Collection and methods for its use
The present disclosure enables collections of variable heavy chain and variable light chain pairs comprising, in part, germline protein sequences that are pre-selected for functional properties relevant to developability, wherein the collections may be used to select against any antigen using, for example, phage display.
1. A collection of synthetic antibodies or fragments thereof, wherein the antibodies or fragments comprise variable heavy chain and variable light chain pairs, wherein the framework regions of the following the variable heavy chain and variable light chain pairs wherein the pairs are listed together comprise germline protein sequences selected from twenty two or more of the variable heavy chain and variable light chain pairs VH1-18 (SEQ ID NO: 204)/VK1-39 (SEQ ID NO: 236); VH1-18 (SEQ ID NO: 204)/VK3-15 (SEQ ID NO: 238); VH1-18 (SEQ ID NO: 204)/VK3-20 (SEQ ID NO: 239); VH1-46 (SEQ ID NO: 205)/VK3-15 (SEQ ID NO: 238); VH1-46 (SEQ ID NO: 205)/VL1-51 (SEQ ID NO: 252); VH1-46 (SEQ ID NO: 205)/VL3-21 (SEQ ID NO: 257); VH1-69*01 (SEQ ID NO: 206)/VL1-51 (SEQ ID NO: 252); VH3-07 (SEQ ID NO: 207)/VK1-12 (SEQ ID NO: 233); VH3-07 (SEQ ID NO: 207)/VK1-16 (SEQ ID NO: 234); VH3-07 (SEQ ID NO: 207)/VK1-27 (SEQ ID NO: 235); VH3-07 (SEQ ID NO: 207)/VK1-39 (SEQ ID NO: 236); VH3-07 (SEQ ID NO: 207)/VK3-15 (SEQ ID NO: 238); VH3-07 (SEQ ID NO: 207)/VL1-47 (SEQ ID NO: 251); VH3-07 (SEQ ID NO: 207)/VL1-51 (SEQ ID NO: 252); VH3-11 (SEQ ID NO: 208)/VK1-05 (SEQ ID NO: 230); VH3-11 (SEQ ID NO: 208)/VK1-39 (SEQ ID NO: 236); VH3-11 (SEQ ID NO: 208)/VK3-15 (SEQ ID NO: 238); VH3-11 (SEQ ID NO: 208)/VL1-40 (SEQ ID NO: 250); VH3-11 (SEQ ID NO: 208)/VL1-47 (SEQ ID NO: 251); VH3-11 (SEQ ID NO: 208)/VL1-51 (SEQ ID NO: 252); VH3-11 (SEQ ID NO: 208)/VL2-23 (SEQ ID NO: 255); VH3-15 (SEQ ID NO: 209)/VK1-05 (SEQ ID NO: 230); VH3-15 (SEQ ID NO: 209)/VK1-06 (SEQ ID NO: 231); VH3-15 (SEQ ID NO: 209)/VK1-12 (SEQ ID NO: 233); VH3-15 (SEQ ID NO: 209)/VK1-16 (SEQ ID NO: 234); VH3-15 (SEQ ID NO: 209)/VK1-27 (SEQ ID NO: 235); VH3-15 (SEQ ID NO: 209)/VK3-11 (SEQ ID NO: 237); VH3-15 (SEQ ID NO: 209)/VL1-40 (SEQ ID NO: 250); VH3-15 (SEQ ID NO: 209)/VL1-47 (SEQ ID NO: 251); VH3-15 (SEQ ID NO: 209)/VL1-51 (SEQ ID NO: 252); VH3-15 (SEQ ID NO: 209)/VL2-14 (SEQ ID NO: 254); VH3-21 (SEQ ID NO: 210)/VK1-12 (SEQ ID NO: 233); VH3-21 (SEQ ID NO: 210)/VK1-27 (SEQ ID NO: 235); VH3-21 (SEQ ID NO: 210)/VL2-11 (SEQ ID NO: 253); VH3-23 (SEQ ID NO: 211)/VK1-39 (SEQ ID NO: 236); VH3-23 (SEQ ID NO: 211)/VK3-15 (SEQ ID NO: 238); VH3-23 (SEQ ID NO: 211)/VL2-23 (SEQ ID NO: 255); VH3-23 (SEQ ID NO: 211)/VL3-1 (SEQ ID NO: 256); VH3-30 (SEQ ID NO: 212)/VK3-20 (SEQ ID NO: 239); VH3-53 (SEQ ID NO: 213)/VK3-15 (SEQ ID NO: 238); VH3-53 (SEQ ID NO: 213)/VL2-11 (SEQ ID NO: 253); VH3-74 (SEQ ID NO: 214)/VK1-05 (SEQ ID NO; 230); VH3-74 (SEQ ID NO: 214)/VK1-06 (SEQ ID NO: 231); VH3-74 (SEQ ID NO: 214)/VK1-12 (SEQ ID NO: 233); VH3-74 (SEQ ID NO: 214)/VK1-27 (SEQ ID NO: 235); VH3-74 (SEQ ID NO: 214)/VK3-20 (SEQ ID NO: 239); VH3-74 (SEQ ID NO: 214)/VL1-51 (SEQ ID NO: 252); VH5-51 (SEQ ID NO: 215)/VK1-39 (SEQ ID NO: 236); VH5-51 (SEQ ID NO: 215)/VL1-40 (SEQ ID NO: 250); VH5-51 (SEQ ID NO: 215)/VL1-51 (SEQ ID NO: 252); VH6-1 (SEQ ID NO: 216)/VK1-09 (SEQ ID NO: 232); VH6-1 (SEQ ID NO: 216)/VK3-15 (SEQ ID NO: 238); VH6-1 (SEQ ID NO: 216)/VK3-20 (SEQ ID NO: 239) and VH6-1 (SEQ ID NO: 216)/VL1-51 (SEQ ID NO: 252).
2. A collection according to claim 1 , wherein the antibodies or fragments comprise variable heavy chain and variable light chain framework regions comprising germline protein sequences of a germline protein pair, wherein said germline protein pair comprises the following properties:
i) an expression yield in Fab format of at least 2.5 mg/L;
ii) thermal stability at 70° C. or above in Fab format;
iii) monomeric content (% monomer) in Fab format of at least 98% as determined by SEC;
iv) an expression yield in IgG1 format of at least 30 mg/L;
v) thermal stability at 73° C. or above in IgG1 format; and
vii) monomeric content (% monomer) in IgG1 format of at least 99% as determined by SEC.
3. A collection according to claim 1 , wherein the antibodies or fragments thereof comprise variable heavy chain and variable light chain pairs, wherein the framework regions of the variable heavy chain and variable light chain pairs comprise the germline protein sequences of the variable heavy chain and variable light chain pairs VH1-18 (SEQ ID NO: 204)/VK3-20 (SEQ ID NO: 239); VH1-46 (SEQ ID NO: 205)/VK3-15 (SEQ ID NO: 238); VH1-46 (SEQ ID NO: 205)/VL1-51 (SEQ ID NO: 252); VH1-69*01 (SEQ ID NO: 206)/VL1-51 (SEQ ID NO: 252); VH3-07 (SEQ ID NO: 207)/VK1-12 (SEQ ID NO: 233); VH3-07 (SEQ ID NO: 207)/VK1-27 (SEQ ID NO: 235); VH3-07 (SEQ ID NO: 207)/VK3-15 (SEQ ID NO: 238); VH3-07 (SEQ ID NO: 207)/VL1-47 (SEQ ID NO: 251); VH3-07 (SEQ ID NO: 207)/VL1-51 (SEQ ID NO: 252); VH3-11 (SEQ ID NO: 208)/VL1-40 (SEQ ID NO: 250); VH3-11 (SEQ ID NO: 208)/VL1-47 (SEQ ID NO: 251); VH3-11 (SEQ ID NO: 208)/VL1-51 (SEQ ID NO: 252); VH3-11 (SEQ ID NO: 208)/VL2-23 (SEQ ID NO: 255); VH3-15 (SEQ ID NO: 209)/VK1-05 (SEQ ID NO: 230); VH3-15 (SEQ ID NO: 209)/VK1-06 (SEQ ID NO: 231); VH3-15 (SEQ ID NO: 209)/VK1-12 (SEQ ID NO: 233); VH3-15 (SEQ ID NO: 209)/VK1-27 (SEQ ID NO: 235); VH3-15 (SEQ ID NO: 209)/VK3-11 (SEQ ID NO: 237); VH3-15 (SEQ ID NO: 209)/VL1-51 (SEQ ID NO: 252); VH3-21 (SEQ ID NO: 210)/VK1-12 (SEQ ID NO: 233); VH3-23 (SEQ ID NO: 211)/VK3-15 (SEQ ID NO: 238); VH3-53 (SEQ ID NO: 213)/VK3-15 (SEQ ID NO: 238); VH3-53 (SEQ ID NO: 213)/VL2-11 (SEQ ID NO: 253); VH3-74 (SEQ ID NO: 214)/VK1-05 (SEQ ID NO: 230); VH3-74 (SEQ ID NO: 214)/VK1-06 (SEQ ID NO: 231); VH3-74 (SEQ ID NO: 214)/VK1-12 (SEQ ID NO: 233); VH3-74 (SEQ ID NO: 214)/VK3-20 (SEQ ID NO: 239); VH5-51 (SEQ ID NO: 215)/VK1-39 (SEQ ID NO: 236); VH5-51 (SEQ ID NO: 215)/VL1-40 (SEQ ID NO: 250); VH5-51 (SEQ ID NO: 215)/VL1-51 (SEQ ID NO: 252); VH6-1 (SEQ ID NO: 216)/VK1-09 (SEQ ID NO: 232); VH6-1 (SEQ ID NO: 216)/VK3-20 (SEQ ID NO: 239) and VH6-1 (SEQ ID NO: 216)/VL1-51 (SEQ ID NO: 252).
4. A collection according to claim 3 , wherein the antibodies or fragments thereof comprise variable heavy chain and variable light chain pairs, wherein the framework regions of the variable heavy chain and variable light chain pairs comprise germline protein sequences further comprising the variable heavy chain and variable light chain pairs VH3-23 (SEQ ID NO: 211)/VK1-39 (SEQ ID NO: 236); VH3-23 (SEQ ID NO: 211)/VL2-23 (SEQ ID NO: 255); and VH3-23 (SEQ ID NO: 211)/VL3-1 (SEQ ID NO: 256).
5. A collection according to claim 3 , wherein the antibodies or fragments comprise variable heavy chain and variable light chain framework regions comprising germline protein sequences of a germline protein pair, wherein said germline protein pair comprises the following properties:
i) an expression yield in Fab format of at least 2.5 mg/L;
ii) thermal stability at 70° C. or above in Fab format;
iii) monomeric content (% monomer) in Fab format of at least 99% as determined by SEC;
iv) an expression yield in IgG1 format of at least 30 mg/L;
v) thermal stability at 73° C. or above in IgG1 format;
vi) monomeric content (% monomer) in IgG1 format of at least 99% as determined by SEC, and
vii) an isoelectric point in IgG1 format of at least 8.3.
6. The collection according to claim 1 , wherein said antibodies or fragments thereof comprise one or more complementarity determining regions comprising germline protein sequences from the respective variable heavy chain and variable light chain pairs.
7. The collection according to claim 6 , wherein said antibodies or fragments thereof comprise CDR1 regions comprising germline protein sequences from the respective variable heavy chain and variable light chain pairs.
8. The collection according claim 6 , wherein said antibodies or fragments thereof comprise CDR2 regions comprising germline protein sequences from the respective variable heavy chain and variable light chain pairs.
9. The collection according to claim 1 , wherein said antibodies or fragments thereof comprise one or more complementarity determining regions comprising amino acid modifications that remove potential post translational modification sites.
10. The collection according to claim 9 , wherein said antibodies or fragments thereof comprise one or more heavy chain complementarity determining regions comprising the complementarity determining region sequences from the respective variable heavy chains depicted in SEQ ID NOs: 266-278.
11. The collection according to claim 10 , wherein said antibodies or fragments thereof comprise HCDR1 regions from the respective HCDR1 region depicted in SEQ ID NOs: 266-278.
12. The collection according to claim 10 , wherein said antibodies or fragments thereof comprise HCDR2 regions from the respective HCDR2 region depicted in SEQ ID NOs: 266-278.
13. The collection according to claim 1 , wherein said antibodies or fragments thereof comprise a FR4 region selected from the group consisting of JH4 (SEQ ID NO:293), Jκ1 (SEQ ID NO:297), and Jλ2/3 (SEQ ID NO:301).
14. The collection according to claim 1 , wherein said antibodies or fragments thereof comprise a diversified HCDR3 region.
15. The collection according to claim 1 , wherein said antibodies or fragments thereof comprise a diversified LCDR3 region.
16. The collection according to claim 1 , wherein the collection comprises at least 1×10 4 antibodies or fragments thereof.
17. The collection according to claim 1 , wherein said antibodies are selected from the groups consisting of human IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM and IgD.
18. The collection according to claim 1 , wherein said fragments of said antibodies are selected from the group consisting of Fab, F(ab′)2, Fab′, Fv, and scFv.
19. A collection of synthetic antibodies or fragments thereof, wherein the antibodies or functional fragments comprise variable heavy chain and variable light chain pairs, wherein the framework regions of the variable heavy chain and variable light chain pairs comprise germline protein sequences of the variable heavy chain and variable light chain pairs wherein the pairs are listed together: VH1-18 (SEQ ID NO: 204)/VK3-20 (SEQ ID NO: 239); VH1-46 (SEQ ID NO: 205)/VK3-15 (SEQ ID NO: 238); VH1-46 (SEQ ID NO: 205)/VL1-51 (SEQ ID NO: 252); VH1-69*01 (SEQ ID NO: 206)/VL1-51 (SEQ ID NO: 252); VH3-07 (SEQ ID NO: 207)/VK1-12 (SEQ ID NO: 233); VH3-07 (SEQ ID NO: 207)/VK1-27 (SEQ ID NO: 235); VH3-07 (SEQ ID NO: 207)/VK3-15 (SEQ ID NO: 238); VH3-07 (SEQ ID NO: 207)/VL1-47 (SEQ ID NO: 251); VH3-07 (SEQ ID NO: 207)/VL1-51 (SEQ ID NO: 252); VH3-11 (SEQ ID NO: 208)/VL1-40 (SEQ ID NO: 250); VH3-11 (SEQ ID NO: 208)/VL1-47 (SEQ ID NO: 251); VH3-11 (SEQ ID NO: 208)/VL1-51 (SEQ ID NO: 252); VH3-11 (SEQ ID NO: 208)/VL2-23 (SEQ ID NO: 255); VH3-15 (SEQ ID NO: 209)/VK1-05 (SEQ ID NO: 230); VH3-15 (SEQ ID NO: 209)/VK1-06 (SEQ ID NO: 231); VH3-15 (SEQ ID NO: 209)/VK1-12 (SEQ ID NO: 233); VH3-15 (SEQ ID NO: 209)/VK1-27 (SEQ ID NO: 235); VH3-15 (SEQ ID NO: 209)/VK3-11 (SEQ ID NO: 237); VH3-15 (SEQ ID NO: 209)/VL1-51 (SEQ ID NO: 252); VH3-21 (SEQ ID NO: 210)/VK1-12 (SEQ ID NO: 233); VH3-23 (SEQ ID NO: 211)/VK1-39 (SEQ ID NO: 236); VH3-23 (SEQ ID NO: 211)/VK3-15 (SEQ ID NO: 238); VH3-23 (SEQ ID NO: 211)/VL2-23 (SEQ ID NO: 255); VH3-23 (SEQ ID NO: 211)/VL3-1 (SEQ ID NO: 256); VH3-53 (SEQ ID NO: 213)/VK3-15 (SEQ ID NO: 238); VH3-53 (SEQ ID NO: 213)/VL2-11 (SEQ ID NO: 253); VH3-74 (SEQ ID NO: 214)/VK1-05 (SEQ ID NO: 230); VH3-74 (SEQ ID NO: 214)/VK1-06 (SEQ ID NO: 231); VH3-74 (SEQ ID NO: 214)/VK1-12 (SEQ ID NO: 233); VH3-74 (SEQ ID NO: 214)/VK3-20 (SEQ ID NO: 239); VH5-51 (SEQ ID NO: 215)/VK1-39 (SEQ ID NO: 236); VH5-51 (SEQ ID NO: 215)/VL1-40 (SEQ ID NO: 250); VH5-51 (SEQ ID NO: 215)/VL1-51 (SEQ ID NO: 252); VH6-1 (SEQ ID NO: 216)/VK1-09 (SEQ ID NO: 232); VH6-1 (SEQ ID NO: 216)/VK3-20 (SEQ ID NO: 239) and VH6-1 (SEQ ID NO: 216)/VL1-51 (SEQ ID NO: 252).
20. A collection according to claim 19 , wherein the antibodies or fragments thereof comprise variable heavy chain and variable light chain framework regions comprising germline protein sequences of a germline protein pair, wherein said germline protein pair comprises the following properties:
i) an expression yield in Fab format of at least 2.5 mg/L;
ii) thermal stability at 70° C. or above in Fab format;
iii) monomeric content (% monomer) in Fab format of at least 98% as determined by SEC;
iv) an expression yield in IgG1 format of at least 30 mg/L;
v) thermal stability at 73° C. or above in IgG1 format; and
vii) monomeric content (% monomer) in IgG1 format of at least 99% as determined by SEC.
21. The collection according to claim 19 , wherein said antibodies or fragments thereof comprise CDR1 regions comprising germline protein sequences from the respective variable heavy chain and variable light chain pairs.
22. The collection according claim 19 , wherein said antibodies or fragments thereof comprise CDR2 regions comprising germline protein sequences from the respective variable heavy chain and variable light chain pairs.
23. The collection according to claim 19 , wherein said antibodies or fragments thereof comprise HCDR1 regions from the respective HCDR1 region depicted in SEQ ID NOs: 266-278.
24. The collection according to claim 19 , wherein said antibodies or fragments thereof comprise HCDR2 regions from the respective HCDR2 region depicted in SEQ ID NOs: 266-278.
25. The collection according to claim 19 , wherein said antibodies or fragments thereof comprise a FR4 region selected from the group consisting of JH4 (SEQ ID NO:293), Jκ1 (SEQ ID NO:297), and Jλ2/3 (SEQ ID NO:301).
26. The collection according to claim 19 , wherein said antibodies or fragments thereof comprise a diversified HCDR3 region.
27. The collection according to claim 19 , wherein said antibodies or fragments thereof comprise a diversified LCDR3 region.
28. The collection according to claim 19 , wherein the collection comprises at least 1×10 4 antibodies or fragments thereof.
29. The collection according to claim 19 , wherein said antibodies are selected from the groups consisting of human IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM and IgD.
30. The collection according to claim 19 , wherein said antibody fragments antibodies are selected from the group consisting of Fab, F(ab′)2, Fab′, Fv, and scFv.