U.S. patent application number 11/416460 was filed with the patent office on 2006-11-16 for focused libraries of genetic packages.
This patent application is currently assigned to Dyax Corp., a Delaware corporation. Invention is credited to Robert Charles Ladner.
Application Number | 20060257937 11/416460 |
Document ID | / |
Family ID | 22972033 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060257937 |
Kind Code |
A1 |
Ladner; Robert Charles |
November 16, 2006 |
Focused libraries of genetic packages
Abstract
Focused libraries of vectors or genetic packages that display,
display and express, or comprise a member of a diverse family of
antibody peptides, polypeptides or proteins and collectively
display, display and express, or comprise at least a portion of the
focused diversity of the family. The libraries have length and
sequence diversities that mimic that found in native human
antibodies.
Inventors: |
Ladner; Robert Charles;
(Ijamsville, MD) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Dyax Corp., a Delaware
corporation
|
Family ID: |
22972033 |
Appl. No.: |
11/416460 |
Filed: |
May 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10026925 |
Dec 18, 2001 |
|
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11416460 |
May 1, 2006 |
|
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60256380 |
Dec 18, 2000 |
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Current U.S.
Class: |
435/7.1 ; 506/14;
506/19 |
Current CPC
Class: |
C40B 40/08 20130101;
C07K 16/00 20130101; C12N 15/1037 20130101; C40B 40/10 20130101;
C07K 16/005 20130101; C40B 40/02 20130101; C07K 2317/565
20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
C40B 30/06 20060101
C40B030/06; C40B 40/10 20060101 C40B040/10; G01N 33/53 20060101
G01N033/53 |
Claims
1. A focused library of vectors or genetic packages that display,
display and express, or comprise a member of a diverse family of
human antibody related peptides, polypeptides and proteins and
collectively display, display and express, or comprise at least a
portion of the diversity of the antibody family, the vectors or
genetic packages being characterized by variegated DNA sequences
that encode a heavy chain CDR1 selected from the group consisting
of: (1)
<1>.sub.1Y.sub.2<1>.sub.3M.sub.4<1>.sub.5,
wherein <1> is an equimolar mixture of each of amino acid
residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and
Y; (2)
(S/T).sub.1(S/G/X).sub.2(S/G/X).sub.3Y.sub.4Y.sub.5W.sub.6(S/G/X).sub.7.
wherein (S/T) is a 1:1 mixture of S and T residues, (S/G/X) is a
mixture of 0.2025 S, 0.2025, G and 0.035 of each of amino acid
residues A, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; (3)
V.sub.1S.sub.2G.sub.3G.sub.4S.sub.5I.sub.6S.sub.7<<1>.sub.8<1-
>.sub.9<1>.sub.10Y.sub.11Y.sub.12W.sub.13<1>.sub.14,
wherein <1> is an equimolar mixture of each of amino acid
residues A, D, E, F, G, H,. I, K, L, M, N, P, Q, R, S, T, V, W, and
Y; and (4) mixtures of vectors or genetic packages characterized by
any of the above DNA sequences.
2. The focused library according to claim 1, wherein HC CDR1s (1),
(2) and (3) are present in the library in the ratio
0.80:0.17:0.02.
3. A focused library of vectors or genetic packages that display,
display and express, or comprise a member of a diverse family of
human antibody related peptides, polypeptides and proteins and
collectively display, display and express, or comprise at least a
portion of the diversity of the antibody facility, the vectors or
genetic packages being characterized by variegated DNA sequences
that encode a heavy chain CDR2 selected from the group consisting
of: (1) <2>I<2><3>SGG<1>T<1>YADSVKG,
wherein <1> is an equimolar mixture of each of amino acid
residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and
Y; <2> is an equimolar mixture of each of amino acid residues
Y, R, W, V, G, and S; and <3> is an equimolar mixture of each
of amino acid residues P, S, and G or an equimolar mixture of P and
S; (2)
<1>I<4><1><1><G><5><1><1>-
<1>YADSVKG, wherein <1> is an equimolar mixture of each
of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, V, W, and Y; <4> is an equimolar mixture of residues D, I,
N, S, W, Y; and <5> is an equimolar mixture of residues S, G,
D and N; (3)
<1>I<4><1><1>G<5><1><1>YNPSLKG,
wherein <1> is an equimolar mixture of each of amino acid
residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and
Y; and <4> and <5> are as defined above; (4)
<1>I<8>S<1><1><1>GGYY<1>YAASVKG,
wherein <1> is an equimolar mixture of each amino acid
residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and
Y; <8> is 0.27 R and 0.027 of each of ADEFGHIKLMNPQSTVWY; and
(5) mixtures of vectors or genetic packages characterized by any of
the above DNA sequences.
4. The focused library according to claim 3 wherein a mixture of HC
CDR2s (1)/(2) (equimolar), (3) and (4) are present in the library
in a ratio of 0.54:0.43:0.03.
5. A focused library of vectors or genetic packages that display,
display and express, or comprise a member of a diverse family of
human antibody related peptides, polypeptides and proteins and
collectively display, display and express, or comprise at least a
portion of the diversity of the antibody family, the vectors or
genetic packages being characterized by variegated DNA sequences
that encode a heavy chain CDR3 selected from the group consisting
of: (1) YYCA21111YFDYWG, wherein 1 is an equimolar mixture of each
amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W and Y; and 2 is an equimolar mixture of K and R; (2)
YYCA2111111YFDYWG, wherein 1 is an equimolar mixture of each amino
acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W
and Y; and 2 is an equimolar mixture of K and R; (3)
YYCA211111111YFDAYTG, wherein 1 is an equimolar mixture of each
amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W and Y; and 2 is an equimolar mixture of K and R; (4)
YYCAR111S2S3111YFDYWG, wherein 1 is an equimolar mixture of each
amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W and Y; and 2 is an equimolar mixture of S and G; and 3 is an
equimolar mixture of Y and W; (5) YYCA2111CSG11CY1YFDYWG, wherein 1
is an equimolar mixture of each amino acid residues A, D, E, F, G,
H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar
mixture of K and R; (6) YYCA211S1TIFG11111YFDYWG, wherein 1 is an
equimolar mixture of each amino acid residues A, D, E, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar
mixture of K and R; (7) YYCAR111YY2S3344111YFDYWG, wherein 1 is an
equimolar mixture of each amino acid residues A, D, E, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W and Y; 2 is an equimolar mixture of
D and S; and 3 is an equimolar mixture of S and G; (8)
YYCAR1111YC2231CY111YFDYWG, wherein 1 is an equimolar mixture of
each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P,. Q, R,
S, T, V, W and Y; 2 is an equimolar mixture of S and G; and 3 is an
equimolar mixture of T, D and G; and (9) mixtures of vectors or
genetic packages characterized by any of the above DNA
sequences.
6. The focused library according to claim 5, wherein 1 in one or
all of HC CDR3s (1) through (8) is 0.095 of each of G and Y and
0.048 of each of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V,
and W.
7. The focused library according to claim 5 or 6, wherein HC CDR3s
(1) through (8) are present in the library in the following
proportions: (1) 0.10 (2) 0.14 (3) 0.25 (4) 0.13 (5) 0.13 (6) 0.11
(7) 0.04 and (8) 0.10
8. The focused library according to claim 5 or 6, wherein the HC
CDR3s (1) through (8) are present in the library in the following
proportions: (1) 0.02 (2) 0.14 (3) 0.25 (4) 0.14 (5) 0.14 (6) 0.12
(7) 0.08 and (8) 0.11
9. A focused library of vectors or genetic packages that display,
display and express, or comprise a member of a diverse family of
human antibody related peptides, polypeptides and proteins and
collectively display, display and express, or comprise at least a
portion of the diversity of the antibody family, the vectors or
genetic packages being characterized by variegated DNA sequences
that encodes a kappa light chain CDR1 selected from the group
consisting of: (1) RASQ<1>V<2><2><3>LA (2)
RASQ<1>V<2><2><2><3>LA; wherein
<1> is an equimolar mixture of amino acid residues
ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.044 of each of
ADEFGHIKLMNPQRTVWY; and <3> is 0.2Y and 0.044 each of
ADEFGHIKLMNPQRTVW and Y; and (3) mixtures of vectors or genetic
packages characterized by any of the above DNA sequences.
10. The focused library of claim 9, wherein CDR1s (1) and (2) are
present in the library in a ratio of 0.68:0.32.
11. A focused library of vectors or genetic packages that display,
display and express, or comprise a member of a diverse family of
human antibody related peptides, polypeptides and proteins and
collectively display, display and express, or comprise at least a
portion of the diversity of the antibody family, the vectors or
genetic packages being characterized by variegated DNA sequences
that encode a kappa light chain CDR2 having the sequence:
<1>AS<2>R<4><1>, wherein <1> is an
equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY;
<2> is 0.2 S and 0.044 of each of ADEFGHIKLMNPQRTVWY; and
<4> is 0.2 A and) 0.044 each of DEFGHIKLMNPQRSTVWY.
12. A focused library of vectors or genetic packages that display,
display and express, or comprise a member of a diverse family of
human antibody related peptides, polypeptides and proteins and
collectively display, display and express, or comprise at least a
portion of the diversity of the antibody family, the vectors or
genetic packages being characterized by variegated DNA sequences
that encode a kappa light chain CDR3 selected from the groups
consisting of: (1)
QQ<3><1><1><1>P<1>T, wherein
<1> is an equimolar mixture of amino acid residues
ADEFGHIKLMNPQRSTVWY; <3> is 0.2 Y and 0.044 each of
ADEFGHIKLMNPQRTVW; (2) QQ33111P, wherein 1 and 3 are as defined in
(1) above; (3) QQ3211PP1T, wherein 1 and 3 are as defined in (1)
above and 2 is 0.2 S and 0.044 each of ADEFGHIKLMNPQRTVWY; and (4)
mixtures of vectors or genetic packages characterized by any of the
above DNA sequences.
13. The focused library according to claim 12, wherein CDR3s (1),
(2) and (3) are present in the library in a ratio of
0.65:0.1:0.25.
14. A focused library of vectors or genetic packages that display,
display and express, or comprise a member of a diverse family of
human antibody related peptides, polypeptides and proteins and
collectively display, display and express, or comprise at least a
portion of the diversity of the antibody family, the vectors or
genetic packages being characterized by variegated DNA sequences
that encode a lambda light chain CDR1 selected from the group
consisting of: (1)
TG<1>SS<2>VG<1><3><2><3>VS,
wherein <1> is 0.27 T, 0.27 G and 0.027 each of
ADEFHIKLMNPQRSVWY, <2> is 0.27 D, 0.27 N and 0.027 each of
AEFGHIKLMPQRSTVWY, and <3> is 0.36 Y and 0.036 each of
ADEFGHIKLMNPQRSTVW; (2)
G<2><4>L<4><4><4><3><4><4>-
;, wherein <2> is as defined in (1) above and <4> is an
equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; and
(3) mixtures of vectors or genetic packages characterized by any of
the above DNA sequences.
15. The focused library according to claim 14, where CDR1s (1) and
(2) are present in the library in a ratio of 0.67:0.33.
16. A focused library of vectors or genetic packages that display,
display and express, or comprise a member of a diverse family of
human antibody related peptides, polypeptides and proteins and
collectively display, display and express, or comprise at least a
portion of the diversity of the antibody family, the vectors or
genetic packages being characterized by variegated DNA sequences
that encode a lambda light chain CDR2 has the sequence:
<4><4><4><2>RPS, wherein <2> is 0.27
D, 0.27 N, and 0.027 each of AEFGHIKLMPQRSTVWY and <4> is an
equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVW.
17. A focused library of vectors or genetic packages that display,
display and express, or comprise a member of a diverse family of
human antibody related peptides, polypeptides and proteins and
collectively display, display and express, or comprise at least a
portion of the diversity of the antibody family, the vectors or
genetic packages being characterized by variegated DNA sequences
that encode a lambda light chain CDR3 selected from the group
consisting of: (1)
<4><5><4><2><4>S<4><4><4>-
<4>V, wherein <2> is 0.27 D, 0.27 N, and 0.027 each of
AEFGHIKLMPQRSTVWY; <4> is an equimolar mixture of amino acid
residues ADEFGHIKLMNPQRSTVW; and <5> is 0.36 S and 0.0355
each of ADEFGHIKLMNPQRTVWY; (2)
<5>SY<1><5>S<5><1><4>V, wherein
<1> is an equimolar mixture of ADEFGHIKLMNPQRSTVWY; and
<4> and <5> are as defined in (1) above; and (3)
mixtures of vectors or genetic packages characterized by any of the
above DNA sequences.
18. The focused library according to claim 17, wherein CDR3s (1)
and (2) are present in the library in an equimolar mixture.
19. The focused library according to claim 1 or 2 further
comprising variegated DNA sequences that encode a heavy chain CDR
selected from the group consisting of: (1) one or more of the heavy
chain CDR2s defined in claim 3 or 4; (2) one or more of the heavy
chain CDR3s defined in claims 5, 6, 7, or 8; and (3) mixtures of
vectors or genetic packages characterized by (1) and (2).
20. The focused library according to claim 3 further comprising
variegated DNA sequences that encodes one or more heavy chain CDR3s
selected from the group defined in claims 5, 6, 7 or 8.
21. The focused library according to claim 19 or 20, further
comprising variegated DNA sequences that encodes a light chain CDR
selected from the group consisting of (1) one or more the kappa
light chain CDR1s defined in claim 9 or 10; (2) the kappa light
chain CDR2 defined in claim 11; (3) one or more of the kappa light
chain CDR3s defined in claim 12 or 13; (4) one or more of the kappa
light chain CDR1s defined in claim 14 or 15; (5) the lambda light
chain CDR2 defined in claim 16; (6) one or more of the lambda light
chain CDR3s defined in claim 17 or 18; and (7) mixtures of vectors
and genetic packages characterized by one or more of (1) through
(6).
22. A population of variegated DNA sequences that encode a heavy
chain CDR1 selected from the group consisting of: (1)
<1>.sub.1Y.sub.2<1>.sub.3M.sub.4<1>.sub.5,
wherein <1> is an equimolar mixture of each of amino acid
residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and
Y; (2) (S/T).sub.1(S/G/X).sub.2
(S/G/X).sub.3Y.sub.4Y.sub.5W.sub.6(S/G/X).sub.7 wherein (S/T) is a
1:1 mixture of S and T residues, (S/G/X) is a mixture of 0.2025 S,
0.2025 G and 0.035 of each of amino acid residues A, D, E, F, H, I,
K, L, M, N, P, Q, R, T, V, W, and Y; (3)
V.sub.1S.sub.2G.sub.3G.sub.4S.sub.5I.sub.6S.sub.7<1>.sub.8<1>-
.sub.9<1>.sub.10Y.sub.11Y.sub.12W.sub.13<1>.sub.14,
wherein <1> is an equimolar mixture of each of amino acid
residues A, D, E, F, G, H, I, K, L, M,. N, P, Q, R, S, T, V, W, and
Y; and (4) mixtures of variegated DNA sequences characterized by
any of the above DNA sequences.
23. The population of variegated DNA sequences according to claim
22, wherein HC CDR1s (1), (2) and (3) are present in the population
in the ratio 0.80:0.17:0.02.
24. A population of variegated DNA sequences that encode a heavy
chain CDR2 selected from the group consisting of: (1)
<2>I<2><3>SGG<1>T<1>YADSVKG, wherein
<1> is an equimolar mixture of each of amino acid residues A,
D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; <2>
is an equimolar mixture of each of amino acid residues Y, R, W, V,
G, and S; and <3> is an equimolar mixture of each of amino
acid residues P, S, and G or an equimolar mixture of P and S; (2)
<1>I<4><1><1><G><5><1><1>-
<1>YADSVKG, wherein <1> is an equimolar mixture of each
of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, V, W, and Y; <4> is an equimolar mixture of residues D, I,
N, S, W, Y; and <5> is an equimolar mixture of residues S, G,
D and N; (3)
<1>I<4><1><1>G<5><1><1>YNPSLKG,
wherein <1> is an equimolar mixture of each of amino acid
residues A, D, E, F, G, H, I, K, L, M, N, P,. Q, R, S, T, V, W and
Y; and <4>and <5> are as defined above; (4)
<1>I<8>S<1><1><1>GGYY<1>YAASVKG,
wherein <1> is an equimolar mixture of each amino acid
residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and
Y; <8> is 0.27 R and 0.027 of each of ADEFGHIKLMNPQSTVWY; and
(5) mixtures of variegated DNA sequences characterized by any of
the above DNA sequences.
25. The population of variegated DNA sequences according to claim
24, wherein a mixture of HC CDR2s (1)/(2) (equimolar), (3) and (4)
are present in the population in a ratio of 0.54:0.43:0.03.
26. A population of variegated DNA sequences that encode a heavy
chain CDR3 selected from the group consisting of: (1)
YYCA21111YFDYWG, wherein 1 is an equimolar mixture of each amino
acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W
and Y; and 2 is an equimolar mixture of K and R; (2)
YYCA2111111YFDYWG, wherein 1 is an equimolar mixture of each amino
acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W
and Y; and 2 is an equimolar mixture of K and R; (3)
YYCA211111111YFDAYTG, wherein 1 is an equimolar mixture of each
amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W and Y; and 2 is an equimolar mixture of K and R; (4)
YYCAR111S2S3111YFDYWG, wherein 1 is an equimolar mixture of each
amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W and Y; and 2 is an equimolar mixture of S and G; and 3 is an
equimolar mixture of Y and W; (5) YYCA2111CSG11CY1YFDYWG, wherein 1
is an equimolar mixture of each amino acid residues A, D, E, F, G,
H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar
mixture of K and R; (6) YYCA211S1TIFG11111YFDYWG, wherein 1 is an
equimolar mixture of each amino acid residues A, D, E, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar
mixture of K and R; (7) YYCAR111YY2S3344111YFDYWG, wherein 1 is an
equimolar mixture of each amino acid residues A, D, E, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W and Y; 2 is an equimolar mixture of
D and S; and 3 is an equimolar mixture of S and G; (8)
YYCAR1111YC2231CY111YFDYWG, wherein 1 is an equimolar mixture of
each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R,
S, T, V, W and Y; 2 is an equimolar mixture of S and G; and 3 is an
equimolar mixture of T, D and G; and (9) mixtures of variegated DNA
sequences characterized by any of the above DNA sequences.
27. The population of variegated DNA according to claim 26, wherein
1 in one or all of HC CDR3s (1) through (8) is 0.095 of each of G
and Y and 0.048 of each of A, D, E, F, H, I, K, L, M, N, P, Q, R,
S, T, V, and W.
28. The population of variegated DNA sequences according to claim
26 or 27, wherein HC CDR3s (1) through (8) are present in the
population in the following proportions: (1) 0.10 (2) 0.14 (3) 0.25
(4) 0.13 (5) 0.13 (6) 0.11 (7) 0.04 and (8) 0.10
29. The population of variegated DNA sequences according to claim
26 or 27, wherein the HC CDR3s (1) through (8) are present in the
population in the following proportions: (1) 0.02 (2) 0.14 (3) 0.25
(4) 0.14 (5) 0.14 (6) 0.12 (7) 0.08 and (8) 0.11
30. A population of variegated DNA sequences that encode a kappa
light chain CDR1 selected from the group consisting of: (1)
RASQ<1>V<2><2><3>LA (2)
RASQ<1>V<2><2><2><3>LA; wherein
<1> is an equimolar mixture of amino acid residues
ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.044 of each of
ADEFGHIKLMNPQRTVWY; and <3> is 0.2Y and 0.044 each of
ADEFGHIKLMNPQRTVW and Y; and (3) mixtures of variegated DNA
sequences characterized by any of the above DNA sequences.
31. The population of variegated DNA sequences of claim 30, wherein
CDR1s (1) and (2) are present in the population in a ratio of
0.68:0.32.
32. A population of variegated DNA sequences that encode a kappa
light chain CDR2 having the sequence:
<1>AS<2>R<4><1>, wherein <1> is an
equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY;
<2> is 0.2 S and 0.044 of each of ADEFGHIKLMNPQRTVWY; and
<4> is 0.2 A and) 0.044 each of DEFGHIKLMNPQRSTVWY.
33. A population of variegated DNA sequences that encode a kappa
light chain CDR3 selected from the groups consisting of: (1)
QQ<3><1><1><1>P<1>T, wherein
<1> is an equimolar mixture of amino acid residues
ADEFGHIKLMNPQRSTVWY; <3> is 0.2 Y and 0.044 each of
ADEFGHIKLMNPQRTVW; (2) QQ33111P, wherein 1 and 3 are as defined in
(1) above; (3) QQ3211PPlT, wherein 1 and 3 are as defined in (1)
above and 2 is 0.2 S and 0.044 each of ADEFGHIKLMNPQRTVWY; and (4)
mixtures of variegated DNA sequences characterized by any of the
above DNA sequences.
34. The population of variegated DNA sequences according to claim
33, wherein CDR3s (1) , (2) and (3) are present in the population
in a ratio of 0.65:0.1:0.25.
35. A population of variegated DNA sequences that encode a lambda
light chain CDR1 selected from the group consisting of: (1)
TG<1>SS<2>VG<1><3><2><3>VS,
wherein <1> is 0.27 T, 0.2.7 G and 0.027 each of
ADEFHIKLMNPQRSVWY, <2> is 0.27 D, 0.27 N and 0.027 each of
AEFGHIKLMPQRSTVWY, and <3> is 0.36 Y and 0.036 each of
ADEFGHIKLMNPQRSTVW; (2)
G<2><4>L<4><4><4><3><4><4>-
;, wherein <2> is as defined in (1) above and <4> is an
equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; and
(3) mixtures of variegated DNA sequences characterized by any of
the above DNA sequences.
36. The population of variegated DNA sequences according to claim
35, where CDR1s (1) and (2) are present in the population in a
ratio of 0.67:0.33.
37. A population of variegated DNA sequences that encode a lambda
light chain CDR2 has the sequence:
<4><4><4><2>RPS, wherein <2> is 0.27
D, 0.27 N, and 0.027 each of AEFGHIKLMPQRSTVWY and <4> is an
equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVW.
38. A population of variegated DNA sequences that encode a lambda
light chain CDR3 selected from the group consisting of: (1)
<4><5><4><2><4>S<4><4><4>-
<4>V, wherein <2> is 0.27 D, 0.27 N, and 0.027 each of
AEFGHIKLMPQRSTVWY; <4> is an equimolar mixture of amino acid
residues ADEFGHIKLMNPQRSTVW; and <5> is 0.36 S and 0.0355
each of ADEFGHIKLMNPQRTVWY; (2)
<5>SY<1><5>S<5><1><4>V, wherein
<1> is an equimolar mixture of ADEFGHIKLMNPQRSTVWY; and
<4> and <5> are as defined in (1) above; and (3)
mixtures of variegated. DNA sequence characterized by any of the
above DNA sequences.
39. The population of variegated DNA sequences according to claim
38, wherein CDR3s (1) and (2) are present in the population in an
equimolar mixture.
40. The population of variegated DNA sequences according to claim
22 or 23 further comprising variegated DNA sequences that encode a
heavy chain CDR selected from the group consisting of: (1) one or
more of the heavy chain CDR2s defined in claim 24 or 25; (2) one or
more of the heavy chain CDR3s defined in claims 26, 27, 28 or 29;
and (3) mixtures of variegated DNA sequence characterized by (1)
and (2).
41. The population of variegated DNA sequences according to claim
24 further comprising variegated DNA sequences that encodes one or
more heavy chain CDR3s selected from the group defined in claims
26, 27, 28 or 29.
42. The population of variegated DNA sequences according to claim
40 or 41 further comprising variegated DNA sequences that encodes a
light chain CDR selected from the group consisting of (1) one or
more the kappa light chain CDR1s defined in claim 30 or 31; (2) the
kappa light chain CDR2 defined in claim 32; (3) one or more of the
kappa light chain CDR3s defined in claim 33 or 34; (4) one or more
of the kappa light chain CDR1s defined in claim 35 or 36; (5) the
lambda light chain CDR2 defined in claim 37; (6) one or more of the
lambda light chain CDR3s defined in claim 38 or 39; and (7)
mixtures of variegated DNA sequences characterized by one or more
of (1) through (6).
43. A population of vectors comprising the variegated DNA sequences
of any one of claims 22-42.
Description
[0001] This application claims the benefit under 35 USC .sctn. 120
of U.S. provisional application 60/256,380, filed Dec. 18, 2001.
The provisional application and the Tables attached to it are
specifically incorporated by reference herein.
[0002] The present invention relates to focused libraries of
genetic packages that each display, display and express, or
comprise a member of a diverse family of peptides, polypeptides or
proteins and collectively display, display and express, or comprise
at least a portion of the focused diversity of the family. The
focused diversity of the libraries of this invention comprises both
sequence diversity and length diversity. In a preferred embodiment,
the focused diversity of the libraries of this invention is biased
toward the natural diversity of the selected family. In a more
preferred embodiment, the libraries are biased toward the natural
diversity of human antibodies and are characterized by variegation
in their heavy chain and light chain complementarity determining
regions ("CDRs").
[0003] The present invention further relates to vectors and genetic
packages (e.g., cells, spores or viruses) for displaying, or
displaying and expressing a focused diverse family of peptides,
polypeptides or proteins. In a preferred embodiment the genetic
packages are filamentous phage or phagemids or yeast. Again, the
focused diversity of the family comprises diversity in sequence and
diversity in length.
[0004] The present invention further relates to methods of
screening the focused libraries of the invention and to the
peptides, polypeptides and proteins identified by such
screening.
BACKGROUND OF THE INVENTION
[0005] It is now common practice in the art to prepare libraries of
genetic packages that individually display, display and express, or
comprise a member of a diverse family of peptides, polypeptides or
proteins and collectively display, display and express, or comprise
at least a portion of the amino acid diversity of the family. In
many common libraries, the peptides, polypeptides or proteins are
related to antibodies (e.g., single chain Fv (scFv), Fv, Fab, whole
antibodies or minibodies (i.e., dimers that consist of V.sub.H
linked to V.sub.L)). Often, they comprise one or more of the CDRs
and framework regions of the heavy and light chains of human
antibodies.
[0006] Peptide, polypeptide or protein libraries have been produced
in several ways in the prior art. See e.g., Knappik et al., J. Mol.
Biol., 296, pp. 57-86 (2000), which is incorporated herein by
references. One method is to capture the diversity of native
donors, either naive or immunized. Another way is to generate
libraries having synthetic diversity. A third method is a
combination of the first two. Typically, the diversity produced by
these methods is limited to sequence diversity, i.e., each member
of the library differs from the other members of the family by
having different amino acids or variegation at a given position in
the peptide, polypeptide or protein chain. Naturally diverse
peptides, polypeptides or proteins, however, are not limited to
diversity only in their amino acid sequences. For example, human
antibodies are not limited to sequence diversity in their amino
acids, they are also diverse in the lengths of their amino acid
chains.
[0007] For antibodies, diversity in length occurs, for example,
during variable region rearrangements. See e.g., Corbett et al., J.
Mol. Biol., 270, pp. 587-97 (1997). The joining of V genes to J
genes, for example, results in the inclusion of a recognizable D
segment in CDR3 in about half of the heavy chain antibody
sequences, thus creating regions encoding varying lengths of amino
acids. The following also may occur during joining of antibody gene
segments: (i) the end of the V gene may have zero to several bases
deleted or changed; (ii) the end of the D segment may have zero to
many bases removed or changed; (iii) a number of random bases may
be inserted between V and D or between D and J; and (iv) the 5' end
of J may be edited to remove or to change several bases. These
rearrangements result in antibodies that are diverse both in amino
acid sequence and in length.
[0008] Libraries that contain only amino acid sequence diversity
are, thus, disadvantaged in that they do not reflect the natural
diversity of the peptide, polypeptide or protein that the library
is intended to mimic. Further, diversity in length may be important
to the ultimate functioning of the protein, peptide or polypeptide.
For example, with regard to a library comprising antibody regions,
many of the peptides, polypeptides, proteins displayed, displayed
and expressed, or comprised by the genetic packages of the library
may not fold properly or their binding to an antigen may be
disadvantaged, if diversity both in sequence and length are not
represented in the library.
[0009] An additional disadvantage of prior art libraries of genetic
packages that display, display and express, or comprise peptides,
polypeptides and proteins is that they are not focused oh those
members that are based on natural occurring diversity and thus on
members that are most likely to be functional. Rather, the prior
art libraries, typically, attempt to include as much diversity or
variegation at every amino acid residue as possible. This makes
library construction time-consuming and less efficient than
possible. The large number of members that are produced by trying
to capture complete diversity also makes screening more cumbersome
than it needs to be. This is particularly true given that many
members of the library will not be functional.
SUMMARY OF THE INVENTION
[0010] One objective of this invention is focused libraries of
vectors or genetic packages that encode members of a diverse family
of peptides, polypeptides or proteins wherein the libraries encode
populations that are diverse in both length and sequence. The
diverse length comprising components that contain motifs that are
likely to fold and function in the context of the parental peptide,
polypeptide or protein.
[0011] Another object of this invention is focused libraries of
genetic packages that display, display and express, or comprise a
member of a diverse family of peptides, polypeptides and proteins
and collectively display, display and express, or comprise at least
a portion of the focused diversity of the family. These libraries
are diverse not only in their amino acid sequences, but also in
their lengths. And, their diversity is focused so as to more
closely mimic or take into account the naturally-occurring
diversity of the specific family that the library represents.
[0012] Another object of this invention is diverse, but focused,
populations of DNA sequences encoding peptides, polypeptides or
proteins suitable for display or display and expression using
genetic packages (such as phage or phagemids) or other regimens
that allow selection of specific binding components of a
library.
[0013] A further object of this invention is focused libraries
comprising the CDRs of human antibodies that are diverse in both
their amino acid sequence and in their length (examples of such
libraries include libraries of single chain Fv (scFv), Fv, Fab,
whole antibodies or minibodies (i.e., dimers that consist of
V.sub.H linked to V.sub.L)). Such regions may be from the heavy or
light chains or both and may include one or more of the CDRs of
those chains. More preferably, the diversity or variegation occurs
in all of the heavy chain and light chain CDRs.
[0014] It is another object of this invention to provide methods of
making and screening the above libraries and the peptides,
polypeptides and proteins obtained in such screening.
[0015] Among the preferred embodiments of this invention are the
following:
[0016] 1. A focused library of vectors or genetic packages that
display, display and express, or comprise a member of a diverse
family of human antibody related peptides, polypeptides and
proteins and collectively display, display and express, or comprise
at least a portion of the diversity of the antibody family, the
vectors or genetic packages being characterized by variegated DNA
sequences that encode a heavy chain CDR1 selected from the group
consisting of: [0017] (1)
<1>.sub.1Y.sub.2<1>.sub.3M.sub.4<1>.sub.5,
wherein <1> is an equimolar mixture of each of amino acid
residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and
Y; [0018] (2)
(S/T).sub.1(S/G/X).sub.2(S/G/X).sub.3Y.sub.4Y.sub.5W.sub.6(S/G/X).sub.7,
wherein (S/T) is a 1:1 mixture of S and T residues, (S/G/X) is a
mixture of 0.2025 S, 0.2025 G and 0.035 of each of amino acid
residues A, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y;
[0019] (3)
V.sub.1S.sub.2G.sub.3G.sub.4S.sub.5I.sub.6S.sub.7<1>.sub.8<1>-
.sub.9<1>.sub.10Y.sub.11Y.sub.12W.sub.13<1>.sub.14,
wherein <1> is an equimolar mixture of each of amino acid
residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and
Y; and [0020] (4) mixtures of vectors or genetic packages
characterized by any of the above DNA sequences, preferably in the
ratio: HC CDR1s (1):(2):(3)::0.80:0.17:0.02.
[0021] 2. A focused library of vectors or genetic packages that
display, display and express, or comprise a member of a diverse
family of human antibody related peptides, polypeptides and
proteins and collectively display, display and express, or comprise
at least a portion of the diversity of the antibody facility, the
vectors or genetic packages being characterized by variegated DNA
sequences that encode a heavy chain CDR2 selected from the group
consisting of: [0022] (1)
<2>I<2><3>SGG<1>T<1>YADSVKG, wherein
<1> is an equimolar mixture of each of amino acid residues A,
D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; <2>
is an equimolar mixture of each of amino acid residues Y, R, W, V,
G, and S; and <3> is an equimolar mixture of each of amino
acid residues P, S, and G or an equimolar mixture of P and S;
[0023] (2)
<1>I<4><1><1><G><5><1><1>-
<1>YADSVKG, herein <1> is an equimolar mixture of each
of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, V, W, and Y; <4> is an equimolar mixture of residues D, I,
N, S, W, Y; and <5> is an equimolar mixture of residues S, G,
D and N; [0024] (3)
<1>I<4><1><1>G<5><1><1>YNPS-
LKG, wnerein <1> is an equimolar mixture of each of amino
acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W
and Y; and <4> and <5> are as defined above; [0025] (4)
<1>I<8>S<1><1><1>GGYY<1>YAASVKG,
wherein <1> is an equimolar mixture of each amino acid
residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and
Y; <8> is 0.27 R and 0.027 of each of ADEFGHIKLMNPQSTVWY; and
[0026] (5) mixtures of vectors or genetic packages characterized by
any of the above DNA sequences, preferably in the ratio: HC CDR2s:
(1)/(2) (equimolar): (3):(4)::0.54:0.43:0.03.
[0027] 3. A focused library of vectors or genetic packages that
display, display and express, or comprise a member of a diverse
family of human antibody related peptides, polypeptides and
proteins and collectively display, display and express, or comprise
at least a portion of the diversity of the antibody family, the
vectors or genetic packages being characterized by variegated DNA
sequences that encode a heavy chain CDR3 selected from the group
consisting of: [0028] (1) YYCA21111YFDYWG, wherein 1 is an
equimolar mixture of each amino acid residues A, D, E, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar
mixture of K and R; [0029] (2) YYCA2111111YFDYWG, wherein 1 is an
equimolar mixture of each amino acid residues A, D, E, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar
mixture of K and R; [0030] (3) YYCA211111111YFDAYTG, wherein 1 is
an equimolar mixture of each amino acid residues A, D, E, F, G, H,
I, K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar
mixture of K and R; [0031] (4) YYCAR111S2S3111YFDYWG, wherein 1 is
an equimolar mixture of each amino acid residues A, D, E, F, G, H,
I, K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar
mixture of S and G; and 3 is an equimolar mixture of Y and W;
[0032] (5) YYCA2111CSG11CY1YFDYWG, wherein 1 is an equimolar
mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M,
N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar mixture of K
and R; [0033] (6) YYCA211S1TIFG11111YFDYWG, wherein 1 is an
equimolar mixture of each amino acid residues A, D, E, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar
mixture of K and R; [0034] (7) YYCAR111YY2S3344111YFDYWG, wherein 1
is an equimolar mixture of each amino acid residues A, D, E, F, G,
H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; 2 is an equimolar
mixture of D and S; and 3 is an equimolar mixture of S and G;
[0035] (8) YYCAR1111YC2231CY111YFDYWG, wherein 1 is an equimolar
mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M,
N, P, Q, R, S, T, V, W and Y; 2 is an equimolar mixture of S and G;
and 3 is an equimolar mixture of T, D and G; and [0036] (9)
mixtures of vectors or genetic packages characterized by any of the
above DNA sequences, preferably the HC CDR3s (1) through (8) are in
the following proportions in the mixture: [0037] (1) 0.10 [0038]
(2) 0.14 [0039] (3) 0.25 [0040] (4) 0.13 [0041] (5) 0.13 [0042] (6)
0.11
[0043] (7) 0.04 and [0044] (8) 0.10; and more preferably the HC
CDR3s (1) through (8) are in the following proportions in the
mixture: [0045] (1) 0.02 [0046] (2) 0.14 [0047] (3) 0.25 [0048] (4)
0.14 [0049] (5) 0.14 [0050] (6) 0.12 [0051] (7) 0.08 and [0052] (8)
0.11.
[0053] Preferably, 1 in one or all of HC CDR3s (1) through (8) is
0.095 of each of G and Y and 0.048 of each of A, D, E, F, H, I, K,
L, M, N, P, Q, R, S, T, V, and W.
[0054] 4. A focused library of vectors or genetic packages that
display, display and express, or comprise a member of a diverse
family of human antibody related peptides, polypeptides and
proteins and collectively display, display and express, or comprise
at least a portion of the diversity of the antibody family, the
vectors or genetic packages being characterized by variegated DNA
sequences that encodes a kappa light chain CDR1 selected from the
group consisting of: [0055] (1)
RASQ<1>V<2><2><3>LA [0056] (2)
RASQ<1>V<2><2><2><3>LA; wherein
<1> is an equimolar mixture of amino acid residues
ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.044 of each of
ADEFGHIKLMNPQRTVWY; and <3> is 0.2Y and 0.044 each of
ADEFGHIKLMNPQRTVW and Y; and [0057] (3) mixtures of vectors or
genetic packages characterized by any of the above DNA sequences,
preferably in the ratio CDR1s (1):(2)::0.68:0.32.
[0058] 5. A focused library of vectors or genetic packages that
display, display and express, or comprise a member of a diverse
family of human antibody related peptides, polypeptides and
proteins and collectively display, display and express, or comprise
at least a portion of the diversity of the antibody family, the
vectors or genetic packages being characterized by variegated DNA
sequences that encode a kappa light chain CDR2 having the sequence:
[0059] <1>AS<2>R<4><1>, wherein <1>
is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY;
<2> is 0.2 S and 0.044 of each of ADEFGHIKLMNPQRTVWY; and
<4> is 0.2 A and) 0.044 each of DEFGHIKLMNPQRSTVWY.
[0060] 6. A focused library of vectors or genetic packages that
display, display and express, or comprise a member of a diverse
family of human antibody related peptides, polypeptides and
proteins and collectively display, display and express, or comprise
at least a portion of the diversity of the antibody family, the
vectors or genetic packages being characterized by variegated DNA
sequences that encode a kappa light chain CDR3 selected from the
groups consisting of: [0061] (1)
QQ<3><1><1><1>P<1>T, wherein
<1> is an equimolar mixture of amino acid residues
ADEFGHIKLMNPQRSTVWY; <3> is 0.2 Y and 0.044 each of
ADEFGHIKLMNPQRTVW; [0062] (2) QQ33111P, wherein 1 and 3 are as
defined in (1) above; [0063] (3) QQ3211PP1T, wherein 1 and 3 are as
defined in (1) above and 2 is 0.2 S and 0.044 each of
ADEFGHIKLMNPQRTVWY; and [0064] (4) mixtures of vectors or genetic
packages characterized by any of the above DNA sequences,
preferably in the ratio CDR3s (1):(2):(3)::0.65:0.1:0.25.
[0065] 7. A focused library of vectors or genetic packages that
display, display and express, or comprise a member of a diverse
family of human antibody related peptides, polypeptides and
proteins and collectively display, display and express, or comprise
at least a portion of the diversity of the antibody family, the
vectors or genetic packages being characterized by variegated DNA
sequences that encode a lambda light chain CDR1 selected from the
group consisting of: [0066] (1)
TG<1>SS<2>VG<1><3><2><3>VS,
wherein <1> is 0.27 T, 0.27 G and 0.027 each of
ADEFHIKLMNPQRSVWY, <2> is 0.27 D, 0.27 N and 0.027 each of
AEFGHIKLMPQRSTVWY, and <3> is 0.36 Y and 0.036 each of
ADEFGHIKLMNPQRSTVW; [0067] (2)
G<2><4>L<4><4><4><3><4><4>-
;, wherein <2> is as defined in (1) above and <4> is an
equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; and
[0068] (3) mixtures of vectors or genetic packages characterized by
any of the above DNA sequences, preferably in the ratio CDR1s
(1):(2)::0.67:0.33.
[0069] 8. A focused library of vectors or genetic packages that
display, display and express, or comprise a member of a diverse
family of human antibody related peptides, polypeptides and
proteins and collectively display, display and express, or comprise
at least a portion of the diversity of the antibody family, the
vectors or genetic packages being characterized by variegated DNA
sequences that encode a lambda light chain CDR2 has the sequence:
[0070] <4><4><4><2>RPS, wherein <2>
is 0.27 D, 0.27 N, and 0.027 each of AEFGHIKLMPQRSTVWY and
<4> is an equimolar mixture of amino acid residues
ADEFGHIKLMNPQRSTVW.
[0071] 9. A focused library of vectors or genetic packages that
display, display and express, or comprise a member of a diverse
family of human antibody related peptides, polypeptides and
proteins and collectively display, display and express, or comprise
at least a portion of the diversity of the antibody family, the
vectors or genetic packages being characterized by variegated DNA
sequences that encode a lambda light chain CDR3 selected from the
group consisting of: [0072] (1)
<4><5><4><2><4>S<4><4><4>-
<4>V, wherein <2> is 0.27 D, 0.27 N, and 0.027 each of
AEFGHIKLMPQRSTVWY; <4> is an equimolar mixture of amino acid
residues ADEFGHIKLMNPQRSTVW; and <5> is 0.36 S and 0.0355
each of ADEFGHIKLMNPQRTVWY; [0073] (2)
<5>SY<1><5>S<5><1><4>V, wherein
<1> is an equimolar mixture of ADEFGHIKLMNPQRSTVWY; and
<4> and <5> are as defined in (1) above; and [0074] (3)
mixtures of vectors or genetic packages characterized by any of the
above DNA sequences, preferably in the ratio CDR3s
(1):(2)::1:1.
[0075] 10. A focused library comprising variegated DNA sequences
that encode a heavy chain CDR selected from the group consisting
of: [0076] (1) one or more of the heavy chain CDR1s of paragraph 1
above; [0077] (2) one or more of the heavy chain CDR2s of paragraph
2 above; [0078] (3) one or more of the heavy chain CDR3s of
paragraph 3 above; and [0079] (4) mixtures of vectors or genetic
packages characterized by (1), (2) and (3).
[0080] 11. The focused library comprising one or more of the
variegated DNA sequences that encodes a heavy chain CDR of
paragraphs 1, 2 and 3 and further comprising variegated DNA
sequences that encodes a light chain CDR selected from the group
consisting of [0081] (1) one or more the kappa light chain CDR1s of
paragraph 4; [0082] (2) the kappa light chain CDR2 of paragraph 5;
[0083] (3) one or more of the kappa light chain CDR3s of paragraph
6; [0084] (4) one or more of the kappa light chain CDR1s of
paragraph 7; [0085] (5) the lambda light chain CDR2 of paragraph 8;
[0086] (6) one or more of the lambda light chain CDR3s of paragraph
9; and [0087] (7) mixtures of vectors and genetic packages
characterized by one or more of (1) through (6).
[0088] 12. A population of variegated DNA sequences as described in
paragraphs 1-11 above.
[0089] 13. A population of vectors comprising the variegated DNA
sequences as described in paragraphs 1-11 above.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0090] Antibodies ("Ab") concentrate their diversity into those
regions that are involved in determining affinity and specificity
of the Ab for particular targets. These regions may be diverse in
sequence or in length. Generally, they are diverse in both ways.
However, within families of human antibodies the diversities, both
in sequence and in length, are not truly random. Rather, some amino
acid residues are preferred at certain positions of the CDRs and
some CDR lengths are preferred. These preferred diversities account
for the natural diversity of the antibody family.
[0091] According to this invention, and as more fully described
below, libraries of vectors and genetic packages that more closely
mirror the natural diversity, both in sequence and in length, of
antibody families, or portions thereof are prepared and used.
Human Antibody Heavy Chain Sequence and Length Diversity
[0092] (a) Framework
[0093] The heavy chain ("HC") Germ-Line Gene (GLG) 3-23 (also known
as VP-47) accounts for about 12% of all human Abs and is preferred
as the framework in the preferred embodiment of the invention. It
should, however, be understood that other well-known frameworks,
such as 4-34, 3-30, 3-30.3 and 4-30.1, may also be used without
departing from the principles of the focused diversities of this
invention.
[0094] In addition, JH4 (YFDYWGQGTLVTUSS) occurs more often than
JH3 in native antibodies. Hence, it is preferred for the focused
libraries of this invention. However, JH3 (AFDIWGQGTMVTVSS) could
as well be used.
[0095] (b) Focused Length Diversity: CDR1, 2 and 3
[0096] (i) CDR1
[0097] For CDR1, GLGs provide CDR1s only of the lengths 5, 6, and
7. Mutations during the maturation of the V-domain gene, however,
can lead to CDR1s having lengths as short as 2 and as long as 16.
Nevertheless, length 5 predominates. Accordingly, in the preferred
embodiment of this invention, the preferred HC CDR1 is 5 amino
acids, with less preferred CDR is having lengths of 7 and 14. In
the most preferred libraries of this invention, all three lengths
are used in proportions similar to those found in natural
antibodies.
[0098] (ii) CDR2
[0099] GLGs provide CDR2s only of the lengths 15-19, but mutations
during maturation may result in CDR2s of lengths from 16 to 28
amino acids. The lengths 16 and 17 predominate in mature Ab genes.
Accordingly, length 17 is the preferred length for HC CDR2 of the
present invention. Less preferred HC CDR2s of this invention have
lengths 16 and 19. In the most preferred focused libraries of this
invention, all three lengths are included in proportions similar to
those found in natural antibody families.
[0100] (iii) CDR3
[0101] HC CDR3s vary in length. About half of human HCs consist of
the components: V::nz::D::ny::JHn where V is a V gene, nz is a
series of bases (mean 12) that are essentially random, D is a D
segment, often with heavy editing at both ends, ny is a series of
bases (mean 6) that are essentially random, and JH is one of the
six JH segments, often with heavy editing at the 5' end. The D
segments appear to provide spacer segments that allow folding of
the IgG. The greatest diversity is at the junctions of V with D and
of D with JH.
[0102] In the preferred libraries of this invention both types of
HC CDR3s are used. In HC CDR3s that have no identifiable D segment,
the structure is V::nz::JHn where JH is usually edited at the 51
end. In HC CDR3s that have an identifiable D segment, the structure
is V::nz::D::ny::JHn.
[0103] (c) Focused Sequence Diversity: CDR1, 2 and 3
[0104] (i) CDR1
[0105] In 5 amino acid length CDR1, examination of a 3D model of a
humanized Ab showed that the side groups of residues 1, 3, and 5
were directed toward the combining pocket. Consequently, in the
focused libraries of this invention, each of these positions may be
selected from any of the native amino acid residues, except
cysteine ("C"). Cysteine can form disulfide bonds, which are an
important component of the canonical Ig fold. Having free thiol
groups could, thus, interfere with proper folding of the HC and
could lead to problems in production or manipulation of selected
Abs. Thus, in the focused libraries of this invention cysteine is
excluded from positions 1, 3 and 5 of the preferred 5 amino acid
CDR1s. The other 19 natural amino acids residues may be used at
positions 1, 3 and 5. Preferably, each is present in equimolar
ratios in the variegated libraries of this invention.
[0106] 3D modeling also suggests that the side groups of residue 2
in a 5 amino acid CDR1 are directed away from the combining-pocket.
Although this position shows substantial diversity, both in GLG and
mature genes, in the focused libraries of this invention this
residue is preferably Tyr (Y) because it occurs in 681/820 mature
antibody genes. However, any of the other native amino acid
residues, except Cys (C), could also be used at this position.
[0107] For position 4, there is also some diversity in GLG and
mature antibody genes. However, almost all mature genes have
uncharged hydrophobic amino acid residues: A, G, L, P, F, M, W, I,
V, at this position. Inspection of a 3D model also shows that the
side group of residue 4 is packed into the innards of the HC. Thus,
in the preferred embodiment of this invention which uses framework
3-23, residue 4 is preferably Met because it is likely to fit very
well into the framework of 3-23. With other frameworks, a similar
fit consideration is used to assign residue 4.
[0108] Thus, the most preferred HC CDR1 of this invention consists
of the amino acid sequence <1>Y<1>M<1>where
<1> can be any one of amino acid residues: A, D, E, F, G, H,
I, K, L, M, N, P, Q, R, S, T,. V, W, Y (not C), preferably present
at each position in an equimolar amount. This diversity is shown in
the context of a framework 3-23:JH4 in Table 1. It has a diversity
of 6859-fold.
[0109] The two less preferred HC CDR1s of this invention have
length 7 and length 14. For length 7, a preferred variegation is
(S/T).sub.1(S/G/<1>).sub.2(S/G/<1>).sub.3Y.sub.4Y.sub.5W.sub.-
6(S/G/<1>).sub.7; where (S/T) indicates an equimolar mixture
of Ser and Thr codons; (S/G/<1>) indicates a mixture of
0.2025 S, 0.2025 G, and 0.035 for each of A, D, E, F, H, I, K, L,
M, N, P, Q, R, T, V, W, Y. This design gives a predominance of Ser
and Gly at positions 2, 3, and 7, as occurs in mature HC genes. For
length 14, a preferred variegation is
VSGGSIS<1><1><1>YYW<1>, where <1> is
an equimolar mixture of the 19 native amino acid residues, except
Cys (C).
[0110] The DNA that encodes these preferred HC CDR1s is preferably
synthesized using trinucleotide building blocks so that each amino
acid residue is present in essentially equimolar or other described
amounts. The preferred codons for the <1> amino acid residues
are gct, gat, gag, ttt, ggt, cat, att, aag, ctt, atg, aat, cct,
cag, cgt, tct, act, gtt, tgg, and tat. Of course, other codons for
the chosen amino acid residue could also be used.
[0111] The diversity oligonucleotide (ON) is preferably synthesized
from BspEI to BstXI (as shown in Table 1) and can, therefore, be
incorporated either by PCR synthesis using overlapping ONs or
introduced by ligation of BspEI/BstXI-cut fragments. Table 2 shows
the oligonucleotides that embody the specified variegations of the
preferred length 5 HC CDR1s of this invention. PCR using ON-R1V1vg,
ON-R1top, and ON-R1bot gives a dsDNA product of 73 base pairs,
cleavage with BspEI and BstXI trims 11 and 13 bases from the ends
and provides cohesive ends that can be ligated to similarly cut
vector having the 3-23 domain shown in Table 1. Replacement of
ON-R1V1vg with either ONR1V2vg or ONR1V3vg (see Table 2) allows
synthesis of the two alternative diversity patterns--the 7 residue
length and the 14 residue length HC CDR1.
[0112] The more preferred libraries of this invention comprise the
3 preferred HC CDR1 length diversities. Most preferably, the 3
lengths should be incorporated in approximately the ratios in which
they are observed in antibodies selected without reference to the
length of the CDRs. For example, one sample of 1095 HC genes have
the three lengths present in the ratio:
L=5:L=7:L=14::820:175:23::0.80:0.17:0.02. This is the preferred
ratio in accordance with this invention.
[0113] (ii) CDR2
[0114] Diversity in HC CDR2 was designed with the same
considerations as for HC CDR1: GLG sequences, mature sequences and
3D structure. A preferred length for CDR2 is 17, as shown in Table
1. For this preferred 17 length CDR2, the preferred variegation in
accordance with the invention is:
<2>I<2><3>SGG<1>T<1>YADSVKG, where
<2>indicates any amino acid residue selected from the group
of Y, R, W, V, G and S (equimolar mixture), <3> is P, S and G
or P and S only (equimolar mixture), and <1> is any native
amino acid residue except C (equimolar mixture).
[0115] ON-R2V1vg shown in Table 3 embodies this diversity pattern.
It is preferably synthesized so that fragments of dsDNA containing
the BstXI and XbaI site can be generated by PCR. PCR with
ON-R2V1vg, ON-R2top, and ONR2bot gives a dsDNA product of 122 base
pairs. Cleavage with BstXI and XbaI removes about 10 bases from
each end and produces cohesive ends that can be ligated to
similarly cut vector that contains the 3-23gene shown in Table
1.
[0116] In an alternative embodiment for a 17 length HC CDR2, the
following variegation may be used:
<1>I<4><1><1>G<5><1><1><1>-
;YADSVKG, where <1> is as described above for the more
preferred alternative of HC CDR2; <4> indicates an equimolar
mixture of DINSWY, and <5> indicates an equimolar mixture of
SGDN. This diversity pattern is embodied in ON-R2V2vg shown in
Table 3. Preferably, the two embodiments are used in equimolar
mixtures in the libraries of this invention.
[0117] Other preferred HC CDR2s have lengths 16 and 19. Length 16:
<1>I<4><1><1>G<5<1><1>YNPSLKG;
Length 19:
<1>I<8>S<1><1><1>GGYY<1>YAASVKG,
wherein <1> is an equimolar mixture of all native amino acid
residues except C; <4> is a equimolar mixture of DINSWY;
<5> is an equimolar mixture of SGDN; and <8> is 0.27 R
and 0.027 of each of residues ADEFGHIKLMNPQSTVWY. Table 3 shows
ON-R2V3vg which embodies a preferred CDR2 variegation of length 16
and ON-R2V4vg which embodies a preferred CDR2 variegation of length
19. To prepare these variegations ON-R2V3vg may be PCR amplified
with ON-R2top and ON-R2bo3 and ON-R2V4vg may be PCR amplified with
ON-R2top and ON-R2-bo4. See Table 3. In the most preferred
embodiment of this invention, all three HC CDR2 lengths are used.
Preferably, they are present in a ratio
17:16:19::579:464:31::0.54:0.43:0.03.
[0118] (iii) CDR3
[0119] The preferred libraries of this invention comprise several
HC CDR3 components. Some of these will have only sequence
diversity. Others will have sequence diversity with embedded D
segments to extend the length, while also incorporating sequences
known to allow Igs to fold. The HC CDR3 components of the preferred
libraries of this invention and their diversities are depicted in
Table 4: Components 1-8.
[0120] This set of components was chosen after studying the
sequences of 1383 human HC sequences. The proposed components are
meant to fulfill the following goals:
[0121] 1) approximately the same distribution of lengths as seen in
native Ab genes;
[0122] 2) high level of sequence diversity at places having high
diversity in native Ab genes; and
[0123] 3) incorporation of constant sequences often seen in native
Ab genes.
[0124] Component 1 represents all the genes having lengths 0 to 8
(counting from the YYCAR motif at the end of FR3 to the WG
dipeptide motif near the start of the J region, i.e., FR4).
Component 2 corresponds the all the genes having lengths 9 or 10.
Component 3 corresponds to the genes having lengths 11 or 12 plus
half the genes having length 13. Component 4 corresponds to those
having length 14 plus half those having length 13. Component 5
corresponds to the genes having length 15 and half of those having
length 16. Component 6 corresponds to genes of length 17 plus half
of those with length 16. Component 7 corresponds to those with
length 18. Component 8 corresponds to those having length 19 and
greater. See Table 4.
[0125] For each HC CDR3 residue having the diversity <1>,
equimolar ratios are preferably not used. Rather, the following
ratios are used 0.095 [G and Y] and 0.048 [A, D, E, F, H, I, K, L,
M, N, P, Q, R, S, T, V, and W]. Thus, there is a double dose of G
and Y with the other residues being in equimolar ratios. For the
other diversities, e.g., KR or SG, the residues are present in
equimolar mixtures.
[0126] In the preferred libraries of this invention the eight
components are present in the following fractions: 1 (0.10), 2
(0.14), 3 (0.25), 4 (0.13), 5 (0.13), 6 (0.11), 7 (0.04) and 8
(0.10). See Table 4.
[0127] In the more preferred embodiment of this invention, the
amounts of the eight components is adjusted because the first
component is not complex enough to justify including it as 10% of
the library. For example, if the final library were to have
1.times.10.sup.9 members, then 1.times.10.sup.8 sequences would
come from component 1, but it has only 2.6.times.10.sup.5 CDR3
sequences so that each one would occur in .about.385 CDR1/2
contexts. Therefore, the more preferred amounts of the eight
components are 1(0.02), 2(0.14), 3(0.25), 4(0.14), 5(0.14),
6(0.12), 7(0.08), 8(0.11). In accordance with the more preferred
embodiment component 1 occurs in .about.77 CDR1/2 contexts and the
other, longer CDR3s occur more often.
[0128] Table 5 shows vgDNA that embodies each of the eight HC CDR3
components shown in Table 4. In Table 5, the oligonucleotides (ON)
Ctop25, CtprmA, CBprmB, and CBot25 allow PCR amplification of each
of the variegated ONs (vgDNA): C1t08, C2t10, C3t12, C4t14, C5t15,
C6t17, C7t18, and C8t19. After amplification, the dsDNA can be
cleaved with AflII and BstEII (or KpnI) and ligated to similarly
cleaved vector that contains the remainder of the 3-23 domain.
Preferably, this vector already contains diversity in one, or both,
of CDR1 and CDR2 as disclosed herein. Most preferably, it contains
diversity in both the CDR1 and CDR2 regions. It is, of course, to
be understood that the various diversities can be incorporated into
the vector in any order.
[0129] Preferably, the recipient vector originally contains a
stuffer in place of CDR1, CDR2 and CDR3 so that there will be no
parental sequence that would then occur in the resulting library.
Table 6 shows a version of the V3-23 gene segment with each CDR
replaced by a short segment that contains both stop codons and
restriction sites that will allow specific cleavage of any vector
that does not have the stuffer removed. The stuffer can either be
short and contain a restriction enzyme site that will not occur in
the finished library, allowing removal of vectors that are not
cleaved by both AflII and BstEII (or KpnI) and religated.
Alternatively, the stuffer could be 200-400 bases long so that
uncleaved or once cleaved vector can be readily separated from
doubly cleaved vector.
Human Antibody Light Chain: Sequence and Length Diversity
[0130] (i) Kappa Chain
[0131] (a) Framework
[0132] In the preferred embodiment of this invention, the kappa
light chain is built in an A27 framework with a JK1 region. These
are the most common V and J regions in the native genes. Other
frameworks, such as 012, L2, and A11, and other J regions, such as
JK4, however, may be used without departing from the scope of this
invention.
[0133] (b) CDR1
[0134] In native human kappa chains, CDR1s with lengths of 11, 12,
13, 16, and 17 were observed with length 11 being predominant and
length 12 being well represented. Thus, in the preferred
embodiments of this invention LC CDR1s of length 11 and 12 are used
in an and mixture similar to that observed in native antibodies),
length 11 being most preferred. Length 11 has the following
sequence: RASQ<1>V<2><2><3>LA and Length 12
has the following sequence:
RASQ<1>V<2><2><2><3>LA, wherein
<1> is an equimolar mixture of all of the native amino acid
residues, except C, <2> is 0.2 S and 0.044 of each of
ADEFGHIKLMNPQRTVWY, and <3> is 0.2 Y and 0.044 each of A, D,
E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W and Y. In the most
preferred embodiment of this invention, both CDR1 lengths are used.
Preferably, they are present in a ratio of
11:12::154:73::0.68:0.32.
[0135] (c) CDR2
[0136] In native kappa, CDR2 exhibits only length 7. This length is
used in the preferred embodiments of this invention. It has the
sequence <1>AS<2>R<4><1>, wherein <1>
is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY;
<2> is 0.2 S and 0.004 of each of ADEFGHIKLMNPQRTVWY; and
<4> is 0.2 A and 0.044 of each of DEFGHIKLMNPQRSTUWY.
[0137] (d) CDR3
[0138] In native kappa, CDR3 exhibits lengths of 1, 4, 6, 7, 8, 9,
10, 11, 12, 13, and 19. While any of these lengths and mixtures of
them can be employed in this invention, we prefer lengths 8, 9 and
10, length 9 being more preferred. For the preferred Length 9, the
sequence is QQ<3><1><1><1>P<1>T,
wherein <1> is an equimolar mixture of amino acid residues
ADEFGHIKLMNPQRSTVWY and <3> is 0.2 Y and 0.044 each of
ADEFGHIKLMNPQRSVW. Length 8 is preferably QQ33111P and Length 10 is
preferably QQ3211PP1T, wherein 1 and 3 are as defined for Length 9
and 2 is S (0.2) and 0.044 each of ADEFGHIKLMNPQRTVWY. A mixture of
all 3 lengths being most preferred (ratios as in native
antibodies), i.e., 8:9:10::28:166:63::0.1:0.65:0.25.
[0139] Table 7 shows a kappa chain gene of this invention,
including a PlacZ promoter, a ribosome-binding site, and signal
sequence (M13 III signal). The DNA sequence encodes the GLG amino
acid sequence, but does not comprise the GLG DNA sequence.
Restriction sites are designed to fall within each framework region
so that diversity can be cloned into the CDRs. XmaI and EspI are in
FR1, SexAI is in FR2, RsrII is in FR3, and KpnI (or Acc65I) are in
FR4. Additional sites are provided in the constant kappa chain to
facilitate construction of the gene.
[0140] Table 7 also shows a suitable scheme of variegation for
kappa. In CDR1, the most preferred length 11 is depicted. However,
most preferably both lengths 11 and 12 are used. Length 12 in CDR1
can be construed by introducing codon 51 as <2> (i.e. a
Ser-biased mixture). CDR2 of kappa is always 7 codons. Table 7
shows a preferred variegation scheme for CDR2. Table 7 shows a
variegation scheme for the most preferred CDR3 (length 9). Similar
variegations can be used for CDRs of length 8 and 10. In the
preferred embodiment of this invention, those three lengths (8, 9
and 10) are included in the libraries of this invention in the
native ratios, as described above.
[0141] Table 9 shows series of diversity oligonucleotides and
primers that may be used to construct the kappa chain diversities
depicted in Table 7.
[0142] (ii) Lambda Chain
[0143] (a) Framework
[0144] The lambda chain is preferably built in a 2a2 framework with
an L2J region. These are the most common V and J regions in the
native genes. Other frameworks, such as 31, 4b, 1a and 6a, and
other J regions, such as L1J, L3J and L7J, however, may be used
without departing from the scope of this invention.
[0145] (b) CDR1
[0146] In native human lambda chains, CDR1s with length 14
predominate, lengths 11, 12 and 13 also occur. While any of these
can be used in this invention, lengths 11 and 14 are preferred. For
length 11 the sequence is:
TG<2><4>L<4><4><4><3><4><-
;4>and for Length 14 the sequence is:
TG<1>5S<2>VG<1><3><2><3>VS,
wherein <1> is 0.27 T, 0.27 G and 0.027 each of
ADEFHIKLMNPQRSVWY; <2> is 0.27 D, 0.27 N and 0.027 each of
AEFGHIKLMPQRSTVWY; <3> is 0.36 Y and 0.0355 each of
ADEFGHIKLMNPQRSTVW; and <4> is an equimolar mixture of amino
acid residues ADEFGHIKLMNPQRSTVWY. Most preferably, mixtures
(similar to those occurring in native antibodies) preferably, the
ratio is 11:14::23:46::0.33: 0.67 of the three lengths are
used.
[0147] (c) CDR2
[0148] In native human lambda chains, CDR2s with length 7 are by
far the most common. This length is preferred in this invention.
The sequence of this Length 7 CDR2 is
<4><4><4><2>RPS, wherein <2> is 0.27
D, 0.27 N, and 0.027 each of AEFGHIKLMPQRSTVWY and <4> is an
equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVW.
[0149] (d) CDR3
[0150] In native human lambda chains, CDR3s of length 10 and 11
predominate, while length 9 is also common. Any of these three
lengths can be used in the invention. Length 11 is preferred and
mixtures of 10 and 11 more preferred. The sequence of Length 11 is
<4><5><4><2><4>S<4><4><4>-
<4>V, where <2> and <4> are as defined for the
lambda CDR1 and <5> is 0.36 S and 0.0355 each of
ADFFGHIKLMNPQRTVWY. The sequence of Length 10 is
<5>SY<1><5>S<5><1><4>V, wherein
<1> is an equimolar mixture of ADEFGHIKLMNPQRSTVWY; and
<4> and <5> are as defined for Length 11. The preferred
mixtures of this invention comprise an equimolar mixture of Length
10 and Length 11. Table 8 shows a preferred focused lambda light
chain diversity in accordance with this invention.
[0151] Table 9 shows a series of diversity oligonucleotides and
primers that may be used to construct the lambda chain diversities
depicted in Table 7.
Method of Construction of the Genetic Package
[0152] The diversities of heavy chain and the kappa and lambda
light chains are best constructed in separate vectors. First a
synthetic gene is designed to embody each of the synthetic variable
domains. The light chains are bounded by restriction sites for
ApaLI (positioned at the very end of the signal sequence) and AscI
(positioned afer the stop codon). The heavy chain is bounded by
SfiI (positioned within the Pe1B signal sequence) and NotI
(positioned in the linker between CH1 and the anchor protein).
Signal sequences other than Pe1B may also need, e.g., a M13 pIII
signal sequence.
[0153] The initial genes are made with "stuffer" sequences in place
of the desired CDRs. A "Stuffer" is a sequence that is to be cut
away and replaced by diverse DNA but which does not allow
expression of a functional antibody gene. For example, the stuffer
may contain several stop codons and restriction sites that will not
occur in the correct finished library vector. For example, in Table
10, the stuffer for CDR1 of kappa A27 contains a StuI site. The
vgDNA for CDR1 is introduced as a cassette from EspI, XmaI, or
AflII to either SexAI or KasI. After the ligation, the DNA is
cleaved with StuI; there should be no StuI sites in the desired
vectors.
[0154] The sequences of the heavy chain gene with stuffers is
depicted in Table 6. The sequences of the kappa light chain gene
with stuffers is depicted in Table 10. The sequence of the lambda
light chain gene with stuffers is depicted in Table 11.
[0155] In another embodiment of the present intention the
diversities of heavy chain and the kappa or lambda light chains are
constructed in a single vector or genetic packages (e.g., for
display or display and expression) having appropriate restriction
sites that allow cloning of these chains. The processes to
construct such vectors are well known and widely used in the art.
Preferably, a heavy chain and Kappa light chain library and a heavy
chain and lambda light chain library would be prepared separately.
The two libraries, most preferably, will then be mixed in equimolar
amounts to attain maximum diversity.
[0156] Most preferably, the display is had on the surface of a
derivative of M13 phage. The most preferred vector contains all the
genes of M13, an antibiotic resistance gene, and the display
cassette. The preferred vector is provided with restriction sites
that allow introduction and excision of members of the diverse
family of genes, as cassettes. The preferred vector is stable
against rearrangement under the growth conditions used to amplify
phage.
[0157] In another embodiment of this invention, the diversity
captured by the methods of the present invention may be displayed
and/or expressed in a phagemid vector (e.g., pCES1) that displays
and/or expresses the peptide, polypeptide or protein. Such vectors
may also be used to store the diversity for subsequent display
and/or expression using other vectors or phage.
[0158] In another embodiment of this invention, the diversity
captured by the methods of the present invention may be displayed
and/or expressed in a yeast vector. TABLE-US-00001 TABLE 1 3-23:JH4
CDR1/2 diversity = 1.78 .times. 10.sup.8 FR1 (VP47/V3-23)....... 20
21 22 23 24 25 26 27 28 29 30 A M A E V Q L L E S G ctgtctgaac cc
atg gcc gaa|gtt|caa|ttg|tta|gag|tct|ggt| Scab...... NcoI.... MfeI
-------------FR1--------------------------------- 31 32 33 34 35 36
37 38 39 40 41 42 43 44 45 G G L V Q P G G S L R L S C A
|ggc|ggt|cct|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gtc| Sites
of variegation <1> <1> <1> <1> 6859-fold
diversity
----FR1------------------->|.....CDR1..................|---FR2------
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 A S G F T F S - Y - M
- W V R |gct|tcc|gga|ttc|act|ttc|tct|-|tac|-|atg|-|tgg|gtt|cgc|
BspEI BsiWI BstXI. Sites of variegation-><2> <2>
<3>
-------FR2-------------------------------->|...CDR2.......... 61
62 63 64 65 66 67 68 69 70 71 72 73 74 75 Q A P G K G L E W V S - I
- - |caa|gct|cct|ggt|aaa|ggt|ttg|gag|tgg|gtt|tct| - |atc| - | - |
...BstXI <1> <1> 25992-fold diversity in CDR2
....CDR2............................................|---FR3--- 76
77 78 79 80 81 82 83 84 85 86 87 88 89 90 S G G - T - Y A D S V K G
R F |tct|ggt|ggc| - |act| - |tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc|
-------FR3--------------------------------------------------- 91 92
93 94 95 96 97 98 99 100 101 102 103 104 105 T I S R D N S K N T L
Y L Q M
|act|atc|tct|aga|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| XbaI
---FR3---------------------------------------------------->| 106
107 108 109 110 111 112 113 114 115 116 117 118 119 120 N S L R A E
D T A V Y Y C A K
|aac|agc|tta|agg|gct|gag|tac|acc|gct|gtc|tac|tac|tgc|gcc|aaa| AflII
.......CDR3.................| Replaced by the various components!
121 122 123 124 125 126 127 D Y E G T G Y
|gac|tat|gaa|ggt|act|ggt|tat|
|------FRV----(JH4)------------------------------------------ Y F D
Y W G Q G T L V T V S S
|tat|ttc|gat|tat|tgg|ggt|caa|ggt|acc|ctg|gtc|acc|gtc|tct|agt|...
KpnI BstEII <1> = Codons for ADEFGHIKLMNPQRSTVWY (equimolar
mixture) <2> = Codons for YRWVGS (equimolar mixture)
<3> = Codons for PS or PS and G (equimolar mixture)
[0159] TABLE-US-00002 TABLE 2 OligoNucleotides used to variegate
CDR1 of human HC CDR1 - 5 residues (ON-R1V1vg):
5'ct|tcc|gga|ttc|act|ttc|tct|<1>|tac|<1>|atg|<1>|tgg|gt-
t|cgc|caa|gct|cct|gg|-3' (ON-R1top):
5'-cctactgtct|tcc|gga|ttc|act|ttc|tct-3' (ON-R1bot) [RC]:
5'-tgg|gtt|cgc|caa|gct|cct|ggttgctcactc-3' CDR1 - 7 residues
(ON-R1V2vg):
5'-ct|tcc|gga|ttc|act|ttc|tct|<6>|<7>|<7>|tac|tac|tgg|&-
lt;7>|tgg|gtt|cgc|caa|gct| cct|gg-3' CDR1 - 14 residues
(ON-R1V3vg):
5'-ct|tcc|gga|ttc|act|ttc|tct|atc|agc|ggt|ggt|tct|atc|tcc|<1>|<1-
>|<1>|-
tac|tac|tgg|<1>|tgg|gtt|cgc|caa|gct|cct|gg-3' <1> =
Codons of ADEFGHIKLMNPQRSTVWY 1:1 <6> = Codons for ST, 1:1
<7> = 0.2025 (Codons for SG) + 0.035 (Codons for
ADEFHIKLMNPQRTVWY) <1> = Codons for ADEFGHIKLMNPQRSTVWY
1:1
[0160] TABLE-US-00003 TABLE 3 Oligonucleotides used to variegate
CDR2 of human HC CDR2 - 17 residues (ON-R2V1vg):
5'-ggt|ttg|gag|tgg|gtt|tct|<2>|atc|<2>|<3>|tct|ggt|ggc|-
<1>|act|<1>|tat|gct|- gac|tcc|gtt|aaa|gg-3' (ON-R2top):
5'-ct|tgg|gtt|cgc|caa|gct|cct|ggt|aaa|ggt|ttg|gag|tgg|gtt|tct-3'
(ON-R2bot) [RC]:
5'-tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc|act|atc|tct|aga|ttcctgtcac-3'
(ON-R2V2vg):
5'-ggt|ttg|gag|tgg|gtt|tct|<1>|atc|<4>|<1>|<1>|gg-
t|<5>|<1>|<1>|<1>|tat|gct|-
gac|tcc|gtt|aaa|gg-3' CDR2 - 16 residues (ON-R2V3vg):
5'-ggt|ttg|gag|tgg|gtt|tct|<1>|atc|<4>|<1>|<1>|gg-
t| <5>|<1>|<1>|tat|aac|cct|tcc|ctt|aag|gg-3'
(ON-R2bo3) [RC]:
5'-tat|aac|cct|tcc|ctt|aag|ggt|cgc|ttc|act|tct|aga|ttcctgtcac-3'
CDR2 - 19 residues (ON-R2V4vg):
5'-fft|ttg|gag|tgg|gtt|tct|<1>|atc|<8|agt|<1>|<1>|
<1>|ggt|ggt|act|act|<1>|tat|gcc|gct|tcc|gtt|aag|gg-3'
(ON-R2bo4) [RC]:
5'-tat|gcc|gct|tcc|gtt|aag|ggt|cgc|ttc|act|atc|tct|aga|ttcctgtcac-3'
<1> = Codons for A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, V, W and Y (equimolar mixture) <2> = Codons for Y, R, W,
V, G and S (equimolar mixture) <3> = Codons for P and S
(equimolar mixture) or P, S and G (equimolar mixture) <4>
=Codons for DINSWY (equimolar mixture) <5> =Codons for SGDN,
(equimolar mixture) <1>, <2>, <3>, <4> and
<5> are as defined above <8> is 0.27 R and 0.027 each
of ADEFGHIKLMNPQSTVWY
[0161] TABLE-US-00004 TABLE 4 Preferred Components of HC CDR3
Preferred Fraction of Adjusted Component Length Complexity Library
Fraction 1 YYCA21111YFDYWG. 8 2.6 .times. 10.sup.5 .10 .02 (1 = any
amino acid residue, except C; 2 = K and R) 2 YYCA2111111YFDYWG. 10
9.4 .times. 10.sup.7 .14 .14 (1 = any amino acid residue, except C;
2 = K and R 3 YYCA211111111YFDYTG. 12 3.4 .times. 10.sup.10 .25 .25
(1 = any amino acid residue, except C; 2 = K and R 4
YYCAR111S2S3111YFDYWG. 14 1.9 .times. 10.sup.8 .13 .14 (1 = any
amino acid residue, except C; 2 = S and G 3 = Y and W) 5
YYCA2111CSG11CY1YFDYWG. 15 9.4 .times. 10.sup.7 .13 .14 (1 = any
amino acid residue, except C; 2 = K and R 6
YYCA211S1TIFG11111YFDYWG. 17 1.7 .times. 10.sup.10 .11 .12 (1 = any
amino acid residue, except C; 2 = K and R 7
YYCAR111YY2S33YY111YFDYWG. 18 3.8 .times. 10.sup.8 .04 .08 (1 = any
amino acid residue, except C; 2 = D or G; 3 = S and G) 8
YYCAR1111YC2231CY111YFDYWG. 19 2.0 .times. 10.sup.11 .10 .11 (1 =
any amino acid residue, except C; 2 = S and G; 3 = T, D and G)
[0162] TABLE-US-00005 TABLE 5 Oligonucleotides used to variegate
the eight components of HC CDR3 (Ctop25):
5'-gctctggtcaac|tta|agg|gct|gag|g-3' (CtprmA):
5'-gctctggtcaac|tta|agg|gct|gag|gac|acc|gct|gtc|tac|tac|tgc|gcc--
3' AflII... (CBprmB) [RC]:
5'-|tac|ttc|gat|tac|tgg|ggc|caa|ggt|acc|ctg|gtc|acc|tcgctccacc-3'
BstEII... (CBot25) [RC]: 5'-|ggt|acc|ctg|gtc|acc|tcgctccacc-3' The
20 bases at 3' end of CtprmA are identical to the most 5' 20 bases
of each of the vgDNA molecules. Ctop25 is identical to the most 5'
25 bases of CtprmA. The 23 most 3' bases of CBprmB are the reverse
complement of the most 3' 23 bases of each of the vgDNA molecules.
CBot25 is identical to the 25 bases at the 5' end of CBprmB.
Component 1 (C1t08):
5'-cc|gct|gtc|tac|tac|tgc|ggc|<2>|<1>|<1>|<1-
>|<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3' Component 2
(C2t10):
5'-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|<1-
>|<1>|<1>|<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3'
Component 3 (C3t12):
5'-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|<1-
>|<1>|<1>|<1>|<1>|<1>|tac|ttc||gat|tac|-
tgg|ggc|caa|gg-3' Component 4 (C4t140):
5'-cc|gct|gtc|tac|tac|tgc|gcc|cgt|<1>|<1>|<1>|-
tct|<2>|tct|<3>|<1>|<1>|<1>|tac|ttc|gat|-
tac|tgg|ggc|caa|gg-3' Component 5 (C5t15):
5'-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|<1-
>|tgc|tct|ggt|<1>|<1>|tgc|tat|<1>|tac|-
ttc|gat|tac|tgg|ggc|caa|gg-3' Component 6 (C6t17):
5'-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|tct&l-
t;1>|act|atc|ttc|ggt|<1>|<1>|<1>|<1>|-
<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3' Component 7 (C7t18):
5'-cc|gct|gtc|tac|tac|tgc|ggc|cgt|<1>|<1>|<1>|t-
at|tac|<2>|tct|<3>|<3>|tac|tat|-
<1>|<1>|<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3'
Component 8 (c8t19):
5'-cc|gct|gtc|tac|tac|tgc|gcc|cgt|<1>|<1>|<1>|&-
lt;1>|tat|tgc|<2>|<2>|<3>|<1>|tgc|tat|-
<1>|<1>|<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3'
<1> = 0.095 Y + 0.095 G + 0.048 each of the residues
ADEFHIKLMNPQRSTVW, no C; <2> = K and R (equimolar mixture)
<1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no
C; <2> = K and R (equimolar mixture) <1> = 0.095 Y +
0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C; <2> = K and
R (equimolar mixture) <1> = 0.095 Y + 0.095 G + 0.048 each of
ADEFHIKLMNPQRSTVW, no C; <2> = S and G (equimolar mixture);
<3> = Y and W (equimolar mixture) <1> = 0.095 Y + 0.095
G + 0.048 each of ADEFHIKLMNPQRSTVW, no C; <2> = K and R
(equimolar mixture) <1> = 0.095 Y + 0.095 G + 0.048 each of
ADEFHIKLMNPQRSTVW, no C; <2> = K and R (equimolar mixture)
<1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no
C; <2> = D and G (equimolar mixture); <3> = S and G
(equimolar mixture) <1> = 0.095 Y + 0.095 G + 0.048 each of
ADEFHIKLMNPQRSTVW, no C; <2> = S and G (equimolar mixture);
<3> = TDG (equimolar mixture);
[0163] TABLE-US-00006 TABLE 6 3-23::JH4 Stuffers in place of CDRs
FR1 (DP47/V3-23) 20 21 22 23 24 25 26 27 28 29 30 A M A E V Q L L E
S G ctgtctgaac cc atg gcc gaa|gtt|caa|ttg|tta|gag|tct|ggt|
Scab...... NcoI.... MfeI
--------------FR1-------------------------------------------- 31 32
33 34 35 36 37 38 39 40 41 42 43 44 45 G G L V Q P G G S L R L S C
A |ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct|
----FR1-------------------->|...CDR1 stuffer....|---FR2------ 46
47 48 49 50 51 52 53 54 55 56 57 58 59 60 A S G F T F S S Y A | | W
V R |gct|tcc|gaa|ttc|act|ttc|tct|tcg|tac|gct|tag|taa|tgg|gtt|cgc|
BspEI BsiWI BstXI.
-------FR2-------------------------------->|...CDR2 stuffer. 61
62 63 64 65 66 67 68 69 70 71 72 73 74 75 Q A P G K G L E W V S | p
r | |caa|gct|cct|ggt|aaa|ggt|ttg|gag|tgg|gtt|tct|taa|cct|agg|tag|
... BstXI AvrII.. ...CDR2
stuffer....................................|---FR3--- 91 92 93 94
95 96 97 98 99 100 101 102 103 104 105 T I S R D N S K N T L Y L Q
M |act|atc|tct|aga|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|
XbaI ---FR3-----------..> CDR3 Stuffer------------->| 106 107
108 109 110 N S L R A |aac|agc|tta|agg|gct|tag taa agg cct taa
AflII StuI... |----- FR4
---(JH4)------------------------------------------ Y F D Y W G Q G
T L V T V S S
|tat|ttc|gat|tat|tgg|ggt|caa|ggt|acc|ctg|gtc|acc|gtc|tct|agt| KpnI
BstEII
[0164] TABLE-US-00007 TABLE 7 A27:JH1 Human Kappa light chain gene
gaggacc attgggcccc ctccgagact ctcgagcgca Scab...... Eco0109I XhoI..
ApaI. acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
tttatgcttc ..-35.. Plac ..-10. cggctcgtat gttgtgtgga attgtgagcg
gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt tggagccttt
tttttggaga ttttcaac Pf1MI....... Hind III M13 III signal sequence
(AA se|)--------------------------> 1 2 3 4 5 6 7 8 9 10 11 12
13 14 15 M K K L L F A I P L V V P F Y gtg aag aag ctc cta ttt gct
atc ccg ctt gtc gtt ccg ttt tac --
Signal-->FR1------------------------------------------> 16 17
18 19 20 21 22 23 24 25 26 27 28 29 30 S H S A Q S V L T Q S P G T
L |agc|cat|agt|gca|caa|tcc|gtc|ctt|act|caa|tct|cct|ggc|act|ctt|
ApaLI... ----- FR1 ------------------------------------->|
CDR1------> 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 S L S P
G E R A T L S C R A S
|tcg|cta|agc|ccg|ggt|gaa|cgt|gct|acc|tta|agt|tgc|cgt|gct|tcc|
EspI..... AflII... XmaI.... (CDR1 installed as AflII-(SexAI or
KasI) cassette.) For the most preferred 11 length codon 51 (XXX) is
omitted; for the preferred 12 length this codon is <2>
-------- CDR1 --------------------->|--- FR2 -------------->
<1> <2> <2> xxx <3> 46 47 48 49 50 51 52 53
54 55 56 57 58 59 60 Q - V - - - - L A W Y Q Q K P |cag| - |gtt| -
| - | - | - |ctt|gct|tgg|tat|caa|cag|aaa|cct| SexAI... CDR2
installed as (SexAI or KasI) to (BamHI or RsrII) cassette.) ----
FR2 ------------------------->|------- CDR2 ---------->
<1> <2> <4> 61 62 63 64 65 66 67 68 69 70 71 72
73 74 75 G Q A P R L L I Y - A S - R -
|ggt|cag|gcg|ccg|cgt|tta|ctt|att|tat| - |gct|tct| - |cgc| - |
SexAI.... KasI.... CDR2-->|--- FR3
-------------------------------------------> <1> 76 77 78
79 80 81 82 83 84 85 86 87 88 89 90 - G I P D R F S G S G S G T D |
- |ggg|atc|ccg|gac|cgt|ttc|tct|ggc|tct|ggt|tca|ggt|act|gac|
BamHI... RsrII..... ------ FR3
------------------------------------------------> 91 92 93 94 95
96 97 98 99 100 101 102 103 104 105 F T L T I S R L E P E D F A V
|ttt|acc|ctt|act|att|tct|aga|ttg|gaa|cct|gaa|gac|ttc|gct|gtt|
XbaI... For CDR3 (Length 8): QQ33111P 1 and 3 as defined for Length
9 For CDR3 (Length 10): QQ3211PP1T 1 and 3 as defined for Length 9
2 S (0.2) and 0.044 each of ADEFGHIKLMNPQRTVWY CDR3 installed as
XbaI to (StyI or BsiWI) cassette.
----------->|----CDR3-------------------------->|-----FR4--->
<3> <1> <1> <1> <1> 106 107 108 109
110 111 112 113 114 115 116 117 118 119 120 Y Y C Q Q - - - - P - T
F G Q |tat|tat|tgc|caa|cag| - | - | - | - |cct| - |act|ttc|ggt|caa|
BstXI.......... -----FR4------------------->| <--------
Ckappa ----------- 121 122 123 124 125 126 127 128 129 130 131 132
133 134 G T K V E I K R T V A A P S |gt|acc|aag|gtt|gaa|atc|aag|
|cgt|acg|gtt|gcc|gct|cct|agt| StyI.... BsiWI.. 135 136 137 138 139
140 141 142 143 144 145 146 147 148 149 V F I F P P S D E Q L K S G
T |gtg|ttt|atc|ttt|cct|cct|tct|gac|gaa|caa|ttg|aag|tca|ggt|act|
MfeI... 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164
A S V V C L L N N F Y P R E A
|gct|tct|gtc|gta|tgt|ttg|ctc|aac|aat|ttc|tac|cct|cgt|gaa|gct|
BssSI... 165 166 167 168 169 170 171 172 173 174 175 176 177 178
179 K V Q W K V D N A L Q S G N S
|aaa|gtt|cag|tgg|aaa|gtc|gat|aac|gcg|ttg|cag|tcg|ggt|aac|agt|
MluI.... 180 181 182 183 184 185 186 187 188 189 190 191 192 193
194 Q E S V T E Q D S K D S T Y S
|caa|gaa|tcc|gtc|act|gaa|cag|gat|agt|aag|gac|tct|acc|tac|tct| 195
196 197 198 199 200 201 202 203 204 205 206 207 208 209 L S S T L T
L S K A D Y E K H
|ttg|tcc|tct|act|ctt|act|tta|tca|aag|gct|gat|tat|gag|aag|cat| 210
211 212 213 214 215 216 217 218 219 220 221 222 223 224 K V Y A C E
V T H Q G L S S P
|aag|gtc|tat|GCt|TGC|gaa|gtt|acc|cac|cag|ggt|ctg|agc|tcc|cct|
SacI.... 225 226 227 228 229 230 231 232 233 234 V T K S F N R G E
C . . |gtt|acc|aaa|agt|ttc|aac|cgt|ggt|gaa|tgc|taa|tag ggcgcgcc
DsaI.... AscI.... BssHII acgcatctctaa gcggccgc aacaggaggag NotI....
For CDR1: <1> ADEFGHIKLMNPQRSTVWY 1:1 <2> S (0.2)
ADEFGHIKLMNPQRTVWY (0.044 each) <3> Y (0.2)
ADEFGHIKLMNPQRSTVW (0.044 each) For CDR2: <1>
ADEFGHIKLMNPQRSTVWY 1:1 <2> S (0.2) ADEFGHIKLMNPQRTVWY (0.044
each) <4> A (0.2) DEFGHIKLMNPQRSTVWY (0.044 each) For CDR3
(Length 9): <1> ADEFGHIKLMNPQRSTVWY 1:1 <3> Y (0.2)
ADEFGHIKLMNPQRTVW (0.044 each)
[0165] TABLE-US-00008 TABLE 8 2a2 JH2 Human lambda-chain gene
gaggaccatt gggcccc ttactccgtgac Scab...... Eco0109I ApaI..
-----------FR1--------------------------------------------> 1 2
3 4 5 6 7 8 9 10 11 12 13 14 15 S A Q S A L T Q P A S V S G S P G
agt|gca|caa|tcc|gct|ctc|act|cag|cct|gct|agc|gtt|tcc|ggg|tca|cct|ggt|
ApaLI... NheI... BstEII... SexAI..... T G <1> S S <2> V
G ------FR1------------------> |-----CDR1-------------------- 16
17 18 19 20 21 22 23 24 25 26 27 28 29 30 Q S I T I S C T G - S S -
V G |caa|agt|atc|act|att|tct|tgt|aca|ggt| - |tct|tct| - gtt|ggc|
BsrGI.. <1> <3> <2> <3> V S = vg Scheme #1,
length = 14
-----CDR1------------->|--------FR2------------------------- 31
32 33 34 35 36 37 38 39 40 41 42 43 44 45 - - - - V S W Y Q Q H P G
K A | - | - | - | - |gtt|tct|tgg|tat|caa|caa|cac|ccg|ggc|aag|gcg|
XmaI.... KasI AvaI.... <4> <4> <4> <2> R P
S --FR2------------------>
|------CDR2--------------->|----FR3- 46 47 48 49 50 51 52 53 54
55 56 57 58 59 60 P K L M I Y - - - - R P S G V
|ccg|aag|ttg|atg|atc|tac| - | - | - | - |cgt|cct|tct|ggt|gtt|
KasI.... FR3 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 S N R F S
G S K S G N T A S L
|agc|aat|cgt|ttc|tcc|gga|tct|aaa|tcc|ggt|aat|acc|gca|agc|tta|
BspEI.. HindIII. BsaBI........(blunt)
-------FR3------------------------------------------------->1 76
77 78 79 80 81 82 83 84 85 86 87 88 89 90 T I S G L Q A E D E A D Y
Y C |act|atc|tct|ggt|ctg|cag|gct|gaa|gac|gag|gct|gac|tac|tat|tgt|
PstI... <4> <5> <4> <2> <4> S
<4> <4> <4> <4> V
-----CDR3-------------------------------->|---FR4---------- 91
92 93 94 95 96 97 98 99 100 101 102 103 104 105 - - - - - S - - - -
V F G G G | - | - | - | - | - |tct| - | - | - | -
|gtc|ttc|ggc|ggt|ggt| KpnI... ------FR4--------------> 106 107
108 109 110 111 112 113 114 115 116 117 118 119 120 T K L T V L G Q
P K A A P S V
|acc|aaa|ctt|act|gtc|ctc|ggt|caa|cct|aag|gct|gct|cct|tcc|gtt|
KpnI... HincII.. Bsu36I... 121 122 123 124 125 126 127 128 129 130
131 132 133 134 135 T L F P P S S E E L Q A N K A
|act|ctc|ttc|cct|cct|agt|tct|gaa|gag|ctt|caa|gct|aac|aag|gct|
SapI...... 136 137 138 139 140 141 142 143 144 145 146 147 148 149
150 T L V C L I S D F Y P G A V T
|act|ctt|gtt|tgc|ttg|atc|agt|gac|ttt|tat|cct|ggt|gct|gtt|act|
BclI.... 151 152 153 154 155 156 157 158 159 160 161 162 163 164
165 V A W K A D S S P V K A G V E
|gtc|gct|tgg|aaa|gcc|gat|tct|tct|cct|gtt|aaa|gct|ggt|gtt|gag|
BsmBI... 166 167 168 169 170 171 172 173 174 175 176 177 178 179
180 T T T P S K Q S N N K Y A A S
|acg|acc|act|cct|tct|aaa|caa|tct|aac|aat|aag|tac|gct|gcg|agc|
BsmBI.... SacI.... 181 182 183 184 185 186 187 188 189 190 191 192
193 194 195 S Y L S L T P E Q W K S H K S
|tct|tat|ctt|tct|ctc|acc|cct|gaa|caa|tgg|aag|tct|cat|aaa|tcc|
SacI... 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
Y S C Q V T H E G S T V E K T
|tat|tcc|tgt|caa|gtt|act|cat|gaa|ggt|tct|acc|gtt|gaa|aag|act|
BspHI... 211 212 213 214 215 216 217 218 219 V A P T E C S . .
|gtt|gcc|cct|act|gag|tgt|tct|tag|tga|ggcgcgcc AscI.... BssHII
aacgatgttc aag gcggccgc aacaggaggag NotI.... Scab....... For CDR1
(length 14): <1> = 0.27 T, 0.27 G, 0.027 each of
ADEFHIKLMNPQRSVWY, no C <2> = 0.27 D, 0.27 N, 0.027 each of
AEFGHIKLMPQRSTVWY, no C <3> = 0.36 Y, 0.0355 each of
ADEFGHIKLMNPQRSTVW, no C A second Vg scheme for CDR1 gives segments
of length 11:
T.sub.22G<2><4>L<4><4><4><3><4>-
<4> where <4> 32 e|uimolar mixture of each of
ADEFGHIKLMNPQRSTVWY, no C <3> 32 as defined above for the
alternative CDR1 For CDR2: <2> and <4> are the same
variegation as for CDR1 CDR3 (Length 11): <2> and
<4>are the same variegation as for CDR1 <5> = 0.36 S,
0.0355 each of ADEFGHIKLMNPQRTVWY no C CDR3 (Length 10): <5>
SY <1> <5> S <5> <1> <4> V <1>
is an equimolar mixture of ADEFGHIKLMNPQRSTVWY, no C <4> and
<5> are as defined for Length 11
[0166] TABLE-US-00009 TABLE 9 Oligonucleotides For Kappa and Lambda
Light Chain Variegation (Ctop25):
5'-gctctggtcaac|tta|agg|gct|gag|g-3' (CtprmA):
5'-gctctggtcaac|tta|agg|gct|gag|gac|acc|gct|gtc|tac|tac|tgc|gcc--
3' AflII... (CBprmB) [RC]:
5'-|tac|ttc|gat|tac|ttg|ggc|caa|ggt|acc|ctg|gtc|acc|tcgctccacc-3'
BstEII... (CBot25) [RC]: 5'-|ggt|acc|ctg|gtc|acc|tcgctccacc-3'
Kappa chains: CDR1 ("1"), CDR2 ("2"), CDR3 ("3") CDR1 (Ka1Top610):
5'-ggtctcagttg|cta|agc|ccg|ggt|gaa|cgt|gct|acc|tta|agt|tgc|cgt|gct|tcc|ca-
g-3' (Ka1STp615): 5'-ggtctcagttg|cta|agc|ccg|ggt|g-3' (Ka1Bot620)
[RC]:
5'-ctt|gct|tgg|tat|caa|cag|aaa|cct|ggt|cag|gcg|ccaagtcgtgtc-3'
(Ka1SB625) [RC]: 5'-cct|ggt|cag|gcg|ccaagtcgtgtc-3' (Ka1vg600):
5'-gct|acc|tta|agt|tgc|cgt|gct|tcc|cag-
|<1>|gtt|<2>|<2>|<3>|ctt|gct|tgg|tat|caa|cag|aaa|-
cc-3' (Ka1vg600-12): 5'-gct|acc|tta|agt|tgc|cgt|gct|tcc|cag-
|<1>|gtt<2>|<2>|<2>|<3>|ctt|gct|tgg|tat|caa-
|cag|aaa|cc-3' CDR2 (Ka2Tshort657):
5'-cacgagtccta|cct|ggt|cag|gc-3' (Ka2Tlong655):
5'-cacgagtccta|cct|ggt|cag|gdg|ccg|cgt|tta|ctt|att|tat-3'
(Ka2Bshort660): [RC]: 5'-|gac|cgt|ttc|tct|ggt|tctcacc-3'
|cgc|<4>|<1>|ggg|atc|ccg|gac|cgt|ttc|tct|ggt|tctcacc-3'
CDR3 (Ka3Tlon672):
5'-gacgagtccttct|aga|ttg|gaa|cct|gaa|gac|ttc|gct|gtt|tat|tat|tgc|caa|c-3'
(Ka3BotL682) [RC]:
5'-act|ttc|ggt|caa|ggt|acc|aag|gtt|gaa|atc|aag|cgt|acg|tcacaggtgag-3'
(Ka3Bsho694) [RC]: 5'-gaa|atc|aag|cgt|acg|tcacaggtgag-3'
(Ka3vg670): 5'-gac|ttc|gct|gtt|-
|tat|tat|tgc|caa|cag|<3>|<1>|<1>a<1>|cct|<1>-
;|act|ttc|ggt|caa|- |ggt|acc|aag|gtt|g-3' (Ka3vg670-8):
5'-gac|ttc|gct|gtt|-
|tat|tat|tgc|caa|cag|<3>|<3>|<1>|<1>|<1>|cc-
t|ttc|ggt|caa|- |ggt|acc|aag|gtt|g-3' (Ka3vg670-10):
5'-gac|ttc|gct|gtt|tat|-
|tat|tgc|caa|cag|<3>|<2>|<1>|<1>|cct|cct|<1>-
;|act|ttc|ggt|caa|- |ggt|acc|aag|gtt|g-3' Lambda Chains: CDR1
("1"), CDR2 ("2"), CDR3 ("3") CDR1 (Lm1TPri75):
5'-gacgagtcctgg|tca|cct|ggt|-3' (Lm1tlo715):
5'-gacgagtcctgg|tca|cct|ggt|caa|agt|atc|act|att|tct|tgt|aca|ggt-3'
(Lm1blo724) [rc]:
5'-gtt|tct|tgg|tat|caa|caa|cac|ccg|ggc|aag|gcg|agatcttcacaggtgag-3'
(Lm1bsh737) [rc]: 5'-gc|aag|gcg|agatcttcacaggtgag-3' (Lm1vh710b):
5'-gt|atc|act|att|tct|tgt|aca|ggt|<2>|<4>|ctc|<4>|<4-
>|<4>|-
|<3>|<4>|<4>|tgg|tat|caa|caa|cac|cc-3-'
(Lm1vh710):
5'-gt|atc|act|att|tct|tgt|aca|ggt|<1>|tct|tct|<2>|gtt|ggc|-
|<1>|<3>|<2>|<3>|gtt|tct|tgg|tat|caa|caa|cac|cc-3-
' CDR2 (Lm2TSh757): 5'-gagcagaggac|ccg|ggc|aag|gc-3' (Lm2TLo753):
5'-gagcagaggac|ccg|ggc|aag|gcg|ccg|aag|ttg|atg|atc|tac|-3'
(Lm2BLo762) [RC]:
5'-cgt|cct|tct|ggt|gtc|agc|aat|cgt|ttc|tcc|gga|tcacaggtgag-3'
(Lm2BSh765) [RC]: 5'-cgt|ttc|tcc|gga|tcacaggtgag-3' (Lm2vg750):
5'-g|ccg|aag|ttg|atg|atc|tac|-
<4>|<4>|<4>|<2>|cgt|cct|tct|ggt|gtc|agc|aat|c-3'
CDR3 (Lm3TSh822): 5'-ctg|cag|gct|gaa|gac|gag|gct|gac-3'
(Lm3TLo819): 5'-ctg|cag|gct|gaa|gac|gag|gct|gac|tac|tat|tgt|-3'
(Lm3BLo825) [RC]:
5'-gtc|ttc|ggc|ggt|ggt|acc|aaa|ctt|act|gtc|ctc|ggt|caa|cct|aag|g-
acacaggtgag-3' (Lm3BSh832) [RC]:
5'-c|ggt|caa|cct|aag|gacacaggtgag-3' (Lm3vg817):
5'-gac|gag|gct|gac|tac|tat|tgt|-
|<4>|<5>|<4>|<2>|<4>|tct|<4>|<4>-
;|<4>|<4>|- Gtc|ttc|ggc|ggt|ggt|acc|aaa|ctt|ac-3'
(Lm3vg817-10): 5'- gac|gag|gct|gac|tac|tat|tgt|-
|<5>|agc|tat|<1>|<5>|tct<5>|<1>|<4>|g-
tc|ttc|ggc|ggt|ggt|- |acc|aaa|ctt|ac-3'
[0167] TABLE-US-00010 TABLE 10 A27:JH1 Kappa light chain gene with
stuffers in place of CDRs Each stuffer contains at least one stop
codon and a restriction site that will be uni|ue within the
diversity vector. gaggacc attgggcccc ctccgagact ctcgagcgca
Scab.....Eco0109I ApaI. XhoI.. acgcaattaa tgtgagttag ctcactcatt
aggcacccca ggctttacac tttatgcttc ..-35.. Plac ..-10. cggctcgtat
gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatgac catgatta
cgccaagctt tggagccttt tttttggaga ttttcaac PflMI....... Hind3. M13
III signal se|uence (AA se|)--------------------------> 1 2 3 4
5 6 7 8 9 10 11 12 13 14 15 M K K L L F A I P L V V P F Y gtg aag
aag ctc cta ttt gct atc ccg ctt gtc gtt ccg ttt tac --Signal-->
FR1-------------------------------------------> 16 17 18 19 20
21 22 23 24 25 26 27 28 29 30 S H S A Q S V L T Q S P G T L
|agc|cat|agt|gca|caa|tcc|gtc|ctt|act|caa|tct|cct|ggc|act|ctt|
ApaLI... ----- FR1
--------------------------------->|-------Stuffer-> 31 32 33
34 35 36 37 38 39 40 41 42 43 S L S P G E R A T L S | |
|tcg|cta|agc|ccg|ggt|gaa|cgt|gct|acc|tta|agt|tag|taa|gct|ccc|
EspI..... AflII... XmaI.... - Stuffer for CDR1-->FR2 ---------
FR2 ------>|-----------Stuffer for CDR2 59 60 61 62 63 64 65 66
K P G Q A P R
|agg|cct|actt|tga|tct|g|aaa|cct|ggt|cag|gcg|ccg|cgt|taa|tga|aagcgctaatggcc-
aacagtg StuI... SexAI... KasI.... AfeI.. MscI.. Stuffer-->|---
FR3 -----------------------------------------> 76 77 78 79 80 81
82 83 84 85 86 87 88 89 90 T G I P D R F S G S G S G T D
|act|ggg|atc|ccg|gac|ccgt|ttc|tct|ggc|tct|ggt|tca|ggt|act|gac|
BamHI... RsrII..... ------ FR3 ----->----------------STUFFER for
CDR3-----------------> 91 92 93 94 95 96 97 F T L T I S R | |
|ttt|acc|ctt|act|att|tct|aga|taa|tga| gttaac tag acc tacgta acc tag
XbaI... HpaI.. SnaBI. -----------------CDR3
stuffer------------------>|-----FR4---> 118 119 120 F G Q
|ttc|ggt|caa| -----FR4------------------->| <------ Ckappa
------------- 121 122 123 124 125 126 127 128 129 130 131 132 133
134 G T K V E I K R T V A A P S |ggt|acc|aag|gtt|gaa|atc|aag|
|cgt|acg|gtt|gcc|gct|cct|agt| StyI.... BsiWI.. 135 136 137 138 139
140 141 142 143 144 145 146 147 148 149 V F I F P P S D E Q L K S G
T |gtg|ttt|atc|ttt|cct|cct|tct|gac|gaa|caa|ttg|aag|tca|ggt|act|
MfeI... acgcatctctaa gcggccgc aacaggaggag NotI.... EagI..
[0168] TABLE-US-00011 TABLE 11 2a2:JH2 Human lambda-chain gene with
stuffers in place of CDRs gaggaccatt gggcccc ttactccgtgac
Scab...... Eco0109I ApaI..
----------FR1--------------------------------------------> 1 2 3
4 5 6 7 8 9 10 11 12 13 14 15 S A Q S A L T Q P A S V S G S P G
agt|gca|caa|tcc|gct|ctc|act|cag|cct|gct|agc|gtt|tcc|ggg|tca|cct|ggt|
ApaLI... NheI... BstEII... SexAI....
------FR1------------------> |-----stuffer for CDR1--------- 16
17 18 19 20 21 22 23 Q S I T I S C T
|caa|agt|atc|act|att|tct|tgt|aca|tct tag tga ctc BsrGI..
-----Stuffer--------------------------->-------FR2---------->
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 R S | | P | H P G K A
aga tct taa tga ccg tag cac|ccg|ggc|aag|gcg| BglII XmaI....
KasI..... AvaI.... --|--------------Stuffer for CDR2
-----------------------------> P |ccg|taa|tga|atc tcg tac g
ct|ggt|gtt| KasI.... BsiWI...
-------FR3---------------------------------------------------- 61
62 63 64 65 66 67 68 69 70 71 72 73 74 75 S N R F S G S K S G N T A
S L |agc|aat|cgt|ttc|tcc|gga|tct|aaa|tcc|ggt|aat|acc|gca|agc|tta|
BspEI.. HindIII. BsaBI........(blunt)
-------FR3------------->|--Stuffer for CDR3---------------->|
76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 T I S G L Q
|act|atc|tct|ggt|ctg|caglgtt ctg tag ttc caattg ctt tag tga ccc
PstI... MfeI..
-----Stuffer------------------------------->|---FR4--------- 103
104 105 G G G |ggc|ggt|ggt| KpnI... ---------FR4-------------->
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 T K L T
V L G Q P K A A P S V
|acc|aaa|ctt|act|gtc|ctc|ggt|caa|cct|aag|gct|gct|cct|tcc|gtt|
KpnI... HincII.. Bsu36I... 121 122 123 124 125 126 127 128 129 130
131 132 133 134 135 T L F P P S S E E L Q A N K A
|act|ctc|ttc|cct|cct|agt|tct|gaa|gag|ctt|caa|gct|aac|aag|gct|
SapI..... 136 137 138 139 140 141 142 143 144 145 146 147 148 149
150 T L V C L I S D F Y P G A V T
|act|ctt|gtt|tgc|ttg|atc|agt|gac|ttt|tat|cct|ggt|gct|gtt|act|
BclI....
[0169]
Sequence CWU 1
1
99 1 14 PRT Artificial Sequence Description of Artificial Sequence
Heavy chain CDR1 vector 1 Val Ser Gly Gly Ser Ile Ser Xaa Xaa Xaa
Tyr Tyr Trp Xaa 1 5 10 2 17 PRT Artificial Sequence Description of
Artificial Sequence Heavy chain CDR2 vector 2 Xaa Ile Xaa Xaa Ser
Gly Gly Xaa Thr Xaa Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 3 17 PRT
Artificial Sequence Description of Artificial Sequence Heavy chain
CDR2 vector 3 Xaa Ile Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Tyr Ala Asp
Ser Val Lys 1 5 10 15 Gly 4 16 PRT Artificial Sequence Description
of Artificial Sequence Heavy chain CDR2 vector 4 Xaa Ile Xaa Xaa
Xaa Gly Xaa Xaa Xaa Tyr Asn Pro Ser Leu Lys Gly 1 5 10 15 5 19 PRT
Artificial Sequence Description of Artificial Sequence Heavy chain
CDR2 vector 5 Xaa Ile Xaa Ser Xaa Xaa Xaa Gly Gly Tyr Tyr Xaa Tyr
Ala Ala Ser 1 5 10 15 Val Lys Gly 6 15 PRT Artificial Sequence
Description of Artificial Sequence Heavy chain CDR3 vector 6 Tyr
Tyr Cys Ala Xaa Xaa Xaa Xaa Xaa Tyr Phe Asp Tyr Trp Gly 1 5 10 15 7
17 PRT Artificial Sequence Description of Artificial Sequence Heavy
chain CDR3 vector 7 Tyr Tyr Cys Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr
Phe Asp Tyr Trp 1 5 10 15 Gly 8 19 PRT Artificial Sequence
Description of Artificial Sequence Heavy chain CDR3 vector 8 Tyr
Tyr Cys Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Phe Asp 1 5 10
15 Tyr Trp Gly 9 21 PRT Artificial Sequence Description of
Artificial Sequence Heavy chain CDR3 vector 9 Tyr Tyr Cys Ala Arg
Xaa Xaa Xaa Ser Xaa Ser Xaa Xaa Xaa Xaa Tyr 1 5 10 15 Phe Asp Tyr
Trp Gly 20 10 22 PRT Artificial Sequence Description of Artificial
Sequence Heavy chain CDR3 vector 10 Tyr Tyr Cys Ala Xaa Xaa Xaa Xaa
Cys Ser Gly Xaa Xaa Cys Tyr Xaa 1 5 10 15 Tyr Phe Asp Tyr Trp Gly
20 11 24 PRT Artificial Sequence Description of Artificial Sequence
Heavy chain CDR3 vector 11 Tyr Tyr Cys Ala Xaa Xaa Xaa Ser Xaa Thr
Ile Phe Gly Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Tyr Phe Asp Tyr Trp Gly
20 12 25 PRT Artificial Sequence Description of Artificial Sequence
Heavy chain CDR3 vector 12 Tyr Tyr Cys Ala Arg Xaa Xaa Xaa Tyr Tyr
Xaa Ser Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Tyr Phe Asp Tyr Trp
Gly 20 25 13 26 PRT Artificial Sequence Description of Artificial
Sequence Heavy chain CDR3 vector 13 Tyr Tyr Cys Ala Arg Xaa Xaa Xaa
Xaa Tyr Cys Xaa Xaa Xaa Xaa Cys 1 5 10 15 Tyr Xaa Xaa Xaa Tyr Phe
Asp Tyr Trp Gly 20 25 14 11 PRT Artificial Sequence Description of
Artificial Sequence Kappa light chain CDR1 vector 14 Arg Ala Ser
Gln Xaa Val Xaa Xaa Xaa Leu Ala 1 5 10 15 12 PRT Artificial
Sequence Description of Artificial Sequence Kappa light chain CDR1
vector 15 Arg Ala Ser Gln Xaa Val Xaa Xaa Xaa Xaa Leu Ala 1 5 10 16
9 PRT Artificial Sequence Description of Artificial Sequence Kappa
light chain CDR3 vector 16 Gln Gln Xaa Xaa Xaa Xaa Pro Xaa Thr 1 5
17 10 PRT Artificial Sequence Description of Artificial Sequence
Kappa light chain CDR3 vector 17 Gln Gln Xaa Xaa Xaa Xaa Pro Pro
Xaa Thr 1 5 10 18 14 PRT Artificial Sequence Description of
Artificial Sequence Lambda light chain CDR1 vector 18 Thr Gly Xaa
Ser Ser Xaa Val Gly Xaa Xaa Xaa Xaa Val Ser 1 5 10 19 10 PRT
Artificial Sequence Description of Artificial Sequence Lambda light
chain CDR3 vector 19 Xaa Ser Tyr Xaa Xaa Ser Xaa Xaa Xaa Val 1 5 10
20 14 PRT Homo sapiens 20 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Ser Ser 1 5 10 21 15 PRT Homo sapiens 21 Ala Phe Asp Ile
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5 10 15 22 5 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 22 Tyr Tyr Cys Ala Arg 1 5 23 323 DNA Artificial Sequence
Description of Artificial Sequence 3-23 JH4 vector with CDR1/2
diversity 23 cc atg gcc gaa gtt caa ttg tta gag tct ggt ggc ggt ctt
gtt cag 47 Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln 1 5 10 15 cct ggt ggt tct tta cgt ctt tct tgc gct gct tcc
gga ttc act ttc 95 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe 20 25 30 tct nnn tac nnn atg nnn tgg gtt cgc caa
gct cct ggt aaa ggt ttg 143 Ser Xaa Tyr Xaa Met Xaa Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 35 40 45 gag tgg gtt tct nnn atc nnn nnn
tct ggt ggc nnn act nnn tat gct 191 Glu Trp Val Ser Xaa Ile Xaa Xaa
Ser Gly Gly Xaa Thr Xaa Tyr Ala 50 55 60 gac tcc gtt aaa ggt cgc
ttc act atc tct aga gac aac tct aag aat 239 Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 65 70 75 80 act ctc tac ttg
cag atg aac agc tta agg gct gag gac acc gct gtc 287 Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 85 90 95 tac tac
tgc gcc aaa gac tat gaa ggt act ggt tat 323 Tyr Tyr Cys Ala Lys Asp
Tyr Glu Gly Thr Gly Tyr 100 105 24 108 PRT Artificial Sequence
Description of Artificial Sequence 3-23 JH4 vector with CDR1/2
diversity 24 Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe 20 25 30 Ser Xaa Tyr Xaa Met Xaa Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Xaa Ile Xaa
Xaa Ser Gly Gly Xaa Thr Xaa Tyr Ala 50 55 60 Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 65 70 75 80 Thr Leu Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 85 90 95 Tyr
Tyr Cys Ala Lys Asp Tyr Glu Gly Thr Gly Tyr 100 105 25 45 DNA Homo
sapiens CDS (1)..(45) 25 tat ttc gat tat tgg ggt caa ggt acc ctg
gtc acc gtc tct agt 45 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 1 5 10 15 26 15 PRT Homo sapiens 26 Tyr Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 15 27 55 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 27 cttccggatt cactttctct nnntacnnna tgnnntgggt
tcgccaagct cctgg 55 28 28 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 28 cctactgtct
tccggattca ctttctct 28 29 30 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 29 tgggttcgcc
aagctcctgg ttgctcactc 30 30 61 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 30 cttccggatt
cactttctct wsnnnnnnnt actactggnn ntgggttcgc caagctcctg 60 g 61 31
82 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 31 cttccggatt cactttctct atcagcggtg
gttctatctc cnnnnnnnnn tactactggn 60 nntgggttcg ccaagctcct gg 82 32
68 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 32 ggtttggagt gggtttctnn natcnnnnsn
tctggtggcn nnactnnnta tgctgactcc 60 gttaaagg 68 33 44 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 33 cttgggttcg ccaagctcct ggtaaaggtt tggagtgggt ttct
44 34 49 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 34 tatgctgact ccgttaaagg tcgcttcact
atctctagat tcctgtcac 49 35 68 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 35 ggtttggagt
gggtttctnn natcdnnnnn nnnggtdvnn nnnnnnnnta tgctgactcc 60 gttaaagg
68 36 65 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 36 ggtttggagt gggtttctnn natcdnnnnn
nnnggtdvnn nnnnntataa cccttccctt 60 aaggg 65 37 49 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 37 tataaccctt cccttaaggg tcgcttcact atctctagat
tcctgtcac 49 38 74 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 38 ggtttggagt
gggtttctnn natcnnnagt nnnnnnnnng gtggtactac tnnntatgcc 60
gcttccgtta aggg 74 39 49 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 39 tatgccgctt
ccgttaaggg tcgcttcact atctctagat tcctgtcac 49 40 25 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 40 gctctggtca acttaagggc tgagg 25 41 48 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 41 gctctggtca acttaagggc tgaggacacc gctgtctact
actgcgcc 48 42 46 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 42 tacttcgatt actggggcca
aggtaccctg gtcacctcgc tccacc 46 43 25 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 43
ggtaccctgg tcacctcgct ccacc 25 44 58 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 44
ccgctgtcta ctactgcgcc mrnnnnnnnn nnnnntactt cgattactgg ggccaagg 58
45 64 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 45 ccgctgtcta ctactgcgcc mrnnnnnnnn
nnnnnnnnnn ntacttcgat tactggggcc 60 aagg 64 46 70 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 46 ccgctgtcta ctactgcgcc mrnnnnnnnn nnnnnnnnnn
nnnnnnntac ttcgattact 60 ggggccaagg 70 47 76 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 47 ccgctgtcta ctactgcgcc cgtnnnnnnn nntctdsntc
ttrbnnnnnn nnntacttcg 60 attactgggg ccaagg 76 48 79 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 48 ccgctgtcta ctactgcgcc mrnnnnnnnn nntgctctgg
tnnnnnntgc tatnnntact 60 tcgattactg gggccaagg 79 49 85 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 49 ccgctgtcta ctactgcgcc mrnnnnnnnt ctnnnactat
cttcggtnnn nnnnnnnnnn 60 nntacttcga ttactggggc caagg 85 50 88 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 50 ccgctgtcta ctactgcgcc cgtnnnnnnn nntattacgr
ntctdsndsn tactatnnnn 60 nnnnntactt cgattactgg ggccaagg 88 51 91
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 51 ccgctgtcta ctactgcgcc cgtnnnnnnn
nnnnntattg cdsndsnrvn nnntgctatn 60 nnnnnnnnta cttcgattac
tggggccaag g 91 52 242 DNA Artificial Sequence Description of
Artificial Sequence 3-23 JH4 vector with stuffers 52 cc atg gcc gaa
gtt caa ttg tta gag tct ggt ggc ggt ctt gtt cag 47 Ala Met Ala Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 cct ggt
ggt tct tta cgt ctt tct tgc gct gct tcc gga ttc act ttc 95 Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25 30
tct tcg tac gct tagtaa tgg gtt cgc caa gct cct ggt aaa ggt ttg 143
Ser Ser Tyr Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45
gag tgg gtt tct taa cct agg tag act atc tct aga gac aac tct aag 191
Glu Trp Val Ser Pro Arg Thr Ile Ser Arg Asp Asn Ser Lys 50 55 60
aat act ctc tac ttg cag atg aac agc tta agg gct tagtaaaggc cttaa
242 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 65 70 53 72 PRT
Artificial Sequence Description of Artificial Sequence 3-23 JH4
vector with stuffers 53 Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe 20 25 30 Ser Ser Tyr Ala Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Pro Arg Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 50 55 60 Leu Gln Met
Asn Ser Leu Arg Ala 65 70 54 952 DNA Homo sapiens CDS (206)..(907)
modified_base (344)..(346) a, c, t, g, other or unknown 54
gaggaccatt gggccccctc cgagactctc gagcgcaacg caattaatgt gagttagctc
60 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
gtgtggaatt 120 gtgagcggat aacaatttca cacaggaaac agctatgacc
atgattacgc caagctttgg 180 agcctttttt ttggagattt tcaac gtg aag aag
ctc cta ttt gct atc ccg 232 Met Lys Lys Leu Leu Phe Ala Ile Pro 1 5
ctt gtc gtt ccg ttt tac agc cat agt gca caa tcc gtc ctt act caa 280
Leu Val Val Pro Phe Tyr Ser His Ser Ala Gln Ser Val Leu Thr Gln 10
15 20 25 tct cct ggc act ctt tcg cta agc ccg ggt gaa cgt gct acc
tta agt 328 Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr
Leu Ser 30 35 40 tgc cgt gct tcc cag nnn gtt nnn nnn nnn nnn ctt
gct tgg tat caa 376 Cys Arg Ala Ser Gln Xaa Val Xaa Xaa Xaa Xaa Leu
Ala Trp Tyr Gln 45 50 55 cag aaa cct ggt cag gcg ccg cgt tta ctt
att tat nnn gct tct nnn 424 Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile Tyr Xaa Ala Ser Xaa 60 65 70 cgc nnn nnn ggg atc ccg gac cgt
ttc tct ggc tct ggt tca ggt act 472 Arg Xaa Xaa Gly Ile Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr 75 80 85 gac ttt acc ctt act att
tct aga ttg gaa cct gaa gac ttc gct gtt 520 Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu Pro Glu Asp Phe Ala Val 90 95 100 105 tat tat tgc
caa cag nnn nnn nnn nnn cct nnn act ttc ggt caa ggt 568 Tyr Tyr Cys
Gln Gln Xaa Xaa Xaa Xaa Pro Xaa Thr Phe Gly Gln Gly 110 115 120 acc
aag gtt gaa atc aag cgt acg gtt gcc gct cct agt gtg ttt atc 616 Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 125 130
135 ttt cct cct tct gac gaa caa ttg aag tca ggt act gct tct gtc gta
664 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
140 145 150 tgt ttg ctc aac aat ttc tac cct cgt gaa gct aaa gtt cag
tgg aaa 712 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys 155 160 165 gtc gat aac gcg ttg cag tcg ggt aac agt caa gaa
tcc gtc act gaa 760 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu 170 175 180 185 cag gat agt aag gac tct acc tac tct
ttg tcc tct act ctt act tta 808 Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu 190 195 200 tca aag gct gat tat gag aag
cat aag gtc tat gct tgc gaa gtt acc 856 Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala Cys Glu Val Thr 205 210 215 cac cag ggt ctg agc
tcc cct gtt acc aaa agt ttc aac cgt ggt gaa 904 His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 220 225 230 tgc
taatagggcg cgccacgcat ctctaagcgg ccgcaacagg aggag 952 Cys 55 234
PRT Homo sapiens MOD_RES (47) Any amino acid 55 Met Lys Lys Leu Leu
Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser 1 5 10 15 His Ser Ala
Gln Ser Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu 20 25 30 Ser
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Xaa Val
35 40 45 Xaa Xaa Xaa Xaa Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro 50 55 60 Arg Leu Leu Ile Tyr Xaa Ala Ser Xaa Arg Xaa Xaa
Gly Ile Pro Asp 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser 85 90 95 Arg Leu Glu Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Xaa Xaa 100 105 110 Xaa Xaa Pro Xaa Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155
160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 225 230 56 732 DNA Homo sapiens CDS (30)..(686)
modified_base (108)..(110) a, c, t, g, other or unknown 56
gaggaccatt gggcccctta ctccgtgac agt gca caa tcc gct ctc act cag 53
Ser Ala Gln Ser Ala Leu Thr Gln 1 5 cct gct agc gtt tcc ggg tca cct
ggt caa agt atc act att tct tgt 101 Pro Ala Ser Val Ser Gly Ser Pro
Gly Gln Ser Ile Thr Ile Ser Cys 10 15 20 aca ggt nnn tct tct nnn
gtt ggc nnn nnn nnn nnn gtt tct tgg tat 149 Thr Gly Xaa Ser Ser Xaa
Val Gly Xaa Xaa Xaa Xaa Val Ser Trp Tyr 25 30 35 40 caa caa cac ccg
ggc aag gcg ccg aag ttg atg atc tac nnn nnn nnn 197 Gln Gln His Pro
Gly Lys Ala Pro Lys Leu Met Ile Tyr Xaa Xaa Xaa 45 50 55 nnn cgt
cct tct ggt gtt agc aat cgt ttc tcc gga tct aaa tcc ggt 245 Xaa Arg
Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly 60 65 70
aat acc gca agc tta act atc tct ggt ctg cag gct gaa gac gag gct 293
Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala 75
80 85 gac tac tat tgt nnn nnn nnn nnn nnn tct nnn nnn nnn nnn gtc
ttc 341 Asp Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa Val
Phe 90 95 100 ggc ggt ggt acc aaa ctt act gtc ctc ggt caa cct aag
gct gct cct 389 Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys
Ala Ala Pro 105 110 115 120 tcc gtt act ctc ttc cct cct agt tct gaa
gag ctt caa gct aac aag 437 Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
Glu Leu Gln Ala Asn Lys 125 130 135 gct act ctt gtt tgc ttg atc agt
gac ttt tat cct ggt gct gtt act 485 Ala Thr Leu Val Cys Leu Ile Ser
Asp Phe Tyr Pro Gly Ala Val Thr 140 145 150 gtc gct tgg aaa gcc gat
tct tct cct gtt aaa gct ggt gtt gag acg 533 Val Ala Trp Lys Ala Asp
Ser Ser Pro Val Lys Ala Gly Val Glu Thr 155 160 165 acc act cct tct
aaa caa tct aac aat aag tac gct gcg agc tct tat 581 Thr Thr Pro Ser
Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr 170 175 180 ctt tct
ctc acc cct gaa caa tgg aag tct cat aaa tcc tat tcc tgt 629 Leu Ser
Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser Tyr Ser Cys 185 190 195
200 caa gtt act cat gaa ggt tct acc gtt gaa aag act gtt gcc cct act
677 Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr
205 210 215 gag tgt tct tagtgaggcg cgccaacgat gttcaaggcg gccgcaacag
gaggag 732 Glu Cys Ser 57 219 PRT Homo sapiens MOD_RES (27) Any
amino acid 57 Ser Ala Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro 1 5 10 15 Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Xaa
Ser Ser Xaa Val Gly 20 25 30 Xaa Xaa Xaa Xaa Val Ser Trp Tyr Gln
Gln His Pro Gly Lys Ala Pro 35 40 45 Lys Leu Met Ile Tyr Xaa Xaa
Xaa Xaa Arg Pro Ser Gly Val Ser Asn 50 55 60 Arg Phe Ser Gly Ser
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser 65 70 75 80 Gly Leu Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Xaa Xaa Xaa Xaa 85 90 95 Xaa
Ser Xaa Xaa Xaa Xaa Val Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105
110 Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser
115 120 125 Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser 130 135 140 Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys
Ala Asp Ser Ser 145 150 155 160 Pro Val Lys Ala Gly Val Glu Thr Thr
Thr Pro Ser Lys Gln Ser Asn 165 170 175 Asn Lys Tyr Ala Ala Ser Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp 180 185 190 Lys Ser His Lys Ser
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr 195 200 205 Val Glu Lys
Thr Val Ala Pro Thr Glu Cys Ser 210 215 58 25 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 58 gctctggtca acttaagggc tgagg 25 59 48 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 59 gctctggtca acttaagggc tgaggacacc gctgtctact
actgcgcc 48 60 46 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 60 tacttcgatt acttgggcca
aggtaccctg gtcacctcgc tccacc 46 61 25 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 61
ggtaccctgg tcacctcgct ccacc 25 62 56 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 62
ggtctcagtt gctaagcccg ggtgaacgtg ctaccttaag ttgccgtgct tcccag 56 63
24 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 63 ggtctcagtt gctaagcccg ggtg 24 64 45
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 64 cttgcttggt atcaacagaa acctggtcag
gcgccaagtc gtgtc 45 65 24 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 65 cctggtcagg
cgccaagtcg tgtc 24 66 65 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 66 gctaccttaa
gttgccgtgc ttcccagnnn gttnnnnnnn nncttgcttg gtatcaacag 60 aaacc 65
67 68 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 67 gctaccttaa gttgccgtgc ttcccagnnn
gttnnnnnnn nnnnncttgc ttggtatcaa 60 cagaaacc 68 68 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 68 cacgagtcct acctggtcag gc 22 69 41 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 69 cacgagtcct acctggtcag gcgccgcgtt tacttattta t 41
70 22 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 70 gaccgtttct ctggttctca cc 22 71 76 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 71 caggcgccgc gtttacttat ttatnnngct tctnnncgcn
nnnnngggat cccggaccgt 60 ttctctggtt ctcacc 76 72 53 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 72 gacgagtcct tctagattgg aacctgaaga cttcgctgtt
tattattgcc aac 53 73 50 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 73 actttcggtc
aaggtaccaa ggttgaaatc aagcgtacgt cacaggtgag 50 74 26 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 74 gaaatcaagc gtacgtcaca ggtgag 26 75 70 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 75 gacttcgctg tttattattg ccaacagnnn nnnnnnnnnc
ctnnnacttt cggtcaaggt 60 accaaggttg 70 76 67 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 76 gacttcgctg tttattattg ccaacagnnn nnnnnnnnnn
nncctttcgg tcaaggtacc 60 aaggttg 67 77 73 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 77
gacttcgctg tttattattg ccaacagnnn nnnnnnnnnc ctcctnnnac tttcggtcaa
60 ggtaccaagg ttg 73 78 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 78 gacgagtcct
ggtcacctgg t 21 79 48 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 79 gacgagtcct
ggtcacctgg tcaaagtatc actatttctt gtacaggt 48 80 50 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 80 gtttcttggt atcaacaaca cccgggcaag gcgagatctt
cacaggtgag 50 81 25 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 81 gcaaggcgag
atcttcacag gtgag 25 82 67 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 82 gtatcactat
ttcttgtaca ggtnnnnnnc tcnnnnnnnn nnnnnnnnnn tggtatcaac 60 aacaccc
67 83 76 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 83 gtatcactat ttcttgtaca ggtnnntctt
ctnnngttgg cnnnnnnnnn nnngtttctt 60 ggtatcaaca acaccc 76 84 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 84 gagcagagga cccgggcaag gc 22 85 41 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 85 gagcagagga cccgggcaag gcgccgaagt tgatgatcta c 41
86 44 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 86 cgtccttctg gtgtcagcaa tcgtttctcc
ggatcacagg tgag 44 87 23 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 87 cgtttctccg
gatcacaggt gag 23 88 53 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 88 gccgaagttg
atgatctacn nnnnnnnnnn ncgtccttct ggtgtcagca atc 53 89 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 89 ctgcaggctg aagacgaggc tgac 24 90 33 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 90 ctgcaggctg aagacgaggc tgactactat tgt 33 91 57
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 91 gtcttcggcg gtggtaccaa acttactgtc
ctcggtcaac ctaaggacac aggtgag 57 92 25 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 92
cggtcaacct aaggacacag gtgag 25 93 77 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 93
gacgaggctg actactattg tnnnnnnnnn nnnnnntctn nnnnnnnnnn ngtcttcggc
60 ggtggtacca aacttac 77 94 74 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 94 gacgaggctg
actactattg tnnnagctat nnnnnntctn nnnnnnnngt cttcggcggt 60
ggtaccaaac ttac 74 95 627 DNA Artificial Sequence Description of
Artificial Sequence A27 JH1 Kappa light chain gene with stuffers 95
gaggaccatt gggccccctc cgagactctc gagcgcaacg caattaatgt gagttagctc
60 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
gtgtggaatt 120 gtgagcggat aacaatttca cacaggaaac agctatgacc
atgattacgc caagctttgg 180 agcctttttt ttggagattt tcaac gtg aag aag
ctc cta ttt gct atc ccg 232 Met Lys Lys Leu Leu Phe Ala Ile Pro 1 5
ctt gtc gtt ccg ttt tac agc cat agt gca caa tcc gtc ctt act caa 280
Leu Val Val Pro Phe Tyr Ser His Ser Ala Gln Ser Val Leu Thr Gln 10
15 20 25 tct cct ggc act ctt tcg cta agc ccg ggt gaa cgt gct acc
tta agt 328 Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr
Leu Ser 30 35 40 tagtaagctc ccaggcctct ttgatctg aaa cct ggt cag gcg
ccg cgt 377 Lys Pro Gly Gln Ala Pro Arg 45 taatgaaagc gctaatggcc
aacagtg act ggg atc ccg gac cgt ttc tct ggc 431 Thr Gly Ile Pro Asp
Arg Phe Ser Gly 50 55 tct ggt tca ggt act gac ttt acc ctt act att
tct aga taatgagtta 480 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg 60 65 70 actagaccta cgtaacctag ttc ggt caa ggt acc aag gtt
gaa atc aag cgt 533 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 75
80 acg gtt gcc gct cct agt gtg ttt atc ttt cct cct tct gac gaa caa
581 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
85 90 95 ttg aag tca ggt act acgcatctct aagcggccgc aacaggagga g 627
Leu Lys Ser Gly Thr 100 96 102 PRT Artificial Sequence Description
of Artificial Sequence A27 JH1 Kappa light chain gene with stuffers
96 Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser
1 5 10 15 His Ser Ala Gln Ser Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu 20 25 30 Ser Pro Gly Glu Arg Ala Thr Leu Ser Lys Pro Gly
Gln Ala Pro Arg 35 40 45 Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe 50 55 60 Thr Leu Thr Ile Ser Arg Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 65 70 75 80 Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 85 90 95 Gln Leu Lys Ser
Gly Thr 100 97 413 DNA Artificial Sequence Description of
Artificial Sequence 2a2 JH2 Human lambda-chain gene with stuffers
in place of CDRs 97 gaggaccatt gggcccctta ctccgtgac agt gca caa tcc
gct ctc act cag 53 Ser Ala Gln Ser Ala Leu Thr Gln 1 5 cct gct agc
gtt tcc ggg tca cct ggt caa agt atc act att tct tgt 101 Pro Ala Ser
Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys 10 15 20 aca
tcttagtgac tc aga tct taatga ccg tag cac ccg ggc aag gcg 149 Thr
Arg Ser Pro His Pro Gly Lys Ala 25 30 ccg taatgaatct cgtacgctgg
tgtt agc aat cgt ttc tcc gga tct aaa 200 Pro Ser Asn Arg Phe Ser
Gly Ser Lys 35 40 tcc ggt aat acc gca agc tta act atc tct ggt ctg
cag gttctgtagt 249 Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
Gln 45 50 55 tccaattgct ttagtgaccc ggc ggt ggt acc aaa ctt act gtc
ctc ggt caa 302 Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 60 65
cct aag gct gct cct tcc gtt act ctc ttc cct cct agt tct gaa gag 350
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 70
75 80 ctt caa gct aac aag gct act ctt gtt tgc ttg atc agt gac ttt
tat 398 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
Tyr 85 90 95 cct ggt gct gtt act 413 Pro Gly Ala Val Thr 100 98 103
PRT Artificial Sequence Description of Artificial Sequence 2a2 JH2
Human lambda-chain gene with stuffers in place of CDRs 98 Ser Ala
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro 1 5
10 15 Gly Gln Ser Ile Thr Ile Ser Cys Thr Arg Ser Pro His Pro Gly
Lys 20 25 30 Ala Pro Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn
Thr Ala Ser 35 40 45 Leu Thr Ile Ser Gly Leu Gln Gly Gly Gly Thr
Lys Leu Thr Val Leu 50 55 60 Gly Gln Pro Lys Ala Ala Pro Ser Val
Thr Leu Phe Pro Pro Ser Ser 65 70 75 80 Glu Glu Leu Gln Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp 85 90 95 Phe Tyr Pro Gly Ala
Val Thr 100 99 10 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 99 ctgtctgaac 10
* * * * *