Collection and methods for its use
The present disclosure enables methods of identifying the VH and VL class pairs in the human immune repertoire, determining the VH and VL class pairs that are most prevalent and those having favorable biophysical properties. More specifically, the collections of the present disclosure comprise the most prevalent and/or preferred VH and VL class pairings with highly diversified CDRs.
1. A method of identifying an antibody or antibody fragment thereof specific for an immunogen comprising
a) generating a collection of antibodies or functional fragments thereof comprising variable heavy chain and variable light chain framework regions,
wherein said variable heavy chain framework regions and variable light chain framework regions comprise germline protein sequences of a germline protein pair,
wherein said germline protein pair comprises the following properties:
i) a relative display rate in Fab format comprising a value within the top 75% of Fabs sampled;
ii) an expression level in Fab format of at least 0.4 as compared to Fab VH 1-69 VLA_V11-40 AYA, the sequences of which are depicted in Table 5, Table 9, FIG. 45A and FIG. 47A ;
iii) thermal stability at 60° C. or more for at least 45 minutes in Fab format;
iv) stability in bovine or mouse serum in Fab format for greater than ten days at 37° C.;
v) an expression level in IgG format of at least 0.4 as compared to MOR03080, the CDR sequences of which are depicted in Table 31; and
vi) stability in bovine serum in IgG format for fourteen days at 37° C.;
wherein said collection of antibodies or functional fragments thereof comprises germline protein sequences of at least two different germline protein pairs,
wherein the germline protein pairs are selected from IGHV1-18 (SEQ ID NO: 65)/IGKV1-05 (SEQ ID NO: 118); IGHV1-18 (SEQ ID NO: 65)/IGLV2-23 (SEQ ID NO: 174); IGHV1-46 (SEQ ID NO: 68)/IGKV1-09 (SEQ ID NO: 121); IGHV1-46 (SEQ ID NO:68)/IGKV1-39 (SEQ ID NO: 129); IGHV1-46 (SEQ ID NO: 68)1IGKV3-15 (SEQ ID NO:153); IGHV1-69*01 (SEQ ID NO: 70)/IGKV1-05 (SEQ ID NO: 118); IGHV3-07 (SEQ ID NO: 77)/IGKV1-09 (SEQ ID NO: 121); IGHV3-07 (SEQ ID NO: 77)/IGKV1-(SEQ ID NO: 122); IGHV3-07 (SEQ ID NO: 77)/IGKV1-(SEQ ID NO: 126); IGHV3-07 (SEQ ID NO: 77)1IGKV3-(SEQ ID NO: 153); IGHV3-07 (SEQ ID NO: 77)/IGLV1-(SEQ ID NO: 167); IGHV3-07 (SEQ ID NO: 77)/IGLV2-(SEQ ID NO: 174); IGHV3-07 (SEQ ID NO: 77)/IGLV3-(SEQ ID NO: 175); IGHV3-11 (SEQ ID NO: 79)/IGKV1-(SEQ ID NO: 118); IGHV3-11 (SEQ ID NO: 79)/IGKV1-(SEQ ID NO: 119); IGHV3-11 (SEQ ID NO: 79)/IGKV1-(SEQ ID NO: 122); IGHV3-11 (SEQ ID NO: 79)/IGKV1-(SEQ ID NO: 124); IGHV3-11 (SEQ ID NO: 79)/IGKV3-(SEQ ID NO: 153); IGHV3-11 (SEQ ID NO: 79)/IGLV1-47 (SEQ ID NO: 167); IGHV3-11 (SEQ ID NO: 79)/IGLV2-23 (SEQ ID NO: 174); IGHV3-15 (SEQ ID NO: 81)/IGKV1-05 (SEQ ID NO: 118); IGHV3-15 (SEQ ID NO: 81)/IGKV1-06 (SEQ ID NO: 119); IGHV3-15 (SEQ ID NO: 81)/IGKV1-09 (SEQ ID NO: 121); IGHV3-15 (SEQ ID NO: 81)/IGKV1-12 (SEQ ID NO: 122); IGHV3-15 (SEQ ID NO: 81)/IGKV1-16 (SEQ ID NO: 124); IGHV3-15 (SEQ ID NO: 81)/IGKV1-27 (SEQ ID NO: 126); IGHV3-15 (SEQ ID NO: 81)/IGKV1-39 (SEQ ID NO: 129); IGHV3-15 (SEQ ID NO: 81)/IGKV3-15 (SEQ ID NO: 153); IGHV3-15 (SEQ ID NO: 81)/IGLV1-40 (SEQ ID NO: 165); IGHV3-15 (SEQ ID NO: 81)/IGLV1-51 (SEQ ID NO: 169); IGHV3-15 (SEQ ID NO: 81)/IGLV2-11 (SEQ ID NO: 171); IGHV3-21 (SEQ ID NO: 84)/IGKV1-06 (SEQ ID NO: 119); IGHV3-21 (SEQ ID NO: 84)/IGKV 1-12 (SEQ ID NO: 122); IGHV3-21 (SEQ ID NO: 84)/IGKV 1-27 (SEQ ID NO: 126); IGHV3-21 (SEQ ID NO: 84)/IGKV 1-39 (SEQ ID NO: 129); IGHV3-21 (SEQ ID NO: 84)/IGLV2-14 (SEQ ID NO: 172); IGHV3-21 (SEQ ID NO: 84)/IGLV2-23 (SEQ ID NO: 174); IGHV3-30 (SEQ ID NO: 86)/IGLV2-23 (SEQ ID NO: 174); IGHV3-30 (SEQ ID NO: 86)/IGLV3-1 (SEQ ID NO: 175); IGHV3-33 (SEQ ID NO: 89)/IGKV3-15 (SEQ ID NO: 153); IGHV3-33 (SEQ ID NO: 89)/IGLV2-23 (SEQ ID NO: 174); IGHV3-48 (SEQ ID NO: 93)/IGKV 1-27 (SEQ ID NO: 126); IGHV3-53 (SEQ ID NO: 95)/IGKV1-09 (SEQ ID NO: 121); IGHV3-53 (SEQ ID NO: 95)/IGKV1-12 (SEQ ID NO: 122); IGHV3-53 (SEQ ID NO: 95)/IGLV1-51 (SEQ ID NO: 169); IGHV3-53 (SEQ ID NO: 95)/IGLV2-23 (SEQ ID NO: 174); IGHV3-53 (SEQ ID NO: 95)/IGLV3-1 (SEQ ID NO: 175); IGHV3-74 (SEQ ID NO: 100)/IGKV1-06 (SEQ ID NO: 119); IGHV3-74 (SEQ ID NO: 100)/IGKV1-09 (SEQ ID NO: 121); IGHV3-74 (SEQ ID NO: 100)/IGKV1-27 (SEQ ID NO: 126); IGHV3-74 (SEO ID NO: 100)/IGKV3-15(SEQ ID NO: 153); IGHV3-74 (SEQ ID NO: 100)/IGLV1-51 (SEQ ID NO: 169); IGHV4-04 (SEQ ID NO: 102)/IGLV2-23 (SEQ ID NO: 174); IGHV4-04 (SEQ ID NO: 102)/IGLV3-1 (SEQ ID NO: 175); IGHV4-04 (SEQ ID NO: 102)/IGLV3-21 (SEQ ID NO: 182); IGHV4-39 (SEQ ID NO: 109)/IGLV2-14 (SEQ ID NO: 172); IGHV4-39 (SEQ ID NO: 109)/IGLV3-1 (SEQ ID NO: 175); IGHV5-51 (SEQ ID NO:113)/IGKV1-09 (SEQ ID NO: 121); IGHV5-51 (SEQ ID NO: 113)/IGLV2-23 (SEQ ID NO: 174); IGHV5-51 (SEQ ID NO: 113)/IGLV3-1 (SEQ ID NO: 175); IGHV6-1 (SEQ ID NO:115)/IGKV1-06 (SEQ ID NO: 119); and IGHV6-1 (SEQ ID NO: 115)/IGLV2-23 (SEQ ID NO: 174),
b) screening the collection against a specific immunogen; and
c) selecting one or more antibodies or fragments thereof specific for said immunogen.
2. The method according to claim 1 , wherein said antibodies or fragments thereof comprise human sequences.
3. The method according to claim 1 , wherein said antibodies or fragments thereof comprises germline protein sequences of at least eight different germline protein pairs.
4. The method according to claim 1 , wherein said antibodies or fragments thereof comprise one or more complementarity determining regions comprising germline protein sequences.
5. The method according to claim 1 , wherein said antibodies or fragments thereof comprise FR1, CDR1, FR2, CDR2, and FR3 comprising germline protein sequences.
6. The method according to claim 1 , where said antibodies or fragments thereof comprise a FR4 region selected from the group consisting of: JH4, JK1, and JA2/3.
7. The method according to claim 1 , wherein the antibodies or fragments thereof comprise a diversified HCDR3 region.
8. The method according to claim 1 , wherein the antibodies or fragments thereof comprise a diversified LCDR3 region.
9. The method according to claim 1 , wherein the collection comprises 1×10 4 antibodies or functional fragments thereof.
10. The method according to claim 1 , wherein said germline protein pairs comprise a relative display in Fab format comprising a value within the top 60% of Fabs sampled.
11. The method according to claim 1 , wherein said germline protein pairs comprise an expression level in Fab format of at least 0.6 as compared to Fab VH 1-69 VLA_V11-40 AYA.
12. The method according to claim 1 , wherein said germline protein pairs comprise thermal stability at 70° C. or more for at least 45 minutes in Fab format.
13. The method according to claim 1 , wherein said germline protein pairs comprise an expression level in IgG format of at least 0.6 as compared to MOR03080.
14. The method according to claim 1 , wherein said functional fragments of said antibodies are selected from the group consisting of Fab, F(ab′)2, Fab′, Fv, and scFv.