Patent Application
20240052051
The Sequence Listing written in file 598926SEQLIST.xml is 573 kilobytes, was created on Jul. 28, 2023, and is hereby incorporated by reference.
Iron delivery to the brain is accomplished via binding and intracellular trafficking of the iron binding protein transferrin (Tf). The Tf receptor (TfR) is a target of some studies to deliver drugs to the brain. For example, approaches include the use of liposomes decorated with Tf used for delivery of imaging agents and DNA (Sharma et al., (2013) Cell penetrating peptide tethered bi-ligand liposomes for delivery to brain in vivo: biodistribution and transfection. J. Control. Release 167, 1-10) or the use of an iron-mimetic peptide as ligand (Staquicini et al., (2011).
A correlation has also been suggested between increased antibody affinity and lysosomal degradation (Bien-Ly et al., (2014) Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants. J. Exp. Med. 211, 233-244) supporting the idea that lower antibody's affinity would help avoid intracellular degradation of the complexes being transported. Bien-Ly et al. found that bispecific antibodies against TfR and beta-secretase (BACE1) traversed the blood-brain barrier (BBB) and effectively reduce brain amyloid beta levels; but also that high-affinity binding to TfR caused a dose-dependent reduction of brain TfR levels. Similarly, Moos & Morgan (2001) compared the ability of anti-TfR antibody, OX26, and transferrin to cross the rat BBB finding that OX26 did not recycle out of the brain as did transferrin because the antibody exhibited a high-affinity antibody-antigen interaction with TfR that is not easily reversed, whereas that of Tf is readily reversed depending on pH and the iron content of Tf (Restricted transport of anti-transferrin receptor antibody (OX26) through the blood-brain barrier in the rat, J Neurochem 2001 October; 79(1):119-29).
In one aspect, provided are antigen-binding proteins that bind specifically to transferrin receptor or an antigenic-fragment thereof or variant thereof. Some such antigen-binding proteins comprise: (i) a HCVR that comprises the HCDR1, HCDR2 and HCDR3 of a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2; 12; 22; 32; 42; 52; 62; 72; 82; 92; 102; 112; 122; 132; 142; 152; 162; 172; 182; 192; 202; 212; 222; 232; 242; 252; 262; 272; 282; 292; 302; or 312 (or a variant thereof); and/or (ii) a LCVR that comprises the LCDR1, LCDR2 and LCDR3 of a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 7; 17; 27; 37; 47; 57; 67; 77; 87; 97; 107; 117; 127; 137; 147; 157; 167; 177; 187; 197; 207; 217; 227; 237; 247; 257; 267; 277; 287; 297; 307; or 317 (or a variant thereof). Optionally, the antigen-binding protein is fused to a payload.
Some such antigen-binding proteins comprise: (1) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 7 (or a variant thereof); (2) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 12 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (3) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (4) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 37 (or a variant thereof); (5) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 47 (or a variant thereof); (6) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 57 (or a variant thereof); (7) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 67 (or a variant thereof); (8) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 72 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 77 (or a variant thereof); (9) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 87 (or a variant thereof); (10) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof); (11) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof); (12) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 117 (or a variant thereof); (13) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 127 (or a variant thereof); (14) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (15) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 142 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 147 (or a variant thereof); (16) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof); (17) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 162 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof); (18) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (19) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 182 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (20) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 192 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof); (21) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 202 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); (22) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof); (23) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (24) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof); (25) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (26) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof); (27) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (28) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (29) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); (30) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (31) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 307 (or a variant thereof); and/or (32) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof).
Some such antigen-binding proteins comprise a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof).
Some such antigen-binding proteins comprise a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof).
Some such antigen-binding proteins comprise: (a) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); (b) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20 (or a variant thereof); (c) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 (or a variant thereof); (d) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 (or a variant thereof); (e) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 48 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50 (or a variant thereof); (f) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 54 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60 (or a variant thereof); (g) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 63 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 64 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 65 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70 (or a variant thereof); (h) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80 (or a variant thereof); (i) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 83 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 84 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90 (or a variant thereof); (j) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 95 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 99 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100 (or a variant thereof); (k) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 103 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 104 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 105 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110 (or a variant thereof); (1) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 118 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 119 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120 (or a variant thereof); (m) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 123 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 124 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 125 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 128 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 129 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 130 (or a variant thereof); (n) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof); (o) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof); (p) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 155 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 158 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 159 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 160 (or a variant thereof); (q) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 163 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 164 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 165 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 168 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 169 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 170 (or a variant thereof); (r) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 178 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180 (or a variant thereof); (s) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 183 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 184 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 185 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 188 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 189 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 190 (or a variant thereof); (t) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 193 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 194 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 195 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 199 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200 (or a variant thereof); (u) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 203 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 205 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 208 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 210 (or a variant thereof); (v) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 213 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 214 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 215 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 218 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 220 (or a variant thereof); (w) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof); (x) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 233 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 234 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 235 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 238 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 239 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 240 (or a variant thereof); (y) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof); (z) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 253 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 254 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 255 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 258 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 259 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 260 (or a variant thereof); (aa) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof); (ab) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof); (ac) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 283 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 284 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 285 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 288 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 289 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 290 (or a variant thereof); (ad) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 293 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 294 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 295 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 298 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 299 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 300 (or a variant thereof); (ae) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 303 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 304 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 305 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 308 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 309 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 310 (or a variant thereof); and/or (af) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 313 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 314 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 315 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 318 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 319 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 320 (or a variant thereof).
Some such antigen-binding proteins comprise a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 178 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof).
Some such antigen-binding proteins comprise a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof).
Some such antigen-binding proteins comprise: (i) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 7 (or a variant thereof); (ii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 12 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (iii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (iv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 37 (or a variant thereof); (v) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 47 (or a variant thereof); (vi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 57 (or a variant thereof); (vii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 67 (or a variant thereof); (viii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 72 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 77 (or a variant thereof); (ix) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 87 (or a variant thereof); (x) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof); (xi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof); (xii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 117 (or a variant thereof); (xiii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 127 (or a variant thereof); (xiv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (xv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 142 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 147 (or a variant thereof); (xvi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof); (xvii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 162 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof); (xviii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (xix) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 182 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (xx) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 192 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof); (xxi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 202 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); (xxii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof); (xxiii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (xxiv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof); (xxv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (xxvi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof); (xxvii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (xxviii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (xxix) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); (xxx) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (xxxi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 307 (or a variant thereof); and/or (xxxii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof).
Some such antigen-binding proteins comprise a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof). Some such antigen-binding proteins comprise a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof).
Some such antigen-binding proteins comprise a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof).
In some such antigen-binding proteins, the transferrin receptor is the human transferrin receptor or a variant thereof. Some such antigen-binding proteins are an antibody or antigen-binding fragment thereof. Some such antigen-binding proteins are a Fab. Some such antigen-binding proteins are an scFv; optionally wherein the scFv and the payload are connected by a peptide linker which is -(GGGGS)m- (SEQ ID NO: 426); wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and optionally, wherein the scFv variable regions are connected by a peptide linker which is -(GGGGS)n- (SEQ ID NO: 426); wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some such antigen-binding proteins, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 443 (or a variant thereof), comprises the amino acid sequence set forth in SEQ ID NO: 440 (or a variant thereof), comprises the amino acid sequence set forth in SEQ ID NO: 429 (or a variant thereof), comprises the amino acid sequence set forth in SEQ ID NO: 433 (or a variant thereof), comprises the amino acid sequence set forth in SEQ ID NO: 442 (or a variant thereof), or comprises the amino acid sequence set forth in SEQ ID NO: 438 (or a variant thereof). In some such antigen-binding proteins, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 443 (or a variant thereof).
Also provided are antigen-binding proteins that bind specifically to transferrin receptor or an antigenic-fragment thereof or variant thereof and bind to one or more epitopes of hTfR selected from: (a) an epitope comprising the sequence LLNE (SEQ ID NO: 525) and/or an epitope comprising the sequence TYKEL (SEQ ID NO: 507); (b) an epitope comprising the sequence DSTDFTGT (SEQ ID NO: 526) and/or an epitope comprising the sequence VKHPVTGQF (SEQ ID NO: 527) and/or an epitope comprising the sequence IERIPEL (SEQ ID NO: 528); (c) an epitope comprising the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 529); (d) an epitope comprising the sequence FEDL (SEQ ID NO: 519); (e) an epitope comprising the sequence IVDKNGRL (SEQ ID NO: 530); (f) an epitope comprising the sequence IVDKNGRLVY (SEQ ID NO: 531); (g) an epitope comprising the sequence DQTKF (SEQ ID NO: 532); (h) an epitope comprising the sequence LVENPGGY (SEQ ID NO: 533) and/or an epitope comprising the sequence PIVNAELSF (SEQ ID NO: 534) and/or an epitope comprising the sequence PYLGTTMDT (SEQ ID NO: 535); (i) an epitope comprising the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope comprising the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope comprising the sequence TYKEL (SEQ ID NO: 507); (j) an epitope comprising the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 508) and/or an epitope comprising the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 509) and/or an epitope comprising the sequence LIERIPELNKVARAAAE (SEQ ID NO: 510); (k) an epitope comprising the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 511); (1) an epitope comprising the sequence GTKKDFEDL (SEQ ID NO: 512); (m) an epitope comprising the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 513); (n) an epitope comprising the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope comprising the sequence DQTKFPIVNAEL (SEQ ID NO: 515) and/or an epitope comprising the sequence TYKELIERIPELNK (SEQ ID NO: 516); (o) an epitope comprising the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope comprising the sequence TYKELIERIPELNK (SEQ ID NO: 516); (p) an epitope comprising the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 517); (q) an epitope comprising the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope comprising the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 518); (r) an epitope comprising the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope comprising the sequence LVENPGGYVAYSKAATVTGKL (SEQ ID NO: 520) and/or an epitope comprising the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 521) and/or an epitope comprising the sequence ISRAAAEKL (SEQ ID NO: 522) and/or an epitope comprising the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 523) and/or an epitope comprising the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 524); (s) an epitope comprised within or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope comprised within or overlapping with the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope comprised within or overlapping with the sequence TYKEL (SEQ ID NO: 507); (t) an epitope comprised within or overlapping with the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 508) and/or an epitope comprised within or overlapping with the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 509) and/or an epitope comprised within or overlapping with the sequence LIERIPELNKVARAAAE (SEQ ID NO: 510); (u) an epitope comprised within or overlapping with the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 511); (v) an epitope comprised within or overlapping with the sequence GTKKDFEDL (SEQ ID NO: 512); (w) an epitope comprised within or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 513); (x) an epitope comprised within or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope comprised within or overlapping with the sequence DQTKFPIVNAEL (SEQ ID NO: 515) and/or an epitope comprised within or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 516); (y) an epitope comprised within or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope comprised within or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 516); (z) an epitope comprised within or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 517); (aa) an epitope comprised within or overlapping with the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope comprised within or overlapping with the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 518); and (ab) an epitope comprised within or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope comprised within or overlapping with the sequence LVENPGGYVAYSKAATVTGKL (SEQ ID NO: 520) and/or an epitope comprised within or overlapping with the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 521) and/or an epitope comprised within or overlapping with the sequence ISRAAAEKL (SEQ ID NO: 522) and/or an epitope comprised within or overlapping with the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 523) and/or an epitope comprised within or overlapping with the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 524). In some such antigen-binding proteins, the antigen binding protein comprises an antibody or antigen-binding fragment thereof which binds to one or more epitopes of hTfR selected from: (a) an epitope consisting of the sequence LLNE (SEQ ID NO: 525) and/or an epitope consisting of the sequence TYKEL (SEQ ID NO: 507); (b) an epitope consisting of the sequence DSTDFTGT (SEQ ID NO: 526) and/or an epitope consisting of the sequence VKHPVTGQF (SEQ ID NO: 527) and/or an epitope consisting of the sequence IERIPEL (SEQ ID NO: 528); (c) an epitope consisting of the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 529); (d) an epitope consisting of the sequence FEDL (SEQ ID NO: 519); (e) an epitope consisting of the sequence IVDKNGRL (SEQ ID NO: 530); (f) an epitope consisting of the sequence IVDKNGRLVY (SEQ ID NO: 531); (g) an epitope consisting of the sequence DQTKF (SEQ ID NO: 532); (h) an epitope consisting of the sequence LVENPGGY (SEQ ID NO: 533) and/or an epitope consisting of the sequence PIVNAELSF (SEQ ID NO: 534) and/or an epitope consisting of the sequence PYLGTTMDT (SEQ ID NO: 535); (i) an epitope consisting of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope consisting of the sequence TYKEL (SEQ ID NO: 507); (j) an epitope consisting of the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 508) and/or an epitope consisting of the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 509) and/or an epitope consisting of the sequence LIERIPELNKVARAAAE (SEQ ID NO: 510); (k) an epitope consisting of the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 511); (1) an epitope consisting of the sequence GTKKDFEDL (SEQ ID NO: 512); (m) an epitope consisting of the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 513); (n) an epitope consisting of the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope consisting of the sequence DQTKFPIVNAEL (SEQ ID NO: 515) and/or an epitope consisting of the sequence TYKELIERIPELNK (SEQ ID NO: 516); (o) an epitope consisting of the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope consisting of the sequence TYKELIERIPELNK (SEQ ID NO: 516); (p) an epitope consisting of the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 517); (q) an epitope consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope consisting of the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 518); and (r) an epitope consisting of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope consisting of the sequence LVENPGGYVAYSKAATVTGKL (SEQ ID NO: 520) and/or an epitope consisting of the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 521) and/or an epitope consisting of the sequence ISRAAAEKL (SEQ ID NO: 522) and/or an epitope consisting of the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 523) and/or an epitope consisting of the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 524).
In some such antigen-binding proteins, the antigen-binding protein is selected from a humanized antibody or antigen binding fragment thereof, human antibody or antigen binding fragment thereof, murine antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monovalent Fab′, divalent Fab2, F(ab)′3 fragments, single-chain fragment variable (scFv), bis-scFv, (scFv)2, diabody, bivalent antibody, one-armed antibody, minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, single heavy chain antibody, bispecific antibody or biding fragment thereof, bi-specific T-cell engager (BiTE), trispecific antibody, or chemically modified derivatives thereof.
In another aspect, provided is a fusion protein comprising any of the above antigen-binding proteins fused to a payload. In another aspect, provided is a fusion protein comprising an antigen-binding protein that binds specifically to transferrin receptor or an antigenic-fragment thereof or variant thereof fused to a payload, wherein the antigen-binding protein binds to human transferrin receptor with a KD of about 41 nM or a stronger affinity. In some such fusion proteins, the antigen-binding protein binds to human transferrin receptor with a KD of about 41 nM or a stronger affinity. In some such fusion proteins, the antigen-binding protein binds to human transferrin receptor with a KD of about 3 nM or a stronger affinity. In some such fusion proteins, the antigen-binding protein binds to human transferrin receptor with a KD of about 3 nM or a stronger affinity, or wherein the antigen-binding protein binds to human transferrin receptor with a KD of about 0.45 nM to 3 nM. In some such fusion proteins, the payload is one or more antibodies or antigen-binding fragments thereof, proteins, enzymes or viral vectors containing one or more polynucleotides or oligonucleotides; or human alpha-glucosidase polypeptide (hGAA) or a variant thereof. In some such fusion proteins, the payload is a lysosomal storage disease therapeutic agent (LSD-TA); or a polypeptide or a polypeptide encoded by a human gene specified in any one of Tables C-N or a variant thereof. In some such fusion proteins, the payload is an LSD-TA which is Miglustat, Eliglustat, α-galactosidase A; ceramidase; β-glucosidase; saposin-C activator; acid sphingomyelinase; β-galactosidase; β-hexosaminidase A and B; β-hexosaminidase A; GM2-activator protein; GM3 synthase; arylsulfatase A; sphingolipid activator; α-iduronidase; iduronidase-2-sulphatase; heparan N-sulphatase; N-acetyl-α-glucosaminidase; acetyl-CoA; α-glucosamide N-acetyltransferase; N-acetylglucosamine-6-sulphatase; N-acetylgalactosamine-6-sulphate sulphatase; β-galactosidase; N-acetylgalactosamine-4-sulphatase (arylsulphatase B); β-glucuronidase; hylauronidase; α-hlucosidase 2; or lysosomal acid lipase.
Some such fusion proteins are a fusion protein comprising an antigen-binding protein that binds specifically to human transferrin receptor, which comprises a heavy chain variable region (HCVR or VH) and a light chain variable region (LCVR or VL), which is fused to an alpha-glucosidase polypeptide (GAA), wherein a Fab having said VH and VL binds to human transferrin receptor with a KD of about 0.65 nM or a greater affinity; and wherein, when said fusion protein is administered to a mouse expressing human transferrin receptor in the brain, the mouse achieves a molar ratio of mature GAA protein in the brain:serum GAA protein, in the mouse, of about 1:1 or greater when normalized against said ratio in mouse expressing mouse transferrin receptor that was administered 8D3.
In some such fusion proteins, the antigen-binding protein is a Fab. In some such fusion proteins, the antigen-binding protein is a single chain fragment variable (scFv). In some such fusion proteins, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 408 (or a variant thereof) or comprises the amino acid sequence set forth in SEQ ID NO: 405 (or a variant thereof). In some such fusion proteins, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 408 (or a variant thereof).
In some such fusion proteins, the antigen-binding protein is an antibody or antigen-binding fragment thereof. In some such fusion proteins, the antigen-binding protein is an scFv comprising domains arranged in the following orientation: N-Heavy chain variable region-Light chain variable region-GAA protein-C. In some such fusion proteins, the antigen-binding protein is an scFv comprising domains arranged in the following orientation: N-Light chain variable region-Heavy chain variable region-GAA protein-C. In some such fusion proteins, the antigen-binding protein is an scFv, wherein said scFv and GAA are connected by a peptide linker. In some such fusion proteins, the scFv and GAA are connected by a peptide linker which is -(GGGGS)m- (SEQ ID NO: 426); wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some such fusion proteins, the antigen-binding protein is an scFv and said scFv variable regions are connected by a peptide linker. In some such fusion proteins, the scFv variable regions are connected by a peptide linker which is -(GGGGS)n- (SEQ ID NO: 426); wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some such fusion proteins, the fusion protein binds to human transferrin receptor with a KD of about 1×10−7 M or a greater affinity.
Some such fusion proteins comprise: (i) a HCVR that comprises the HCDR1, HCDR2 and HCDR3 of a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2; 462; 12; 463; 22; 464; 32; 42; 52; 467; 62; 492; 72; 470; 82; 92; 472; 102; 112; 473; 122; 132; 142; 475; 152; 162; 477; 172; 182; 478; 192; 480; 202; 481; 212; 222; 232; 242; 252; 482; 262; 272; 282; 292; 302; 483 or 312 (or a variant thereof); and/or (ii) a LCVR that comprises the LCDR1, LCDR2 and LCDR3 of a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 7; 17; 27; 37; 465; 47; 466; 57; 468; 67; 469; 77; 471; 87; 97; 107; 117; 474; 127; 137; 147; 476; 157; 167; 177; 187; 479; 197; 207; 217; 227; 237; 247; 257; 267; 277; 287; 297; 307; 488; 317 or 484 (or a variant thereof).
In some such fusion proteins, the fusion protein comprises an scFv that comprises a heavy chain variable region (VH) and a light chain variable region (VL), and an alpha-glucosidase polypeptide (GAA), wherein said VH, VL and GAA are arranged as follows: (i) VL-VH-GAA; (ii) VH-VL-GAA; (iii) VL-[(GGGGS)3 (SEQ ID NO: 538)]-VH-[(GGGGS)2 (SEQ ID NO: 537)]-GAA; or (iv) VH-[(GGGGS)3 (SEQ ID NO: 538)]-VL-[(GGGGS)2 (SEQ ID NO: 537)]-GAA.
Some such fusion proteins comprise the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs: 392-423; SEQ ID NO: 321 (optionally lacking the N-terminal MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 500) signal sequence); SEQ ID NO: 322 (optionally lacking the N-terminal MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 500) signal sequence); SEQ ID NO: 323 (optionally lacking the N-terminal MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 500) signal sequence); and SEQ ID NO: 324 (optionally lacking the N-terminal MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 500) signal sequence); or a variant thereof.
In some such fusion proteins, the antigen-binding protein, which when not fused to a GAA polypeptide, does not block more than 50% of binding of a human transferrin receptor C-terminal fragment to human holo-transferrin that occurs in the absence of such single chain fragment variable (scFv), antibody or an antigen-binding fragment. In some such fusion proteins, the blocking is as measured in an Enzyme Linked Immunosorbent Assay (ELISA) plate assay wherein binding of human transferrin receptor extracellular domain that is fused to a His6-myc-myc tag is pre-bound to said scFv, antibody or antigen-binding fragment and then contacted with holo-transferrin which is immobilized to the surface of the plate by binding of an anti-holo-transferrin antibody that is bound to the plate. In some such fusion proteins, binding of the holo-transferrin and human transferrin receptor extracellular domain in the absence of the scFv, antibody or antigen-binding fragment is measured at a concentration of about 300 pM human transferrin receptor extracellular domain.
Some such fusion proteins or antigen-binding proteins bind specifically to human transferrin receptor which has one or more of the following characteristics:
In another aspect, provided are pharmaceutical compositions comprising any of the above fusion proteins or antigen-binding proteins and a pharmaceutically acceptable carrier.
In another aspect, provided are compositions or kits comprising any of the above fusion proteins or antigen-binding proteins or pharmaceutical compositions in association with a further therapeutic agent. In some such compositions or kits, the further therapeutic agent is selected from: alglucosidase alfa, rituximab, methotrexate, intravenous immunoglobulin (IVIG), avalglucosidase alfa, levalbuterol, an antibiotic, cortisone, prednisone, a bisphosphonate, and palivizumab. In some such compositions or kits, the further therapeutic agent is selected from: a beta2-adrenergic agonist, a steroid, a bisphosphonate, an infectious disease treatment, a vaccine, and a Pneumococcal vaccine.
In another aspect, provided is a complex comprising any of the above fusion proteins or antigen-binding proteins bound to a human transferrin receptor polypeptide or antigenic fragment thereof.
In another aspect, provided are isolated polynucleotide encoding any of the above fusion proteins or antigen-binding proteins. Some such polynucleotides comprise the nucleotide sequence set forth in SEQ ID NO: 1; 6; 11; 16; 21; 26; 31; 36; 41; 46; 51; 56; 61; 66; 71; 76; 81; 86; 91; 96; 101; 106; 111; 116; 121; 126; 131; 136; 141; 146; 151; 156; 161; 166; 171; 176; 181; 186; 191; 196; 201; 206; 211; 216; 221; 226; 231; 236; 241; 246; 251; 256; 261; 266; 271; 276; 281; 286; 291; 296; 301; 306; 311; and/or 316. Some such polynucleotides comprise: (1) the nucleotide sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 6; (2) the nucleotide sequence set forth in SEQ ID NO: 11 and SEQ ID NO: 16; (3) the nucleotide sequence set forth in SEQ ID NO: 21 and SEQ ID NO: 26; (4) the nucleotide sequence set forth in SEQ ID NO: 31 and SEQ ID NO: 36; (5) the nucleotide sequence set forth in SEQ ID NO: 41 and SEQ ID NO: 46; (6) the nucleotide sequence set forth in SEQ ID NO: 51 and SEQ ID NO: 56; (7) the nucleotide sequence set forth in SEQ ID NO: 61 and SEQ ID NO: 66; (8) the nucleotide sequence set forth in SEQ ID NO: 71 and SEQ ID NO: 76; (9) the nucleotide sequence set forth in SEQ ID NO: 81 and SEQ ID NO: 86; (10) the nucleotide sequence set forth in SEQ ID NO: 91 and SEQ ID NO: 96; (11) the nucleotide sequence set forth in SEQ ID NO: 101 and SEQ ID NO: 106; (12) the nucleotide sequence set forth in SEQ ID NO: 111 and SEQ ID NO: 116; (13) the nucleotide sequence set forth in SEQ ID NO: 121 and SEQ ID NO: 126; (14) the nucleotide sequence set forth in SEQ ID NO: 131 and SEQ ID NO: 136; (15) the nucleotide sequence set forth in SEQ ID NO: 141 and SEQ ID NO: 146; (16) the nucleotide sequence set forth in SEQ ID NO: 151 and SEQ ID NO: 156; (17) the nucleotide sequence set forth in SEQ ID NO: 161 and SEQ ID NO: 166; (18) the nucleotide sequence set forth in SEQ ID NO: 171 and SEQ ID NO: 176; (19) the nucleotide sequence set forth in SEQ ID NO: 181 and SEQ ID NO: 186; (20) the nucleotide sequence set forth in SEQ ID NO: 191 and SEQ ID NO: 196; (21) the nucleotide sequence set forth in SEQ ID NO: 201 and SEQ ID NO: 206; (22) the nucleotide sequence set forth in SEQ ID NO: 211 and SEQ ID NO: 216; (23) the nucleotide sequence set forth in SEQ ID NO: 221 and SEQ ID NO: 226; (24) the nucleotide sequence set forth in SEQ ID NO: 231 and SEQ ID NO: 236; (25) the nucleotide sequence set forth in SEQ ID NO: 241 and SEQ ID NO: 246; (26) the nucleotide sequence set forth in SEQ ID NO: 251 and SEQ ID NO: 256; (27) the nucleotide sequence set forth in SEQ ID NO: 261 and SEQ ID NO: 266; (28) the nucleotide sequence set forth in SEQ ID NO: 271 and SEQ ID NO: 276; (29) the nucleotide sequence set forth in SEQ ID NO: 281 and SEQ ID NO: 286; (30) the nucleotide sequence set forth in SEQ ID NO: 291 and SEQ ID NO: 296; (31) the nucleotide sequence set forth in SEQ ID NO: 301 and SEQ ID NO: 306; and/or (32) the nucleotide sequence set forth in SEQ ID NO: 311 and SEQ ID NO: 316.
In another aspect, provided are vectors comprising any of the above polynucleotides.
In another aspect, provided are host cells comprising any of the above fusion proteins, antigen-binding proteins, polynucleotides, or vectors. Some such host cells are a Chinese hamster ovary (CHO) cell.
In another aspect, provided are methods for making any of the above fusion proteins or antigen-binding proteins, comprising culturing a host cell comprising a polynucleotide that encodes the fusion protein or antigen-binding protein in a culture medium under conditions favorable to expression of the fusion protein or antigen-binding protein. Some such methods comprise the steps: (a) introducing said polynucleotide into a host cell; (b) culturing the host cell under conditions favorable to expression of the fusion protein or antigen-binding protein; and (c) optionally, isolating the fusion protein or antigen-binding protein from the culture medium and/or host cell; and (d) optionally, chemically conjugating the antigen-binding protein to a payload. In another aspect, provided are fusion proteins or antigen-binding proteins which are the product of such methods.
In another aspect, provided are vessels or injection devices comprising any of the above fusion proteins or antigen-binding proteins.
In another aspect, provided are methods for administering any of the above fusion proteins or antigen-binding proteins to a subject comprising introducing the protein into the body of the subject. In some such methods, the fusion protein or antigen-binding protein is introduced into the body of the subject parenterally.
In another aspect, provided are methods for treating or preventing a lysosomal storage disease in a subject in need thereof comprising administering, to the subject, an effective amount of any of the above fusion proteins, wherein the payload is a lysosomal storage disease therapeutic agent (LSD-TA). In some such methods, the lysosomal storage disease is: Fabry disease; Farber lipogranulomatosis; Gaucher disease type I; Gaucher disease (type II or III); Niemann-Pick diseases (type A or B); GM1-gangliosidosis; GM2-gangliosidosis (Sandhoff); GM2-gangliosidosis (Tay-Sachs); GM2-gangliosidosis (GM2-activator deficiency); GM3-gangliosidosis; Metachromatic leukodystrophy; Sphingolipid-activator deficiency; MPS I (Scheie, Hurler-Scheie, or Hurler disease); MPS II (Hunter); MPS IIIA (Sanfilippo A); MPS IIIB (Sanfilippo B); MPS IIIC (Sanfilippo C); MPS IIID (Sanfilippo D); MPS IVA (Morquio syndrome A); MPS IVB (Morquio syndrome B); MPS VI (Maroteaux-Lamy); MPS VII (Sly disease); MPS IX; Pompe (glycogen storage disease type II); or Lysosomal acid lipase deficiency (LAL-D; Wolman disease). In some such methods, one or more signs or symptoms of the LSD in the subject are alleviated after the fusion protein or antigen-binding protein is administered.
In another aspect, provided are methods for treating or preventing a glycogen storage disease (GSD)) in a subject in need thereof comprising administering, to the subject, an effective amount of any of the above fusion proteins. In some such methods, the glycogen storage disease is Pompe disease. In some such methods, the Pompe disease is classic infantile-onset form Pompe disease. In some such methods, the Pompe disease is non-classic infantile form Pompe disease. In some such methods, the Pompe disease is late onset form Pompe disease. In some such methods, the subject has a GAA genotype selected from the group consisting of: ASP91ASN; MET318THR; GLU521LYS; GLY643ARG; ARG725TRP; IVS1AS, T-G, −13; LYS903DEL; LEU299ARG; SER529VAL; ASP645GLU; GLU689LYS; EX18DEL; PRO545LEU; 1-BP DEL, 525T; ARG854TER; ALA237VAL; GLY293ARG; and IVS6AS, G-C, −1.
In some such methods, the subject is administered the fusion protein in association with a further therapeutic agent. In some such methods, the further therapeutic agent is selected from: alglucosidase alfa, rituximab, methotrexate, intravenous immunoglobulin (IVIG), avalglucosidase alfa, levalbuterol, an antibiotic, cortisone, prednisone, a bisphosphonate, and palivizumab. In some such methods, the further therapeutic agent is selected from: a beta2-adrenergic agonist, a steroid, a bisphosphonate, an infectious disease treatment, a vaccine, and a pneumococcal vaccine. In some such methods, the subject is 1 year of age or less and experiences a symptom selected from:
In some such methods, the subject is an adult and experiences a symptom selected from:
In some such methods, one or more signs or symptoms of the GSD in the subject are alleviated after the fusion protein or antigen-binding protein is administered.
In another aspect, provided are methods for delivering a payload to a tissue or cell type in the body of a subject comprising administering, to the subject, an antigen-binding protein that binds specifically to transferrin receptor or an antigenic-fragment thereof or variant thereof to the subject fused to the payload. In some such methods, the payload is one or more antibodies or antigen-binding fragments thereof, proteins, enzymes or viral vectors containing one or more polynucleotides or oligonucleotides. In some such methods, the payload is human GAA protein or a variant thereof. In some such methods, the tissue is brain/spinal cord/CNS; eye; skeletal muscle; adipose tissue; blood/bone marrow; breast; lung/bronchus; colon; uterus; esophagus; heart; kidney; liver; lymph node; ovary; pancreas; placenta; prostate; rectum; skin; peripheral blood mononuclear cell (PBMC); small intestine; spleen; stomach; testis; peripheral nervous system; and/or bone/cartilage/joint. In some such methods, the cell type and tissue that is associate with the cell type is as follows:
In some such methods, the method comprises piercing the body of the subject with a needle of a syringe and injecting the antigen-binding protein that binds specifically to transferrin receptor or an antigenic-fragment thereof or variant thereof to the subject fused to the payload into the body of the subject. In some such methods, the subject suffers from a muscle atrophy condition, metabolic disease, sarcopenia or cachexia.
In another aspect, provided are methods of expressing in a cell a fusion protein comprising an antigen-binding protein that binds specifically to transferrin receptor or an antigenic-fragment thereof or variant thereof fused to a payload comprising: (a) administering to the cell a gene therapy vector comprising any of the above polynucleotides, wherein the isolated polynucleotide encodes the fusion protein; (b) allowing the isolated polynucleotide to integrate into a genomic locus of the cell; and (c) allowing the cell to produce the fusion protein.
In some such methods, the method further comprises administering a nuclease agent or one or more polynucleotides encoding the nuclease agent to the cell, wherein the nuclease agent cleaves a nuclease target site in the genomic locus, and the isolated polynucleotide is integrated into the genomic locus. In some such methods, the nuclease agent comprises a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system, a zinc finger nuclease (ZFN), or a Transcription Activator-Like Effector Nuclease (TALEN). In some such methods, the cell is in vivo in a subject. In some such methods, the cell is ex vivo. In some such methods, the gene therapy vector is a viral vector, a naked polynucleotide, or a polynucleotide complex, optionally wherein the polynucleotide complex is a lipid nanoparticle comprising the polynucleotide. In some such methods, the gene therapy vector is a viral vector selected from the group consisting of a retrovirus, an adenovirus, a herpes simplex virus, a pox virus, a vaccinia virus, a lentivirus, or an adeno-associated virus. In some such methods, the gene therapy vector is an adeno-associated virus (AAV) vector, optionally wherein the gene therapy vector is an AAV2/8 chimera and/or an AAV pseudotyped to the liver. In some such methods, the genomic locus is a safe harbor locus. In some such methods, the genomic locus is at or proximal to a locus selected from the group consisting of an EESYR locus, a SARS locus, position 188,083,272 of human chromosome 1 or its non-human mammalian orthologue, position 3,046,320 of human chromosome 10 or its non-human mammalian orthologue, position 67,328,980 of human chromosome 17 or its non-human mammalian orthologue, an adeno-associated virus site 1 (AAVS1) on chromosome, a naturally occurring site of integration of AAV virus on human chromosome 19 or its non-human mammalian orthologue, a chemokine receptor 5 (CCR5) gene, a chemokine receptor gene encoding an HIV-1 coreceptor, a mouse Rosa26 locus or its non-murine mammalian orthologue, and an albumin (alb) locus. In some such methods, the cell is a human cell. In some such methods, the cell is a liver cell.
Provided herein are antigen-binding proteins that can be fused to a payload having one or more of the following characteristics: (1) Affinity (KD) for binding to human TfR at 25° C. in surface plasmon resonance format of about 41 nM or a higher affinity; (2) Affinity (KD) for binding to monkey TfR at 25° C. in surface plasmon resonance format of about 0 nM (no detectable binding) or a higher affinity; (3) Ratio of [KD for binding to monkey TfR/KD for binding to human TfR] at 25° C. in surface plasmon resonance format of from 0 to 278; (4) Blocks about 3-13% hTfR binding to Human Holo-Tf when in Fab format (IgG1); (5) Blocks about 6-13% hTfR binding to Human Holo-Tf when in scFv (VK-VH) format; (6) Blocks about 11-26% hTfR binding to Human Holo-Tf when in scFv (VH-VL) format; (7) When comprising the antigen-binding protein fused to GAA, exhibits a ratio of about 1-2 mature hGAA protein in brain (normalized to that of positive control 8D3:GAA scFv) when administered to mice expressing human transferrin receptor (optionally, lacking functional endogenous GAA) when in anti-hTfR scFv:hGAA format; (8) When comprising the antigen-binding protein fused to GAA, exhibits a ratio of about 0.1-1.2 mature hGAA protein in brain parenchyma (normalized to that of positive control 8D3:GAA scFv) when administered to mice expressing human transferrin receptor (optionally, lacking functional endogenous GAA) when in anti-hTfR scFv:hGAA format; (9) When comprising the antigen-binding protein fused to GAA, exhibits a ratio of about 0.67, 1.80, 1.78 or 7.74 (about 1-2) mature hGAA protein in quadriceps (normalized to that of positive control 8D3:GAA scFv) when administered to mice expressing human transferrin receptor (optionally, lacking functional endogenous GAA) when in anti-hTfR scFv:hGAA format; (10) When comprising the antigen-binding protein fused to GAA, exhibits a ratio of about 0.1-1.2 mature hGAA protein in brain parenchyma (normalized to that of positive control 8D3:GAA scFv) when administered to mice expressing human transferrin receptor (optionally, lacking functional endogenous GAA) when in anti-hTfR scFv:hGAA format; (11) When comprising the antigen-binding protein fused to GAA, delivers mature hGAA protein to serum, liver, cerebrum, cerebellum, spinal cord, heart and/or quadricep when administered to mice expressing human transferrin receptor (optionally, lacking functional endogenous GAA) when in anti-hTfR scFv:hGAA format; (12) When comprising the antigen-binding protein fused to GAA, reduces glycogen stored in cerebrum, cerebellum, spinal cord, heart and/or quadricep when administered to mice expressing human transferrin receptor (optionally, lacking functional endogenous GAA) when in anti-hTfR scFv:hGAA format; (13) When comprising the antigen-binding protein fused to GAA, reduces glycogen levels in cerebellum of mice expressing human transferrin receptor but lacking functional endogenous GAA by at least about 90% relative to that of untreated mice; (14) When comprising the antigen-binding protein fused to GAA, reduces glycogen levels in quadricep of mice expressing human transferrin receptor but lacking functional endogenous GAA by at least about 89% relative to that of untreated mice; and/or (15) Does not cause abnormal iron homeostasis when administered to mice expressing human transferrin receptor.
Provided herein is an antibody or antigen-binding fragment thereof that binds specifically to transferrin receptor (e.g., human transferrin receptor) that comprises: (i) a heavy chain variable region that comprises the HCDR1, HCDR2 and HCDR3 of a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2; 462; 12; 463; 22; 464; 32; 42; 52; 467; 62; 492; 72; 470; 82; 92; 472; 102; 112; 473; 122; 132; 142; 475; 152; 162; 477; 172; 182; 478; 192; 480; 202; 481; 212; 222; 232; 242; 252; 482; 262; 272; 282; 292; 302; 483 or 312 (or a variant thereof); and/or (ii) a light chain variable region that comprises the LCDR1, LCDR2 and LCDR3 of a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 7; 17; 27; 37; 465; 47; 466; 57; 468; 67; 469; 77; 471; 87; 97; 107; 117; 474; 127; 137; 147; 476; 157; 167; 177; 187; 479; 197; 207; 217; 227; 237; 247; 257; 267; 277; 287; 297; 307; 488; 317 or 484 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment thereof comprises: (1) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 7 (or a variant thereof); (2) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 12 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (3) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (4) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 37 (or a variant thereof); (5) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 47 (or a variant thereof); (6) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 57 (or a variant thereof); (7) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 67 (or a variant thereof); (8) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 72 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 77 (or a variant thereof); (9) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 87 (or a variant thereof); (10) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof); (11) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof); (12) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 117 (or a variant thereof); (13) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 127 (or a variant thereof); (14) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (15) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 142 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 147 (or a variant thereof); (16) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof); (17) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 162 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof); (18) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (19) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 182 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (20) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 192 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof); (21) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 202 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); (22) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof); (23) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (24) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof); (25) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (26) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof); (27) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (28) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (29) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); (30) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (31) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 307 or 488 (or a variant thereof); and/or (32) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment thereof comprises: (23) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); or (25) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment thereof comprises: (23) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment thereof comprises: (25) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment thereof comprises: (14) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment thereof comprises: (18) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment thereof comprises: (27) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment thereof comprises: (28) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof).
In an embodiment, the antibody or antigen-binding fragment comprises: (a) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); (b) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20 (or a variant thereof); (c) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 (or a variant thereof); (d) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 (or a variant thereof); (e) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 48 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50 (or a variant thereof); (f) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 54 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60 (or a variant thereof); (g) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 63 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 64 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 65 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70 (or a variant thereof); (h) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80 (or a variant thereof); (i) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 83 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 84 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90 (or a variant thereof); (j) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 95 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 99 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100 (or a variant thereof); (k) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 103 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 104 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 105 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110 (or a variant thereof); (1) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 118 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 119 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120 (or a variant thereof); (m) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 123 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 124 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 125 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 128 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 129 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 130 (or a variant thereof); (n) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof); (o) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof); (p) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 155 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 158 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 159 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 160 (or a variant thereof); (q) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 163 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 164 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 165 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 168 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 169 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 170 (or a variant thereof); (r) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 178 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180 (or a variant thereof); (s) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 183 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 184 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 185 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 188 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 189 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 190 (or a variant thereof); (t) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 193 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 194 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 195 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 199 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200 (or a variant thereof); (u) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 203 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 205 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 208 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 210 (or a variant thereof); (v) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 213 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 214 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 215 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 218 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 220 (or a variant thereof); (w) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof); (x) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 233 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 234 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 235 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 238 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 239 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 240 (or a variant thereof); (y) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof); (z) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 253 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 254 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 255 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 258 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 259 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 260 (or a variant thereof); (aa) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof); (ab) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof); (ac) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 283 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 284 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 285 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 288 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 289 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 290 (or a variant thereof); (ad) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 293 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 294 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 295 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 298 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 299 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 300 (or a variant thereof); (ae) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 303 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 304 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 305 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 308 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 309 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 310 (or a variant thereof); and/or (af) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 313 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 314 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 315 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 318 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 319 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 320 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment comprises: (w) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof); or (y) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof. In an embodiment, the antibody or antigen-binding fragment comprises: (w) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment comprises: (y) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof. In an embodiment, the antibody or antigen-binding fragment comprises: (n) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment comprises: (r) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 178 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment comprises: (aa) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof). In an embodiment, the antibody or antigen-binding fragment comprises: (ab) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof).
In an embodiment, the antibody or antigen-binding fragment comprises: (i) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 7 (or a variant thereof); (ii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 12 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (iii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (iv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 37 (or a variant thereof); (v) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 47 (or a variant thereof); (vi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 57 (or a variant thereof); (vii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 67 (or a variant thereof); (viii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 72 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 77 (or a variant thereof); (ix) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 87 (or a variant thereof); (x) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof); (xi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof); (xii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 117 (or a variant thereof); (xiii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 127 (or a variant thereof); (xiv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (xv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 142 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 147 (or a variant thereof); (xvi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof); (xvii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 162 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof); (xviii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (xix) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 182 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (xx) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 192 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof); (xxi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 202 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); (xxii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof); (xxiii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (xxiv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof); (xxv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (xxvi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof); (xxvii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (xxviii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (xxix) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); (xxx) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (xxxi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 307 or 488 (or a variant thereof); and/or (xxxii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof). See, e.g.,
In an embodiment, the antigen-binding fragment comprises an scFv. In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 443 (or a variant thereof) or comprises the amino acid sequence set forth in SEQ ID NO: 440 (or a variant thereof). In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 443 (or a variant thereof). In an embodiment, the antigen-binding fragment comprises an scFv. In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 440 (or a variant thereof). In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 429 (or a variant thereof). In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 433 (or a variant thereof). In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 442 (or a variant thereof). In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 438 (or a variant thereof).
Provided herein is an anti-hTfR:Payload fusion protein comprising a single chain fragment variable (scFv), an antibody (e.g., an IgG, e.g., IgG1, IgG2, IgG3 or IgG4) or an antigen-binding fragment thereof (e.g., a Fab) that binds specifically to human transferrin receptor (or a vessel (e.g., vial) or injection device (e.g., syringe) containing such a fusion) (e.g., that binds to human transferrin receptor with a KD of about 1×10−7 M or a greater affinity), which comprises a heavy chain variable region (VH) and a light chain variable region (VL), which is fused to a payload such as an alpha-glucosidase polypeptide (GAA), wherein a Fab having said VH and VL binds to human transferrin receptor with a KD of about 0.65 nM or a greater affinity; and wherein, when said fusion protein is an anti-hTfR:GAA and is administered to a mouse expressing human transferrin receptor in the brain, the mouse achieves a molar ratio of mature GAA protein in the brain:serum GAA protein, in the mouse, of about 1:1 or greater when normalized against said ratio in mouse expressing mouse transferrin receptor that was administered 8D3. For example, where, when the fusion protein is an scfV, the scFv comprises domains arranged in the following orientation: N-Heavy chain variable region-Light chain variable region-GAA protein-C or N-Light chain variable region-Heavy chain variable region-Payload protein-C (“N-” denotes the amino terminus of the polypeptide and “C-” denotes the carboxy terminus of the polypeptide). In an embodiment, the scFv and the payload, e.g., GAA, are connected by a peptide linker such as -(GGGGS)m- (SEQ ID NO: 426); wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In an embodiment, the scFv variable regions are connected by a peptide linker such as -(GGGGS)n- (SEQ ID NO: 426); wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In an embodiment, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 408 (or a variant thereof) or comprises the amino acid sequence set forth in SEQ ID NO: 405 (or a variant thereof). In an embodiment, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 408 (or a variant thereof). In an embodiment, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 405 (or a variant thereof).
In an embodiment, the anti-hTfR:Payload fusion protein comprises: (i) a heavy chain variable region that comprises the HCDR1, HCDR2 and HCDR3 of a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2; 462; 12; 463; 22; 464; 32; 42; 52; 467; 62; 492; 72; 470; 82; 92; 472; 102; 112; 473; 122; 132; 142; 475; 152; 162; 477; 172; 182; 478; 192; 480; 202; 481; 212; 222; 232; 242; 252; 482; 262; 272; 282; 292; 302; 483 or 312 (or a variant thereof); and/or (ii) a light chain variable region that comprises the LCDR1, LCDR2 and LCDR3 of a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 7; 17; 27; 37; 465; 47; 466; 57; 468; 67; 469; 77; 471; 87; 97; 107; 117; 474; 127; 137; 147; 476; 157; 167; 177; 187; 479; 197; 207; 217; 227; 237; 247; 257; 267; 277; 287; 297; 307; 488; 317 or 484 (or a variant thereof). In an embodiment, the fusion protein comprises: (1) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 7 (or a variant thereof); (2) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 12 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (3) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (4) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 37 (or a variant thereof); (5) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 47 (or a variant thereof); (6) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 57 (or a variant thereof); (7) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 67 (or a variant thereof); (8) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 72 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 77 (or a variant thereof); (9) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 87 (or a variant thereof); (10) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof); (11) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof); (12) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 117 (or a variant thereof); (13) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 127 (or a variant thereof); (14) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (15) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 142 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 147 (or a variant thereof); (16) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof); (17) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 162 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof); (18) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (19) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 182 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (20) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 192 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof); (21) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 202 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); (22) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof); (23) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (24) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof); (25) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (26) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof); (27) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (28) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (29) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); (30) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (31) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 307 or 488 (or a variant thereof); and/or (32) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof). In an embodiment, the fusion protein comprises: (23) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); or (25) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). In an embodiment, the fusion protein comprises: (23) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof). In an embodiment, the fusion protein comprises: (25) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). In an embodiment, the fusion protein comprises: (14) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof). In an embodiment, the fusion protein comprises: (18) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof). In an embodiment, the fusion protein comprises: (27) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof). In an embodiment, the fusion protein comprises: (28) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof).
In an embodiment, the fusion protein comprises: (a) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); (b) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20 (or a variant thereof); (c) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 (or a variant thereof); (d) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 (or a variant thereof); (e) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 48 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50 (or a variant thereof); (f) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 54 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60 (or a variant thereof); (g) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 63 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 64 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 65 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70 (or a variant thereof); (h) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80 (or a variant thereof); (i) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 83 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 84 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90 (or a variant thereof); (j) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 95 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 99 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100 (or a variant thereof); (k) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 103 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 104 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 105 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110 (or a variant thereof); (1) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 118 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 119 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120 (or a variant thereof); (m) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 123 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 124 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 125 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 128 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 129 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 130 (or a variant thereof); (n) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof); (o) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof); (p) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 155 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 158 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 159 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 160 (or a variant thereof); (q) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 163 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 164 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 165 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 168 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 169 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 170 (or a variant thereof); (r) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 178 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180 (or a variant thereof); (s) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 183 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 184 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 185 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 188 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 189 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 190 (or a variant thereof); (t) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 193 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 194 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 195 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 199 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200 (or a variant thereof); (u) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 203 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 205 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 208 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 210 (or a variant thereof); (v) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 213 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 214 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 215 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 218 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 220 (or a variant thereof); (w) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof); (x) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 233 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 234 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 235 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 238 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 239 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 240 (or a variant thereof); (y) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof); (z) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 253 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 254 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 255 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 258 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 259 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 260 (or a variant thereof); (aa) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof); (ab) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof); (ac) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 283 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 284 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 285 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 288 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 289 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 290 (or a variant thereof); (ad) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 293 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 294 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 295 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 298 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 299 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 300 (or a variant thereof); (ae) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 303 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 304 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 305 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 308 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 309 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 310 (or a variant thereof); and/or (af) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 313 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 314 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 315 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 318 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 319 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 320 (or a variant thereof). In an embodiment, the fusion protein comprises: (w) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof); or (y) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof. In an embodiment, the fusion protein comprises: (w) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof). In an embodiment, the fusion protein comprises: (y) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof. In an embodiment, the fusion protein comprises: (n) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof). In an embodiment, the fusion protein comprises: (r) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 178 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180 (or a variant thereof). In an embodiment, the fusion protein comprises: (aa) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof). In an embodiment, the fusion protein comprises: (ab) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof).
In an embodiment, the fusion protein comprises: (i) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 7 (or a variant thereof); (ii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 12 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (iii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (iv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 37 (or a variant thereof); (v) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 47 (or a variant thereof); (vi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 57 (or a variant thereof); (vii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 67 (or a variant thereof); (viii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 72 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 77 (or a variant thereof); (ix) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 87 (or a variant thereof); (x) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof); (xi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof); (xii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 117 (or a variant thereof); (xiii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 127 (or a variant thereof); (xiv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (xv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 142 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 147 (or a variant thereof); (xvi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof); (xvii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 162 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof); (xviii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (xix) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 182 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (xx) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 192 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof); (xxi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 202 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); (xxii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof); (xxiii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (xxiv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof); (xxv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (xxvi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof); (xxvii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (xxviii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (xxix) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); (xxx) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (xxxi) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 307 or 488 (or a variant thereof); and/or (xxxii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof). In an embodiment, the fusion protein comprises: (xxiii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); or (xxv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). In an embodiment, the fusion protein comprises: (xxiii i) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof). In an embodiment, the fusion protein comprises: (xxv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). In an embodiment, the fusion protein comprises: (xiv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof). In an embodiment, the fusion protein comprises: (xviii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof). In an embodiment, the fusion protein comprises: (xxvii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof). In an embodiment, the fusion protein comprises: (xxviii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof).
Provided herein is a fusion protein that is an scFv that comprises a heavy chain variable region (VH) and a light chain variable region (VL), and a payload such as an alpha-glucosidase polypeptide (GAA), wherein said VH, VL and payload, e.g., GAA, are arranged as follows: (i) VL-VH-Payload; (ii) VH-VL-Payload; (iii) VL-[(GGGGS)3 (SEQ ID NO: 538)]-VH-[(GGGGS)2 (SEQ ID NO: 537)]-Payload, or (iv) VH-[(GGGGS)3 (SEQ ID NO: 538)]-VL-[(GGGGS)2 (SEQ ID NO: 537)]-Payload. For example, in an embodiment, the fusion protein comprises (i) the amino acid sequence set forth in SEQ ID NO: 321 (or a mature polypeptide thereof), (ii) the amino acid sequence set forth in SEQ ID NO: 322 (or a mature polypeptide thereof), (iii) the amino acid sequence set forth in SEQ ID NO: 323 (or a mature polypeptide thereof), (iv) the amino acid sequence set forth in SEQ ID NO: 324 (or a mature polypeptide thereof), (v) amino acids 30-1168 of SEQ ID NO: 321, (vi) amino acids 30-1171 of SEQ ID NO: 322, (vii) amino acids 30-1164 of SEQ ID NO: 323, or (viii) amino acids 30-1166 of SEQ ID NO: 324. In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 443 (or a variant thereof) or comprises the amino acid sequence set forth in SEQ ID NO: 440 (or a variant thereof). In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 443 (or a variant thereof). In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 440 (or a variant thereof). In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 429 (or a variant thereof). In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 433 (or a variant thereof). In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 442 (or a variant thereof). In an embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 438 (or a variant thereof). In an embodiment, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 408 (or a variant thereof) or comprises the amino acid sequence set forth in SEQ ID NO: 405 (or a variant thereof). In an embodiment, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 408 (or a variant thereof). In an embodiment, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 405 (or a variant thereof). In an embodiment, the fusion protein (e.g., a Fab of said fusion protein) comprises the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs: 392-423, e.g., which is fused to a payload such as a GAA polypeptide. In an embodiment, an anti-TfR:GAA fusion protein single chain fragment variable (scFv), antibody or an antigen-binding fragment thereof, which is not fused to a GAA polypeptide, does not block more than 50% of binding of a human transferrin receptor C-terminal fragment to human holo-transferrin that occurs in the absence of such single chain fragment variable (scFv), antibody or an antigen-binding fragment; for example, wherein said blocking is as measured in an Enzyme Linked Immunosorbent Assay (ELISA) plate assay wherein binding of human transferrin receptor C-terminal fragment that is fused to a His6-myc-myc tag is pre-bound to said scFv, antibody or antigen-binding fragment and then contacted with holo-transferrin which is immobilized to the surface of the plate by binding of an anti-holo-transferrin antibody that is bound to the plate, e.g., wherein binding of the holotransferrin and human transferrin receptor C in the absence of the scFv, antibody or antigen-binding fragment is measured at a concentration of about 300 pM human transferrin receptor C-terminal fragment.
Also provided is a composition that includes an anti-hTfR:GAA fusion protein provided herein, e.g., pharmaceutical composition comprising a fusion protein as disclosed herein and a pharmaceutically acceptable carrier. Kits including a fusion protein as disclosed herein are also provided. Such a composition or kit further including a further therapeutic agent (e.g., alglucosidase alfa, rituximab, methotrexate, Intravenous immunoglobulin (IVIG), avalglucosidase alfa, levalbuterol, an antibiotic, cortisone, prednisone, a bisphosphonate, and palivizumab, a Beta2-adrenergic agonist, a steroid, a bisphosphonate, an infectious disease treatment, a vaccine, and/or a Pneumococcal vaccine) is also provided.
Complexes comprising an anti-hTfR:GAA fusion protein as disclosed herein bound to a human transferrin receptor polypeptide or antigenic fragment thereof are also provided.
Also provided is an isolated polynucleotide encoding an anti-hTfR:GAA fusion protein disclosed herein, e.g., that comprises the nucleotide sequence set forth in SEQ ID NO: 1; 6; 11; 16; 21; 26; 31; 36; 41; 46; 51; 56; 61; 66; 71; 76; 81; 86; 91; 96; 101; 106; 111; 116; 121; 126; 131; 136; 141; 146; 151; 156; 161; 166; 171; 176; 181; 186; 191; 196; 201; 206; 211; 216; 221; 226; 231; 236; 241; 246; 251; 256; 261; 266; 271; 276; 281; 286; 291; 296; 301; 306; 311; and/or 316. For example, polynucleotides comprising any one or more of the following are provided: (1) the nucleotide sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 6; (2) the nucleotide sequence set forth in SEQ ID NO: 11 and SEQ ID NO: 16; (3) the nucleotide sequence set forth in SEQ ID NO: 21 and SEQ ID NO: 26; (4) the nucleotide sequence set forth in SEQ ID NO: 31 and SEQ ID NO: 36; (5) the nucleotide sequence set forth in SEQ ID NO: 41 and SEQ ID NO: 46; (6) the nucleotide sequence set forth in SEQ ID NO: 51 and SEQ ID NO: 56; (7) the nucleotide sequence set forth in SEQ ID NO: 61 and SEQ ID NO: 66; (8) the nucleotide sequence set forth in SEQ ID NO: 71 and SEQ ID NO: 76; (9) the nucleotide sequence set forth in SEQ ID NO: 81 and SEQ ID NO: 86; (10) the nucleotide sequence set forth in SEQ ID NO: 91 and SEQ ID NO: 96; (11) the nucleotide sequence set forth in SEQ ID NO: 101 and SEQ ID NO: 106; (12) the nucleotide sequence set forth in SEQ ID NO: 111 and SEQ ID NO: 116; (13) the nucleotide sequence set forth in SEQ ID NO: 121 and SEQ ID NO: 126; (14) the nucleotide sequence set forth in SEQ ID NO: 131 and SEQ ID NO: 136; (15) the nucleotide sequence set forth in SEQ ID NO: 141 and SEQ ID NO: 146; (16) the nucleotide sequence set forth in SEQ ID NO: 151 and SEQ ID NO: 156; (17) the nucleotide sequence set forth in SEQ ID NO: 161 and SEQ ID NO: 166; (18) the nucleotide sequence set forth in SEQ ID NO: 171 and SEQ ID NO: 176; (19) the nucleotide sequence set forth in SEQ ID NO: 181 and SEQ ID NO: 186; (20) the nucleotide sequence set forth in SEQ ID NO: 191 and SEQ ID NO: 196; (21) the nucleotide sequence set forth in SEQ ID NO: 201 and SEQ ID NO: 206; (22) the nucleotide sequence set forth in SEQ ID NO: 211 and SEQ ID NO: 216; (23) the nucleotide sequence set forth in SEQ ID NO: 221 and SEQ ID NO: 226; (24) the nucleotide sequence set forth in SEQ ID NO: 231 and SEQ ID NO: 236; (25) the nucleotide sequence set forth in SEQ ID NO: 241 and SEQ ID NO: 246; (26) the nucleotide sequence set forth in SEQ ID NO: 251 and SEQ ID NO: 256; (27) the nucleotide sequence set forth in SEQ ID NO: 261 and SEQ ID NO: 266; (28) the nucleotide sequence set forth in SEQ ID NO: 271 and SEQ ID NO: 276; (29) the nucleotide sequence set forth in SEQ ID NO: 281 and SEQ ID NO: 286; (30) the nucleotide sequence set forth in SEQ ID NO: 291 and SEQ ID NO: 296; (31) the nucleotide sequence set forth in SEQ ID NO: 301 and SEQ ID NO: 306; and/or (32) the nucleotide sequence set forth in SEQ ID NO: 311 and SEQ ID NO: 316. A vector, e.g., an expression vector, comprising a polynucleotide encoding a fusion protein disclosed herein is provided. Also provided is a host cell (Chinese hamster ovary (CHO) cell) comprising the fusion protein, polynucleotide and/or vector.
Also provided is a method for making an anti-hTfR:GAA fusion protein as disclosed herein comprises the steps of culturing a host cell (e.g., CHO cell) comprising a polynucleotide that encodes the fusion protein in a culture medium under conditions favorable to expression of the fusion protein, e.g., (a) introducing said polynucleotide into a host cell; (b) culturing the host cell under conditions favorable to expression of the fusion protein; and (c) optionally, isolating the fusion protein from the culture medium and/or host cell. Such fusion proteins which are a product of such a method are provided.
Also provided herein is a method for administering (e.g., parenterally, e.g., intravenously) an anti-hTfR:GAA fusion protein as disclosed herein, optionally in association with a further therapeutic agent, to a subject (e.g., having a GSD and/or a GAA genotype selected from the group consisting of: ASP91ASN; MET318THR; GLU521LYS; GLY643ARG; ARG725TRP; IVS1AS, T-G, −13; LYS903DEL; LEU299ARG; SER529VAL; ASP645GLU; GLU689LYS; EX18DEL; PRO545LEU; 1-BP DEL, 525T; ARG854TER; ALA237VAL; GLY293ARG; and IVS6AS, G-C, −1) comprising introducing the protein into the body of the subject. Also provided herein is a method for treating or preventing a glycogen storage disease (e.g., Pompe disease, for example, classic infantile-onset form Pompe disease; non-classic infantile form Pompe disease; or late onset form Pompe disease), in a subject (e.g., having a GAA genotype selected from the group consisting of: ASP91ASN; MET318THR; GLU521LYS; GLY643ARG; ARG725TRP; IVS1AS, T-G, −13; LYS903DEL; LEU299ARG; SER529VAL; ASP645GLU; GLU689LYS; EX18DEL; PRO545LEU; 1-BP DEL, 525T; ARG854TER; ALA237VAL; GLY293ARG; and IVS6AS, G-C, −1) in need thereof comprising administering, to the subject, an effective amount of the fusion protein as disclosed herein, optionally in association with a further therapeutic agent. In an embodiment, the subject is 1 year of age or less and experiences a symptom selected from: Trouble eating and not gaining weight; Poor head and neck control; Rolling over and sitting up later than expected; Breathing problems; Lung infection; Enlarged and thickening heart; Heart defect; Enlarged liver; and Enlarged tongue. In an embodiment, the subject is an adult and experiences a symptom selected from: Weakness in the legs, trunk, and/or arms; Shortness of breath; Lung infection; Trouble breathing while sleeping; Spine curvature; Enlarged liver; Enlarged tongue; and Stiff joints.
Also provided herein is a method for delivering a payload (e.g., one or more antibodies or antigen-binding fragments thereof, proteins, enzymes or viral vectors containing one or more polynucleotides or oligonucleotides), e.g., GAA, to a tissue in the body of a subject (e.g., cartilage, brain, cerebral cortex; cerebellum; hippocampus; caudate; parathyroid gland; adrenal gland; bronchus; lung; oral mucosa; esophagus; stomach; duodenum; small intestine; colon; rectum; liver; gallbladder; pancreas; kidney; urinary bladder; testis; epididymis; prostate; vagina; ovary; fallopian tube; endometrium; cervix; placenta; breast; muscle, heart muscle; skeletal muscle, smooth muscle, muscle endothelial vasculature; soft tissue; skin; appendix; lymph node; tonsil; and/or bone marrow) including administering an antigen-binding protein that binds specifically to transferrin receptor or an antigenic-fragment thereof or variant thereof to the subject fused to the payload. For example, the method can include the steps of piercing the body of the subject with a needle of a syringe and injecting antigen-binding protein that binds specifically to transferrin receptor or an antigenic-fragment thereof or variant thereof to the subject fused to the payload into the body of the subject.
Provided herein are anti-transferrin receptor antigen-binding proteins. Also provided are anti-transferrin receptor antigen-binding proteins that are fused to a payload. Such fusions are useful, for example, for delivery of the payload to various tissues in the body, including the brain. For example, anti-TfR:GAA fusion proteins exhibiting high affinity to the transferrin receptor and superior blood-brain barrier crossing are provided. Surprisingly, fusions exhibiting high binding affinity to TfR crossed the BBB more efficiently than that of low affinity binders. We found that high affinity antibodies impart the best delivery to the CNS and muscle in the anti-hTFRscfv:payload (e.g., GAA) format. This is in contrast to previous findings with mono- and bivalent anti-TFR antibodies, where low affinity antibodies crossed the BBB more effectively. The fusions disclosed herein have an ability to efficiently deliver GAA to the brain and, thus, are an effective treatment of glycogen storage diseases such as Pompe Disease.
There may be employed herein conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook, et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
A polynucleotide includes DNA and RNA. Provided herein is any polynucleotide disclosed herein which is operably linked to a promoter or other expression control sequence.
A symptom is a manifestation of disease apparent to the patient himself, while a sign is a manifestation of disease that the physician perceives. Reduction, fully or in part, of a sign or symptom may be referred to as alleviation of the sign or symptom.
An oligonucleotide is a polynucleotide of up to about 30 nucleotides in length, e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
Transferrin receptor 1 (TfR) is a membrane receptor involved in the control of iron supply to the cell through the binding of transferrin, the major iron-carrier protein. Transferrin receptor 1 is expressed from the TFRC gene. Transferrin receptor 1 may be referred to, herein, at TFRC. This receptor plays a key role in the control of cell proliferation because iron is essential for sustaining ribonucleotide reductase activity, and is the only enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides. Preferably, the TfR is human TfR (hTfR). See e.g., Accession numbers NP_001121620.1; BAD92491.1; and NP_001300894.1; and e!Ensembl entry: ENSG00000072274. The human transferrin receptor 1 is expressed in several tissues, including but not limited to: cerebral cortex; cerebellum; hippocampus; caudate; parathyroid gland; adrenal gland; bronchus; lung; oral mucosa; esophagus; stomach; duodenum; small intestine; colon; rectum; liver; gallbladder; pancreas; kidney; urinary bladder; testis; epididymis; prostate; vagina; ovary; fallopian tube; endometrium; cervix; placenta; breast; heart muscle; smooth muscle; soft tissue; skin; appendix; lymph node; tonsil; and bone marrow. See also tissues and cell types of Table B herein. A related transferrin receptor is transferrin receptor 2 (TfR2). Human transferrin receptor 2 bears about 45% sequence identity to human transferrin receptor 1. Trinder & Baker, Transferrin receptor 2: a new molecule in iron metabolism. Int J Biochem Cell Biol. 2003 March; 35(3):292-6. Unless otherwise stated, transferrin receptor as used herein generally refers to transferrin receptor 1 (e.g., human transferrin receptor 1) (CD71).
Human Transferrin (Tf) is a single chain, 80 kDa member of the anion-binding superfamily of proteins. Transferrin is a 698 amino acid precursor that is divided into a 19 aa signal sequence plus a 679 aa mature segment that typically contains 19 intrachain disulfide bonds. The N- and C-terminal flanking regions (or domains) bind ferric iron through the interaction of an obligate anion (e.g., bicarbonate) and four amino acids (His, Asp, and two Tyr). Apotransferrin (or iron-free) will initially bind one atom of iron at the C-terminus, and this is followed by subsequent iron binding by the N-terminus to form holotransferrin (diferric Tf, Holo-Tf). Through its C-terminal iron-binding domain, holotransferrin will interact with the TfR on the surface of cells where it is internalized into acidified endosomes. Iron dissociates from the Tf molecule within these endosomes, and is transported into the cytosol as ferrous iron. In addition to TfR, transferrin is reported to bind to cubulin, IGFBP3, microbial iron-binding proteins and liver-specific TfR2.
The blood-brain barrier (BBB) is located within the microvasculature of the brain, and it regulates passage of molecules from the blood to the brain. Burkhart et al., Accessing targeted nanoparticles to the brain: the vascular route. Curr Med Chem. 2014; 21(36):4092-9. The transcellular passage through the brain capillary endothelial cells can take place via 1) cell entry by leukocytes; 2) carrier-mediated influx of e.g., glucose by glucose transporter 1 (GLUT-1), amino acids by e.g., the L-type amino acid transporter 1 (LAT-1) and small peptides by e.g., organic anion-transporting peptide-B (OATP-B); 3) paracellular passage of small hydrophobic molecules; 4) adsorption-mediated transcytosis of e.g., albumin and cationized molecules; 5) passive diffusion of lipid soluble, non-polar solutes, including CO2 and O2; and 5) receptor-mediated transcytosis of e.g., insulin by the insulin receptor and Tf by the TfR. Johnsen et al., Targeting the transferrin receptor for brain drug delivery, Prog Neurobiol. 2019 October; 181:101665.
Provided are antigen-binding proteins, such as antibodies, antigen-binding fragments thereof, such as Fabs and scFvs, that bind specifically to the transferrin receptor, preferably the human transferrin receptor 1 (anti-hTfR). For example, in an embodiment, the anti-hTfR is in the form of a fusion protein. The fusion protein includes the anti-hTfR antigen-binding protein fused to a particular payload (anti-hTfR:Payload). The anti-hTfRs disclosed herein efficiently cross the blood-brain barrier (BBB) and can, thereby, deliver the fused payload to the brain.
An antigen-binding protein that specifically binds to transferrin receptor and fusions thereof, for example, a tag such as His6 and/or myc (e.g., human transferrin receptor (e.g., REGN2431) or monkey transferrin receptor (e.g., REGN2054)) binds at about 25° C., e.g., in a surface plasmon resonance assay, with a KD of about 20 nM or a higher affinity. Such an antigen-binding protein may be referred to as “anti-TfR”. In some embodiments, the antigen-binding protein binds to human transferrin receptor with a KD of about 0.41 nM or a stronger affinity. In some embodiments, the antigen-binding protein binds to human transferrin receptor with a KD of about 3 nM or a stronger affinity. In some embodiments, the antigen-binding protein binds to human transferrin receptor with a KD of about 0.45 nM to 3 nM. In some embodiments, a Fab having an HCVR and LCVR binds to human transferrin receptor with a KD of about 0.65 nM or a stronger affinity. In some embodiments, a fusion protein disclosed herein binds to human transferrin receptor with a KD of about 1×10−7 M or a stronger affinity.
In an embodiment, an anti-hTfR scFv:Payload or anti-TfR:Payload scFv fusion protein includes an scFv comprising the arrangement of variable regions as follows LCVR-HCVR or HCVR-LCVR, wherein the HCVR and LCVR are optionally connected by a linker and the scFv is connected, optionally by a linker, to a payload (e.g., GAA or variant thereof) (e.g., LCVR-(Gly4Ser)3 (SEQ ID NO: 538)-HCVR-(Gly4Ser)2 (SEQ ID NO: 537))-Payload (e.g., mature human GAA); or LCVR-(Gly4Ser)3 (SEQ ID NO: 538)-HCVR-(Gly4Ser)2 (SEQ ID NO: 537))-Payload (e.g., mature human GAA)) (Gly4Ser=SEQ ID NO: 426).
An anti-hTfR:Payload optionally comprises a signal peptide, connected to the antigen-binding protein that binds specifically to transferrin receptor (TfR), preferably, human transferrin receptor (hTfR) which is fused (optionally by a linker) to a payload such as GAA or a variant thereof. In an embodiment, the signal peptide is the mROR signal sequence (e.g., mROR signal sequence-LCVR-(Gly4Ser)3 (SEQ ID NO: 538)-HCVR-(Gly4Ser)2 (SEQ ID NO: 537))-Payload (e.g., mature human GAA); or LCVR-(Gly4Ser)3 (SEQ ID NO: 538)-HCVR-(Gly4Ser)2 (SEQ ID NO: 537))-Payload (e.g., mature human GAA)) (Gly4Ser=SEQ ID NO: 426).
The term “fused” or “tethered” with regard to fused polypeptides refers to polypeptides joined directly or indirectly (e.g., via a linker or other polypeptide).
In an embodiment, the assignment of amino acids to each framework or CDR domain in an immunoglobulin is in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342: 878-883. Thus, provided herein are antibodies and antigen-binding fragments including the CDRs of a VH and the CDRs of a VL, which VH and VL comprise amino acid sequences as set forth herein (see e.g., sequences of Table A, or variants thereof), wherein the CDRs are as defined according to Kabat and/or Chothia.
Provided herein are antibodies that bind specifically to the human transferrin receptor 1. The term “antibody”, as used herein, refers to immunoglobulin molecules comprising four polypeptide chains, two heavy chains (HCs) and two light chains (LCs), inter-connected by disulfide bonds. In an embodiment, each antibody heavy chain (HC) comprises a heavy chain variable region (“HCVR” or “VH”) (e.g., comprising SEQ ID NO: 2; 462; 12; 463; 22; 464; 32; 42; 52; 467; 62; 492; 72; 470; 82; 92; 472; 102; 112; 473; 122; 132; 142; 475; 152; 162; 477; 172; 182; 478; 192; 480; 202; 481; 212; 222; 232; 242; 252; 482; 262; 272; 282; 292; 302; 483 and/or 312, or a variant thereof) and a heavy chain constant region (e.g., human IgG, human IgG1 or human IgG4); and each antibody light chain (LC) comprises a light chain variable region (“LCVR or “VL”) (e.g., SEQ ID NO: 7; 17; 27; 37; 465; 47; 466; 57; 468; 67; 469; 77; 471; 87; 97; 107; 117; 474; 127; 137; 147; 476; 157; 167; 177; 187; 479; 197; 207; 217; 227; 237; 247; 257; 267; 277; 287; 297; 307; 488; 317 and/or 484, or a variant thereof) and a light chain constant region (e.g., human kappa or human lambda). In an embodiment, each antibody heavy chain (HC) comprises a heavy chain variable region (“HCVR” or “VH”) (e.g., comprising SEQ ID NO: 222 or 242, or a variant thereof) and a heavy chain constant region (e.g., human IgG, human IgG1 or human IgG4); and each antibody light chain (LC) comprises a light chain variable region (“LCVR or “VL”) (e.g., SEQ ID NO: 227 or 247, or a variant thereof) and a light chain constant region (e.g., human kappa or human lambda). In an embodiment, each antibody heavy chain (HC) comprises a heavy chain variable region (“HCVR” or “VH”) (e.g., comprising SEQ ID NO: 222, or a variant thereof) and a heavy chain constant region (e.g., human IgG, human IgG1 or human IgG4); and each antibody light chain (LC) comprises a light chain variable region (“LCVR or “VL”) (e.g., SEQ ID NO: 227, or a variant thereof) and a light chain constant region (e.g., human kappa or human lambda). In an embodiment, each antibody heavy chain (HC) comprises a heavy chain variable region (“HCVR” or “VH”) (e.g., comprising SEQ ID NO: 242, or a variant thereof) and a heavy chain constant region (e.g., human IgG, human IgG1 or human IgG4); and each antibody light chain (LC) comprises a light chain variable region (“LCVR or “VL”) (e.g., SEQ ID NO: 247, or a variant thereof) and a light chain constant region (e.g., human kappa or human lambda). In an embodiment, each antibody heavy chain (HC) comprises a heavy chain variable region (“HCVR” or “VH”) (e.g., comprising SEQ ID NO: 132, or a variant thereof) and a heavy chain constant region (e.g., human IgG, human IgG1 or human IgG4); and each antibody light chain (LC) comprises a light chain variable region (“LCVR or “VL”) (e.g., SEQ ID NO: 137, or a variant thereof) and a light chain constant region (e.g., human kappa or human lambda). In an embodiment, each antibody heavy chain (HC) comprises a heavy chain variable region (“HCVR” or “VH”) (e.g., comprising SEQ ID NO: 172, or a variant thereof) and a heavy chain constant region (e.g., human IgG, human IgG1 or human IgG4); and each antibody light chain (LC) comprises a light chain variable region (“LCVR or “VL”) (e.g., SEQ ID NO: 177, or a variant thereof) and a light chain constant region (e.g., human kappa or human lambda). In an embodiment, each antibody heavy chain (HC) comprises a heavy chain variable region (“HCVR” or “VH”) (e.g., comprising SEQ ID NO: 262, or a variant thereof) and a heavy chain constant region (e.g., human IgG, human IgG1 or human IgG4); and each antibody light chain (LC) comprises a light chain variable region (“LCVR or “VL”) (e.g., SEQ ID NO: 267, or a variant thereof) and a light chain constant region (e.g., human kappa or human lambda). In an embodiment, each antibody heavy chain (HC) comprises a heavy chain variable region (“HCVR” or “VH”) (e.g., comprising SEQ ID NO: 272, or a variant thereof) and a heavy chain constant region (e.g., human IgG, human IgG1 or human IgG4); and each antibody light chain (LC) comprises a light chain variable region (“LCVR or “VL”) (e.g., SEQ ID NO: 277, or a variant thereof) and a light chain constant region (e.g., human kappa or human lambda). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs. Anti-TfR antibodies disclosed herein can also be fused to a payload such as GAA or a variant thereof.
An anti-TfR antigen-binding protein provided herein may be an antigen-binding fragment of an antibody which may be tethered to a payload. The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody, as used herein, refers to an immunoglobulin molecule that binds antigen but that does not include all of the sequences of a full antibody (preferably, the full antibody is an IgG). Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; and (vi) dAb fragments; consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies and small modular immunopharmaceuticals (SMIPs), are also encompassed within the expression “antigen-binding fragment,” as used herein.
As mentioned, an anti-TfR antigen-binding protein provided herein may be an scFv which may be tethered to a payload. An scFv (single chain fragment variable) has variable regions of heavy (VH) and light (VL) domains (in either order), which, preferably, are joined together by a flexible linker (e.g., peptide linker). The length of the flexible linker used to link both of the V regions may be important for yielding the correct folding of the polypeptide chain. Previously, it has been estimated that the peptide linker must span 3.5 nm (35 A) between the carboxy terminus of the variable domain and the amino terminus of the other domain without affecting the ability of the domains to fold and form an intact antigen-binding site (Huston et al., Protein engineering of single-chain Fv analogs and fusion proteins. Methods in Enzymology. 1991; 203:46-88). In an embodiment, the linker comprises an amino acid sequence of such length to separate the variable domains by about 3.5 nm.
In some embodiments, an anti-TfR antigen-binding protein described herein comprises a monovalent or “one-armed” antibody. The monovalent or “one-armed” antibodies as used herein refer to immunoglobulin proteins comprising a single variable domain. For example, the one-armed antibody may comprise a single variable domain within a Fab wherein the Fab is linked to at least one Fc fragment. In certain embodiments, the one-armed antibody comprises: (i) a heavy chain comprising a heavy chain constant region and a heavy chain variable region, (ii) a light chain comprising a light chain constant region and a light chain variable region, and (iii) a polypeptide comprising a Fc fragment or a truncated heavy chain. In certain embodiments, the Fc fragment or a truncated heavy chain comprised in the separate polypeptide is a “dummy Fc,” which refers to an Fc fragment that is not linked to an antigen binding domain. The one-armed antibodies of the present disclosure may comprise any of the HCVR/LCVR pairs or CDR amino acid sequences as set forth in Table A herein. One-armed antibodies comprising a full-length heavy chain, a full-length light chain and an additional Fc domain polypeptide can be constructed using standard methodologies (see, e.g., WO2010151792, which is incorporated herein by reference in its entirety), wherein the heavy chain constant region differs from the Fc domain polypeptide by at least two amino acids (e.g., H95R and Y96F according to the IMGT exon numbering system; or H435R and Y436F according to the EU numbering system). Such modifications are useful in purification of the monovalent antibodies (see WO2010151792).
An antigen-binding fragment of an antibody will, in an embodiment, comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody described herein include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody described herein may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). The present disclosure includes an antigen-binding fragment of an antigen-binding protein such as an antibody set forth herein.
Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) may be monospecific or multi-specific (e.g., bispecific). Multispecific antigen-binding proteins are discussed further herein. The present disclosure includes monospecific as well as multispecific (e.g., bispecific) antigen-binding fragments comprising one or more variable domains from an antigen-binding protein that is specifically set forth herein.
The term “specifically binds” or “binds specifically” refers to those antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof) having a binding affinity to an antigen, such as human TfR protein, mouse TfR protein or monkey TfR protein, expressed as KD, of at least about 10−9 M (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 nM), as measured by real-time, label free bio-layer interferometry assay, for example, at 25° C. or 37° C., e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™, or by solution-affinity ELISA. The present disclosure includes antigen-binding proteins that specifically bind to TfR protein. “Anti-TfR” refers to an antigen-binding protein (or other molecule), for example an antibody or antigen-binding fragment thereof, that binds specifically to TfR.
“Isolated” antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof), polypeptides, polynucleotides and vectors, are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antigen-binding protein may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules (e.g., minor or insignificant amounts of impurity may remain) or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antigen-binding proteins (e.g., antibodies or antigen-binding fragments).
The present disclosure includes antigen-binding proteins, e.g., antibodies or antigen-binding fragments, that bind to the same epitope as an antigen-binding protein described herein. In some embodiments, provided is an antigen-binding protein that binds specifically to transferrin receptor or an antigenic-fragment thereof or variant thereof which binds to one or more epitopes of hTfR selected from: (a) an epitope comprising the sequence LLNE (SEQ ID NO: 525) and/or an epitope comprising the sequence TYKEL (SEQ ID NO: 507); (b) an epitope comprising the sequence DSTDFTGT (SEQ ID NO: 526) and/or an epitope comprising the sequence VKHPVTGQF (SEQ ID NO: 527) and/or an epitope comprising the sequence IERIPEL (SEQ ID NO: 528); (c) an epitope comprising the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 529); (d) an epitope comprising the sequence FEDL (SEQ ID NO: 519); (e) an epitope comprising the sequence IVDKNGRL (SEQ ID NO: 530); (f) an epitope comprising the sequence IVDKNGRLVY (SEQ ID NO: 531); (g) an epitope comprising the sequence DQTKF (SEQ ID NO: 532); (h) an epitope comprising the sequence LVENPGGY (SEQ ID NO: 533) and/or an epitope comprising the sequence PIVNAELSF (SEQ ID NO: 534) and/or an epitope comprising the sequence PYLGTTMDT (SEQ ID NO: 535); (i) an epitope comprising the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope comprising the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope comprising the sequence TYKEL (SEQ ID NO: 507); (j) an epitope comprising the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 508) and/or an epitope comprising the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 509) and/or an epitope comprising the sequence LIERIPELNKVARAAAE (SEQ ID NO: 510); (k) an epitope comprising the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 511); (1) an epitope comprising the sequence GTKKDFEDL (SEQ ID NO: 512); (m) an epitope comprising the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 513); (n) an epitope comprising the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope comprising the sequence DQTKFPIVNAEL (SEQ ID NO: 515) and/or an epitope comprising the sequence TYKELIERIPELNK (SEQ ID NO: 516); (o) an epitope comprising the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope comprising the sequence TYKELIERIPELNK (SEQ ID NO: 516); (p) an epitope comprising the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 517); (q) an epitope comprising the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope comprising the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 518); (r) an epitope comprising the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope comprising the sequence LVENPGGYVAYSKAATVTGKL (SEQ ID NO: 520) and/or an epitope comprising the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 521) and/or an epitope comprising the sequence ISRAAAEKL (SEQ ID NO: 522) and/or an epitope comprising the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 523) and/or an epitope comprising the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 524); (s) an epitope comprised within or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope comprised within or overlapping with the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope comprised within or overlapping with the sequence TYKEL (SEQ ID NO: 507); (t) an epitope comprised within or overlapping with the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 508) and/or an epitope comprised within or overlapping with the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 509) and/or an epitope comprised within or overlapping with the sequence LIERIPELNKVARAAAE (SEQ ID NO: 510); (u) an epitope comprised within or overlapping with the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 511); (v) an epitope comprised within or overlapping with the sequence GTKKDFEDL (SEQ ID NO: 512); (w) an epitope comprised within or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 513); (x) an epitope comprised within or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope comprised within or overlapping with the sequence DQTKFPIVNAEL (SEQ ID NO: 515) and/or an epitope comprised within or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 516); (y) an epitope comprised within or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope comprised within or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 516); (z) an epitope comprised within or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 517); (aa) an epitope comprised within or overlapping with the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope comprised within or overlapping with the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 518); and (bb) an epitope comprised within or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope comprised within or overlapping with the sequence LVENPGGYVAYSKAATVTGKL (SEQ ID NO: 520) and/or an epitope comprised within or overlapping with the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 521) and/or an epitope comprised within or overlapping with the sequence ISRAAAEKL (SEQ ID NO: 522) and/or an epitope comprised within or overlapping with the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 523) and/or an epitope comprised within or overlapping with the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 524). In some embodiments, provided is an antigen-binding protein, wherein the antigen binding protein comprises an antibody or antigen-binding fragment thereof which binds to one or more epitopes of hTfR selected from: (a) an epitope consisting of the sequence LLNE (SEQ ID NO: 525) and/or an epitope consisting of the sequence TYKEL (SEQ ID NO: 507); (b) an epitope consisting of the sequence DSTDFTGT (SEQ ID NO: 526) and/or an epitope consisting of the sequence VKHPVTGQF (SEQ ID NO: 527) and/or an epitope consisting of the sequence IERIPEL (SEQ ID NO: 528); (c) an epitope consisting of the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 529); (d) an epitope consisting of the sequence FEDL (SEQ ID NO: 519); (e) an epitope consisting of the sequence IVDKNGRL (SEQ ID NO: 530); (f) an epitope consisting of the sequence IVDKNGRLVY (SEQ ID NO: 531); (g) an epitope consisting of the sequence DQTKF (SEQ ID NO: 532); (h) an epitope consisting of the sequence LVENPGGY (SEQ ID NO: 533) and/or an epitope consisting of the sequence PIVNAELSF (SEQ ID NO: 534) and/or an epitope consisting of the sequence PYLGTTMDT (SEQ ID NO: 535); (i) an epitope consisting of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope consisting of the sequence TYKEL (SEQ ID NO: 507); (j) an epitope consisting of the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 508) and/or an epitope consisting of the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 509) and/or an epitope consisting of the sequence LIERIPELNKVARAAAE (SEQ ID NO: 510); (k) an epitope consisting of the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 511); (1) an epitope consisting of the sequence GTKKDFEDL (SEQ ID NO: 512); (m) an epitope consisting of the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 513); (n) an epitope consisting of the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope consisting of the sequence DQTKFPIVNAEL (SEQ ID NO: 515) and/or an epitope consisting of the sequence TYKELIERIPELNK (SEQ ID NO: 516); (o) an epitope consisting of the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 514) and/or an epitope consisting of the sequence TYKELIERIPELNK (SEQ ID NO: 516); (p) an epitope consisting of the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 517); (q) an epitope consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 506) and/or an epitope consisting of the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 518); and (r) an epitope consisting of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 505) and/or an epitope consisting of the sequence LVENPGGYVAYSKAATVTGKL (SEQ ID NO: 520) and/or an epitope consisting of the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 521) and/or an epitope consisting of the sequence ISRAAAEKL (SEQ ID NO: 522) and/or an epitope consisting of the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 523) and/or an epitope consisting of the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 524).
An antigen is a molecule, such as a peptide (e.g., TfR or a fragment thereof (an antigenic fragment)), to which, for example, an antibody or antigen-binding fragment thereof binds. The specific region on an antigen that an antibody recognizes and binds to is called the epitope. Antigen-binding proteins (e.g., antibodies) described herein that specifically bind to such antigens are part of the present disclosure.
The term “epitope” refers to an antigenic determinant (e.g., on TfR) that interacts with a specific antigen-binding site of an antigen-binding protein, e.g., a variable region of an antibody, known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” may also refer to a site on an antigen to which B and/or T cells respond and/or to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may be linear or conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Epitopes to which antigen-binding proteins described herein bind may be included in fragments of TfR, for example the extracellular domain thereof. Antigen-binding proteins (e.g., antibodies) described herein that bind to such epitopes are part of the present disclosure.
Methods for determining the epitope of an antigen-binding protein, e.g., antibody or fragment or polypeptide, include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis, crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding protein (e.g., antibody or fragment or polypeptide) interacts is hydrogen/deuterium exchange detected by mass spectrometry. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.
The present disclosure includes antigen-binding proteins that compete for binding to a TfR epitope as discussed herein, with an antigen-binding protein described herein. The term “competes” as used herein, refers to an antigen-binding protein (e.g., antibody or antigen-binding fragment thereof) that binds to an antigen (e.g., TfR) and inhibits or blocks the binding of another antigen-binding protein (e.g., antibody or antigen-binding fragment thereof) to the antigen. Unless otherwise stated, the term also includes competition between two antigen-binding proteins e.g., antibodies, in both orientations, i.e., a first antibody that binds antigen and blocks binding by a second antibody and vice versa. Thus, in an embodiment, competition occurs in one such orientation. In certain embodiments, the first antigen-binding protein (e.g., antibody) and second antigen-binding protein (e.g., antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different, but, for example, overlapping or non-overlapping epitopes, wherein binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance. Competition between antigen-binding proteins (e.g., antibodies) may be measured by methods known in the art, for example, by a real-time, label-free bio-layer interferometry assay. Also, binding competition between TfR-binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using a real time, label-free bio-layer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp.).
Typically, an antibody or antigen-binding fragment described herein which is modified in some way retains the ability to specifically bind to TfR, e.g., retains at least 10% of its TfR binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment described herein retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the TfR binding affinity as the parental antibody. It is also intended that an antibody or antigen-binding fragment described herein may include conservative or non-conservative amino acid substitutions (referred to as “conservative variants” or “function conserved variants” of the antibody) that do not substantially alter its biologic activity.
An anti-TfR antigen-binding protein provided herein may be a monoclonal antibody or an antigen-binding fragment of a monoclonal antibody which may be tethered to a payload. Provided herein are monoclonal anti-TfR antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, as well as monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins. The term “monoclonal antibody” or “mAb”, as used herein, refers to a member of a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. A “plurality” of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., as discussed above, in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts) antibodies and fragments which is above that which would normally occur in nature, e.g., in the blood of a host organism such as a mouse or a human.
In an embodiment, an anti-TfR antigen-binding protein, e.g., antibody or antigen-binding fragment (which may be tethered to a payload) comprises a heavy chain constant domain, e.g., of the type IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 and IgG4) or IgM. In an embodiment, an antigen-binding protein, e.g., antibody or antigen-binding fragment, comprises a light chain constant domain, e.g., of the type kappa or lambda. In an embodiment, a VH as set forth herein is linked to a human heavy chain constant domain (e.g., IgG) and a VL as set forth herein is linked to a human light chain constant domain (e.g., kappa). The present disclosure includes antigen-binding proteins comprising the variable domains set forth herein, which are linked to a heavy and/or light chain constant domain, e.g., as set forth herein.
Provided herein are human anti-TfR antigen-binding proteins which may be tethered to a payload. The term “human” antigen-binding protein, such as an antibody or antigen-binding fragment, as used herein, includes antibodies and fragments having variable and constant regions derived from human germline immunoglobulin sequences whether in a human cell or grafted into a non-human cell, e.g., a mouse cell. See e.g., U.S. Pat. Nos. 8,502,018, 6,596,541 or U.S. Pat. No. 5,789,215. The anti-TfR human mAbs provided herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody”, as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal. The term is not intended to include natural antibodies directly isolated from a human subject. The present disclosure includes human antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof described herein).
Provided herein are anti-TfR chimeric antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof (which may be tethered to a payload), and methods of use thereof. As used herein, a “chimeric antibody” is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species. (see e.g., U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855). The present disclosure includes chimeric antibodies comprising the variable domains which are set forth herein and a non-human constant domain.
The term “recombinant” anti-TfR antigen-binding proteins, such as antibodies or antigen-binding fragments thereof (which may be tethered to a payload), refers to such molecules created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression. The term includes antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) such as a cellular expression system or isolated from a recombinant combinatorial human antibody library. The present disclosure includes recombinant antigen-binding proteins, such as antibodies and antigen-binding fragments as set forth herein.
An antigen-binding fragment of an antibody will, in an embodiment, comprise less than a full antibody but still binds specifically to antigen, e.g., TfR, e.g., including at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one (e.g., 3) CDR(s), which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH and/or VL domain which are bound non-covalently.
In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody described herein include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody described herein may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). The present disclosure includes an antigen-binding fragment of an antigen-binding protein such as an antibody set forth herein.
Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) may be monospecific or multi-specific (e.g., bispecific). Multispecific antigen-binding proteins are discussed further herein. The present disclosure includes monospecific as well as multispecific (e.g., bispecific) antigen-binding fragments comprising one or more variable domains from an antigen-binding protein that is specifically set forth herein.
A “variant” of a polypeptide, such as an immunoglobulin chain, refers to a polypeptide comprising an amino acid sequence that is at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a referenced amino acid sequence that is set forth herein (e.g., any of SEQ ID NOs: 2; 3; 4; 5; 7; 8; 9; 10; 12; 13; 14; 15; 17; 18; 19; 20; 22; 23; 24; 25; 27; 28; 29; 30; 32; 33; 34; 35; 37; 38; 39; 40; 42; 43; 44; 45; 47; 48; 49; 50; 52; 53; 54; 55; 57; 58; 59; 60; 62; 63; 64; 65; 67; 68; 69; 70; 72; 73; 74; 75; 77; 78; 79; 80; 82; 83; 84; 85; 87; 88; 89; 90; 92; 93; 94; 95; 97; 98; 99; 100; 102; 103; 104; 105; 107; 108; 109; 110; 112; 113; 114; 115; 117; 118; 119; 120; 122; 123; 124; 125; 127; 128; 129; 130; 132; 133; 134; 135; 137; 138; 139; 140; 142; 143; 144; 145; 147; 148; 149; 150; 152; 153; 154; 155; 157; 158; 159; 160; 162; 163; 164; 165; 167; 168; 169; 170; 172; 173; 174; 175; 177; 178; 179; 180; 182; 183; 184; 185; 187; 188; 189; 190; 192; 193; 194; 195; 197; 198; 199; 200; 202; 203; 204; 205; 207; 208; 209; 210; 212; 213; 214; 215; 217; 218; 219; 220; 222; 223; 224; 225; 227; 228; 229; 230; 232; 233; 234; 235; 237; 238; 239; 240; 242; 243; 244; 245; 247; 248; 249; 250; 252; 253; 254; 255; 257; 258; 259; 260; 262; 263; 264; 265; 267; 268; 269; 270; 272; 273; 274; 275; 277; 278; 279; 280; 282; 283; 284; 285; 287; 288; 289; 290; 292; 293; 294; 295; 297; 298; 299; 300; 302; 303; 304; 305; 307; 488; 308; 309; 310; 312; 313; 314; 315; 317; 318; 319; 320; 321 (optionally not including the N-terminal MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 500)), 322 (optionally not including the N-terminal MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 500)), 323 (optionally not including the N-terminal MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 500)), 324 (optionally not including the N-terminal MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 500)); 328-423 or 427-458); when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expect threshold: 10; word size: 3; max matches in a query range: 0; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment) and/or comprising the amino acid sequence but having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations (e.g., point mutation, insertion, truncation, and/or deletion).
Moreover, a variant of a polypeptide may include a polypeptide such as an immunoglobulin chain which may include the amino acid sequence of the reference polypeptide whose amino acid sequence is specifically set forth herein but for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations, e.g., one or more missense mutations (e.g., conservative substitutions), non-sense mutations, deletions, or insertions. For example, the present disclosure includes TfR-binding proteins which include an immunoglobulin light chain (or VL) variant comprising the amino acid sequence set forth in SEQ ID NO: 7, 17, 27, 37, 465, 47, 466, 57, 468, 67, 469, 77, 471, 87, 97, 107, 117, 474, 127, 137, 147, 476, 157, 167, 177, 187, 479, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 488, 317, or 484 but having one or more of such mutations and/or an immunoglobulin heavy chain (or VH) variant comprising the amino acid sequence set forth in SEQ ID NO: 2, 462, 12, 463, 22, 464, 32, 42, 52, 467, 62, 492, 72, 470, 82, 92, 472, 102, 112, 473, 122, 132, 142, 475, 152, 162, 477, 172, 182, 478, 192, 480, 202, 481, 212, 222, 232, 242, 252, 482, 262, 272, 282, 292, 302, 483, or 312 but having one or more of such mutations. In an embodiment, a TfR-binding protein includes an immunoglobulin light chain variant comprising CDR-L1, CDR-L2 and CDR-L3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions) and/or an immunoglobulin heavy chain variant comprising CDR-H1, CDR-H2 and CDR-H3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions).
The following references relate to BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J. 272(20): 5101-5109; Altschul, S. F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, T. L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, J. C., et al., (1993) Comput. Chem. 17:149-163; Hancock, J. M. et al., (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al., “Matrices for detecting distant relationships.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J., et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S. F. “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, N.Y.
A “conservatively modified variant” or a “conservative substitution”, e.g., of an immunoglobulin chain set forth herein, refers to a variant wherein there is one or more substitutions of amino acids in a polypeptide with other amino acids having similar characteristics (e.g., charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.). Such changes can frequently be made without significantly disrupting the biological activity of the antibody or fragment. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt biological activity. The present disclosure includes TfR-binding proteins comprising such conservatively modified variant immunoglobulin chains.
Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-45.
Antibodies and antigen-binding fragments described herein comprise immunoglobulin chains including the amino acid sequences specifically set forth herein (and variants thereof) as well as cellular and in vitro post-translational modifications to the antibody or fragment. For example, the present disclosure includes antibodies and antigen-binding fragments thereof that specifically bind to TfR comprising heavy and/or light chain amino acid sequences set forth herein as well as antibodies and fragments wherein one or more asparagine, serine and/or threonine residues is glycosylated, one or more asparagine residues is deamidated, one or more residues (e.g., Met, Trp and/or His) is oxidized, the N-terminal glutamine is pyroglutamate (pyroE) and/or the C-terminal lysine or other amino acid is missing.
In an embodiment, an anti-hTfR:Payload or anti-hTfR:Payload (e.g., in scFv, Fab, antibody or antigen-binding fragment thereof format), e.g., wherein the payload is human GAA, exhibits one or more of the following characteristics:
The amino acid sequences of domains in anti-human transferrin receptor antigen-binding proteins provided herein are summarized below in Table A. The amino acid sequences of domains in anti-human transferrin receptor antigen-binding proteins of fusions provided herein are also summarized below in Table A. For example, anti-human transferrin receptor 1 antibodies and antigen-binding fragments thereof (e.g., scFvs and Fabs) comprising the HCVR and LCVR of the molecules in Table A; or comprising the CDRs thereof, fused to a payload, are provided herein. In a specific example, the anti-human transferrin receptor 1 antibodies and antigen-binding fragments thereof (e.g., scFvs and Fabs) comprise the HCVR and LCVR of or comprise the CDRs of #23 or #25 in Table A. In a specific example, the anti-human transferrin receptor 1 antibodies and antigen-binding fragments thereof (e.g., scFvs and Fabs) comprise the HCVR and LCVR of or comprise the CDRs of #23 in Table A. In a specific example, the anti-human transferrin receptor 1 antibodies and antigen-binding fragments thereof (e.g., scFvs and Fabs) comprise the HCVR and LCVR of or comprise the CDRs of #14 in Table A. In a specific example, the anti-human transferrin receptor 1 antibodies and antigen-binding fragments thereof (e.g., scFvs and Fabs) comprise the HCVR and LCVR of or comprise the CDRs of #18 in Table A. In a specific example, the anti-human transferrin receptor 1 antibodies and antigen-binding fragments thereof (e.g., scFvs and Fabs) comprise the HCVR and LCVR of or comprise the CDRs of #27 in Table A. In a specific example, the anti-human transferrin receptor 1 antibodies and antigen-binding fragments thereof (e.g., scFvs and Fabs) comprise the HCVR and LCVR of or comprise the CDRs of #28 in Table A.
DIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIHDVSNRATGIPARFSGSGSGTDFTLTISS
DILMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISS
QVQLVESGGGLVQPGGSLRLSCAVSGFIFSSYEMNWVRQAPGKGLEWVSYISSSGSTIFYADSVKGRFTISRDNAKN
DIVMTQSPATLSVSPGERATLSCRASQSVSSNFAWYQQKPGQAPRLLIYSASSRATGIPVRFSGSGSGTEFTLTISS
QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMTWVRQAPGKGLEWVANIKEDGSEKDYVDSVKGRFTISRDNAKN
EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDISKN
EVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVIFISYDGSDKYYADSVKGRFAISRDSSKN
EVQLVESGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISVYHGNTNYAQKFQGRVTMTTDTSTS
EVQLVESGGGLVKPGGSLRLSCAASGFTFSVYYMNWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKN
As discussed, an anti-hTfR:Payload scFv fusion protein (e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263) comprises an optional signal peptide, connected to an scFv (e.g., including a VL and a VH optionally connected by a linker), connected to an optional linker, connected to a payload such as GAA or variant thereof wherein, for example:
Also provided are antigen-binding proteins or antibodies or antigen-binding fragments thereof comprising any of the heavy chain variable regions and/or light chain variable regions or any of the heavy chain variable region and light chain variable region combinations or any of the HCDR and LCDR combinations described above in the context of anti-hTFR:Payload scFv fusion proteins.
In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (23) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); or (25) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (23) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (25) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (14) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (18) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (27) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (28) a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof).
In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (w) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof); or (y) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (w) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (y) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (n) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (r) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 178 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (aa) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (ab) a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof).
In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (xxiii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); or (xxv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (xxiii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (xxv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (xiv) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (xviii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (xxvii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof). In an embodiment, the scFv or antigen-binding protein or antibody or antigen-binding fragment thereof comprises: (xxviii) a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof).
In an embodiment, an anti-hTfR scFv provided herein, in VL-(Gly4Ser)3 (SEQ ID NO: 538)-VH format (Gly4Ser=SEQ ID NO: 426), comprises an amino acid sequence as set forth below (optionally, an anti-hTfR scFv provided herein further includes an N-terminal LLQGSG (SEQ ID NO: 501) and/or a C-terminal HHHHHH (SEQ ID NO: 502)):
In an embodiment, an anti-hTfR scFv comprises an amino acid sequence as set forth in SEQ ID NO: 443 or SEQ ID NO: 440. In an embodiment, an anti-hTfR scFv comprises an amino acid sequence as set forth in SEQ ID NO: 443. In an embodiment, an anti-hTfR scFv comprises an amino acid sequence as set forth in SEQ ID NO: 440. In an embodiment, an anti-hTfR scFv comprises an amino acid sequence as set forth in SEQ ID NO: 429. In an embodiment, an anti-hTfR scFv comprises an amino acid sequence as set forth in SEQ ID NO: 433. In an embodiment, an anti-hTfR scFv comprises an amino acid sequence as set forth in SEQ ID NO: 442. In an embodiment, an anti-hTfR scFv comprises an amino acid sequence as set forth in SEQ ID NO: 438.
In an embodiment, an anti-hTfR scFv:GAA fusion provided herein comprises the amino acid sequence:
In an embodiment, an anti-hTfR scFv:GAA fusion provided herein comprises the amino acid sequence set forth in SEQ ID NO: 408 or SEQ ID NO: 405. In an embodiment, an anti-hTfR scFv:GAA fusion provided herein comprises the amino acid sequence set forth in SEQ ID NO: 408. In an embodiment, an anti-hTfR scFv:GAA fusion provided herein comprises the amino acid sequence set forth in SEQ ID NO: 405.
In an embodiment, the anti-hTfR:GAA scFv fusion protein comprises the amino acid sequence:
Fab fragments that bind specifically to human transferrin receptor, optionally fused to a payload such as GAA (or variant thereof) (anti-TfR Fab:Payload fusion proteins), are provided herein. Fab fragments typically contain one complete light chain, VL, and a constant light domain, e.g., kappa (e.g., RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 424)) and the VH and IgG1 CH1 portion (e.g., ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH (SEQ ID NO: 425)) or IgG4 CH1 (e.g., ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPLLQGSG (SEQ ID NO: 459); or ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPP (SEQ ID NO: 493)) of one heavy chain. Fab fragment antibodies can be generated by papain digestion of whole IgG antibodies to remove the entire Fc fragment, including the hinge region. For example, provided herein are Fab proteins comprising:
For example, provided herein are Fab proteins comprising: (23) a heavy chain variable region (HCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 222, or a heavy chain variable region that includes HCDR1, HCDR2 and HCDR3 of such a HCVR-linked to the CH1 domain-and comprising a light chain variable region (LCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 227, or LCDR1, LCDR2 and LCDR3 of such a LCVR-linked to the CL domain; or (25) a heavy chain variable region (HCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 242, or a heavy chain variable region that includes HCDR1, HCDR2 and HCDR3 of such a HCVR-linked to the CH1 domain-and a light chain variable region (LCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 247, or LCDR1, LCDR2 and LCDR3 of such a LCVR-linked to the CL domain. For example, provided herein are Fab proteins comprising: (23) a heavy chain variable region (HCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 222, or a heavy chain variable region that includes HCDR1, HCDR2 and HCDR3 of such a HCVR-linked to the CH1 domain-and comprising a light chain variable region (LCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 227, or LCDR1, LCDR2 and LCDR3 of such a LCVR-linked to the CL domain. For example, provided herein are Fab proteins comprising: (25) a heavy chain variable region (HCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 242, or a heavy chain variable region that includes HCDR1, HCDR2 and HCDR3 of such a HCVR-linked to the CH1 domain-and a light chain variable region (LCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 247, or LCDR1, LCDR2 and LCDR3 of such a LCVR-linked to the CL domain. For example, provided herein are Fab proteins comprising: (14) a heavy chain variable region (HCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 132, or a heavy chain variable region that includes HCDR1, HCDR2 and HCDR3 of such a HCVR-linked to the CH1 domain-and a light chain variable region (LCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 137, or LCDR1, LCDR2 and LCDR3 of such a LCVR-linked to the CL domain. For example, provided herein are Fab proteins comprising: (18) a heavy chain variable region (HCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 172, or a heavy chain variable region that includes HCDR1, HCDR2 and HCDR3 of such a HCVR-linked to the CH1 domain-and a light chain variable region (LCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 177, or LCDR1, LCDR2 and LCDR3 of such a LCVR-linked to the CL domain. For example, provided herein are Fab proteins comprising: (27) a heavy chain variable region (HCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 262, or a heavy chain variable region that includes HCDR1, HCDR2 and HCDR3 of such a HCVR-linked to the CH1 domain-and a light chain variable region (LCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 267, or LCDR1, LCDR2 and LCDR3 of such a LCVR-linked to the CL domain. For example, provided herein are Fab proteins comprising: (28) a heavy chain variable region (HCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 272, or a heavy chain variable region that includes HCDR1, HCDR2 and HCDR3 of such a HCVR-linked to the CH1 domain-and a light chain variable region (LCVR) that comprises the amino acid sequence set forth in SEQ ID NO: 277, or LCDR1, LCDR2 and LCDR3 of such a LCVR-linked to the CL domain.
In an embodiment, the antigen-binding fragment comprises a Fab protein. In an embodiment, the Fab protein comprises the amino acid sequences set forth in SEQ ID NO: 372 and SEQ ID NO: 496, 487, or 373 (or variants thereof) or comprises the amino acid sequences set forth in SEQ ID NO: 376 and SEQ ID NO: 497, 489, or 377 (or variants thereof). In an embodiment, the Fab protein comprises the amino acid sequences set forth in SEQ ID NO: 372 and SEQ ID NO: 496, 487, or 373 (or variants thereof). In an embodiment, the Fab protein comprises the amino acid sequences set forth in SEQ ID NO: 376 and SEQ ID NO: 497, 489, or 377 (or variants thereof).
Heavy and light chains of anti-hTfR Fabs in exemplary anti-hTfR:Payload fusion proteins provided herein are set forth below.
In an embodiment, an anti-TfR antigen-binding protein, e.g., antibody or antigen-binding fragment (which may be tethered to a payload) comprises an IgG1 heavy chain constant domain comprising the sequence set forth in SEQ ID NO: 571: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (see, e.g., sequences of Table B, or variants thereof). In an embodiment, an antigen-binding protein, e.g., antibody or antigen-binding fragment, comprises a light chain constant domain, e.g., of the type kappa or lambda. In an embodiment, a VH as set forth herein is linked to a human heavy chain constant domain (e.g., IgG) and a VL as set forth herein is linked to a human light chain constant domain (e.g., kappa). The present disclosure includes antigen-binding proteins comprising the variable domains set forth herein, which are linked to a heavy and/or light chain constant domain, e.g., as set forth herein.
“31874B”; “31863B”; “69348”; “69340”; “69331”; “69332”; “69326”; “69329”; “69323”; “69305”; “69307”; “12795B”; “12798B”; “12799B”; “12801B”; “12802B”; “12808B”; “12812B”; “12816B”; “12833B”; “12834B”; “12835B”; “12847B”; “12848B”; “12843B”; “12844B”; “12845B”; “12839B”; “12841B”; “12850B”; “69261”; and “69263” refer to anti-TfR:Payload fusion proteins, e.g., anti-TfR scFv:GAA or anti-TfR Fab:GAA, comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7; 17; 27; 37; 465; 47; 466; 57; 468; 67; 469; 77; 471; 87; 97; 107; 117; 474; 127; 137; 147; 476; 157; 167; 177; 187; 479; 197; 207; 217; 227; 237; 247; 257; 267; 277; 287; 297; 307; 488; 317 or 484 (or a variant thereof), and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; 462; 12; 463; 22; 464; 32; 42; 52; 467; 62; 492; 72; 470; 82; 92; 472; 102; 112; 473; 122; 132; 142; 475; 152; 162; 477; 172; 182; 478; 192; 480; 202; 481; 212; 222; 232; 242; 252; 482; 262; 272; 282; 292; 302; 483 or 312 (or a variant thereof); which, in the case of an scFv, can be fused together (in either order), e.g., by a peptide linker (e.g., (G4S)3 (SEQ ID NO: 538)), respectively; or that comprise a VH that comprises the CDRs thereof (CDR-H1 (or a variant thereof), CDR-H2 (or a variant thereof) and CDR-H3 (or a variant thereof)) and/or a VL that comprises the CDRs thereof (CDR-L1 (or a variant thereof), CDR-L2 (or a variant thereof) and CDR-L3 (or a variant thereof)), wherein the VH fused to the VL or the VL fused to the VH, in the case of an scFv, can be fused, e.g., by a peptide linker (e.g., (G4S)2 (SEQ ID NO: 537)), to a payload such as GAA polypeptide or variant thereof.
In some embodiments, the anti-TfR antigen-binding protein described herein comprises a humanized antibody or antigen binding fragment thereof, murine antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof (e.g., monovalent Fab′, divalent Fab2, F(ab)′3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)2, diabody, bivalent antibody, one-armed antibody, minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or antigen binding fragment thereof, bispecific antibody or biding fragment thereof, (e.g., bisscFv, or a bi-specific T-cell engager (BiTE)), trispecific antibody (e.g., F(ab)′3 fragments or a triabody), or a chemically modified derivative thereof. In some embodiments, the anti-TfR antigen-binding protein can be bivalent. In some embodiments, the anti-TfR antigen-binding protein can be monovalent (e.g., one-arm antibody).
The term “humanized antibody,” as used herein, includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences, or otherwise modified to increase their similarity to antibody variants produced naturally in humans.
In some cases, the anti-TfR antigen-binding protein is an antibody which comprises one or more mutations in a framework region, e.g., in the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some embodiments, the one or more mutations are to stabilize the antibody and/or to increase half-life. In some embodiments, the one or more mutations are to modulate Fc receptor interactions, to reduce or eliminate Fc effector functions such as FcγR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In additional embodiments, the one or more mutations are to modulate glycosylation.
In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody described herein (e.g., in a CH2 domain (residues 231-340 of human IgG1) and/or CH3 domain (residues 341-447 of human IgG1) and/or the hinge region, with numbering according to the Kabat numbering system (e.g., the EU index in Kabat)) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity. In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine residues in the hinge region are altered (e.g., increased or decreased) as described in, e.g., U.S. Pat. No. 5,677,425. The number of cysteine residues in the hinge region of the CH1 domain can be altered to, e.g., facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.
In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into an IgG constant domain, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) half-life of the antibody in vivo. See, e.g., PCT Publication Nos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples of mutations that will alter (e.g., decrease or increase) the half-life of an antibody in vivo. In some embodiments, the Fc region comprises a mutation at residue position L234, L235, or a combination thereof. In some embodiments, the mutations comprise L234 and L235. In some embodiments, the mutations comprise L234A and L235A.
The anti-TfR antibodies and antigen-binding fragments described herein may be modified after translation, e.g., glycosylated.
For example, antibodies and antigen-binding fragments described herein may be glycosylated (e.g., N-glycosylated and/or O-glycosylated) or aglycosylated. Typically, antibodies and antigen-binding fragments are glycosylated at the conserved residue N297 of the IgG Fc domain. Some antibodies and fragments include one or more additional glycosylation sites in a variable region. In an embodiment, the glycosylation site is in the following context: FN297S or YN297S.
In an embodiment, said glycosylation is any one or more of three different N-glycan types: high mannose, complex and/or hybrid that are found on IgGs with their respective linkage. Complex and hybrid types exist with core fucosylation, addition of a fucose residue to the innermost N-acetylglucosamine, and without core fucosylation.
In some cases, the anti-TfR antigen-binding protein is an aglycosylated antibody, i.e., an antibody that does not comprise a glycosylation sequence that might interfere with a transglutamination reaction, for instance an antibody that does not have a saccharide group at N180 and/or N297 on one or more heavy chains. In particular embodiments, an antibody heavy chain has an N180 mutation. In other words, the antibody is mutated to no longer have an asparagine residue at position 180 according to the EU numbering system as disclosed by Kabat et al. In particular embodiments, an antibody heavy chain has an N180Q mutation. In particular embodiments, an antibody heavy chain has an N297 mutation. In particular embodiments, an antibody heavy chain has an N297Q or an N297D mutation. Antibodies comprising such above-described mutations can be prepared by site-directed mutagenesis to remove or disable a glycosylation sequence or by site-directed mutagenesis to insert a glutamine residue at site apart from any interfering glycosylation site or any other interfering structure. Such antibodies also can be isolated from natural or artificial sources. Aglycosylated antibodies also include antibodies comprising a T299 or S298P or other mutations, or combinations of mutations that result in a lack of glycosylation.
In some cases, the antigen-binding protein is a deglycosylated antibody, i.e., an antibody in which a saccharide group at is removed to facilitate transglutaminase-mediated conjugation. Saccharides include, but are not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation is performed at residue N180. In some embodiments, deglycosylation is performed at residue N297. In some embodiments, removal of saccharide groups is accomplished enzymatically, included but not limited to via PNGase.
In an embodiment, an antibody or fragment described herein is afucosylated.
The antibodies and antigen-binding fragments described herein may also be post-translationally modified in other ways including, for example: Glu or Gln cyclization at N-terminus; Loss of positive N-terminal charge; Lys variants at C-terminus; Deamidation (Asn to Asp); Isomerization (Asp to isoAsp); Deamidation (Gln to Glu); Oxidation (Cys, His, Met, Tyr, Trp); and/or Disulfide bond heterogeneity (Shuffling, thioether and trisulfide formation).
In some embodiments, an antibody disclosed herein comprises Q295 which can be native to the antibody heavy chain sequence. In some embodiments, an antibody heavy chain disclosed herein may comprise Q295. In some embodiments, an antibody heavy chain disclosed herein may comprise Q295 and an amino acid substitution N297D.
According to certain embodiments of the present disclosure, anti-TfR antibodies and antigen-binding fragments are provided comprising an Fc domain comprising one or more mutations which enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, the present disclosure includes anti-TfR antibodies comprising a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal.
Non-limiting examples of such Fc modifications include, e.g., a modification at position:
In an embodiment, the modification comprises:
For example, the present disclosure includes anti-TfR antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of:
In yet another embodiment, the modification comprises a 265A (e.g., D265A) and/or a 297A (e.g., N297A) modification.
In an embodiment, the heavy chain constant domain is gamma4 comprising an S228P and/or S108P mutation. See Angal et al., A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody, Mol Immunol. 1993 January; 30(1):105-108.
All possible combinations of the foregoing Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present disclosure.
The anti-TfR antibodies described herein may comprise a modified Fc domain having reduced effector function. As used herein, a “modified Fc domain having reduced effector function” means any Fc portion of an immunoglobulin that has been modified, mutated, truncated, etc., relative to a wild-type, naturally occurring Fc domain such that a molecule comprising the modified Fc exhibits a reduction in the severity or extent of at least one effect selected from the group consisting of cell killing (e.g., ADCC and/or CDC), complement activation, phagocytosis and opsonization, relative to a comparator molecule comprising the wild-type, naturally occurring version of the Fc portion. In certain embodiments, a “modified Fc domain having reduced effector function” is an Fc domain with reduced or attenuated binding to an Fc receptor (e.g., FcTR).
In certain embodiments, the modified Fc domain is a variant IgG1 Fc or a variant IgG4 Fc comprising a substitution in the hinge region. For example, a modified Fc for use in the context of the present disclosure may comprise a variant IgG1 Fc wherein at least one amino acid of the IgG1 Fc hinge region is replaced with the corresponding amino acid from the IgG2 Fc hinge region. Alternatively, a modified Fc for use in the context of the present disclosure may comprise a variant IgG4 Fc wherein at least one amino acid of the IgG4 Fc hinge region is replaced with the corresponding amino acid from the IgG2 Fc hinge region. Non-limiting, exemplary modified Fc regions that can be used in the context of the present disclosure are set forth in US Patent Application Publication No. 2014/0243504, the disclosure of which is hereby incorporated by reference in its entirety, as well as any functionally equivalent variants of the modified Fc regions set forth therein.
Also provided herein are antigen-binding proteins, antibodies or antigen-binding fragments, comprising a HCVR set forth herein and a chimeric heavy chain constant (CH) region, wherein the chimeric CH region comprises segments derived from the CH regions of more than one immunoglobulin isotype. For example, the antibodies of the disclosure may comprise a chimeric CH region comprising part or all of a CH2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule, combined with part or all of a CH3 domain derived from a human IgG1, human IgG2 or human IgG4 molecule. According to certain embodiments, the antibodies provided herein comprise a chimeric CH region having a chimeric hinge region. For example, a chimeric hinge may comprise an “upper hinge” amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from a human IgG1, a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge” sequence (amino acid residues from positions 228 to 236 according to EU numbering) derived from a human IgG1, a human IgG2 or a human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. An antibody comprising a chimeric CH region as described herein may, in certain embodiments, exhibit modified Fe effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. See, e.g., WO2014/022540.
Other modified Fc domains and Fc modifications that can be used in the context of the present disclosure include any of the modifications as set forth in US2014/0171623; U.S. Pat. No. 8,697,396; US2014/0134162; WO2014/043361, the disclosures of which are hereby incorporated by reference in their entireties. Methods of constructing antibodies or other antigen-binding fusion proteins comprising a modified Fc domain as described herein are known in the art.
In some embodiments, the anti-TfR antibodies and antigen-binding fragments described herein comprise an Fc domain comprising one or more mutations in the CH2 and/or CH3 regions that generate a separate TfR binding site.
In an embodiment, the CH2 region comprises one or more amino acid mutations, or a combination thereof, selected from the following: a) position 47 is Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp; position 49 is Ile, Val, Asp, Glu, Thr, Ala, or Tyr; position 56 is Asp, Pro, Met, Leu, Ala, Asn, or Phe; position 58 is Arg, Ser, Ala, or Gly; position 59 is Tyr, Trp, Arg, or Val; position 60 is Glu; position 61 is Trp or Tyr; position 62 is Gln, Tyr, His, Ile, Phe, Val, or Asp; and position 63 is Leu, Trp, Arg, Asn, Tyr, or Val; b) position 39 is Pro, Phe, Ala, Met, or Asp; position 40 is Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr; position 41 is Thr, Ser, Gly, Met, Val, Phe, Trp, or Leu; position 42 is Pro, Val, Ala, Thr, or Asp; position 43 is Pro, Val, or Phe; position 44 is Trp, Gln, Thr, or Glu; position 68 is Glu, Val, Thr, Leu, or Trp; position 70 is Tyr, His, Val, or Asp; position 71 is Thr, His, Gln, Arg, Asn, or Val; and position 72 is Tyr, Asn, Asp, Ser, or Pro; c) position 41 is Val or Asp; position 42 is Pro, Met, or Asp; position 43 is Pro or Trp; position 44 is Arg, Trp, Glu, or Thr; position 45 is Met, Tyr, or Trp; position 65 is Leu or Trp; position 66 is Thr, Val, Ile, or Lys; position 67 is Ser, Lys, Ala, or Leu; position 69 is His, Leu, or Pro; and position 73 is Val or Trp; or d) position 45 is Trp, Val, Ile, or Ala; position 47 is Trp or Gly; position 49 is Tyr, Arg, or Glu; position 95 is Ser, Arg, or Gln; position 97 is Val, Ser, or Phe; position 99 is Ile, Ser, or Trp; position 102 is Trp, Thr, Ser, Arg, or Asp; position 103 is Trp; and position 104 is Ser, Lys, Arg, or Val; wherein the substitutions and the positions are determined with reference to amino acids 4-113 of
In an embodiment, the CH3 region comprises one or more amino acid mutations, or a combination thereof, selected from the following: position 153 is Trp, Leu, or Glu; position 157 is Tyr or Phe; position 159 is Thr; position 160 is Glu; position 161 is Trp; position 162 is Ser, Ala, Val, or Asn; position 163 is Ser or Asn; position 186 is Thr or Ser; position 188 is Glu or Ser; position 189 is Glu; and position 194 is Phe; or b) position 118 is Phe or Ile; position 119 is Asp, Glu, Gly, Ala, or Lys; position 120 is Tyr, Met, Leu, Ile, or Asp; position 122 is Thr or Ala; position 210 is Gly; position 211 is Phe; position 212 is His, Tyr, Ser, or Phe; and position 213 is Asp; wherein the substitutions and the positions are determined with reference to amino acids 114-220 of SEQ ID NO: 536.
In some embodiments, the CH3 region comprises one or more mutations, or a combination thereof, selected from the following: position 384 is Leu, Tyr, Met, or Val; position 386 is Leu, Thr, His, or Pro; position 387 is Val, Pro, or an acidic amino acid; position 388 is Trp; position 389 is Val, Ser, or Ala; position 413 is Glu, Ala, Ser, Leu, Thr, or Pro; position 416 is Thr or an acidic amino acid; and position 421 is Trp, Tyr, His, or Phe, according to EU numbering. In an embodiment, the CH3 region comprises one or more amino acid mutations, or a combination thereof, selected from the following: a) position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.
In some embodiments, the CH3 region comprises one or more mutations, or a combination thereof, selected from the following: a) Phe at position 382, Tyr at position 383, Asp at position 384, Asp at position 385, Ser at position 386, Lys at position 387, Leu at position 388, Thr at position 389, Pro at position 419, Arg at position 420, Gly at position 421, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440, Gly at position 442, and Glu at position 443; b) Phe at position 382, Tyr at position 383, Gly at position 384, N at position 385, Ala at position 386, Lys at position 387, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; c) Phe at position 382, Tyr at position 383, Glu at position 384, Ala at position 385, Lys at position 387, Leu at position 388, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; d) Phe at position 382, Glu at position 384, Ser at position 386, Lys at position 387, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; e) Phe at position 382, Gly at position 384, Ala at position 385, Lys at position 387, Ser at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; f) Phe at position 382, Gly at position 384, Ala at position 385, Lys at position 387, Leu at position 388, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; wherein the positions are determined according to EU numbering.
Additional mutations in CH2 and/or CH3 regions that can introduce non-native TfR binding sites into the antigen-binding proteins descried herein include those described in US Patent Application Publication Nos. 2020/0223935, 2020/0369746, 2021/0130485, 2022/0017634; and PCT application Publications Nos. WO2023/279099, WO2023/114499 and WO2023/114510, which are incorporated herein by reference in their entireties.
Provided herein is a vessel (e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising an anti-TfR:Payload fusion protein, e.g., anti-TfR scFv:GAA or anti-TfR Fab:GAA, provided herein, e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263.
Also provided is an injection device comprising an anti-TfR:Payload fusion protein, e.g., anti-TfR scFv:GAA or anti-TfR Fab:GAA disclosed herein, e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, or a pharmaceutical composition thereof. The injection device may be packaged into a kit. An injection device is a device that introduces a substance into the body of a subject via a parenteral route, e.g., intramuscular, subcutaneous or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., comprising the fusion protein or a pharmaceutical composition thereof), a needle for piercing skin and/or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore.
Further provided are methods for administering an anti-TfR:Payload fusion protein, e.g., anti-TfR scFv:GAA or anti-TfR Fab:GAA provided herein, e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, to a subject, comprising introducing the fusion protein into the body of the subject (e.g., a human), for example, parenterally. For example, the method comprises piercing the body of the subject with a needle of a syringe and injecting the fusion protein into the body of the subject, e.g., into the vein, artery, tumor, muscular tissue or subcutis of the subject.
Further provided herein are methods for delivering a payload wherein the payload is fused to, e.g., an antigen-binding protein provided herein, e.g., anti-TfR scFv:GAA or anti-TfR Fab:GAA provided herein, e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, to a targeted tissue in a subject (e.g., any of the tissues or cell types or cell types in or associated with the corresponding tissues as set forth in Table C below; or brain, cerebral cortex; cerebellum; hippocampus; caudate; parathyroid gland; adrenal gland; bronchus; lung; oral mucosa; esophagus; stomach; duodenum; small intestine; colon; rectum; liver; gallbladder; pancreas; kidney; urinary bladder; testis; epididymis; prostate; vagina; ovary; fallopian tube; endometrium; cervix; placenta; breast; muscle, (e.g., heart muscle; skeletal muscle, smooth muscle and/or endothelial vasculature thereof); soft tissue; skin; appendix; lymph node; tonsil; and/or bone marrow), comprising introducing the fusion protein into the body of the subject (e.g., a human), for example, parenterally. For example, the method comprises piercing the body of the subject with a needle of a syringe and injecting the fusion protein into the body of the subject, e.g., into the vein, artery, tumor, muscular tissue or subcutis of the subject.
Provided are anti-TfR:Payload fusion proteins (e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; and 69263), or polynucleotides encoding anti-TfR Payload fusion proteins, wherein the payload is a GAA polypeptide or variant thereof, which can be used, for example, for delivering GAA peptide to the body of a subject, e.g., for treating or preventing a disease or disorder mediated, at least in part, by deficiency of GAA protein and/or activity in the body (e.g., the brain) of the subject (a Glycogen Storage Disease (GSD)). Pompe disease is an example of a GSD. For example, a GSD is a glycogen storage disease that is mediated by deficiency in GAA.
Glycogen storage disease (GSD) type 2, also known as Pompe disease or acid maltase deficiency disease, is an example of GSD which is an inherited metabolic disorder. While glycogen storage disease type 2 is a single disease, it may be classified in 2 forms according to the rates of disease progression, its severity and the age at which symptoms start. The classic infantile-onset starts before 12 month of age and involves the heart muscle (myocardiopathy). The later-onset form may start before 12 months of age (non-classic infantile-onset), or after 12 months of age, but does not affect the heart. Muscle weakness is a main symptom in all forms. The infantile-onset is the most severe form and, if untreated, it may lead to death from heart failure in the first year of life. The late-onset form is usually milder, but if untreated may lead to severe breathing problems.
Glycogen storage disease type 2 is caused by variants (mutations) in the GAA gene which have instructions to produce the enzyme acid alpha-glucosidase (acid maltase), needed to break down glycogen, a substance that is a source of energy for the body. The enzyme deficiency results in the accumulation of glycogen inside lysosomes, structures within cells that break down waste products within the cell. Accumulation of glycogen in certain tissues, especially muscles, impairs their function.
The classic infantile form of glycogen storage disease type 2 is characterized by severe muscle weakness (myopathy) and abnormally diminished muscle tone (hypotonia) without muscle wasting, and usually manifests within the first few months of life. Additional abnormalities may include enlargement of the heart (cardiomegaly), the liver (hepatomegaly), and/or the tongue (macroglossia). Affected infants may also have poor feeding, failure to gain weight and grow at the expected rate (failure to thrive), breathing problems, and hearing loss. Most infants with glycogen storage disease type 2 cannot hold up their heads or move normally. Without treatment, progressive cardiac failure usually causes life-threatening complications by the age of 12 to 18 months.
The non-classic infantile form of glycogen storage disease type 2 usually presents within the first year of life. Initial symptoms may include delayed motor skills (crawling, sitting) and myopathy. Cardiomegaly may be present, but unlike the classic infantile form, cardiac failure does not typically occur. Muscle weakness may lead to serious, life-compromising breathing problems by early childhood.
In the late onset form of glycogen storage disease type 2, symptoms may not be evident until childhood, adolescence, or adulthood. This form is usually milder than the infantile-onset form of the disorder. Most individuals experience progressive muscle weakness, especially in the legs and the trunk, including the muscles that control breathing.
Thus, provided herein are methods for treating or preventing a glycogen storage disease (e.g., the classic infantile form, the non-classic infantile form or the late onset form of glycogen storage disease type 2), in a subject in need thereof, by administering a therapeutically effective amount of anti-TfR:GAA fusion protein (e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; and 69263) or polynucleotides encoding anti-TfR Payload fusion proteins, to the subject, e.g., wherein one or more signs or symptoms of the GSD are alleviated.
In an embodiment, a subject having Pompe disease has one or more of the following GAA mutations (e.g., homozygous or heterozygous):
Thus, provided herein are methods for treating or preventing a GSD (e.g., the classic infantile form, the non-classic infantile form or the late onset form of glycogen storage disease type 2), in a subject in need thereof, wherein the subject has GAA comprising one or more of said mutations, by administering a therapeutically effective amount of anti-TfR:GAA fusion protein (e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; and 69263) or polynucleotides encoding anti-TfR Payload fusion proteins, to the subject, e.g., wherein one or more signs or symptoms of the GSD are alleviated.
As used herein, the term “subject” refers to a mammal (e.g., rat, mouse, cat, dog, cow, sheep, horse, goat, rabbit), preferably a human, for example, in need of prevention and/or treatment of a GAA-deficiency disease or disorder. In an embodiment, a subject has been diagnosed as suffering from a GSD such as Pompe Disease.
Provided herein are combinations including an anti-TfR:Payload fusion protein provided herein (e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; and 69263) or polynucleotides encoding anti-TfR Payload fusion proteins, in association with one or more further therapeutic agents. The anti-TfR:Payload fusion protein and the further therapeutic agent can be in a single composition or in separate compositions. For example, in an embodiment, the further therapeutic agent is alglucosidase alfa (e.g., Myozyme or Lumizyme), Rituximab, Methotrexate, Intravenous immunoglobulin (IVIG), avalglucosidase alfa-ngpt (e.g., Nexviazyme), a selective beta agonist (e.g., levalbuterol), an antibiotic, a steroid (e.g., cortisone or prednisone), a bisphosphonate, an infectious disease treatment (e.g., an antibiotic, a vaccine (e.g., Pneumococcal vaccine), palivizumab).
Methods for treating or preventing a GSD (e.g., Pompe Disease) in a subject in need of said treatment or prevention by administering an anti-TfR:GAA fusion protein, e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; and 69263, or polynucleotides encoding anti-TfR Payload fusion proteins, in association with a further therapeutic agent are provided herein. Compositions comprising the anti-TfR:GAA fusion protein in association with one or more further therapeutic agents are also provided herein.
The term “in association with” indicates that components, an anti-TfR:Payload fusion protein provided herein, along with another agent such as methotrexate, can be formulated into a single composition, e.g., for simultaneous delivery, or formulated separately into two or more compositions (e.g., a kit including each component). Each component can be administered to a subject at a different time than when the other component is administered; for example, each administration may be given non-simultaneously (e.g., separately or sequentially) at intervals over a given period of time. Moreover, the separate components may be administered to a subject by the same or by a different route.
An effective or therapeutically effective dose of anti-TfR:Payload fusion protein provided herein for treating or preventing a GAA-deficiency disease or disorder refers to the amount of anti-TfR:Payload sufficient to alleviate one or more signs and/or symptoms of the disease or condition in the treated subject, whether by inducing the regression or elimination of such signs and/or symptoms or by inhibiting the progression of such signs and/or symptoms. In an embodiment, an effective or therapeutically effective dose of anti-TfR:GAA is about 1 mg/kg to about 50 mg/kg. The dose amount may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of antigen-binding protein in an amount that can be approximately the same or less or more than that of the initial dose, wherein the subsequent doses are separated by days or weeks.
The diagnosis of Pompe disease can be based on a thorough clinical evaluation, a detailed patient and family history, and a variety of biochemical tests including the measuring of GAA activity. In individuals suspected of having Pompe disease, blood can be drawn and the function/activity of GAA can be measured in white blood cells (leukocytes). Proper assay conditions should be used and acarbose should be added to the reaction mixture to inhibit the activity of glucoamylase. Alternatively, the GAA activity/functional assay can also be performed on dried blood spots, although this method is not any quicker, less reliable, and also requires the use of acarbose to inhibit the glucoamylase activity.
Each diagnosis performed with the dried blood spot test method should be confirmed through molecular genetic testing (GAA gene copy analysis) or by measuring the GAA activity with another method. Leukocytes can be used for this purpose, but cultured skin fibroblasts obtained by a skin biopsy are the very best material. More invasive muscle biopsies are not needed and not optimal either for measuring the GAA activity.
The application of a skin biopsy and the initiation of a culture of skin fibroblasts might not be feasible in every diagnostic setting, but should be considered as there are important advantages with this procedure. The GAA activity/functional test using skin fibroblasts is superior to all other methods for its high sensitivity and for discriminating between classic-infantile, childhood and adult Pompe disease (IOPD vs LOPD) in almost all cases.
A variety of other tests can be performed to detect or assess symptoms potentially associated with Pompe disease such as sleep studies, tests that measure lung function, and tests that measure muscle function. Muscle MRI (imaging by magnetic resonance) is used to visualize the degree of muscle damage.
Specific tests may also be performed to assess the heart function, including chest x-ray, electrocardiography (ECG), and echocardiography (imaging by ultrasound). Chest x-rays allow physicians to assess the size of the heart, which is enlarged in classic infantile Pompe disease. Electrocardiography (ECG) measures the electric activity of the heart and detects abnormal heart rhythms. Echocardiography uses reflected sound waves to create a picture of the heart and can reveal abnormal thickening of the walls of the heart.
The anti-TfR antigen-binding proteins set forth herein are useful for delivering any of many types of payload to a targeted tissue (e.g., brain)—for example, therapeutic agents (TAs). Such payloads include proteins, enzymes and viral vectors containing polynucleotides. The delivery of any payload by fusion to an anti-TfR antigen-binding protein may be referred to as anti-TfR-mediated delivery.
Payloads include polypeptides, e.g., enzymes and antigen-binding proteins (e.g., antibodies and antigen-binding fragments thereof). In some embodiments, the enzyme is a hydrolase, including esterases, glycosylases, hydrolases that act on ether bonds, peptidases, linear amidases, diphosphatases, ketone hydrolases, halogenases, phosphoamidases, sulfohydrolases, sulfinases, desulfinases, and the like. In some embodiments, the enzyme is a glycosylase, including glycosidases and N-glycosylases. In some embodiments, the enzyme is a glycosidase, including alpha-amylase, beta-amylase, glucan 1,4-alpha-glucosidase, cellulose, endo-1,3(4)-beta-glucanase, inulinase, endo-1,4-beta-xylanase, endo-1,4-b-xylanase, dextranase, chitinase, polygalacturonidase, lysozyme, exo-alpha-sialidase, alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, alpha-mannosidase, beta-mannosidase, beta-fructofuranosidase, alpha,alpha-trehalose, beta-glucuronidase, xylan endo-1,3-beta-xylosidase, amylo-alpha-1,6-glucosidase, hyaluronoglucosaminidase, hyaluronoglucuronidase, and the like.
In some embodiments, the payload is a alpha-glucosidase (GAA) polypeptide. GAA is described in more detail elsewhere herein.
In some embodiments, the payload is an alpha-galactosidase A (GLA) polypeptide. “Alpha-galactosidase A” (GLA or “α-galactosidase A”) facilitates the hydrolysis of terminal α-galactosyl moieties from glycolipids and glycoproteins, and also hydrolyses α-D-fucosides. GLA is also known inter alia as EC 3.2.1.22, melibiase, α-D-galactosidase, α-galactosidase A, α-galactoside galactohydrolase, α-D-galactoside galactohydrolase. Fabry disease is caused by defective lysosomal enzyme alpha-galactosidase A (GLA), which results in the accumulation of globotriaosylceramide within the blood vessels and other tissues and organs. Symptoms associated with Fabry disease include pain from nerve damage and/or small vascular obstruction, renal insufficiency and eventual failure, cardiac complications such as high blood pressure and cardiomyopathy, dermatological symptoms such as formation of angiokeratomas, anhidrosis or hyperhidrosis, and ocular problems such as cornea verticillata, spoke-like cataract, and conjunctival and retinal vascular abnormalities. Treatments include FABRAZYME (agalsidase beta), REPLAGAL (agalsidase alfa) and GALAFOLD. Thus, provided herein are anti-TfR:Payload fusion proteins wherein the payload is alpha-galactosidase A, agalsidase beta, agalsidase alfa or miglastat as well as methods for treating Fabry disease in a patient by administering an effective amount of such a fusion protein to the patient.
In some embodiments, the payload is an acid sphingomyelinase (ASM) polypeptide. “Acid sphingomyelinase” (ASM, sphingomyelin phosphodiesterase, or SMPD1) converts sphingomyelin to ceramide. ASMD (acid sphingomyelinase deficiency) is historically known as Niemann-Pick disease types A, A/B, and B. In people with ASMD, the body is unable to make enough of the ASM enzyme, and sphingomyelin cannot be broken down efficiently, and instead builds up in major organs such as the liver, lungs, and spleen. This can lead to complications over time, as key organs in the body may not be able to function properly. Niemann-Pick disease A (NPDA) is an early-onset lysosomal storage disorder caused by failure to hydrolyze sphingomyelin to ceramide. It results in the accumulation of sphingomyelin and other metabolically related lipids in reticuloendothelial and other cell types throughout the body, leading to cell death. Niemann-Pick disease type A is a primarily neurodegenerative disorder characterized by onset within the first year of life, intellectual disability, digestive disorders, failure to thrive, major hepatosplenomegaly, and severe neurologic symptoms. The severe neurological disorders and pulmonary infections lead to an early death, often around the age of four. Clinical features are variable. A phenotypic continuum exists between type A (basic neurovisceral) and type B (purely visceral) forms of Niemann-Pick disease, and the intermediate types encompass a cluster of variants combining clinical features of both types A and B. Niemann-Pick disease B (NPDB) is a late-onset lysosomal storage disorder caused by failure to hydrolyze sphingomyelin to ceramide. It results in the accumulation of sphingomyelin and other metabolically related lipids in reticuloendothelial and other cell types throughout the body, leading to cell death. Clinical signs involve only visceral organs. The most constant sign is hepatosplenomegaly which can be associated with pulmonary symptoms. Patients remain free of neurologic manifestations. However, a phenotypic continuum exists between type A (basic neurovisceral) and type B (purely visceral) forms of Niemann-Pick disease, and the intermediate types encompass a cluster of variants combining clinical features of both types A and B. In Niemann-Pick disease type B, onset of the first symptoms occurs in early childhood and patients can survive into adulthood.
In some embodiments, the payload is a lysosomal acid glucosylceramidase (GBA) polypeptide. “Lysosomal acid glucosylceramidase” (GBA, glucocerebrosidase, lysosomal acid GCase, acid beta-glucosidase, alglucerase, beta-glucocerebrosidase, beta-GC, beta-glucosylceramidase 1, cholesterol glucosyltransferase, cholesteryl-beta-glucosidase, D-glucosyl-N-acylsphingosine glucohydrolase, glucosylceramidase beta 1, imiglucerase, lysosomal cholesterol glycosyltransferase, lysosomal galactosylceramidase, lysosomal glycosylceramidase, GBA, GBA1, GC, or GLUC) hydrolyzes glucosylceramide (GlcCer) to glucose and ceramide. In addition, GCase catalyzes the transfer of glucose from GlcCer to cholesterol to contribute to in the synthesis of 0-cholesteryl glucoside. Homozygous GBA mutations result in the most common lysosomal storage disorder, Gaucher disease (GD), which is classified according to the presence (neuronopathic types, type 2 and 3 GD) or absence (non-neuronopathic type, type 1 GD) of neurological symptoms.
A. Alpha-Glucosidase (GAA) Payload
Provided herein are methods and compositions for delivering the payload, alpha-glucosidase (GAA) mature peptide, preferably human GAA, to the brain. Alpha-glucosidases are enzymes that catalyze the exohydrolysis of 1,4-alpha-glucosidic linkages with release of alpha-glucose. Preferably, the TfR to which an antigen-binding protein (e.g., scFv) binds is from the same species from which the GAA polypeptide is obtained; for example, anti-human TfR is fused to a human GAA (or a variant thereof).
“Acid alpha-glucosidase” or “alpha-glucosidase” or “GAA” is intended to refer to the mature peptide of the human enzyme. The enzyme hydrolyzes alpha-1,4 linkages between the D-glucose units of glycogen, maltose, and isomaltose. Alternative names include but are not limited to lysosomal alpha-glucosidase (EC:3.2.1.20); glucoamylase; 1,4-alpha-D-glucan glucohydrolase; amyloglucosidase; gamma-amylase and exo-1,4-alpha-glucosidase. Human acid alpha-glucosidase is encoded by the GAA gene (National Centre for Biotechnology Information (NCBI) Gene ID 2548), which has been mapped to the long arm of chromosome 17 (location 17q25.2-q25.3). More than 500 mutations have currently been identified in the human GAA gene, many of which are associated with Pompe disease. Mutations resulting in misfolding or misprocessing of the acid alpha-glucosidase enzyme include T1064C (Leu355Pro) and C2104T (Arg702Cys). In addition, GAA mutations which affect maturation and processing of the enzyme include Leu405Pro and Met519Thr. The conserved hexapeptide WIDMNE at amino acid residues 516-521 is required for activity of the acid alpha-glucosidase protein. As used herein, the abbreviation “GAA” is intended to refer to the acid alpha-glucosidase enzyme, while the italicized abbreviation “GAA” is intended to refer to the human gene coding for the human acid alpha-glucosidase enzyme.
In an embodiment, the mature peptide of human alpha-glucosidase comprises the amino acid sequence:
Provided herein are anti-TfR:Payload fusion proteins wherein the payload is a lysosomal storage disease therapeutic agent (LSD-TA), e.g., an LSD protein (which may be referred to as an anti-TfR:LSD protein fusion protein or anti-TfR:LSD protein fusion); e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263. Methods for treating or preventing an LSD in a patient by administering an effective amount of anti-TfR:LSD-TA to the patient.
An LSD-therapeutic agent in an agent that, when delivered to a subject having an LSD, can treat such a disease. An LSD protein is any protein, e.g., an enzyme, that, when delivered to the cells of a patient having an LSD, treats or prevents the LSD. Preferably, the LSD protein is the enzyme for which the patient's lysosomes are deficient.
“Lysosomal storage diseases” (LSDs) include any disorder resulting from a defect in lysosome function. The most well-known lysosomal disease includes Tay-Sachs, Gaucher, and Niemann-Pick disease. The pathogeneses of the diseases are ascribed to the buildup of incomplete degradation products in the lysosome, sometimes due to loss of protein function. Lysosomal storage diseases may be caused by loss-of-function or attenuating variants in the proteins whose normal function is to degrade or coordinate degradation of lysosomal contents. The proteins affiliated with lysosomal storage diseases include enzymes, receptors and other transmembrane proteins (e.g., NPC1), post-translational modifying proteins (e.g., sulfatase), membrane transport proteins, and non-enzymatic cofactors and other soluble proteins (e.g., GM2 ganglioside activator). Thus, lysosomal storage diseases encompass more than those disorders caused by defective enzymes per se, and include any disorder caused by any molecular defect.
LSDs include sphingolipidoses (heterogeneous group of inherited disorders of lipid metabolism affecting primarily the central nervous system), a mucopolysaccharidoses (a group of inherited lysosomal storage disorders), and glycogen storage diseases. In some embodiments, the LSD is any one or more of Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Niemann-Pick disease type A, Niemann-Pick disease type BGM1-gangliosidosis, Sandhoff disease, Tay-Sachs disease, GM2-activator deficiency, GM3-gangliosidosis, metachromatic leukodystrophy, sphingolipid-activator deficiency, Scheie disease, Hurler-Sceie disease, Hurler disease, Hunter disease, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Morquio syndrome A, Morquio syndrome B, Maroteaux-Lamy disease, Sly disease, MPS IX, and Pompe disease. In a specific embodiment, the LSD is Fabry disease. In another embodiment, the LSD is Pompe disease. Thus, provided herein are methods for treating or preventing any such LSD in a patient by administering an effective amount of anti-TfR:LSD-TA to the patient.
The nature of the molecular lesion in a lysosomal storage disease affects the severity of the disease in many cases, i.e., complete loss-of-function may be associated with pre-natal or neo-natal onset, and involves severe symptoms; partial loss-of-function may be associated with milder (relatively) and later-onset disease. Only a small percentage of activity may need to be restored to have to correct metabolic defects in deficient cells. Table D-1 and D-2 lists some lysosomal storage diseases and their associated loss-of-function proteins. Lysosomal storage diseases are generally described in Desnick & Schuchman, “Enzyme replacement therapy for lysosomal diseases: lessons from 20 years of experience and remaining challenges,” 13 Annu. Rev. Genomics Hum. Genet. 307-35, 2012).
Thus, provided herein are anti-TfR:LSD protein fusions wherein the LSD fusion protein is as set forth in Table D-1 and D-2 as well as methods for treating or preventing the corresponding LSD in Table D-1 and D-2 in a patient by administering an effective amount of anti-TfR:LSD protein fusion to the patient.
Options for the treatment of lysosomal storage diseases include enzyme replacement therapy (ERT), substrate reduction therapy, pharmacological chaperone-mediated therapy, hematopoietic stem cell transplant therapy, and gene therapy. An example of substrate reduction therapy is Miglustat or Eliglustat for treating Gaucher Type 1. These drugs act by blocking synthase activity, which reduces subsequent substrate production. Hematopoietic stem cell therapy (HSCT), for example, is used to ameliorate and slow-down the negative central nervous system phenotype in patients with some forms of MPS. See R. M. Boustany, “Lysosomal storage diseases—the horizon expands,” 9(10) Nat. Rev. Neurol. 583-98, October 2013; which reference is incorporated herein in its entirety by reference. Thus, provided herein are anti-TfR:Payload fusion proteins wherein the payload is an enzyme replacement therapy (ERT) agent, substrate reduction therapy agent (e.g., Miglustat), pharmacological chaperone-mediated therapy agent, or gene therapy agent as well as methods for treating LSDs in a patient by administering an effective amount of such a fusion protein to the patient.
Two LSDs are Pompe disease and Fabry disease. As discussed herein, Pompe disease is caused by defective lysosomal enzyme alpha-glucosidase (GAA), which results in the deficient processing of lysosomal glycogen. Thus, Pompe disease may also be referred to as a glycogen storage disease. Thus, provided herein are anti-TfR:Payload fusion proteins wherein the payload is GAA as well as methods for treating Pompe disease in a patient by administering an effective amount of such a fusion protein to the patient.
Fabry disease is caused by defective lysosomal enzyme alpha-galactosidase A (GLA), which results in the accumulation of globotriaosylceramide within the blood vessels and other tissues and organs. Symptoms associated with Fabry disease include pain from nerve damage and/or small vascular obstruction, renal insufficiency and eventual failure, cardiac complications such as high blood pressure and cardiomyopathy, dermatological symptoms such as formation of angiokeratomas, anhidrosis or hyperhidrosis, and ocular problems such as cornea verticillata, spoke-like cataract, and conjunctival and retinal vascular abnormalities. Treatments include FABRAZYIE (agalsidase beta), REPLAGAL (agalsidase alfa) and GALAFOLD. Thus, provided herein are anti-TfR:Payload fusion proteins wherein the payload is alpha-galactosidase A, agalsidase beta, agalsidase alfa or miglastat as well as methods for treating Fabry disease in a patient by administering an effective amount of such a fusion protein to the patient.
“Alpha-galactosidase A” (GLA or “α-galactosidase A”) facilitates the hydrolysis of terminal α-galactosyl moieties from glycolipids and glycoproteins, and also hydrolyses α-D-fucosides. GLA is also known inter alia as EC 3.2.1.22, melibiase, α-D-galactosidase, α-galactosidase A, α-galactoside galactohydrolase, α-D-galactoside galactohydrolase.
Provided herein are anti-TfR:Payload fusion proteins e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, and wherein the payload is a heart disease or disorder therapeutic agent, e.g., a protein that is set forth below in Table E or a variant thereof. Methods for treating or preventing a heart disease or disorder that is listed below in Table E, in a patient in need thereof, by administering an effective amount of an anti-TfR:Payload to the patient wherein the payload is a protein encoded by a gene corresponding to the particular heart disease or disorder in Table E.
Provided herein are anti-TfR:Payload fusion proteins e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, and wherein the payload is a CNS disease or disorder therapeutic agent, e.g., a protein that is set forth below in Table F or a variant thereof. Methods for treating or preventing a CNS disease or disorder that is listed below in Table F, in a patient in need thereof, by administering an effective amount of an anti-TfR:Payload to the patient wherein the payload is a protein encoded by a gene corresponding to the particular CNS disease or disorder in Table F.
Provided herein are anti-TfR:Payload fusion proteins e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, and wherein the payload is an eye disease or disorder therapeutic agent, e.g., a protein that is set forth below in Table G or a variant thereof. Methods for treating or preventing an eye disease or disorder that is listed below in Table G, in a patient in need thereof, by administering an effective amount of an anti-TfR:Payload to the patient wherein the payload is a protein encoded by a gene corresponding to the particular eye disease or disorder in Table G.
Provided herein are anti-TfR:Payload fusion proteins e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, and wherein the payload is a brain disease or disorder therapeutic agent, e.g., a protein that is set forth below in Table H or a variant thereof. Methods for treating or preventing a brain disease or disorder that is listed below in Table H, in a patient in need thereof, by administering an effective amount of an anti-TfR:Payload to the patient wherein the payload is a protein encoded by a gene corresponding to the particular brain disease or disorder in Table H.
Provided herein are anti-TfR:Payload fusion proteins e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, and wherein the payload is a spinal cord disease or disorder therapeutic agent, e.g., a protein that is set forth below in Table I or a variant thereof. Methods for treating or preventing a spinal cord disease or disorder that is listed below in Table I, in a patient in need thereof, by administering an effective amount of an anti-TfR:Payload to the patient wherein the payload is a protein encoded by a gene corresponding to the particular spinal cord disease or disorder in Table I.
Provided herein are anti-TfR:Payload fusion proteins e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, and wherein the payload is a PNS disease or disorder therapeutic agent, e.g., a protein that is set forth below in Table J or a variant thereof. Methods for treating or preventing a PNS disease or disorder that is listed below in Table J, in a patient in need thereof, by administering an effective amount of an anti-TfR:Payload to the patient wherein the payload is a protein encoded by a gene corresponding to the particular PNS disease or disorder in Table J.
Provided herein are anti-TfR:Payload fusion proteins e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, and wherein the payload is a skeletal muscle disease or disorder therapeutic agent, e.g., a protein that is set forth below in Table K or a variant thereof. Methods for treating or preventing a skeletal muscle disease or disorder that is listed below in Table K, in a patient in need thereof, by administering an effective amount of an anti-TfR:Payload to the patient wherein the payload is a protein encoded by a gene corresponding to the particular skeletal muscle disease or disorder in Table K.
Provided herein are anti-TfR:Payload fusion proteins e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, and wherein the payload is a cartilage disease or disorder therapeutic agent, e.g., a protein that is set forth below in Table L or a variant thereof. Methods for treating or preventing a cartilage disease or disorder that is listed below in Table L, in a patient in need thereof, by administering an effective amount of an anti-TfR:Payload to the patient wherein the payload is a protein encoded by a gene corresponding to the particular cartilage disease or disorder in Table L.
Provided herein are anti-TfR:Payload fusion proteins e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, and wherein the payload is a bone growth plate disease or disorder therapeutic agent, e.g., a protein that is set forth below in Table M or a variant thereof. Methods for treating or preventing a bone growth plate disease or disorder that is listed below in Table M, in a patient in need thereof, by administering an effective amount of an anti-TfR:Payload to the patient wherein the payload is a protein encoded by a gene corresponding to the particular bone growth plate disease or disorder in Table M.
Provided herein are anti-TfR:Payload fusion proteins e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, and wherein the payload is a kidney disease or disorder therapeutic agent, e.g., a protein that is set forth below in Table N or a variant thereof. Methods for treating or preventing a kidney disease or disorder that is listed below in Table N, in a patient in need thereof, by administering an effective amount of an anti-TfR:Payload to the patient wherein the payload is a protein encoded by a gene corresponding to the particular kidney disease or disorder in Table N.
Provided herein are anti-TfR:Payload fusion proteins e.g., wherein the antigen-binding protein of the fusion (e.g., scFv) is 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263, and wherein the payload is a blood disease or disorder therapeutic agent, e.g., a protein that is set forth below in Table O or a variant thereof. Methods for treating or preventing a blood disease or disorder that is listed below in Table O, in a patient in need thereof, by administering an effective amount of an anti-TfR:Payload to the patient wherein the payload is a protein encoded by a gene corresponding to the particular blood
A polynucleotide includes DNA and RNA. Provided herein is any polynucleotide disclosed herein, for example, encoding an anti-TfR:Payload fusion protein, e.g., anti-TfR scFv:GAA or anti-TfR Fab:GAA (e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; or 69263), optionally, which is operably linked to a promoter or other expression control sequence. Polypeptides encoded by such polynucleotides are also provided herein.
Nucleotide sequences of HCVRs and LCVRs of anti-hTfR:Payload fusion proteins set forth herein are summarized below in Table P. Polynucleotides encoding an anti-hTfR:Payload fusion protein that includes one or more of the HCVRs and/or LCVRs set forth in Table P are provided herein.
For example, provided herein is a polynucleotide encoding an anti-hTfR:Payload fusion protein that includes:
In general, a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter may be operably linked to other expression control sequences, including enhancer and repressor sequences and/or with a polynucleotide provided herein. Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist, et al., (1981) Nature 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., (1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner, et al., (1981) Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster, et al., (1982) Nature 296:39-42); prokaryotic expression vectors such as the beta-lactamase promoter (VIIIa-Komaroff, et al., (1978) Proc. Natl. Acad. Sci. USA 75:3727-3731), or the tac promoter (DeBoer, et al., (1983) Proc. Natl. Acad. Sci. USA 80:21-25); see also “Useful proteins from recombinant bacteria” in Scientific American (1980) 242:74-94; and promoter elements from yeast or other fungi such as the Ga4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or the alkaline phosphatase promoter.
A polynucleotide encoding a polypeptide is “operably linked” to a promoter or other expression control sequence when, in a cell or other expression system, the sequence directs RNA polymerase mediated transcription of the coding sequence into RNA, preferably mRNA, which then may be RNA spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence.
Provided herein are polynucleotides encoding polypeptide chains which are variants of those whose nucleotide sequence is specifically set forth herein. A “variant” of a polynucleotide refers to a polynucleotide comprising a nucleotide sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical to a referenced nucleotide sequence that is set forth herein (see e.g., the nucleotide sequences of Table P); when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expect threshold: 10; word size: 28; max matches in a query range: 0; match/mismatch scores: 1, −2; gap costs: linear). In an embodiment, a variant of a nucleotide sequence specifically set forth herein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) point mutations, insertions (e.g., in frame insertions) or deletions (e.g., in frame deletions) of one or more nucleotides. Such mutations may, in an embodiment, be missense or nonsense mutations.
Eukaryotic and prokaryotic host cells, including mammalian cells, may be used as hosts for expression of an anti-TfR:Payload fusion protein. Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia, Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Provided herein is an isolated host cell (e.g., a CHO cell or any type of host cell set forth above) comprising an anti-TfR:Payload such as 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; and 69263.
In addition, also provided herein is a complex comprising an anti-TfR:Payload, as discussed herein complexed with a transferrin receptor polypeptide or an antigenic fragment thereof or fusion thereof and/or with a secondary antibody or antigen-binding fragment thereof (e.g., detectably labeled secondary antibody) that binds specifically to the anti-TfR:Payload. In an embodiment, the complex is in vitro (e.g., is immobilized to a solid substrate) or is in the body of a subject.
Recombinant anti-TfR:Payload fusion proteins disclosed herein may also be produced in an E. coli/T7 expression system. In this embodiment, polynucleotides encoding the anti-TfR:Payload fusion proteins disclosed herein (e.g., 31874B; 31863B; 69348; 69340; 69331; 69332; 69326; 69329; 69323; 69305; 69307; 12795B; 12798B; 12799B; 12801B; 12802B; 12808B; 12812B; 12816B; 12833B; 12834B; 12835B; 12847B; 12848B; 12843B; 12844B; 12845B; 12839B; 12841B; 12850B; 69261; and 69263) may be inserted into a pET-based plasmid and expressed in the E. coli/T7 system. For example, provided herein are methods for expressing anti-TfR:Payload fusion proteins in a host cell (e.g., bacterial host cell such as E. coli such as BL21 or BL21DE3) comprising expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding the anti-TfR:Payload fusion protein that is operably linked to a T7 promoter. For example, in an embodiment, a bacterial host cell, such as an E. coli, includes a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside). See U.S. Pat. Nos. 4,952,496 and 5,693,489 or Studier & Moffatt, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol. 1986 May 5; 189(1): 113-30.
Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, polynucleotides may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, for example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455. Thus, provided herein are recombinant methods for making an anti-TfR:Payload fusion protein disclosed herein comprising (i) introducing, into a host cell, one or more polynucleotides encoding the anti-TfR:Payload, for example, wherein the polynucleotide is in a vector; and/or integrates into the host cell chromosome and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) under conditions favorable to expression of the polynucleotide and, (iii) optionally, isolating the anti-TfR:Payload fusion protein from the host cell and/or medium in which the host cell is grown. Also provided are anti-TfR:Payload fusion protein which are the product of the production methods set forth herein, and, optionally, the purification methods set forth herein.
In an embodiment, a method for making an anti-TfR:Payload fusion protein, includes a method of purifying the anti-TfR:Payload fusion protein, e.g., by column chromatography, precipitation and/or filtration. As discussed, the product of such a method are also provided herein.
In one aspect, provided is a method of producing an anti-TfR:Payload fusion protein in a cell. In one embodiment, the anti-TfR:Payload fusion protein is produced by administering to the cell a gene therapy vector comprising a polynucleotide encoding the anti-TfR:Payload fusion protein. In one embodiment, the polynucleotide subsequently integrates at a genomic locus (e.g., in the liver) and the encoded fusion protein is produced (i.e., the polynucleotide is transcribed and translated). In another embodiment, the polynucleotide is transcribed episomally (e.g., in the liver) and the encoded fusion protein is produced.
In some embodiments, the methods further comprise administering a nuclease agent or one or more polynucleotides encoding the nuclease agent to the cell, wherein the nuclease agent cleaves the genomic locus, and the polynucleotide encoding the anti-TfR:Payload fusion protein is integrated into the genomic locus. Suitable nuclease agents include, for example, Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems, zinc finger nuclease (ZFN) systems, or Transcription Activator-Like Effector Nuclease (TALEN) systems.
In some embodiments, the polynucleotide encoding the anti-TfR:Payload fusion protein can comprise flanking homology arms for integration into the genomic locus by homology-directed repair. In other embodiments, the polynucleotide does not include homology arms, such as for integration into the genomic locus by non-homologous end joining.
In some embodiments, the gene therapy vector is one that is commonly used in cell transfection, such as an adeno-associated virus (AAV) vector. In some embodiments, the gene therapy vector is selected from the group consisting of a viral vector, optionally wherein the viral vector is a natural virus, an engineered virus, or a chimeric virus, and a naked polynucleotide comprising a polynucleotide described herein, a polynucleotide complex, optionally wherein the polynucleotide complex is a lipid nanoparticle comprising the polynucleotide and lipids, and any combination thereof. In some embodiments, the gene therapy vector is a viral vector, optionally selected from the group consisting of a retrovirus, adenovirus, a herpes simplex virus, a pox virus, a vaccinia virus, a lentivirus, or an adeno-associated virus. In some embodiments, the gene therapy vector is AAV9, Anc80, a recombinant AAV8 (e.g., an AAV2/8 chimera) and/or an AAV pseudotyped to a specific tissue, e.g., the liver or neuronal tissue.
In some embodiments, the genomic locus into which the polynucleotide encoding the anti-TfR:Payload fusion protein is integrated is a “safe harbor locus.” In one embodiment, a “safe harbor locus” enables high expression of the anti-TfR:Payload fusion protein, while not interfering with the expression of essential genes or promoting the expression of oncogenes or other deleterious genes. In one embodiment, the genomic locus is at or proximal to the liver-expressed albumin (Alb) locus, a EESYR locus, a SARS locus, position 188,083,272 of human chromosome 1 or its non-human mammalian orthologue, position 3,046,320 of human chromosome 10 or its non-human mammalian orthologue, position 67,328,980 of human chromosome 17 or its non-human mammalian orthologue, an adeno-associated virus site 1 (AAVS1) on chromosome, a naturally occurring site of integration of AAV virus on human chromosome 19 or its non-human mammalian orthologue, a chemokine receptor 5 (CCR5) gene, a chemokine receptor gene encoding an HIV-1 coreceptor, or a mouse Rosa26 locus or its non-murine mammalian orthologue. In one embodiment, the genomic locus is an adeno-associated virus site. In one embodiment, the genomic locus for integration is selected according to the method of Papapetrou and Schambach, J. Molecular Therapy, vol. 24 (4):678-684, April 2016, which is herein incorporated by reference for the stepwise selection of a safe harbor genomic locus for gene therapy vector integration; see also Barzel et al. Nature, vol. 517:360-364, incorporated herein by reference in its entirety, for the promoterless gene targeting into the liver-expressed albumin (Alb) locus.
In some embodiments, the polynucleotide, e.g., DNA, also contains a promoter operably linked to the anti-TfR:Payload fusion protein encoding sequence. In a specific embodiment, the promoter is a tissue-specific promotor that drives gene expression in a particular tissue. In one embodiment, the tissue specific promoter is a liver-specific enhancer/promoter derived from serpinal and/or is a TTR promoter. In other embodiments, the promoter is a CMV promoter. In other embodiments, the promoter is a ubiquitin C promoter.
In one embodiment, the cell is a mammalian cell, such as a human cell or a mouse cell. In one embodiment, the cell is a liver cell, such as a mammalian liver cell, a human liver cell, or a mouse liver cell. In one embodiment, the cell is ex vivo. In another embodiment, the cell is in vivo, and the gene therapy vector containing the polynucleotide encoding the anti-TfR:Payload fusion protein is administered to a subject (e.g., a human or non-human subject).
In one embodiment, the polynucleotide encoding the anti-TfR:Payload fusion protein is used to treat a subject in need of enzyme replacement therapy (e.g., in a method of delivering a therapeutic protein to the central nervous system (CNS) of a subject), comprising administering to the subject a gene therapy vector comprising a polynucleotide encoding the anti-TfR:Payload fusion protein (e.g., via a liver-targeted delivery method sufficient to provide a therapeutically effective amount of the anti-TfR:Payload fusion protein in the CNS). In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In one embodiment, the polynucleotide subsequently integrates at a genomic locus in the liver and the encoded fusion protein is produced. In another embodiment, the polynucleotide is transcribed episomally in the liver and the encoded fusion protein is produced.
All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.
The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5′ end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3′ end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. When a nucleotide sequence encoding an amino acid sequence is provided, it is understood that codon degenerate variants thereof that encode the same amino acid sequence are also provided. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.
Anti-human transferrin receptor (hTfR) antibodies were generated and screened for the ability to bind hTfR and for lack of strong blocking of human transferrin-hTfR binding.
Anti-hTfR Generation. VelocImmune mice were immunized with a recombinant protein comprising human transferrin receptor extracellular domain fused at N-terminus to a 6-His tag (referred to as human 6×His-TfR) as immunogen via subcutaneous footpad injection with Alum:CpG adjuvant. Mice bleeds were collected prior to the initial immunization injection and post-boost injections, and the immune sera were subjected to antibody titer determination using a human TfR specific enzyme-linked immunosorbent assay (ELISA). In this assay serum samples in serial dilutions were added to the immunogen coated plates and plate-bound mouse IgG were detected using an HRP-conjugated anti-mouse IgG antibody. Titer of a tested serum sample is defined as the extrapolated dilution factor of the sample that produces a binding signal two times of the signal of the buffer alone control sample. The mice with optimal anti-TfR antibody titers were selected and subjected to a final boost 3-5 days prior to euthanasia and splenocytes from these mice were harvested and subject to antibody isolation using B cell sorting technology (BST).
TfR specific antibodies of isolated antibodies were isolated and characterized. Two hundred and fourteen TfR-binding antibodies were cloned into single chain fragment variables (scFvs) in complementary orientations with either the variable heavy chain followed by the variable light chain (VH-VK), or the variable light chain followed by the variable heavy chain (VK-VH), and as fragment antigen-binding regions (Fabs). Conditioned media of CHO cell culture containing the scFvs or Fabs were tested for the ability to bind hTfR proteins and hTfR-expressing cells.
Biacore binding kinetics assays were conducted for the interaction of 32 anti-human TfR IgG1 monoclonal antibodies from CHO supernatants with TfR reagents at 25° C.
Reagents Used:
Equilibrium dissociation constants (KD) for the interaction of anti-TfR monoclonal antibodies with human and fascicularis monkey TfR ecto domain recombinant proteins were determined using a real-time surface plasmon resonance (SPR) based Biacore S200 biosensor. All binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v surfactant Tween-20, pH 7.4 (HBS-EP) running buffer at 37° C. The Biacore CM5 sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567) followed by a step to capture anti-TfR monoclonal antibodies in CHO conditioned media. Human TfR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hTFR-mmh; REGN2431) or monkey TfR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mfTFR-mmh; REGN2054) at concentrations of 100 nM in HBS-EP running buffer were injected at a flow rate of 50 μL/min for 2 minutes. The dissociation of TfR bound to anti-TfR monoclonal antibodies was monitored for 3 minutes in HBS-EP running buffer. At the end of each cycle, the anti-TfR monoclonal antibodies capture surface was regenerated using a 12-sec injection of 20 mM H3PO4. The association rate (ka) and dissociation rate (kd) were determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c software. The dissociation equilibrium constant (KD) and dissociative half-life (t½) were calculated from the kinetic rate constants as:
The equilibrium binding constant and the kinetic binding constants are summarized in Tables 2-2 and Table 2-3 for human TfR and monkey TfR, respectively. At 25° C., anti-TfR monoclonal antibodies bound to hTfR-mmh with KD values ranging from 65.6 pM to 41 nM, as shown in Table 2-2. Anti-TfR monoclonal antibodies bound to mfTfR-mmh with KD values ranging from 1.16 nM to 20.5 nM, as shown in Table 2-3.
Results are set forth below.
#indicates no dissociation was observed under the current experimental conditions and the kd value was manually fixed at 1.00E−05 s−1 while fitting the real time binding sensorgrams.
An ELISA-based blocking assay was developed to determine the ability of anti-Transferrin Receptor (TfR) antibodies to block the binding of human Transferrin Receptor to human holo-transferrin ligand.
The human Transferrin Receptor recombinant protein, hTFRC, used in the experiment was comprised of hTfR extracellular domain (amino acids C89-F760) expressed with an N-terminal 6-Histidine-myc-myc tag (Hmm.hTfrc (REGN2431): Monomeric human Tfrc ectodomain (amino acids C89-F760, Accession #: NP_001121620.1) with an N-terminal hexahistidine-myc-myc-tag containing a GG linker (underlined) between the 2 myc epitope sequences (HHHHHHEQKLISEEDLGGEQKLISEEDL) (amino acids 1-28 of SEQ ID NO: 460)). The human holo-transferrin ligand protein (holo-Tf) isolated from human plasma was purchased from R&D Systems.
In the blocking assay, the anti-human Transferrin goat IgG polyclonal antibody (anti-hTf pAb) was passively absorbed at a concentration of 2 micrograms/mL in PBS on a 96-well microtiter plate overnight at 4° C. Nonspecific binding sites were subsequently blocked using a 0.5% (w/v) solution of BSA in PBS for 1 hour at room temperature. To the same plate, human holo-Tf was then added at a concentration of 1 micrograms/mL in PBS+0.5% BSA for 2 hours at room temperature. In a separate set of 96-well microtiter plates, solutions of 300 pM Hmm-hTFRC were mixed with TFRC antibody supernatants at 2-fold dilution. After a 1-hour incubation, the mixtures were transferred to the human holo-Tf microtiter plates. After another hour incubation at room temperature, plates were washed, and plate-bound Hmm-hTFRC was detected with horseradish peroxidase (HRP) conjugated rabbit anti-Myc polyclonal antibody. The plates were developed using TMB substrate solution according to the manufacturer's recommended procedure and absorbance at 450 nm was measured on a Victor™ Multilabel Plate Reader.
Percent blocking for the tested anti-TfR antibodies was calculated using the formula below:
Antibodies that blocked binding of Hmm-hTFRC to human holo-Tf equal or more than 50% were classified as blockers.
The ability of the anti-TfR antibody to block human TFRC binding to human holo-Tf was evaluated using an ELISA-based blocking assay. In this assay, a fixed concentration of Hmm-hTFRC was pre-incubated with anti-TfR antibody containing supernatant before binding to plate immobilized human holo-Tf protein, and the plate-bound Hmm-hTFRC was detected with HRP-conjugated c-Myc specific rabbit polyclonal antibodies.
Thirty-two anti-TfR antibodies cloned into single chain fragment variables (scFvs) in complementary orientations with either the variable heavy chain followed by the variable light chain (VH-VK), or the variable light chain followed by the variable heavy chain (VK-VH) and also as fragment antigen-binding regions (Fabs). All ninety-six anti-TfR antibody supernatants were tested for the ability to block human TFRC binding to human holo-Tf. Ninety-four anti-TfR antibody supernatants showed no or low blocking activity with percentage blocking ranging from 0% to 45%, and these antibodies (Fabs or scFvs formats) were classified as non-blockers (Table 3-2). Only two Fab supernatants had blocking activity greater than 50%, with % blocking values of 64% and 78% respectively.
In this example, the ability of various anti-TFRC molecules to cross the blood-brain barrier and localize to the parenchyma of the brain was evaluated. Delivery of the molecules via episomal AAV liver depot was also evaluated along with rescue of the glycogen storage phenotype in various tissues.
To further evaluate the anti-human TFRC antibodies that were screened for binding in vitro, in vivo mouse studies in Tfrchum/hum knock-in mice were performed to evaluate blood-brain-barrier (BBB) crossing. This screen of 31 antibodies revealed 11 that had mature hGAA protein in brain homogenate detected by Western blot.
Human TFRC knock-in mice were injected with DNA plasmids expressing the various anti-hTFRC antibodies in the anti-hTFRC scfv:2×G4S(SEQ ID NO: 537):hGAA format under the liver-specific mouse TTR promoter. Mice received 50 ug of DNA in 0.9% sterile saline diluted to 10% of the mouse's body weight (0.1 mL/g body weight). Forty-eight hours post-injection, tissues were dissected from mice immediately after sacrifice by CO2 asphyxiation, snap frozen in liquid nitrogen, and stored at −80° C.
Tissue lysates were prepared by lysis in RIPA buffer with protease inhibitors (1861282, Thermo Fisher, Waltham, MA, USA). Tissue lysates were homogenized with a bead homogenizer (FastPrep5, MP Biomedicals, Santa Ana, CA, USA). Cells or tissue lysates were run on SDS-PAGE gels using the Novex system (LifeTech Thermo, XPO4200BOX, LC2675, LC3675, LC2676). Gels were transferred to low-fluorescence polyvinylidene fluoridev (PVDF) membrane (IPFLO7810, LI-COR, Lincoln, NE, USA) and stained with Revert 700 Total Protein Stain (TPS; 926-11010 LI-COR, Lincoln, NE, USA), followed by blocking with Odyssey blocking buffer (927-500000, LI-COR, Lincoln, NE, USA) in Tris buffer saline with 0.1% Tween 20 and staining with antibodies against GAA (ab137068, Abcam, Cambridge, MA, USA), or anti-GAPDH (ab9484, Abcam, Cambridge, MA, USA) and the appropriate secondary (926-32213 or 925-68070, LI-COR, Lincoln, NE, USA). Blots were imaged with a LI-COR Odyssey CLx.
Protein band intensity was quantified in LI-COR Image Studio software. The quantification of the mature 77 kDa GAA band for each sample was determined by first normalizing to the lane's TPS signal, then normalizing to GAA levels in the serum (loading control and liver expression control, respectively). Values were then compared to the positive control group anti-mouse TFRC scfv:hGAA in Wt mice, and negative control group anti-mTFRC scfv:hGAA in Tfrchum/hum mice (
The control anti-mTRFC that was conjugated to GAA was 8D3 scFv. The 8D3 scFv has the heavy chain amino acid sequence:
and the light chain amino acid sequence:
Capillary Depletion of Brain Samples Following HDD of Anti-hTFRC Scfv:hGAA Plasmids
Anti-hTFRC scfv:hGAA molecules from Table 4-1 were tested in a secondary screen in Tfrchum mice to determine whether hGAA was present in the brain parenchyma, and not trapped in the BBB endothelial cells. Four molecules (12799, 12839, 12843, and 12847) identified in screen as being present in parenchyma based on mature hGAA in the parenchyma fraction on Western blot, as well as high affinity to cyno TFRC.
Animals were treated HDD as detailed above. Forty-eight hours post-injection, mice were perfused with 30 mL 0.9% saline immediately after sacrifice by CO2 asphyxiation. A 2 mm coronal slice of cerebrum was taken between bregma and −2 mm bregma and placed in 700 uL physiological buffer (10 mM HEPES, 4 mM KCl, 2.8 mM CaCl2), 1 mM MgSO4, 1 mM NaH2PO4, 10 mM D-glucose in 0.9% saline pH 7.4) on ice. Brain slices were gently homogenized on ice with a glass dounce homogenizer. An equivalent volume of 26% dextran (MW 70,000 Da) in physiological buffer was added (final 13% dextran) and homogenized 10 more strokes. Parenchyma (supernatant) and endothelial (pellet) fractions were separated by centrifugation at 5,400 g for 15 min at 4° C. Anti-hGAA western blot was performed on fractions as detailed above (
Capillary Depletion of Mouse Brain Samples Following Liver-Depot AAV8 Anti-hTFRC Scfv:hGAA Treatment
To confirm our HDD screen findings in a more long-term treatment model, we treated Tfrchum mice with anti-hTFRC scfv:hGAA molecules delivered as episomal liver depot AAV8 anti-hTFRC scfv:GAA under the TTR promoter. We found that all 4 molecules (12799, 12843, 12847 and 12839) delivered mature hGAA to the brain parenchyma when delivered as AAV8.
Recombinant AAV8 (AAV2/8) was produced in HEK293 cells. Cells were transfected with three plasmids encoding adenovirus helper genes, AAV8 rep and cap genes, and recombinant AAV genomes containing transgenes flanked by AAV2 inverted terminal repeats (ITRs). On day 5, cells and medium were collected, centrifuged, and processed for AAV purification. Cell pellets were lysed by freeze-thaw and cleared by centrifugation. Processed cell lysates and medium were overlaid onto iodixanol gradients columns and centrifuged in an ultracentrifuge. Virus fractions were removed from the interface between the 40% and 60% iodixanol solutions and exchanged into 1×PBS with desalting columns. AAV vg (vg=viral genomes) were quantified by ddPCR. AAVs were diluted in PBS+0.001% F-68 Pluronic immediately prior to injection. Tfrchum mice were dosed with 3e12vg/kg body weight in a volume of −100 uL. Mice were sacrificed 4 weeks post injection and capillary depletion and western blotting were performed as described above (
Rescue of Glycogen Storage Phenotype in Gaa−/−/Tfrchum Mice with AAV8 Episomal Liver Depot Anti-hTFRC Scfv:GAA
Anti-hTFRC scfv:GAA molecules in Pompe disease model mice were tested to determine whether anti-hTFRCscfv:GAA rescued the glycogen storage phenotype. The molecules, 12839, 12843, 12847, normalized glycogen to Wt levels. (12799 not tested).
AAV production and in vivo transduction were performed as above. Gaa−/−/Tfrchum mice were dosed with 2e12vg/kg AAV8. Tissues were harvested 4 weeks post-injection and flash-frozen as above. hGAA Western blot was performed as above (
Tissues were dissected from mice immediately after sacrifice by CO2 asphyxiation, snap frozen in liquid nitrogen, and stored at −80° C. Tissues were lysed on a benchtop homogenizer with stainless steel beads in distilled water for glycogen measurements or RIPA buffer for protein analyses. Glycogen analysis lysates were boiled and centrifuged to clear debris. Glycogen measurements were performed fluorometrically with a commercial kit according to manufacturer's instructions (K646, BioVision, Milpitas, CA, USA). See Table 4-6 and
Rescue of Glycogen Storage in Brain and Muscle in Gaa−/−/Tfrchum Mice with AAV8 Episomal Liver Depot Anti-hTFRC Scfv:GAA
Anti-hTFRCscfv:GAA molecules, 12799, 12843, and 12847, were tested in Pompe disease model mice to determine whether they rescued the glycogen storage phenotype. Histology was performed on brain and muscle sections to visualize glycogen in the tissues. All 3 molecules reduced glycogen staining in the brain and muscle.
AAV production and in vivo transduction were performed as above. Three month old Gaa−/−/Tfrchum mice were dosed with 4e11vg/kg AAV8. Four weeks post-injection, tissues were frozen for glycogen analysis as above (Table 4-7). For histology, animals were perfused with saline (0.9% NaCl), and tissues were drop-fixed overnight in 10% Normal Buffered Formalin. Tissues were washed 3× in PBS and stored in PBS/0.01% sodium azide until embedding. Tissues were embedded in paraffin and Sum sections were cut from brain (coronal, −2 mm bregma) and quadricep (fiber cross-section). Sections were stained with Periodic Acid-Schiff and Hematoxylin using standard protocols (
This Example evaluated the effect of anti-TfR antigen-binding proteins on iron homeostasis in mice.
To validate that Tfrchum mice expressed TFRC at physiological levels and had normal iron homeostasis, we compared Tfrchum mice to Wt mice and quantified expression of TFRC in tissues, serum markers, tissue iron content, and transferrin in tissues. Overall, TFRC expression and iron homeostasis was normal in the Tfrchum mice.
Six month old Wt mice (11 males, 4 females) and Tfrchum mice (10 males, 8 females) were analyzed in this experiment. Tissues were dissected from mice immediately after sacrifice by CO2 asphyxiation, snap frozen in liquid nitrogen, and stored at −80° C.
Tfrc RNA Quantification by qPCR
Total RNA was isolated from tissues with Trizol following manufacturer protocol (ThermoFisher 15596026). Tfrc RNA was quantified by Taqman qPCR (ThermoFisher) following standard protocols using universal primers to exon 1 that amplify from both Wt and and Tfrchum mice (GCTGCATTGCGGACTGTAGA (SEQ ID NO: 503)/TCCATCATTCTCAGCTGCTACAA (SEQ ID NO: 504)). ΔΔCT values were calculated relative to the Wt male group. Data in Table 5-1.
Blood was collected from mice by cardiac puncture immediately following CO2 asphyxiation and serum was separated using serum separator tubes (BD Biosciences, 365967). Serum iron and Total Iron Binding Content (TIBC) were quantified using standard protocols. Serum hepcidin was quantified by ELISA kit (Intrinsic Life Sciences SKU HMC-001). Data in Table 5-2.
Wet tissue was weighed to achieve uniformity and then dried for 72 hours in an open tube at 56° C. Tissue was then placed in digestion buffer (10% Tricloroacetic acid and 37% HCL) and heated at 65° C. for 48 hours. To assay iron content, the supernatant was placed in a 96 well plate and incubated in a color development solution (Thioglycolic acid, bathophenanthroline acid and sodium acetate). Absorbance was read on a Spectramax i3 by Molecular Devices and Graph Pad Prism was used to interpolate the sample absorbance values read against a standard curve to calculate iron content in the whole piece of tissue. Iron content was then calculated based on dry weight. Data in Table 5-3.
All tissues were homogenized using a Fastprep-24 5G from MP Biomedicals. Prior to homogenization, tissues were placed in RIPA buffer with phosphatase and HALT protease inhibitors (ThermoFisher), homogenized with their organ specific protocol and then centrifuged to pellet debris. The supernatant was collected and assayed for total protein using a Pierce BCA Protein Assay Kit. Absorbance was measured on a Spectramax i3 by Molecular Devices. Once total protein was measured, all samples were diluted to match the least concentrated sample so loading would be uniform for the ELISA. Kits obtained from Abcam were used to measure the presence of total transferrin in tissue homogenate (Abcam ab157724). Plates were run in accordance with the supplied protocol using the provided reagents and absorbance was read on a Spectramax i3 by Molecular Devices. Graph Pad Prism was used to interpolate the sample absorbance values read against a standard curve. Data in Table 5-4.
Rescue of Glycogen Storage in Brain and Muscle in Gaa−/−/Tfrchum Mice with AAV8 Episomal Liver Depot Anti-hTFRC Scfv:GAA
We tested the anti-hTFRC scfv:GAA leads 12799, 12843, and 12847 in Pompe disease model mice to determine whether anti-hTFRC scfv:GAA rescued the glycogen storage phenotype (glycogen data in other data package). Here we also tested whether treatment with anti-TFRCscfv:GAA leads altered iron homeostasis (Tables 5-5, 5-6 and 5-7). We found that 4-week treatment did not affect iron homeostasis with any of the leads.
We tested our lead anti-hTFRC 12847scfv:GAA in Pompe disease model mice by albumin insertion to determine whether we could replicate the results we saw with episomal AAV8 liver depot expression. Albumin insertion of 12847scfv:GAA delivered mature hGAA protein to the brain and muscle, and rescued the glycogen storage phenotype in Gaa−/−/Tfrchum/hum mice. These data were produced with the native 12847scfv:GAA sequence that is not optimized.
We compared 12847scfv:GAA to the muscle-targeted anti-hCD63scfv:GAA in Gaa−/−/Cd63hum mice. In this particular experiment, the expression of anti-hCD63scfv:GAA was lower than usual and does not deliver as much GAA protein to the muscle nor normalize glycogen as it usually does. This may make it appear that anti-hCD63scfv:GAA is less effective than 12847scfv:GAA in the muscle but in most experiments we found them to be comparable in the muscle.
A promoterless AAV genome plasmid was created with the 12847scfv:GAA sequence and the mouse albumin exon 1 splice acceptor site at the 3′ end. Recombinant AAV8 (AAV2/8) was produced in HEK293 cells. Cells were transfected with three plasmids encoding adenovirus helper genes, AAV8 rep and cap genes, and recombinant AAV genomes containing transgenes flanked by AAV2 inverted terminal repeats (ITRs). On day 5, cells and medium were collected, centrifuged, and processed for AAV purification. Cell pellets were lysed by freeze-thaw and cleared by centrifugation. Processed cell lysates and medium were overlaid onto iodixanol gradients columns and centrifuged in an ultracentrifuge. Virus fractions were removed from the interface between the 40% and 60% iodixanol solutions and exchanged into 1×PBS with desalting columns. AAV vg were quantified by ddPCR.
In Vivo CRISPR/Cas9 Insertion into the Albumin Locus
Three month old Gaa−/−/Tfrchum/hum mice were dosed via tail vein injection with 3e12vg/kg AAV8 12847scfv:GAA and 3 mg/kg LNP gRNA/Cas9 mRNA diluted in PBS+0.001% F-68 Pluronic. Mice were sacrificed 3 weeks post injection. Negative control mice received insertion AAV8 without LNP. Positive control mice were dosed with 4e11vg/kg episomal liver depot AAV8 12847scfv:GAA under the TTR promoter (phenotype rescue data previously shown). Tissues were dissected from mice immediately after sacrifice by CO2 asphyxiation, snap frozen in liquid nitrogen, and stored at −80° C. Blood was collected from mice by cardiac puncture immediately following CO2 asphyxiation and serum was separated using serum separator tubes (BD Biosciences, 365967).
Tissue lysates were prepared by lysis in RIPA buffer with protease inhibitors (1861282, Thermo Fisher, Waltham, MA, USA). Tissue lysates were homogenized with a bead homogenizer (FastPrep5, MP Biomedicals, Santa Ana, CA, USA). Cells or tissue lysates were run on SDS-PAGE gels using the Novex system (LifeTech Thermo, XPO4200BOX, LC2675, LC3675, LC2676). Gels were transferred to low-fluorescence polyvinylidene fluoridev (PVDF) membrane (IPFL07810, LI-COR, Lincoln, NE, USA) and stained with Revert 700 Total Protein Stain (TPS; 926-11010 LI-COR, Lincoln, NE, USA), followed by blocking with Odyssey blocking buffer (927-500000, LI-COR, Lincoln, NE, USA) in Tris buffer saline with 0.1% Tween 20 and staining with antibodies against GAA (ab137068, Abcam, Cambridge, MA, USA), or anti-GAPDH (ab9484, Abcam, Cambridge, MA, USA) and the appropriate secondary (926-32213 or 925-68070, LI-COR, Lincoln, NE, USA). Blots were imaged with a LI-COR Odyssey CLx.
Protein band intensity was quantified in LI-COR Image Studio software. The quantification of the mature 77 kDa GAA band for each sample was determined by normalizing to the lane's TPS signal (loading control).
Tissues were dissected from mice immediately after sacrifice by CO2 asphyxiation, snap frozen in liquid nitrogen, and stored at −80° C. Tissues were lysed on a benchtop homogenizer with stainless steel beads in distilled water for glycogen measurements or RIPA buffer for protein analyses. Glycogen analysis lysates were boiled and centrifuged to clear debris. Glycogen measurements were performed fluorometrically with a commercial kit according to manufacturer's instructions (K646, BioVision, Milpitas, CA, USA).
§§p < 0.001 vs. AAV only negative control group.
§non-significant vs. Wt untreated group.
We tested our lead anti-hTFRC 12847scfv:GAA in cynomolgus monkeys by albumin insertion to determine whether we could replicate the results we saw in mice. We compared 12847scfv:GAA to the muscle-targeted anti-hCD63scfv:GAA in cynomolgus monkeys. As shown in
Albumin insertion of anti-hCD63scfv:GAA or 12847scfv:GAA resulted in similar serum GAA levels with two different gRNAs, regardless of what gRNA was used (data not shown). Insertion did not negatively affect serum iron panel or creatinine (data not shown).
A promoterless AAV genome plasmid was created with the 12847scfv:GAA sequence and the mouse albumin exon 1 splice acceptor site at the 3′ end. Recombinant AAV8 (AAV2/8) was produced in HEK293 cells. Cells were transfected with three plasmids encoding adenovirus helper genes, AAV8 rep and cap genes, and recombinant AAV genomes containing transgenes flanked by AAV2 inverted terminal repeats (ITRs). On day 5, cells and medium were collected, centrifuged, and processed for AAV purification. Cell pellets were lysed by freeze-thaw and cleared by centrifugation. Processed cell lysates and medium were overlaid onto iodixanol gradients columns and centrifuged in an ultracentrifuge. Virus fractions were removed from the interface between the 40% and 60% iodixanol solutions and exchanged into 1×PBS with desalting columns. AAV vg were quantified by ddPCR.
In Vivo CRISPR/Cas9 Insertion into the Albumin Locus
Cynomolgus monkeys age 2-3 years were dosed intravenously with 1.5e13vg/kg AAV8 12847scfv:GAA (or anti-CD63scfv:GAA) and 3 mg/kg LNP gRNA/Cas9 mRNA. Negative control monkeys received insertion AAV8 without LNP or vehicle control only. Serum and flash-frozen tissues were collected 90 days post-injection.
Serum was collected prior to dosing and at indicated timepoints post-injection. GAA activity in the serum was quantified using Lysosomal alpha-Glucosidase Activity Assay Kit (Abcam ab252887). Serum GAA activity in CD63scfv:GAA and 12847scfv:GAA treated animals was above the vehicle controls and activity was similar between the treatment groups. Serum GAA activity corresponded with liver GAA protein expression and serum GAA protein levels (
Tissue lysates were prepared by lysis in RIPA buffer with protease inhibitors (1861282, Thermo Fisher, Waltham, MA, USA). Tissue lysates were homogenized with a bead homogenizer (FastPrep5, MP Biomedicals, Santa Ana, CA, USA). Cells or tissue lysates were run on SDS-PAGE gels using the Novex system (LifeTech Thermo, XPO4200BOX, LC2675, LC3675, LC2676). Gels were transferred to low-fluorescence polyvinylidene fluoridev (PVDF) membrane (IPFL07810, LI-COR, Lincoln, NE, USA) and stained with Revert 700 Total Protein Stain (TPS; 926-11010 LI-COR, Lincoln, NE, USA), followed by blocking with Odyssey blocking buffer (927-500000, LI-COR, Lincoln, NE, USA) in Tris buffer saline with 0.1% Tween 20 and staining with antibodies against GAA (ab137068, Abcam, Cambridge, MA, USA), or anti-GAPDH (ab9484, Abcam, Cambridge, MA, USA) and the appropriate secondary (926-32213 or 925-68070, LI-COR, Lincoln, NE, USA). Blots were imaged with a LI-COR Odyssey CLx.
Protein band intensity was quantified in LI-COR Image Studio software. The quantification of the mature 77 kDa GAA band for each sample was determined by normalizing to the lane's TPS signal (loading control).
Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) was performed to delineate regions in mouse and human Transferrin (m/hTfR) involved in binding of anti-Transferrin Receptor (TfR) antibodies. The anti-TfR monoclonal antibodies tested are described in Table 8-1. The reagents used and corresponding lot numbers are set forth in Table 8-2.
A general description of the HDX-MS method is set forth in, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A. The experiment was performed on a customized HDX automation system (NovaBioAssays, MA) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, MA).
PBS-D2O buffer was prepared by dissolving one PBS tablet in 100 mL 99.9% D2O to form solution of 10 mM sodium phosphate, 137 mM NaCl, 3 mM KCl, pD 7.0 (equivalent to pH 7.4 at 25° C.). To initiate deuterium exchange, 10 μL of protein sample (hTfR alone, or hTfR in mixture with either of the monoclonal mAbs listed above, see, e.g., Table 8-1) was diluted with 90 μL PBS-D2O buffer. After 5 minutes or 10 minutes, deuterium exchange was quenched by adding 100 μL quenching buffer (0.5 M TCEP, 4 M guanidine hydrochloride, pH 2.08) followed by 90 second incubation at 20° C. The quenched samples were digested by online pepsin/protease XIII column (NovaBioAssays, MA) at room temperature with 100 μL/min 0.1% formic acid in water. Peptic peptides were trapped by an ACQUITY UPLC Peptide BEH C18 VanGuard Pre-column (2.1×5 mm, Waters, MA) and further separated by an ACQUITY UPLC Peptide BEH C18 column (2.1×50 mm, Waters, MA) at −5° C., using 10-minute or 15-minute gradients with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as mobile phases at 200 μL/min. Eluted peptides were analyzed by the mass spectrometer in LC-MS/MS or LC-MS mode.
A set of non-deuterated samples was prepared in PBS—H2O buffer and analyzed with the method described above to identify peptide sequences and determine peptide masses without deuterium exchange. The LC-MS/MS data of non-deuterated samples were searched against a database containing sequences of hTfR, pepsin and protease XIII using the Byonic search engine (Protein Metrics, CA) with parameters for non-specific enzymatic digestion. The identified peptide list was then imported into the HDExaminer software (Sierra Analytics, CA) together with LC-MS data from all deuterated samples to calculate the deuterium uptake percentage (D %) of individual peptides from hTfR. Differences in deuterium uptake were calculated as ΔD %=D % of hTfR-mAb−D % of hTfR. Differences were considered significant if ΔD %<−5% (equivalent to |ΔD|>5% and ΔD %<0, averaged from 2 replicates). Mass spectra of peptides showing significant differences were examined manually to ensure that correct isotopic patterns were used for D % calculations by the software.
Two TfR protein constructs were used in HDX-MS experiments by reason of reagent availability and antibody specificity: hTfR(C89-F763).mmh, and hmm.hTfR(C89-F763). HDX data were obtained on 88%-95% of amino acids in hTfR with mmh tag. The numerical range provided before each amino acid sequence in the list below indicates the amino acid (aa) residue positions in hTfR which are protected by the indicated antibody. These amino acid residue positions are indicative of antibody binding sites on hTfR and does not provide residue-level contacts between them. Due to the nature of HDX-MS technique, the regions obtained by HDX-MS may be larger or smaller than actual contacts determined by high-resolution structural studies such as X-ray crystallography and cryogenic electron microscopy methods.
REGN17507 (H1H12798B) protects the following regions in hTfR:
REGN17508 (H1H12799B) protects the following regions in hTfR:
REGN17509 (H1H12835B) protects the following region in hTfR:
REGN17510 (H1H12839B) protects the following region in hTfR:
REGN17511 (H1H12841B) protects the following region in hTfR:
REGN17512 (H1H12843B) protects the following region in hTfR:
REGN17513 (H1H12845B) protects the following region in hTfR:
REGN17514 (H1H12847B) protects the following region in hTfR:
REGN17515 (H1H12848B) protects the following region in hTfR:
REGN17516 (H1H12850B) protects the following region in hTfR:
REGN17517 (H1H1874B) protects the following region in hTfR:
The minimal amino acid sequence in hTfR which is protected by the above-listed anti-TfR antibodies (i.e., the minimal epitope sequence), numerical range indicating the amino acid (aa) residue positions in hTfR which are protected each antibody, as well as the conformational or linear nature of each minimal epitope are described in Table 8-3. Each of the minimal epitopes is bound by its corresponding antibody at one or more amino acid residues within the minimal epitope sequence.
The extracellular unit of hTfR is structurally categorized into three domains, the helical, protease-like and apical domains (PDB 1 SUV).
Structural studies of TfR in complex with a variety of molecules that have identified TfR binding sites, including Mammarenavirus machupoense GP1 protein (PDB 3KAS), canine parvovirus (PDB 2NSU), human ferritin (PDB 6GSR), Plasmodium vivax Sal-1 PvRBP2b (PDB 61D04), human HFE protein (PDB 1DE4), human transferrin (PDB 1 SUV), etc.
HDX protections for the antibodies tested in HDX-MS experiments can be assigned to 5 regions in TfR (PDB 1SUV) as depicted in
Tabulated summaries of data of the present Example are described in Tables 8-4 to Table 8-8.
Mammarenavirus machupoense GP1, canine
vivax Sal-1 PvRBP2b binding sites.
vivax Sal-1 PvRBP2b.
All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants to relate to each and every individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.
This application claims the benefit of U.S. Application No. 63/393,719, filed Jul. 29, 2022, which is herein incorporated by reference in its entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63393719 | Jul 2022 | US |
