Compositions and methods related to engineered Fc constructs
11623964 · 2023-04-11
Assignee
Inventors
Cpc classification
A61K39/395
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
C07K2317/60
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
A61P43/00
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
International classification
A61K39/395
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
Abstract
The present disclosure relates to compositions and methods of engineered IgG Fc constructs, wherein the Fc constructs include one or more Fc domains.
Claims
1. An Fc construct comprising: a) a first polypeptide having the formula A-L-B; wherein i) A comprises a first Fc domain monomer; ii) L is a linker; and iii) B comprises a second Fc domain monomer; b) a second polypeptide having the formula A′-L′-B′; wherein i) A′ comprises a third Fc domain monomer; ii) L′ is a linker; and iii. B′ comprises a fourth Fc domain monomer; c) a third polypeptide comprises a fifth Fc domain monomer; and d) a fourth polypeptide comprises a sixth Fc domain monomer; wherein the A of first polypeptide and the A′ of second polypeptide combine to form a first Fc domain, the B of first polypeptide and fifth Fc domain monomer combine to form a second Fc domain, and the B′ of second polypeptide and sixth Fc domain monomer combine to form a third Fc domain; and wherein the first Fc domain monomer and the third Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the first Fc domain monomer and the third Fc domain monomer; second Fc domain monomer and the fifth Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the second Fc domain monomer and the fifth Fc domain monomer; the fourth Fc domain monomer and the sixth Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the fourth Fc domain monomer and the sixth Fc domain monomer; wherein each of A, B, A′, B′, the third polypeptide and the fourth polypeptide comprises an Fc domain monomer; and each Fc domain monomer comprises a hinge domain, a C.sub.H2 domain and a C.sub.H3 domain; and wherein at least one of the Fc domain monomers comprises both a S267E mutation and a L328F mutation that together enhance binding to the FcγRIIb receptor.
2. A method of treating inflammation in a subject, the method comprising administering to the subject a pharmaceutical composition comprising therapeutically effective amount of an Fc construct of claim 1.
3. The Fc construct of claim 1, further comprising an albumin-binding peptide comprising SEQ ID NO: 28.
4. The Fc construct of claim 1, wherein the first polypeptide and the second polypeptide have the same amino acid sequence and wherein the third polypeptide and the fourth polypeptide have the same amino acid sequence.
5. The Fc construct of claim 1, wherein each Fc domain monomers comprises both a S267E mutation and a L328F mutation.
6. The Fc construct of claim 1, wherein each of the first, second, third and fourth polypeptides lack a carboxy-terminal lysine.
7. The Fc construct of claim 1, wherein each dimerization selectivity module independently comprises: (a) an engineered cavity in the C.sub.H3 domain of one of the Fc domain monomers and an engineered protuberance in the C.sub.H3 domain of the other of the Fc domain monomers, wherein the engineered cavity and the engineered protuberance are positioned to form a protuberance-into-cavity pair of Fc domain monomers; or (b) a negatively-charged amino acid in the C.sub.H3 domain of one of the domain monomers and a positively-charged amino acid in the C.sub.H3 domain of the other of the Fc domain monomers, wherein the negatively-charged amino acid and the positively-charged amino acid are positioned to promote formation of an Fc domain.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(23) Therapeutic proteins that include Fc domains of IgG can be used to treat inflammation and immunological and inflammatory diseases. The present disclosure features compositions and methods for preparing various Fc constructs containing two or more (e.g., 2-10) Fc domains.
(24) I. Fc Domain Monomers
(25) An Fc domain monomer includes a hinge domain, a C.sub.H2 antibody constant domain, and a C.sub.H3 antibody constant domain. The Fc domain monomer can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. The Fc domain monomer may also be of any immunoglobulin antibody isotype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). A dimer of Fc domain monomers is an Fc domain (further defined herein) that can bind to an Fc receptor, e.g., FcγRIIIa, which is a receptor located on the surface of leukocytes. In the present disclosure, the C.sub.H3 antibody constant domain of an Fc domain monomer may contain amino acid substitutions at the interface of the C.sub.H3-C.sub.H3 antibody constant domains to promote their association with each other. In some embodiments, an Fc domain monomer includes two other constant domains, e.g., C.sub.L and C.sub.H1 antibody constant domains, attached to the N-terminus (
(26) II. Fc Domains
(27) As defined herein, an Fc domain includes two Fc domain monomers that are dimerized by the interaction between the C.sub.H3 antibody constant domains. In the present disclosure, an Fc domain does not include a variable region of an antibody, e.g., V.sub.H, V.sub.L, CDR, or HVR. An Fc domain forms the minimum structure that binds to an Fc receptor, e.g., Fc-gamma receptors (i.e., Fcγ receptors (FcγR)), Fc-alpha receptors (i.e., Fcα receptors (FcαR)), Fc-epsilon receptors (i.e., Fcε receptors (FcεR)), and/or the neonatal Fc receptor (FcRn). In some embodiments, an Fc domain of the present disclosure binds to an Fcγ receptor (e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and/or FcγRIIIB (CD16b)) and/or the neonatal Fc receptor (FcRn).
(28) III. Fc Domain Modifications
(29) An unmodified Fc domain monomer can be a naturally occurring human Fc domain monomer or a WT human Fc domain monomer. An Fc domain monomer can be a naturally occurring human Fc domain monomer comprising a hinge, a C.sub.H2 domain, and a C.sub.H3 domain; or a variant thereof having up to 16 (e.g., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) amino acid modifications (e.g., single amino acid modifications) to accommodate or promote directed dimerization. In some cases, the Fc domain includes at least one amino acid modification, wherein the amino acid modifications alter one or more of (i) binding affinity to one or more Fc receptors, (ii) effector functions, (iii) the level of Fc domain sulfation, (iv) half-life, (v) protease resistance, (vi) Fc domain stability, and/or (vii) susceptibility to degradation (e.g., when compared to the unmodified Fc domain). In some cases, the Fc domain includes no more than 16 amino acid modifications (e.g., no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid modifications in the C.sub.H3 domain).
(30) The Fc domains of the disclosure include at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) or more amino acid modifications at residues selected from positions 40, 92-103, 113-116, 118, 131, 137-146, 169-175, 196, 199, 203, 206, 211, 214, 217, 219-341, 344-346, 349-370, 372-374, 376-380, 382-405, 407-422, 424, 426-442, and/or 445-447. In some embodiments, the amino acid modification is an amino acid substitution, wherein the substituted amino acid is a natural or non-natural amino acid. In some embodiments, an amino acid modification is an amino acid deletion, wherein at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues are deleted from the Fc domain. In some embodiments, an Fc domain modification is an amino acid insertion, wherein at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues are inserted into the Fc domain. The amino acid modification can be a combination of multiple modifications, for example, a combination of one or more amino acid substitutions, deletions, and/or insertions.
(31) Fc Receptor Binding Affinity
(32) Amino acid modifications of the present disclosure may alter (i.e., increase or decrease) the binding affinity of an Fc domain to one (e.g., 1, 2, 3, 4, 5, or 6) or more Fc receptors (e.g., Fc-gamma receptors (FcγR), Fc-alpha receptors (FcαR), Fc-epsilon receptors (FcεR), and/or to the neonatal Fc receptor (FcRn)). A modified Fc domain may bind to an FcγR (e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and/or FcγRIIIB (CD16b)) and/or to the neonatal Fc receptor (FcRn) with an altered (i.e., increased or decreased) affinity as compared to an unmodified Fc domain. A modified Fc domain may have an altered (i.e., increased or decreased) dissociation constant (Kd) for one (e.g., 1, 2, 3, 4, 5, or 6) or more Fc receptors as compared to an unmodified Fc domain. Additionally, a modified Fc domain may have an altered (i.e., increased or decreased) level of glycosylation (e.g., glygan modification (e.g., mannose, sialic acids, fucose (Fuc), and/or galactose (Gal))) as compared to an unmodified Fc domain. An Fc modification may alter the affinity of an Fc domain to one (e.g., 1, 2, 3, 4, 5, or 6) or more Fc receptors, while inversely altering the affinity to at least one (e.g., 1, 2, 3, 4, 5, or 6) or more other Fc receptors.
(33) Table 1 lists exemplary Fc domain residues that may be modified to alter Fc receptor binding affinity. In some embodiments, one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 1 are modified, wherein the modified Fc domain has an altered binding affinity to an Fc receptor as compared to an unmodified Fc domain. In some embodiments, the Fc domain modification is an amino acid substitution occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 1. In some embodiments, an Fc domain modification is an amino acid deletion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 1. In some embodiments, an Fc domain modification is an amino acid insertion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 1. The Fc domain modification can be a combination of multiple modifications, for example, the modification can comprise amino acid substitutions, deletions, and/or insertions.
(34) Table 2 lists exemplary amino acid modifications that alter Fc domain binding affinity to Fc receptors. A modified Fc domain may include one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more of the modifications listed in Table 2. In addition, modifications in Table 2 may be combined with modifications of any one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more of the residues listed in Table 1.
(35) An Fc domain modification may increase the affinity of a modified Fc domain binding to one (e.g., two, three, four, five, or six) or more Fc receptors by at least 1×, (e.g., 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, 300×, 400×, 500×), as compared to an unmodified Fc domain. In some embodiments, an Fc domain modification increases binding affinity to an Fcγ receptor (e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and/or FcγRIIIB (CD16b)) and/or to the neonatal Fc receptor (FcRn). In some embodiments, an Fc domain modification increases binding affinity to FcγRIIIA (CD16a).
(36) An Fc domain modification may decrease the affinity of a modified Fc domain binding to one (e.g., two, three, four, five, or six) or more Fc receptors by at least 1×, (e.g., 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, 300×, 400×, 500×), as compared to an unmodified Fc domain. In some embodiments, an Fc domain modification decreases binding affinity to an Fcγ receptor (e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and/or FcγRIIIB (CD16b)) and/or to the neonatal Fc receptor (FcRn). In some embodiments, an Fc domain modification decreases binding affinity to FcγRIIB (CD32). In some embodiments, an Fc domain modification decreases binding affinity to FcRn.
(37) Exemplary Fc domains with altered binding affinity to Fc receptors include Fc monomers containing the double mutants S267E/L328F. S267E/L328F mutations have been previously shown to significantly and specifically enhance IgG1 binding to the FcγRIIb receptor (Chu et al. Molecular Immunology. 2008 September; 45(15):3926-33).
(38) TABLE-US-00001 TABLE 1 Fc domain residues that may be modified to alter Fc receptor binding affinity Fc Domain Residues 92 172 240 272 304 336 376 413 93 173 241 273 305 337 377 414 94 174 242 274 306 338 378 415 95 175 243 275 307 339 379 416 96 196 244 276 308 340 380 417 97 199 245 277 309 341 382 418 98 203 246 278 310 343 383 419 99 206 247 279 311 344 384 420 100 211 248 280 312 345 385 421 101 214 249 281 313 346 386 422 102 217 250 282 314 350 387 424 103 219 251 283 315 351 388 426 113 220 252 284 316 352 389 427 114 221 253 285 317 353 390 428 115 222 254 286 318 354 391 429 116 223 255 287 319 355 392 430 118 224 256 288 320 356 393 431 131 225 257 289 321 357 394 432 133 226 258 290 322 358 396 433 137 227 259 291 323 359 397 434 138 228 260 292 324 360 398 435 139 229 261 293 325 361 399 436 140 230 262 294 326 362 400 437 141 231 263 295 327 363 401 438 142 232 264 296 328 365 402 439 143 233 265 297 329 366 404 440 144 234 266 298 330 367 405 441 145 235 267 299 331 369 408 442 146 236 268 300 332 370 409 445 169 237 269 301 333 372 410 446 170 238 270 302 334 373 411 447 171 239 271 303 335 374 412
(39) TABLE-US-00002 TABLE 2 Fc domain modifications altering Fc receptor binding affinity Amino Acid Modifications 100ins + 250; 291 322; 299 A63A5: A85A5: A5: A42 101ins 250; 299 323; 275 102ins 250T; 251L; 323; 281 252M; 253I; 254S 103ins 250X; 314X 323; 284 137ins 250X; 314X; 323; 291 428X 138ins 250X; 428X 323; 299 139ins 252Y; 254T; 324; 275 256E; 433K; 434F; 140ins 252Y; 254T; 324; 281 256E; 433K; 434F; 436Y 141ins 252Y; 254T; 324; 284 256E; 433K; 434F; 446del 142ins 252Y; 428L 324; 291 143ins 252Y; 434S 324; 299 144ins 255L; 396L 325; 275 145ins 256T; 257P 325; 281 146ins 258; 275 325; 284 169ins 258; 281 325; 291 170ins 258; 284 325; 299 171ins 258; 291 325S; 326A; 327A; 328F 172ins 258; 299 325S; 326K; 327A; 328F 173ins 259I; 308F 326; 275 174ins 259I; 308F 326; 281 175ins 259I; 308F; 326; 284 428L 235insA 262; 275 326; 291 235insD 262; 281 326; 299 235insG 262; 284 326A; I332E; 333A 235insL 262; 291 326A; 333A 235insN 262; 299 327; 275 235insS 263; 275 327; 281 235insT 263; 281 327; 284 235insV 263; 284 327; 291 254insN 263; 291 327; 299 281insA 263; 299 328; 275 281insD 264; 275 328; 281 281insS 264; 281 328; 284 281insT 264; 284 328; 291 297insA 264; 291 328; 299 297insD 264; 299 328D; 332E 297insG 264E; 297D; 328E; 332E 332E 297insS 264I; 298A; 328H; 332E 332E 326insA 264I; 330L; 328I; 332E 332E 326insD 264I; 330Y; 328M; 332E 332E 326insE 2641; 332E 328N; 332E 326insG 265; 275 328Q; 332E 326insT 265; 281 328R; 236insR 92ins 265; 284 328R; 236R 93ins 265; 291 328T; 332E 94ins 265; 299 328V; 332E 95ins 265A; 269A 329; 275 96ins 265F; 297E; 329; 281 332E 97ins 265X; 269X 329; 284 98ins 265X; 270X 329; 291 99ins 265X; 297X 329; 299 281insA 265X; 327X 330; 275 281insD 265Y; 297D; 330; 281 299L; 332E 281insS 265Y; 297D; 330; 284 332E 281insT 266; 275 330; 291 131C; 133R; 266; 281 330; 299 137E; 138S; 196K; 199T; 203D; 214R; 217S; 219Y; 220G; 221del; 222del; 223del; 224P; 233D; 237D; 238D; 268D; 271G; 296D; 330R; 439E 221; 275 266; 284 330A; 331P; 339T 221; 281 266; 291 330L; 332E 221; 284 266; 299 330Y; 332E 221; 291 267; 275 331; 275 221; 299 267; 281 331; 281 221del; 267; 284 331; 284 222del; 223del; 224del; 225del; 252Y; 254T; 256E; 433K; 434F; 446del 221E; 270E; 267; 291 331; 291 308A; 311H; 396L; 402D 222; 275 267; 299 331; 299 222; 281 267; 328 332; 275 222; 284 267E; 268F; 332; 281 324T; I332E 222; 291 267E; 268F; 332; 284 324T; 332E 222; 299 267E; 328F 332; 291 223; 275 267E; 332E 332; 299 223; 281 267L; 327S 332X; 221X 223; 284 267Q; 327S 332X; 222X 223; 291 268; 275 332X; 223X 223; 299 268; 281 332X; 224X 224; 275 268; 284 332X; 227X 224; 281 268; 291 332X; 228X 224; 284 268; 299 332X; 230X 224; 291 268F; 324T; 332X; 231X I332E 224; 299 268F; 324T; 332X; 233X 332E 227; 275 268H; 355R; 332X; 234X 419Q; 434N 227; 281 268H; 355R; 332X; 235X 419Q; 434N; 131C; 133R; 137E; 138S; 220C 227; 284 268N; 396L 332X; 236X 227; 291 268P; 294K; 332X; 237X 361S; 382V; 428L 227; 299 268Q; 309L; 332X; 238X 330S; 331S 228; 275 269; 275 332X; 239X 228; 281 269; 281 332X; 240X 228; 284 269; 284 332X; 241X 228; 291 269; 291 332X; 243X 228; 299 269; 299 332X; 244X 230; 275 269X; 270X 332X; 245X 230; 281 269X; 297X 332X; 246X 230; 284 269X; 327X 332X; 247X 230; 291 270 332X; 249X 230; 299 270E 332X; 250X 231; 275 270; 275 332X; 258X 231; 281 270; 281 332X; 262X 231; 284 270; 284 332X; 263X 231; 291 270; 291 332X; 264X 231; 299 270; 299 332X; 265X 233; 275 270X; 297X 332X; 266X 233; 281 270X; 327X 332X; 268X 233; 284 271; 275 332X; 269X 233; 291 271; 281 332X; 270X 233; 299 271; 284 332X; 271X 233D; 271; 291 332X; 272X 237D; 238D; 264I; 267A; 268E; 271G; 272D; 296D; 439E 233D; 271; 299 332X; 273X 237D; 238D; 264I; 267A; 268E; 271G; 272P; 330R; 439E 233D; 272; 275 332X; 274X 237D; 238D; 264I; 267A; 268E; 271G; 296D; 327G; 330R; 396M; 439E 233D; 272; 281 332X; 275X 237D; 238D; 264I; 267A; 268E; 271G; 296D; 330R; 396L; 439E 233D; 272; 284 332X; 276X 237D; 238D; 264I; 267A; 268E; 271G; 296D; 330R; 396M; 439E 233D; 272; 291 332X; 278X 237D; 238D; 264I; 267A; 268E; 271G; 296D; 330R; 439E 233D; 272; 299 332X; 280X 237D; 238D; 264I; 267A; 268E; 271G; 330R; 396L; 439E 233D; 273; 275 332X; 281X 237D; 238D; 264I; 267A; 268E; 271G; 330R; 396M; 439E 233D; 237D; 273; 281 332X; 283X 238D; 264I; 267A; 268E; 271G; 330R; 439E 233D; 237D; 273; 284 332X; 285X 238D; 264I; 267A; 268E; 271G; 439E 233D; 237D; 273; 291 332X; 286X 238D; 264I; 267G; 268E; 271G; 330R; 439E 233D; 237D; 273; 299 332X; 288X 238D; 267A; 268E; 271G; 296D; 330R; 332T; 439E 233D; 237D; 274; 275 332X; 290X 238D; 268D; 271G; 296D; 327G; 330R; 439E 233D; 237D; 274; 281 332X; 291X 238D; 268D; 271G; 296D; 330R; 332T; 439E 233D; 237D; 274; 284 332X; 293X 238D; 268D; 271G; 296D; 330R; 439E 233D; 238D; 274; 291 332X; 294X 264I; 267A; 268E; 271G 233D; 238D; 274; 299 332X; 295X 264I; 267A; 268E; 271G; 296D; 439E 233P; 234A; 275; 275 332X; 296X 235A; 237A; 238P 233P; 234A; 275; 281 332X; 297X 235A; 237A; 238S 233P; 234V; 275; 284 332X; 298X 235A; 236del 233S; 234A; 275; 291 332X; 299X 235A; 237A; 238S 234; 275 275; 299 332X; 300X 234; 281 276; 275 332X; 302X 234; 284 276; 281 332X; 313X 234; 291 276; 284 332X; 317X 234; 299 276; 291 332X; 318X 234A; 237A; 276; 299 332X; 320X 238S; 268A; 309L; 330S; 331S 234F; 235L; 278; 275 332X; 322X 409R 234L; 235L; 278; 281 332X; 323X 297N 234L; 235L; 278; 284 332X; 324X 297N; 327A; 330A; 331P 234L; 235L; 278; 291 332X; 325X 327A; 330A; 331P 234L; 235L; 278; 299 332X; 326X 327A; 330A; 331P; 268H; 274L; 355R; 356D; 358L; 419Q 234L; 235L; 280; 275 332X; 327X 327A; 330A; 331P; 434N 234V; 237G; 280; 281 332X; 328X 297N 234V; 237G; 280; 284 332X; 329X 330A 234V; 237G; 280; 291 332X; 330X 330A; 331P; 339T; 297N 235; 275 280; 299 332X; 331X 235; 281 281; 275 332X; 333X 235; 284 281; 281 332X; 334X 235; 291 281; 284 332X; 335X 235; 299 281; 291 332X; 336X 236; 275 281; 299 332X; 428X 236; 281 283; 275 333; 275 236; 284 283; 281 333; 281 236; 291 283; 284 333; 284 236; 299 283; 291 333; 291 236A; 239D 283; 299 333; 299 236N; 267E 284M; 298N; 334; 275 334E; 355W; 416T 236S; 239D 285; 275 334; 281 237; 275 285; 281 334; 284 237; 281 285; 284 334; 291 237; 284 285; 291 334; 299 237; 291 285; 299 334E; 292L 237; 299 286; 275 335; 275 237A; 239D; 286; 281 335; 281 I332E 237A; 239D; 286; 284 335; 284 332E 237D; 238D; 286; 291 335; 291 264I; 267A; 268E; 271G; 272P; 296D; 330R; 439E 237D; 238D; 286; 299 335; 299 264I; 267A; 268E; 271G; 296D; 330R; 439E 237D; 238D; 288; 275 336; 275 264I; 267A; 268E; 271G; 330R; 439E 237D; 238D; 288; 281 336; 281 267A; 268E; 271G; 296D; 330R; 332T; 439E 237D; 238D; 288; 284 336; 284 267A; 268E; 271G; 296D; 330R; 439E 237D; 238D; 288; 291 336; 291 267G; 268D; 271G; 296D; 330R; 439E 237D; 238D; 288; 299 336; 299 267G; 268E; 271G; 296D; 330R; 439E 237D; 238D; 288M; 334E 370E; 396L 268D; 271G; 296D; 330R; 439E 237D; 238D; 288N; 330S; 378T; 226G 268E; 271G; 396L 296D; 330R; 439E 237D; 239D; 290; 275 378T; 230L I332E 237D; 239D; 290; 281 378T; 230S 332E 237P; 239D; 290; 284 378T; 230T I332E 237P; 239D; 290; 291 378T; 241L 332E 237Q; 239D; 290; 299 378T; 264E I332E 237Q; 239D; 291; 275 378T; 307P 332E 237S; 239D; 291; 281 378T; 315D I332E 237S; 239D; 291; 284 378T; 330V 332E 238 291; 291 378T; 362R 238; 271 291; 299 378T; 389K 238; 275 292; 305 378T; 389T 238; 281 292P 378T; 434S 238; 284 292P; 305I 378T; 434Y 238; 291 293; 275 378T; P228L 238; 299 293; 281 378T; P228R 238; 271; 293; 284 378V; 226G 233; 330 238; 271; 293; 291 378V; 230L 237; 330 238; 271; 293; 299 378V; 230S 237; 268 238; 271; 293del; 378V; 230T 237; 268; 294del 330 238D 293V; 295E 378V; 241L 238D; 271G 294; 275 378V; 264E 238D; 271G; 294; 281 378V; 307P 233D; 330R 238D; 271G; 294; 284 378V; 315D 237D; 330R 238D; 271G; 294; 291 378V; 330V 237D; 268D 238D; 271G; 294; 299 378V; 362R 237D; 268D; 330R 238D; 264I; 295; 275 378V; 389K 267A; 268E; 271G 238D; 264I; 295; 281 378V; 389T 267A; 268E; 271G; 272D; 296D; 439E 238D; 264I; 295; 284 378V; 434S 267A; 268E; 271G; 296D; 439E 238D; 264I; 295; 291 378V; 434Y 267A; 268E; 271G; 439E 239; 275 295; 299 378V; P228L 239; 281 296; 275 378V; P228R 239; 284 296; 281 380X; 434X 239; 291 296; 284 382V; 263E 239; 299 296; 291 382V; 390D; 428L 239; 332 296; 299 392E; 382V; 397M; 428L 239; 330; 296D; 297D; 392E; 396L 332 332E 239D; I332E 296E; 297D; 392T; 396L 332E 239D; 264I; 296H; 297D; 428; 275 298A; 332E 332E 239D; 264I; 296N; 297D; 428; 281 330L; 332E 332E 239D; 264I; 296Q; 297D; 428; 284 332E 332E 239D; 265F; 296T; 297D; 428; 291 297D; 332E 332E 239D; 265H; 297; 275 428; 299 297D; 332E 239D; 265I; 297; 281 428L; 252X 297D; 332E 239D; 265L; 297; 284 428L; 308F 297D; 332E 239D; 265T; 297; 291 428L; 434S 297D; 332E 239D; 265V; 297; 299 428L; 434X 297D; 332E 239D; 265Y; 297D; 298A; 434S; 226G 297D; 332E 330Y; 332E 239D; 268F; 297D; 299E; 434S; 230L 324T; I332E 332E 239D; 268F; 297D; 299F; 434S; 230S 324T; 332E 332E 239D; 297D; 297D; 299H; 434S; 230T 332E 332E 239D; 298A; 297D; 299I; 434S; 241L 332E 332E 239D; 326A; 297D; 299L; 434S; 264E 333A 332E 239D; 330L; 297D; 299V; 434S; 307P 332E 332E 239D; 330Y; 297D; 330Y; 434S; 311I 332E 332E 239D; 332D 297D; 332E 434S; 311V 239D; 332E 297E; 332E 434S; 315D 239D; 332E; 297N; 298X; 434S; 330V 330L 299S 239D; 332N 297N; 298X; 434S; 362R 299T 239D; 332Q 297S; 332E 434S; 378T 239E; 264I; 297X; 327X 434S; 378V 298A; 330Y; 332E 239E; 264I; 298; 275 434S; 389K 330Y; 332E 239E; 264I; 298; 281 434S; 389T 332E 239E; 265G 298; 284 434S; 436I 239E; 265N 298; 291 434S; 436V 239E; 265Q 298; 299 434S; P228L 239E; 297D; 298A; 332E 434S; P228R 332E 239E; 332D 298G; 299A 434Y; 226G 239E; 332E 298X; 299X; 434Y; 230L 268X; 294X; 361X; 382X; 428X 239E; 332N 298X; 299X; 434Y; 230S 382X 239E; 332Q 298X; 299X; 434Y; 230T 382X; 263X 239N; 298A; 298X; 299X; 434Y; 241L 332E 382X; 390X; 428X 239N; 330L; 298X; 299X; 434Y; 264E 332E 392X; 382X; 397X; 428X 239N; 330Y; 298X; 333X; 434Y; 307P 332E 334X 239N; 332D 298X; 334X 434Y; 315D 239N; 332E 299; 275 434Y; 330V 239N; 332N 299; 281 434Y; 362R 239N; 332Q 299; 284 434Y; 378T 239Q; 264I; 299; 291 434Y; 378V 332E 239Q; 332D 299; 299 434Y; 389K 239Q; 332E 300; 275 434Y; 389T 239Q; 332N 300; 281 434Y; P228L 239Q; 332Q 300; 284 434Y; P228R 240; 275 300; 291 436I; 434S 240; 281 300; 299 436I; 428L 240; 284 302; 275 436I; 434S 240; 291 302; 281 436V; 428L 240; 299 302; 284 436V; 434S 241; 275 302; 291 A330S; P331S; T339A 241; 281 302; 299 D265Y; N297D; T299L; I332E 241; 284 304D; 290D F234A; L235A; R409L 241; 291 304D; 284D F241E; F243Q; V262T; V264E 241; 299 304D; 284E F241E; F243Q; V262T; V264E; I332E 241E; 243; 304D; 285D F241E; F243R; V262E; V264R R1262E; 264R; 332E 241E; 243Q; 304D; 285E F241E; F243R; V262E; V264R; I332E 262T; 264E 241E; 243Q; 304D; 286D F241E; F243Y; V262T; V264R 262T; 264E; 332E 241E; 243R; 304D; 286E F241E; F243Y; V262T; V264R; I332E 262E; 264R 241E; 243Y; 304D; 288D 262T; 264R 241E; 243Y; 304D; 288E F241L; F243L; V262I; V264I 262T; 264R; 332E 241L; 243L; 304D; 290E F241L; V262I 262I; 264I 241L; 262I 304D; 305D F241R; F243Q; V262T; V264R 241R; 243Q; 304D; 305E F241R; F243Q; V262T; V264R; I332E 262T; 264R 241R; 243Q; 304E; 284D F241W; F243W; V262A; V264A 262T; 264R; 332E 241W; 243W 304E; 284E F241Y; F243Y; V262T; V264T 241W; 243W; 304E; 285D F241Y; F243Y; V262T; V264T; N297D; 262A; 264A I332E 241Y; 243Y; 304E; 285E F243L; V262I; V264W 262T; 264T 241Y; 243Y; 304E; 286D R292P 262T; 264T; 297D; 332E 243; 275 304E; 286E F243L; R292P 243; 281 304E; 288D F243L; R292P; Y300L 243; 284 304E; 288E F243L; R292P; P396L 243; 291 304E; 290D H268G; R355Q; Q419E; N434A 243; 292 304E; 290E H268Q; R355Q; Q419E; N434A; C131S; R133K; E137G; S138G; C220S 243; 299 304E; 305D D270E 243; 292; 304E; 305E H433K; N434F 300 243; 292; 305I; 292P K326I; A327E; L328A 396 243I; 379L V305I; R292P K326I; A327Y; L328G 243I; 379L; 306L; 307T; L234A; L235A; A327G; A330S; 420V 308V; 309L; P331S 310H; 311Q; 312D 243L; 255L 307Q; 434S L234A; L235A; A327G; A330S; P331S; H268Q; K274Q; R355Q; D356E; L358M; Q419E 243L; 262I; 307X; 380X L234A; L235A; A327G; A330S; P331S; 264W N434A 243L; 264I 307X; 380X; L234A; L235A; N297A; 434X 243L; 292P 307X; 434X L234A; L235A; N297A; A327G; A330S; P331S 243L; 292P; 311I; 434S L234A; L235D; A327G; A330S; P331S 300L 243L; 292P; 311V; 434S K267E; L328F 396L 243L; 292P; 313; 275 S267E; L328F 300L; 305I; 396L 243L; 292P; 313; 281 N297D; S298A 300L; 396L 243L; 305I; 313; 284 N297D; S298T 378D; 404S; 396L 244; 275 313; 291 N297D; T299E; I332E 244; 281 313; 299 N297D; T299F; I332E 244; 284 314L; 315N; N297D; T299H; I332E 316G 244; 291 314X; 428X N297D; T299I; I332E 244; 299 315D; 382V; N297D; T299V; I332E 428L 244H; 315I; 379M; N297H; S298A 245A; 399E 247V 245; 275 316D; 378V; N315D; A330V; A378V; N434Y 399E 245; 281 317; 275 N315D; A330V; N361D; A378V; N434Y 245; 284 317; 281 N315D; A378V; N434Y 245; 291 317; 284 N315D; K334E; A378V; N434Y 245; 299 317; 291 P113E; V114L; A115L; InG115/116; S118D; G206A; I211E 246; 275 317; 299 P228L; N315D; A330V; N361D; A378V; N434Y 246; 281 318; 275 P228L; P230S; N315D; A330V; N361D; A378V; N434Y 246; 284 318; 281 P228R; N315D; A330V; N361D; A378V; N434Y 246; 291 318; 284 P228R; P230S; N315D; A330V; N361D; A378V; N434Y 246; 299 318; 291 P230A; E233D 247; 275 318; 299 P230A; E233D; I332E 247; 281 319F; P230S; N315D; A330V; N361D; A378V; 352L; N434Y 396L 247; 284 320; 275 P230T; V264E; N315D; K370R; A378V 247; 291 320; 281 P244H; P245A; P247V 247; 299 320; 284 S239D; I332E 248M; 320; 291 S239D; A330L; I332E 247L; 420V 249; 275 320; 299 S239D; N297D; I332E; A330Y; F241S; F243H; V262T; V264T 249; 281 322; 275 V234A; G237A; A330S; P331S; T339A 249; 284 322; 281 V234A; G237A; A330S; P331S; T339A; N297A 249; 291 322; 284 V234A; G237A; N297A 249; 299 322; 291 P238D 250; 275 252Y; P238D; P271G 254T; 256E 250; 281 V264E; P238D; P271G; E233D; A330R N315D; A378V; N390S; G420R; N434Y 250; 284 V264E; P238D; P271G; G237D; A330R N315D; A378V R292P; P238D; P271G; G237D; H268D V305I P238D; P271G; G237D; H268D; A330R ins = insertion del = deletion X = any amino acid
Half-Life
(40) Fc domain modifications that alter half-life may alter the binding of a modified Fc domain to FcRn, for example, by altering the affinity of the interaction at pH 6.0 and/or pH 7.4. Amino acid modifications that alter half-life may alter the pH dependence of the binding of and Fc domain to the FcRn receptor. Table 3 lists exemplary Fc domain residues that may be modified to alter the half-life (e.g., serum half-life) of Fc domains. In some embodiments, one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 3 may be modified, wherein the modified Fc domain has an altered half-life as compared to an unmodified Fc domain. In some embodiments, the Fc domain modification is an amino acid substitution occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 3. In some embodiments, an Fc domain modification is an amino acid deletion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 3. In some embodiments, an Fc domain modification is an amino acid insertion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 3. The Fc domain modification can be a combination of multiple modifications, for example, the modification can comprise amino acid substitutions, deletions, and/or insertions.
(41) Table 4 lists exemplary modifications that alter Fc domain half-life. A modified Fc domain may include one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more of the modifications listed in Table 4. In addition, modifications in Table 4 may be combined with modifications of residue positions listed in Table 3.
(42) In some embodiments, an Fc domain modification may increase the half-life of a modified Fc domain at least 0.5×, (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×), as compared to an unmodified Fc domain.
(43) In some embodiments, an Fc domain modification may decrease the half-life of a modified Fc domain by at least 0.5×, (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×), as compared to an unmodified Fc domain.
(44) TABLE-US-00003 TABLE 3 Fc domain residues that may be modified to alter half-life Fc Domain Residues 92 141 248 282 314 380 436 93 142 249 284 315 383 438 94 143 250 285 316 385 446 95 144 251 286 317 386 447 96 145 252 287 322 387 97 146 253 288 330 388 98 169 254 289 331 389 99 170 255 290 335 419 100 171 256 304 339 424 101 172 257 305 340 426 102 173 258 306 343 428 103 174 259 307 344 429 131 175 260 308 345 430 133 219 268 309 346 431 137 220 277 310 355 432 138 234 279 311 374 433 139 235 280 312 376 434 140 236 281 313 378 435
(45) TABLE-US-00004 TABLE 4 Fc domain modifications altering half-life Amino Acid Modifications 259I; 308F 258N 345T 434S; 311I 258Q 345N 434S; 311V 279R 345Q 434S; 436I 279H 376S 434S; 436V 279K 376T H433K; N434F 279D 376N 428L; 434S 279E 376Q C131X; R133X; C220X; E137X; S138X; 280R 376R H268X; R355X; Q419X C131X; R133X; C220X; E137X; S138X; 280H 376H H268X; R355X; Q419X; G446del; 447Kdel C131X; R133X; C220X; E137X; S138X; 280D 376K H268X; R355X; Q419X; A330X; P331X; T339X C131X; R133X; C220X; E137X; S138X; 280E 376D H268X; R355X; Q419X; A330X; P331X; T339X; G446del; K447del C219X; C220X 281R 376E C219X; C220X; H268X; R335X; Q419X 281H 378S C219X; C220X; H268X; R335X; Q419X; 281K 383R G446del; K447del C219X; C220X; G446del; K447del 281D 383H C219S; C220S 281E 383K C219X; C220X; H268X; R355X; Q419X 282R 383D C219S; C220S; H268X; R355X; Q419X 282H 383E C219S; C220S; H268Q; R355Q; Q419E 282D 385R C219X; C220X; H268X; R355X; Q419X; 282E 385H G446del; K447del C219S; C220S; G446del; K447del 284S 385K C219S; C220S; H268X; R355X; Q419X; 284T 385D G446del; K447del C219S; C220S; H268Q; R355Q; Q419E; 284N 385E G446del; K447del 428L 284Q 389D 434S 284R 389E 251del 284H 424R 253del 284D 424H 255del 284E 424K 285del 285R 424D 286del 285H 424E 287del 285K 426R 288del 285D 426H 289del 285S 426K 290del 285T 426D 308del 285N 426E 309del 285Q 430R 310del 286E 430H 322del 286T 430D 312del 286M 430E 313del 287S 430S 314del 287T 430T 385del 287N 430N 386del 287Q 430Q 387del 287R 431R 388del 287H 431H 389del 287K 431K 428del 287D 431D 429del 287E 431E 430del 288R 432S 431del 288H 432T 432del 288K 432N 433del 288D 432Q 434del 288E 434K 435del 304S 434R 436del 304T 434L 251ins 304N 436D 253ins 304Q 436E 255ins 304R 438R 285ins 304H 438H 286ins 304K 438K 287ins 304D 438D 288ins 304E 438E 289ins 305S 285E 290ins 305T 286D 308ins 305N 290E 309ins 305Q 250R 310ins 305R 250K 322ins 305H 251R 312ins 305K 251K 313ins 305D 254S 314ins 305E 255L 385ins 307S 255D 386ins 307T 255M 387ins 307N 260K 388ins 307R 257K 389ins 307H 277R 428ins 307K 277D 429ins 307D 277Q 430ins 307E 277K 431ins 308R 281Q 432ins 308H 282K 433ins 308K 287P 434ins 308D 285F 435ins 308E 290D 436ins 309R 306R 435L 309H 306D 252Y; 428L 309K 306E 252Y; 434S 309D 306K 428L; 252X 309E 310L 428L; 434X 310R 374R 433K; 434F; 436H 310H 374K 255V 310K 374L 309N 310D 428R 312I 310E 428Q 386L 310S 428K 252Y 310T 431P 252F 310N 432R 252S 310Q 308F 252W 311R 259I 252T 311H 259I; 308F 254T 311K 436I; 428L 256S 312R 4361; 434S 256R 312H 436V; 434S 256Q 312K 436V; 428L 256E 312S 259I; 308F; 428L 256D 312T 436I; 434S 309P 312N 252Y; 254T; 256E 311S 312Q 308C 311E 313R 308Y 311L 313H 308W 433R 313K 428L; 308F 433S 313D 308P; 434A 433I 313E 234F 433P 315R 235A 433Q 315H 235N 434H 315K 235F 434F 315D 235Q 434Y 315E 235V 251D 316R 322A 251E 316H 322D 307Q 316K 322E 308P 317R 322H 378V 317H 322N 430A 317K 322Q 430K 317D 331A 434A 317E 331G 436I 317S 92ins 380A 317T 93ins 250E 317N 94ins 250Q 317Q 95ins 428F 340R 96ins 248R 340H 97ins 248H 340K 98ins 248K 340D 99ins 248D 340E 100ins 248E 343R 101ins 249R 343H 102ins 249K 343K 103ins 251S 343D 137ins 251T 343E 138ins 251N 343S 139ins 251Q 343T 140ins 252N 343N 141ins 252Q 343Q 142ins 255S 344L 143ins 255T 345R 144ins 255N 345H 145ins 255Q 345K 146ins 256K 345D 169ins 257R 345E 170ins 257H 345S 171ins 257D 258T 172ins 257E 175ins 173ins 258S 174ins 252Y; 254T; 256E; 433K; 434F; 436Y ins = insertion del = deletion X = any amino acid
Effector Function
(46) Table 5 lists exemplary Fc domain residues that may be modified to alter Fc domain effector function (e.g., cell lysis (e.g., antibody-dependent cell-mediated toxicity (ADCC) and/or complement dependent cytotoxicity activity (CDC)), phagocytosis (e.g., antibody dependent cellular phagocytosis (ADCP) and/or complement-dependent cellular cytotoxicity (CDCC)), immune activation, and T-cell activation). In some embodiments, one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 5 may be modified, wherein the modified Fc domain has an altered effector function (e.g., ADCC, CDC, ADCP, CDCC, immune activation, and/or T-cell activation) as compared to an unmodified Fc domain. In some embodiments, the Fc domain modification is an amino acid substitution occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 5. In some embodiments, an Fc domain modification is an amino acid deletion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 5. In some embodiments, an Fc modification is an amino acid insertion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 5.
(47) Table 6 lists exemplary modifications that alter Fc domain effector function (e.g., ADCC, CDC, ADCP, CDCC, immune activation, and/or T-cell activation). A modified Fc domain may include one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more of the modifications listed in Table 6. In addition, modifications in Table 6 may be combined with modifications of residues listed in Table 5.
(48) An Fc domain modification may increase the effector function (e.g., ADCC, CDC, ADCP, CDCC, immune activation, and/or T-cell activation) of a modified Fc domain at least 0.5×, (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×), as compared to an unmodified Fc domain.
(49) An Fc domain modification may decrease the effector functions (e.g., cell lysis (e.g., ADCC, CDC, ADCP, CDCC, immune activation, and/or T-cell activation) of a modified Fc domain by at least 0.5×, (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×), as compared to an unmodified Fc domain.
(50) TABLE-US-00005 TABLE 5 Fc domain residues that may be modified to alter effector function Fc Domain Residues 40 264 297 332 390 436 224 265 298 333 391 439 225 266 299 334 393 440 228 267 300 339 394 441 230 268 301 345 399 442 234 269 304 355 404 445 235 273 305 356 408 446 236 276 306 358 409 447 239 278 307 359 411 240 279 309 361 412 241 280 311 362 414 243 283 312 365 421 244 285 315 370 422 246 288 320 372 426 247 289 322 376 427 251 290 323 377 428 252 291 324 378 430 253 292 325 382 431 254 293 328 383 432 258 294 329 386 433 261 295 330 388 434 262 296 331 389 435
(51) TABLE-US-00006 TABLE 6 Fc domain modifications altering effector function Amino Acid Modifications 224N 301H 411I 224Y 301K 412A 225A 301N 414M 228L 301Q 421S 228P; 235E 301R 422I 230S 301S 426F 234A; 235A 301T 426P 234F 304G 427F 235A 305A 428T 235F 306F 430K 235N 306I 431S 235Q 307P 432P 235V 309K 433P 236A 309M 435A 236R; 328R 309P 435G 239D; 378F 311R 435N 239D; 378G 312N 435Q 239D; 378S 315D 435S 239D; 378W 315K 435T 239D; 378Y 315S 435Y 239E; 378F 320R 439E 239E; 378G 322A 439R 239E; 378S 322D 440G 239E; 378W 322E 441F 239E; 378Y 322H 442T 239P 322N 445R 240A 322Q 446A 241H 323A 447E 241L 323F A330H 241Q 324T A330L 243G 325A A378F 243H 328D A378K 243I 328G A378T 243L 328K A378W 244L 328T D265E 246E 329G D356 247A 329R E293D 247L 330H E294S 251A 330L E294T 251F 331A E345 251G 331G E356 251I 332D E382 251L 332D; 261A E430 251M 332D; 378F F241H 251P 332D; 378K F241Q 251S 332D; 378W G236A 251V 332D; 378Y H435A 251W 332D; 435G H435G 252S 332D; 435S H435S 252T 332E I253 252W 332K I332D 252Y 332Q I332E 254P 333X; 334X I332K 254T 334R I332Q 258K 355W K334L 261A 356G K334R 261Y 356W K439D; S440H 262L 358T K439D; S440K 264T 361D K439D; S440R 265D 361Y K439E; S440K 265E 362L K447 265V 364C L251A 266A 365P L251G 266F 365Q L261A 267G 370R L268P 267N 372L N376F 268D 376C N376H 268E 376D N376K 268N 376E N376R 268P 376F N376W 269G 376H N434A 269K 376K N434F 273A 376N N434H 276D 376Q N434W 278H 376R N434Y 279M 376S P247 280N 376T Q311 283G 376W Q386 285R 376Y R301K 288R 377V R301N 289A 378D R301Q 290E 378E R301S 291L 378F R301T 292Q 378H S239P 293C 378K S254 293D 378Q S440W 294C 378R S440Y 294R 378T T299A; 297Z 294S 378W T299C; 297Z 294T 378Y T299C; N297Z 295C 383N T299D; 297Z 296C 389S T299E; 297Z 297C 390D T299F; 297Z 297D 391C T299G; 297Z 297G; 356E; 358M 393A T299H; 297Z 297Q 394A T299I; 297Z 298C 399G T299K; 297Z 298N; 300S 399S T299L; 297Z 298N; 300T 404S T299M; 297Z 298X; 333X 408G T299N; 297Z 298X; 334X 409R T299P; 297Z 299A 40F T299R; 297Z 299K 301E T299V; 297Z 300C Y436 T299W; 297Z 300H T359 T299X; N297Z 301C 301D T299Y; 297Z ins = insertion del = deletion X = any amino acid
Alters Stability
(52) Altering Fc domain stability can impact thermal stability (e.g., a melting temperature or Tm) and aggregate formation (e.g., aggregate formation under acidic, or low-pH, conditions).
(53) In some embodiments, the thermal stability of a modified Fc domain may be altered (i.e., increased or decreased) by at least about 0.1° C. (e.g., about 0.25° C., about 0.5° C., about 0.75° C., about 1° C., about 1.25° C., about 1.5° C., about 1.75° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 20° C., about 30° C., about 40° C., or about 50° C.) or as compared to an unmodified Fc domain. In some embodiments, the thermal stability of a modified Fc domain is increased as compared to an unmodified Fc domain. In some embodiments, the thermal stability of a modified Fc domain is decreased as compared to an unmodified Fc domain. In certain embodiments, a modified Fc domain has altered (i.e., increased or decreased) aggregation properties of at least 1% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or more as compared to an unmodified Fc domain. In some embodiments, the aggregation properties of a modified Fc domain are increased as compared to an unmodified Fc domain. In some embodiments, the aggregation properties are decreased as compared to an unmodified Fc domain.
(54) Table 7 lists exemplary Fc domain residues that may be modified to alter Fc domain stability. In some embodiments, one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 7 may be modified, wherein the modified Fc domain has an altered stability as compared to an unmodified Fc domain. In some embodiments, the Fc domain modification is an amino acid substitution occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 7. In some embodiments, an Fc domain modification is an amino acid deletion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 7. In some embodiments, an Fc domain modification is an amino acid insertion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues listed in Table 7.
(55) Table 8 lists exemplary modifications that alter Fc domain stability. A modified Fc domain may include one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more of the modifications listed in Table 8. In addition, modifications in Table 8 may be combined with modifications of residues listed in Table 7.
(56) An Fc domain modification may increase the stability of a modified Fc domain at least 0.5×, (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×), as compared to an unmodified Fc domain.
(57) An Fc domain modification may decrease the stability of a modified Fc domain by at least 0.5×, (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×), as compared to an unmodified Fc domain.
(58) TABLE-US-00007 TABLE 7 Fc domain residues that may be modified to alter stability Fc Domain Residues 40 252 300 354 368 402 217 262 307 355 370 403 219 264 309 356 392 404 225 266 322 357 394 405 228 267 323 358 395 407 232 273 331 359 396 409 234 275 339 360 397 427 235 277 349 361 398 236 279 351 362 399 238 297 352 364 400 250 299 353 366 401
(59) TABLE-US-00008 TABLE 8 Fc domain modifications altering stability Amino Acid Modifications 217R 228P; 235E 273R 359del 235Q 309L 219N 228P; 235E; 409K 273Y 360del 235V 309M 219Q 228P; 235E; 409L 275F 361del 236S 309P 225I 228P; 235E; 409M 275K 362del 236T 322A 225T 228P; 235E; 409T 275Q 392K 238R 322D 225V 228P; 235P 277E 397del 250E 322E 228E 228P; 235P; 409K 279D 397V 250Q 322H 228E; 228P; 235P; 409L 279N 398del 252S 322N 235E 228E; 228P; 235P; 409M 279V 399del 252T 322Q 235E; 409K 228E; 228P; 235P; 409T 297D 399S 252W 323F 235E; 409L 228E; 232K 297G; 400del 252Y 331A 235E; 356E; 409M 358M 228E; 232R 297Q 401del 262L 331G 235E; 409T 228E; 234F 299K 402del 264T 339A 235P 228E; 234K 300Y 403del 266F 354del 235P; 409K 228E; 234N 307P 404del 267S 355del 235P; 409L 228E; 234R 309K 409K 267T 356del 235P; 409M 228E; 235A 427F 409L 273K 357del 235P; 409T 228P 235E 235P 409M 273Q 235N 235F 409T 40F 358del ins = insertion del = deletion X = any amino acid
Alters Susceptibility to Degradation
(60) Susceptibility to degradation can impact how an Fc domain containing molecule can be stored and transported. Reducing an Fc domain's susceptibility to environmental conditions (e.g., temperature, humidity, pH), such as temperature, can make an Fc domain comprising molecule more readily transportable and/or storable over longer periods of time. Exemplary Fc residues that may be modified to alter Fc domain susceptibility to degradation include 233, 234, 235, 236, 237, 239, 241, and 249. In some embodiments, one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) residues selected from the group consisting of residues 233, 234, 235, 236, 237, 239, 241, and 249 may be modified, wherein the modified Fc domain has an altered susceptibility to degradation as compared to an unmodified Fc domain. In some embodiments, the Fc modification is an amino acid substitution occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) residue positions selected from the groups consisting of 233, 234, 235, 236, 237, 239, 241, and 249. In some embodiments, an Fc modification is an amino acid deletion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) residue positions selected from the group consisting of 233, 234, 235, 236, 237, 239, 241, and 249. In some embodiments, an Fc modification is an amino acid insertion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) residue positions selected from the group consisting of 233, 234, 235, 236, 237, 239, 241, and 249.
(61) Exemplary modifications that alter Fc domain susceptibility to degradation may include one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) modifications selected from the group consisting of C233X, D234X, K235X, S236X, T236X, H237X, C239X, S241X, and G249X, in which X is any amino acid.
(62) An Fc domain modification may decrease the degradation of a modified Fc domain by at least 1%, (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or more as compared to an unmodified Fc domain. In some embodiments, an Fc domain modification may decrease the degradation of a modified Fc domain upon heating by at least 1%, (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or more as compared to an unmodified Fc domain. In some embodiments, the Fc domain is heated over a period of at least one hour (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week) or more. In some embodiments, the temperature to which an Fc domain is heated is at least 45° C. (e.g., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 85° C., or 95° C.) or higher. The level of degradation a modified Fc domain is susceptible to may be measured by assessing the degrees of aggregation, degradation, or fragmentation by methods known to those skilled in the art, including but not limited to reduced Capillary Gel Electrophoresis (rCGE), Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and high performance size exclusion chromatography (HPSEC).
(63) Sulfation
(64) Exemplary Fc residues that may be modified to alter Fc domain sulfation include residues 241, 243, 246, 260, and 301. In some embodiments, one (e.g., 1, 2, 3, 4, or 5) Fc domain residues selected from the group consisting of 241, 243, 246, 260, and 301 may be modified, wherein the modified Fc domain has altered sulfation as compared to an unmodified Fc domain. In some embodiments, the Fc domain modification is an amino acid substitution occurring at one (e.g., 1, 2, 3, 4, or 5) residues selected from the group consisting of residues 241, 243, 246, 260, and 301. In some embodiments, an Fc domain modification is an amino acid deletion occurring at one (e.g., 1, 2, 3, 4, or 5) residues selected from the group consisting of residues 241, 243, 246, 260, and/or 301. In some embodiments, an Fc modification is an amino acid insertion occurring at one (e.g., 1, 2, 3, 4, or 5) residue positions selected from the group consisting of Fc domain residues 241, 243, 246, 260, and 301.
(65) Exemplary modifications that alter Fc domain sulfation include 241F, 243F, 246K, 260T, and/or 301 R. A modified Fc domain may include one (e.g., 1, 2, 3, 4, or 5) modifications selected from the group consisting of 241F, 243F, 246K, 260T, and 301R. Any one of these modifications may be combined with additional modifications of residues 241, 243, 246, 260, and/or 301.
(66) An Fc domain modification may increase the sulfation of a modified Fc domain at least 0.5×, (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×), as compared to an unmodified Fc domain.
(67) An Fc domain modification may decrease the sulfation of a modified Fc domain by at least 0.5×, (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×), as compared to an unmodified Fc domain.
(68) Protease Resistance
(69) The Fc domain may be modified to increase protease resistance, for example, resistance to endosomal proteases, extracellular proteases (e.g., trypsin, chymotypsin, plasmin), digestive proteases (e.g., pepsin), serum proteases (e.g., clotting factors), proteases released from leukocytes (e.g., elastase and cathepsin G) and tissue-specific proteases (e.g., tumor-specific proteases (e.g. matrix metalloproteinases). Susceptibility to protease degradation can play an important role in regulating the half-life of an Fc domain, with increased susceptibility contributing to a shorter half-life and reduced susceptibility contributing to a longer half-life. To alter protease resistance, amino acid modifications of may be made within regions of the Fc domain that comprise or affect protease cleavage sites. Alternatively, amino acid modifications that alter the glycosylation state of the Fc domain may alter the protease resistance and/or susceptibility characteristics of an Fc domain.
(70) Exemplary Fc residues that may be modified to alter protease resistance comprise 233, 234, 235, 236, 237, 239, 243, 267, 268, 292, 300, 324, 326, 332, and 333. In some embodiments, one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) Fc domain residues selected from 233, 234, 235, 236, 237, 239, 243, 267, 268, 292, 300, 324, 326, 332, and 333 may be modified, wherein the modified Fc domain has an altered protease resistance as compared to an unmodified Fc domain. An Fc domain modification may be an amino acid substitution occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) residues selected from residues 233, 234, 235, 236, 237, 239, 243, 267, 268, 292, 300, 324, 326, 332, and 333. An Fc modification may also be an amino acid deletion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16) residues selected from 233, 234, 235, 236, 237, 239, 243, 267, 268, 292, 300, 324, 326, 332, and 333. In some embodiments, an Fc modification is an amino acid insertion occurring at one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) residue positions selected from 233, 234, 235, 236, 237, 239, 243, 267, 268, 292, 300, 324, 326, 332, and 333. In some embodiments, the Fc domain modification may be a combination of any one of the above (e.g., a combination of an amino acid substitution, deletion, and/or insertion).
(71) Exemplary modifications that alter Fc domain protease resistance may comprise any one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the following 233P, 234V, 235A, and 236del; 237A, 239D, and 332E; 237D, 239D, and 332E; 237P, 239D, and 332E; 237Q, 239D, and 332E; 237S, 239D, and 332E; 239D, 268F, 324T, and 332E; 239D, 326A, and 333A; 239D and 332E; 243L, 292P, and 300L; 267E, 268F, 324T, and 332E; 267E and 332E; 268F, 324T, and 332E; 326A, 332E, and 333A; and 326A and 333A.
(72) An Fc domain modification may increase the protease resistance of a modified Fc domain at least 1%, (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or more, as compared to an unmodified Fc domain.
(73) IV. Dimerization Selectivity Modules
(74) In the present disclosure, a dimerization selectivity module is the part of the Fc domain monomer that facilitates the preferred pairing of two Fc domain monomers to form an Fc domain. Specifically, a dimerization selectivity module is that part of the C.sub.H3 antibody constant domain of an Fc domain monomer which includes amino acid substitutions positioned at the interface between interacting C.sub.H3 antibody constant domains of two Fc monomers. In a dimerization selectivity module, the amino acid substitutions make favorable the dimerization of the two C.sub.H3 antibody constant domains as a result of the compatibility of amino acids chosen for those substitutions. The ultimate formation of the favored Fc domain is selective over other Fc domains which form from Fc domain monomers lacking dimerization selectivity modules or with incompatible amino acid substitutions in the dimerization selectivity modules. This type of amino acid substitution can be made using conventional molecular cloning techniques well-known in the art, such as QuikChange® mutagenesis.
(75) In some embodiments, a dimerization selectivity module includes an engineered cavity (described further herein) in the C.sub.H3 antibody constant domain. In other embodiments, a dimerization selectivity module includes an engineered protuberance (described further herein) in the C.sub.H3 antibody constant domain. To selectively form an Fc domain, two Fc domain monomers with compatible dimerization selectivity modules, e.g., one C.sub.H3 antibody constant domain containing an engineered cavity and the other C.sub.H3 antibody constant domain containing an engineered protuberance, combine to form a protuberance-into-cavity pair of Fc domain monomers.
(76) In other embodiments, an Fc domain monomer with a dimerization selectivity module containing positively-charged amino acid substitutions and an Fc domain monomer with a dimerization selectivity module containing negatively-charged amino acid substitutions may selectively combine to form an Fc domain through the favorable electrostatic steering (described further herein) of the charged amino acids. Specific dimerization selectivity modules are further listed, without limitation, in Tables 1 and 2 described further below.
(77) In other embodiments, two Fc domain monomers include dimerization selectivity modules containing identical reverse charge mutations in at least two positions within the ring of charged residues at the interface between C.sub.H3 domains. By reversing the charge of both members of two or more complementary pairs of residues in the two Fc domain monomers, mutated Fc domain monomers remain complementary to Fc domain monomers of the same mutated sequence, but have a lower complementarity to Fc domain monomers without those mutations. In one embodiment, an Fc domain includes Fc monomers including the double mutants K409D/D339K, K392D/D399K, E357K/K370E, D356K/K439D, K409E/D339K, K392E/D399K, E357K/K370D, or D356K/K439E. In another embodiment, an Fc domain includes Fc monomers including quadruple mutants combining any pair of the double mutants, e.g., K409D/D399K/E357K/K370E.
(78) The formation of such Fc domains is promoted by the compatible amino acid substitutions in the C.sub.H3 antibody constant domains. Two dimerization selectivity modules containing incompatible amino acid substitutions, e.g., both containing engineered cavities, both containing engineered protuberances, or both containing the same charged amino acids at the C.sub.H3-C.sub.H3 interface, will not promote the formation of an Fc domain.
(79) Furthermore, other methods used to promote the formation of Fc domains with defined Fc domain monomers include, without limitation, the LUZ-Y approach (U.S. Patent Application Publication No. WO2011034605) which includes C-terminal fusion of a monomer α-helices of a leucine zipper to each of the Fc domain monomers to allow heterodimer formation, as well as strand-exchange engineered domain (SEED) body approach (Davis et al., Protein Eng Des Sel. 23:195-202, 2010) that generates Fc domain with heterodimeric Fc domain monomers each including alternating segments of IgA and IgG C.sub.H3 sequences.
(80) V. Engineered Cavities and Engineered Protuberances
(81) The use of engineered cavities and engineered protuberances (or the “knob-into-hole” strategy) is described by Carter and co-workers (Ridgway et al., Protein Eng. 9:617-612, 1996; Atwell et al., J Mol Biol. 270:26-35, 1997; Merchant et al., Nat Biotechnol. 16:677-681, 1998). The knob and hole interaction favors heterodimer formation, whereas the knob-knob and the hole-hole interaction hinder homodimer formation due to steric clash and deletion of favorable interactions. The “knob-into-hole” technique is also disclosed in U.S. Pat. No. 5,731,168.
(82) In the present disclosure, engineered cavities and engineered protuberances are used in the preparation of the Fc constructs described herein. An engineered cavity is a void that is created when an original amino acid in a protein is replaced with a different amino acid having a smaller side-chain volume. An engineered protuberance is a bump that is created when an original amino acid in a protein is replaced with a different amino acid having a larger side-chain volume. Specifically, the amino acid being replaced is in the C.sub.H3 antibody constant domain of an Fc domain monomer and is involved in the dimerization of two Fc domain monomers. In some embodiments, an engineered cavity in one C.sub.H3 antibody constant domain is created to accommodate an engineered protuberance in another C.sub.H3 antibody constant domain, such that both C.sub.H3 antibody constant domains act as dimerization selectivity modules (described above) that promote or favor the dimerization of the two Fc domain monomers. In other embodiments, an engineered cavity in one C.sub.H3 antibody constant domain is created to better accommodate an original amino acid in another C.sub.H3 antibody constant domain. In yet other embodiments, an engineered protuberance in one C.sub.H3 antibody constant domain is created to form additional interactions with original amino acids in another C.sub.H3 antibody constant domain.
(83) An engineered cavity can be constructed by replacing amino acids containing larger side chains such as tyrosine or tryptophan with amino acids containing smaller side chains such as alanine, valine, or threonine. Specifically, some dimerization selectivity modules (described further above) contain engineered cavities such as Y407V mutation in the C.sub.H3 antibody constant domain. Similarly, an engineered protuberance can be constructed by replacing amino acids containing smaller side chains with amino acids containing larger side chains. Specifically, some dimerization selectivity modules (described further above) contain engineered protuberances such as T366W mutation in the C.sub.H3 antibody constant domain. In the present disclosure, engineered cavities and engineered protuberances are also combined with inter-C.sub.H3 domain disulfide bond engineering to enhance heterodimer formation. Specifically, the cavity Fc contains an Y349C mutation, and the protuberance Fc contains an S354C mutation. Other engineered cavities and engineered protuberances, in combination with either disulfide bond engineering or structural calculations (mixed HA-TF) are included, without limitation, in Table 9.
(84) TABLE-US-00009 TABLE 9 CH.sub.3 CH.sub.3 antibody antibody constant constant domain domain of Fc of Fc domain domain Strategy monomer 1 monomer 2 Reference Engineered Y407T T366Y U.S. Pat. No. 8,216,805 cavities Y407A T366W U.S. Pat. No. 8,216,805 and pro- F405A T394W U.S. Pat. No. 8,216,805 tuberances Y407T T366Y U.S. Pat No. 8,216,805 (“knob- T394S F405W U.S. Pat. No. 8,216,805 into-hole”) T394W:Y407T T366Y:F405A U.S. Pat. No. 8,216,805 T394S:Y407A T366W:F405W U.S. Pat No. 8,216,805 T366W:T394S F405W:Y4074 U.S. Pat. No. 8,216,805 Engineered T366S:L368A: T366W:S354C Zeidler et al., cavities Y407V:Y349C J Immunol. and pro- 163: 1246- tuberances 52, 1999 (“knob- into-hole”), S-S engineering Mixed S364H:F405A Y349T:T394F WO2006106905 HA-TF
(85) Replacing an original amino acid residue in the C.sub.H3 antibody constant domain with a different amino acid residue can be achieved by altering the nucleic acid encoding the original amino acid residue. The upper limit for the number of original amino acid residues that can be replaced is the total number of residues in the interface of the C.sub.H3 antibody constant domains, given that sufficient interaction at the interface is still maintained.
(86) VI. Electrostatic Steering
(87) Electrostatic steering is the utilization of favorable electrostatic interactions between oppositely charged amino acids in peptides, protein domains, and proteins to control the formation of higher ordered protein molecules. A method of using electrostatic steering effects to alter the interaction of antibody domains to reduce for formation of homodimer in favor of heterodimer formation in the generation of bi-specific antibodies is disclosed in U.S. Patent Application Publication No. 2014-0024111.
(88) In the present disclosure, electrostatic steering is used to control the dimerization of Fc domain monomers and the formation of Fc constructs. In particular, to control the dimerization of Fc domain monomers using electrostatic steering, one or more amino acid residues that make up the C.sub.H3-C.sub.H3 interface are replaced with positively- or negatively-charged amino acid residues such that the interaction becomes electrostatically favorable or unfavorable depending on the specific charged amino acids introduced. In some embodiments, a positively-charged amino acid in the interface, such as lysine, arginine, or histidine, is replaced with a negatively-charged amino acid such as aspartic acid or glutamic acid. In other embodiments, a negatively-charged amino acid in the interface is replaced with a positively-charged amino acid. The charged amino acids may be introduced to one of the interacting C.sub.H3 antibody constant domains, or both. By introducing charged amino acids to the interacting C.sub.H3 antibody constant domains, dimerization selectivity modules (described further above) are created that can selectively form dimers of Fc domain monomers as controlled by the electrostatic steering effects resulting from the interaction between charged amino acids.
(89) In one particular example, to create a dimerization selectivity module including reversed charges, amino acid Asp399 in the C.sub.H3 antibody constant domain is replaced with Lys, and amino acid Lys409 is replaced with Asp. Heterodimerization of Fc domain monomers can be promoted by introducing different, but compatible, mutations in the two Fc domain monomers, such as the charge residue pairs included, without limitation, in Table 10, Homodimerization of Fc domain monomers can be promoted by introducing the same mutations in both Fc domain monomers in a symmetric fashion, such as the double mutants K409D/D339K or K392D/D399K.
(90) TABLE-US-00010 TABLE 10 CH.sub.3 antibody CH.sub.3 antibody constant constant domain of domain of Fc domain Fc domain monomer 1 monomer 2 Reference K408D D399K US 2014/0024111 K409D D399R US 2014/0024111 K409E D399K US 2014/0024111 K409E D399R US 2014/0024111 K392D D399K US 2014/0024111 K392D D399R US 2014/0024111 K392E D399K US 2014/0024111 K392E D399R US 2014/0024111 K409D:K392D D399K:E356K Gunasekaran et al., J Biol Chem. 285: 19637-46, 2010 K370E:K409D: E356K:E357K: Martens et al., K439E D399K Clin Cancer Res. 12: 6144-52, 2006
VII. Linkers
(91) In the present disclosure, a linker is used to describe a linkage or connection between polypeptides or protein domains and/or associated non-protein moieties. In some embodiments, a linker is a linkage or connection between at least two Fc domain monomers, for which the linker connects the C-terminus of the C.sub.H3 antibody constant domain of a first Fc domain monomer to the N-terminus of the hinge domain of a second Fc domain monomer, such that the two Fc domain monomers are joined to each other in tandem series. In other embodiments, a linker is a linkage between an Fc domain monomer and any other protein domains that are attached to it. For example, a linker can attach the C-terminus of the C.sub.H3 antibody constant domain of an Fc domain monomer to the N-terminus of an albumin-binding peptide. In another example, a linker can connect the C-terminus of a C.sub.H1 antibody constant domain to the N-terminus of the hinge domain of an Fc domain monomer. In yet other embodiments, a linker can connect two individual protein domains (not including an Fc domain), for example, the C-terminus of a C.sub.L antibody constant domain can be attached to the N-terminus of a C.sub.H1 antibody constant domain by way of a linker.
(92) A linker can be a simple covalent bond, e.g., a peptide bond, a synthetic polymer, e.g., a polyethylene glycol (PEG) polymer, or any kind of bond created from a chemical reaction, e.g. chemical conjugation. In the case that a linker is a peptide bond, the carboxylic acid group at the C-terminus of one protein domain can react with the amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. Specifically, the peptide bond can be formed from synthetic means through a conventional organic chemistry reaction well-known in the art, or by natural production from a host cell, wherein a polynucleotide sequence encoding the DNA sequences of both proteins, e.g., two Fc domain monomer, in tandem series can be directly transcribed and translated into a contiguous polypeptide encoding both proteins by the necessary molecular machineries, e.g., DNA polymerase and ribosome, in the host cell.
(93) In the case that a linker is a synthetic polymer, e.g., a PEG polymer, the polymer can be functionalized with reactive chemical functional groups at each end to react with the terminal amino acids at the connecting ends of two proteins.
(94) In the case that a linker (except peptide bond mentioned above) is made from a chemical reaction, chemical functional groups, e.g., amine, carboxylic acid, ester, azide, or other functional groups commonly used in the art, can be attached synthetically to the C-terminus of one protein and the N-terminus of another protein, respectively. The two functional groups can then react to through synthetic chemistry means to form a chemical bond, thus connecting the two proteins together. Such chemical conjugation procedures are routine for those skilled in the art.
(95) Spacer
(96) In the present disclosure, a linker between two Fc domain monomers can be an amino acid spacer including 3-200 amino acids (e.g., 3-150, 3-100, 3-60, 3-50, 3-40, 3-30, 3-20, 3-10, 3-8, 3-5, 4-30, 5-30, 6-30, 8-30, 10-20, 10-30, 12-30, 14-30, 20-30, 15-25, 15-30, 18-22, and 20-30 amino acids). Suitable peptide spacers are known in the art, and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. In certain embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of GS, GGS, GGGGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2), or SGGG (SEQ ID NO: 3). In certain embodiments, a spacer can contain 2 to 12 amino acids including motifs of GS, e.g., GS, GSGS (SEQ ID NO: 4), GSGSGS (SEQ ID NO: 5), GSGSGSGS (SEQ ID NO: 6), GSGSGSGSGS (SEQ ID NO: 7), or GSGSGSGSGSGS (SEQ ID NO: 8). In certain other embodiments, a spacer can contain 3 to 12 amino acids including motifs of GGS, e.g., GGS, GGSGGS (SEQ ID NO: 9), GGSGGSGGS (SEQ ID NO: 10), and GGSGGSGGSGGS (SEQ ID NO: 11). In yet other embodiments, a spacer can contain 4 to 12 amino acids including motifs of GGSG (SEQ ID NO: 12), e.g., GGSG (SEQ ID NO: 13), GGSGGGSG (SEQ ID NO: 14), or GGSGGGSGGGSG (SEQ ID NO: 15). In other embodiments, a spacer can contain motifs of GGGGS (SEQ ID NO: 16), e.g., GGGGSGGGGSGGGGS (SEQ ID NO: 17). In other embodiments, a spacer can also contain amino acids other than glycine and serine, e.g., GENLYFQSGG (SEQ ID NO: 18), SACYCELS (SEQ ID NO: 19), RSIAT (SEQ ID NO: 20), RPACKIPNDLKQKVMNH (SEQ ID NO: 21), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 22), AAANSSIDLISVPVDSR (SEQ ID NO: 23), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 24). In certain embodiments in the present disclosure, a 12- or 20-amino acid peptide spacer is used to connect two Fc domain monomers in tandem series (
(97) VIII. Serum Protein-Binding Peptides
(98) Binding to serum protein peptides can improve the pharmacokinetics of protein pharmaceuticals, and in particular the Fc constructs described here may be fused with serum protein-binding peptides
(99) As one example, albumin-binding peptides that can be used in the methods and compositions described here are generally known in the art. In one embodiment, the albumin binding peptide comprises, consists of, or consists essentially of the sequence DICLPRWGCLW (SEQ ID NO: 28). In one embodiment, the albumin binding peptide comprises, consists of, or consists essentially of the sequence DICLPRWGCLW (SEQ ID NO: 28) with up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions).
(100) In the present disclosure, albumin-binding peptides may be attached to the N- or C-terminus of certain polypeptides in the Fc construct. In one embodiment, an albumin-binding peptide may be attached to the C-terminus of one or more polypeptides in constructs 1, 2, 3, or 7A (
(101) IX. Fc Constructs
(102) In general, the disclosure features Fc constructs having 2-10 Fc domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains; 2-8 Fc domains, 2-6 Fc domains, 2-4 Fc domains, 2-3 Fc domains, 5-10 Fc domains, 5-8 Fc domains, or 5-6 Fc domains). These may have greater binding affinity and/or avidity than a single wild-type Fc domain for an Fc receptor, e.g., FcγRIIIa. The disclosure discloses methods of engineering amino acids at the interface of two interacting C.sub.H3 antibody constant domains such that the two Fc domain monomers of an Fc domain selectively form a dimer with each other, thus preventing the formation of unwanted multimers or aggregates. An Fc construct includes an even number of Fc domain monomers, with each pair of Fc domain monomers forming an Fc domain. An Fc construct includes, at a minimum, one functional Fc domain formed from a dimer of two Fc domain monomers.
(103) In some embodiments, an Fc construct contains one Fc domain including a dimer of two Fc domain monomers (
(104) In other embodiments, an Fc construct contains two Fc domains (
(105) Furthermore, in other embodiments, an Fc construct can contain three Fc domains formed from four polypeptides (
(106) In yet other embodiments, a single polypeptide can form dimers (e.g., construct 7A;
(107) In yet other embodiments, Fc constructs can contain five Fc domains formed from six polypeptides. Two examples are depicted in
(108) In another embodiment, an Fc construct containing two or more Fc domains can be formed from two polypeptides having the same primary sequence. Such a construct can be formed from expression of a single polypeptide sequence in a host cell. An example is depicted in
(109) In some embodiments, one or more Fc polypeptides in an Fc construct contain a terminal lysine residue. In some embodiments, one or more Fc polypeptides in an Fc construct do not contain a terminal lysine residue. In some embodiments, all of the Fc polypeptides in an Fc construct contain a terminal lysine residue. In some embodiments, all of the Fc polypeptides in an Fc construct do not contain a terminal lysine residue. In one example, the terminal lysine residue in an Fc polypeptide comprises, consists of, or consists essentially of the sequence of any one of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 45, and 46 (see Example 1) may be removed to generate a corresponding Fc polypeptide that does not contain a terminal lysine residue. In another example, a terminal lysine residue may be added to an Fc polypeptide comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO: 48 or 50 (see Example 1) to generate a corresponding Fc polypeptide that contains a terminal lysine residue. In another embodiment, a terminal lysine residue may be added to an Fc polypeptide comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO: 48 or 50 (see Example 1) to generate a corresponding Fc polypeptide that contains a terminal lysine residue with up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions).
(110) X. Host Cells and Protein Production
(111) In the present disclosure, a host cell refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express the polypeptides and constructs described herein from their corresponding nucleic acids. The nucleic acids may be included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). Host cells can be of either mammalian or bacterial origin. Mammalian host cells include, but are not limited to, CHO (or CHO-derived cell strains, e.g., CHO-K1, CHO-DXB11 CHO-DG44), murine host cells (e.g., NS0, Sp2/0), VERY, HEK (e.g., HEK293), BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7O3O and HsS78Bst cells. Host cells can also be chosen that modulate the expression of the protein constructs, or modify and process the protein product in the specific fashion desired. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of protein products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the protein expressed.
(112) For expression and secretion of protein products from their corresponding DNA plasmid constructs, host cells may be transfected or transformed with DNA controlled by appropriate expression control elements known in the art, including promoter, enhancer, sequences, transcription terminators, polyadenylation sites, and selectable markers. Methods for expression of therapeutic proteins are known in the art. See, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 edition (Jul. 20, 2004); Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012 edition (Jun. 28, 2012).
(113) XI. Purification
(114) An Fc construct can be purified by any method known in the art of protein purification, for example, by chromatography (e.g., ion exchange, affinity (e.g., Protein A affinity), and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. For example, an Fc construct can be isolated and purified by appropriately selecting and combining affinity columns such as Protein A column with chromatography columns, filtration, ultra filtration, salting-out and dialysis procedures (see, e.g., Process Scale Purification of Antibodies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009; and Subramanian (ed.) Antibodies—Volume I—Production and Purification, Kluwer Academic/Plenum Publishers, New York (2004)). In some instances, an Fc construct can be conjugated to marker sequences, such as a peptide to facilitate purification. An example of a marker amino acid sequence is a hexa-histidine peptide, which binds to nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767).
(115) For the Fc constructs, Protein A column chromatography may be employed as a purification process. Protein A ligands interact with Fc constructs through the Fc region, making Protein A chromatography a highly selective capture process that is able to remove most of the host cell proteins. In the present disclosure, Fc constructs may be purified using Protein A column chromatography as described in Example 2.
(116) XII. Pharmaceutical Compositions/Preparations
(117) The disclosure features pharmaceutical compositions that include one or more Fc constructs described herein. In one embodiment, a pharmaceutical composition includes a substantially homogenous population of Fc constructs that are identical or substantially identical in structure. In various examples, the pharmaceutical composition includes a substantially homogenous population of any one of constructs 1-10 and 5*.
(118) A therapeutic protein construct, e.g., an Fc construct, of the present disclosure can be incorporated into a pharmaceutical composition. Pharmaceutical compositions including therapeutic proteins can be formulated by methods know to those skilled in the art. The pharmaceutical composition can be administered parenterally in the form of an injectable formulation including a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, the pharmaceutical composition can be formulated by suitably combining the Fc construct with pharmaceutically acceptable vehicles or media, such as sterile water for injection (WFI), physiological saline, emulsifier, suspension agent, surfactant, stabilizer, diluent, binder, excipient, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices. The amount of active ingredient included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided.
(119) The sterile composition for injection can be formulated in accordance with conventional pharmaceutical practices using distilled water for injection as a vehicle. For example, physiological saline or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, alcohol such as ethanol and polyalcohol such as propylene glycol or polyethylene glycol, and a nonionic surfactant such as polysorbate 80™ HCO-50, and the like commonly known in the art. Formulation methods for therapeutic protein products are known in the art, see e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2d ed.) Taylor & Francis Group, CRC Press (2006).
(120) XIII. Dosage
(121) The pharmaceutical compositions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an improvement or remediation of the symptoms. The pharmaceutical compositions are administered in a variety of dosage forms, e.g., intravenous dosage forms, subcutaneous dosage forms, oral dosage forms such as ingestible solutions, drug release capsules, and the like. The appropriate dosage for the individual subject depends on the therapeutic objectives, the route of administration, and the condition of the patient. Generally, recombinant proteins are dosed at 1-200 mg/kg, e.g., 1-100 mg/kg, e.g., 20-100 mg/kg. Accordingly, it will be necessary for a healthcare provider to tailor and titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.
(122) XIV. Indications
(123) The pharmaceutical compositions and methods of the disclosure are useful to reduce inflammation in a subject, to promote clearance of autoantibodies in a subject, to suppress antigen presentation in a subject, to reduce the immune response, e.g., to block immune complex-based activation of the immune response in a subject, and to treat immunological and inflammatory conditions or diseases in a subject. Exemplary conditions and diseases include rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid antibody syndrome; autoimmune hemolytic anemia; chronic inflammatory demyelinating neuropathy; clearance of anti-allo in transplant, anti-self in GVHD, anti-replacement, IgG therapeutics, IgG paraproteins; dermatomyositis; Goodpasture's Syndrome; organ system-targeted type II hypersensitivity syndromes mediated through antibody-dependent cell-mediated cytotoxicity, e.g., Guillain Barre syndrome, CIDP, dermatomyositis, Felty's syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenia purpura; Myasthenia Gravis, neuromyelitis optica; pemphigus and other autoimmune blistering disorders; Sjogren's Syndrome; autoimmune cytopenias and other disorders mediated through antibody-dependent phagocytosis; other FcR-dependent inflammatory syndromes e.g., synovitis, dermatomyositis, systemic vasculitis, glomerulitis and vasculitis.
EXAMPLES
Example 1—Design and Cloning of DNA Plasmid Constructs
(124) A total of eight DNA plasmid constructs were used to assemble eight Fc constructs (
(125) A. wt Fc: wild-type Fc domain monomer (
(126) B. protuberance Fc: Fc domain monomer with engineered protuberance in C.sub.H3 antibody constant domain (
(127) C. cavity Fc: Fc domain monomer with engineered cavity in C.sub.H3 antibody constant domain (
(128) C. cavity Fc*: Fc domain monomer with engineered cavity in C.sub.H3 antibody constant domain (
(129) D. charges Fc: Fc domain monomer with reversed charges in C.sub.H3 antibody constant domain (
(130) E. wt-12-wt Fc2: Two Fc domain monomers joined in series by way of a 12-amino acid GGGS peptide linker (
(131) F. protuberance-20-charges Fc2: Fc domain monomer with reversed charges in C.sub.H3 antibody constant domain and Fc domain monomer with engineered protuberance in C.sub.H3 antibody constant domain joined in series by way of a 20-amino acid SGGG peptide linker (
F*. protuberance-20-charges Fc2*: Fc domain monomer with reversed charges in C.sub.H3 antibody constant domain and Fc domain monomer with engineered protuberance in C.sub.H3 antibody constant domain joined in series by way of a 20-amino acid SGGG peptide linker (
G. protuberance-20-protuberance Fc2: Two Fc domain monomers both with engineered protuberance in C.sub.H3 antibody constant domain joined in series by way of a 20-amino acid GGGS peptide linker (
H. C.sub.HC.sub.L Fc+: Fc domain monomer with C.sub.H1 and C.sub.L constant domains attached to the hinge domain (
(132) Fc DNA sequences were derived from human IgG1 Fc. Protuberance, cavity and charges mutations were substituted in the parental Fc sequence. DNA encoding a leader peptide derived from the human immunoglobulin Kappa Light chain was attached to the 5′ region. All but one of the polypeptides (C.sub.HC.sub.L Fc+) contained this encoded peptide on the amino terminus to direct protein translocation into the endoplasmic reticulum for assembly and secretion. It will be understood that any one of a variety of leader peptides may be used in connection with the present disclosure. The leader peptide is usually clipped off in the ER lumen. An 11 nucleotide sequence containing a 5′ terminal EcoR1 site was added upstream of the ATG start codon. A 30 nucleotide sequence containing a 3′ terminal Xho1 site was added downstream of the 3′ terminal TGA translation termination codon. The DNA sequences were optimized for expression in mammalian cells and cloned into the pcDNA3.4 mammalian expression vector.
(133) Mutations are denoted by the wild-type amino acid residue followed by the position using the EU Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., ed. 5, 1991) and then the replacement residue in single-letter code. The nucleotide and amino acid sequences of secreted polypeptides A-H described above are provided below (except for cavity Fc* and protuberance-20-charges Fc2*, for which only the amino acid sequences are provided).
(134) Wt Fc
(135) SEQ ID NO: 29:
(136) GACAAGACCCACACCTGTCCGCCTTGCCCTGCCCCTGAGCTG CTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTG GTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATT GGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC CCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCG TGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAAT ACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCCATCGA GAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCA GGTGTACACACTGCCCCCCAGCCGGGACGAGCTGACCAAGAA CCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCAGC GATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAAC AACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCAT TCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGC AGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCC TGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCG GCAAG
SEQ ID NO: 30: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK
protuberance Fc
SEQ ID NO: 31: GACAAGACCCACACCTGTCCGCCTTGCCCTGCCCCTGAGCTG CTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTG GTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATT GGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC CCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCG TGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAAT ACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCCATCGA GAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCA GGTGTACACACTGCCCCCCTGCCGGGACGAGCTGACCAAGAA CCAGGTGTCCCTGTGGTGCCTGGTGAAAGGCTTCTACCCCAG CGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAA CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTC ATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCC CTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCC GGCAAG
SEQ ID NO: 32: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDVVLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPC RDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK
Cavity Fc
SEQ ID NO: 33: GACAAGACCCACACCTGTCCGCCTTGCCCTGCCCCTGAGCTG CTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTG GTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATT GGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC CCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCG TGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAAT ACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCCATCGA GAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCA AGTGTGTACACTGCCCCCCAGCCGGGACGAGCTGACCAAGAA CCAGGTGTCCCTGAGCTGCGCCGTGAAAGGCTTCTACCCCAG CGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAA CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTC ATTCTTCCTGGTTAGCAAGCTGACCGTGGACAAGAGCCGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCC CTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCC GGCAAG
SEQ ID NO: 34: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDVVLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSR DELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK
Cavity Fc*
SEQ ID NO: 45: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDVVLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPS RDELTKNQVSLSCAVEGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK
Cavity Fc
SEQ ID NO: 47: GACAAGACCCACACCTGTCCGCCTTGCCCTGCCCCTGAGCTGCTGGGAGG CCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCA GCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGAC CCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGC CAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGT CCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAG TGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAG CAAGGCCAAGGGCCAGCCCCGCGAGCCCCAAGTGTGTACACTGCCCCCCA GCCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGAGCTGCGCCGTGGAC GGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCC CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCAT TCTTCCTGGTTAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGC AACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACAC CCAGAAGTCCCTGAGCCTGAGCCCCGGCTAG
SEQ ID NO: 48: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDVVLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVD GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG
Charges Fc
SEQ ID NO: 35: GACAAGACCCACACCTGTCCGCCTTGCCCTGCCCCTGAGCTG CTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTG GTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATT GGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC CCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCG TGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAAT ACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCCATCGA GAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCA GGTGTACACACTGCCCCCCAGCCGGGACGAGCTGACCAAGAA CCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCAGC GATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAAC AACTACAAGACCACCCCCCCTGTGCTGAAAAGCGACGGCTCAT TCTTCCTGTACAGCGACCTGACCGTGGACAAGAGCCGGTGGC AGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCC TGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCG GCAAG
SEQ ID NO: 36: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDVVLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS DGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK
wt-12-wt Fc2
SEQ ID NO: 37: GACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAGCTG CTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTG GTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATT GGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC CCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCG TGCTGACCGTGCTGCACCAGGACTGGCTCAACGGCAAAGAGT ACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCG AGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCC AGGTCTACACACTGCCCCCCAGCCGGGACGAGCTGACCAAGA ACCAGGTCTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCAG CGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAA CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTC ATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCC CTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCC GGCAAAGGCGGGGGATCTGGGGGAGGAAGCGGAGGCGGCAG CGATAAGACCCATACCTGCCCTCCCTGTCCCGCTCCCGAACTG CTGGGGGGACCCTCCGTGTTTCTGTTTCCACCTAAGCCTAAGG ATACGCTCATGATCTCCAGAACCCCTGAAGTCACATGTGTGGT GGTCGATGTGTCTCATGAAGATCCCGAAGTCAAGTTTAACTGG TATGTGGATGGGGTCGAGGTCCACAATGCCAAAACAAAGCCTC GGGAAGAACAGTATAACTCCACCTACAGAGTCGTCAGCGTGCT GACAGTCCTTCATCAGGATTGGCTGAATGGGAAAGAGTACAAA TGTAAAGTGTCTAACAAAGCTCTGCCCGCTCCTATCGAAAAGA CCATCTCCAAAGCCAAAGGGCAGCCCAGAGAACCTCAGGTGTA CACCCTGCCACCCTCCAGAGATGAGCTGACAAAAAATCAGGTG TCACTGACATGTCTGGTGAAAGGGTTTTATCCCTCCGACATTGC TGTGGAATGGGAATCCAATGGGCAGCCTGAAAACAATTATAAG ACAACACCTCCCGTGCTGGACTCCGATGGCTCATTTTTTCTGTA CTCTAAACTGACAGTGGATAAGTCCAGATGGCAGCAGGGAAAT GTGTTTTCCTGCTCTGTGATGCATGAAGCTCTGCATAATCACTA TACACAGAAAAGCCTGTCCCTGTCCCCCGGCAAG
SEQ ID NO: 38: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDVVLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKGGGSGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
Protuberance-20-Charges Fc2
SEQ ID NO: 39: GACAAGACCCACACCTGTCCCCCTTGCCCAGCCCCTGAGCTG CTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTG GTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATT GGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC CCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCG TGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGT ACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCG AGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCC AGGTGTACACCCTGCCCCCTTGCAGAGATGAGCTGACCAAGAA CCAGGTGTCCCTGTGGTGCCTGGTCAAGGGCTTCTACCCCAG CGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAA CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTC ATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCC CTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCC GGCAAGTCTGGGGGAGGATCAGGGGGTGGAAGTGGCGGTGG ATCTGGTGGTGGAAGCGGAGGCGGCGATAAGACACACACATG CCCCCCCTGTCCAGCTCCCGAACTGCTGGGGGGACCCTCCGT GTTTCTGTTTCCACCTAAGCCTAAGGATACGCTCATGATCTCCA GAACCCCTGAAGTCACATGTGTGGTGGTCGATGTGTCTCATGA AGATCCCGAAGTCAAGTTTAATTGGTATGTCGATGGGGTCGAG GTGCACAATGCCAAAACAAAACCTCGGGAAGAACAGTATAACT CCACATACAGAGTGGTGTCTGTCCTCACAGTCCTGCATCAGGA TTGGCTCAATGGGAAAGAGTACAAATGTAAAGTCTCTAACAAG GCTCTCCCCGCTCCGATCGAAAAGACCATCTCCAAAGCCAAAG GGCAGCCCAGAGAACCTCAGGTCTACACACTGCCTCCCAGCC GGGACGAGCTGACAAAAAATCAAGTGTCTCTGACCTGCCTCGT GAAGGGCTTTTATCCCTCCGACATTGCCGTCGAGTGGGAGTCC AATGGACAGCCGGAAAACAATTATAAGACCACGCCTCCAGTGC TGAAGTCCGACGGCAGCTTCTTTCTGTACTCCGACCTGACAGT GGATAAGTCCAGATGGCAGCAAGGGAATGTGTTCTCCTGTTCC GTGATGCATGAAGCCCTCCATAATCACTATACCCAGAAAAGCC TGTCCCTGTCCCCTGGCAAG
SEQ ID NO: 40: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDVVLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSGGGSGGGSGGGSGGGSGGGDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Protuberance-20-Charges Fc2*
SEQ ID NO: 46: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDVVLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCR DKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSGGGSGGGSGGGSGGGSGGGDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK protuberance-20-protuberance Fc2
SEQ ID NO: 41: GACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAGCTG CTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTG GTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATT GGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC CCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCG TGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGT ACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCG AGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCC AGGTGTACACCCTGCCCCCTTGCAGAGATGAACTGACCAAGAA CCAGGTGTCCCTGTGGTGCCTGGTCAAGGGCTTCTACCCCAG CGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAA CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTC ATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCC CTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCC GGCAAGTCTGGGGGAGGATCAGGGGGTGGAAGTGGCGGTGG ATCTGGTGGTGGAAGCGGAGGCGGCGATAAGACACACACATG CCCCCCCTGTCCAGCTCCCGAACTGCTGGGGGGACCCTCCGT GTTTCTGTTTCCACCTAAGCCTAAGGATACGCTCATGATCTCCA GAACCCCTGAAGTCACATGTGTGGTGGTCGATGTGTCTCATGA AGATCCCGAAGTCAAGTTTAACTGGTATGTGGATGGGGTCGAG GTCCACAATGCCAAAACAAAGCCTCGGGAAGAACAGTATAACT CCACCTACAGAGTCGTCAGCGTGCTGACAGTCCTGCATCAAGA TTGGCTCAATGGGAAAGAGTATAAGTGTAAAGTCTCGAACAAA GCCCTCCCCGCTCCTATCGAAAAGACCATCTCCAAAGCCAAAG GGCAGCCCAGAGAACCTCAGGTCTACACACTGCCTCCATGTCG GGACGAGCTGACAAAAAATCAGGTGTCACTGTGGTGTCTGGTG AAGGGGTTTTACCCTTCCGACATTGCTGTGGAATGGGAATCCA ATGGGCAGCCTGAAAACAATTATAAGACAACACCTCCCGTGCT GGACTCCGATGGCTCATTTTTTCTGTACTCTAAACTGACAGTGG ATAAGTCCAGATGGCAGCAGGGAAATGTGTTTTCCTGCTCTGT GATGCATGAAGCTCTGCATAATCACTATACACAGAAAAGCCTGT CCCTGTCCCCTGGCAAG
SEQ ID NO: 42: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDVVLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSGGGSGGGSGGGSGGGSGGGDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Protuberance-20-Charges Fc2
SEQ ID NO: 49: GACAAGACCCACACCTGTCCCCCTTGCCCAGCCCCTGAGCTGCTGGGAGG CCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCA GCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGAC CCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGC CAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGT CCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAG TGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAG CAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCTT GCAGAGATAAGCTGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTCAAG GGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCC CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCAT TCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGC AACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACAC CCAGAAGTCCCTGAGCCTGAGCCCCGGCAAGGGAGGGGGAGGAGGAGGGG GTGGAGGTGGCGGTGGAGGCGGTGGTGGAGGCGGAGGCGGCGATAAGACA CACACATGCCCCCCCTGTCCAGCTCCCGAACTGCTGGGGGGACCCTCCGT GTTTCTGTTTCCACCTAAGCCTAAGGATACGCTCATGATCTCCAGAACCC CTGAAGTCACATGTGTGGTGGTCGATGTGTCTCATGAAGATCCCGAAGTC AAGTTTAATTGGTATGTCGATGGGGTCGAGGTGCACAATGCCAAAACAAA ACCTCGGGAAGAACAGTATAACTCCACATACAGAGTGGTGTCTGTCCTCA CAGTCCTGCATCAGGATTGGCTCAATGGGAAAGAGTACAAATGTAAAGTC TCTAACAAGGCTCTCCCCGCTCCGATCGAAAAGACCATCTCCAAAGCCAA AGGGCAGCCCAGAGAACCTCAGGTCTACACACTGCCTCCCAGCCGGGACG AGCTGACAAAAAATCAAGTGTCTCTGACCTGCCTCGTGAAGGGCTTTTAT CCCTCCGACATTGCCGTCGAGTGGGAGTCCAATGGACAGCCGGAAAACAA TTATAAGACCACGCCTCCAGTGCTGAAGTCCGACGGCAGCTTCTTTCTGT ACTCCGACCTGACAGTGGATAAGTCCAGATGGCAGCAAGGGAATGTGTTC TCCTGTTCCGTGATGCATGAAGCCCTCCATAATCACTATACCCAGAAAAG CCTGTCCCTGTCCCCTGGCTAG
SEQ ID NO: 50: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDVVLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGGGDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPG
C.sub.HC.sub.L Fc+
SEQ ID NO: 43: AGGACAGTGGCCGCTCCCAGCGTGTTCATCTTCCCACCCAGCGACGAGCA GCTGAAGTCCGGCACAGCCAGCGTGGTCTGCCTGCTGAACAACTTCTACC CCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGC AACAGCCAGGAAAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAG CCTGTCTAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGG TGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAG AGCTTCAACAGAGGCGAGTGCGGCGGCTCTGGCGGAGGATCCGGGGGAGG ATCAGGCGGCGGAAGCGGAGGCAGCGCTAGCACAAAGGGCCCCTCCGTGT TCCCCCTGGCCCCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTG GGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAA CTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGA GCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACAGTGCCTAGCAGCAGC CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACAC CAAAGTGGACAAGCGGGTGGAACCCAAGAGCTGCGACAAGACCCACACGT GTCCCCCCTGCCCAGCCCCTGAACTGCTGGGCGGACCTAGCGTGTTCCTG TTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGT GACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCA ATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCCAGA GAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCT GCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACA AGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAG CCCCGCGAGCCCCAGGTGTACACACTGCCCCCCAGCCGGGACGAGCTGAC CAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTACCCCTCCG ATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAA GCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCT CCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGC CTGAGCCCCGGCAAA
SEQ ID NO: 44: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGECGGSGGGSGGGSGGGSGGSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK
(137) In some embodiments, the Fc polypeptides in the Fc constructs described herein comprise, consist of, or consist essentially of any of the sequences described herein with up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions).
(138) In some embodiments, one or more Fc polypeptides in an Fc construct contain a terminal lysine residue. In some embodiments, one or more Fc polypeptides in an Fc construct do not contain a terminal lysine residue. In some embodiments, all of the Fc polypeptides in an Fc construct contain a terminal lysine residue. In some embodiments, all of the Fc polypeptides in an Fc construct do not contain a terminal lysine residue. In one example, the terminal lysine residue in an Fc polypeptide comprising, consisting of, or consisting essentially of the sequence of any one of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 45, and 46 may be removed to generate a corresponding Fc polypeptide that does not contain a terminal lysine residue. In another example, a terminal lysine residue may be added to an Fc polypeptide comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO: 48 or 50 to generate a corresponding Fc polypeptide that contains a terminal lysine residue.
Example 2—Expression of Fc Construct Proteins
(139) For protein expression of the Fc constructs, two of the DNA plasmid constructs selected from A-H described in Example 1 were transfected into EXPI293 cells (LifeTechnologies). Liposome transfection was used to introduce plasmid DNA into EXPI293 cells. The total amount of transfected plasmid constructs was fixed whereas the ratio of different plasmid constructs was varied to maximize the yield of desired constructs (see Table 11 below). For each Fc construct, the ratio (by mass) of the two transfected DNA plasmid constructs is shown in Table 11. Illustrations of the constructs are shown
(140) After protein expression, the expressed constructs were purified from the cell culture supernatant by Protein A-based affinity column chromatography. Media supernatants were loaded onto a Poros MabCapture A (LifeTechnologies) column using an AKTA Avant preparative chromatography system (GE Healthcare Life Sciences). Captured Fc constructs were then washed with phosphate buffered saline (low-salt wash) followed by phosphate buffered saline supplemented with 500 mM NaCl (high-salt wash). Fc constructs are eluted with 100 mM glycine, 150 mM NaCl, pH 3 buffer. The protein solution emerging from the column is neutralized by addition of 1M TRIS pH 7.4 to a final concentration of 100 mM. The Fc constructs were further fractionated by ion exchange chromatography using Poros® XS resin (Applied Biosciences Cat. #4404336). The column was pre-equilibrated with 10 mM MES, pH 6 (buffer A), and the sample was eluted with a gradient against 10 mM MES, 500 mM sodium chloride, pH 6 (buffer B).
(141) We obtained a total of seven Fc constructs (see Table 11 below and
(142) TABLE-US-00011 TABLE 11 Approx. Approx. MW MW in Ratio of in kDa KDa (non- Plasmids Plasmids (reducing reducing Construct Transfected A:B SDS-Page) SDS-Page) 1 A: Wt Fc n/a 25 50 2 A: Protuberance 1:1 25 50 Fc B: Cavity Fc 3 A: Charges Fc 25 50 4 A: Wt-12-Wt Fc2 1:2 25, 50 100 B: Wt FC 5 A: Protuberance- 2:1 25, 50 150 20-Charges Fc2 B: Cavity Fc 5* A: Protuberance- 2:1 25, 50 150 20-Charges Fc2* B: Cavity Fc* 6 A: Protuberance- 1:1 25, 50 100 20-Protuberance Fc2 B: Cavity Fc 7 and 8 A: Ch, Cl, Fc+ n/a 50 100
Example 3—Preparation and SDS-PAGE Analysis of Construct 4
(143) Two DNA plasmid constructs, wt-12-wt Fc2 (DNA plasmid construct E in Example 1) and wt Fc (DNA plasmid construct A in Example 1), were used to express construct 4 (
(144) In addition, we observed higher molecular weight bands at approximately 150 kDa, 200 kDa and 250 kDa (lanes 2 and 3,
Example 4—Preparation and SDS-PAGE Analysis of Construct 6
(145) Two plasmid constructs, protuberance-20-protuberance Fc2 (DNA plasmid construct G in Example 1) and cavity Fc (DNA plasmid construct C in Example 1), were used to express construct 6 (
(146) A similar experiment was performed with construct 5 (
(147)
(148) These findings demonstrate that the selectivity dimerization module containing either an engineered protuberance or an engineered cavity in the C.sub.H3 antibody constant domain reduces self-association and prevents uncontrolled Fc-mediated aggregate or multimer formation, indicating that the use of dimerization selectivity modules in the constructs described herein can be used to produce substantially homogenous preparations of the Fc constructs. This observation has significant implications for advantages in manufacturing, yield, and purity of the constructs, e.g., in order to control biological activity and potency.
Example 5—Binding Affinity and Avidity
(149) The binding of constructs to multiple Fcγ receptors was assessed using cell-based FRET competition assays (Cisbio Bioassays). Constructs 5 and 6 showed at least a ten-fold decrease in IC50 (i.e. increased binding) to FcγRIIa, FcγRIIb, and FcγRIIIa relative to the wild type Fc domain (construct 1).
Example 6—Monocyte Activation and Blocking Assays
(150) Three Fc constructs, constructs 1, 5, and 6, containing one, three, and two Fc domains, respectively, were tested for their ability to activate THP-1 monocytes on their own. IL-8 release was used as an indicator of monocyte activation. Constructs 1, 5, and 6 were expressed and purified as described in Examples 1 and 2. Each of the purified Fc constructs was added to THP-1 monocytes. No substantial IL-8 release was observed for any of the three constructs. The data are provided in
(151) The same three Fc constructs were then tested for their ability to inhibit Fc receptor-mediated monocyte activation. IgG1 (100 μg/mL) was immobilized on a 96 well plate and used to induce IL-8 release by THP-1 monocytes. Serial dilutions of constructs 1, 5 and 6 or control substances (intravenous immunoglobulin (IVIg), human serum albumin (HSA), and glycine buffer) were subsequently performed in the tissue culture plate. THP-1 monocytes (1.5×10.sup.5 cells) were immediately added with thorough mixing. The cultures were incubated for 18 h and the supernatants analyzed for IL-8. Constructs 5 and 6 were found to inhibit IL-8 release more effectively than construct 1 at low doses. The data are provided in
Example 7—K/B×N Arthritis Model
(152) Fc constructs 1, 5, and 6 and IVIg were tested for their ability to protect mice from joint inflammation in the K/B×N serum transfer model using a method described in Anthony, Proc. Natl. Acad. Sci. U.S.A. 105:19571-19578 (2008). Twelve-week old K/B×N mice were generated/purchased from Jackson Laboratories. A total of thirty C57BL mice were separated into five groups of six mice each. Each group was injected intravenously (i.v.) with 200 μl construct 6 at 0.1 g/kg, 200 μl construct 5 at 0.1 g/kg, 200 μl IVIg at 0.1 g/kg, 230 μl IVIg at 1 g/kg, or 200 μl phosphate-buffered saline (PBS) one hour before injection of 200 μl K/B×N serum (an arthritis inducing serum) (Day 0). Inflammation was scored by clinical examination of paw swelling and ankle thickness. For paw swelling, each paw was scored 0-3 (0, no swelling; 3, maximal swelling). Scores of four paws were added for total clinical score per individual mouse. For ankle thickness, caliper measurement was used. Each mouse was scored daily from Day 0 to Day 10. The daily average clinical score for each group of six mice was plotted in
Example 8—Chronic ITP Model
(153) Constructs 1 and 5, as well as IVIg, were tested for their ability to treat mice undergoing immune thrombocytopenia (ITP). ITP was induced by an anti-platelet Ab that causes platelet depletion. Forty five C57BL/6 mice (18-22 g, Charles Rivers Labs, MA) were injected i.p. with 1.5 pg/mouse of rat anti-CD41 antibody (Ab) (clone MWReg30 BioLegend cat #133910) once daily for 4 days (on days 1, 2, 3 and 4). Five mice were injected with 1.5 pg/mouse of a rat IgG1, k isotype control Ab (BioLegend cat #400414) to determine normal platelet levels. Abs were injected in 100 μl of PBS. All mice were dosed once intravenously with 200 μl of either saline control, IVIg at 1 g/kg, construct 1 at 0.02, 0.03, 0.1, and 0.3 g/kg, and construct 5 at 0.004, 0.02, and 0.1 g/kg 2 h after the third anti-CD41 Ab injection on day 3. Mice were bled on day 5 (24 h after the forth anti-CD41 Ab injection) to quantitate total platelet levels by the VetScan Instrument. All procedures were performed in compliance with the Animal Welfare Act and with the Guide for the Care and Use of Laboratory Animals.
(154) As shown in
Example 9—Construct 5* Shows Augmented Binding and Avidity to FcγR Compared to IVIg
(155) Following the same protocol as described in Example 8, two plasmid constructs, encoding protuberance-20-charges Fc2* (construct F* in Example 1) and cavity Fc* (construct C* in Example 1), were used to express and purify construct 5*. The binding profile of this construct to various Fc receptors was compared to that of IVIG in a fluorescence resonance energy transfer (FRET) competitive binding assay.
(156) Construct 5* displayed an overall binding profile to the different Fcγ-receptors similar to that of IVIg (with the lowest binding affinity observed for FcγRIIb) but with greatly enhanced binding to all low affinity FcγRs when compared to IVIg. Augmented binding to FcγR corresponds to higher avidity, which refers to the cumulative effect of the accumulated affinities of each individual binding interaction. IC50 values for construct 5* were consistently lower than those of IVIg, indicating striking increases in binding to low affinity FcγRs compared to individual IgG molecules. For example, compared to IVIg, construct 5* displayed approximately 170 fold increased affinity FcγRIIa (H131 variant), 55 fold increased affinity for FcγRIIb.
Example 10—Inhibition of Phagocytosis in THP-1 Monocytic Cells
(157) Construct 5* and IVIg were Tested in a Model of Phagocytosis.
(158) Phagocytosis is the process by which cells (phagocytes) engulf solid particles such as bacteria, to form an internal vesicle known as a phagosome. In the immune system, phagocytosis is a major mechanism used to remove pathogens and cell debris. Monocytes and macrophages are among the cells specialized in clearing opsonized (antibody coated) particles from the immune system through phagocytosis, a mechanism largely dependent on FcγR mediated engagement. However, in autoimmune diseases, phagocytes can become activated leading to the detrimental release of pro-inflammatory cytokines or the phagocytosis of other critical cells in the body. IVIg, containing pooled, polyvalent, IgG antibodies extracted from the plasma of over one thousand blood donors, is used to treat autoimmune disease.
(159) In this assay system, fluorescently labeled antibody-coated latex beads, a mimic of opsonized bacteria or viruses, were fed to THP-1 cells and allowed to be phagocytosed in the presence and absence of construct 5* and IVIg. At the end of the incubation period, any external fluorescence was quenched with trypan blue, and the amount of intracellular fluorescence quantified by flow cytometry. All groups were normalized to their non-treated control (THP-1 cells and latex beads only). Results are representative of two separate experiments.
(160) As shown in
Example 11—Enhancement of Fc Construct Binding to FcγRIIb
(161) S267E/L328F mutations have been previously shown to significantly and specifically enhance IgG1 binding to the FcγRIIb receptor (Chu et al. Molecular Immunology 45 2008). The S267E/L328F mutations were incorporated into the Construct 5 (SIF) backbone. This Construct 5-FcγRIIb+ mutant expresses and assembles well (see
(162) Binding of the Construct 5-FcγRIIb+ mutant to the inhibitory FcγRIIb receptor was greatly enhanced when compared to the Construct 5 (SIF3) control (over 300 fold increase in binding). Conversely, binding to the activating FcγRIIa is relatively unaffected, whereas binding to FcγRIIIa is reduced (see
(163)
Example 12—Inhibition of Monocyte Derived Dendritic Cells (moDCs) Activation
(164) Construct 5-FcγRIIb+ mutant greatly potentiates activation of monocyte derived dendritic cells (moDCs). Dendritic cells (DCs), which are the most important population of professional antigen presenting cells, process antigen material and present it on the cell surface with the aim of initiating T-cell responses. FcγRs can play a major role in regulating moDC function. Immune complex engagement of activating FcγRs can trigger maturation and activation of immature human moDCs. Conversely, engagement of inhibitory FcγRIIb can suppress maturation and activation (Boruchov A M et al. J Clin Invest. 2005 115(10)). We had previously shown that Fc constructs can inhibit maturation and activation of moDCs (Ortiz et al Sci Transl Med 2016 and see
(165) Immature human moDCs were generated from negatively selected CD14+ monocytes in the presence of 100 ng/mL GM-CSF and 50 ng/mL IL-4. Harvested DCs were incubated with either PBS, anti-CD32a antibody IV.3, Construct 5 (SIF3), or Construct 5-FcγRIIb+ at 37° C. for 20 min in medium. After blocking, the cell suspension was transferred to the IgG1 coated plates and an additional GM-CSF and IL-4 supplemented medium was added. After a 48 h incubation, lightly adherent cells were harvested by washing plates twice with ice cold PBS. Harvested cells were stained with anti-HLA-DR FITC and anti-CD86-PE Cy7 Abs. Cells were analyzed with a FACSCanto (BD) and FlowJo Software (TreeStar).
(166)
(167)