FC-DERIVED POLYPEPTIDES
The present disclosure pertains to polypeptides comprising a transmembrane domain and an FcRn binding site (e.g., a modified Fc domain) and nanovesicles (e.g, extracellular vesicles (EVs) and hybridosomes) comprising such polypeptides. Said polypeptides can facilitate isolation and purification of nanovesicles comprising such polypeptides. The polypeptides and nanovesicles can be used in therapeutic and/or diagnostic applications. Also provided are nucleic acids and expression vectors encoding such polypeptides as well as cells expressing said polypeptides. Further provided are methods for producing nanovesicles comprising such polypeptides and methods for purifying these nanovesicles. Compositions comprising such polypeptides or nanovesicles as well as their uses are also described.
1 . A polypeptide, wherein the polypeptide comprises:
a. a transmembrane domain; and
b. a modified Fe domain of an immunoglobulin that
i. is capable of specifically binding to the Fc binding site of an FcRn; and
ii. lacks the ability to form homodimers.
2 . The polypeptide of claim 1 , wherein the equilibrium dissociation constant of the modified Fc domain bound to FcRn at a pH of 6.5 has a value of at most 10 −4 M.
3 . The polypeptide of claim 1 or 2 , wherein the equilibrium dissociation constant of the modified Fc domain bound to FcRn at a pH of 7.4 has a value of at least 10 −4 M.
4 . The polypeptide of any one of claims 1-3 , wherein the modified Fc domain is capable of specifically binding to the amino acid sequence between position 135-158 of human FcRn (SEQ ID NO: 7) and/or mouse FcRn (SEQ ID NO: 8).
5 . The polypeptide of any one of claims 1-4 , wherein the modified Fc domain is capable of specifically binding to the amino acid sequence LNGEEFMX 1 FX 2 X 3 X 4 X 5 GX 6 WX 7 GX 8 W (SEQ ID NO:6), wherein X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 and X 8 each is any amino acid.
6 . The polypeptide of any one of claims 1-5 , wherein said polypeptide does not substantially bind to C1q, FcγRI, FcγRII or FcγRIII.
7 . The polypeptide of any one of claims 1-6 , wherein:
a. the complement dependent cytotoxicity (CDC) activity of the modified Fc domain;
b. the antibody dependent cell mediated cytotoxicity (ADCC) activity of the modified Fc domain;
c. the antibody dependent cell mediated phagocytosis (ADCP) activity of the modified Fc domain; and/or
d. the antibody dependent intracellular neutralization (ADIN) activity of the modified Fc domain
is decreased by at least 10%, 20%, 30%, 40%, or 50% compared to the unmodified Fc domain.
8 . The polypeptide of any one of claims 1-7 , wherein:
a. the complement dependent cytotoxicity (CDC) activity of the modified Fc domain;
b. the antibody dependent cell mediated cytotoxicity (ADCC) activity of the modified Fc domain;
c. the antibody dependent cell mediated phagocytosis (ADCP) activity of the modified Fc domain; and/or
d. the antibody dependent intracellular neutralization (ADIN) activity of the modified Fc domain
is decreased by at least 1.5, 2, 3, 4, or 5-fold, compared to the unmodified Fc domain.
9 . The polypeptide of any one of claims 1-8 , wherein the FcRn binding polypeptide comprises from N-terminus to C-terminus:
a. a modified CH2 domain that is modified relative to the unmodified CH2 domain to decrease effector function;
b. a modified CH3 domain that is modified relative to the unmodified CH3 domain to lack the homodimerize;
c. a linker sequence; and
d. a transmembrane domain.
10 . The polypeptide of any one of claims 1-9 , wherein the FcRn binding polypeptide comprises from C-terminus to N-terminus:
a. a modified CH3 domain that is modified relative to the unmodified CH3 domain to lack the homodimerize;
b. a modified CH2 domain that is modified relative to the unmodified CH2 domain to decrease effector function;
c. a linker sequence; and
d. a transmembrane domain.
11 . The polypeptide of any one of claims 1-10 , wherein the transmembrane domain is a multipass transmembrane domain.
12 . The polypeptide of any one of the claims 1-11 , further comprising a targeting domain selected from the group consisting of: scFv, (scFv) 2 , Fab, Fab′, F(ab′) 2 , F(abl) 2 , Fv, dAb, Fd fragments, diabodies, F(ab)2, F(ab′), F(ab′)3, Fd, Fv, disulfide linked Fv, dAb, sdAb, nanobody, CDR, di-scFv, bi-scFv, tascFv (tandem scFv), AVIBODY (e.g., diabody, triabody, tetrabody), T-cell engager (BiTE), V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, intrabody, DARPin, Centyrin, affibody, affilin, affitin, anticalin, avimer, Fynomer, Kunitz domain peptide, monobody, adnectin, tribody, and nanofitin.
13 . A nucleic acid encoding the polypeptide of any one of claims 1-12 .
14 . An expression vector comprising the nucleic acid of claim 13 .
15 . A cell comprising the nucleic acid of claim 13 or the expression vector of claim 14 .
16 . An extracellular vesicle comprising the polypeptide of any one of claims 1 to 12 .
17 . A hybridosome comprising the polypeptide of any one of claims 1 to 12 .
18 . A method for purifying an extracellular vesicle (EV), wherein said method comprises:
a. providing the EV wherein the EV is associated with a first binding partner, wherein the first binding partner is capable of binding to the Fc binding site of an FcRn in a pH dependent manner; and
b. contacting at a first pH the EV associated with the first binding partner with a second binding partner, wherein the second binding partner comprises the Fc binding site of the FcRn and is associated with a solid matrix; and
c. eluting the EV associated with the first binding partner from the solid matrix at a second pH.
19 . The method of claim 18 , wherein the method comprises a washing step at the first pH.
20 . The method of claim 18 or 19 , wherein the first pH is below 6.5.
21 . The method of any one of claims 18 to 20 , wherein the second pH is above 7.4.
22 . A method for purifying an EV, wherein said method comprises:
a. providing the EV wherein the EV is associated with a first binding partner, wherein the first binding partner is capable of binding to the Fc binding site of an FcRn in a pH dependent manner and comprises or consists of the polypeptide of any one of claims 1-12 ; and
b. contacting at a first pH the EV associated with the first binding partner with a second binding partner, wherein the second binding partner comprises the Fc binding site of the FcRn and is associated with a solid matrix; and
c. eluting the EV associated with the first binding partner from the solid matrix at a second pH.
23 . The method of claim 22 , wherein the method comprises a washing step at the first pH.
24 . The method of claim 22 or 23 , wherein the first pH is below 6.5.
25 . The method of any one of claims 22 to 24 , wherein the second pH is above 7.4.