Antibodies selective for cells presenting EGFR at high density
Herein described are antibodies to epidermal growth factor receptor (EGER) having an EGER binding affinity that is sufficient to kill disease cells presenting EGFR at high density, but is insufficient for binding to normal cells. A therapeutic effect is thus achieved while avoiding adverse events that result from unintended binding to normal cells.
1. An epidermal growth factor receptor (EGFR) antibody that binds preferentially to disease cells having an EGFR density greater than a normal EGFR density as compared to cells comprising the normal EGFR density, wherein the normal EGFR density is less than 10,000 EGFR molecules per cell, the EGFR antibody comprising a heavy chain and a light chain, each chain having a constant region and a variable region, each variable region comprising framework regions and complementarity determining regions (CDRs), wherein the CDRs have an amino acid sequence set forth below:
For the heavy chain:
For the heavy chain:
CDR1
(SEQ ID No. 1)
NYGVH
CDR2
(SEQ ID No. 2)
VIWSGGNTD 58 YNTPFTS
CDR3
(SEQ ID No. 3)
ALTY 101 Y 102 D 103 YE 105 FAY
For the light chain:
CDR1
(SEQ ID No. 4)
RASQSIGTNIH
CDR2
(SEQ ID No. 5)
ASE 53 SIS
CDR3
(SEQ ID No. 6)
QQNNNW 94 PTT
wherein Y 101 , Y 102 or D 103 is/are substituted with at least one of A 101 , A 102 or N 103 , and D 58 , Y 101 , Y 102 , D 103 , or E 105 is/are substituted with up to three of N 58 , A 101 , A 102 , N 103 and/or Q 105 , and/or wherein E 53 or W 94 is/are wild type or substituted with at least one of K 53 or A 94 , wherein the substituted amino acid(s) confer(s) on said antibody a reduced EGFR binding affinity (Kd) that is 1.0 nM or weaker.
2. The antibody according to claim 1 , wherein Y 101 is substituted by A 101 .
3. The antibody according to claim 1 , wherein Y 102 is substituted by A 102 .
4. The antibody according to claim 1 , wherein D 103 is replaced by N 103 .
5. The antibody according to claim 1 , wherein at least one of said substitutions is in the heavy chain and at least one of said substitutions is in the light chain.
6. The antibody according to claim 5 , wherein E 53 and Y 102 are both substituted.
7. The antibody according to claim 6 , wherein E 53 is substituted by K 53 and Y 102 is substituted by A 102 .
8. The antibody according to claim 1 , wherein D 58 and D 103 are both substituted.
9. The antibody according to claim 8 , wherein D 58 is substituted by N 58 and D 103 is substituted by N 103 .
10. The antibody according to claim 8 , further wherein E 105 is substituted.
11. The antibody according to claim 10 , wherein E 105 is substituted by Q 105 .
12. The antibody according to claim 1 , wherein E 53 is substituted by K 53 .
13. The antibody according to claim 1 , wherein W 94 is substituted by A 94 .
14. An antibody according to claim 1 , the antibody having the framework region sequences of cetuximab and/or the constant region sequence of cetuximab.
15. A bivalent fragment of an antibody according to claim 1 .
16. A conjugate comprising a cytotoxin or a detectable label and, conjugated thereto, an antibody according to claim 1 .
17. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the EGFR antibody of claim 1 in an amount useful to control the growth of cells presenting EGFR at a density greater than the normal EGFR density, wherein the EGFR antibody has an affinity (Kd) for EGFR that is within the range from about 1.0 nM to about 1 uM, said antibody having insignificant binding affinity for a cell presenting EGFR at a normal EGFR density, wherein the normal EGFR density is less than 10,000 EGFR molecules per cell.
18. A pharmaceutical composition comprising the antibody according to claim 1 .
19. An epidermal growth factor receptor (EGFR) antibody comprising a heavy chain and a light chain, each chain having a variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID No. 7 and Y 101 , Y 102 or D 103 is/are substituted by at least one of A 101 , A 102 or N 103 or a conservative amino acid thereof, and D 58 , Y 101 , Y 102 , D 103 , or E 105 is/are substituted with up to three of N 58 , A 101 , A 102 , N 103 or Q 105 or a conservative amino acid thereof, and/or wherein the light chain variable region comprises the sequence of SEQ ID No. 8 and E 53 or W 94 is/are wild type or substituted by at least one of K 53 or A 94 or a conservative amino acid thereof.
20. A bivalent fragment of an antibody according to claim 19 .
21. The antibody according to claim 15 , wherein the fragment is a single chain Fv (scFv), a diabody, a F(ab′) 2 fragment.
22. The antibody according to claim 19 , wherein the fragment is a single chain Fv (scFv), a diabody, a F(ab′) 2 fragment.
23. The conjugate according to claim 16 , wherein the cytotoxin is a radioactive isotope, diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain from Pseudomonas aeruginosa , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca Americana proteins PAPI, PAPII, or PAP-S, Momordica charantia inhibitor, curcin, crotin, Sapaonaria, officinalis inhibitor, gclonin, saporin, restrictocin, phenomycin, or a tricothecene.
24. A conjugate comprising a cytotoxin or a detectable label and, conjugated thereto, an antibody according to claim 19 .
25. The conjugate according to claim 24 , wherein the cytotoxin is a radioactive isotope, diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain from Pseudomonas aeruginosa , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca Americana proteins PAPI, PAPII, or PAP-S, Momordica charantia inhibitor, curcin, crotin, Sapaonaria, officinalis inhibitor, gclonin, saporin, restrictocin, phenomycin, or a tricothecene.