Hinge-modified IgG antibody compositions for protease resistance and fc-γ receptor binding and methods of making the same
Provided herein are antibodies, recombinant proteins, and methods of use thereof including, for example, immunoglobulin G (IgG) antibodies and recombinant proteins including a Fab region and an Fc region connected through a hinge region, where the hinge region is resistant to cleavage by a protease. Also, provided herein, inter alia, are immunoglobulin G (IgG) antibodies and recombinant proteins including a Fab region and an Fc region connected through a hinge region, and where the Fc region has higher affinity for a ligand compared to a wildtype Fc.
1 . An immunoglobulin G (IgG) antibody comprising a Fab region and an Fc region connected through a hinge region, wherein the hinge region comprises a protease resistant sequence comprising the amino acid sequence of CWDW (SEQ ID NO: 648), EETCWDW (SEQ ID NO:545), EDSCWDW (SEQ ID NO:546), EETCWSW (SEQ ID NO: 547), ETCWDW (SEQ ID NO:647), DSCWDW (SEQ ID NO:646), YDCWDW (SEQ ID NO: 645), DDCWDW (SEQ ID NO:644), DMCWDW (SEQ ID NO:643), EHCWDW (SEQ ID NO: 642), IICWDW (SEQ ID NO:641), DVCWDW (SEQ ID NO:640), EFCWDW (SEQ ID NO: 639), FNCWDW (SEQ ID NO:638), EETCWDW (SEQ ID NO:545), or EDSCWDW (SEQ ID NO: 546).
2 . The IgG antibody of claim 1 , wherein the IgG antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.
3 . The IgG antibody of claim 1 , wherein the hinge region is resistant to cleavage by a protease.
4 . The IgG antibody of claim 1 , wherein the protease resistant sequence is between amino acid positions 246-252 according to Kabat numbering.
5 . The IgG antibody of claim 1 , wherein the protease resistant sequence comprises the amino acid sequence CWDW (SEQ ID NO:648).
6 . The IgG antibody of claim 1 , wherein the protease resistant sequence comprises the amino acid sequence of EETCWDW (SEQ ID NO:545).
7 . The IgG antibody of claim 1 , wherein the Fc region has a dissociation constant of about 1×10 −5 M to about 1×10 −13 M to one or more of CD32 (FcγRII), CD16 (FcγRIII), and CD64 (FcγRI).
8 . The IgG antibody of claim 1 , wherein the protease is selected from the group consisting of IdeS, IdeZ, pepsin, matrix metalloproteinase 7, matrix metalloproteinase 3, matrix metalloproteinase, cathepsin G, and glutamyl endopeptidase V8.
9 . The IgG antibody of claim 1 , wherein the Fc region comprises an amino acid substitution selected from S252D, 1351E, and A349L according to Kabat numbering and wherein the substitution confers higher binding affinity for CD16 (FcγRIII) compared to wild type IgG antibody.
10 . The IgG antibody of claim 1 , wherein hinge region consists of the protease resistant sequence.
11 . The IgG antibody of claim 10 , wherein the protease resistance sequence is the amino acid sequence of CWDW (SEQ ID NO:648).
12 . The IgG antibody of claim 10 , wherein the protease resistance sequence is the amino acid sequence of EETCWDW (SEQ ID NO:545).
13 . A recombinant protein comprising:
(i) a Type I transmembrane domain; and
(ii) the IgG immunoglobulin G (IgG) antibody of claim 1 ;
wherein the Type I transmembrane domain is fused to the C-terminus of the IgG antibody; and wherein the recombinant protein is resistant to cleavage by a protease.
14 . The recombinant protein of claim 13 , wherein the Type I transmembrane domain is capable of dimerization.
15 . The recombinant protein of any one of claim 13 , wherein the Type I transmembrane domain is selected from the group consisting of an EGFR, a PDGFR-alpha, a PDGFR-beta, a HER2, a HER3, a HER4, a FGFR1, a FGFR2, a FGFR3, a FGFR4, a VEGFR1, a VEGFR2, a VEGFR3, a Trk-A, a Trk-B, a Trk-C, and an insulin receptor transmembrane domain.
16 . The recombinant protein of claim 13 , wherein the Type I transmembrane domain is an EGFR transmembrane domain.
17 . An isolated nucleic acid encoding the recombinant protein of claim 13 .
18 . An expression vector comprising a nucleic acid encoding the recombinant protein of claim 13 .
19 . A cell comprising the expression vector of claim 18 .
20 . A method of binding a ligand to a Chinese hamster ovary (CHO) cell surface recombinant protein, the method comprising contacting the ligand with the CHO cell surface recombinant protein, wherein the CHO cell surface recombinant protein comprises:
(i) an EGFR-transmembrane domain; and
(ii) the IgG immunoglobulin G (IgG) antibody of claim 1 ;
wherein the EGFR-transmembrane domain is fused to the C-terminus of the IgG antibody; and wherein the IgG antibody is capable of binding the ligand; and
wherein the CHO cell surface protein is resistant to cleavage by an IgG-specific protease or has higher binding affinity for one or more of CD32 (FcγRII), CD16 (FcγRIII), and CD64 (FcγRI).