RADICALLY DIVERSE HUMAN ANTIBODY LIBRARY
Disclosed is an antibody library comprising a plurality of antibodies with non-naturally occurring combinations of complementary determining regions from memory and naïve B-cells naturally occurring in humans, and wherein the antibody library comprises a high number of functional and non-redundant antibodies. Further disclosed are methods of preparing antibody libraries with a high level of functional diversity.
1 . An antibody library that comprises a plurality of antibodies,
wherein each antibody of the plurality of antibodies comprises:
a) a VH domain that comprises a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence; and
b) a VL domain that comprises a VL-CDR1 sequence, a VL-CDR2 sequence, a VL-CDR3 sequence; and
wherein:
a) at least one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from a naïve B-cell;
b) if only one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from the naïve B-cell, then the VH-CDR3 sequence or VL-CDR3 sequence not derived from the naïve B-cell is derived from a memory cell; and
c) the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence, and VL-CDR2 sequence are derived from a memory B-cell.
2 . The antibody library of claim 1 , wherein the at least one of the VH-CDR3 sequence and the VL-CDR3 sequence derived from a naïve B-cell is a naturally occurring sequence.
3 . The antibody library of claim 1 , wherein the VH-CDR3 sequence or VL-CDR3 sequence derived from a memory cell is a naturally occurring sequence.
4 . The antibody library of claim 1 , wherein the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence, and VL-CDR2 sequence derived from a memory B cell are naturally occurring sequences.
5 . (canceled)
6 . (canceled)
7 . (canceled)
8 . The antibody library of claim 1 , wherein the VL domain is a Vκ domain or a Vλ domain.
9 . The antibody library of claim 1 , wherein the naïve B-cell is a CD27−/IgM+ B-cell or a CD27−/IgD+ B-cell.
10 . The antibody library of claim 1 , wherein the memory B-cell is selected from the group consisting of: a CD27+/IgG+ B-cell, a CD27+/IgM+ B-cell, an IgA+ B-cell, and a combination thereof.
11 . The antibody library of claim 1 , wherein the naïve B-cell and memory B-cell are from a sample comprising a plurality of naïve B-cells and memory B-cells sampled from a plurality of individuals.
12 . The antibody library of claim 11 , wherein the plurality of individuals is at least 50 individuals.
13 . The antibody library of claim 1 , wherein the plurality of antibodies are expressed on a surface of a plurality of phages.
14 . The antibody library of claim 13 , wherein the plurality of phages comprise bacteriophages or phagemids.
15 . The antibody library of claim 13 , wherein each phage of the plurality of phages comprises a nucleic acid sequence that encodes: i) an antibody of the plurality of antibodies, and ii) a gene encoding a phage coat protein.
16 . The antibody library of claim 15 , wherein the phage coat protein is a protein gIII.
17 . The antibody library of claim 15 , wherein expression of the nucleic acid sequence of each phage produces an antibody fused to a phage coat protein.
18 . The antibody library of claim 1 , wherein the VH domain further comprises framework regions selected from the group consisting of: IGHJ4, IGHV1-46, IGHV1-69, IGHV3-15, and IGHV3-23.
19 . The antibody library of claim 1 , wherein the VL domain further comprises framework regions selected from the group consisting of: IGKV1-39, IGKV2-28, IGKV3-15, and IGKV4-1.
20 . The antibody library of claim 1 , wherein the plurality of antibodies comprises at least 7.6×10 10 antibodies.
21 . The antibody library of claim 1 , wherein at least 95% of the plurality of antibodies are functional.
22 . A method of preparing an antibody library, comprising:
a) obtaining sequence information for a plurality of VH-CDR3 and VL-CDR3 sequences from a pool of naïve B-cells and sequence information for a plurality of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 sequences from a pool of memory B-cells;
b) assembling a plurality of variable light (VL) domain sequences, each VL domain sequence comprising: a VL-CDR1 sequence derived from the sequence information from memory B-cells determined in step a., a VL-CDR2 sequence derived from the sequence information from memory B-cells determined in step a., and a VL-CDR3 sequence derived from the sequence information from memory B-cells or naïve B-cells determined in step a.,
c) assembling a plurality of first nucleic acid sequences encoding a plurality of first antibodies, each first antibody comprising:
i. a variable light (VL) domain sequence assembled in step b.; and
ii. a single fixed heavy chain sequence;
d) inserting the plurality of first nucleic acid sequences into a plurality of phages;
e) expressing the plurality of first antibodies on the surface of the plurality of phages;
f) applying at least one selective pressure to the plurality of phages to produce a subset of phages comprising a subset of first nucleic acid sequences;
g) assembling a plurality of a variable heavy (VH) domain sequences, each VH domain sequence comprising: a VH-CDR1 sequence derived from the sequence information from memory B-cells determined in step a., a VH-CDR2 sequence derived from the sequence information from memory B-cells determined in step a., and a VH-CDR3 sequence derived from the sequence information from memory B-cells or naïve B-cells determined in step a., wherein at least one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from the sequence information from naïve B-cells;
h) replacing the single fixed heavy chain sequences from the subset of first nucleic acid sequences with the plurality of VH domain sequences assembled in step g. to produce a plurality of second nucleic acid sequences, each second nucleic acid sequence comprising:
i. a variable light (VL) domain sequence assembled in step b., and
ii. a variable heavy (VH) domain sequence assembled in step g.
wherein the plurality of second nucleic acid sequences encodes a plurality of second antibodies;
i) transforming a plurality of microbes with the plurality of phages to produce a plurality of transformants.
23 - 51 . (canceled)
52 . An antibody library that comprises a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises:
a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and
b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein
c) a CDR sequence is selected from the group consisting of: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence, wherein the CDR sequence is the same for each antibody of the plurality of antibodies; and
d) a unique combination of remaining CDR sequences are selected from the group consisting of: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence.
53 . The antibody library of claim 52 , wherein the CDR sequence of (c) is a VH-CDR3 sequence.
54 . The antibody library of claim 53 , wherein remaining CDR sequences of (d) are a VH-CDR1 sequence, a VH-CDR2 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence.
55 . The antibody library of claim 52 , wherein the CDR sequence of (c) is the same as a CDR sequence derived from an initial antibody clone.
56 . The antibody library of claim 54 , wherein each one of the remaining CDR sequences of (d) is present in the antibody library at a high degree of diversity.
57 . The antibody library of claim 56 , wherein the high degree of diversity comprises at least 1×10 3 different CDR sequences.
58 . (canceled)
59 . The antibody library of claim 58 , wherein the naturally occurring CDR sequence is derived from a human population.
60 . The antibody library of claim 58 , wherein the remaining CDR sequences of (d) are present in non-naturally occurring combinations for each antibody of the plurality of antibodies.
61 . The antibody library of claim 55 , wherein at least one antibody of the plurality of antibodies has at least one of the following: a higher melting temperature (Tm) as compared to an initial antibody clone, a higher affinity for a target epitope as compared to an initial antibody clone, or a higher cross-reactivity for a target epitope across two or more species as compared to an initial antibody clone.
62 . The antibody library of claim 52 , wherein at least one antibody of the plurality of antibodies has a melting temperature (Tm) that is from 50° C. to 90° C.
63 . The antibody library of claim 52 , wherein at least one antibody of the plurality of antibodies binds to a target epitope with a K d of 100 nM or less.
64 . A method for generating an antibody library, the method comprising:
(a) selecting a CDR sequence, wherein the CDR sequence is selected from the group consisting of: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence;
(b) replacing a CDR sequence for each antibody of a first antibody library with the CDR sequence selected in (a), thereby generating a second antibody library comprising a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises:
(i) the CDR sequence selected in (a); and
(ii) a unique combination of remaining CDR sequences not selected in (a), wherein the remaining CDR sequences are selected from the group consisting of: a VH-CDR1 sequence, a VH-CDR sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence.
65 - 79 . (canceled)