variant via a flexible linker (e.g., a gly / ser linker) at its N-terminus or C-terminus. In another aspect, a tetravalent antibody can be prepared by fusing an ScFv molecule with a linker peptide to construct an ScFv-Fab tetravalent molecule. In one aspect, the binding peptide of this disclosure comprises an ScFv-Fab tetravalent containing an Fc domain variant peptide.

[0290] In one aspect, the Fc domain variant peptide of this disclosure is a modified microantibody. The modified microantibody of this disclosure is a dimer molecule composed of two polypeptide chains, each containing an ScFv molecule fused to the Fc domain variant peptide via a linker peptide. The microantibody can be prepared by constructing the ScFv component and linking the peptide component using methods described in the art (see, for example, U.S. Patent 5,837,821 or WO 94 / 09817A1). In another aspect, a tetravalent microantibody can be constructed. The tetravalent microantibody can be constructed in the same manner as the microantibody, except that the two ScFv molecules are linked using a flexible linker. This linked scFv-scFv construct is then linked to the Fc domain variant peptide. In one aspect, the binding peptide of this disclosure comprises a modified microantibody containing an Fc domain variant peptide.

[0291] In another aspect, the Fc domain variant peptides of this disclosure comprise biantibodies, triantibodies, or tetraantibodies. Biantibodies are polymeric, tetravalent molecules, each having a polypeptide similar to an scFv molecule, but typically possessing a short (less than 10, e.g., about 1 to about 5) amino acid residue linker connecting the two variable domains, such that the VL and VH domains on the same polypeptide chain do not interact. Instead, the VL and VH domains of one polypeptide chain interact (respectively) with the VH and VL domains of a second polypeptide chain (see, for example, WO 2002002781A1). Reducing the linker length to less than three residues can force scFv to bind as a trimer (tria, about 90 kDa) or a tetramer (about 120 kDa), depending on the linker length, composition, and V domain orientation. The biantibodies, triantibodies, or tetraantibodies of this disclosure may comprise scFv-like molecules fused to an Fc domain variant. In another aspect, the binding peptides of this disclosure comprise both a biantibody and an Fc domain variant peptide. In one aspect, the binding peptide of this disclosure comprises a triantibody and an Fc domain variant peptide. In another aspect, the binding peptide of this disclosure comprises a tetraantibody and an Fc domain variant peptide.

[0292] In one aspect, the Fc domain variant peptide of this disclosure is a VHH. The term “VHH” or “VHH antibody” is a type of single-domain antibody containing a variable heavy chain domain that does not contain a light chain. Similar to the conventional VH domain, VHH contains four FRs and three CDRs. VHH has advantages over conventional antibodies. Because they are about ten times smaller than IgG molecules, properly folded functional VHHs can be generated through in vitro expression while achieving high yields. In addition, VHH is very stable and resistant to the action of proteases. The properties and generation of VHH have been reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov; 77(1):13-22). In one aspect, the Fc domain variant peptide is present in or complexed with VHH (e.g., fused). In one aspect, the binding peptide of this disclosure comprises an Fc domain variant peptide fused with one or more VHHs.

[0293] In one aspect, the Fc domain variant peptides of this disclosure comprise an immunoglobulin single variable domain (ISV), such as a domain antibody fused to an Fc domain variant, a "dAb", a VHH (including humanized VHH), a camelified VHH, other single variable domains, or any suitable fragment thereof. In another aspect, the binding peptides of this disclosure comprise both an ISV and an Fc domain variant peptide.

[0294] The term "immunoglobulin single variable domain" (ISV or ISVD) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which an antigen-binding site is located on and formed by a single immunoglobulin domain. This distinguishes the immunoglobulin single variable domain from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V... H ) and light chain variable structural domain (V L The interaction between VH and VL forms the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of VH and VL will facilitate the formation of the antigen-binding site; that is, a total of 6 CDRs will participate in the formation of the antigen-binding site. The term "V" can be used in this paper. H ISVs belonging to the "Class 3" (i.e., ISVs with high sequence homology to VH3 phylogenetic sequences, such as DP-47, DP-51, or DP-29) or the so-called "VH4" ISVs (i.e., ISVs with high sequence homology to VH3 phylogenetic sequences, such as DP-47, DP-51, or DP-29) or those belonging to the so-called "VH4" H Class 4 ethnological sequences have highly sequence-homological ISVs (such as DP-78), as described in WO 2007 / 118670A1.

[0295] ISVs offer several advantages over conventional antibodies: they are approximately ten times smaller than IgG molecules, allowing for the generation of correctly folded functional ISVs through in vitro expression in high yields. Furthermore, ISVs are highly stable and resistant to protease activity. The characteristics and generation of ISVs have been reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov; 77(1):13-22).

[0296] In particular, ISVs (especially V) HH The sequence and partially humanized VHH) characteristics may be characterized by the presence of one or more "hallmark residues" (as described in Table 1 of this document and in the paragraphs describing the single variable domain of NANOBODY® immunoglobulins), making the ISV a NANOBODY® ISV.

[0297] Therefore, generally, NANOBODY® ISV (especially VHH, including (partially or fully) humanized VH and camelified VH) can be defined as an amino acid sequence with a (universal) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, where FR1 to FR4 refer to framework regions 1 to 4, and where CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, and where one or more of these marker residues are further defined as shown in Table 1. In particular, NANOBODY® ISV (especially VHH) HH Including (partial) humanized V HH and camel-like V H The ISV can be an amino acid sequence with the (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, where FR1 to FR4 refer to framework regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, as further defined herein. More specifically, the ISV can be an amino acid sequence with the following (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, where FR1 to FR4 refer to framework regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3.

[0298] The term “immunoglobulin single variable domain (ISV)” encompasses NANOBODY® VHH as described in WO 08 / 020079 or WO 09 / 138519, and therefore in one aspect denotes VHH, humanized VHH, or camelified VH (e.g., camelified human VH) or generally sequence-optimized VHH (e.g., optimized for chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression).

[0299] Typically, NANOBODY® immunoglobulin single variable domain (ISV) (especially V) HH Sequence, including (partially) humanized V HH Sequence and Camelization V H A sequence characterized by the presence of one or more “signature residues” (as described herein) in one or more frame sequences (also as further described herein). Therefore, generally, NANOBODY® ISV can be defined as an immunoglobulin sequence having the following (general) structure. FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, wherein one or more marker residues are further defined as in this paper.

[0300] Specifically, NANOBODY® ISV can be an immunoglobulin sequence having the following (general) structure. FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to frame regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively. The frame sequence is further defined in this paper.

[0301] More specifically, NANOBODY® ISV can be an immunoglobulin sequence having the following (universal) structure. FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to frame regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. According to the Kabat number, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 are selected from the marker residues mentioned in Table 1 below. Table 1: Key Residues in Nanobody® ISV

[0302] In one aspect, Fc domain variant peptides comprise multispecific or multivalent antibodies containing one or more variable domains tandemly linked on the same polypeptide chain, such as tandem variable domain (TVD) peptides. Exemplary TVD peptides include the “double-headed” or “double-Fv” configuration described in U.S. Patent No. 5,989,830. In the double-Fv configuration, the variable domains of two different antibodies are expressed in a tandem orientation on two separate chains (one heavy chain and one light chain), wherein one polypeptide chain has two tandem VH domains separated by a peptide linker (VH1-linker-VH2) and the other polypeptide chain consists of complementary VL domains tandemly linked by a peptide linker (VL1-linker-VL2). In a cross-headed 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), one polypeptide chain having two tandem VH domains separated by peptide linkers (VH1-linker-VH2) and the other polypeptide chain consisting of complementary VL domains tandemly linked by oppositely oriented peptide linkers (VL2-linker-VL1). Other antibody variants based on the “double Fv” format include bispecific antibodies with dual variable domains (DVD-IgG) (see U.S. Patent No. 7,612,181) and the TBTI format (see US 2010 / 0226923A1). In one aspect, the Fc domain variant polypeptide comprises a multispecific or multivalent antibody containing one or more variable domains tandemly on the same polypeptide chain fused to the Fc domain variant polypeptide. In another aspect, the binding polypeptide of this disclosure comprises a TVD polypeptide and an Fc domain variant polypeptide.

[0303] In one aspect, the Fc domain variant peptide comprises a bispecific antibody against a cross-linked dual variable domain IgG (CODV-IgG) based on a “two-headed” configuration (see US 20120251541A1, which is incorporated herein by reference in its entirety). In another aspect, the binding peptide of this disclosure comprises CODV-IgG containing an Fc domain variant peptide.

[0304] In one aspect, the Fc domain variant peptide comprises a CrossMab or CrossMab-Fab multispecific format (see WO 2009080253 and Schaefer et al., PNAS (2011), 108: 11187-1191). Antibody variants based on the CrossMab format have cross-links of antibody domains within one arm of a bispecific IgG antibody, thereby achieving proper chain association. In another aspect, the binding peptide of this disclosure comprises a CrossMab containing an Fc domain variant peptide.

[0305] In one aspect, the Fc domain variant peptide may comprise a glycosylated effector-capable peptide comprising a multispecific antibody in the form of a T-cell binder. A “T-cell binder” refers to a binding protein targeting both the host immune system (more specifically, the cytotoxic activity of T cells) and tumor target proteins. In some embodiments, the isolated effector-capable peptide comprises a multispecific antibody in the form of an NK-cell binder. An “NK-cell binder” refers to a binding protein comprising a monoclonal antibody fragment targeting an activated NK cell receptor, an antigen-specific targeting region, and an Fc region (Gauthier et al., Cell (2019), 177: 1701-13). In one aspect, the binding peptide of this disclosure comprises a T-cell binder containing an Fc domain variant peptide. In another aspect, the binding peptide of this disclosure comprises an NK-cell binder containing an Fc domain variant peptide.

[0306] The Fc domain variant peptides of this disclosure, which include the Fc domain variants described herein, may include the CDR sequence of a known “parental” antibody or a variable domain sequence. In some embodiments, the parental antibody and the antibody of this disclosure may share similar or identical sequences, except for modifications to the Fc domain disclosed herein.

[0307] In one aspect, the Fc domain variant polypeptide comprises a therapeutic polypeptide. In another aspect, the therapeutic polypeptide may be a receptor, ligand, or enzyme. In another aspect, the therapeutic polypeptide may be a coagulation factor. In another aspect, the coagulation factor is selected from the group consisting of: FI, FII, FIII, FIV, FV, FVI, FVII, FVIII, FIX, FX, FXI, FXII, FXIII), VWF, prokaloplasmin, high molecular weight kininogen, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-associated protease inhibitor (ZPI), plasminogen, α2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI-2), any of their proenzymes, any of their active forms, and any combination thereof. In another aspect, the therapeutic polypeptide may be a growth factor. The growth factor may be selected from any growth factor known in the art. In another aspect, the growth factor is a hormone; in another aspect, the growth factor is a cytokine; in another aspect, the growth factor is a chemokine; in another aspect, the binding peptide comprises a therapeutic molecule or therapeutic peptide linked to the N-terminus and / or C-terminus of the Fc domain variant peptide described herein; in another aspect, the Fc domain variant peptide is an Fc fusion peptide. Fc receptor binding site

[0308] Typically, Fc domain variant peptides, as described herein, function by binding to Fc receptors and target proteins with high affinity or strong affinity. In some respects, Fc domain variant peptides can bind to Fc receptors and / or target proteins, whose K... D Measured by surface plasmon resonance, the value is less than about 1 μM (e.g., at 25°C or 37°C). In some aspects, Fc domain variant peptides bind to Fc receptors and / or target proteins, K... D Less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, or less than about 1 nM, as measured by surface plasmon resonance.

[0309] In some aspects, the Fc domain variant peptides described herein bind to Fc receptors with a dissociation half-life (t½) greater than about 1.1 minutes, as measured by surface plasmon resonance at about 25°C or 37°C. In some aspects, the Fc domain variant peptides bind to Fc receptors and target proteins with t½ times greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1200 minutes, as measured by surface plasmon resonance at 25°C or 37°C.

[0310] In some aspects, Fc domain variant peptides bind to Fc receptors on cell surfaces with an affinity of about 100 pM to about 1 µM (e.g., about 100 pM to about 1,000 pM, about 1,000 pM to about 0.01 µM, about 0.01 µM to about 0.1 µM, or about 0.1 µM to about 1.0 µM). Sugar Project Transformation

[0311] As used herein, the term "glycoengineering" refers to any method for altering the glycoform profile of an Fc domain polypeptide (an Fc domain variant or an Fc domain parent polypeptide) to produce a "modified glycan". In some embodiments, glycoengineered Fc domain polypeptides and / or binding proteins comprising Fc domains, as well as methods for preparing glycoengineered binding proteins, are provided or incorporated herein by reference to WO 2014164503A1, WO 2015143091A1, WO 2016057769A2 and U.S. Provisional Patent Application Serial No. 63 / 419,188.

[0312] In one aspect, the Fc domain variant peptide is glycoengineered. In another aspect, the Fc domain variant peptide contains modified glycans. In one aspect, the Fc domain variant peptide is about 20%, 30%, 40%, 50%, 60%, 70%, 90% or more unfucosylated. In another aspect, the Fc domain variant peptide substantially does not contain fucose.

[0313] On the other hand, compared to the parental Fc domain peptide, the Fc domain variant peptide contains a modified number of fucosylated residues. For example, the Fc domain variant peptide may have a reduced number of fucosylated residues on the N-glycan at Asn297 in the Fc region, i.e., it is unfucosylated. Unfucosylation can increase FcγRII binding on NK cells and effectively increase ADCC. Glycosylated Fc domain variant peptides

[0314] In some respects, Fc domain peptides (Fc domain variants or Fc domain parent peptides) are glycosylated. Glycosylation at conserved sites in the constant region of an antibody can have a profound impact on antibody function, particularly effector function (as described above), see, for example, Boyd et al. (Mol. Immunol, 32: 1311-1318, 1996). Glycosylation of Fc domain variants described herein is considered, wherein one or more carbohydrate moieties are added, substituted, deleted, or modified. In some embodiments, Fc domain glycosylation is N-linked glycosylation. The introduction of asparagine-X-serine or asparagine-X-threonine motifs creates potential sites for enzymatic attachment of carbohydrate moieties and can therefore be used to manipulate the glycosylation of Fc domain variant peptides. In Raju et al. (Biochemistry 40: 8868-8876, 2001), the terminal sialylation of TNFR-IgG immunoadhesins was increased through regalactosylation and / or resialylation processes using β-1,4-galactosyltransferase and / or α,2,3-sialyltransferase. They suggested that increased terminal sialylation could increase the half-life of immunoglobulins.

[0315] Like most glycoproteins, antibodies are typically produced as a mixture of glycoforms. This mixture is particularly evident when antibodies are produced in eukaryotic cells, especially mammalian cells. Several methods have been developed to produce specific glycoforms (see Zhang et al., 2004, Science 303: 371; Sears et al., 2001, Science 291:2344; Wacker et al., 2002, Science 298: 1790; Davis et al., 2002, Chem. Rev. 102:579; Hang et al., 2001, Acc. Chem. Res. 34: 727). In one aspect, according to EU numbering, the glycosylated Fc domain contains a native glycan at amino acid position 297. Glycosylation of asparagine at amino acid position 297 in the CH2 domain of IgG1 can promote the interaction between the Fc domain and FcγR. Eliminating this glycosylation site eliminates effector function (Leabman et al., 2013, MAbs 5:896-903). On the other hand, according to EU designations, Fc domain variant peptides contain glycosylation at amino acid position 297 at a wild-type level or near-wild-type level.

[0316] In one aspect, the glycosylated Fc domain variant peptide contains engineered or non-natural glycans. In another aspect, the engineered or non-natural glycans are modified glycans that can be coupled to therapeutic molecules (e.g., antibody-drug conjugates). N-linked glycosylation modified by engineering

[0317] Exemplary aspects of modified glycans connected to the Fc domain via engineered glycosylation sites present on the Fc domain are described in WO 2014043361A1 (which is incorporated herein by reference in its entirety).

[0318] In some aspects, the Fc domain variant peptides of this disclosure, comprising an Fc domain, have engineered N-linked glycosylation sites. In some aspects, the Fc domain variant peptides of this disclosure contain one or more mutations or glycan modifications to regulate Fc-mediated effector functions. In some aspects, the Fc domain variant peptides may contain one or more mutations to regulate serum half-life.

[0319] In some aspects, the Fc domain variant peptides of this disclosure contain mutations or glycan modifications to regulate Fc-mediated effector function. In some aspects, the Fc domain variant peptides contain one or more mutations or glycan modifications to regulate serum half-life. Oligomannose-type N-glycans

[0320] Exemplary aspects of modified glycans linked to Fc domain variant peptides comprising oligomannose and methods for their preparation are described in U.S. Provisional Patent Application Serial No. 63 / 419,188 (which is incorporated herein by reference in its entirety).

[0321] As used herein, the term "oligomannose-type N-glycan" or "oligomannose" refers to any one or a combination of the following mannose-rich structures: Man5(GlcNAc)2, Man6(GlcNAc)2, Man7(GlcNAc)2, Man8(GlcNAc)2, and Man9(GlcNAc)2 (see Schachter et al., Mannose Oligosaccharide, 4.06.3.3.1, Comprehensive Glycoscience, 2007.). As used herein, Man5 refers to the structure Man5(GlcNAc)2; Man6 refers to the structure Man6(GlcNAc)2; Man7 refers to the structure Man7(GlcNAc)2; Man8 refers to the structure Man8(GlcNAc)2; and Man9 refers to the structure Man9(GlcNAc)2. In some respects, the oligomannose is Man8(GlcNAc)2 or Man9(GlcNAc)2.

[0322] In one aspect, the Fc domain variant polypeptide of this disclosure may contain a modified glycan. In another aspect, the modified glycan is mannose. In another aspect, mannose is an oligomannose-type N-linked glycan. In another aspect, the oligomannose-type N-glycan comprises oligosaccharides selected from the group consisting of: Man9(GlcNAc)2, Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2, and Man5(GlcNAc)2.

[0323] In one aspect, the Fc domain variant peptides of this disclosure comprise 20%, 30%, 40%, 50%, 60%, 70%, 90% or more of Man in a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 polysaccharide.

[0324] In one aspect, the Fc domain variant polypeptide of this disclosure comprises greater than 70%, 75%, 80%, 85%, 90%, or 95% Man in a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 N-glycan.

[0325] In one aspect, the Fc domain variant peptide of this disclosure contains Man8 and Man9 together as Man 5-9 The main types of (GlcNAc)2 N-glycans.

[0326] In one aspect, the Fc domain variant polypeptide of this disclosure comprises at least 97% Mann based on a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 N-glycan.

[0327] In one aspect, the Fc domain variant peptides of this disclosure comprise less than 30%, 20%, 10%, 5%, and 1% Man, in molar ratios relative to all N-glycans. 5-9 (GlcNAc)2 polysaccharide, or essentially no man in molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 polysaccharide.

[0328] In one aspect, the Fc domain variant polypeptide of this disclosure further includes cysteine ​​(C) at amino acid position 292 and cysteine ​​(C) at amino acid position 302. Chifu base

[0329] In one aspect, this paper discloses Fc domain variant peptides comprising oligomannose-type N-glycans. In another aspect, the oligomannose-type N-glycans are produced by culturing engineered cells to express binding peptides in the presence of a mannosidase inhibitor (e.g., α-mannosidase I inhibitor chifine or a derivative or functional homolog thereof). In yet another aspect, treatment of cells with a mannosidase inhibitor results in the production of Fc domain variant peptides carrying oligomannose-type N-glycans (e.g., antibodies or antigenic fragments thereof comprising Fc domain variants) while preventing the formation of complex N-glycans.

[0330] Cells engineered to express Fc domain variant peptides may lack one or more glycosidases required for early processing of N-glycans. In some respects, culture conditions can inhibit the activity of one or more of these glycosidases. Due to the effect of one or both of these conditions, oligosaccharide synthesis shifts to the oligomannose class. For example, cells may lack one or more glycosidases selected from the group consisting of α-glucosidase I, α-glucosidase II, and α-mannosidase I. Cells lacking the glycosidase of interest can be engineered using methods as described below: e.g., Tymms et al., Gene Knockout Protocols (Methods in Molecular Biology), Human Press, 1st ed., 2001; and Joyner, Gene Targeting: A Practical Approach, Oxford University Press, 2nd ed., 2000. For example, lectin selection can be used to engineer cells lacking the glycosidase. See Stanley et al., (1975) Biochemistry, 72 (9):3323-3327.

[0331] In some respects, Fc domain variant peptides containing oligomannose-type N-glycans can be generated by chemically linking an antibody to an unglycosylated Fc domain variant peptide or an Fc fusion protein containing an Fc domain variant and a separately synthesized oligosaccharide moiety.

[0332] In another approach, cells can be engineered to prevent the expression of one or more glycosidases selected from the group consisting of α-glucosidase I, α-glucosidase II, and α-mannosidase I. Glycosidase genes can be disrupted through targeted mutagenesis, for example, by targeting CRISPR (clustered regularly spaced short palindromic repeats) sites within the glycosidase gene. In yet another approach, cells can be engineered to prevent the expression of glycosidase genes using one or more expression vectors that encode at least the target RNA and a CRISPR-associated nuclease (e.g., Cas9).

[0333] In other respects, cells engineered to express Fc domain variant peptides can be contacted with inhibitors of one or more glycosidases selected from the group consisting of α-glucosidase I, α-glucosidase II, and α-mannosidase I. In some respects, these enzyme inhibitors may be, for example, small molecules or small interfering RNAs (siRNAs). siRNAs are short (20-25 nt) double-stranded RNAs that inhibit the glycosidase of interest by post-transcriptional gene silencing. Glycosidase-specific siRNAs may be prepared as described and used as in U.S. Patent No. 6,506,559 and / or using other suitable methods (see Appasani, RNA Interference Technology From BasiCScience to Drug Development, Cambridge University Press, 1st ed., 2005; and Uei-Ti et al. (2004) Nucleic Acids Res., 32(3):936948). Examples of small molecule α-glucosidase I inhibitors include castanospermine (see Pan et al. (1983) Biochemistry, 22:3975-3984), deoxynojirimycin (see DNJ; Hettkamp et al. (1984) Eur. J. Biochem., 142:85-90) and its N-alkyl and N-alkenyl derivatives (e.g., N-butyl-DNJ); 2,5-dihydromethyl-3,4-dihydroxypyrrolidine (see DMDP; Elbein et al. (1984) J. Biol. Chem., 259:12409-12413); and australine (see Molyneux et al. (1988) J. Nat. Prod., 51:1198-1206). Examples of small molecule α-glucosidase II inhibitors include DNJ and its N-alkyl and N-alkenyl derivatives; and MDL 25637 (see Hettkamp et al. (1984) Eur. J. Biochem., 142: 85-90; and Kaushal et al. (1988) J. Biol. Chem., 263: 17278-17283).Examples of small molecule α-mannosidase I inhibitors include mannojirimycin (DMJ) (see Legler et al. (1984) Carbohydr. Res., 128:61-72) and its derivatives (e.g., N-methyl derivatives, such as those described in Bosch et al. (1985) Virology, 143:342-346), 1,4-dideoxy-1,4-imino-D-mannitol (DIM) (see Fleet et al. (1984) J. Chem. Soc. Chem. Commun., 1240-1241 and Palmarzyk et al. (1985) Arch. Biochem. Biophys., 243:35-45) and chiffon bases (see Elbein (1990) J. Biol. Chem., 265:15599-15605).

[0334] In one aspect, the Fc domain variant polypeptide comprising the modified glycan is produced by culturing cells expressing the Fc domain variant polypeptide in the presence of an α-mannosidase I inhibitor (e.g., chifine). In another aspect, chifine can be used at concentrations of 0.01 μg / ml to 100 μg / ml, 0.01 μg / ml to 75 μg / ml, 0.01 μg / ml to 50 μg / ml, 0.01 μg / ml to 40 μg / ml, 0.01 μg / ml to 30 μg / ml, 0.01 μg / ml to 20 μg / ml, 0.1 μg / ml to 10 μg / ml, 0.1 μg / ml to 2.0 μg / ml, or 1 μg / ml to 0.5 μg / ml for at least 12 hours, 24 hours, 48 ​​hours, 72 hours, or 4 days, 7 days, 10 days, 20 days, or longer, or continuously. In another embodiment, CHO or fusion tumor cells are incubated with chifrine at a concentration of approximately 0.5 μg / ml to 10 μg / ml for more than 10 days. In another embodiment, chifrine is used at a concentration of approximately 60 ng / ml to about 2500 ng / ml. In yet another embodiment, chifrine is used at a concentration of 2000 ng / ml. Oligomannose-type N-glycan-bound peptides

[0335] In another aspect, the compositions produced by the methods disclosed herein contain at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more (in molar ratio relative to all N-glycans) of oligomannose-type glycans. 5-9(GlcNAc)2. In another aspect, a composition comprising isolated glycosylated Fc domain variant polypeptide groups, each comprising an N-glycan, wherein the composition comprises at least 50% Mann based on a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2N-polysaccharide.

[0336] In another aspect, compositions comprising the Fc domain variant peptides disclosed herein contain Man8 and Man9 N-glycans as the predominant type of N-glycan. In yet another aspect, compositions comprising a group of isolated glycosylated Fc domain variant peptides containing N-glycans are provided, wherein the composition comprises at least 50% Man8 and Man9 N-glycans in a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 N-glycan, and Man8 and Man9, which contain N-glycan, are the main types.

[0337] In one aspect, the Fc domain variant peptides disclosed herein primarily contain Man9(GlcNAc)2 N-glycan. In another aspect, compositions comprising isolated groups of glycosylated Fc domain variant peptides, each comprising an Fc domain variant containing an N-glycan, are provided, wherein the composition comprises at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 99% Man9 in a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 N-glycan. In one aspect, a composition comprising a group of isolated glycosylated Fc domain variant peptides comprising N-glycans is provided, wherein the composition comprises at least 97% Mann based on a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2N-polysaccharide.

[0338] In one aspect, the binding peptide containing the Fc domain variants disclosed herein contains reduced or undetectable amounts of oligomannose-type N-glycans Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2, and Man5(GlcNAc)2, while also containing trace amounts (e.g., less than 10% relative to all N-glycans) or undetectable amounts of complex N-glycans (e.g., G0, C1, G2, G0F, G1F, G2F, and G0F-Gn).

[0339] In another aspect, the composition contains less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% (in molar ratio relative to all N-glycans) or less of Man5(GlcNAc)2 and / or Man6(GlcNAc)2 N-glycans. In another aspect, the composition contains a small amount (i.e., less than 10% in molar ratio relative to all N-glycans) or an undetectable amount of Man4(GlcNAc)2. In another aspect, the composition contains less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% Man. 5-9 (GlcNAc)2-glycan (in molar ratio relative to all N-glycans) or substantially free of Man in molar ratio relative to all N-glycans 5-9 (GlcNAc)2. In another aspect, the composition contains less than 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (in molar ratio relative to all N-glycans) or less of a complex polysaccharide.

[0340] The glycan compositions can be evaluated using, for example, lectin blotting, HPLC and / or mass spectrometry (such as MALDI-TOF) (see, for example, Townsend et al. (1997) Techniques in Glybiology, CRC Press).

[0341] In one aspect, Fc domain variant peptides carrying oligomannose-type glycans exhibited enhanced ADCC activity compared to the same Fc domain variant peptides produced without treatment with mannosidase inhibitors (e.g., chiffonines). In another aspect, Fc domain variant peptides carrying oligomannose-type glycans exhibited enhanced binding to Fc receptors. In one aspect, Fc domain variant peptides carrying oligomannose-type glycans exhibited enhanced binding to Fcγ receptors. In another aspect, Fc domain variant peptides carrying oligomannose-type glycans exhibited enhanced binding to FcγRIIIa.

[0342] In another instance, Fc domain variant peptides carrying oligomannose-type glycans exhibited substantially the same or better binding specificity to the target. In yet another instance, Fc domain variant peptides carrying oligomannose-type glycans exhibited substantially the same or higher binding affinity to the target. In yet another instance, Fc domain variant peptides carrying oligomannose-type glycans exhibited substantially the same or lower binding affinity to the mannose receptor. Other Fc mutations and functions

[0343] In some aspects, the Fc domain variant peptides of this disclosure comprise a modified Fc domain and may further comprise one or more (e.g., two or more, three or more, or four or more) amino acid substitutions that impart one or more biochemical characteristics to the Fc domain variant peptides in addition to glycan modification.

[0344] Exemplary modified Fc domain amino acid substitutions that may impart additional biochemical characteristics to the Fc domain variant peptides described herein are disclosed in WO 2021016571A2 (which is incorporated herein by reference in its entirety).

[0345] In some respects, the modified Fc region of the Fc domain variant peptides of this disclosure contains one or more mutations to regulate half-life (see, for example, Dall'Acqu A et al. (2006) J Biol Chem 281: 23514-24, Zalevsky et al. (2010) Nat Biotechnol 28: 157-9, Hinton et al. (2004) J Biol Chem 279:6213-6, Hinton et al. (2006) J Immunol 176: 346-56, Shields et al. (2001) J Biol Chem 276: 6591-604, Petkov A et al. (2006) Int Immunol 18:1759-69, Datta-Mannan et al. (2007) Drug MetaBDispos 35: 869-874, Vaccaro et al. (2005) Nat Biotechnol 23:1283-8, Yeung et al. (2010) Cancer Res 70: 3269-77 and Kim et al. (1999) Eur J Immunol 29: 2819-25. (e.g., T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and / or H435R).

[0346] In some respects, the Fc domain variant peptides of this disclosure may exhibit enhanced FcRn binding affinity at both acidic pH (e.g., less than about 7.0, not greater than about 6.5, or not greater than about 6.0) and non-acidic pH (e.g., not less than about 7.0, or not less than about 7.4) compared to the corresponding Fc domain parent peptide (e.g., a molecule having the same structure as an FcRn antagonist, except that it has a wild-type Fc domain). In another example, the Fc domain variant peptide may contain one or more amino acid mutations (e.g., substitutions) that alter the effector function (e.g., ADCC or CDC function) of the Fc domain. In another example, the Fc domain variant peptide may contain a modified Fc domain comprising one or more amino acid mutations (e.g., substitutions) that alter (e.g., increase or decrease) the circulating half-life (e.g., serum half-life) of the FcRn antagonist compared to the corresponding Fc domain parent peptide.

[0347] In some aspects, the Fc domain variant peptides described herein may comprise a modified Fc domain that alters the serum half-life compared to the parent Fc domain peptide. In some aspects, the Fc domain variant peptide has an increased serum half-life compared to the parent Fc domain peptide. In some aspects, the Fc domain variant peptide is modified to alter the FcRn binding affinity compared to the parent Fc domain peptide. In some aspects, the modified Fc domain has enhanced FcRn binding affinity compared to the parent Fc domain peptide. In some aspects, the Fc domain variant peptide is modified to enhance FcRn binding affinity at acidic pH compared to the parent Fc domain peptide. Fc domain variant peptide expression and purity

[0348] Despite numerous advances in recombinant and cell culture methodologies, ensuring proper protein folding remains a major obstacle to the most widespread attempts to produce commercially useful quantities of desired Fc domain variant peptides. All Fc domain variant peptides must achieve appropriate two-dimensional and three-dimensional conformations to function while retaining the ability to modulate functional properties, such as effector function compared to the Fc domain parent peptide. Therefore, in addition to the regulation of functional properties, the protein expression and purity of the Fc domain variant peptides disclosed herein can also be evaluated.

[0349] Standard techniques can be used to express and purify the Fc domain variant peptides of this disclosure. For example, recombinant DNA, oligonucleotide synthesis, cell transfection (e.g., but not limited to electroporation, liposome-mediated transfection, transformation methods), and cell and tissue culture methods can all be used to express the Fc domain variants of this disclosure. Enzymatic reactions and purification techniques can be performed as described in the manufacturer's instructions or as commonly performed as described herein. The foregoing techniques and procedures can be performed according to the methods described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., eds., *Molecular Cloning: A Laboratory Manual*, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) and Ausubel et al., eds., *Current Protocols in Molecular Biology* (John Wiley & Sons, New York, 2012), which are incorporated herein by reference.

[0350] In one aspect, the Fc domain variant peptides of this disclosure comprise production titrations ranging from about 5 mg / L, 10 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L to about 500 mg / L. In another aspect, the Fc domain variant peptides comprise a purity of at least 60%, 70%, 80%, 90%, or 100% when measured by size exclusion chromatography (SEC).

[0351] In one aspect, this disclosure provides a method for generating Fc domain variant peptides, the method comprising screening for Fc domain variants, wherein the Fc domain variant peptide is selected if: (a) the Fc domain variant peptide has at least a 2-fold increased binding affinity to the Fc receptor relative to the Fc domain parent peptide; (b) the Fc domain variant peptide production titration is at least 10 mg / L; and (c) the Fc domain variant peptide has at least 95% purity when measured by SEC. thermal stability

[0352] It remains difficult to predict a priori which Fc domain variant peptides will be affected by protein instability, such as the tendency of proteins to unfold or form aggregates due to temperature changes. Therefore, thermal stability screening of Fc domain variant peptides can serve as an alternative to assessing conformational stability under in vivo and long-term storage conditions. Thus, in addition to analyses concerning the regulation of protein expression, protein purity, and functional properties, thermal stability screening can also evaluate the Fc domain variant peptides of this disclosure.

[0353] Melting temperature (Tm) is a measure of protein thermal stability, where an increase in Tm reflects the stabilizing effect of selected conditions on protein thermal stability. Methods that can be used to obtain Tm values ​​include, for example, differential scanning calorimetry (DSC), differential scanning fluorescence (DSF), or circular dichroism (CD).

[0354] In one aspect, the Fc domain variant peptide exhibits a Tm greater than 44°C. In another aspect, the Fc domain variant peptide exhibits a Tm greater than at least 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, and 75°C.

[0355] In one aspect, a method for generating Fc domain variant peptides is provided, the method comprising screening for Fc domain variants, wherein the Fc domain variant peptide is selected if: (a) the Fc domain variant peptide exhibits at least a 2-fold increased binding affinity to the Fc receptor relative to the Fc domain parent peptide; (b) the Fc domain variant peptide production titration is at least 10 mg / L; and (c) the Fc domain variant peptide has a purity of at least 95% when measured by SEC; and / or (d) the Fc domain variant peptide is selected if it exhibits a Tm greater than 44°C. target protein

[0356] The Fc domain variant polypeptides disclosed herein may include a binding moiety that binds to a target protein. As used herein, a "target protein" may be a protein with a harmful function and whose degradation may be therapeutically beneficial. In some aspects, the target protein is a membrane-associated target protein, a soluble target protein, or both. In some aspects, the target protein is a pathogenic protein or a peptide that causes disease or disease symptoms. In some aspects, the target protein is an immune checkpoint protein, a cancer antigen, and / or an immunomodulatory protein. In some aspects, the target protein is associated with diseases selected from the group consisting of: cancer, autoimmune diseases, inflammatory disorders, infectious diseases, and neurodegenerative disorders.

[0357] In other respects, the target protein is a soluble protein. Exemplary target proteins or peptides include proteins or peptides secreted by the tumor, inflammatory proteins or peptides; signaling molecules, including cytokines, interleukins, interferons, tumor necrosis factor, growth factors, hormones, neurotransmitters, lipid mediators, activators, extracellular matrix (ECM) proteins, Wnt proteins, members of the transforming growth factor β (TGF-β) family, and Notch ligands. In some respects, the target protein is selected from the group consisting of antibodies, autoantibodies, inflammatory proteins, interleukins, cytokines, interferons, tumor necrosis factor (TNF), growth factors, hormones, neurotransmitters, lipid mediators, activators, extracellular matrix (ECM) proteins, Wnt proteins, members of the transforming growth factor β (TGF-β) family, Notch ligands, and immune checkpoint proteins.

[0358] In some respects, the target protein is an antigen. An antigen is a molecule that can trigger an immune response. In some respects, the antigen is a self-antigen or autoantigen produced in the cells of a subject. For example, an antigen may be a surface marker expressed on a specific cell type, thereby allowing the multispecific binding protein of this disclosure to selectively target and modulate those cells. By binding to CD25+ cells via the CD25-binding moiety, the antigen-targeting multispecific binding protein of this disclosure can enhance immune responses, promote cell-mediated cytotoxicity, or modulate immune cell function in immunotherapy.

[0359] In other respects, the target protein is an antibody (e.g., an autoantibody) or a fragment thereof. An autoantibody is an antibody that specifically binds to one or more antigens prepared or formed by the subject's own body. Autoantibodies mistakenly recognize and target their own antigens, leading to autoimmune diseases. By binding an autoantibody as a second binding component, a multispecific binding protein or its binding fragment can specifically bind to autoantigens associated with autoimmune disorders. This approach offers potential for targeted therapy by redirecting the immune response toward self-reactive cells or molecules involved in the autoimmune process. Disease indications

[0360] In one aspect, this disclosure provides a method for treating a disease or condition in a subject in need, the method comprising administering to the subject an effective amount of the disclosed Fc domain variant polypeptide. In some embodiments, this disclosure provides kits and methods for treating diseases and disorders (e.g., cancer) in mammalian subjects in need of such treatment.

[0361] The Fc domain variant peptides disclosed herein can be used for a wide range of applications. For example, in one aspect, the subject Fc domain variant peptides can be used to reduce or eliminate cells carrying epitopes recognized by the binding domain of the Fc domain variant. In another aspect, the subject Fc domain variant peptides are effective in reducing or eliminating circulating soluble antigens. In yet another aspect, the subject Fc domain variant peptides are effective as T cell binders. In one aspect, the Fc domain variant peptides can reduce tumor size, inhibit tumor growth, and / or prolong the survival time of tumor-bearing animals. Therefore, this disclosure also relates to a method of treating tumors in humans or other animals by administering an effective and non-toxic amount of the Fc domain variant peptide to such humans or animals.

[0362] In another respect, the Fc domain variant peptides can be used to treat other disorders, including but not limited to infectious diseases, autoimmune disorders, inflammatory disorders, lung diseases, neuronal or neurodegenerative disorders, liver diseases, spinal diseases, uterine diseases, depression, etc. Non-limiting examples of infectious diseases include infectious diseases caused by RNA viruses (e.g., orthomyxoviruses (e.g., influenza), paramyxoviruses (e.g., respiratory syncytial virus, parainfluenza virus, interstitial pneumonia virus), rhabdoviruses (e.g., rabies virus), coronaviruses (e.g., SARS-CoV), alpha viruses (e.g., chikungunya virus), lentiviruses (e.g., HIV), etc.) or DNA viruses. Examples of infectious diseases include, but are not limited to, bacterial infectious diseases caused by, for example, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus, Streptococcus, and Escherichia coli, and other infectious diseases, including, for example, those caused by Candida albicans. Other infectious diseases include, but are not limited to, malaria, SARS, yellow fever, Lymeborreliosis, leishmaniasis, anthrax, and meningitis. Exemplary autoimmune disorders include, but are not limited to, psoriasis and lupus. Therefore, this disclosure relates to methods for treating various conditions, which will benefit from the use of Fc domain variant peptides with effector regulatory functions (e.g., extended half-life).

[0363] Those skilled in the art can determine an effective and non-toxic dose of the Fc domain variant peptide for treating malignant tumors through routine experiments. For example, the therapeutically active dose of the Fc domain variant peptide of this disclosure can vary depending on factors such as disease stage (e.g., stage I or IV), age, sex, medical complications (e.g., immunosuppressive status or disease), and the subject's weight, as well as the ability of the modified antibody to elicit the desired response in the subject. Dosing regimens can be adjusted to provide optimal therapeutic response. For example, as indicated by an emergency situation in the treatment context, several fractional doses may be administered daily or the dose may be proportionally reduced.

[0364] Typically, the compositions provided in this disclosure can be used for preventative or therapeutic treatment of any neoplasm containing an antigenic marker that allows Fc domain variants to target cancer cells. Other terms (including polynucleotide / carrier / host cell / drug composition) Kabat and EU numbering

[0365] Amino acid positions in the heavy chain constant region (including those in the CH1, hinge, CH2, CH3, and CL domains) can be numbered according to the Kabat indexing system (see Kabat et al., "Sequences of Proteins of Immunological Interest," US Dept. Health and Human Services, 5th ed., 1991). Alternatively, antibody amino acid positions can be numbered according to the EU indexing system. See Kabat et al. Specific binding

[0366] As used herein, the term "specific binding" refers to an antibody, its antigen-binding fragment, or an Fc domain variant polypeptide up to approximately 1 × 10⁻⁶. -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 The ability to bind antigens or Fc receptors with a dissociation constant (M) or lower (KD), and / or the ability to bind antigens or Fc receptors with an affinity at least twice that of nonspecific antigens or receptors. Antibodies can specifically bind to target antigens via the CDR sequence. Antibodies containing Fc domain variant peptides can also specifically bind to FcRs (such as FcRn or FcγRIIIa) via the Fc region. dissociation constant

[0367] The dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance (SPR). Typically, SPR analysis measures the instantaneous binding interaction between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) using a Biacore system (CytivALife Sciences, Marlborough, Massachusetts) or a CarterrALSA platform (Carterra, Salt Lake City, UT). SPR analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). As used herein, the term “KD” refers to the dissociation constant of the interaction between a specific binding protein and a target antigen or Fc receptor. price

[0368] As used herein, the term "valence" refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to one target molecule or a specific site on a target molecule. When a polypeptide contains more than one target binding site, each target binding site may specifically bind to the same or different molecules (e.g., it may bind to more than one target protein, or different epitopes on the same target protein). Subject Fc domain variant polypeptides may have at least one Fc receptor-specific binding site (e.g., 1, 2, 3 or more). Subject Fc domain variant polypeptides may have at least one target protein binding site (e.g., 1, 2, 3, 4 or more). Specificity

[0369] The term "specificity" refers to the ability to specifically bind to a given target antigen (e.g., an Fc receptor) (e.g., to induce an immune response). In some respects, Fc domain variant peptides are specific to different types of Fc receptors.

[0370] For example, Fc domain variant peptides are specific for Fc-γ receptors (FcγR), Fc-α receptors (FcαR), Fc-ε receptors (FcεR), and / or neonatal Fc receptors (FcRn). In one aspect, Fc domain variant peptides are specific for FcγR and FcRn, but not for FcαR or FcεR. In another aspect, Fc domain variant peptides are specific for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb.

[0371] In some aspects, the Fc domain variant peptides of this disclosure employ an Fc receptor-binding portion that binds to human Fc receptors but not to Fc receptors from other species. Alternatively, the Fc domain variant peptides bind to human Fc receptors and to Fc receptors from one or more non-human species. For example, the Fc domain variant peptides may bind to human Fc receptors and may bind or not (as appropriate) to one or more of the Fc receptors of mice, rats, guinea pigs, hamsters, gerbils, pigs, cats, dogs, rabbits, goats, sheep, cattle, horses, camels, cynomolgus monkeys, marmosets, rhesus monkeys, or chimpanzees. In some aspects, the Fc domain variant peptides may bind to human Fc receptors but not to rat and mouse Fc receptors. In other aspects, the Fc domain variant peptides bind to human Fc receptors as well as rat and mouse Fc receptors with similar binding affinity. about or approximately

[0372] The terms “about” or “approximately” mean about 20% of a given value or range (such as about 10%, about 5%, or about 1% or less).

[0373] As used herein, “administer or administration” means the act of injecting or otherwise physically delivering a substance present outside the body (e.g., the Fc domain variant peptide provided herein) to a patient via injection, through methods such as, but not limited to, pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When managing or treating a disease or its symptoms, the substance is typically administered after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the substance is typically administered before the onset of the disease or its symptoms and may continue for an extended period to delay or reduce the appearance or severity of disease-related symptoms. Composition

[0374] As used herein, the term "composition" is intended to cover products containing optionally specified amounts of specified ingredients (e.g., Fc domain variant peptide compositions provided herein), and any products directly or indirectly produced from combinations of optionally specified amounts of specified ingredients. Effective amount

[0375] "Effective amount" means the amount of an active pharmaceutical ingredient (e.g., an Fc domain variant peptide of this disclosure) sufficient to achieve the desired physiological outcome in an individual in need of the agent. Effective amounts can vary between individuals depending on the health and physical condition of the individual being treated, the taxonomy of the individual being treated, the formulation of the composition, the assessment of the individual's medical symptoms, and other relevant factors. Subject or patient

[0376] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject may be a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). In some respects, as used herein, the term “subject” refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, untamed animals, farm animals, sporting animals, and pets. Therapeutic and pharmaceutical compositions

[0377] As used herein, the term "therapy" means any protocol, method, and / or agent that can be used to prevent, manage, treat, and / or improve a disease or its associated symptoms. In one aspect, the term "therapy" means any protocol, method, and / or agent that can be used to modulate or deplete a target protein circulating in a subject. In some aspects, the term "therapies" means biological therapies, supportive therapies, and / or other therapies known to those skilled in the art (such as medical personnel) that can be used to prevent, manage, treat, and / or improve a disease or its associated symptoms. In other aspects, the term "therapy" means biological therapies, supportive therapies, and / or other therapies known to those skilled in the art (such as medical personnel) that can be used to modulate the immune response in a subject to an inflammatory disease or an autoimmune disease or its associated symptoms.

[0378] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or improvement in the progression, severity, and / or duration of disease or related symptoms resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as the administration of Fc domain variant peptides or binding peptides containing Fc domain variants provided herein). As used herein, the term "treatment" can also refer to altering the disease course in a subject undergoing treatment. Therapeutic effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of one or more symptoms, attenuation of the direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement or mitigation of the disease state, and remission or improvement of prognosis.

[0379] Fc domain variant peptides (e.g., included in the Fc-containing binding peptides of this disclosure) can be used for a wide range of applications. In some aspects, Fc domain variant peptides (e.g., included in the Fc-containing binding peptides of this disclosure) are effective in reducing the concentration of circulating target proteins or eliminating circulating target proteins. In some aspects, Fc domain variant peptides (e.g., included in the Fc-containing binding peptides) can alleviate inflammatory symptoms. In some aspects, Fc domain variant peptides (e.g., included in the binding peptides of this disclosure) can reduce tumor size. Methods for preparing and administering Fc domain variant peptides (e.g., included in the binding peptides of this disclosure) to subjects are well known or readily determined by those skilled in the art.

[0380] The Fc domain variant peptides of this disclosure can be administered orally, parenterally, by inhalation, topically, or by other suitable methods. As used herein, parenterally includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all these forms of administration are explicitly considered within the scope of this disclosure, the form of administration will be a solution for injection, particularly a solution for intravenous or intra-arterial injection or infusion. Typically, pharmaceutical compositions suitable for injection may contain buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), optionally stabilizers (e.g., human albumin), etc. However, in other methods compatible with the teachings herein, the Fc domain variant peptides can be delivered directly to the site of diseased tissue, thereby increasing the exposure of the diseased tissue to the therapeutic agent. In some aspects, the pharmaceutical composition comprises the Fc domain variant peptide described herein and a pharmaceutically acceptable carrier or diluent. In some aspects, methods of consuming target proteins include administering to a subject an effective amount of an Fc-containing binding polypeptide or a pharmaceutical composition containing an Fc domain variant polypeptide of the present disclosure.

[0381] Formulations intended for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffer media. In the compositions and methods disclosed herein, pharmaceutically acceptable carriers include, but are not limited to, 0.01 M–0.1 M, such as 0.05 M phosphate buffer or 0.8% saline. Other common parenteral media include sodium phosphate solutions, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements, such as those based on Ringer's dextran. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders of sterile injectable solutions or dispersions for ad hoc preparation. In such cases, the composition must be sterile and should be a fluid present to a degree of ease of injection. It should be stable under manufacturing and storage conditions and will typically be preserved against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0382] In many cases, isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride, will be included in the composition. Extended absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0383] In any case, a sterile injectable solution can be prepared as needed by incorporating the desired amount of an active compound (e.g., an Fc domain variant peptide or an antibody containing an Fc domain variant peptide or an antibody fragment thereof, or in combination with other active agents) with one or a combination of the ingredients listed herein in a suitable solvent, followed by filtration and sterilization. Typically, a dispersion is prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, exemplary preparation methods include vacuum drying and freeze-drying, which produce a powder of the active ingredient plus any other desired ingredients from a previously sterile filtered solution. Injectable formulations are processed according to methods known in the art, filled into containers (such as ampoules, bags, bottles, syringes, or vials), and sealed under sterile conditions. Such articles will typically have labels or instructions for use indicating that the relevant composition may be used to treat subjects who have or are susceptible to autoimmune disorders or neoplastic disorders.

[0384] The effective dosage of the Fc domain variant peptide compositions of this disclosure for treating the aforementioned conditions varies depending on many different factors, including the route of administration, target site, patient's physiological state, whether the patient is human or another animal, other agents administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. The therapeutic dosage can be gradually adjusted using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0385] The disclosed Fc domain variant peptides can be administered at multiple times. The intervals between single doses can be weekly, monthly, or yearly. Intervals can also be irregular, as indicated by measuring the blood levels of the target protein in the patient. Alternatively, the Fc domain variant peptides can be administered as a sustained-release formulation, in which case less frequent administration is required. For Fc domain variant peptides, the dose and frequency vary based on the half-life of the Fc domain variant peptide in the patient.

[0386] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, a composition containing the Fc domain variant peptide of the present invention or a mixture thereof is administered to a patient who is not yet in a disease state to enhance the patient's resistance. Such an amount is defined as a "prophylactic effective dose". In this use, the precise amount again depends on the patient's health status and general immunity, but typically ranges from about 0.1 mg / dose to about 25 mg / dose, particularly from about 0.5 mg / dose to about 2.5 mg / dose. Relatively low doses are administered at relatively low frequency intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic use, it is sometimes necessary to administer relatively high doses (e.g., about 1 mg / kg to 400 mg / kg of Fc domain variant peptide per dose) at relatively short intervals until disease progression is reduced or terminated, or until the patient shows partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen can be administered to the patient.

[0387] The pharmaceutical compositions according to this disclosure may contain pharmaceutically acceptable non-toxic and sterile carriers, such as physiological saline, non-toxic buffers, preservatives, etc. For the purposes of this application, the pharmaceutically effective amount of the Fc domain variant peptides disclosed herein should be considered as an amount sufficient to achieve effective binding to the target protein and to provide benefits (e.g., improvement of symptoms of a disease or disorder). Determine specificity

[0388] The binding affinity of Fc domain variant peptides that bind to Fc receptors (e.g., FcγRIIIa or FcRn) can be assessed using, for example, surface plasmon resonance, ELISA or other suitable methods (see Shields et al. (2001) J. Biol. Chem., 276:6591-6604).

[0389] In one aspect, the binding constant K of the Fc domain variant peptide to the Fc receptor is... D The binding constant of the Fc domain variant peptide can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times higher than that of the Fc domain parent peptide. The Fc domain parent peptide can be an antibody containing an Fc domain (e.g., the wild-type FcIgG domain) or its antigen-binding fragment. The binding constant K of the Fc domain variant peptide to the Fc receptor is... D It can be substantially the same as (i.e., ±50%) or higher than that of the Fc domain parent peptide. In one respect, the Fc domain variant peptide has an increased binding affinity to the Fc receptor by approximately 1.5 to 20 times compared to the Fc domain parent peptide.

[0390] In one aspect, the binding constant K of the Fc domain variant peptide to FcγRIIIa is... D The binding constant can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times higher than that of the wild-type control. The wild-type control group can be an antibody containing a parent peptide with an Fc domain (e.g., the wild-type Fc IgG domain) or its antigen-binding fragment. The binding constant K of the Fc domain variant peptide with FcγRIIIa is... D It can be substantially the same as (i.e., ±50%) or higher than that of the parent polypeptide with the Fc domain.

[0391] In one aspect, the binding constant K of the Fc domain variant peptide to FcRn is... D The binding constant can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times higher than that of the wild-type control. The wild-type control group can be an antibody containing a parent peptide with an Fc domain (e.g., the wild-type Fc IgG domain) or its antigen-binding fragment. The binding constant K of the Fc domain variant peptide with FcRn is... D It can be substantially the same as (i.e., ±50%) or higher than that of the parent polypeptide with the Fc domain.

[0392] In one aspect, the binding constant K of the Fc domain variant peptide disclosed herein to the Fc receptor is... D It can be substantially the same as (i.e., ±50%) or lower than the wild-type control. In one aspect, the binding constant K of the Fc domain variant peptide of this disclosure to FcγRIIIa is... D It can be substantially the same as (i.e., ±50%) or lower than the wild-type control. In one aspect, the binding constant K of the Fc domain variant peptide of this disclosure to FcRn is... D It can be substantially the same as (i.e., ±50%) or lower than the wild-type control.

[0393] The binding specificity of Fc domain variant peptides to Fc receptors or FcγRIIIa can be determined by, for example, flow cytometry, Western blotting, or other suitable methods. In some respects, Fc domain variant peptides bind specifically to Fc receptors (e.g., FcγRIIIa and / or FcRn) and target proteins.

[0394] In one aspect, certain pharmacokinetic parameters of the Fc domain variant peptides of this disclosure are the same as, better than, or higher than those of the Fc domain parent peptide (e.g., wild-type IgG Fc domain). For example, in one aspect, the elimination half-life (t1 / 2) and / or area under the concentration curve (AUC) may be substantially the same as (i.e., ±50%) or higher than those of the wild-type control group. Pharmacokinetic parameters may be measured in humans or using appropriate animal models (see, for example, Shargel et al. (1995) Applied Biopharmaceutics and Pharmacokinetics, 4th edition, McGraw-Hill / Appleton). Polynucleotides

[0395] In one aspect, a polynucleotide (i.e., nucleic acid molecule) encoding the Fc domain variant polypeptide or a variant thereof described herein is provided. The polynucleotide variant used herein shares approximately 50%, 75%, 80%, 85%, 90%, 93%, 95%, 98%, 99% or more identity with the polynucleotide encoding the Fc domain variant polypeptide described herein.

[0396] Methods for preparing Fc domain variant peptides are also provided, which include expressing these polynucleotides.

[0397] Polynucleotide insertion expression vectors, typically encoding Fc domain variant polypeptides disclosed herein, are introduced into host cells, which can then be used to produce the desired quantity. Therefore, in some aspects, this disclosure provides expression vectors comprising the polynucleotides disclosed herein, and host cells comprising these vectors and the polynucleotides.

[0398] In one aspect, the nucleic acid molecule encodes the amino acid sequence of an Fc domain variant polypeptide. Expression vectors and host cells

[0399] For the purposes of this specification and the claims, the term "vector" or "expression vector" as used herein means a vector used as a medium according to this disclosure for introducing a polynucleotide sequence encoding an Fc domain variant polypeptide into a cell and expressing that polynucleotide sequence in the cell. As is well known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, bacteriophages, viruses, and retroviruses. Typically, vectors compatible with this disclosure will include selection markers, appropriate restriction sites to facilitate the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0400] In vitro production allows for scaling up to produce large quantities of the desired peptide. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include homogeneous suspension culture (e.g., in an airlift reactor or a continuous stirred reactor), or immobilized or embedded cell culture (e.g., in hollow fibers, microcapsules, agarose beads, or ceramic casks). Solutions of the peptide can be purified by conventional chromatography (e.g., gel filtration, ion exchange chromatography, DEAE-cellulose chromatography, and / or (immuno)affinity chromatography) if necessary and / or desired.

[0401] One or more polynucleotides encoding Fc domain variant polypeptides can also be expressed in non-mammalian cells such as bacteria, yeast, or plant cells. In this respect, it should be understood that various single-celled non-mammalian microorganisms, such as bacteria, can also be transformed; that is, those capable of growth in culture or fermentation. Transformation-sensitive bacteria include members of the Enterobacteriaceae family, such as *Escherichia coli* or *Salmonella* strains; members of the Bacillaceae family, such as *Bacillus subtilis*; *Pneumococcus*; *Streptoococcus*; and *Haemophilus influenzae*. It should also be understood that when expressed in bacteria, the polypeptide can become part of an integrity. It must be possible to isolate, purify, and then assemble the polypeptide into a functional molecule.

[0402] In addition to prokaryotes, eukaryotic cells can also be used. Saccharomyces cerevisiae or common baker's yeast are the most commonly used, although many other strains are usually available.

[0403] In some respects, the vector contains a nucleic acid molecule that encodes an amino acid sequence of an Fc domain variant polypeptide.

[0404] The contents of any articles, patents, and patent applications mentioned or cited herein, as well as all other documents and electronically available information, are hereby incorporated in their full text by reference, to the extent that each individual publication expressly and individually indicates its inclusion by reference. The applicant reserves the right to actually incorporate any and all material and information from any such articles, patents, patent applications, or other physical and electronic documents into this application.

[0405] While this disclosure has been described with reference to specific aspects thereof, those skilled in the art will understand that various changes and equivalent substitutions can be made without departing from the true spirit and scope of this application. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations can be made to the methods described herein using appropriate equivalents without departing from the scope of the aspects disclosed herein. Furthermore, numerous modifications can be made to adapt particular circumstances, materials, composition, methods, or one or more method steps to the purpose, spirit, and scope of this disclosure. All such modifications are intended to fall within the scope of the appended claims. Having now described certain aspects in detail, these embodiments will become clearer with reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. Example

[0406] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The sequence listings, figures, and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. Example 1. Engineering, expression, and purification of Fc domain variants introduction One hundred and twenty-five (125) Fc domain variant peptides were designed, each with one or more mutations relative to the wild-type Fc domain parent peptide. Each mutation was introduced to alter amino acid residues at the protein-coding sequence level. The Fc domain variant peptides were then screened for the following three different parameters: (1) protein expression profile; (2) protein purity; and (3) FcγRIIIa binding affinity (discussed in Example 2). Materials and methods

[0407] The designed Fc domain variant peptides were expressed and purified using the following methods. Expression and purification

[0408] Point mutations were introduced into the nucleic acid sequence encoding the human IgG1 Fc domain. These nucleic acid sequences were then fused with the coding sequence for the variable domain of mAb1 (an IgG1 antibody targeting the protein of interest) and cloned into a mammalian expression plasmid containing a cytomegalovirus (CMV) enhancer / promoter and an SV40 polyA signal. The resulting plasmid was transfected into HEK293 cells according to the manufacturer's instructions. The Fc domain variant peptide was then purified from the transfected HEK293 cell line using purification techniques according to the manufacturer's instructions. result

[0409] Of the 125 expressed Fc domain variant peptides, only 47 (approximately 38% of the total) exhibited protein yield levels greater than 0.2 mg, such as... Figure 1 As shown. In protein purity assessment, 36 of the 125 Fc domain variant peptides (approximately 29% of the total) exhibited purity levels greater than 80%, as shown. Figure 2 As shown. in conclusion

[0410] Obtaining expression and purity profiles for all 125 Fc domain variant peptides helps exclude peptides that express incorrect folding and / or form protein aggregates. Therefore, Fc domain variant peptides expressing more than 0.2 mg of protein and with a minimum purity of 80% are valuable criteria for selecting Fc domain variant peptides for downstream analysis. Example 2: Variant of Fc domain with regulated Fc function introduction

[0411] The 125 Fc domain variant peptides described in Example 1 were tested for their ability to enhance FcγR binding (especially hFcγRIIIa binding) relative to the Fc domain parent peptide and other control Fc domain parent peptides. method

[0412] The Fc fragment was captured onto the immobilized protein AG and flowed together with multiple concentrations of hFcγRIIIa-V158 used to measure binding.

[0413] Sample preparation

[0414] PEPP samples were filtered at 0.22 µM in 96-well plates. A280 was measured on a Stunner. Samples were normalized to 100 µg / mL in PBS at pH 7.2 and then to 10 µg / mL on a Hamiltonian. Samples were diluted to 0.25 µg / mL in duplicate in 384-well plates using Benchsmart.

[0415] Programs on Carterra LSA

[0416] Protein AG was immobilized onto an HC30M sensor chip using amine chemistry. During multiple printings, the loaded antibody was diluted to 0.25 μg / mL every 3 minutes. Multiple concentrations of hFcγRIIIa-V158 were injected over 2 minutes, followed by dissociation over 5 minutes, including twelve consecutive 2-fold dilutions from 4000 nM to 1.9 nM in HBS-EP+ at pH 7.4 (concentrations decreased by 4 μM and 2 μM due to affinity). The chip was regenerated using 0.425% phosphate for 3 x 60-second cycles and stabilized for 1 minute. Finally, a 1:1 kinetic binding model was used to fit the sensor map. result

[0417] Sensing data were collected on two plates with corresponding controls. The sensing data measured the hFcγRIIIa binding affinity for each of the 125 Fc domain variant peptides collected, such as... Figure 3 and Figure 4 As shown in the figure. Example sensor data collected from the control group are as follows: Figure 5 As shown. Comparative studies: (1) A commercially available antibody with a wild-type IgG domain (adalimumab; top left); (2) The same antibody as (1) but with a glycoengineering-enhancing Fc domain mutation that eliminates effector functions, including hFcγRIIIa binding (antibody with an S298N / T299A / Y300S Fc domain mutation; top right); (3) An antibody with an Fc domain mutation that enhances hFcγRIIIa binding (bottom left); and (4) The antibody in (3) further enhances hFcγRIIIa binding through glycoengineering. in conclusion

[0418] Using sensor data from all 125 Fc domain variant peptides and controls, an is affinity plot (K-map) of the binding affinity of the Fc domain variant peptide hFcγRIIIa was constructed. a 1 / Ms, y-axis; Kd, 1 / s, x-axis, e.g. Figure 6 As shown, Fc domain variant peptides exhibit a range of affinities. The Fc domain variant peptides were also classified based on a comparison of their binding affinity to the hFcγRIIIa of the wild-type IgG Fc domain (adalimumab). Figure 7As shown, 68 Fc domain variant peptides exhibited >2-fold binding affinity relative to the wild-type IgG Fc domain. When protein expression (greater than 10 mg / L) and protein purity profile (minimum purity of 95%) of the 68 Fc domain variant peptides were evaluated as described in Example 1, the number of candidate Fc domain variant peptides decreased to 16. The 16 candidate Fc domain variant peptides that met all three criteria (high protein expression, protein purity profile, and binding kinetics) are shown in the table below. Figure 8A Among them Figure 8B This is a diagram illustrating the standard used. Table 2 provides a summary of affinity assessments for a range of Fc domain variant peptides tested.

[0419] Table 2: FcgRIIIa binding kinetics of some Fc domain variant peptides

[0420] In further studies, thermal stability assessments were performed to determine the melting temperatures of some Fc domain variant peptides in Table 2, which contain mutations at position L251.

[0421] Table 3: Thermal stability of some Fc domain variant peptides containing mutations at position L251

[0422] To further explore the role of the mutation at position L251, other Fc domain variants were tested, with or without the mutation at position L251.

[0423] As shown in Table 4, all tested Fc domain variant peptides exhibited enhanced binding affinity relative to the wild-type IgG Fc domain (WT). It was observed that the inclusion of a mutation at position L251 did indeed enhance FcγRIIIa binding (e.g., while the H268D, L251Q, S298A combination showed approximately 8.7-fold enhanced binding affinity compared to WT, mutation reversal at position L251 was observed, as seen in the H268D, S298A combination, where the fold increase relative to WT decreased to approximately 5-fold). When comparing these combinations, similar effects were observed: (A) H268D, L251Q, Y373W, S298A (approximately 5.8x enhancement) compared to H268D, Y373W, S298A; and (B) H268D, L251Q, L314M, S298A (approximately 7.8x enhancement) compared to WT compared to H268D, L314M, S298A (approximately 5.6x enhancement).

[0424] However, in terms of thermal stability, variants containing wild-type residues at position 251 (L251) exhibit higher melting temperatures (Tm, in °C) compared to the corresponding combinations with the L251X mutation. The results are summarized in Table 4. See, for example, the comparison between the H268D, L251Q, Y373W, S298A combination with a Tm of 44.9 °C and the H268D, Y373W, S298A combination with a Tm of 62 °C.

[0425] Table 4: Summary of Fc domain variant peptides

Claims

1. An Fc domain variant polypeptide, wherein, according to EU designation, the Fc domain variant polypeptide contains at least two substitutions at amino acid positions 251, 267, 268, 298, 314, 330, 339, 373 and 376, compared to the Fc domain parent polypeptide.

2. The Fc domain variant polypeptide of claim 1, wherein the Fc domain variant polypeptide exhibits increased binding to the Fc receptor relative to the parent Fc domain polypeptide.

3. The Fc domain variant polypeptide of claim 1 or 2, wherein the at least two amino acid substitutions are selected from: (i) Alanine (A), histidine (H), isoleucine (I), phenylalanine (F), glutamine (Q), or tryptophan (W) at amino acid position 251. (ii) Alanine (A) or aspartic acid (D) at amino acid position 267, (iii) Aspartic acid (D) or glutamic acid (E) at amino acid position 268, (iv) Alanine (A) at amino acid position 298, (v) Glycine (G), lysine (K), asparagine (N), methionine (M), serine (S), threonine (T), valine (V), glutamic acid (E), or tryptophan (W) at amino acid position 314. (vi) Phenylalanine (F), methionine (M), or tyrosine (Y) at amino acid position 330. (vii) Threonine (T) is present at amino acid position 339. (viii) Tryptophan (W) at amino acid position 373, or (ix) Valine (V) at amino acid position 376, The amino acid positions are specified according to EU designations.

4. The Fc domain variant polypeptide according to any one of claims 1 to 3, wherein the Fc domain parent polypeptide is a wild-type Fc domain.

5. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, tryptophan (W) at amino acid position 373, and alanine (A) at amino acid position 298.

6. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and alanine (A) at amino acid position 298.

7. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, isoleucine (I) at amino acid position 251, phenylalanine (F) at amino acid position 330, and alanine (A) at amino acid position 298.

8. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, threonine (T) at amino acid position 339, and alanine (A) at amino acid position 298.

9. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises glutamic acid (E) at amino acid position 268, valine (V) at amino acid position 376, alanine (A) at amino acid position 251, and methionine (M) at amino acid position 330.

10. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, aspartic acid (D) at amino acid position 267, isoleucine (I) at amino acid position 251, and threonine (T) at amino acid position 339.

11. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, threonine (T) at amino acid position 339, and tryptophan (W) at amino acid position 373.

12. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and tryptophan (W) at amino acid position 373.

13. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and threonine (T) at amino acid position 314.

14. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and valine (V) at amino acid position 314.

15. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and valine (V) at amino acid position 376.

16. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises alanine (A) at amino acid position 298, tyrosine (Y) at amino acid position 330, threonine (T) at amino acid position 339, and tryptophan (W) at amino acid position 373.

17. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and alanine (A) at amino acid position 251.

18. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 267, tyrosine (Y) at amino acid position 330, and threonine (T) at amino acid position 339.

19. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 267, tyrosine (Y) at amino acid position 330, and tryptophan (W) at amino acid position 373.

20. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises glutamic acid (E) at amino acid position 268, histidine (H) at amino acid position 251, and phenylalanine (F) at amino acid position 330.

21. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 373, and alanine (A) at amino acid position 298.

22. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and alanine (A) at amino acid position 298.

23. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 330, and alanine (A) at amino acid position 298.

24. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises glutamic acid (E) at amino acid position 268, valine (V) at amino acid position 314, and methionine (M) at amino acid position 330.

25. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, aspartic acid (D) at amino acid position 267, and threonine (T) at amino acid position 339.

26. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and tryptophan (W) at amino acid position 373.

27. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and valine (V) at amino acid position 314.

28. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises glutamic acid (E) at amino acid position 268 and phenylalanine (F) at amino acid position 330.

29. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and glutamic acid (E) at amino acid position 314.

30. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

31. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, and serine (S) at amino acid position 314.

32. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, and asparagine (N) at amino acid position 314.

33. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

34. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 314, tryptophan (W) at amino acid position 373, and methionine (M) at amino acid position 330.

35. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises glutamic acid (E) at amino acid position 268, isoleucine (I) at amino acid position 251, threonine (T) at amino acid position 314, and valine (V) at amino acid position 376.

36. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, and glycine (G) at amino acid position 314.

37. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, glycine (G) at amino acid position 314, and alanine (A) at amino acid position 298.

38. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and lysine (K) at amino acid position 314.

39. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, threonine (T) at amino acid position 314, and alanine (A) at amino acid position 298.

40. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises glutamic acid (E) at amino acid position 268, valine (V) at amino acid position 314, alanine (A) at amino acid position 251, and methionine (M) at amino acid position 330.

41. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises alanine (A) at amino acid position 298, tyrosine (Y) at amino acid position 330, and threonine (T) at amino acid position 339.

42. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and lysine (K) at amino acid position 314.

43. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and glycine (G) at amino acid position 314.

44. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, tryptophan (W) at amino acid position 251, and valine (V) at amino acid position 376.

45. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises tyrosine (Y) at amino acid position 330, alanine (A) at amino acid position 267, valine (V) at amino acid position 376, and alanine (A) at amino acid position 298.

46. ​​The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, phenylalanine (F) at amino acid position 251, and threonine (T) at amino acid position 314.

47. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

48. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and tryptophan (W) at amino acid position 373.

49. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and glutamic acid (E) at amino acid position 314.

50. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

51. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tryptophan (W) at amino acid position 373, and alanine (A) at amino acid position 298.

52. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and serine (S) at amino acid position 314.

53. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and asparagine (N) at amino acid position 314.

54. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises glutamic acid (E) at amino acid position 268, threonine (T) at amino acid position 314, and valine (V) at amino acid position 376.

55. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and glycine (G) at amino acid position 314.

56. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, threonine (T) at amino acid position 314, and alanine (A) at amino acid position 298.

57. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises glutamic acid (E) at amino acid position 268, valine (V) at amino acid position 314, and methionine (M) at amino acid position 330.

58. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and lysine (K) at amino acid position 314.

59. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and valine (V) at amino acid position 376.

60. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, glutamine (Q) at amino acid position 251, methionine (M) at amino acid position 314, and alanine (A) at amino acid position 298.

61. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, aspartic acid (D) at amino acid position 267, and threonine (T) at amino acid position 339.

62. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and threonine (T) at position 314.

63. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and glutamic acid (E) at position 314.

64. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and glycine (G) at position 314.

65. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268 and serine (S) at position 314.

66. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (D) at amino acid position 268, tyrosine (Y) at amino acid position 330, and valine (V) at position 376.

67. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises glutamic acid (E) at amino acid position 268, threonine (T) at amino acid position 314, and valine (V) at position 376.

68. The Fc domain variant polypeptide according to any one of claims 1 to 4, wherein, according to EU designation, the Fc domain variant comprises aspartic acid (E) at amino acid position 268, valine (V) at amino acid position 314, and methionine (M) at amino acid position 330.

69. The Fc domain variant polypeptide of any one of claims, wherein the Fc domain variant is of mammalian origin.

70. The Fc domain variant polypeptide of any one of claims 1 to 69, wherein the Fc domain variant is of human origin.

71. The Fc domain variant polypeptide of claim 69 or 70, wherein the Fc domain variant is derived from an immunoglobulin class selected from the group consisting of IgM, IgG, IgD, IgA, and IgE.

72. The Fc domain variant polypeptide of claim 71, wherein the Fc domain variant is derived from the IgG Fc domain.

73. The Fc domain variant polypeptide of claim 71, wherein the Fc domain variant is derived from the IgG1 Fc domain or the IgG4 Fc domain.

74. The Fc domain variant polypeptide according to any one of claims 1 to 73, further comprising aspartic acid (D) at amino acid position 239.

75. The Fc domain variant polypeptide of any one of claims 1 to 73, further comprising glutamic acid (E) at amino acid position 332.

76. The Fc domain variant polypeptide according to any one of claims 1 to 73, further comprising aspartic acid (D) at amino acid position 239 and glutamic acid (E) at amino acid position 332.

77. The Fc domain variant polypeptide of any one of claims 1 to 76, wherein the Fc domain variant is about 20%, 30%, 40%, 50%, 60%, 70%, 90% or more unfucosylated.

78. The Fc domain variant polypeptide according to any one of claims 1 to 77, further comprising cysteine ​​(C) at amino acid position 292 and cysteine ​​(C) at amino acid position 302.

79. The Fc domain variant polypeptide of any one of claims 1 to 73, wherein the Fc domain variant comprises at least one N-glycan.

80. The Fc domain variant polypeptide of claim 79, wherein the N-glycan comprises mannose and / or GlcNAc.

81. The Fc domain variant polypeptide of claim 79 or 80, wherein the N-glycan is an oligomannose.

82. The Fc domain variant polypeptide of claim 81, wherein the oligomannose-type N-glycan comprises oligosaccharides selected from the group consisting of: Man9(GlcNAc)2, Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2 and Man5GlcNAc2.

83. The Fc domain variant polypeptide of claim 81 or 82, wherein the Fc domain variant polypeptide comprises 20%, 30%, 40%, 50%, 60%, 70%, 90% or more of Mann based on a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 polysaccharide.

84. The Fc domain variant polypeptide of any one of claims 81 to 83, wherein the Fc domain variant polypeptide comprises greater than 70%, 75%, 80%, 85%, 90%, or 95% Mann based on a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 N-glycan.

85. The Fc domain variant polypeptide of any one of claims 81 to 84, wherein the Fc domain variant polypeptide comprises Man8 and Man9 together as Man 5-9 The main types of (GlcNAc)2 N-glycans.

86. The Fc domain variant polypeptide of any one of claims 83 to 85, wherein the Fc domain variant polypeptide comprises at least 97% Mann based on a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 N-glycan.

87. The Fc domain variant polypeptide of any one of claims 1 to 73, wherein the Fc domain variant polypeptide comprises less than 30%, 20%, 10%, 5%, or 1% Mann based on a molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 polysaccharide, or essentially no man in molar ratio relative to all N-glycans. 5-9 (GlcNAc)2 polysaccharide.

88. The Fc domain variant polypeptide according to any one of claims 79 to 87, further comprising cysteine ​​(C) at amino acid position 292 and cysteine ​​(C) at amino acid position 302.

89. The Fc domain variant polypeptide of any one of claims 79 to 88, wherein the Fc domain variant polypeptide is generated by culturing cells expressing the Fc domain variant polypeptide in the presence of a mannosidase inhibitor.

90. The Fc domain variant polypeptide of claim 89, wherein the mannosidase inhibitor is kifunensine.

91. The Fc domain variant polypeptide of claim 90, wherein the concentration of chiffon base is from about 60 ng / mL to about 2500 ng / mL.

92. The Fc domain variant polypeptide of claim 91, wherein the concentration of chiffon base is about 2000 ng / mL.

93. A binding polypeptide comprising at least one binding site for selectively binding a target antigen of interest and an Fc domain variant polypeptide as described in any one of claims 1 to 92.

94. The binding polypeptide of claim 93, wherein the binding polypeptide is a multispecific binding polypeptide.

95. The binding polypeptide of claim 93 or 94, wherein the binding polypeptide is an antibody or an antigen fragment thereof.

96. The binding polypeptide of claim 95, wherein the binding polypeptide is a multispecific antibody.

97. The binding polypeptide of any one of claims 93 to 96, wherein the binding polypeptide comprises VHH.

98. The binding polypeptide of claim 97, wherein the binding polypeptide comprises at least one antigen-binding fragment selected from the group consisting of: variable fragment (Fv), Fab, Fab', F(ab')2, microantibody, biantibody, triantibody, tetraantibody, tandem di-scFv, tandem tri-scFv, and immunoglobulin single variable domain (ISV).

99. The Fc domain variant polypeptide of any one of claims 1 to 92, wherein the Fc receptor is a human Fc receptor.

100. The Fc domain variant polypeptide of claim 99, wherein the Fc receptor is an Fcγ receptor (FcγR).

101. The Fc domain variant polypeptide of claim 100, wherein the FcγR is FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA or FcγRIIIB.

102. The Fc domain variant polypeptide of claim 101, wherein, according to EU designation, the FcγRIIIa is hFcγRIIIA (FcγRIIIa) having a valine residue at amino acid position 158. V158 ).

103. The Fc domain variant polypeptide of claim 101, wherein, according to EU designation, the FcγRIIIa is hFcγRIIIA (FcγRIIIa) having a phenylalanine residue at amino acid position 158. F158 ).

104. The Fc domain variant polypeptide of claim 100, wherein the Fc receptor is the neonatal Fc receptor (FcRn).

105. The Fc domain variant polypeptide of any one of claims 1 to 92 or 99 to 104, wherein the parent Fc domain polypeptide is a wild-type Fc domain.

106. The Fc domain variant polypeptide of any one of claims 1 to 92 or 99 to 104, wherein the Fc domain parent polypeptide comprises a modified Fc domain.

107. The Fc domain variant polypeptide of claim 105 or 106, wherein the Fc domain parent polypeptide comprises at least one modified glycan.

108. The Fc domain variant polypeptide of claim 107, wherein the modified polysaccharide is bis-mannose-6-phosphate (bisM6P), disaccharide-mannose-6-phosphate, or mannose-6-phosphate monosaccharide.

109. The Fc domain variant polypeptide of claim 108, wherein the modified glycan is an oligomannose-type N-glycan comprising oligosaccharides selected from the group consisting of: Man9(GlcNAc)2, Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2, or Man5GlcNAc2.

110. The Fc domain variant polypeptide according to any one of claims 1 to 92 or 99 to 109, wherein the Fc domain variant polypeptide has an increased binding affinity to the Fc receptor by about 1.5 to 20 times relative to the parent Fc domain polypeptide.

111. The Fc domain variant polypeptide of any one of claims 1 to 92 or 99 to 110, wherein the production titration of the polypeptide comprising the Fc domain variant is in the range of about 5 mg / L to about 500 mg / L.

112. The Fc domain variant polypeptide of any one of claims 1 to 92 or 99 to 111, wherein the polypeptide containing the Fc domain variant has a purity of at least 60%, 70%, 80%, 90%, or 100% when measured by size exclusion chromatography (SEC).

113. The Fc domain variant polypeptide of any one of claims 1 to 92 or 99 to 112, wherein the polypeptide comprising the Fc domain variant exhibits a melting temperature (Tm) greater than about 44°C.

114. A pharmaceutical composition comprising an Fc domain variant polypeptide as described in any of the preceding claims and a pharmaceutically acceptable carrier or diluent.

115. A nucleic acid molecule encoding an Fc domain variant polypeptide as described in any one of claims 1 to 92 or 99 to 113.

116. The nucleic acid molecule of claim 115, wherein the vector comprises a nucleic acid molecule encoding the Fc domain variant polypeptide.

117. A production cell that expresses the nucleic acid molecule as described in claim 115 or 116.

Images