US2018258178A1PendingUtilityA1
Molecules with Reduced Effector Function and Extended Half-Lives, Compositions, and Uses Thereof
Est. expiryApr 30, 2032(~5.8 yrs left)· nominal 20-yr term from priority
A61P 43/00C07K 16/11A61K 39/39591C07K 16/18C07K 2317/55C07K 2317/52C07K 2317/524C07K 16/2887C07K 2317/71C07K 2317/54C07K 2317/94C07K 16/2866C07K 16/1027
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Claims
Abstract
Provided are polypeptides comprising a variant IgG Fc domain, wherein the polypeptides exhibit reduced or ablated effector functions (e.g., ADCC and/or CDC) and increased stability and plasma half-life compared to a parent polypeptide. Also provided are compositions, methods of treatment, and methods to diminish Fc-induced effector function in a parent polypeptide.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . An isolated polypeptide comprising a variant IgG Fc domain, wherein the variant IgG Fc domain comprises:
(a) a Phenylalanine (F) amino acid at position 234; (b) an Alanine (A), Asparagine (N), Phenylalanine (F), Glutamine (Q), or Valine (V) amino acid at position 235; and, (c) an Alanine (A), Aspartic acid (D), Glutamic acid (E), Histidine (H), Asparagine (N), or Glutamine (Q) amino acid at position 322; or, an Alanine (A) or Glycine (G) amino acid at position 331,
wherein the amino acid numbering is according to the EU index as in Kabat.
2 . The polypeptide of claim 1 , comprising a Phenylalanine (F) amino acid at position 234; a Glutamine (Q) amino acid at position 235; and a Glutamine (Q) amino acid at position 322, wherein the amino acid numbering is according to the EU index as in Kabat.
3 . The polypeptide of claim 1 , comprising a Phenylalanine (F) amino acid at position 234; a Glutamine (Q) amino acid at position 235; and a Glycine (G) amino acid at position 331, wherein the amino acid numbering is according to the EU index as in Kabat.
4 . The polypeptide of claim 1 , comprising a Phenylalanine (F) amino acid at position 234; an Alanine (A) amino acid at position 235; and a Glutamine (Q) amino acid at position 322, wherein the amino acid numbering is according to the EU index as in Kabat.
5 . The polypeptide of any one of claims 1 - 4 , further comprising:
(a) a Tyrosine (Y) amino acid at position 252, or a Serine (S) amino acid at position 252, or a Tryptophan (W) amino acid at position 252 or a Threonine (T) amino acid at position 252; and/or (b) a Threonine (T) amino acid at position 254; and/or (c) a Glutamic acid (E) amino acid at position 256, or a Serine (S) amino acid at position 256, or a Arginine (R) amino acid at position 256, or a Glutamine (Q) amino acid at position 256, or an Aspartate (D) amino acid at position 256,
wherein the amino acid numbering is according to the EU index as in Kabat.
6 . The polypeptide of any one of claims 1 - 4 , further comprising:
(a) a Tyrosine (Y) amino acid at position 252; and/or (b) a Threonine (T) amino acid at position 254; and/or (c) a Glutamic acid (E) amino acid at position 256,
wherein the amino acid numbering is according to the EU index as in Kabat.
7 . The polypeptide of any one of claims 1 - 4 , further comprising:
(a) a Tyrosine (Y) amino acid at position 252, or a Serine (S) amino acid at position 252, or a Tryptophan (W) amino acid at position 252 or a Threonine (T) amino acid at position 252; and (b) a Threonine (T) amino acid at position 254,
wherein the amino acid numbering is according to the EU index as in Kabat.
8 . The polypeptide of any one of claims 1 - 4 , further comprising:
(a) a Threonine (T) amino acid at position 254; and (b) a Glutamic acid (E) amino acid at position 256, or a Serine (S) amino acid at position 256, or a Arginine (R) amino acid at position 256, or a Glutamine (Q) amino acid at position 256, or an Aspartate (D) amino acid at position 256,
wherein the amino acid numbering is according to the EU index as in Kabat.
9 . The polypeptide of any one of claims 1 - 4 , further comprising:
(a) a Tyrosine (Y) amino acid at position 252, or a Serine (S) amino acid at position 252, or a Tryptophan (W) amino acid at position 252 or a Threonine (T) amino acid at position 252; and (b) a Glutamic acid (E) amino acid at position 256, or a Serine (S) amino acid at position 256, or a Arginine (R) amino acid at position 256, or a Glutamine (Q) amino acid at position 256, or an Aspartate (D) amino acid at position 256,
wherein the amino acid numbering is according to the EU index as in Kabat.
10 . The polypeptide of any one of claims 1 - 4 , further comprising:
(a) a Tyrosine (Y) amino acid at position 252, and a Threonine (T) amino acid at position 254; or, (b) a Threonine (T) amino acid at position 254 and a Glutamic acid (E) amino acid at position 256; or, (c) a Tyrosine (Y) amino acid at position 252 and a Glutamic acid (E) amino acid at position 256
wherein the amino acid numbering is according to the EU index as in Kabat.
11 . The polypeptide of any one of claims 1 - 4 , further comprising a Tyrosine (Y) amino acid at position 252, a Threonine (T) amino acid at position 254, and, a Glutamic acid (E) amino acid at position 256, wherein the amino acid numbering is according to the EU index as in Kabat.
12 . The polypeptide of claim 1 , comprising:
(a) a Phenylalanine (F) amino acid at position 234; (b) a Glutamine (Q) amino acid at position 235; (c) a Glutamine (Q) amino acid at position 322; (d) a Tyrosine (Y) amino acid at position 252; (e) a Threonine (T) amino acid at position 254; and, (f) a Glutamic acid (E) amino acid at position 256,
wherein the amino acid numbering is according to the EU index as in Kabat.
13 . The polypeptide of claim 1 , comprising:
(a) a Phenylalanine (F) amino acid at position 234; (b) a Glutamine (Q) amino acid at position 235; (c) a Glycine (G) amino acid at position 331; (d) a Tyrosine (Y) amino acid at position 252; (e) a Threonine (T) amino acid at position 254; and, (f) a Glutamic acid (E) amino acid at position 256,
wherein the amino acid numbering is according to the EU index as in Kabat.
14 . The polypeptide of claim 1 , comprising:
(a) a Phenylalanine (F) amino acid at position 234; (b) an Alanine (A) amino acid at position 235; (c) a Glutamine (Q) amino acid at position 322; (d) a Tyrosine (Y) amino acid at position 252; (e) a Threonine (T) amino acid at position 254; and, (f) a Glutamic acid (E) amino acid at position 256,
wherein the amino acid numbering is according to the EU index as in Kabat.
15 . The polypeptide of any one of claims 1 - 14 , wherein the polypeptide has an improved pharmacokinetic (PK) property when compared to the same polypeptide comprising a wild-type Fc domain.
16 . The polypeptide of claim 15 , wherein the PK property is half-life.
17 . The polypeptide of any one of claims 1 - 16 , wherein the polypeptide has improved FcRn binding when compared to the same polypeptide comprising a wild-type Fc domain.
18 . The polypeptide of any of claims 1 - 17 , wherein the IgG Fc domain is non-human.
19 . The polypeptide of any of claims 1 - 17 , wherein the IgG Fc domain is human.
20 . The polypeptide of claim 18 , wherein the non-human IgG Fc domain is from rodent, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken.
21 . The polypeptide of claim 19 , wherein the IgG Fc domain is selected from the group consisting of human immunoglobulin G class 1 (IgG 1 ) Fc domain, human immunoglobulin G class 2 (IgG 2 ) Fc domain, human immunoglobulin G class 3 (IgG 3 ) Fc domain, and human immunoglobulin G class 4 (IgG 4 ) Fc domain.
22 . The polypeptide of any one of claims 1 - 21 , wherein the polypeptide further comprises an antigen binding domain.
23 . The polypeptide of claim 22 , wherein the antigen-binding domain is derived from a monoclonal antibody or an antigen-binding fragment thereof.
24 . The polypeptide of claim 22 , wherein the antigen-binding domain is derived from a human antibody, a humanized antibody, or a chimeric antibody.
25 . The polypeptide of claim 22 , wherein the antigen-binding domain comprises:
(a) a single chain antibody; (b) a diabody; (c) a polypeptide chain of an antibody; (d) an F(ab′) 2 fragment; or, (e) and F(ab) fragment.
26 . The polypeptide of any one of claims 1 - 21 , wherein the polypeptide has reduced Fc-mediated effector function when compared to the same polypeptide comprising a wild-type Fc domain.
27 . The polypeptide of claim 26 , wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
28 . The polypeptide of claim 26 , wherein the effector function is complement-dependent cytotoxicity (CDC).
29 . The polypeptide of any one of claims 1 - 28 , wherein the polypeptide has lower affinity for an Fc gamma receptor (FcγR) when compared to the same polypeptide comprising a wild-type Fc domain.
30 . The polypeptide of claim 29 , wherein the FcγR is a human FcγR.
31 . The polypeptide of claim 29 , wherein the FcγR is selected from the group consisting of FcγRI, FcγRII, and FcγRIII.
32 . The polypeptide of claim 31 , wherein the FcγRI is FcγRIα.
33 . The polypeptide of claim 31 , wherein the FcγRII is FcγRIIa or FcγRIIb.
34 . The polypeptide of claim 31 , wherein the FcγRIII is FcγRIII (158V) or FcγRIII (158F).
35 . The polypeptide of any one of claims 1 - 34 , wherein the polypeptide binds with improved affinity to FcRn when compared to the same polypeptide comprising a wild-type Fc domain.
36 . The polypeptide of claim 35 , wherein the polypeptide has a higher affinity for FcRn at pH 6.0 than at pH 7.4.
37 . The polypeptide of any one of claims 1 - 36 , wherein the polypeptide binds with reduced affinity to C1q when compared to the same polypeptide comprising a wild-type Fc domain.
38 . The polypeptide of any one of claims 1 - 37 , wherein the polypeptide displays an increase in thermal stability when compared to the same polypeptide comprising a FES-YTE IgG Fc domain.
39 . The polypeptide of claim 38 , wherein thermal stability is measured by Differential Scanning calorimetry (DSC).
40 . The polypeptide of claim 39 , wherein the increase in thermal stability is at least 4° C.
41 . The polypeptide of claim 38 , wherein thermal stability is measured by Differential Scanning Fluorimetry (DSF).
42 . The polypeptide of claim 41 , wherein the DSF fluorescent probe is Sypro Orange.
43 . The polypeptide of claim 42 , wherein the increase in thermal stability increases is at least 5° C.
44 . The polypeptide of any one of claims 1 - 43 , wherein the polypeptide displays an increase in apparent solubility as measured using a polyethylene glycol (PEG) precipitation assay when compared to the same polypeptide comprising a FES-YTE IgG Fc domain.
45 . The polypeptide of any one of claims 1 - 44 , wherein the polypeptide displays an increase in stability as measured using an accelerated stability assay when compared to the same polypeptide comprising a FES-YTE IgG Fc domain.
46 . The polypeptide of claim 45 , wherein the accelerated stability assay comprises: (i) incubation of the polypeptide for an extended time period, and (ii) incubation at high temperature.
47 . The polypeptide of claim 46 , wherein the accelerated stability assay is performed by incubation at a high concentration.
48 . The polypeptide of claim 46 , wherein the extended time period is at least one month.
49 . The polypeptide of claim 47 , wherein the high concentration is at least 25 mg/ml.
50 . The polypeptide of claim 46 , wherein the high temperature is at least 40° C.
51 . The polypeptide of any one of claims 45 - 50 , wherein the accelerated stability assay is performed using High Performance Size Exclusion Chromatography (HPSEC) or Dynamic Light Scattering (DLS).
52 . An isolated nucleic acid comprising a sequence encoding the polypeptide according to any one of claims 1 - 51 .
53 . A composition comprising the nucleic acid according to claim 52 .
54 . An expression vector comprising the nucleic acid according to claim 52 .
55 . A host cell comprising the nucleic acid sequence according to claim 52 , the composition according to claim 53 , or the vector according to claim 54 .
56 . A method of making the polypeptide of any one of claims 1 - 51 comprising (a) culturing the cell of claim 55 ; and, (b) isolating the polypeptide.
57 . A composition comprising the polypeptide according to any one of claims 1 - 51 and a carrier.
58 . A diagnostic reagent comprising the polypeptide according to any one of claims 1 - 51 .
59 . The diagnostic reagent of claim 58 , wherein the polypeptide is labeled.
60 . A conjugate comprising the polypeptide according to any one of claims 1 - 51 and a therapeutic moiety.
61 . A kit comprising the comprising the polypeptide according to any one of claims 1 - 51 , or the composition of any one of claim 52 - 55 or 57 - 60 .
62 . A method of treating a mammal, comprising administering to a mammal in need of treatment an effective amount of the polypeptide according to any one of claims 1 - 51 , or the composition of any one of claim 52 - 55 or 57 - 60 .
63 . A method to diminish Fc-induced effector function in a parent polypeptide comprising an Fc domain comprising:
(a) substituting the amino acid at position 234 in the Fc domain with Phenylalanine (F); (b) substituting the amino acid at position 235 in the Fc domain with Alanine (A), Asparagine (N), Phenylalanine (F), Glutamine (Q), or Valine (V); and, (c) substituting the amino acid at position 322 of the Fc domain with Alanine (A), Aspartic acid (D), Glutamic acid (E), Histidine (H), Asparagine (N), or Glutamine (Q); or substituting the amino acid at position 331 of the Fc domain with Alanine (A) or Glycine (G),
wherein the amino acid numbering of the Fc domain is according to the EU index as in Kabat.
64 . The method of claim 63 , wherein the Fc domain of the parent polypeptide comprises:
(a) a Tyrosine (Y) amino acid at position 252, or a Serine (S) amino acid at position 252, or a Tryptophan (W) amino acid at position 252 or a Threonine (T) amino acid at position 252; and/or (b) a Threonine (T) amino acid at position 254; and/or (c) a Glutamic acid (E) amino acid at position 256, or a Serine (S) amino acid at position 256, or an Arginine (R) amino acid at position 256, or a Glutamine (Q) amino acid at position 256, or an Aspartate (D) amino acid at position 256,
wherein the amino acid numbering of the Fc domain is according to the EU index as in Kabat.
65 . The method of claim 63 , wherein the Fc domain of the parent polypeptide comprises:
(a) a Tyrosine (Y) at position 252; and/or (b) a Threonine (T) at position 254; and/or, (c) a Glutamic acid (E) at position 256;
wherein the amino acid numbering of the Fc domain is according to the EU index as in Kabat.
66 . A method to diminish Fc-induced effector function and increase the half-life of a parent polypeptide comprising an Fc domain, the method comprising:
(a) substituting the amino acid at position 234 in the Fc domain with Phenylalanine (F); (b) substituting the amino acid at position 235 in the Fc domain with Alanine (A), Asparagine (N), Phenylalanine (F), Glutamine (Q), or Valine (V); and, (c) substituting the amino acid at position 322 of the Fc domain with Alanine (A), Aspartic acid (D), Glutamic acid (E), Histidine (H), Asparagine (N), or Glutamine (Q); or substituting the amino acid at position 331 of the Fc domain with Alanine (A) or Glycine (G); and (d) substituting the amino acid at position 252 with Tyrosine (Y) or Serine (S) or Tryptophan (W) or Threonine (T);
wherein the amino acid numbering of the Fc domain is according to the EU index as in Kabat.
67 . The method of claim 66 , wherein the amino acid at position 252 is substituted with Tyrosine (Y), wherein the amino acid numbering is according to the EU index as in Kabat.
68 . The method of claim 66 or 67 , further comprising:
(a) substituting the amino acid at position 254 with Threonine (T); and,
(b) substituting the amino acid at position 256 with Glutamic acid (E) or Serine (S), or Arginine (R), or Glutamine (Q),
wherein the amino acid numbering of the Fc domain is according to the EU index as in Kabat.
69 . The method of claim 68 , wherein the amino acid at position 256 is substituted with Glutamic acid (E), wherein the amino acid numbering is according to the EU index as in Kabat.
70 . The method of any one of claims 63 - 69 , wherein the amino acid at position 234 is substituted with Phenylalanine (F); the amino acid at position 235 is substituted with Glutamine (Q); and the amino acid at position 322 is substituted with Glutamine (Q), wherein the amino acid numbering is according to the EU index as in Kabat.
71 . The method of any one of claims 63 - 60 , wherein the amino acid at position 234 substituted with Phenylalanine (F); the amino acid at position 235 is substituted with Glutamine (Q); and the amino acid at position 331 is substituted with Glycine (G), wherein the amino acid numbering is according to the EU index as in Kabat.
72 . The method of any one of claims 63 - 60 , wherein the amino acid at position 234 is substituted with Phenylalanine (F); the amino acid at position 235 is substituted with Alanine (A); and the amino acid at position 322 is substituted with Glutamine (Q), wherein the amino acid numbering is according to the EU index as in Kabat.
73 . The method of any one of claims 63 - 72 , wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
74 . The method of any one of claims 63 - 72 , wherein the effector function is complement-dependent cytotoxicity (CDC).
75 . The method of any one of claims 63 - 72 , wherein the polypeptide displays an increase in thermal stability when compared to the same polypeptide comprising a FES-YTE IgG Fc domain.
76 . The method of claim 75 , wherein thermal stability is measured by Differential Scanning calorimetry (DSC).
77 . The method of claim 75 , wherein the increase in thermal stability is at least 4° C.
78 . The method of claim 75 , wherein thermal stability is measured by Differential Scanning Fluorimetry (DSF) using a DSF fluorescent probe.
79 . The method of claim 78 , wherein the DSF fluorescent probe is Sypro Orange.
80 . The method of claim 78 , wherein the increase in thermal stability is at least 5° C.
81 . The method of any one of claims 63 - 72 , wherein the polypeptide displays an increase in apparent solubility as measured using a polyethylene glycol (PEG) precipitation assay when compared to the same polypeptide comprising a FES-YTE IgG Fc domain.
82 . The method of any one of claims 63 - 72 , wherein the polypeptide displays an increase in stability as measured using an accelerated stability assay when compared to the same polypeptide comprising a FES-YTE IgG Fc domain.
83 . The method of claim 82 , wherein the accelerated stability assay comprises: (i) incubation of the polypeptide for an extended time period, and (ii) incubation at high temperature.
84 . The method of claim 83 , wherein the accelerated stability assay is performed by incubation at a high concentration.
85 . The method of claim 83 , wherein the extended time period is at least one month.
86 . The method of claim 84 , wherein the high concentration is at least 25 mg/ml.
87 . The method of claim 83 , wherein the high temperature is at least 40° C.Join the waitlist — get patent alerts
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