U.S. patent application number 16/735070 was filed with the patent office on 2020-07-16 for humanized antibodies.
The applicant listed for this patent is The Scripps Research Institute. Invention is credited to Omar Bazirgan, Hongyuan Helen Mao, Vaughn Smider.
Application Number | 20200223944 16/735070 |
Document ID | / |
Family ID | 47604214 |
Filed Date | 2020-07-16 |
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United States Patent
Application |
20200223944 |
Kind Code |
A1 |
Smider; Vaughn ; et
al. |
July 16, 2020 |
HUMANIZED ANTIBODIES
Abstract
The present disclosure provides humanized antibodies, including
antibodies comprising an ultralong CDR3 and uses thereof.
Inventors: |
Smider; Vaughn; (San Diego,
CA) ; Bazirgan; Omar; (San Diego, CA) ; Mao;
Hongyuan Helen; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Scripps Research Institute |
La Jolla |
CA |
US |
|
|
Family ID: |
47604214 |
Appl. No.: |
16/735070 |
Filed: |
January 6, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15801251 |
Nov 1, 2017 |
10562980 |
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16735070 |
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14371170 |
Jul 8, 2014 |
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PCT/US13/20910 |
Jan 9, 2013 |
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15801251 |
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61584680 |
Jan 9, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C40B 40/10 20130101;
C07K 14/5421 20130101; C07K 16/1081 20130101; C07K 2317/20
20130101; C07K 2319/33 20130101; C07K 2318/10 20130101; C07K
2319/00 20130101; A61P 35/00 20180101; C07K 2317/565 20130101; C07K
16/005 20130101; C07K 16/00 20130101; C07K 16/46 20130101; C07K
2317/24 20130101 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 14/54 20060101 C07K014/54; C07K 16/00 20060101
C07K016/00; C07K 16/10 20060101 C07K016/10 |
Claims
1. A polynucleotide encoding a humanized antibody or binding
fragment thereof, wherein the humanized antibody or binding
fragment thereof comprises a variable heavy chain comprising a
human acceptor framework comprising a CDR3 that is an ultralong
CDR3 in which the CDR3 of the human acceptor framework is replaced
by a CDR3 scaffold which is 35 amino acids in length or longer and
comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n(X.sup.aX.sup.b).sub.z
motif and at least a portion of a human JH sequence or derivative
thereof, wherein the X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif is
TTVHQ (SEQ ID NO: 153) or TSVHQ (SEQ ID NO: 154), wherein, for
X.sub.n, X is any amino acid and n is 27-54, and wherein the
(X.sup.aX.sup.b).sub.z motif is YXYXYX with X being any amino
acid.
2. The polynucleotide of claim 1, wherein the ultralong CDR3 is 45
amino acids in length or longer.
3. The polynucleotide of claim 1, wherein the ultralong CDR3 is 50
amino acids in length or longer.
4. The polynucleotide of claim 1, wherein the human acceptor
framework is from a human heavy chain germline sequence that is a
VH4 family member germline sequence.
5. The polynucleotide of claim 1, wherein the human acceptor
framework is from a VH4-34 germline sequence or is derived from a
VH4-34 germline sequence.
6. The polynucleotide of claim 5, wherein the VH4-34 germline
sequence comprises a CDR1 comprising a Gly31Asp and Tyr32Lys
substitution based on Kabat numbering and/or a CDR2 comprising a
Glu50Ser substitution based on Kabat numbering.
7. The polynucleotide of claim 6, wherein the VH4-34 germline
sequence comprises a CDR1 comprising Ala23Thr, Val24Ala, Tyr25Ser,
Gly27Phe, Phe29Leu, Gly31Asp, Tyr32Lys, Tyr33Ala, Trp34Val, and
Ser35Gly substitutions, and a CDR2 comprising Ile48Leu, Glu50Ser,
Asn52Asp, His53Thr, Ser54Gly, Ser56Asn, and Asn58Gly substitutions,
based on Kabat numbering.
8. The polynucleotide of claim 1, wherein the humanized antibody or
antigen binding fragment comprises the amino acid sequence set
forth in SEQ ID NO: 460 and the ultralong CDR3.
9. The polynucleotide of claim 1, wherein the humanized antibody or
antigen binding fragment comprises the amino acid sequence set
forth in SEQ ID NO: 461, 462, 463, 464, 465, 466, 467, or 468 and
the ultralong CDR3.
10. The polynucleotide of claim 1, wherein the ultralong CDR3
comprises a non-bovine sequence or a non-antibody sequence.
11. The polynucleotide of claim 11, wherein ultralong CDR3
comprises a non-antibody sequence that is a cytokine sequence, a
lymphokine sequence, a chemokine sequence, a growth factor
sequence, a hormone sequence, or a toxin sequence.
12. The polynucleotide of claim 1, wherein the at least a portion
of a human JH sequence or derivative thereof comprises the amino
acid sequence set forth in SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, or SEQ ID NO:17.
13. The polynucleotide of claim 1, further comprising a variable
light chain.
14. The polynucleotide of claim 13, wherein the variable heavy
chain and the variable light chain are encoded on the same
vector.
15. A vector comprising the polynucleotide of claim 1.
16. A vector comprising the polynucleotide of claim 13.
17. A host cell comprising the vector of claim 15.
18. A host cell comprising the vector of claim 16.
19. A nucleic acid library comprising a plurality of
polynucleotides comprising sequences coding for humanized
antibodies or binding fragments thereof of claim 1.
20. A nucleic acid library comprising a plurality of
polynucleotides comprising sequences coding for humanized
antibodies or binding fragments thereof of claim 13.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/801,251 (filed Nov. 1, 2017; now pending), which is a
continuation of U.S. patent application Ser. No. 14/371,170 (filed
Jul. 8, 2014; now abandoned), which is a U.S. National Stage
Application under 35 U.S.C. .sctn. 371 of International Patent
Application No. PCT/US2013/020910 (filed Jan. 9, 2013; now
expired), which claims the benefit of U.S. Provisional Application
No. 61/584,680 (filed Jan. 9, 2012; now expired). The entire
contents of the afore-mentioned priority applications are each
incorporated by reference in their entirety and for all
purposes.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] Incorporated herein by reference is the Sequence Listing
being concurrently submitted via EFS-Web as an ASCII text file
named 792472000510SeqList.txt, of file size 354,796 bytes, created
on Mar. 20, 2020.
FIELD
[0003] The present disclosure relates to humanized antibodies,
including antibodies comprising an ultralong CDR3.
BACKGROUND
[0004] Antibodies are natural proteins that the vertebrate immune
system forms in response to foreign substances (antigens),
primarily for defense against infection. For over a century,
antibodies have been induced in animals under artificial conditions
and harvested for use in therapy or diagnosis of disease
conditions, or for biological research. Each individual antibody
producing cell produces a single type of antibody with a chemically
defined composition, however, antibodies obtained directly from
animal serum in response to antigen inoculation actually comprise
an ensemble of non-identical molecules (e.g., polyclonal
antibodies) made from an ensemble of individual antibody producing
cells.
[0005] Some bovine antibodies have unusually long VH CDR3 sequences
compared to other vertebrates. For example, about 10% of IgM
contains "ultralong" CDR3 sequences, which can be up to 61 amino
acids long. These unusual CDR3s often have multiple cysteines.
Functional VH genes form through a process called V(D)J
recombination, wherein the D-region encodes a significant
proportion of CDR3. A unique D-region encoding an ultralong
sequence has been identified in cattle. Ultralong CDR3s are
partially encoded in the cattle genome, and provide a unique
characteristic of their antibody repertoire in comparison to
humans. Kaushik et al. (U.S. Pat. Nos. 6,740,747 and 7,196,185)
disclose several bovine germline D-gene sequences unique to cattle
stated to be useful as probes and a bovine VDJ cassette stated to
be useful as a vaccine vector.
SUMMARY
[0006] The present disclosure provides humanized antibodies,
including antibodies comprising an ultralong CDR3, methods of
making same, and uses thereof.
[0007] The present disclosure provides a humanized antibody or
binding fragment thereof comprising an ultralong CDR3.
[0008] In some embodiments, the humanized antibody or binding
fragment thereof comprises one or more human variable region
framework sequences.
[0009] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 is 35 amino acids in
length or longer, 40 amino acids in length or longer, 45 amino
acids in length or longer, 50 amino acids in length or longer, 55
amino acids in length or longer, or 60 amino acids in length or
longer.
[0010] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 is 35 amino acids in
length or longer.
[0011] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises 3 or more
cysteine residues, 4 or more cysteine residues, 5 or more cysteine
residues, 6 or more cysteine residues, 7 or more cysteine residues,
8 or more cysteine residues, 9 or more cysteine residues, 10 or
more cysteine residues, 11 or more cysteine residues, or 12 or more
cysteine residues.
[0012] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises 3 or more
cysteine residues.
[0013] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
comprise a cysteine motif.
[0014] In some embodiments of each or any of the above or below
mentioned embodiments, the cysteine motif is selected from the
group consisting of: CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID
NO: 41), CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95).
[0015] In some embodiments of each or any of the above or below
mentioned embodiments, the cysteine motif is selected from the
group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152).
[0016] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises 2 to 6
disulfide bonds.
[0017] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises SEQ ID NO: 40
or a derivative thereof.
[0018] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises amino acid
residues 3-6 of any of one SEQ ID NO: 1-4.
[0019] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a non-human DH
or a derivative thereof.
[0020] In some embodiments of each or any of the above or below
mentioned embodiments, the non-human DH is SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11, or SEQ ID NO: 12.
[0021] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a JH sequence
or a derivative thereof.
[0022] In some embodiments of each or any of the above or below
mentioned embodiments, the JH sequence is SEQ ID NO: 13, SEQ ID NO:
14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
[0023] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises: a non-human or
human VH sequence (e.g., a germline VH) or a derivative thereof; a
non-human DH sequence or a derivative thereof; and/or JH sequence
or derivative thereof.
[0024] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises an additional
amino acid sequence comprising two to six amino acid residues or
more positioned between the VH sequence and the DH sequence.
[0025] In some embodiments of each or any of the above or below
mentioned embodiments, the additional amino acid sequence is
selected from the group consisting of: IR, IF, SEQ ID NO: 18, SEQ
ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
[0026] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a sequence
derived from or based on SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:
28.
[0027] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a bovine
sequence, a non-bovine sequence, an antibody sequence, or a
non-antibody sequence.
[0028] In some embodiments of each or any of the above or below
mentioned embodiments, the non-antibody sequence is a synthetic
sequence.
[0029] In some embodiments of each or any of the above or below
mentioned embodiments, the non-antibody sequence is a cytokine
sequence, a lymphokine sequence, a chemokine sequence, a growth
factor sequence, a hormone sequence, or a toxin sequence.
[0030] In some embodiments of each or any of the above or below
mentioned embodiments, the non-antibody sequence is an IL-8
sequence, an IL-21 sequence, an SDF-1 (alpha) sequence, a
somatostatin sequence, a chlorotoxin sequence, a Pro-TxII sequence,
or a ziconotide sequence.
[0031] In some embodiments of each or any of the above or below
mentioned embodiments, the non-antibody sequence is any one of SEQ
ID NOS: 475-481.
[0032] In some embodiments of each or any of the above or below
mentioned embodiments, the non-antibody sequence replaces at least
a portion of the ultralong CDR3.
[0033] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q).
[0034] In some embodiments of each or any of the above or below
mentioned embodiments, the X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif is TTVHQ (SEQ ID NO: 153), TSVHQ (SEQ ID NO: 154), SSVTQ (SEQ
ID NO: 155), STVHQ (SEQ ID NO: 156), ATVRQ (SEQ ID NO: 157), TTVYQ
(SEQ ID NO: 158), SPVHQ (SEQ ID NO: 159), ATVYQ (SEQ ID NO: 160),
TAVYQ (SEQ ID NO: 161), TNVHQ (SEQ ID NO: 162), ATVHQ (SEQ ID NO:
163), STVYQ (SEQ ID NO: 164), TIVHQ (SEQ ID NO: 165), AIVYQ (SEQ ID
NO: 166), TTVFQ (SEQ ID NO: 167), AAVFQ (SEQ ID NO: 168), GTVHQ
(SEQ ID NO: 169), ASVHQ (SEQ ID NO: 170), TAVFQ (SEQ ID NO: 171),
ATVFQ (SEQ ID NO: 172), AAAHQ (SEQ ID NO: 173), VVVYQ (SEQ ID NO:
174), GTVFQ (SEQ ID NO: 175), TAVHQ (SEQ ID NO: 176), ITVHQ (SEQ ID
NO: 177), ITAHQ (SEQ ID NO: 178), VTVHQ (SEQ ID NO: 179); AAVHQ
(SEQ ID NO: 180), GTVYQ (SEQ ID NO: 181), TTVLQ (SEQ ID NO: 182),
TTTHQ (SEQ ID NO: 183), or TTDYQ (SEQ ID NO: 184).
[0035] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif.
[0036] In some embodiments of each or any of the above or below
mentioned embodiments, the CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif is CTTVHQ (SEQ ID NO: 185), CTSVHQ (SEQ ID NO: 186), CSSVTQ
(SEQ ID NO: 187), CSTVHQ (SEQ ID NO: 188), CATVRQ (SEQ ID NO: 189),
CTTVYQ (SEQ ID NO: 190), CSPVHQ (SEQ ID NO: 191), CATVYQ (SEQ ID
NO: 192), CTAVYQ (SEQ ID NO: 193), CTNVHQ (SEQ ID NO: 194), CATVHQ
(SEQ ID NO: 195), CSTVYQ (SEQ ID NO: 196), CTIVHQ (SEQ ID NO: 197),
CAIVYQ (SEQ ID NO: 198), CTTVFQ (SEQ ID NO: 199), CAAVFQ (SEQ ID
NO: 200), CGTVHQ (SEQ ID NO: 201), CASVHQ (SEQ ID NO: 202), CTAVFQ
(SEQ ID NO: 203), CATVFQ (SEQ ID NO: 204), CAAAHQ (SEQ ID NO: 205),
CVVVYQ (SEQ ID NO: 206), CGTVFQ (SEQ ID NO: 207), CTAVHQ (SEQ ID
NO: 208), CITVHQ (SEQ ID NO: 209), CITAHQ (SEQ ID NO: 210), CVTVHQ
(SEQ ID NO: 211); CAAVHQ (SEQ ID NO: 212), CGTVYQ (SEQ ID NO: 213),
CTTVLQ (SEQ ID NO: 214), CTTTHQ (SEQ ID NO: 215), or CTTDYQ (SEQ ID
NO: 216).
[0037] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
(X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is any amino acid
residue, X.sup.b is an aromatic amino acid selected from the group
consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and
histidine (H), and wherein z is 1-4.
[0038] In some embodiments of each or any of the above or below
mentioned embodiments, the (X.sup.aX.sup.b).sub.z motif is CYTYNYEF
(SEQ ID NO: 217), HYTYTYDF (SEQ ID NO: 218), HYTYTYEW (SEQ ID NO:
219), KHRYTYEW (SEQ ID NO: 220), NYIYKYSF (SEQ ID NO: 221),
PYIYTYQF (SEQ ID NO: 222), SFTYTYEW (SEQ ID NO: 223), SYIYIYQW (SEQ
ID NO: 224), SYNYTYSW (SEQ ID NO: 225), SYSYSYEY (SEQ ID NO: 226),
SYTYNYDF (SEQ ID NO: 227), SYTYNYEW (SEQ ID NO: 228), SYTYNYQF (SEQ
ID NO: 229), SYVWTHNF (SEQ ID NO: 230), TYKYVYEW (SEQ ID NO: 231),
TYTYTYEF (SEQ ID NO: 232), TYTYTYEW (SEQ ID NO: 233), VFTYTYEF (SEQ
ID NO: 234), AYTYEW (SEQ ID NO: 235), DYIYTY (SEQ ID NO: 236),
IHSYEF (SEQ ID NO: 237), SFTYEF (SEQ ID NO: 238), SHSYEF (SEQ ID
NO: 239), THTYEF (SEQ ID NO: 240), TWTYEF (SEQ ID NO: 241), TYNYEW
(SEQ ID NO: 242), TYSYEF (SEQ ID NO: 243), TYSYEH (SEQ ID NO: 244),
TYTYDF (SEQ ID NO: 245), TYTYEF (SEQ ID NO: 246), TYTYEW (SEQ ID
NO: 247), AYEF (SEQ ID NO: 248), AYSF (SEQ ID NO: 249), AYSY (SEQ
ID NO: 250), CYSF (SEQ ID NO: 251), DYTY (SEQ ID NO: 252), KYEH
(SEQ ID NO: 253), KYEW (SEQ ID NO: 254), MYEF (SEQ ID NO: 255),
NWIY (SEQ ID NO: 256), NYDY (SEQ ID NO: 257), NYQW (SEQ ID NO:
258), NYSF (SEQ ID NO: 259), PYEW (SEQ ID NO: 260), RYNW (SEQ ID
NO: 261), RYTY (SEQ ID NO: 262), SYEF (SEQ ID NO: 263), SYEH (SEQ
ID NO: 264), SYEW (SEQ ID NO: 265), SYKW (SEQ ID NO: 266), SYTY
(SEQ ID NO: 267), TYDF (SEQ ID NO: 268), TYEF (SEQ ID NO: 269),
TYEW (SEQ ID NO: 270), TYQW (SEQ ID NO: 271), TYTY (SEQ ID NO:
272), or VYEW (SEQ ID NO: 273).
[0039] In some embodiments of each or any of the above or below
mentioned embodiments, the (X.sup.aX.sup.b).sub.z motif is
YXYXYX.
[0040] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n motif, wherein X.sub.1
is threonine (T), glycine (G), alanine (A), serine (S), or valine
(V), wherein X.sub.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sub.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sub.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), wherein X.sub.5 is
glutamine (Q), and wherein n is 27-54.
[0041] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises
X.sub.n(X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is any amino
acid residue, X.sup.b is an aromatic amino acid selected from the
group consisting of: tyrosine (Y), phenylalanine (F), tryptophan
(W), and histidine (H), wherein n is 27-54, and wherein z is
1-4.
[0042] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n(X.sup.aX.sup.b).sub.z
motif, wherein X.sup.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sup.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sup.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sup.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), and wherein X.sup.5 is glutamine (Q), wherein
X.sup.a is any amino acid residue, X.sup.b is an aromatic amino
acid selected from the group consisting of: tyrosine (Y),
phenylalanine (F), tryptophan (W), and histidine (H), wherein n is
27-54, and wherein z is 1-4.
[0043] In some embodiments of each or any of the above or below
mentioned embodiments, the X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif is TTVHQ (SEQ ID NO: 153) or TSVHQ (SEQ ID NO: 154), and
wherein the (X.sup.aX.sup.b).sub.z motif is YXYXYX.
[0044] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises: a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q); a cysteine motif selected from the group
consisting of: CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO:
41), CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95), and a (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
[0045] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises: a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q); a cysteine motif selected from the group
consisting of: wherein the cysteine motif is selected from the
group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152); and a (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
[0046] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises an additional
sequence that is a linker.
[0047] In some embodiments of each or any of the above or below
mentioned embodiments, the linker is linked to a C-terminus, a
N-terminus, or both C-terminus and N-terminus of the non-antibody
sequence.
[0048] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 is a ruminant CDR3.
[0049] In some embodiments of each or any of the above or below
mentioned embodiments, the ruminant is a cow.
[0050] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a human heavy chain variable region framework
sequence.
[0051] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a human heavy chain germline sequence or is a
derived from a human heavy chain germline sequence.
[0052] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a human heavy chain germline sequence that is a
VH4 family member germline sequence.
[0053] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a VH4-34 germline sequence.
[0054] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a human heavy chain germline sequence that is
derived from the VH4-34 germline sequence.
[0055] In some embodiments of each or any of the above or below
mentioned embodiments, the VH4-34 germline sequence comprises a
CDR1 comprising a Gly31Asp and Tyr32Lys substitution based on Kabat
numbering.
[0056] In some embodiments of each or any of the above or below
mentioned embodiments, the VH4-34 germline sequence comprises a
CDR2 comprising a Glu50Ser substitution based on Kabat
numbering.
[0057] In some embodiments of each or any of the above or below
mentioned embodiments, the VH4-34 germline sequence comprises a
CDR1 comprising Ala23Thr, Val24Ala, Tyr25Ser, Gly27Phe, Phe29Leu,
Gly31Asp, Tyr32Lys, Tyr33Ala, Trp34Val, and Ser35Gly substitutions,
and a CDR2 comprising Ile48Leu, Glu50Ser, Asn52Asp, His53Thr,
Ser54Gly, Ser56Asn, and Asn58Gly substitutions, based on Kabat
numbering.
[0058] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises the amino acid sequence set forth in SEQ ID NO:
461, 462, 463, 464, 465, 466, 467, 468, or 469.
[0059] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises the amino acid sequence set forth in SEQ ID NO:
470.
[0060] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises the amino acid sequence set forth in SEQ ID NO:
461, 462, 463, 464, 465, 466, 467, 468, or 469; and the amino acid
sequence set forth in SEQ ID NO: 470.
[0061] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises the amino acid sequence set forth in SEQ ID NO:
461, 462, 463, 464, 465, 466, 467, 468, or 469; the amino acid
sequence set forth in SEQ ID NO: 470; and a peptide sequence,
wherein the peptide sequence is positioned between SEQ ID NO: 461,
462, 463, 464, 465, 466, 467, 468, or 469, and SEQ ID NO: 470.
[0062] In some embodiments of each or any of the above or below
mentioned embodiments, the peptide sequence is a bovine sequence, a
non-bovine sequence, an antibody sequence, or a non-antibody
sequence.
[0063] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a human light chain variable region framework
sequence.
[0064] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a light chain variable region sequence that is a
lambda light chain variable region sequence.
[0065] In some embodiments of each or any of the above or below
mentioned embodiments, the lambda light chain variable region
sequence is a bovine lambda light chain variable region sequence or
derived from a bovine lambda light chain variable region
sequence.
[0066] In some embodiments of each or any of the above or below
mentioned embodiments, the lambda light chain variable region
sequence is a human lambda light chain variable region sequence or
derived from a human lambda light chain variable region
sequence.
[0067] In some embodiments of each or any of the above or below
mentioned embodiments, the human lambda light chain variable region
sequence is a VL1-51 germline sequence.
[0068] In some embodiments of each or any of the above or below
mentioned embodiments, the human lambda light chain variable region
sequence is derived from a VL1-51 germline sequence.
[0069] In some embodiments of each or any of the above or below
mentioned embodiments, the VL1-51 germline sequence comprises a
CDR1 comprising Ile29Val and Asn32Gly substitution based on Kabat
numbering.
[0070] In some embodiments of each or any of the above or below
mentioned embodiments, the VL1-51 germline sequence comprises a
CDR2 comprising a substitution of DNN to GDT.
[0071] In some embodiments of each or any of the above or below
mentioned embodiments, the VL1-51 germline sequence comprises a
CDR2 comprising a substitution of DNNKRP (SEQ ID NO: 471) to GDTSRA
(SEQ ID NO: 472).
[0072] In some embodiments of each or any of the above or below
mentioned embodiments, the VL1-51 germline sequence comprises a
S2A, T5N, P8S, A12G, A13S, and P14L substitution based on Kabat
numbering.
[0073] In some embodiments of each or any of the above or below
mentioned embodiments, the VL1-51 germline sequence comprises a
S2A, T5N, P8S, A12G, A13S, and P14L substitution based on Kabat
numbering, and a CDR2 comprising a substitution of DNN to GDT.
[0074] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprising the amino acid sequence set forth in SEQ ID NO:
440, 441, 442, 443, or 444.
[0075] The present disclosure also provides polynucleotides
encoding the humanized antibody or binding fragment thereof
disclosed herein.
[0076] The present disclosure also provides polynucleotides
encoding a humanized antibody or binding fragment thereof that
comprises an ultralong CDR3.
[0077] The present disclosure also provides polynucleotides
encoding a humanized variable region that comprises an ultralong
CDR3.
[0078] The present disclosure also provides vectors that comprise
the polynucleotides disclosed herein.
[0079] The present disclosure also provides host cells comprising
the vectors disclosed herein.
[0080] The present disclosure also provides a nucleic acid library
comprising a plurality of polynucleotides comprising sequences
coding for humanized antibodies or binding fragments thereof,
wherein the antibodies or binding fragments thereof comprise an
ultralong CDR3.
[0081] The present disclosure also provides a library of humanized
antibodies or binding fragments thereof, wherein the antibodies or
binding fragments thereof comprise an ultralong CDR3.
[0082] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 is 35 amino acids in
length or longer, 40 amino acids in length or longer, 45 amino
acids in length or longer, 50 amino acids in length or longer, 55
amino acids in length or longer, or 60 amino acids in length or
longer.
[0083] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 is 35 amino acids in
length or longer.
[0084] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises 3 or more
cysteine residues, 4 or more cysteine residues, 5 or more cysteine
residues, 6 or more cysteine residues, 7 or more cysteine residues,
8 or more cysteine residues, 9 or more cysteine residues, 10 or
more cysteine residues, 11 or more cysteine residues, or 12 or more
cysteine residues.
[0085] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises 3 or more
cysteine residues.
[0086] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
comprise a cysteine motif.
[0087] In some embodiments of each or any of the above or below
mentioned embodiments, the cysteine motif is selected from the
group consisting of: CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID
NO: 41), CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95).
[0088] In some embodiments of each or any of the above or below
mentioned embodiments, the cysteine motif is selected from the
group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152).
[0089] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises 2 to 6
disulfide bonds.
[0090] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises SEQ ID NO: 40
or a derivative thereof.
[0091] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises amino acid
residues 3-6 of any of one SEQ ID NO: 1-4.
[0092] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a non-human DH
or a derivative thereof.
[0093] In some embodiments of each or any of the above or below
mentioned embodiments, the non-human DH is SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11, or SEQ ID NO: 12.
[0094] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a JH sequence
or a derivative thereof.
[0095] In some embodiments of each or any of the above or below
mentioned embodiments, the JH sequence is SEQ ID NO: 13, SEQ ID NO:
14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
[0096] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises: a non-human VH
sequence or a derivative thereof; a non-human DH sequence or a
derivative thereof; and/or a JH sequence or derivative thereof.
[0097] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises an additional
amino acid sequence comprising two to six amino acid residues or
more positioned between the VH sequence and the DH sequence.
[0098] In some embodiments of each or any of the above or below
mentioned embodiments, the additional amino acid sequence is
selected from the group consisting of: IR, IF, SEQ ID NO: 18, SEQ
ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
[0099] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a sequence
derived from or based on SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:
28.
[0100] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a non-bovine
sequence or a non-antibody sequence.
[0101] In some embodiments of each or any of the above or below
mentioned embodiments, the non-antibody sequence is a synthetic
sequence.
[0102] In some embodiments of each or any of the above or below
mentioned embodiments, the non-antibody sequence is a cytokine
sequence, a lymphokine sequence, a chemokine sequence, a growth
factor sequence, a hormone sequence, or a toxin sequence.
[0103] In some embodiments of each or any of the above or below
mentioned embodiments, the non-antibody sequence is an IL-8
sequence, an IL-21 sequence, an SDF-1 (alpha) sequence, a
somatostatin sequence, a chlorotoxin sequence, a Pro-TxII sequence,
or a ziconotide sequence.
[0104] In some embodiments of each or any of the above or below
mentioned embodiments, the non-antibody sequence is any one of SEQ
ID NOS: 475-481.
[0105] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q).
[0106] In some embodiments of each or any of the above or below
mentioned embodiments, the X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif is TTVHQ (SEQ ID NO: 153), TSVHQ (SEQ ID NO: 154), SSVTQ (SEQ
ID NO: 155), STVHQ (SEQ ID NO: 156), ATVRQ (SEQ ID NO: 157), TTVYQ
(SEQ ID NO: 158), SPVHQ (SEQ ID NO: 159), ATVYQ (SEQ ID NO: 160),
TAVYQ (SEQ ID NO: 161), TNVHQ (SEQ ID NO: 162), ATVHQ (SEQ ID NO:
163), STVYQ (SEQ ID NO: 164), TIVHQ (SEQ ID NO: 165), AIVYQ (SEQ ID
NO: 166), TTVFQ (SEQ ID NO: 167), AAVFQ (SEQ ID NO: 168), GTVHQ
(SEQ ID NO: 169), ASVHQ (SEQ ID NO: 170), TAVFQ (SEQ ID NO: 171),
ATVFQ (SEQ ID NO: 172), AAAHQ (SEQ ID NO: 173), VVVYQ (SEQ ID NO:
174), GTVFQ (SEQ ID NO: 175), TAVHQ (SEQ ID NO: 176), ITVHQ (SEQ ID
NO: 177), ITAHQ (SEQ ID NO: 178), VTVHQ (SEQ ID NO: 179); AAVHQ
(SEQ ID NO: 180), GTVYQ (SEQ ID NO: 181), TTVLQ (SEQ ID NO: 182),
TTTHQ (SEQ ID NO: 183), or TTDYQ (SEQ ID NO: 184).
[0107] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif.
[0108] In some embodiments of each or any of the above or below
mentioned embodiments, the CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif is CTTVHQ (SEQ ID NO: 185), CTSVHQ (SEQ ID NO: 186), CSSVTQ
(SEQ ID NO: 187), CSTVHQ (SEQ ID NO: 188), CATVRQ (SEQ ID NO: 189),
CTTVYQ (SEQ ID NO: 190), CSPVHQ (SEQ ID NO: 191), CATVYQ (SEQ ID
NO: 192), CTAVYQ (SEQ ID NO: 193), CTNVHQ (SEQ ID NO: 194), CATVHQ
(SEQ ID NO: 195), CSTVYQ (SEQ ID NO: 196), CTIVHQ (SEQ ID NO: 197),
CAIVYQ (SEQ ID NO: 198), CTTVFQ (SEQ ID NO: 199), CAAVFQ (SEQ ID
NO: 200), CGTVHQ (SEQ ID NO: 201), CASVHQ (SEQ ID NO: 202), CTAVFQ
(SEQ ID NO: 203), CATVFQ (SEQ ID NO: 204), CAAAHQ (SEQ ID NO: 205),
CVVVYQ (SEQ ID NO: 206), CGTVFQ (SEQ ID NO: 207), CTAVHQ (SEQ ID
NO: 208), CITVHQ (SEQ ID NO: 209), CITAHQ (SEQ ID NO: 210), CVTVHQ
(SEQ ID NO: 211); CAAVHQ (SEQ ID NO: 212), CGTVYQ (SEQ ID NO: 213),
CTTVLQ (SEQ ID NO: 214), CTTTHQ (SEQ ID NO: 215), or CTTDYQ (SEQ ID
NO: 216).
[0109] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
(X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is any amino acid
residue, X.sup.b is an aromatic amino acid selected from the group
consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and
histidine (H), and wherein z is 1-4.
[0110] In some embodiments of each or any of the above or below
mentioned embodiments, the (X.sup.aX.sup.b).sub.z motif is CYTYNYEF
(SEQ ID NO: 217), HYTYTYDF (SEQ ID NO: 218), HYTYTYEW (SEQ ID NO:
219), KHRYTYEW (SEQ ID NO: 220), NYIYKYSF (SEQ ID NO: 221),
PYIYTYQF (SEQ ID NO: 222), SFTYTYEW (SEQ ID NO: 223), SYIYIYQW (SEQ
ID NO: 224), SYNYTYSW (SEQ ID NO: 225), SYSYSYEY (SEQ ID NO: 226),
SYTYNYDF (SEQ ID NO: 227), SYTYNYEW (SEQ ID NO: 228), SYTYNYQF (SEQ
ID NO: 229), SYVWTHNF (SEQ ID NO: 230), TYKYVYEW (SEQ ID NO: 231),
TYTYTYEF (SEQ ID NO: 232), TYTYTYEW (SEQ ID NO: 233), VFTYTYEF (SEQ
ID NO: 234), AYTYEW (SEQ ID NO: 235), DYIYTY (SEQ ID NO: 236),
IHSYEF (SEQ ID NO: 237), SFTYEF (SEQ ID NO: 238), SHSYEF (SEQ ID
NO: 239), THTYEF (SEQ ID NO: 240), TWTYEF (SEQ ID NO: 241), TYNYEW
(SEQ ID NO: 242), TYSYEF (SEQ ID NO: 243), TYSYEH (SEQ ID NO: 244),
TYTYDF (SEQ ID NO: 245), TYTYEF (SEQ ID NO: 246), TYTYEW (SEQ ID
NO: 247), AYEF (SEQ ID NO: 248), AYSF (SEQ ID NO: 249), AYSY (SEQ
ID NO: 250), CYSF (SEQ ID NO: 251), DYTY (SEQ ID NO: 252), KYEH
(SEQ ID NO: 253), KYEW (SEQ ID NO: 254), MYEF (SEQ ID NO: 255),
NWIY (SEQ ID NO: 256), NYDY (SEQ ID NO: 257), NYQW (SEQ ID NO:
258), NYSF (SEQ ID NO: 259), PYEW (SEQ ID NO: 260), RYNW (SEQ ID
NO: 261), RYTY (SEQ ID NO: 262), SYEF (SEQ ID NO: 263), SYEH (SEQ
ID NO: 264), SYEW (SEQ ID NO: 265), SYKW (SEQ ID NO: 266), SYTY
(SEQ ID NO: 267), TYDF (SEQ ID NO: 268), TYEF (SEQ ID NO: 269),
TYEW (SEQ ID NO: 270), TYQW (SEQ ID NO: 271), TYTY (SEQ ID NO:
272), or VYEW (SEQ ID NO: 273).
[0111] In some embodiments of each or any of the above or below
mentioned embodiments, the (X.sup.aX.sup.b).sub.z motif is
YXYXYX.
[0112] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n motif, wherein X.sup.1
is threonine (T), glycine (G), alanine (A), serine (S), or valine
(V), wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q), and wherein n is 27-54.
[0113] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises
X.sub.n(X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is any amino
acid residue, X.sup.b is an aromatic amino acid selected from the
group consisting of: tyrosine (Y), phenylalanine (F), tryptophan
(W), and histidine (H), wherein n is 27-54, and wherein z is
1-4.
[0114] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n(X.sup.aX.sup.b).sub.z
motif, wherein X.sub.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sub.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sub.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sub.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), wherein X.sub.5 is glutamine (Q), X.sup.a is any
amino acid residue, X.sup.b is an aromatic amino acid selected from
the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), wherein n is 27-54, and wherein
z is 1-4.
[0115] In some embodiments of each or any of the above or below
mentioned embodiments, the X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif is TTVHQ (SEQ ID NO: 153) or TSVHQ (SEQ ID NO: 154), and
wherein the (X.sup.aX.sup.b).sub.z motif is YXYXYX.
[0116] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises: a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q), a cysteine motif selected from the group
consisting of: CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO:
41), CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.8CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95); and a (X.sup.aX.sup.b).sub.z motif, X.sup.a is any
amino acid residue, X.sup.b is an aromatic amino acid selected from
the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
[0117] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises: a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q); a cysteine motif selected from the group
consisting of: wherein the cysteine motif is selected from the
group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152); andba (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
[0118] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises an additional
sequence that is a linker.
[0119] In some embodiments of each or any of the above or below
mentioned embodiments, the linker is linked to a C-terminus, a
N-terminus, or both C-terminus and N-terminus of the non-antibody
sequence.
[0120] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 is a ruminant CDR3.
[0121] In some embodiments of each or any of the above or below
mentioned embodiments, the ruminant is a cow.
[0122] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a human heavy chain variable region framework
sequence.
[0123] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a human heavy chain germline sequence or is a
derived from a human heavy chain germline sequence.
[0124] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a human heavy chain germline sequence that is a
VH4 family member germline sequence.
[0125] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a VH4-34 germline sequence.
[0126] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a human heavy chain germline sequence that is
derived from VH4-34.
[0127] In some embodiments of each or any of the above or below
mentioned embodiments, the VH4-34 germline sequence comprises a
CDR1 comprising a Gly31Asp and Tyr32Lys substitution based on Kabat
numbering.
[0128] In some embodiments of each or any of the above or below
mentioned embodiments, the VH4-34 germline sequence comprises a
CDR2 comprising a Glu50Ser substitution based on Kabat
numbering.
[0129] In some embodiments of each or any of the above or below
mentioned embodiments, the VH4-34 germline sequence comprises a
CDR1 comprising Ala23Thr, Val24Ala, Tyr25Ser, Gly27Phe, Phe29Leu,
Gly31Asp, Tyr32Lys, Tyr33Ala, Trp34Val, and Ser35Gly substitutions,
and a CDR2 comprising Ile48Leu, Glu50Ser, Asn52Asp, His53Thr,
Ser54Gly, Ser56Asn, and Asn58Gly substitutions, based on Kabat
numbering.
[0130] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises the amino acid sequence set forth in SEQ ID NO:
470.
[0131] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises the amino acid sequence set forth in SEQ ID NO:
461, 462, 463, 464, 465, 466, 467, 468, or 469; and the amino acid
sequence set forth in SEQ ID NO: 470.
[0132] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises the amino acid sequence set forth in SEQ ID NO:
461, 462, 463, 464, 465, 466, 467, 468, or 469; the amino acid
sequence set forth in SEQ ID NO: 470; and a peptide sequence,
wherein the peptide sequence is positioned between SEQ ID NO: 461,
462, 463, 464, 465, 466, 467, 468, or 469, and SEQ ID NO: 470.
[0133] In some embodiments of each or any of the above or below
mentioned embodiments, the peptide sequence is a bovine sequence, a
non-bovine sequence, an antibody sequence, or a non-antibody
sequence.
[0134] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a lambda light chain variable region
sequence.
[0135] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprises a light chain variable region sequence that is a
lambda light chain variable region sequence.
[0136] In some embodiments of each or any of the above or below
mentioned embodiments, the lambda light chain variable region
sequence is a bovine lambda light chain variable region
sequence.
[0137] In some embodiments of each or any of the above or below
mentioned embodiments, the lambda light chain variable region
sequence is a human lambda light chain variable region
sequence.
[0138] In some embodiments of each or any of the above or below
mentioned embodiments, the human lambda light chain variable region
sequence is VL1-51.
[0139] In some embodiments of each or any of the above or below
mentioned embodiments, the human lambda light chain variable region
sequence is derived from VL1-51.
[0140] In some embodiments of each or any of the above or below
mentioned embodiments, the VL1-51 germline sequence comprises a
CDR1 comprising Ile29Val and Asn32Gly substitution based on Kabat
numbering.
[0141] In some embodiments of each or any of the above or below
mentioned embodiments, the VL1-51 germline sequence comprises a
CDR2 comprising a substitution of DNN to GDT.
[0142] In some embodiments of each or any of the above or below
mentioned embodiments, the VL1-51 germline sequence comprises a
CDR2 comprising a substitution of DNNKRP (SEQ ID NO: 471) to GDTSRA
(SEQ ID NO: 472).
[0143] In some embodiments of each or any of the above or below
mentioned embodiments, the VL1-51 germline sequence comprises a
S2A, T5N, P8S, A12G, A13S, and P14L substitution based on Kabat
numbering.
[0144] In some embodiments of each or any of the above or below
mentioned embodiments, the VL1-51 germline sequence comprises a
S2A, T5N, P8S, A12G, A13S, and P14L substitution based on Kabat
numbering, and a CDR2 comprising a substitution of DNN to GDT.
[0145] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibody or binding fragment
thereof comprising the amino acid sequence set forth in SEQ ID NO:
440, 441, 442, 443, or 444.
[0146] In some embodiments of each or any of the above or below
mentioned embodiments, the humanized antibodies or binding
fragments thereof are present in a spatially addressed format.
[0147] The present disclosure also provides a method of humanizing
an antibody variable region comprising the step of genetically
combining a nucleic acid sequence encoding a non-human ultralong
CDR3 with a nucleic acid sequence encoding a human variable region
framework (FR) sequence.
[0148] The present disclosure also provides a method of generating
a library of humanized antibodies that comprises an ultralong CDR3,
the method comprising: combining a nucleic acid sequence encoding
an ultralong CDR3 with a nucleic acid sequence encoding a human
variable region framework (FR) sequence to produce nucleic acids
encoding for humanized antibodies that comprises an ultralong CDR3;
and expressing the nucleic acids encoding for humanized antibodies
that comprises an ultralong CDR3 to generate a library of humanized
antibodies that comprises an ultralong CDR3.
[0149] The present disclosure also provides a method of generating
a library of humanized antibodies or binding fragments thereof
comprising an ultralong CDR3 and which comprises a non-antibody
sequence, the method comprising: combining a nucleic acid sequence
encoding an ultralong CDR3, a nucleic acid sequence encoding a
human variable region framework (FR) sequence, and a nucleic acid
sequence encoding a non-antibody sequence to produce nucleic acids
encoding humanized antibodies or binding fragments thereof
comprising an ultralong CDR3 and a non-antibody sequence, and
expressing the nucleic acids encoding humanized antibodies or
binding fragments thereof comprising an ultralong CDR3 and a
non-antibody sequence to generate a library of humanized antibodies
or binding fragments thereof comprising an ultralong CDR3 and a
non-antibody sequence. In some embodiments, the ultralong CDR3
comprises a bovine, a non-bovine, an antibody, or a non-antibody
sequence.
[0150] The present disclosure also provides a library of humanized
antibodies or binding fragments thereof comprising an ultralong
CDR3 which comprises a non-bovine or a non-antibody sequence.
[0151] The present disclosure also provides a method of generating
a library of humanized antibodies or binding fragments thereof
comprising an ultralong CDR3 which comprises a cysteine motif, the
method comprising: combining a human variable region framework (FR)
sequence, and a nucleic acid sequence encoding an ultralong CDR3
and a cysteine motif; introducing one or more nucleotide changes to
the nucleic acid sequence encoding one or more amino acid residues
that are positioned between one or more cysteine residues in the
cysteine motif for nucleotides encoding different amino acid
residues to produce nucleic acids encoding humanized antibodies or
binding fragments thereof comprising an ultralong CDR3 and a
cysteine motif with one or more nucleotide changes introduced
between one or more cysteine residues in the cysteine domain; and
expressing the nucleic acids encoding humanized antibodies or
binding fragments thereof comprising an ultralong CDR3 and a
cysteine motif with one or more nucleotide changes introduced
between one or more cysteine residues in the cysteine domain to
generate a library of humanized antibodies or binding fragments
thereof comprising an ultralong CDR3 and a cysteine motif with one
or more amino acid changes introduced between one or more cysteine
residues in the cysteine domain.
[0152] The present disclosure also provides a library of humanized
antibodies or binding fragments thereof comprising an ultralong
CDR3 which comprises a cysteine motif, wherein the antibodies or
binding fragments comprise one or more substitutions of amino acid
residues that are positioned between cysteine residues in the
cysteine motif.
[0153] The present disclosure also provides a method of generating
a library of humanized antibodies or binding fragments thereof
comprising a bovine ultralong CDR3, the method comprising:
combining a nucleic acid sequence encoding a human variable region
framework (FR) sequence and a nucleic acid encoding a bovine
ultralong CDR3, and expressing the nucleic acids encoding a human
variable region framework (FR) sequence and a nucleic acid encoding
a bovine ultralong CDR3 to generate a library of humanized
antibodies or binding fragments thereof comprising a bovine
ultralong CDR3.
[0154] The present disclosure provides humanized antibodies,
including antibodies comprising a CDR3 scaffold, methods of making
same, and uses thereof.
[0155] The present disclosure provides a humanized antibody or
binding fragment thereof comprising a CDR3 scaffold.
[0156] In some embodiments, the CDR3 scaffold comprises one or more
human variable region framework sequences.
[0157] The present disclosure also provides a library of humanized
antibodies or binding fragments thereof comprising a bovine
ultralong CDR3.
[0158] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q).
[0159] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold is 35 amino acids in
length or longer, 40 amino acids in length or longer, 45 amino
acids in length or longer, 50 amino acids in length or longer, 55
amino acids in length or longer, or 60 amino acids in length or
longer.
[0160] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold is 35 amino acids in
length or longer.
[0161] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises 3 or more
cysteine residues, 4 or more cysteine residues, 5 or more cysteine
residues, 6 or more cysteine residues, 7 or more cysteine residues,
8 or more cysteine residues, 9 or more cysteine residues, 10 or
more cysteine residues, 11 or more cysteine residues, or 12 or more
cysteine residues.
[0162] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises 3 or more
cysteine residues.
[0163] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises a cysteine
motif.
[0164] In some embodiments of each or any of the above or below
mentioned embodiments, the cysteine motif is selected from the
group consisting of: CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID
NO: 41), CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95).
[0165] In some embodiments of each or any of the above or below
mentioned embodiments, the cysteine motif is selected from the
group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152).
[0166] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises 2 to 6 disulfide
bonds.
[0167] In some embodiments of each or any of the above or below
mentioned embodiments, the X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif in the CDR3 scaffold is TTVHQ (SEQ ID NO: 153), TSVHQ (SEQ ID
NO: 154), SSVTQ (SEQ ID NO: 155), STVHQ (SEQ ID NO: 156), ATVRQ
(SEQ ID NO: 157), TTVYQ (SEQ ID NO: 158), SPVHQ (SEQ ID NO: 159),
ATVYQ (SEQ ID NO: 160), TAVYQ (SEQ ID NO: 161), TNVHQ (SEQ ID NO:
162), ATVHQ (SEQ ID NO: 163), STVYQ (SEQ ID NO: 164), TIVHQ (SEQ ID
NO: 165), AIVYQ (SEQ ID NO: 166), TTVFQ (SEQ ID NO: 167), AAVFQ
(SEQ ID NO: 168), GTVHQ (SEQ ID NO: 169), ASVHQ (SEQ ID NO: 170),
TAVFQ (SEQ ID NO: 171), ATVFQ (SEQ ID NO: 172), AAAHQ (SEQ ID NO:
173), VVVYQ (SEQ ID NO: 174), GTVFQ (SEQ ID NO: 175), TAVHQ (SEQ ID
NO: 176), ITVHQ (SEQ ID NO: 177), ITAHQ (SEQ ID NO: 178), VTVHQ
(SEQ ID NO: 179); AAVHQ (SEQ ID NO: 180), GTVYQ (SEQ ID NO: 181),
TTVLQ (SEQ ID NO: 182), TTTHQ (SEQ ID NO: 183), or TTDYQ (SEQ ID
NO: 184).
[0168] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif.
[0169] In some embodiments of each or any of the above or below
mentioned embodiments, the CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif is CTTVHQ (SEQ ID NO: 185), CTSVHQ (SEQ ID NO: 186), CSSVTQ
(SEQ ID NO: 187), CSTVHQ (SEQ ID NO: 188), CATVRQ (SEQ ID NO: 189),
CTTVYQ (SEQ ID NO: 190), CSPVHQ (SEQ ID NO: 191), CATVYQ (SEQ ID
NO: 192), CTAVYQ (SEQ ID NO: 193), CTNVHQ (SEQ ID NO: 194), CATVHQ
(SEQ ID NO: 195), CSTVYQ (SEQ ID NO: 196), CTIVHQ (SEQ ID NO: 197),
CAIVYQ (SEQ ID NO: 198), CTTVFQ (SEQ ID NO: 199), CAAVFQ (SEQ ID
NO: 200), CGTVHQ (SEQ ID NO: 201), CASVHQ (SEQ ID NO: 202), CTAVFQ
(SEQ ID NO: 203), CATVFQ (SEQ ID NO: 204), CAAAHQ (SEQ ID NO: 205),
CVVVYQ (SEQ ID NO: 206), CGTVFQ (SEQ ID NO: 207), CTAVHQ (SEQ ID
NO: 208), CITVHQ (SEQ ID NO: 209), CITAHQ (SEQ ID NO: 210), CVTVHQ
(SEQ ID NO: 211); CAAVHQ (SEQ ID NO: 212), CGTVYQ (SEQ ID NO: 213),
CTTVLQ (SEQ ID NO: 214), CTTTHQ (SEQ ID NO: 215), or CTTDYQ (SEQ ID
NO: 216).
[0170] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises a
(X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is any amino acid
residue, X.sup.b is an aromatic amino acid selected from the group
consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and
histidine (H), and wherein z is 1-4.
[0171] In some embodiments of each or any of the above or below
mentioned embodiments, the (X.sup.aX.sup.b).sub.z motif is CYTYNYEF
(SEQ ID NO: 217), HYTYTYDF (SEQ ID NO: 218), HYTYTYEW (SEQ ID NO:
219), KHRYTYEW (SEQ ID NO: 220), NYIYKYSF (SEQ ID NO: 221),
PYIYTYQF (SEQ ID NO: 222), SFTYTYEW (SEQ ID NO: 223), SYIYIYQW (SEQ
ID NO: 224), SYNYTYSW (SEQ ID NO: 225), SYSYSYEY (SEQ ID NO: 226),
SYTYNYDF (SEQ ID NO: 227), SYTYNYEW (SEQ ID NO: 228), SYTYNYQF (SEQ
ID NO: 229), SYVWTHNF (SEQ ID NO: 230), TYKYVYEW (SEQ ID NO: 231),
TYTYTYEF (SEQ ID NO: 232), TYTYTYEW (SEQ ID NO: 233), VFTYTYEF (SEQ
ID NO: 234), AYTYEW (SEQ ID NO: 235), DYIYTY (SEQ ID NO: 236),
IHSYEF (SEQ ID NO: 237), SFTYEF (SEQ ID NO: 238), SHSYEF (SEQ ID
NO: 239), THTYEF (SEQ ID NO: 240), TWTYEF (SEQ ID NO: 241), TYNYEW
(SEQ ID NO: 242), TYSYEF (SEQ ID NO: 243), TYSYEH (SEQ ID NO: 244),
TYTYDF (SEQ ID NO: 245), TYTYEF (SEQ ID NO: 246), TYTYEW (SEQ ID
NO: 247), AYEF (SEQ ID NO: 248), AYSF (SEQ ID NO: 249), AYSY (SEQ
ID NO: 250), CYSF (SEQ ID NO: 251), DYTY (SEQ ID NO: 252), KYEH
(SEQ ID NO: 253), KYEW (SEQ ID NO: 254), MYEF (SEQ ID NO: 255),
NWIY (SEQ ID NO: 256), NYDY (SEQ ID NO: 257), NYQW (SEQ ID NO:
258), NYSF (SEQ ID NO: 259), PYEW (SEQ ID NO: 260), RYNW (SEQ ID
NO: 261), RYTY (SEQ ID NO: 262), SYEF (SEQ ID NO: 263), SYEH (SEQ
ID NO: 264), SYEW (SEQ ID NO: 265), SYKW (SEQ ID NO: 266), SYTY
(SEQ ID NO: 267), TYDF (SEQ ID NO: 268), TYEF (SEQ ID NO: 269),
TYEW (SEQ ID NO: 270), TYQW (SEQ ID NO: 271), TYTY (SEQ ID NO:
272), or VYEW (SEQ ID NO: 273).
[0172] In some embodiments of each or any of the above or below
mentioned embodiments, the (X.sup.aX.sup.b).sub.z motif is
YXYXYX.
[0173] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n motif, wherein X.sup.1
is threonine (T), glycine (G), alanine (A), serine (S), or valine
(V), wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q), and wherein n is 27-54.
[0174] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises
X.sub.n(X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is any amino
acid residue, X.sup.b is an aromatic amino acid selected from the
group consisting of: tyrosine (Y), phenylalanine (F), tryptophan
(W), and histidine (H), wherein n is 27-54, and wherein z is
1-4.
[0175] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n(X.sup.aX.sup.b).sub.z
motif, herein X.sub.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sub.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sub.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sub.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), wherein X.sub.5 is glutamine (Q), X.sup.a is any
amino acid residue, X.sup.b is an aromatic amino acid selected from
the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), wherein n is 27-54, and wherein
z is 1-4.
[0176] In some embodiments of each or any of the above or below
mentioned embodiments, the X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif in the CDR3 scaffold is TTVHQ (SEQ ID NO: 153) or TSVHQ (SEQ
ID NO: 154), and wherein the (X.sup.aX.sup.b).sub.z motif is
YXYXYX.
[0177] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises: a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q), a cysteine motif selected from the group
consisting of: CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO:
41), CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95); and (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
[0178] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises: a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q); a cysteine motif selected from the group
consisting of: wherein the cysteine motif is selected from the
group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152); and a (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
[0179] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises SEQ ID NO: 40 or
a derivative thereof.
[0180] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises amino acid
residues 3-6 of any of one SEQ ID NO: 1-4.
[0181] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises a non-human DH
or a derivative thereof.
[0182] In some embodiments of each or any of the above or below
mentioned embodiments, the non-human DH is SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11, or SEQ ID NO: 12
[0183] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises a JH sequence or
a derivative thereof.
[0184] In some embodiments of each or any of the above or below
mentioned embodiments, the JH sequence is SEQ ID NO: 13, SEQ ID NO:
14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
[0185] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises: a non-human VH
sequence or a derivative thereof; a non-human DH sequence or a
derivative thereof; and/or a JH sequence or derivative thereof.
[0186] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises an additional
amino acid sequence comprising two to six amino acid residues or
more positioned between the VH sequence and the DH sequence.
[0187] In some embodiments of each or any of the above or below
mentioned embodiments, the additional amino acid sequence is
selected from the group consisting of: IR, IF, SEQ ID NO: 18, SEQ
ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
[0188] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold comprises a sequence
derived from or based on SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:
28.
[0189] In some embodiments of each or any of the above or below
mentioned embodiments, the CDR3 scaffold is a ruminant CDR3
scaffold.
[0190] In some embodiments of each or any of the above or below
mentioned embodiments, the ruminant is a cow.
[0191] The present disclosure also provides a library comprising
the CDR3 scaffold disclosed herein.
[0192] The present disclosure also provides a polynucleotide
encoding the CDR3 scaffold disclosed herein.
[0193] The present disclosure also provides a vector comprising any
of the polynucleotides disclosed herein.
[0194] The present disclosure also provides a host cell comprising
any of the vectors disclosed herein.
[0195] The present disclosure also provides
[0196] In some embodiments of each or any of the above or below
mentioned embodiments, the cell is a bacteria, virus, or
bacteriophage.
BRIEF DESCRIPTION OF THE DRAWINGS
[0197] The foregoing summary, as well as the following detailed
description of the disclosure, will be better understood when read
in conjunction with the appended figures. For the purpose of
illustrating the disclosure, shown in the figures are embodiments
which are presently preferred. It should be understood, however,
that the disclosure is not limited to the precise arrangements,
examples and instrumentalities shown.
[0198] FIG. 1 shows a sequence alignment of exemplary
bovine-derived antibody variable region sequences designated
BLV1H12, BLV5B8, BLV5D3, BLV8C11, BF4E9, BF1H1, or F18 that
comprise an ultralong CDR3 sequence.
[0199] FIG. 2A-C depicts ultralong CDR3 sequences. (Top)
Translation from the germline V.sub.HBUL, D.sub.H2, and J.sub.H.
The 5 full length ultralong CDR H3s reported in the literature
contain between four and eight cysteines and are not highly
homologous to one another; however, some conservation of cysteine
residues with D.sub.H2 could be found when the first cysteine of
these CDR H3s was "fixed" prior to alignment. Four of the seven
sequences (BLV1H12, BLV5D3, BLV8C11, and BF4E9) contain four
cysteines in the same positions as D.sub.H2, but also have
additional cysteines. BLV5B8 has two cysteines in common with the
germline D.sub.H2. This limited homology with some cysteine
conservation suggests that mutation of D.sub.H2 could generate
these sequences. B-L1 and B-L2 are from initial sequences from
bovine spleen, and the remaining are selected ultralong CDR H3
sequences from deep sequencing data. The first group contains the
longest CDR H3s identified, and appear clonally related. The *
indicates a sequence represented 167 times, suggesting it was
strongly selected for function. Several of the eight-cysteine
sequences appear selected for function as they were represented
multiple times, indicated in parentheses. Other representative
sequences of various lengths are indicated in the last group. The
framework cysteine and tryptophan residues that define the CDR H3
boundaries are double-underlined. The sequences BLV1H12 through
UL-77 (left-most column) presented in Tables 2A-C are depicted
broken apart into four segments to identify the segments of amino
acid residues that are derived from certain germline sequences and
V/D/J joining sequences. Moving from left to right, the first
segment is derived from the VH germline and is represented in the
disclosure as a X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif. The
second segment represents sequences from V-D joining and is
represented in the disclosure as X.sub.n. The third segment is a
string of amino acid residues derived from D.sub.H2 germline, and
the fourth segment is a string of amino acid residues derived from
J.sub.H1 germline region.
[0200] FIG. 3 depicts a sequence alignment of exemplary
bovine-derived ultralong CDR3 sequences designated BLV1H12, BLV5B8,
BLV5D3, BLV8C11, BF4E9, BF1H1, or F18.
[0201] FIG. 4 shows an exemplary bovine germline heavy chain
variable region (VH) sequence designated VH-UL suitable for
modification or use with an ultralong CDR3 sequence.
[0202] FIG. 5A-B shows exemplary human germline heavy chain
variable region sequences designated 4-39, 4-59*03, 4-34*09, and
4-34*02 that are suitable for modification or use with an ultralong
CDR3 sequence (A) and an alignment of these sequences (B).
[0203] FIG. 6 shows an exemplary bovine light chain variable region
sequence designated BLV1H12 suitable for modification or use with
an ultralong CDR 3 sequence (e.g., a heavy chain variable region
sequence comprising an ultralong CDR3 sequence).
[0204] FIG. 7A-B shows exemplary light chain variable region
sequences designated VI1-47, VI1-40*1, VI1-51*01, and VI2-18*02
that are suitable for modification or use with an ultralong CDR 3
sequence (A) and an alignment of these sequences (B).
DETAILED DESCRIPTION
[0205] The present disclosure provides humanized antibodies
comprising ultralong CDR3 sequences, along with materials for
(e.g., protein sequences, genetic sequences, cells, libraries) and
methods of making the antibodies (e.g., humanizing methods, library
methods). Such humanized antibodies may be useful for the treatment
or prevention of a variety of disease states or disorders.
[0206] The present disclosure also provides humanized antibodies
comprising ultralong CDR3 sequences, wherein the CDR3 sequences are
35 amino acids in length or longer (e.g., 40 or longer, 45 or
longer, 50 or longer, 55 or longer, 60 or longer) and/or wherein
the CDR3 sequences have at least 3 cysteine residues or more (e.g.,
3 or more cysteine residues, 4 or more cysteine residues, 5 or more
cysteine residues, 6 or more cysteine residues, 7 or more cysteine
residues, 8 or more cysteine residues, 9 or more cysteine residues,
10 or more cysteine residues, 11 or more cysteine residues, or 12
or more cysteine residues). Such antibodies, as described herein,
bind (e.g., specifically or selectively bind) a variety of targets,
including, for example protein targets such as transmembrane
proteins (e.g., GPCRs, ion channels, transporter, cell surface
receptors).
[0207] The present disclosure also provides methods and materials
for the preparation or making of humanized antibodies comprising
ultralong CDR3 sequences. Such materials include proteins, genetic
sequences, cells and libraries. Such methods include methods of
humanization and method of making and screening libraries.
[0208] The present disclosure provides a humanized antibody or
binding fragment thereof comprising an ultralong CDR3. In some
embodiments, the ultralong CDR3 may be 35 amino acids in length or
longer, 40 amino acids in length or longer, 45 amino acids in
length or longer, 50 amino acids in length or longer, 55 amino
acids in length or longer, or 60 amino acids in length or longer.
In some embodiments, the ultralong CDR3 may comprise 3 or more
cysteine residues, 4 or more cysteine residues, 5 or more cysteine
residues, 6 or more cysteine residues, 7 or more cysteine residues,
8 or more cysteine residues, 9 or more cysteine residues, 10 or
more cysteine residues, 11 or more cysteine residues, or 12 or more
cysteine residues. The ultralong CDR3 may comprise a cysteine motif
including, for example, where the cysteine motif is selected from
the group consisting of: CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ
ID NO: 41), CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95). Alternatively, the ultralong CDR3 may comprise a
cysteine motif including, for example, where the cysteine motif is
selected from the group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152).
[0209] The present disclosure provides a humanized antibody or
binding fragment thereof comprising an ultralong CDR3, wherein the
ultralong CDR3 comprises a X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif, wherein X.sup.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sup.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sup.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sup.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), and wherein X.sup.5 is glutamine (Q). In some
embodiments, the X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif may be
TTVHQ (SEQ ID NO: 153), TSVHQ (SEQ ID NO: 154), SSVTQ (SEQ ID NO:
155), STVHQ (SEQ ID NO: 156), ATVRQ (SEQ ID NO: 157), TTVYQ (SEQ ID
NO: 158), SPVHQ (SEQ ID NO: 159), ATVYQ (SEQ ID NO: 160), TAVYQ
(SEQ ID NO: 161), TNVHQ (SEQ ID NO: 162), ATVHQ (SEQ ID NO: 163),
STVYQ (SEQ ID NO: 164), TIVHQ (SEQ ID NO: 165), AIVYQ (SEQ ID NO:
166), TTVFQ (SEQ ID NO: 167), AAVFQ (SEQ ID NO: 168), GTVHQ (SEQ ID
NO: 169), ASVHQ (SEQ ID NO: 170), TAVFQ (SEQ ID NO: 171), ATVFQ
(SEQ ID NO: 172), AAAHQ (SEQ ID NO: 173), VVVYQ (SEQ ID NO: 174),
GTVFQ (SEQ ID NO: 175), TAVHQ (SEQ ID NO: 176), ITVHQ (SEQ ID NO:
177), ITAHQ (SEQ ID NO: 178), VTVHQ (SEQ ID NO: 179); AAVHQ (SEQ ID
NO: 180), GTVYQ (SEQ ID NO: 181), TTVLQ (SEQ ID NO: 182), TTTHQ
(SEQ ID NO: 183), or TTDYQ (SEQ ID NO: 184).
[0210] The present disclosure provides a humanized antibody or
binding fragment thereof comprising an ultralong CDR3, wherein the
ultralong CDR3 comprises a (X.sup.aX.sup.b).sub.z motif, wherein
X.sup.a is any amino acid residue, X.sup.b is an aromatic amino
acid selected from the group consisting of: tyrosine (Y),
phenylalanine (F), tryptophan (W), and histidine (H), and wherein z
is 1-4. In some embodiments, the (X.sup.aX.sup.b).sub.z motif may
be CYTYNYEF (SEQ ID NO: 217), HYTYTYDF (SEQ ID NO: 218), HYTYTYEW
(SEQ ID NO: 219), KHRYTYEW (SEQ ID NO: 220), NYIYKYSF (SEQ ID NO:
221), PYIYTYQF (SEQ ID NO: 222), SFTYTYEW (SEQ ID NO: 223),
SYIYIYQW (SEQ ID NO: 224), SYNYTYSW (SEQ ID NO: 225), SYSYSYEY (SEQ
ID NO: 226), SYTYNYDF (SEQ ID NO: 227), SYTYNYEW (SEQ ID NO: 228),
SYTYNYQF (SEQ ID NO: 229), SYVWTHNF (SEQ ID NO: 230), TYKYVYEW (SEQ
ID NO: 231), TYTYTYEF (SEQ ID NO: 232), TYTYTYEW (SEQ ID NO: 233),
VFTYTYEF (SEQ ID NO: 234), AYTYEW (SEQ ID NO: 235), DYIYTY (SEQ ID
NO: 236), IHSYEF (SEQ ID NO: 237), SFTYEF (SEQ ID NO: 238), SHSYEF
(SEQ ID NO: 239), THTYEF (SEQ ID NO: 240), TWTYEF (SEQ ID NO: 241),
TYNYEW (SEQ ID NO: 242), TYSYEF (SEQ ID NO: 243), TYSYEH (SEQ ID
NO: 244), TYTYDF (SEQ ID NO: 245), TYTYEF (SEQ ID NO: 246), TYTYEW
(SEQ ID NO: 247), AYEF (SEQ ID NO: 248), AYSF (SEQ ID NO: 249),
AYSY (SEQ ID NO: 250), CYSF (SEQ ID NO: 251), DYTY (SEQ ID NO:
252), KYEH (SEQ ID NO: 253), KYEW (SEQ ID NO: 254), MYEF (SEQ ID
NO: 255), NWIY (SEQ ID NO: 256), NYDY (SEQ ID NO: 257), NYQW (SEQ
ID NO: 258), NYSF (SEQ ID NO: 259), PYEW (SEQ ID NO: 260), RYNW
(SEQ ID NO: 261), RYTY (SEQ ID NO: 262), SYEF (SEQ ID NO: 263),
SYEH (SEQ ID NO: 264), SYEW (SEQ ID NO: 265), SYKW (SEQ ID NO:
266), SYTY (SEQ ID NO: 267), TYDF (SEQ ID NO: 268), TYEF (SEQ ID
NO: 269), TYEW (SEQ ID NO: 270), TYQW (SEQ ID NO: 271), TYTY (SEQ
ID NO: 272), or VYEW (SEQ ID NO: 273).
[0211] The present disclosure provides a humanized antibody or
binding fragment thereof comprising an ultralong CDR3, wherein the
ultralong CDR3 comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n(X.sup.aX.sup.b).sub.z
motif, wherein X.sup.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sup.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sup.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sup.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), and wherein X.sup.5 is glutamine (Q), wherein
X.sup.a is any amino acid residue, X.sup.b is an aromatic amino
acid selected from the group consisting of: tyrosine (Y),
phenylalanine (F), tryptophan (W), and histidine (H), wherein n is
27-54, and wherein z is 1-4.
[0212] The present disclosure provides a humanized antibody or
binding fragment thereof comprising an ultralong CDR3, wherein the
ultralong CDR3 comprises: a CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif, wherein X.sup.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sup.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sup.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sup.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), and wherein X.sup.5 is glutamine (Q), a cysteine
motif selected from the group consisting of:
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 41),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95), and a (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
[0213] The present disclosure provides a humanized antibody or
binding fragment thereof comprising an ultralong CDR3, wherein the
ultralong CDR3 comprises: a CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif, wherein X.sup.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sup.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sup.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sup.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), and wherein X.sup.5 is glutamine (Q); a cysteine
motif selected from the group consisting of: wherein the cysteine
motif is selected from the group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152); and a (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
[0214] The present disclosure also provides methods of generating a
library of humanized antibodies that comprises an ultralong CDR3,
comprising: combining a nucleic acid sequence encoding an ultralong
CDR3 with a nucleic acid sequence encoding a human variable region
framework (FR) sequence to produce nucleic acids encoding for
humanized antibodies that comprises an ultralong CDR3; and
expressing the nucleic acids encoding for humanized antibodies that
comprises an ultralong CDR3 to generate a library of humanized
antibodies that comprises an ultralong CDR3.
[0215] The present disclosure also provides methods of generating a
library of humanized antibodies or binding fragments thereof
comprising an ultralong CDR3 that comprises a non-antibody
sequence, comprising: combining a nucleic acid sequence encoding an
ultralong CDR3, a nucleic acid sequence encoding a human variable
region framework (FR) sequence, and a nucleic acid sequence
encoding a non-antibody sequence to produce nucleic acids encoding
humanized antibodies or binding fragments thereof comprising an
ultralong CDR3 and a non-antibody sequence, and expressing the
nucleic acids encoding humanized antibodies or binding fragments
thereof comprising an ultralong CDR3 and a non-antibody sequence to
generate a library of humanized antibodies or binding fragments
thereof comprising an ultralong CDR3 and a non-antibody
sequence.
[0216] The present disclosure also provides libraries of humanized
antibodies or binding fragments thereof comprising an ultralong
CDR3 that comprises a non-antibody sequence.
[0217] The present disclosure also provides methods of generating a
library of humanized antibodies or binding fragments thereof
comprising an ultralong CDR3 that comprises a cysteine motif,
comprising: combining a human variable region framework (FR)
sequence, and a nucleic acid sequence encoding an ultralong CDR3
and a cysteine motif; introducing one or more nucleotide changes to
the nucleic acid sequence encoding one or more amino acid residues
that are positioned between one or more cysteine residues in the
cysteine motif for nucleotides encoding different amino acid
residues to produce nucleic acids encoding humanized antibodies or
binding fragments thereof comprising an ultralong CDR3 and a
cysteine motif with one or more nucleotide changes introduced
between one or more cysteine residues in the cysteine domain; and
expressing the nucleic acids encoding humanized antibodies or
binding fragments thereof comprising an ultralong CDR3 and a
cysteine motif with one or more nucleotide changes introduced
between one or more cysteine residues in the cysteine domain to
generate a library of humanized antibodies or binding fragments
thereof comprising an ultralong CDR3 and a cysteine motif with one
or more amino acid changes introduced between one or more cysteine
residues in the cysteine domain.
[0218] The present disclosure also provides libraries of humanized
antibodies or binding fragments thereof comprising an ultralong
CDR3 that comprises a cysteine motif, wherein the antibodies or
binding fragments comprise one or more substitutions of amino acid
residues that are positioned between cysteine residues in the
cysteine motif.
[0219] The present disclosure also provides methods of generating a
library of humanized antibodies or binding fragments thereof
comprising a bovine ultralong CDR3, comprising: combining a nucleic
acid sequence encoding a human variable region framework (FR)
sequence and a nucleic acid encoding a bovine ultralong CDR3, and
expressing the nucleic acids encoding a human variable region
framework (FR) sequence and a nucleic acid encoding a bovine
ultralong CDR3 to generate a library of humanized antibodies or
binding fragments thereof comprising a bovine ultralong CDR3.
[0220] The present disclosure also provides a CDR3 scaffold
comprising a X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein
X.sup.1 is threonine (T), glycine (G), alanine (A), serine (S), or
valine (V), wherein X.sup.2 is serine (S), threonine (T), proline
(P), isoleucine (I), alanine (A), valine (V), or asparagine (N),
wherein X.sup.3 is valine (V), alanine (A), threonine (T), or
aspartic acid (D), wherein X.sup.4 is histidine (H), threonine (T),
arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and
wherein X.sup.5 is glutamine (Q).
Proteins
[0221] The present disclosure provides humanized antibodies
comprising ultralong CDR3 sequences.
[0222] In an embodiment, the present disclosure provides a
humanized antibody comprising an ultralong CDR3, wherein the CDR3
is 35 amino acids in length or more (e.g., 40 or more, 45 or more,
50 or more, 55 or more, 60 or more). Such a humanized antibody may
comprise at least 3 cysteine residues or more (e.g., 4 or more, 6
or more, 8 or more) within the ultralong CDR3.
[0223] In another embodiment, the present disclosure provides a
humanized antibody comprising an ultralong CDR3, wherein the CDR3
is 35 amino acids in length or more and is derived from or based on
a non-human sequence. The ultralong CDR3 sequence may be derived
from any species that naturally produces ultralong CDR3 antibodies,
including ruminants such as cattle (Bos taurus).
[0224] In another embodiment, the present disclosure provides a
humanized antibody comprising an ultralong CDR3, wherein the CDR3
is 35 amino acids in length or more and is derived from a
non-antibody sequence. The non-antibody sequence may be derived
from any protein family including, but not limited to, chemokines,
growth factors, peptides, cytokines, cell surface proteins, serum
proteins, toxins, extracellular matrix proteins, clotting factors,
secreted proteins, etc. The non-antibody sequence may be of human
or non-human origin and may comprise a portion of a non-antibody
protein such as a peptide or domain. The non-antibody sequence of
an ultralong CDR3 may contain mutations from its natural sequence,
including amino acid changes (e.g., substitutions), insertions or
deletions. Engineering additional amino acids at the junction
between the non-antibody sequence may be done to facilitate or
enhance proper folding of the non-antibody sequence within the
humanized antibody.
[0225] In another embodiment, the present disclosure provides a
humanized antibody comprising an ultralong CDR3, wherein the CDR3
is 35 amino acids in length or more and comprises at least 3
cysteine residues or more, including, for example, 4 or more, 6 or
more, and 8 or more.
[0226] In another embodiment, the present disclosure provides for a
humanized antibody comprising an ultralong CDR3 wherein the CDR3 is
35 amino acids in length or more and comprises at least 3 cysteine
residues or more and wherein the ultralong CDR3 is a component of a
multispecific antibody. The multispecific antibody may be
bispecific or comprise greater valencies.
[0227] In another embodiment, the present disclosure provides a
humanized antibody comprising an ultralong CDR3, wherein the CDR3
is 35 amino acids in length or more and comprises at least 3
cysteine residues or more, wherein the partially human ultralong
CDR3 is a component of an immunoconjugate.
[0228] In another embodiment, the present disclosure provides a
humanized antibody comprising an ultralong CDR3, wherein the CDR3
is 35 amino acids in length or more and comprises at least 3
cysteine residues or more, wherein the humanized antibody
comprising an ultralong CDR3 binds to a transmembrane protein
target. Such transmembrane targets may include, but are not limited
to, GPCRs, ion channels, transporters, and cell surface
receptors.
Genetic Sequences
[0229] The present disclosure provides genetic sequences (e.g.,
genes, nucleic acids, polynucleotides) encoding humanized
antibodies comprising ultralong CDR sequences.
[0230] The present disclosure also provides genetic sequences
(e.g., genes, nucleic acids, polynucleotides) encoding an ultralong
CDR3.
[0231] In an embodiment, the present disclosure provides genetic
sequences encoding a humanized antibody comprising an ultralong
CDR3, wherein the CDR3 is 35 amino acids in length or more (e.g.,
40 or more, 45 or more, 50 or more, 55 or more, 60 or more). Such a
humanized antibody may comprise at least 3 cysteine residues or
more (e.g., 4 or more, 6 or more, 8 or more) within the ultralong
CDR3.
[0232] In another embodiment, the present disclosure provides
genetic sequences encoding a humanized antibody comprising an
ultralong CDR3, wherein the CDR3 is 35 amino acids in length or
more and is derived from or based on a non-human sequence. The
genetic sequences encoding the ultralong CDR3 may be derived from
any species that naturally produces ultralong CDR3 antibodies,
including ruminants such as cattle (Bos taurus).
[0233] In another embodiment, the present disclosure provides
genetic sequences encoding a humanized antibody comprising an
ultralong CDR3, wherein the CDR3 is 35 amino acids in length or
more and is derived from a non-antibody protein sequence. The
genetic sequences encoding the non-antibody protein sequences may
be derived from any protein family including, but not limited to,
chemokines, growth factors, peptides, cytokines, cell surface
proteins, serum proteins, toxins, extracellular matrix proteins,
clotting factors, secreted proteins, etc. The non-antibody protein
sequence may be of human or non-human origin and may comprise a
portion of a non-antibody protein such as a peptide or domain. The
non-antibody protein sequence of an ultralong CDR3 may contain
mutations from its natural sequence, including amino acid changes
(e.g., substitutions), insertions or deletions. Engineering
additional amino acids at the junction between the non-antibody
sequence may be done to facilitate or enhance proper folding of the
non-antibody sequence within the humanized antibody.
[0234] In another embodiment, the present disclosure provides
genetic sequences encoding a humanized antibody comprising an
ultralong CDR3, wherein the CDR3 is 35 amino acids in length or
more and comprises at least 3 cysteine residues or more, including,
for example, 4 or more, 6 or more, and 8 or more.
[0235] In another embodiment, the present disclosure provides
genetic sequences encoding a humanized antibody comprising an
ultralong CDR3 wherein the CDR3 is 35 amino acids in length or more
and comprises at least 3 cysteine residues or more and wherein the
ultralong CDR3 is a component of a multispecific antibody. The
multispecific antibody may be bispecific or comprise greater
valencies.
[0236] In another embodiment, the present disclosure provides
genetic sequences encoding a humanized antibody comprising an
ultralong CDR3, wherein the CDR3 is 35 amino acids in length or
more and comprises at least 3 cysteine residues or more, wherein
the ultralong CDR3 is a component of an immunoconjugate.
[0237] In another embodiment, the present disclosure provides
genetic sequences encoding a humanized antibody comprising an
ultralong CDR3 wherein the CDR3 is 35 amino acids in length or more
and comprises at least 3 cysteine residues or more and wherein the
humanized antibody comprising an ultralong CDR3 binds to a
transmembrane protein target. Such transmembrane targets may
include, but are not limited to, GPCRs, ion channels, transporters,
and cell surface receptors.
Libraries and Arrays
[0238] The present disclosure provides collections, libraries, and
arrays of humanized antibodies comprising ultralong CDR3
sequences.
[0239] In an embodiment, the present disclosure provides a library
or an array of humanized antibodies comprising ultralong CDR3
sequences wherein at least two members of the library or array
differ in the positions of at least one of the cysteines in the
ultralong CDR3 sequence. Structural diversity may be enhanced
through different numbers of cysteines in the ultralong CDR3
sequence (e.g., at least 3 or more cysteine residues such as 4 or
more, 6 or more and 8 or more) and/or through different disulfide
bond formation, and hence different loop structures.
[0240] In another embodiment, the present disclosure provides for a
library or an array of humanized antibodies comprising ultralong
CDR3 sequences wherein at least two members of the library or the
array differ in at least one amino acid located between cysteines
in the ultralong CDR3. In this regard, members of the library or
the array can contain cysteines in the same positions of CDR3,
resulting in similar overall structural folds, but with fine
differences brought about through different amino acid side chains.
Such libraries or arrays may be useful for affinity maturation.
[0241] In another embodiment, the present disclosure provides
libraries or arrays of humanized antibodies comprising ultralong
CDR3 sequences wherein at least two of the ultralong CDR3 sequences
differ in length (e.g., 35 amino acids in length or more such as 40
or more, 45 or more, 50 or more, 55 or more and 60 or more). The
amino acid and cysteine content may or may not be altered between
the members of the library or the array. Different lengths of
ultralong CDR3 sequences may provide for unique binding sites,
including, for example, due to steric differences, as a result of
altered length.
[0242] In another embodiment, the present disclosure provides
libraries or arrays of humanized antibodies comprising ultralong
CDR3 sequences wherein at least two members of the library differ
in the human framework used to construct the humanized antibody
comprising an ultralong CDR3.
[0243] In another embodiment, the present disclosure provides
libraries or arrays of humanized antibodies comprising ultralong
CDR3 sequences wherein at least two members of the library or the
array differ in having a non-antibody protein sequence that
comprises a portion of the ultralong CDR3. Such libraries or arrays
may contain multiple non-antibody protein sequences, including for
chemokines, growth factors, peptides, cytokines, cell surface
proteins, serum proteins, toxins, extracellular matrix proteins,
clotting factors, secreted proteins, viral or bacterial proteins,
etc. The non-antibody protein sequence may be of human or non-human
origin and may be comprised of a portion of a non-antibody protein
such as a peptide or domain. The non-antibody protein sequence of
the ultralong CDR3 may contain mutations from its natural sequence,
including amino acid changes (e.g., substitutions), or insertions
or deletions. Engineering additional amino acids at the junction
between the non-antibody sequence within the ultralong CDR3 may be
done to facilitate or enhance proper folding of the non-antibody
sequence within the humanized antibody.
[0244] The libraries or the arrays of the present disclosure may be
in several formats well known in the art. The library or the array
may be an addressable library or an addressable array. The library
or array may be in display format, for example, the antibody
sequences may be expressed on phage, ribosomes, mRNA, yeast, or
mammalian cells.
Cells
[0245] The present disclosure provides cells comprising genetic
sequences encoding humanized antibodies comprising ultralong CDR3
sequences.
[0246] In an embodiment, the present disclosure provides cells
expressing a humanized antibody comprising an ultralong CDR3. The
cells may be prokaryotic or eukaryotic, and a humanized antibody
comprising an ultralong CDR3 may be expressed on the cell surface
or secreted into the media. When displayed on the cell surface a
humanized antibody preferentially contains a motif for insertion
into the plasmid membrane such as a membrane spanning domain at the
C-terminus or a lipid attachment site. For bacterial cells, a
humanized antibody comprising an ultralong CDR3 may be secreted
into the periplasm. When the cells are eukaryotic, they may be
transiently transfected with genetic sequences encoding a humanized
antibody comprising an ultralong CDR3. Alternatively, a stable cell
line or stable pools may be created by transfecting or transducing
genetic sequences encoding a humanized antibody comprising an
ultralong CDR3 by methods well known to those of skill in the art.
Cells can be selected by fluorescence activated cell sorting (FACS)
or through selection for a gene encoding drug resistance. Cells
useful for producing humanized antibodies comprising ultralong CDR3
sequences include prokaryotic cells like E. coli, eukaryotic cells
like the yeasts Saccharomyces cerevisiae and Pichia pastoris,
chinese hamster ovary (CHO) cells, monkey cells like COS-1, or
human cells like HEK-293, HeLa, SP-1.
Humanization Methods
[0247] The present disclosure provides methods for making humanized
antibodies comprising ultralong CDR3 sequences, comprising the
steps of engineering an ultralong CDR3 sequence derived from a
non-human CDR3 into a human framework. The human framework may be
of germline origin, or may be derived from non-germline (e.g.
mutated or affinity matured) sequences. Genetic engineering
techniques well known to those in the art, including as disclosed
herein, may be used to generate a hybrid DNA sequence containing a
human framework and a non-human ultralong CDR3. Unlike human
antibodies which may be encoded by V region genes derived from one
of seven families, bovine antibodies which produce ultralong CDR3
sequences appear to utilize a single V region family which may be
considered to be most homologous to the human VH4 family. In a
preferred embodiment where ultralong CDR3 sequences derived from
cattle are to be humanized to produce an antibody comprising an
ultralong CDR3, human V region sequences derived from the VH4
family may be genetically fused to a bovine-derived ultralong CDR3
sequence. Exemplary VH4 germline gene sequences in the human
antibody locus are shown in FIG. 5A (e.g., SEQ ID NOS: 31-34; and
368-371).
[0248] The present disclosure also provides methods of humanizing
an antibody variable region comprising the step of genetically
combining a nucleic acid sequence encoding a non-human ultralong
CDR3 (ULCDR3) with a nucleic acid sequence encoding a human
variable region framework (FR) sequence. Also provided are methods
of making a humanized antibody variable region comprising selecting
a human framework sequence comprising FR1, FR2, and FR3; selecting
a CDR1 sequence; selecting a CDR2 sequence; selecting an ultralong
CDR3 sequence; and combining the sequences as
FR1-CDR1-FR2-CDR2-FR3-ULCDR3. Also provided are methods of making a
humanized antibody variable region sequence comprising selecting a
human antibody variable region sequence comprising a sequence
encoding FR1-CDR1-FR2-CDR2-FR3; selecting a sequence encoding a
non-human ultralong CDR3 (ULCDR3); and genetically fusing the human
sequence of step (a) in frame with the non-human sequence of step
(b) to generate a sequence encoding
FR1-CDR1-FR2-CDR2-FR3-ULCDR3.
[0249] In an embodiment, the present disclosure provides a fusion
of a human VH4 framework sequence to a bovine-derived ultralong
CDR3, for example, as may be accomplished through the following
steps. First, the second cysteine of a V region genetic sequence is
identified along with the nucleotide sequence encoding the second
cysteine. Generally, the second cysteine marks the boundary of the
framework and CDR3 two residues upstream (N-terminal) of the CDR3.
Second, the second cysteine in a bovine-derived V region sequence
is identified which similarly marks 2 residues upstream
(N-terminal) of the CDR3. Third, the genetic material encoding the
human V region is combined with the genetic sequence encoding the
ultralong CDR3. Thus, a genetic fusion may be made, wherein the
ultralong CDR3 sequence is placed in frame of the human V region
sequence. Preferably a humanized antibody comprising an ultralong
CDR3 is as near to human in amino acid composition as possible.
Optionally, a J region sequence may be mutated from bovine-derived
sequence to a human sequence. Also optionally, a humanized heavy
chain may be paired with a human light chain.
[0250] In another embodiment, the present disclosure provides
pairing of a human ultralong CDR3 heavy chain with a non-human
light chain.
[0251] In another embodiment, the present disclosure provides
pairing of a humanized heavy chain comprising an ultralong CDR3
with a human light chain. Preferably the light chain is homologous
to a bovine light chain known to pair with a bovine ultralong CDR3
heavy chain. An exemplary bovine light chain is shown in FIG. 7A
(e.g., SEQ ID NO: 36-39; and 373-376).
Library Methods
[0252] The present disclosure provides methods for making libraries
comprising humanized antibodies comprising ultralong CDR3
sequences. Methods for making libraries of spatially addressed
libraries are described in WO 2010/054007. Methods of making
libraries in yeast, phage, E. coli, or mammalian cells are well
known in the art.
[0253] The present disclosure also provides methods of screening
libraries of humanized antibodies comprising ultralong CDR3
sequences.
Definitions
[0254] An "ultralong CDR3" or an "ultralong CDR3 sequence", used
interchangeably herein, comprises a CDR3 or CDR3 sequence that is
not derived from a human antibody sequence. An ultralong CDR3 may
be 35 amino acids in length or longer, for example, 40 amino acids
in length or longer, 45 amino acids in length or longer, 50 amino
acids in length or longer, 55 amino acids in length or longer, or
60 amino acids in length or longer. The length of the ultralong
CDR3 may include a non-antibody sequence. An ultralong CDR3 may
comprise a non-antibody sequence, including, for example, an
interleukin sequence, a hormone sequence, a cytokine sequence, a
toxin sequence, a lymphokine sequence, a growth factor sequence, a
chemokine sequence, a toxin sequence, or combinations thereof.
Preferably, the ultralong CDR3 is a heavy chain CDR3 (CDR-H3 or
CDRH3). Preferably, the ultralong CDR3 is a sequence derived from
or based on a ruminant (e.g., bovine) sequence. An ultralong CDR3
may comprise at least 3 or more cysteine residues, for example, 4
or more cysteine residues, 6 or more cysteine residues, 8 or more
cysteine residues, 10 or more cysteine residues, or 12 or more
cysteine residues (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more).
An ultralong CDR3 may comprise one or more of the following motifs:
a cysteine motif, a X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, or a
(X.sup.aX.sup.b).sub.z motif. A "cysteine motif" is a segment of
amino acid residues in an ultralong CDR3 that comprises 3 or more
cysteine residues including, 4 or more cysteine residues, 5 or more
cysteine residues, 6 or more cysteine residues, 7 or more cysteine
residues, 8 or more cysteine residues, 9 or more cysteine residues,
10 or more cysteine residues, 11 or more cysteine residues, or 12
or more cysteine residues. A cysteine motif may comprise an amino
acid sequence selected from the group consisting of:
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 41),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95). Alternatively, a cysteine motif may comprise an amino
acid sequence selected from the group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152). A cysteine motif is preferably positioned within an
ultralong CDR3 between a X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif
and a (X.sup.aX.sup.b).sub.z motif. A
"X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif" is a series of five
consecutive amino acid residues in an ultralong CDR3, wherein
X.sup.1 is threonine (T), glycine (G), alanine (A), serine (S), or
valine (V), wherein X.sup.2 is serine (S), threonine (T), proline
(P), isoleucine (I), alanine (A), valine (V), or asparagine (N),
wherein X.sup.3 is valine (V), alanine (A), threonine (T), or
aspartic acid (D), wherein X.sup.4 is histidine (H), threonine (T),
arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and
wherein X.sup.5 is glutamine (Q). In some embodiments, the
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif may be TTVHQ (SEQ ID NO:
153), TSVHQ (SEQ ID NO: 154), SSVTQ (SEQ ID NO: 155), STVHQ (SEQ ID
NO: 156), ATVRQ (SEQ ID NO: 157), TTVYQ (SEQ ID NO: 158), SPVHQ
(SEQ ID NO: 159), ATVYQ (SEQ ID NO: 160), TAVYQ (SEQ ID NO: 161),
TNVHQ (SEQ ID NO: 162), ATVHQ (SEQ ID NO: 163), STVYQ (SEQ ID NO:
164), TIVHQ (SEQ ID NO: 165), AIVYQ (SEQ ID NO: 166), TTVFQ (SEQ ID
NO: 167), AAVFQ (SEQ ID NO: 168), GTVHQ (SEQ ID NO: 169), ASVHQ
(SEQ ID NO: 170), TAVFQ (SEQ ID NO: 171), ATVFQ (SEQ ID NO: 172),
AAAHQ (SEQ ID NO: 173), VVVYQ (SEQ ID NO: 174), GTVFQ (SEQ ID NO:
175), TAVHQ (SEQ ID NO: 176), ITVHQ (SEQ ID NO: 177), ITAHQ (SEQ ID
NO: 178), VTVHQ (SEQ ID NO: 179); AAVHQ (SEQ ID NO: 180), GTVYQ
(SEQ ID NO: 181), TTVLQ (SEQ ID NO: 182), TTTHQ (SEQ ID NO: 183),
or TTDYQ (SEQ ID NO: 184). A "CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif" is a series of six consecutive amino acid residues in an
ultralong CDR3, wherein the first amino acid residue is cysteine,
wherein X.sup.1 is threonine (T), glycine (G), alanine (A), serine
(S), or valine (V), wherein X.sup.2 is serine (S), threonine (T),
proline (P), isoleucine (I), alanine (A), valine (V), or asparagine
(N), wherein X.sup.3 is valine (V), alanine (A), threonine (T), or
aspartic acid (D), wherein X.sup.4 is histidine (H), threonine (T),
arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and
wherein X.sup.5 is glutamine (Q). In some embodiments, the
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif is CTTVHQ (SEQ ID NO:
185), CTSVHQ (SEQ ID NO: 186), CSSVTQ (SEQ ID NO: 187), CSTVHQ (SEQ
ID NO: 188), CATVRQ (SEQ ID NO: 189), CTTVYQ (SEQ ID NO: 190),
CSPVHQ (SEQ ID NO: 191), CATVYQ (SEQ ID NO: 192), CTAVYQ (SEQ ID
NO: 193), CTNVHQ (SEQ ID NO: 194), CATVHQ (SEQ ID NO: 195), CSTVYQ
(SEQ ID NO: 196), CTIVHQ (SEQ ID NO: 197), CAIVYQ (SEQ ID NO: 198),
CTTVFQ (SEQ ID NO: 199), CAAVFQ (SEQ ID NO: 200), CGTVHQ (SEQ ID
NO: 201), CASVHQ (SEQ ID NO: 202), CTAVFQ (SEQ ID NO: 203), CATVFQ
(SEQ ID NO: 204), CAAAHQ (SEQ ID NO: 205), CVVVYQ (SEQ ID NO: 206),
CGTVFQ (SEQ ID NO: 207), CTAVHQ (SEQ ID NO: 208), CITVHQ (SEQ ID
NO: 209), CITAHQ (SEQ ID NO: 210), CVTVHQ (SEQ ID NO: 211); CAAVHQ
(SEQ ID NO: 212), CGTVYQ (SEQ ID NO: 213), CTTVLQ (SEQ ID NO: 214),
CTTTHQ (SEQ ID NO: 215), or CTTDYQ (SEQ ID NO: 216). A
"(X.sup.aX.sup.b).sub.z" motif is a repeating series of two amino
acid residues in an ultralong CDR3, wherein X.sup.a is any amino
acid residue, X.sup.b is an aromatic amino acid selected from the
group consisting of: tyrosine (Y), phenylalanine (F), tryptophan
(W), and histidine (H), and wherein z is 1-4. In some embodiments,
the (X.sup.aX.sup.b).sub.z motif may comprise CYTYNYEF (SEQ ID NO:
217), HYTYTYDF (SEQ ID NO: 218), HYTYTYEW (SEQ ID NO: 219),
KHRYTYEW (SEQ ID NO: 220), NYIYKYSF (SEQ ID NO: 221), PYIYTYQF (SEQ
ID NO: 222), SFTYTYEW (SEQ ID NO: 223), SYIYIYQW (SEQ ID NO: 224),
SYNYTYSW (SEQ ID NO: 225), SYSYSYEY (SEQ ID NO: 226), SYTYNYDF (SEQ
ID NO: 227), SYTYNYEW (SEQ ID NO: 228), SYTYNYQF (SEQ ID NO: 229),
SYVWTHNF (SEQ ID NO: 230), TYKYVYEW (SEQ ID NO: 231), TYTYTYEF (SEQ
ID NO: 232), TYTYTYEW (SEQ ID NO: 233), VFTYTYEF (SEQ ID NO: 234),
AYTYEW (SEQ ID NO: 235), DYIYTY (SEQ ID NO: 236), IHSYEF (SEQ ID
NO: 237), SFTYEF (SEQ ID NO: 238), SHSYEF (SEQ ID NO: 239), THTYEF
(SEQ ID NO: 240), TWTYEF (SEQ ID NO: 241), TYNYEW (SEQ ID NO: 242),
TYSYEF (SEQ ID NO: 243), TYSYEH (SEQ ID NO: 244), TYTYDF (SEQ ID
NO: 245), TYTYEF (SEQ ID NO: 246), TYTYEW (SEQ ID NO: 247), AYEF
(SEQ ID NO: 248), AYSF (SEQ ID NO: 249), AYSY (SEQ ID NO: 250),
CYSF (SEQ ID NO: 251), DYTY (SEQ ID NO: 252), KYEH (SEQ ID NO:
253), KYEW (SEQ ID NO: 254), MYEF (SEQ ID NO: 255), NWIY (SEQ ID
NO: 256), NYDY (SEQ ID NO: 257), NYQW (SEQ ID NO: 258), NYSF (SEQ
ID NO: 259), PYEW (SEQ ID NO: 260), RYNW (SEQ ID NO: 261), RYTY
(SEQ ID NO: 262), SYEF (SEQ ID NO: 263), SYEH (SEQ ID NO: 264),
SYEW (SEQ ID NO: 265), SYKW (SEQ ID NO: 266), SYTY (SEQ ID NO:
267), TYDF (SEQ ID NO: 268), TYEF (SEQ ID NO: 269), TYEW (SEQ ID
NO: 270), TYQW (SEQ ID NO: 271), TYTY (SEQ ID NO: 272), or VYEW
(SEQ ID NO: 273). In some embodiments, the (X.sup.aX.sup.b).sub.z
motif is YXYXYX. An ultralong CDR3 may comprise an amino acid
sequence that is derived from or based on SEQ ID NO: 40 (see, e.g.,
amino acid residues 3-6 of SEQ ID NO: 1-4; see also, e.g., VH
germline sequences in FIGS. 2A-C). A variable region that comprises
an ultralong CDR3 may include an amino acid sequence that is SEQ ID
NO: 1 (CTTVHQ), SEQ ID NO:2 (CTSVHQ), SEQ ID NO:3 (CSSVTQ) or SEQ
ID NO: 4 (CTTVHP). Such a sequence may be derived from or based on
a bovine germline VH gene sequence (e.g., SEQ ID NO: 1). An
ultralong CDR3 may comprise a sequence derived from or based on a
non-human DH gene sequence, for example, SEQ ID NO: 5 (see also,
e.g., Koti, et al. (2010) Mol. Immunol. 47: 2119-2128), or
alternative sequences such as SEQ ID NO: 6, 7, 8, 9, 10, 11 or 12
(see also, e.g., DH2 germline sequences in FIGS. 2A-C). An
ultralong CDR3 may comprise a sequence derived from or based on a
JH sequence, for example, SEQ ID NO: 13 (see also, e.g., Hosseini,
et al. (2004) Int. Immunol. 16: 843-852), or alternative sequences
such as SEQ ID NO: 14, 15, 16 or 17 (see also, e.g., JH1 germline
sequences in FIGS. 2A-C). In an embodiment, an ultralong CDR3 may
comprise a sequence derived from or based on a non-human VH
sequence (e.g., SEQ ID NO: 1, 2, 3 or 4; alternatively VH sequences
in FIGS. 2A-C) and/or a sequence derived from or based on a
non-human DH sequence (e.g., SEQ ID NO: 5, 6, 7, 8, 9, 10, 11 or
12; alternatively DH sequences in FIGS. 2A-C) and/or a sequence
derived from or based on a JH sequence (e.g., SEQ ID NO: 13, 14,
15, 16, or 17; alternatively JH sequences in
FIGS. 2A-C), and optionally an additional sequence comprising two
to six amino acids or more (e.g., IR, IF, SEQ ID NO: 18, 19, 20 or
21) such as, for example, between the VH derived sequence and the
DH derived sequence. In another embodiment, an ultralong CDR3 may
comprise a sequence derived from or based on SEQ ID NO: 22, 23, 24,
25, 26, 27, or 28 (see also, e.g., SEQ ID NOs: 276-359 in FIGS.
2A-C).
[0255] An "isolated" biological molecule, such as the various
polypeptides, polynucleotides, and antibodies disclosed herein,
refers to a biological molecule that has been identified and
separated and/or recovered from at least one component of its
natural environment.
[0256] "Antagonist" refers to any molecule that partially or fully
blocks, inhibits, or neutralizes an activity (e.g., biological
activity) of a polypeptide. Also encompassed by "antagonist" are
molecules that fully or partially inhibit the transcription or
translation of mRNA encoding the polypeptide. Suitable antagonist
molecules include, e.g., antagonist antibodies or antibody
fragments; fragments or amino acid sequence variants of a native
polypeptide; peptides; antisense oligonucleotides; small organic
molecules; and nucleic acids that encode polypeptide antagonists or
antagonist antibodies. Reference to "an" antagonist encompasses a
single antagonist or a combination of two or more different
antagonists.
[0257] "Agonist" refers to any molecule that partially or fully
mimics a biological activity of a polypeptide. Also encompassed by
"agonist" are molecules that stimulate the transcription or
translation of mRNA encoding the polypeptide. Suitable agonist
molecules include, e.g., agonist antibodies or antibody fragments;
a native polypeptide; fragments or amino acid sequence variants of
a native polypeptide; peptides; antisense oligonucleotides; small
organic molecules; and nucleic acids that encode polypeptides
agonists or antibodies. Reference to "an" agonist encompasses a
single agonist or a combination of two or more different
agonists.
[0258] An "isolated" antibody refers to one which has been
identified and separated and/or recovered from a component of its
natural environment. Contaminant components of its natural
environment are materials which would interfere with diagnostic or
therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or nonproteinaceous solutes. In
preferred embodiments, the antibody will be purified (1) to greater
than 95% by weight of antibody (e.g., as determined by the Lowry
method), and preferably to more than 99% by weight, (2) to a degree
sufficient to obtain at least 15 residues of N-terminal or internal
amino acid sequence (e.g., by use of a spinning cup sequenator), or
(3) to homogeneity by SDS-PAGE under reducing or nonreducing
conditions (e.g., using Coomassie.TM. blue or, preferably, silver
stain). Isolated antibody includes the antibody in situ within
recombinant cells since at least one component of the antibody's
natural environment will not be present. Similarly, isolated
antibody includes the antibody in medium around recombinant cells.
An isolated antibody may be prepared by at least one purification
step.
[0259] An "isolated" nucleic acid molecule refers to a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the antibody nucleic acid. An
isolated nucleic acid molecule is other than in the form or setting
in which it is found in nature. Isolated nucleic acid molecules
therefore are distinguished from the nucleic acid molecule as it
exists in natural cells. However, an isolated nucleic acid molecule
includes a nucleic acid molecule contained in cells that express an
antibody where, for example, the nucleic acid molecule is in a
chromosomal location different from that of natural cells.
[0260] Variable domain residue numbering as in Kabat or amino acid
position numbering as in Kabat, and variations thereof, refers to
the numbering system used for heavy chain variable domains or light
chain variable domains of the compilation of antibodies in Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991). Using this numbering system, the actual linear amino acid
sequence may contain fewer or additional amino acids corresponding
to a shortening of, or insertion into, a FR or CDR of the variable
domain. For example, a heavy chain variable domain may include a
single amino acid insert (e.g., residue 52a according to Kabat)
after residue 52 of H2 and inserted residues (e.g., residues 82a,
82b, and 82c, etc according to Kabat) after heavy chain FR residue
82. The Kabat numbering of residues may be determined for a given
antibody by alignment at regions of homology of the sequence of the
antibody with a "standard" Kabat numbered sequence.
[0261] "Substantially similar," or "substantially the same", refers
to a sufficiently high degree of similarity between two numeric
values (generally one associated with an antibody disclosed herein
and the other associated with a reference/comparator antibody) such
that one of skill in the art would consider the difference between
the two values to be of little or no biological and/or statistical
significance within the context of the biological characteristic
measured by said values (e.g., Kd values). The difference between
said two values is preferably less than about 50%, preferably less
than about 40%, preferably less than about 30%, preferably less
than about 20%, preferably less than about 10% as a function of the
value for the reference/comparator antibody.
[0262] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, "binding affinity" refers to
intrinsic binding affinity which reflects a 1:1 interaction between
members of a binding pair (e.g., antibody and antigen). The
affinity of a molecule X for its partner Y can generally be
represented by the dissociation constant. Affinity can be measured
by common methods known in the art, including those described
herein. Low-affinity antibodies generally bind antigen slowly and
tend to dissociate readily, whereas high-affinity antibodies
generally bind antigen faster and tend to remain bound longer. A
variety of methods of measuring binding affinity are known in the
art, any of which can be used for purposes of the present
disclosure.
[0263] An "on-rate" or "rate of association" or "association rate"
or "k.sub.on" can be determined with a surface plasmon resonance
technique such as Biacore (e.g., Biacore A100, Biacore.TM.-2000,
Biacore.TM.-3000, Biacore, Inc., Piscataway, N.J.)
carboxymethylated dextran biosensor chips (CMS, Biacore Inc.) and
according to the suppliers instructions.
[0264] "Vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One
type of vector is a "plasmid", which refers to a circular double
stranded DNA loop into which additional DNA segments may be
ligated. Another type of vector is a phage vector. Another type of
vector is a viral vector, wherein additional DNA segments may be
ligated into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) can be integrated into the genome
of a host cell upon introduction into the host cell, and thereby
are replicated along with the host genome. Moreover, certain
vectors are capable of directing the expression of genes to which
they are operatively linked. Such vectors are referred to herein as
"recombinant expression vectors" (or simply, "recombinant
vectors"). In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids. Accordingly,
"plasmid" and "vector" may, at times, be used interchangeably as
the plasmid is a commonly used form of vector.
[0265] "Gene" refers to a nucleic acid (e.g., DNA) sequence that
comprises coding sequences necessary for the production of a
polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide
can be encoded by a full length coding sequence or by any portion
of the coding sequence so long as the desired activity or
functional properties (e.g., enzymatic activity, ligand binding,
signal transduction, immunogenicity, etc.) of the full-length or
fragment are retained. The term also encompasses the coding region
of a structural gene and the sequences located adjacent to the
coding region on both the 5' and 3' ends for a distance of about 1
kb or more on either end such that the gene corresponds to the
length of the full-length mRNA. Sequences located 5' of the coding
region and present on the mRNA are referred to as 5' non-translated
sequences. Sequences located 3' or downstream of the coding region
and present on the mRNA are referred to as 3' non-translated
sequences. The term "gene" encompasses both cDNA and genomic forms
of a gene. A genomic form or clone of a gene contains the coding
region interrupted with non-coding sequences termed "introns" or
"intervening regions" or "intervening sequences." Introns are
segments of a gene that are transcribed into nuclear RNA (hnRNA);
introns can contain regulatory elements such as enhancers. Introns
are removed or "spliced out" from the nuclear or primary
transcript; introns therefore are absent in the messenger RNA
(mRNA) transcript. The mRNA functions during translation to specify
the sequence or order of amino acids in a nascent polypeptide. In
addition to containing introns, genomic forms of a gene can also
include sequences located on both the 5' and 3' end of the
sequences that are present on the RNA transcript. These sequences
are referred to as "flanking" sequences or regions (these flanking
sequences are located 5' or 3' to the non-translated sequences
present on the mRNA transcript). The 5' flanking region can contain
regulatory sequences such as promoters and enhancers that control
or influence the transcription of the gene. The 3' flanking region
can contain sequences that direct the termination of transcription,
post transcriptional cleavage and polyadenylation.
[0266] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refers to polymers of nucleotides of any length, and
include DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase, or by a synthetic reaction. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after synthesis, such as by conjugation with a
label. Other types of modifications include, for example, "caps",
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as, for example,
those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.) and with
charged linkages (e.g., phosphorothioates, phosphorodithioates,
etc.), those containing pendant moieties, such as, for example,
proteins (e.g., nucleases, toxins, antibodies, signal peptides,
poly-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid or semi-solid supports.
The 5' and 3' terminal OH can be phosphorylated or substituted with
amines or organic capping group moieties of from 1 to 20 carbon
atoms. Other hydroxyls may also be derivatized to standard
protecting groups. Polynucleotides can also contain analogous forms
of ribose or deoxyribose sugars that are generally known in the
art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-
or 2'-azido-ribose, carbocyclic sugar analogs, alpha-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and a basic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by
P(O)S("thioate"), P(S)S ("dithioate"), "(O)NR.sub.2 ("amidate"),
P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or
R' is independently H or substituted or unsubstituted alkyl (1-20
C) optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0267] "Oligonucleotide" refers to short, generally single
stranded, generally synthetic polynucleotides that are generally,
but not necessarily, less than about 200 nucleotides in length. The
terms "oligonucleotide" and "polynucleotide" are not mutually
exclusive. The description above for polynucleotides is equally and
fully applicable to oligonucleotides.
[0268] "Stringent hybridization conditions" refer to conditions
under which a probe will hybridize to its target subsequence,
typically in a complex mixture of nucleic acids, but to no other
sequences. Stringent conditions are sequence-dependent and will be
different in different circumstances. Longer sequences hybridize
specifically at higher temperatures. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic
Probes, "Overview of principles of hybridization and the strategy
of nucleic acid assays" (1993). Generally, stringent conditions are
selected to be about 5-10.degree. C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength
pH. The Tm is the temperature (under defined ionic strength, pH,
and nucleic concentration) at which 50% of the probes complementary
to the target hybridize to the target sequence at equilibrium (as
the target sequences are present in excess, at Tm, 50% of the
probes are occupied at equilibrium). Stringent conditions may also
be achieved with the addition of destabilizing agents such as
formamide. For selective or specific hybridization, a positive
signal is at least two times background, preferably 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or, 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C.
[0269] "Recombinant" when used with reference to a cell, nucleic
acid, protein or vector indicates that the cell, nucleic acid,
protein or vector has been modified by the introduction of a
heterologous nucleic acid or protein, the alteration of a native
nucleic acid or protein, or that the cell is derived from a cell so
modified. For example, recombinant cells express genes that are not
found within the native (non-recombinant) form of the cell or
express native genes that are overexpressed or otherwise abnormally
expressed such as, for example, expressed as non-naturally
occurring fragments or splice variants. By the term "recombinant
nucleic acid" herein is meant nucleic acid, originally formed in
vitro, in general, by the manipulation of nucleic acid, e.g., using
polymerases and endonucleases, in a form not normally found in
nature. In this manner, operably linkage of different sequences is
achieved. Thus an isolated nucleic acid, in a linear form, or an
expression vector formed in vitro by ligating DNA molecules that
are not normally joined, are both considered recombinant for the
purposes of this disclosure. It is understood that once a
recombinant nucleic acid is made and introduced into a host cell or
organism, it will replicate non-recombinantly, e.g., using the in
vivo cellular machinery of the host cell rather than in vitro
manipulations; however, such nucleic acids, once produced
recombinantly, although subsequently replicated non-recombinantly,
are still considered recombinant for the purposes disclosed herein.
Similarly, a "recombinant protein" is a protein made using
recombinant techniques, e.g., through the expression of a
recombinant nucleic acid as depicted above.
[0270] "Percent (%) amino acid sequence identity" with respect to a
peptide or polypeptide sequence refers to the percentage of amino
acid residues in a candidate sequence that are identical with the
amino acid residues in the specific peptide or polypeptide
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent
amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN or
MegAlign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared.
[0271] "Polypeptide," "peptide," "protein," and "protein fragment"
may be used interchangeably to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymers.
[0272] "Amino acid" refers to naturally occurring and synthetic
amino acids, as well as amino acid analogs and amino acid mimetics
that function similarly to the naturally occurring amino acids.
Naturally occurring amino acids are those encoded by the genetic
code, as well as those amino acids that are later modified, e.g.,
hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino
acid analogs refers to compounds that have the same basic chemical
structure as a naturally occurring amino acid, e.g., an alpha
carbon that is bound to a hydrogen, a carboxyl group, an amino
group, and an R group, e.g., homoserine, norleucine, methionine
sulfoxide, methionine methyl sulfonium. Such analogs can have
modified R groups (e.g., norleucine) or modified peptide backbones,
but retain the same basic chemical structure as a naturally
occurring amino acid. Amino acid mimetics refers to chemical
compounds that have a structure that is different from the general
chemical structure of an amino acid, but that functions similarly
to a naturally occurring amino acid.
[0273] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. "Amino acid variants" refers to
amino acid sequences. With respect to particular nucleic acid
sequences, conservatively modified variants refers to those nucleic
acids which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical or associated (e.g., naturally
contiguous) sequences. Because of the degeneracy of the genetic
code, a large number of functionally identical nucleic acids encode
most proteins. For instance, the codons GCA, GCC, GCG and GCU all
encode the amino acid alanine. Thus, at every position where an
alanine is specified by a codon, the codon can be altered to
another of the corresponding codons described without altering the
encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of conservatively modified
variations. Every nucleic acid sequence herein which encodes a
polypeptide also describes silent variations of the nucleic acid.
One of skill will recognize that in certain contexts each codon in
a nucleic acid (except AUG, which is ordinarily the only codon for
methionine, and TGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, silent variations of a nucleic acid which
encodes a polypeptide is implicit in a described sequence with
respect to the expression product, but not with respect to actual
probe sequences. As to amino acid sequences, one of skill will
recognize that individual substitutions, deletions or additions to
a nucleic acid, peptide, polypeptide, or protein sequence which
alters, adds or deletes a single amino acid or a small percentage
of amino acids in the encoded sequence is a "conservatively
modified variant" including where the alteration results in the
substitution of an amino acid with a chemically similar amino acid.
Conservative substitution tables providing functionally similar
amino acids are well known in the art. Such conservatively modified
variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles disclosed herein.
Typically conservative substitutions include: 1) Alanine (A),
Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine
(N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0274] "Antibodies" (Abs) and "immunoglobulins" (Igs) are
glycoproteins having similar structural characteristics. While
antibodies may exhibit binding specificity to a specific antigen,
immunoglobulins may include both antibodies and other antibody-like
molecules which generally lack antigen specificity. Polypeptides of
the latter kind are, for example, produced at low levels by the
lymph system and at increased levels by myelomas.
[0275] "Antibody" and "immunoglobulin" are used interchangeably in
the broadest sense and include monoclonal antibodies (e.g., full
length or intact monoclonal antibodies), polyclonal antibodies,
multivalent antibodies, multispecific antibodies (e.g., bispecific
antibodies so long as they exhibit the desired biological activity)
and may also include certain antibody fragments (as described in
greater detail herein). An antibody can be human, humanized and/or
affinity matured. An antibody may refer to immunoglobulins and
immunoglobulin portions, whether natural or partially or wholly
synthetic, such as recombinantly produced, including any portion
thereof containing at least a portion of the variable region of the
immunoglobulin molecule that is sufficient to form an antigen
binding site. Hence, an antibody or portion thereof includes any
protein having a binding domain that is homologous or substantially
homologous to an immunoglobulin antigen binding site. For example,
an antibody may refer to an antibody that contains two heavy chains
(which can be denoted H and H') and two light chains (which can be
denoted L and L'), where each heavy chain can be a full-length
immunoglobulin heavy chain or a portion thereof sufficient to form
an antigen binding site (e.g. heavy chains include, but are not
limited to, VH, chains VH-CH1 chains and VH-CH1-CH2-CH3 chains),
and each light chain can be a full-length light chain or a thereof
sufficient to form an antigen binding site (e.g. light chains
include, but are not limited to, VL chains and VL-CL chains). Each
heavy chain (H and H') pairs with one light chain (L and L',
respectively). Typically, antibodies minimally include all or at
least a portion of the variable heavy (VH) chain and/or the
variable light (VL) chain. The antibody also can include all or a
portion of the constant region. For example, a full-length antibody
is an antibody having two full-length heavy chains (e.g.
VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light
chains (VL-CL) and hinge regions, such as antibodies produced by
antibody secreting B cells and antibodies with the same domains
that are produced synthetically. Additionally, an "antibody" refers
to a protein of the immunoglobulin family or a polypeptide
comprising fragments of an immunoglobulin that is capable of
noncovalently, reversibly, and in a specific manner binding a
corresponding antigen. An exemplary antibody structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD), connected through a
disulfide bond. The recognized immunoglobulin genes include the
.kappa., .lamda., .alpha., .gamma., .delta., .epsilon., and .mu.
constant region genes, as well as the myriad immunoglobulin
variable region genes. Light chains are classified as either
.kappa. or .lamda.. Heavy chains are classified as .gamma., .mu.,
.alpha., .delta., or .epsilon. which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
The N-terminus of each chain defines a variable region of about 100
to 110 or more amino acids primarily responsible for antigen
recognition. The terms variable light chain (VL) and variable heavy
chain (VH) refer to these regions of light and heavy chains
respectively.
[0276] "Variable" refers to the fact that certain portions of the
variable domains (also referred to as variable regions) differ
extensively in sequence among antibodies and are used in the
binding and specificity of each particular antibody for its
particular antigen. However, the variability is not evenly
distributed throughout the variable domains of antibodies. It is
concentrated in three segments called complementarity-determining
regions (CDRs) or hypervariable regions (HVRs) both in the
light-chain and the heavy-chain variable domains. CDRs include
those specified as Kabat, Chothia, and IMGT as shown herein within
the variable region sequences. The more highly conserved portions
of variable domains are called the framework (FR). The variable
domains of native heavy and light chains each comprise four FR
regions, largely adopting a .beta.-sheet configuration, connected
by three CDRs, which form loops connecting, and in some cases
forming part of, the .beta.-sheet structure. The CDRs in each chain
are held together in close proximity by the FR regions and, with
the CDRs from the other chain, contribute to the formation of the
antigen-binding site of antibodies (see Kabat et al., Sequences of
Proteins of Immunological Interest, Fifth Edition, National
Institute of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody-dependent cellular toxicity.
[0277] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0278] "Fv" refers to an antibody fragment which contains an
antigen-recognition and antigen-binding site. In a two-chain Fv
species, this region consists of a dimer of one heavy and one light
chain variable domain in non-covalent association. In a single
chain Fv (scFv) species, one heavy chain and one light chain
variable domain can be covalently linked by a flexible peptide
linker such that the light and heavy chains can associate in a
"dimeric" structure analogous to that in a two-chain Fv (scFv)
species. It is in this configuration that the three CDRs of each
variable domain interact to define an antigen-binding site on the
surface of the VH-VL dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0279] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CHI) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0280] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0281] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these can be further divided into
subclasses (isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA.sub.1, and IgA.sub.2. The heavy-chain constant
domains that correspond to the different classes of immunoglobulins
are called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0282] "Antibody fragments" comprise only a portion of an intact
antibody, wherein the portion preferably retains at least one,
preferably most or all, of the functions normally associated with
that portion when present in an intact antibody. Examples of
antibody fragments include Fab, Fab', F(ab')2, single-chain Fvs
(scFv), Fv, dsFv, diabody, Fd and Fd' fragments Fab fragments, Fd
fragments, scFv fragments, linear antibodies, single-chain antibody
molecules, and multispecific antibodies formed from antibody
fragments (see, for example, Methods in Molecular Biology, Vol 207:
Recombinant Antibodies for Cancer Therapy Methods and Protocols
(2003); Chapter 1; p 3-25, Kipriyanov). Other known fragments
include, but are not limited to, scFab fragments (Hust et al., BMC
Biotechnology (2007), 7:14). In one embodiment, an antibody
fragment comprises an antigen binding site of the intact antibody
and thus retains the ability to bind antigen. In another
embodiment, an antibody fragment, for example one that comprises
the Fc region, retains at least one of the biological functions
normally associated with the Fc region when present in an intact
antibody, such as FcRn binding, antibody half life modulation, ADCC
function and complement binding. In one embodiment, an antibody
fragment is a monovalent antibody that has an in vivo half life
substantially similar to an intact antibody. For example, such an
antibody fragment may comprise on antigen binding arm linked to an
Fc sequence capable of conferring in vivo stability to the
fragment. For another example, an antibody fragment or antibody
portion refers to any portion of a full-length antibody that is
less than full length but contains at least a portion of the
variable region of the antibody sufficient to form an antigen
binding site (e.g. one or more CDRs) and thus retains the a binding
specificity and/or an activity of the full-length antibody;
antibody fragments include antibody derivatives produced by
enzymatic treatment of full-length antibodies, as well as
synthetically, e.g. recombinantly produced derivatives.
[0283] A "dsFv" refers to an Fv with an engineered intermolecular
disulfide bond, which stabilizes the VH-VL pair.
[0284] A "Fd fragment" refers to a fragment of an antibody
containing a variable domain (VH) and one constant region domain
(CH1) of an antibody heavy chain.
[0285] A "Fab fragment" refers to an antibody fragment that
contains the portion of the full-length antibody that would results
from digestion of a full-length immunoglobulin with papain, or a
fragment having the same structure that is produced synthetically,
e.g. recombinantly. A Fab fragment contains a light chain
(containing a VL and CL portion) and another chain containing a
variable domain of a heavy chain (VH) and one constant region
domain portion of the heavy chain (CH1); it can be recombinantly
produced.
[0286] A "F(ab')2 fragment" refers to an antibody fragment that
results from digestion of an immunoglobulin with pepsin at pH
4.0-4.5, or a synthetically, e.g. recombinantly, produced antibody
having the same structure. The F(ab')2 fragment contains two Fab
fragments but where each heavy chain portion contains an additional
few amino acids, including cysteine residues that form disulfide
linkages joining the two fragments; it can be recombinantly
produced.
[0287] A "Fab' fragment" refers to a fragment containing one half
(one heavy chain and one light chain) of the F(ab')2 fragment.
[0288] A "Fd" fragment refers to a fragment of an antibody
containing one heavy chain portion of a F(ab')2 fragment.
[0289] A "Fv' fragment" refers to a fragment containing only the VH
and VL domains of an antibody molecule.
[0290] A "scFv fragment" refers to an antibody fragment that
contains a variable light chain (VL) and variable heavy chain (VH),
covalently connected by a polypeptide linker in any order. The
linker is of a length such that the two variable domains are
bridged without substantial interference. Exemplary linkers are
(Gly-Ser)n residues with some Glu or Lys residues dispersed
throughout to increase solubility.
[0291] Diabodies are dimeric scFv; diabodies typically have shorter
peptide linkers than scFvs, and they preferentially dimerize.
[0292] "HsFv" refers to antibody fragments in which the constant
domains normally present in a Fab fragment have been substituted
with a heterodimeric coiled-coil domain (see, e.g., Arndt et al.
(2001) J Mol Biol. 7:312:221-228).
[0293] "Hypervariable region", "HVR", or "HV", as well as
"complementary determing region" or "CDR", may refer to the regions
of an antibody variable domain which are hypervariable in sequence
and/or form structurally defined loops. Generally, antibodies
comprise six hypervariable or CDR regions; three in the VH (H1, H2,
H3), and three in the VL (L1, L2, L3). A number of hypervariable
region or CDR delineations are in use and are encompassed herein.
The Kabat Complementarity Determining Regions (Kabat CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM
hypervariable regions represent a compromise between the Kabat CDRs
and Chothia structural loops, (Chothia "CDRs") and are used by
Oxford Molecular's AbM antibody modeling software. The "contact"
hypervariable regions are based on an analysis of the available
complex crystal structures. The residues from each of these
hypervariable regions are noted below.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
IMGT referes to the international ImMunoGeneTics Information
System, as described by Lefrace et al., Nucl. Acids, Res. 37;
D1006-D1012 (2009), including for example, IMGT designated CDRs for
antibodies.
[0294] Hypervariable regions may comprise "extended hypervariable
regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and
89-97 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and
93-102, 94-102 or 95-102 (H3) in the VH. The variable domain
residues are numbered according to Kabat et al., Supra for each of
these definitions.
[0295] "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined. "Framework regions" (FRs) are the domains within the
antibody variable region domains comprising framework residues that
are located within the beta sheets; the FR regions are
comparatively more conserved, in terms of their amino acid
sequences, than the hypervariable regions.
[0296] "Monoclonal antibody" refers to an antibody from a
population of substantially homogeneous antibodies, that is, for
example, the individual antibodies comprising the population are
identical and/or bind the same epitope(s), except for possible
variants that may arise during production of the monoclonal
antibody, such variants generally being present in minor amounts.
Such monoclonal antibody typically includes an antibody comprising
a polypeptide sequence that binds a target, wherein the
target-binding polypeptide sequence was obtained by a process that
includes the selection of a single target binding polypeptide
sequence from a plurality of polypeptide sequences. For example,
the selection process can be the selection of a unique clone from a
plurality of clones, such as a pool of hybridoma clones, phage
clones or recombinant DNA clones. It should be understood that the
selected target binding sequence can be further altered, for
example, to improve affinity for the target, to humanize the target
binding sequence, to improve its production in cell culture, to
reduce its immunogenicity in vivo, to create a multispecific
antibody, etc., and that an antibody comprising the altered target
binding sequence is also a monoclonal antibody of this disclosure.
In contrast to polyclonal antibody preparations which typically
include different antibodies directed against different
determinants (e.g., epitopes), each monoclonal antibody of a
monoclonal antibody preparation is directed against a single
determinant on an antigen. In addition to their specificity, the
monoclonal antibody preparations are advantageous in that they are
typically uncontaminated by other immunoglobulins. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present disclosure may be made by a
variety of techniques, including, for example, the hybridoma method
(e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies
and T-Cell Hybridomas 563-681, (Elsevier, N.Y., 1981)), recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display
technologies (see, e.g., Clackson et al., Nature, 352:624-628
(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Sidhu et
al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol.
340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA
101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods
284(1-2):119-132 (2004), and technologies for producing human or
human-like antibodies in animals that have parts or all of the
human immunoglobulin loci or genes encoding human immunoglobulin
sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735;
WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA,
90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);
Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos.
5,545,806; 5,569,825; 5,591,669; 5,545,807; WO 1997/17852; U.S.
Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
and U.S. Pat. No. 5,661,016; Marks et al., Bio/Technology, 10:
779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994);
Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature
Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology,
14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13:
65-93 (1995)).
[0297] "Humanized" or "Human engineered" forms of non-human (e.g.,
murine) antibodies are chimeric antibodies that contain amino acids
represented in human immunoglobulin sequences, including, for
example, wherein minimal sequence is derived from non-human
immunoglobulin. For example, humanized antibodies may be human
antibodies in which some hypervariable region residues and possibly
some FR residues are substituted by residues from analogous sites
in non-human (e.g., rodent) antibodies. Alternatively, humanized or
human engineered antibodies may be non-human (e.g., rodent)
antibodies in which some residues are substituted by residues from
analogious sites in human antibodies (see, e.g., U.S. Pat. No.
5,766,886). Humanized antibodies include human immunoglobulins
(recipient antibody) in which residues from a hypervariable region
of the recipient are replaced by residues from a hypervariable
region of a non-human species (donor antibody) such as mouse, rat,
rabbit or nonhuman primate having the desired specificity,
affinity, and capacity. In some instances, framework region (FR)
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Furthermore, humanized antibodies may comprise
residues that are not found in the recipient antibody or in the
donor antibody, including, for example non-antibody sequences such
as a chemokine, growth factor, peptide, cytokine, cell surface
protein, serum protein, toxin, extracellular matrix protein,
clotting factor, or secreted protein sequence. These modifications
may be made to further refine antibody performance. Humanized
antibodies include human engineered antibodies, for example, as
described by U.S. Pat. No. 5,766,886, including methods for
preparing modified antibody variable domains. A humanized antibody
may comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the
hypervariable loops correspond to those of a non-human
immunoglobulin and all or substantially all of the FRs are those of
a human immunoglobulin sequence. A humanized antibody optionally
may also comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details, see Jones et al., Nature 321:522-525 (1986); Riechmann et
al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992). See also the following review articles and
references cited therein: Vaswani and Hamilton, Ann. Allergy,
Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc.
Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op.
Biotech. 5:428-433 (1994).
[0298] "Hybrid antibodies" refer to immunoglobulin molecules in
which pairs of heavy and light chains from antibodies with
different antigenic determinant regions are assembled together so
that two different epitopes or two different antigens can be
recognized and bound by the resulting tetramer.
[0299] "Chimeric" antibodies (immunoglobulins) have a portion of
the heavy and/or light chain identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (see e.g., Morrison et al., Proc. Natl.
Acad. Sci. USA 81:6851-6855 (1984)). Humanized antibody refers to a
subset of chimeric antibodies.
[0300] "Single-chain Fv" or "scFv" antibody fragments may comprise
the VH and VL domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally, the scFv
polypeptide further comprises a polypeptide linker between the VH
and VL domains which enables the scFv to form the desired structure
for antigen binding. For a review of scFv, see e.g., Pluckthun, in
The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
[0301] An "antigen" refers to a predetermined antigen to which an
antibody can selectively bind. The target antigen may be
polypeptide, carbohydrate, nucleic acid, lipid, hapten or other
naturally occurring or synthetic compound. Preferably, the target
antigen is a polypeptide.
[0302] "Epitope" or "antigenic determinant", used interchangeably
herein, refer to that portion of an antigen capable of being
recognized and specifically bound by a particular antibody. When
the antigen is a polypeptide, epitopes can be formed both from
contiguous amino acids and noncontiguous amino acids juxtaposed by
tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained upon protein denaturing, whereas
epitopes formed by tertiary folding are typically lost upon protein
denaturing. An epitope typically includes at least 3, and more
usually, at least 5 or 8-10 amino acids in a unique spatial
conformation. Antibodies may bind to the same or a different
epitope on an antigen. Antibodies may be characterized in different
epitope bins. Whether an antibody binds to the same or different
epitope as another antibody (e.g., a reference antibody or
benchmark antibody) may be determined by competition between
antibodies in assays (e.g., competitive binding assays).
[0303] Competition between antibodies may be determined by an assay
in which the immunoglobulin under test inhibits specific binding of
a reference antibody to a common antigen. Numerous types of
competitive binding assays are known, for example: solid phase
direct or indirect radioimmunoassay (RIA), solid phase direct or
indirect enzyme immunoassay or enzyme-linked immunosorbent assay
(EIA or ELISA), sandwich competition assay including an ELISA assay
(see Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid
phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol.
137:3614-3619 (1986)); solid phase direct labeled assay, solid
phase direct labeled sandwich assay (see Harlow and Lane,
"Antibodies, A Laboratory Manual," Cold Spring Harbor Press
(1988)); solid phase direct label RIA using 1-125 label (see Morel
et al., Molec. Immunol. 25(1):7-15 (1988)); solid phase direct
biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and
direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.,
32:77-82 (1990)). Competition binding assays may be performed using
Surface Plasmon Resonance (SPR), for example, with a Biacore.RTM.
instrument for kinetic analysis of binding interactions. In such an
assay, a humanized antibody comprising an ultralong CDR3 of unknown
epitope specificity may be evaluated for its ability to compete for
binding against a comparator antibody (e.g., a BA1 or BA2 antibody
as described herein). An assay may involve the use of purified
antigen bound to a solid surface or cells bearing either of these,
an unlabeled test immunoglobulin and a labeled reference
immunoglobulin. Competitive inhibition may be measured by
determining the amount of label bound to the solid surface or cells
in the presence of the test immunoglobulin. Usually the test
immunoglobulin is present in excess. An assay (competing
antibodies) may include antibodies binding to the same epitope as
the reference antibody and antibodies binding to an adjacent
epitope sufficiently proximal to the epitope bound by the reference
antibody for steric hindrance to occur. Usually, when a competing
antibody is present in excess, it will inhibit specific binding of
a reference antibody to a common antigen by at least 50%, or at
least about 70%, or at least about 80%, or least about 90%, or at
least about 95%, or at least about 99% or about 100% for a
competitor antibody.
[0304] That an antibody "selectively binds" or "specifically binds"
means that the antibody reacts or associates more frequently, more
rapidly, with greater duration, with greater affinity, or with some
combination of the above to an antigen or an epitope than with
alternative substances, including unrelated proteins. "Selectively
binds" or "specifically binds" may mean, for example, that an
antibody binds to a protein with a K.sub.D of at least about 0.1
mM, or at least about 1 .mu.M or at least about 0.1 .mu.M or
better, or at least about 0.01 .mu.M or better. Because of the
sequence identity between homologous proteins in different species,
specific binding can include an antibody that recognizes a given
antigen in more than one species.
[0305] "Non-specific binding" and "background binding" when used in
reference to the interaction of an antibody and a protein or
peptide refer to an interaction that is not dependent on the
presence of a particular structure (e.g., the antibody is binding
to proteins in general rather that a particular structure such as
an epitope).
[0306] "Diabodies" refer to small antibody fragments with two
antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161; and Hollinger et. al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993).
[0307] A "human antibody" refers to one which possesses an amino
acid sequence which corresponds to that of an antibody produced by
a human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues.
[0308] An "affinity matured" antibody refers to one with one or
more alterations in one or more CDRs thereof which result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s).
Preferred affinity matured antibodies will have nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies are produced by procedures known in the art. Marks et
al., Bio/Technology 10:779-783 (1992) describes affinity maturation
by VH and VL domain shuffling. Random mutagenesis of CDR and/or
framework residues is described by: Barbas et al., Proc Nat. Acad.
Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155
(1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et
al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol.
Biol. 226:889-896 (1992).
[0309] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: Clq binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g. B cell receptor); and B cell activation.
[0310] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, may be
performed. Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA
95:652-656 (1998).
[0311] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source, e.g., from blood.
[0312] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)) and regulates homeostasis of immunoglobulins. For
example, antibody variants with improved or diminished binding to
FcRs have been described (see, e.g., Shields et al. J. Biol. Chem.
9(2): 6591-6604 (2001)).
[0313] Methods of measuring binding to FcRn are known (see, e.g.,
Ghetie 1997, Hinton 2004). Binding to human FcRn in vivo and serum
half life of human FcRn high affinity binding polypeptides can be
assayed, e.g., in transgenic mice or transfected human cell lines
expressing human FcRn, or in primates administered with the Fc
variant polypeptides.
[0314] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (Clq) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, for example, as
described in Gazzano-Santoro et al., J. Immunol. Methods 202:163
(1996), may be performed.
[0315] Polypeptide variants with altered Fc region amino acid
sequences and increased or decreased Clq binding capability have
been described (e.g., see, also, Idusogie et al. J. Immunol. 164:
4178-4184 (2000)).
[0316] "Fc region-comprising polypeptide" refers to a polypeptide,
such as an antibody or immunoadhesin (see definitions below), which
comprises an Fc region. The C-terminal lysine (residue 447
according to the EU numbering system) of the Fc region may be
removed, for example, during purification of the polypeptide or by
recombinant engineering the nucleic acid encoding the
polypeptide.
[0317] "Blocking" antibody or an "antagonist" antibody refers to
one which inhibits or reduces biological activity of the antigen it
binds. Preferred blocking antibodies or antagonist antibodies
substantially or completely inhibit the biological activity of the
antigen.
[0318] "Agonist" antibody refers to an antibody which mimics (e.g.,
partially or fully) at least one of the functional activities of a
polypeptide of interest.
[0319] "Acceptor human framework" refers to a framework comprising
the amino acid sequence of a VL or VH framework derived from a
human immunoglobulin framework, or from a human consensus
framework. An acceptor human framework "derived from" a human
immunoglobulin framework or human consensus framework may comprise
the same amino acid sequence thereof, or may contain pre-existing
amino acid sequence changes. Where pre-existing amino acid changes
are present, preferably no more than 5 and preferably 4 or less, or
3 or less, pre-existing amino acid changes are present.
[0320] A "human consensus framework" refers to a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0321] "Disorder" or "disease" refers to any condition that would
benefit from treatment with a substance/molecule (e.g., a humanized
antibody comprising an ultralong CDR3 as disclosed herein) or
method disclosed herein. This includes chronic and acute disorders
or diseases including those pathological conditions which
predispose the mammal to the disorder in question.
[0322] "Treatment" refers to clinical intervention in an attempt to
alter the natural course of the individual or cell being treated,
and can be performed either for prophylaxis or during the course of
clinical pathology. Desirable effects of treatment include
preventing occurrence or recurrence of disease, alleviation of
symptoms, diminishment of any direct or indirect pathological
consequences of the disease, preventing metastasis, decreasing the
rate of disease progression, amelioration or palliation of the
disease state, and remission or improved prognosis. In some
embodiments, antibodies disclosed herein are used to delay
development of a disease or disorder.
[0323] "Individual" (e.g., a "subject") refers to a vertebrate,
preferably a mammal, more preferably a human. Mammals include, but
are not limited to, farm animals (such as cows), sport animals,
pets (such as cats, dogs and horses), primates, mice and rats.
[0324] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, rodents (e.g., mice and
rats), and monkeys; domestic and farm animals; and zoo, sports,
laboratory, or pet animals, such as dogs, cats, cattle, horses,
sheep, pigs, goats, rabbits, etc. In some embodiments, the mammal
is selected from a human, rodent, or monkey.
[0325] "Pharmaceutically acceptable" refers to approved or
approvable by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, including humans.
[0326] "Pharmaceutically acceptable salt" refers to a salt of a
compound that is pharmaceutically acceptable and that possesses the
desired pharmacological activity of the parent compound.
[0327] "Pharmaceutically acceptable excipient, carrier or adjuvant"
refers to an excipient, carrier or adjuvant that can be
administered to a subject, together with at least one antibody of
the present disclosure, and which does not destroy the
pharmacological activity thereof and is nontoxic when administered
in doses sufficient to deliver a therapeutic amount of the
compound.
[0328] "Pharmaceutically acceptable vehicle" refers to a diluent,
adjuvant, excipient, or carrier with which at least one antibody of
the present disclosure is administered.
[0329] "Providing a prognosis", "prognostic information", or
"predictive information" refer to providing information, including
for example the presence of cancer cells in a subject's tumor,
regarding the impact of the presence of cancer (e.g., as determined
by the diagnostic methods of the present disclosure) on a subject's
future health (e.g., expected morbidity or mortality, the
likelihood of getting cancer, and the risk of metastasis).
[0330] Terms such as "treating" or "treatment" or "to treat" or
"alleviating" or "to alleviate" refer to both 1) therapeutic
measures that cure, slow down, lessen symptoms of, and/or halt
progression of a diagnosed pathologic condition or disorder and 2)
prophylactic or preventative measures that prevent and/or slow the
development of a targeted pathologic condition or disorder. Thus
those in need of treatment include those already with the disorder;
those prone to have the disorder; and those in whom the disorder is
to be prevented.
[0331] "Providing a diagnosis" or "diagnostic information" refers
to any information, including for example the presence of cancer
cells, that is useful in determining whether a patient has a
disease or condition and/or in classifying the disease or condition
into a phenotypic category or any category having significance with
regards to the prognosis of or likely response to treatment (either
treatment in general or any particular treatment) of the disease or
condition. Similarly, diagnosis refers to providing any type of
diagnostic information, including, but not limited to, whether a
subject is likely to have a condition (such as a tumor), whether a
subject's tumor comprises cancer stem cells, information related to
the nature or classification of a tumor as for example a high risk
tumor or a low risk tumor, information related to prognosis and/or
information useful in selecting an appropriate treatment. Selection
of treatment can include the choice of a particular
chemotherapeutic agent or other treatment modality such as surgery
or radiation or a choice about whether to withhold or deliver
therapy.
[0332] A "human consensus framework" refers to a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0333] An "acceptor human framework" for the purposes herein refers
to a framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0334] "Antigen-binding site" refers to the interface formed by one
or more complementary determining regions. An antibody molecule has
two antigen combining sites, each containing portions of a heavy
chain variable region and portions of a light chain variable
region. The antigen combining sites can contain other portions of
the variable region domains in addition to the CDRs.
[0335] An "antibody light chain" or an "antibody heavy chain"
refers to a polypeptide comprising the VL or VH, respectively. The
VL is encoded by the minigenes V (variable) and J (junctional), and
the VH by minigenes V, D (diversity), and J. Each of VL or VH
includes the CDRs as well as the framework regions. In this
application, antibody light chains and/or antibody heavy chains
may, from time to time, be collectively referred to as "antibody
chains."
[0336] These terms encompass antibody chains containing mutations
that do not disrupt the basic structure of VL or VH, as one skilled
in the art will readily recognize.
[0337] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide bonded. From N- to
C-terminus, each heavy chain has a variable region (V H), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (V
L), also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.kappa.), based on the amino acid sequence of
its constant domain.
[0338] "Combinatorial library" refers to collections of compounds
formed by reacting different combinations of interchangeable
chemical "building blocks" to produce a collection of compounds
based on permutations of the building blocks. For an antibody
combinatorial library, the building blocks are the component V, D
and J regions (or modified forms thereof) from which antibodies are
formed. For purposes herein, the terms "library" or "collection"
are used interchangeably.
[0339] A "combinatorial antibody library" refers to a collection of
antibodies (or portions thereof, such as Fabs), where the
antibodies are encoded by nucleic acid molecules produced by the
combination of V, D and J gene segments, particularly human V, D
and J germline segments. The combinatorial libraries herein
typically contain at least 50 different antibody (or antibody
portions or fragment) members, typically at or about 50, 100, 500,
103, 1.times.103, 2.times.103, 3.times.103, 4.times.103,
5.times.103, 6.times.103, 7.times.103, 8.times.103, 9.times.103,
1.times.104, 2.times.104, 3.times.104, 4.times.104, 5.times.104,
6.times.104, 7.times.104, 8.times.104, 9.times.104, 1.times.105,
2.times.105, 3.times.105, 4.times.105, 5.times.105, 6.times.105,
7.times.105, 8.times.105, 9.times.105, 106, 107, 108, 109, 1010, or
more different members. The resulting libraries or collections of
antibodies or portions thereof, can be screened for binding to a
target protein or modulation of a functional activity.
[0340] A "human combinatorial antibody library" refers to a
collection of antibodies or portions thereof, whereby each member
contains a VL and VH chains or a sufficient portion thereof to form
an antigen binding site encoded by nucleic acid containing human
germline segments produced as described herein.
[0341] A "variable germline segment" refers to V, D and J groups,
subgroups, genes or alleles thereof. Gene segment sequences are
accessible from known database (e.g., National Center for
Biotechnology Information (NCBI), the international ImMunoGeneTics
information System.RTM. (IMGT), the Kabat database and the
Tomlinson's VBase database (Lefranc (2003) Nucleic Acids Res.,
31:307-310; Martin et al., Bioinformatics Tools for Antibody
Engineering in Handbook of Therapeutic Antibodies, Wiley-VCH
(2007), pp. 104-107). Tables 3-5 list exemplary human variable
germline segments. Sequences of exemplary VH, DH, JH, V.kappa.,
J.kappa., V.lamda. and or J.lamda., germline segments are set forth
in SEQ ID NOS: 10-451 and 868. For purposes herein, a germline
segment includes modified sequences thereof, that are modified in
accord with the rules of sequence compilation provided herein to
permit practice of the method. For example, germline gene segments
include those that contain one amino acid deletion or insertion at
the 5' or 3' end compared to any of the sequences of nucleotides
set forth in SEQ ID NOS:10-451, 868.
[0342] "Compilation," "compile," "combine," "combination,"
"rearrange," "rearrangement," or other similar terms or grammatical
variations thereof refers to the process by which germline segments
are ordered or assembled into nucleic acid sequences representing
genes. For example, variable heavy chain germline segments are
assembled such that the VH segment is 5' to the DH segment which is
5' to the JH segment, thereby resulting in a nucleic acid sequence
encoding a VH chain. Variable light chain germline segments are
assembled such that the VL segment is 5' to the JL segment, thereby
resulting in a nucleic acid sequence encoding a VL chain. A
constant gene segment or segments also can be assembled onto the 3'
end of a nucleic acid encoding a VH or VL chain.
[0343] "Linked," or "linkage" or other grammatical variations
thereof with reference to germline segments refers to the joining
of germline segments. Linkage can be direct or indirect. Germline
segments can be linked directly without additional nucleotides
between segments, or additional nucleotides can be added to render
the entire segment in-frame, or nucleotides can be deleted to
render the resulting segment in-frame. It is understood that the
choice of linker nucleotides is made such that the resulting
nucleic acid molecule is in-frame and encodes a functional and
productive antibody.
[0344] "In-frame" or "linked in-frame" with reference to linkage of
human germline segments means that there are insertions and/or
deletions in the nucleotide germline segments at the joined
junctions to render the resulting nucleic acid molecule in-frame
with the 5' start codon (ATG), thereby producing a "productive" or
functional full-length polypeptide. The choice of nucleotides
inserted or deleted from germline segments, particularly at joints
joining various VD, DJ and VJ segments, is in accord with the rules
provided in the method herein for V(D)J joint generation. For
example, germline segments are assembled such that the VH segment
is 5' to the DH segment which is 5' to the JH segment. At the
junction joining the VH and the DH and at the junction joining the
DH and JH segments, nucleotides can be inserted or deleted from the
individual VH, DH or JH segments, such that the resulting nucleic
acid molecule containing the joined VDJ segments are in-frame with
the 5' start codon (ATG).
[0345] A portion of an antibody includes sufficient amino acids to
form an antigen binding site.
[0346] A "reading frame" refers to a contiguous and non-overlapping
set of three-nucleotide codons in DNA or RNA. Because three codons
encode one amino acid, there exist three possible reading frames
for given nucleotide sequence, reading frames 1, 2 or 3. For
example, the sequence ACTGGTCA will be ACT GGT CA for reading frame
1, A CTG GTC A for reading frame 2 and AC TGG TCA for reading frame
3. Generally for practice of the method described herein, nucleic
acid sequences are combined so that the V sequence has reading
frame 1.
[0347] A "stop codon" refers to a three-nucleotide sequence that
signals a halt in protein synthesis during translation, or any
sequence encoding that sequence (e.g. a DNA sequence encoding an
RNA stop codon sequence), including the amber stop codon (UAG or
TAG)), the ochre stop codon (UAA or TAA)) and the opal stop codon
(UGA or TGA)). It is not necessary that the stop codon signal
termination of translation in every cell or in every organism. For
example, in suppressor strain host cells, such as amber suppressor
strains and partial amber suppressor strains, translation proceeds
through one or more stop codon (e.g. the amber stop codon for an
amber suppressor strain), at least some of the time.
[0348] A "variable heavy" (VH) chain or a "variable light" (VL)
chain (also termed VH domain or VL domain) refers to the
polypeptide chains that make up the variable domain of an antibody.
For purposes herein, heavy chain germline segments are designated
as VH, DH and JH, and compilation thereof results in a nucleic acid
encoding a VH chain. Light chain germline segments are designated
as VL or JL, and include kappa and lambda light chains (V.kappa.
and J.kappa.; V.lamda. and J.lamda.) and compilation thereof
results in a nucleic acid encoding a VL chain. It is understood
that a light chain is either a kappa or lambda light chain, but
does not include a kappa/lambda combination by virtue of
compilation of a V.kappa. and J.lamda..
[0349] A "degenerate codon" refers to three-nucleotide codon that
specifies the same amino acid as a codon in a parent nucleotide
sequence. One of skill in the art is familiar with degeneracy of
the genetic code and can identify degenerate codons.
[0350] "Diversity" with respect to members in a collection refers
to the number of unique members in a collection. Hence, diversity
refers to the number of different amino acid sequences or nucleic
acid sequences, respectively, among the analogous polypeptide
members of that collection. For example, a collection of
polynucleotides having a diversity of 104 contains 104 different
nucleic acid sequences among the analogous polynucleotide members.
In one example, the provided collections of polynucleotides and/or
polypeptides have diversities of at least at or about 102, 103,
104, 105, 106, 107, 108, 109, 1010 or more.
[0351] "Sequence diversity" refers to a representation of nucleic
acid sequence similarity and is determined using sequence
alignments, diversity scores, and/or sequence clustering. Any two
sequences can be aligned by laying the sequences side-by-side and
analyzing differences within nucleotides at every position along
the length of the sequences. Sequence alignment can be assessed in
silico using Basic Local Alignment Search Tool (BLAST), an NCBI
tool for comparing nucleic acid and/or protein sequences. The use
of BLAST for sequence alignment is well known to one of skill in
the art. The Blast search algorithm compares two sequences and
calculates the statistical significance of each match (a Blast
score). Sequences that are most similar to each other will have a
high Blast score, whereas sequences that are most varied will have
a low Blast score.
[0352] A "polypeptide domain" refers to a part of a polypeptide (a
sequence of three or more, generally 5 or 7 or more amino acids)
that is a structurally and/or functionally distinguishable or
definable. Exemplary of a polypeptide domain is a part of the
polypeptide that can form an independently folded structure within
a polypeptide made up of one or more structural motifs (e.g.
combinations of alpha helices and/or beta strands connected by loop
regions) and/or that is recognized by a particular functional
activity, such as enzymatic activity or antigen binding. A
polypeptide can have one, typically more than one, distinct
domains. For example, the polypeptide can have one or more
structural domains and one or more functional domains. A single
polypeptide domain can be distinguished based on structure and
function. A domain can encompass a contiguous linear sequence of
amino acids. Alternatively, a domain can encompass a plurality of
non-contiguous amino acid portions, which are non-contiguous along
the linear sequence of amino acids of the polypeptide. Typically, a
polypeptide contains a plurality of domains. For example, each
heavy chain and each light chain of an antibody molecule contains a
plurality of immunoglobulin (Ig) domains, each about 110 amino
acids in length.
[0353] An "Ig domain" refers to a domain, recognized as such by
those in the art, that is distinguished by a structure, called the
Immunoglobulin (Ig) fold, which contains two beta-pleated sheets,
each containing anti-parallel beta strands of amino acids connected
by loops. The two beta sheets in the Ig fold are sandwiched
together by hydrophobic interactions and a conserved intra-chain
disulfide bond. Individual immunoglobulin domains within an
antibody chain further can be distinguished based on function. For
example, a light chain contains one variable region domain (VL) and
one constant region domain (CL), while a heavy chain contains one
variable region domain (VH) and three or four constant region
domains (CH). Each VL, CL, VH, and CH domain is an example of an
immunoglobulin domain.
[0354] A "variable domain" with reference to an antibody refers to
a specific Ig domain of an antibody heavy or light chain that
contains a sequence of amino acids that varies among different
antibodies. Each light chain and each heavy chain has one variable
region domain (VL, and, VH). The variable domains provide antigen
specificity, and thus are responsible for antigen recognition. Each
variable region contains CDRs that are part of the antigen binding
site domain and framework regions (FRs).
[0355] A "constant region domain" refers to a domain in an antibody
heavy or light chain that contains a sequence of amino acids that
is comparatively more conserved among antibodies than the variable
region domain. Each light chain has a single light chain constant
region (CL) domain and each heavy chain contains one or more heavy
chain constant region (CH) domains, which include, CH1, CH2, CH3
and CH4. Full-length IgA, IgD and IgG isotypes contain CH1, CH2 CH3
and a hinge region, while IgE and IgM contain CH1, CH2 CH3 and CH4.
CH1 and CL domains extend the Fab arm of the antibody molecule,
thus contributing to the interaction with antigen and rotation of
the antibody arms. Antibody constant regions can serve effector
functions, such as, but not limited to, clearance of antigens,
pathogens and toxins to which the antibody specifically binds, e.g.
through interactions with various cells, biomolecules and
tissues.
[0356] An "antibody or portion thereof that is sufficient to form
an antigen binding site" means that the antibody or portion thereof
contains at least 1 or 2, typically 3, 4, 5 or all 6 CDRs of the VH
and VL sufficient to retain at least a portion of the binding
specificity of the corresponding full-length antibody containing
all 6 CDRs. Generally, a sufficient antigen binding site at least
requires CDR3 of the heavy chain (CDRH3). It typically further
requires the CDR3 of the light chain (CDRL3). As described herein,
one of skill in the art knows and can identify the CDRs based on
Kabat or Chothia numbering (see, e.g., Kabat, E. A. et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).
For example, based on Kabat numbering, CDR-LI corresponds to
residues L24-L34; CDR-L2 corresponds to residues L50-L56; CDR-L3
corresponds to residues L89-L97; CDR-H1 corresponds to residues
H31-H35, 35a or 35b depending on the length; CDR-H2 corresponds to
residues H50-H65; and CDR-H3 corresponds to residues H95-H102.
[0357] A "peptide mimetic" refers to a peptide that mimics the
activity of a polypeptide. For example, an erythropoietin (EPO)
peptide mimetic is a peptide that mimics the activity of Epo, such
as for binding and activation of the EPO receptor.
[0358] An "address" refers to a unique identifier for each locus in
a collection whereby an addressed member (e.g. an antibody) can be
identified. An addressed moiety is one that can be identified by
virtue of its locus or location. Addressing can be effected by
position on a surface, such as a well of a microplate. For example,
an address for a protein in a microwell plate that is F9 means that
the protein is located in row F, column 9 of the microwell plate.
Addressing also can be effected by other identifiers, such as a tag
encoded with a bar code or other symbology, a chemical tag, an
electronic, such RF tag, a color-coded tag or other such
identifier.
[0359] An "array" refers to a collection of elements, such as
antibodies, containing three or more members.
[0360] A "spatial array" refers to an array where members are
separated or occupy a distinct space in an array. Hence, spatial
arrays are a type of addressable array. Examples of spatial arrays
include microtiter plates where each well of a plate is an address
in the array. Spacial arrays include any arrangement wherein a
plurality of different molecules, e.g., polypeptides, are held,
presented, positioned, situated, or supported. Arrays can include
microtiter plates, such as 48-well, 96-well, 144-well, 192-well,
240-well, 288-well, 336-well, 384-well, 432-well, 480-well,
576-well, 672-well, 768-well, 864-well, 960-well, 1056-well,
1152-well, 1248-well, 1344-well, 1440-well, or 1536-well plates,
tubes, slides, chips, flasks, or any other suitable laboratory
apparatus. Furthermore, arrays can also include a plurality of
sub-arrays. A plurality of sub-arrays encompasses an array where
more than one arrangement is used to position the polypeptides. For
example, multiple 96-well plates could constitute a plurality of
sub-arrays and a single array.
[0361] An "addressable library" or "spatially addressed library"
refers to a collection of molecules such as nucleic acid molecules
or protein agents, such as antibodies, in which each member of the
collection is identifiable by virtue of its address.
[0362] An "addressable array" refers to one in which the members of
the array are identifiable by their address, the position in a
spatial array, such as a well of a microtiter plate, or on a solid
phase support, or by virtue of an identifiable or detectable label,
such as by color, fluorescence, electronic signal (i.e. RF,
microwave or other frequency that does not substantially alter the
interaction of the molecules of interest), bar code or other
symbology, chemical or other such label. Hence, in general the
members of the array are located at identifiable loci on the
surface of a solid phase or directly or indirectly linked to or
otherwise associated with the identifiable label, such as affixed
to a microsphere or other particulate support (herein referred to
as beads) and suspended in solution or spread out on a surface.
[0363] "An addressable combinatorial antibody library" refers to a
collection of antibodies in which member antibodies are
identifiable and all antibodies with the same identifier, such as
position in a spatial array or on a solid support, or a chemical or
RF tag, bind to the same antigen, and generally are substantially
the same in amino acid sequence. For purposes herein, reference to
an "addressable arrayed combinatorial antibody library" means that
the antibody members are addressed in an array.
[0364] "In silico" refers to research and experiments performed
using a computer. In silico methods include, but are not limited
to, molecular modeling studies, biomolecular docking experiments,
and virtual representations of molecular structures and/or
processes, such as molecular interactions. For purposes herein, the
antibody members of a library can be designed using a computer
program that selects component V, D and J germline segments from
among those input into the computer and joins them in-frame to
output a list of nucleic acid molecules for synthesis. Thus, the
recombination of the components of the antibodies in the
collections or libraries provided herein, can be performed in
silico by combining the nucleotide sequences of each building block
in accord with software that contains rules for doing so. The
process could be performed manually without a computer, but the
computer provides the convenience of speed.
[0365] A "database" refers to a collection of data items. For
purposes herein, reference to a database is typically with
reference to antibody databases, which provide a collection of
sequence and structure information for antibody genes and
sequences. Exemplary antibody databases include, but are not
limited to, IMGT.RTM., the international ImMunoGeneTics information
system (imgt.cines.fr; see e.g., Lefranc et al. (2008) Briefings in
Bioinformatics, 9:263-275), National Center for Biotechnology
Information (NCBI), the Kabat database and the Tomlinson's VBase
database (Lefranc (2003) Nucleic Acids Res., 31:307-310; Martin et
al., Bioinformatics Tools for Antibody Engineering in Handbook of
Therapeutic Antibodies, Wiley-VCH (2007), pp. 104-107). A database
also can be created by a user to include any desired sequences. The
database can be created such that the sequences are inputted in a
desired format (e.g., in a particular reading frame; lacking stop
codons; lacking signal sequences). The database also can be created
to include sequences in addition to antibody sequences.
[0366] "Screening" refers to identification or selection of an
antibody or portion thereof from a collection or library of
antibodies and/or portions thereof, based on determination of the
activity or property of an antibody or portion thereof. Screening
can be performed in any of a variety of ways, including, for
example, by assays assessing direct binding (e.g. binding affinity)
of the antibody to a target protein or by functional assays
assessing modulation of an activity of a target protein.
[0367] "Activity towards a target protein" refers to binding
specificity and/or modulation of a functional activity of a target
protein, or other measurements that reflects the activity of an
antibody or portion thereof towards a target protein.
[0368] A "target protein" refers to candidate proteins or peptides
that are specifically recognized by an antibody or portion thereof
and/or whose activity is modulated by an antibody or portion
thereof. A target protein includes any peptide or protein that
contains an epitope for antibody recognition. Target proteins
include proteins involved in the etiology of a disease or disorder
by virtue of expression or activity. Exemplary target proteins are
described herein.
[0369] "Hit" refers to an antibody or portion thereof identified,
recognized or selected as having an activity in a screening
assay.
[0370] "Iterative" with respect to screening means that the
screening is repeated a plurality of times, such as 2, 3, 4, 5 or
more times, until a "Hit" is identified whose activity is optimized
or improved compared to prior iterations.
[0371] "High-throughput" refers to a large-scale method or process
that permits manipulation of large numbers of molecules or
compounds, generally tens to hundred to thousands of compounds. For
example, methods of purification and screening can be rendered
high-throughput. High-throughput methods can be performed manually.
Generally, however, high-throughput methods involve automation,
robotics or software.
[0372] Basic Local Alignment Search Tool (BLAST) is a search
algorithm developed by Altschul et al. (1990) to separately search
protein or DNA databases, for example, based on sequence identity.
For example, blastn is a program that compares a nucleotide query
sequence against a nucleotide sequence database (e.g. GenBank).
BlastP is a program that compares an amino acid query sequence
against a protein sequence database.
[0373] A BLAST bit score is a value calculated from the number of
gaps and substitutions associated with each aligned sequence. The
higher the score, the more significant the alignment.
[0374] A "human protein" refers to a protein encoded by a nucleic
acid molecule, such as DNA, present in the genome of a human,
including all allelic variants and conservative variations thereof.
A variant or modification of a protein is a human protein if the
modification is based on the wildtype or prominent sequence of a
human protein.
[0375] "Naturally occurring amino acids" refer to the 20 L-amino
acids that occur in polypeptides. The residues are those 20
.alpha.-amino acids found in nature which are incorporated into
protein by the specific recognition of the charged tRNA molecule
with its cognate mRNA codon in humans.
[0376] "Non-naturally occurring amino acids" refer to amino acids
that are not genetically encoded. For example, a non-natural amino
acid is an organic compound that has a structure similar to a
natural amino acid but has been modified structurally to mimic the
structure and reactivity of a natural amino acid. Non-naturally
occurring amino acids thus include, for example, amino acids or
analogs of amino acids other than the 20 naturally-occurring amino
acids and include, but are not limited to, the D-isostereomers of
amino acids. Exemplary non-natural amino acids are known to those
of skill in the art.
[0377] "Nucleic acids" include DNA, RNA and analogs thereof,
including peptide nucleic acids (PNA) and mixtures thereof. Nucleic
acids can be single or double-stranded. When referring to probes or
primers, which are optionally labeled, such as with a detectable
label, such as a fluorescent or radiolabel, single-stranded
molecules are contemplated. Such molecules are typically of a
length such that their target is statistically unique or of low
copy number (typically less than 5, generally less than 3) for
probing or priming a library. Generally a probe or primer contains
at least 14, 16 or 30 contiguous nucleotides of sequence
complementary to or identical to a gene of interest. Probes and
primers can be 10, 20, 30, 50, 100 or more nucleic acids long.
[0378] A "peptide" refers to a polypeptide that is from 2 to 40
amino acids in length.
[0379] The amino acids which occur in the various sequences of
amino acids provided herein are identified according to their
known, three-letter or one-letter abbreviations (Table 1). The
nucleotides which occur in the various nucleic acid fragments are
designated with the standard single-letter designations used
routinely in the art.
[0380] An "amino acid" is an organic compound containing an amino
group and a carboxylic acid group. A polypeptide contains two or
more amino acids. For purposes herein, amino acids include the
twenty naturally-occurring amino acids, non-natural amino acids and
amino acid analogs (i.e., amino acids wherein the .alpha.-carbon
has a side chain).
[0381] "Amino acid residue" refers to an amino acid formed upon
chemical digestion (hydrolysis) of a polypeptide at its peptide
linkages. The amino acid residues described herein are presumed to
be in the "L" isomeric form. Residues in the "D" isomeric form,
which are so designated, can be substituted for any L-amino acid
residue as long as the desired functional property is retained by
the polypeptide. NH2 refers to the free amino group present at the
amino terminus of a polypeptide. COOH refers to the free carboxy
group present at the carboxyl terminus of a polypeptide. In keeping
with standard polypeptide nomenclature described in J. Biol. Chem.,
243: 3552-3559 (1969), and adopted 37 C.F.R.
.quadrature..sctn..sctn. 1.821-1.822, abbreviations for amino acid
residues are shown below:
TABLE-US-00002 SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr Tyrosine G
Gly Glycine F Phe Phenylalanine M Met Methionine A Ala Alanine S
Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine V Val
Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine E
Glu Glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg
Arginine D Asp Aspartic acid N Asn Asparagine B Asx Asn and/or Asp
C Cys Cysteine X Xaa Unknown or other
[0382] It should be noted that all amino acid residue sequences
represented herein by formulae have a left to right orientation in
the conventional direction of amino-terminus to carboxyl-terminus.
In addition, the phrase "amino acid residue" is broadly defined to
include the amino acids listed in the Table of Correspondence
(Table 1) and modified and unusual amino acids, such as those
referred to in 37 C.F.R. .sctn..sctn. 1.821-1.822, and incorporated
herein by reference. Furthermore, it should be noted that a dash at
the beginning or end of an amino acid residue sequence indicates a
peptide bond to a further sequence of one or more amino acid
residues, to an amino-terminal group such as NH2 or to a
carboxyl-terminal group such as COOH. The abbreviations for any
protective groups, amino acids and other compounds, are, unless
indicated otherwise, in accord with their common usage, recognized
abbreviations, or the IUPAC-IUB Commission on Biochemical
Nomenclature (see, (1972) Biochem. 11:1726). Each naturally
occurring L-amino acid is identified by the standard three letter
code (or single letter code) or the standard three letter code (or
single letter code) with the prefix "L-"; the prefix "D-" indicates
that the stereoisomeric form of the amino acid is D.
[0383] An "immunoconjugate" refers to an antibody conjugated to one
or more heterologous molecule(s), including but not limited to a
cytotoxic agent. An immunoconjugate may include non-antibody
sequences.
General Techniques
[0384] The present disclosure relies on routine techniques in the
field of recombinant genetics. Basic texts disclosing the general
methods of use in this present disclosure include Sambrook and
Russell, Molecular Cloning: A Laboratory Manual 3d ed. (2001);
Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990);
and Ausubel et al., Current Protocols in Molecular Biology
(1994).
[0385] For nucleic acids, sizes are given in either kilobases (Kb)
or base pairs (bp). These are estimates derived from agarose or
polyacrylamide gel electrophoresis, from sequenced nucleic acids,
or from published DNA sequences. For proteins, sizes are given in
kilo-Daltons (kD) or amino acid residue numbers. Proteins sizes are
estimated from gel electrophoresis, from sequenced proteins, from
derived amino acid sequences, or from published protein
sequences.
[0386] Oligonucleotides that are not commercially available can be
chemically synthesized according to the solid phase phosphoramidite
triester method first described by Beaucage and Caruthers,
Tetrahedron Letters, 22:1859-1862 (1981), using an automated
synthesizer, as described in Van Deventer et al., Nucleic Acids
Res., 12:6159-6168 (1984). Purification of oligonucleotides is by
either native polyacrylamide gel electrophoresis or by
anion-exchange chromatography as described in Pearson &
Reanier, J. Chrom., 255:137-149 (1983). The sequence of the cloned
genes and synthetic oligonucleotides can be verified after cloning
using, e.g., the chain termination method for sequencing
double-stranded templates of Wallace et al., Gene, 16:21-26
(1981).
[0387] The nucleic acids encoding recombinant polypeptides of the
present disclosure may be cloned into an intermediate vector before
transformation into prokaryotic or eukaryotic cells for replication
and/or expression. The intermediate vector may be a prokaryote
vector such as a plasmid or shuttle vector.
Humanized Antibodies with Ultralong CDR3 Sequences
[0388] To date, cattle are the only species where ultralong CDR3
sequences have been identified. However, other species, for example
other ruminants, may also possess antibodies with ultralong CDR3
sequences.
[0389] Exemplary antibody variable region sequences comprising an
ultralong CDR3 sequence identified in cattle include those
designated as: BLV1H12 (see, SEQ ID NO: 22), BLV5B8 (see, SEQ ID
NO: 23), BLV5D3 (see, SEQ ID NO: 24) and BLV8C11 (see, SEQ ID NO:
25) (see, e.g., Saini, et al. (1999) Eur. J. Immunol. 29:
2420-2426; and Saini and Kaushik (2002) Scand. J. Immunol. 55:
140-148); BF4E9 (see, SEQ ID NO: 26) and BF1H1 (see, SEQ ID NO: 27)
(see, e.g., Saini and Kaushik (2002) Scand. J. Immunol. 55:
140-148); and F18 (see, SEQ ID NO: 28) (see, e.g., Berens, et al.
(1997) Int. Immunol. 9: 189-199).
[0390] In an embodiment, bovine antibodies are identified and
humanized. Multiple techniques exist to identify antibodies.
[0391] Antibodies of the present disclosure may be isolated by
screening combinatorial libraries for antibodies with the desired
activity or activities. For example, a variety of methods are known
in the art for generating phage display libraries and screening
such libraries for antibodies possessing the desired binding
characteristics. Such methods are reviewed, e.g., in Hoogenboom et
al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed.,
Human Press, Totowa, N.J., 2001) and further described, e.g., in
the McCafferty et al., Nature 348:552-554; Clackson et al., Nature
352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597
(1992); Marks and Bradbury, in Methods in Molecular Biology
248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et
al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol.
Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci.
USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.
Methods 284(1-2): 119-132 (2004).
[0392] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Phage display libraries of bovine antibodies may be a
source of bovine antibody gene sequences, including ultralong CDR3
sequences.
[0393] Typically, a non-human antibody is humanized to reduce
imrnunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which CDRs (or
portions thereof) are derived from a non-human antibody, and FRs
(or portions thereof) are derived from human antibody sequences. A
humanized antibody optionally will also comprise at least a portion
of a human constant region. In some embodiments, some FR residues
in a humanized antibody are substituted with corresponding residues
from a non-human antibody (e.g., the antibody from which the CDR
residues are derived), e.g., to restore or improve antibody
specificity or affinity.
[0394] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol.
Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua
et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and
Osbourn et al., Methods 36:61-68 (2005); Klimka et al., Br. J.
Cancer, 83:252-260 (2000) (describing the "guided selection"
approach to FR shuffling); and Studnicka et al., U.S. Pat. No.
5,766,886.
[0395] Human variable region framework sequences that may be used
for humanization include but are not limited to: framework
sequences selected using the "best-fit" method (see, e.g., Sims et
al. J. Immunol. 151:2296 (1993)); framework sequences derived from
the consensus sequence of human antibodies of a particular subgroup
of light or heavy chain variable regions (see, e.g., Carter et al.
Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J.
Immunol., 151:2623 (1993)); human mature (somatically mutated)
framework sequences or human germline framework sequences (see,
e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008));
and framework sequences derived from screening FR libraries (see,
e.g., Baca et al., Biol. Chem. 272:10678-10684 (1997) and Rosok et
al., J. Biol. Chem. 271:22611-22618 (1996)).
[0396] Humanized antibodies with ultralong CDR3 sequences may also
include engineered non-antibody sequences, such as cytokines or
growth factors, into the CDR3 region, such that the resultant
humanized antibody is effective, for example, in inhibiting tumor
metastasis. Non-antibody sequences may include an interleukin
sequence, a hormone sequence, a cytokine sequence, a toxin
sequence, a lymphokine sequence, a growth factor sequence, a
chemokine sequence, a toxin sequence, or combinations thereof.
Non-antibody sequences may be human, non-human, or synthetic. In
some embodiments, the cytokine or growth factor may be shown to
have an antiproliferative effect on at least one cell population.
Such cytokines, lymphokines, growth factors, or other hematopoietic
factors include M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,
IL-16, IL-17, IL-18, IFN, TNFo1, TNF1, TNF2, G-CSF, Meg-CSF,
GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.
Additional growth factors for use in humanized antibodies and/or
pharmaceutical compositions of the present disclosure include:
angiogenin, bone morphogenic protein-1, bone morphogenic protein-2,
bone morphogenic protein-3, bone morphogenic protein-4, bone
morphogenic protein-5, bone morphogenic protein-6, bone morphogenic
protein-7, bone morphogenic protein-8, bone morphogenic protein-9,
bone morphogenic protein-10, bone morphogenic protein-11, bone
morphogenic protein-12, bone morphogenic protein-13, bone
morphogenic protein-14, bone morphogenic protein-15, bone
morphogenic protein receptor IA, bone morphogenic protein receptor
IB, brain derived neurotrophic factor, ciliary neutrophic factor,
ciliary neutrophic factor receptor, cytokine-induced neutrophil
chemotactic factor 1, cytokine-induced neutrophil, chemotactic
factor 2, cytokine-induced neutrophil chemotactic factor 2,
endothelial cell growth factor, endothelin 1, epidermal growth
factor, epithelial-derived neutrophil attractant, fibroblast growth
factor 4, fibroblast growth factor 5, fibroblast growth factor 6,
fibroblast growth factor 7, fibroblast growth factor 8, fibroblast
growth factor 8b, fibroblast growth factor 8c, fibroblast growth
factor 9, fibroblast growth factor 10, fibroblast growth factor
acidic, fibroblast growth factor basic, glial cell line-derived
neutrophic factor receptor-1, glial cell line-derived neutrophic
factor receptor-2, growth related protein, growth related
protein-1, growth related protein-2, growth related protein-3,
heparin binding epidermal growth factor, hepatocyte growth factor,
hepatocyte growth factor receptor, insulin-like growth factor I,
insulin-like growth factor receptor, insulin-like growth factor II,
insulin-like growth factor binding protein, keratinocyte growth
factor, leukemia inhibitory factor, leukemia inhibitory factor
receptor-1, nerve growth factor nerve growth factor receptor,
neurotrophin-3, neurotrophin-4, placenta growth factor, placenta
growth factor 2, platelet-derived endothelial cell growth factor,
platelet derived growth factor, platelet derived growth factor A
chain, platelet derived growth factor AA, platelet derived growth
factor AB, platelet derived growth factor B chain, platelet derived
growth factor BB, platelet derived growth factor receptor-1,
platelet derived growth factor receptor-2, pre-B cell growth
stimulating factor, stem cell factor, stem cell factor receptor,
transforming growth factor-1, transforming growth factor-2,
transforming growth factor-1, transforming growth factor-1.2,
transforming growth factor-2, transforming growth factor-3,
transforming growth factor-S, latent transforming growth factor-1,
transforming growth factor-1 binding protein I, transforming growth
factor-1 binding protein II, transforming growth factor-1 binding
protein III, tumor necrosis factor receptor type I, tumor necrosis
factor receptor type II, urokinase-type plasminogen activator
receptor, vascular endothelial growth factor, and chimeric proteins
and biologically or immunologically active fragments thereof.
Exemplary non-antibody sequences include interleukin 8 (IL-8, SEQ
ID NO: 475), interleukin 21 (IL-21, SEQ ID NO: 480),
CXCL12/SDF-1alpha (SEQ ID NO: 479), somatostain (SEQ ID NO: 477),
ProTx-II (SEQ ID NO: 481), chlorotoxin (SEQ ID NO: 478), and
ziconotide (SEQ ID NO: 476).
[0397] A non-human antibody may be humanized to reduce
imrnunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0398] The humanized antibodies comprising an ultralong CDR3 as
disclosed herein are preferably monoclonal. Also encompassed within
the scope of the disclosure are Fab, Fab', Fab'-SH and F(ab').sup.2
fragments of the humanized antibodies comprising an ultralong CDR3
as provided herein. These antibody fragments can be created by
traditional means, such as enzymatic digestion, or may be generated
by recombinant techniques. Such antibody fragments may be chimeric
or humanized. These fragments are useful for the diagnostic and
therapeutic purposes set forth below.
[0399] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, e.g., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
[0400] The humanized antibodies comprising an ultralong CDR3 as
disclosed herein can be made using a hybridoma cell-based method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods.
[0401] Hybridoma cells can be generated by fusing B cells producing
a desired antibody with an immortalized cell line, usually a
myeloma cell line, so that the resulting fusion cells will be an
immortalized cell line that secrets a particular antibody. By the
same principle, myeloma cells can be first transfected with a
nucleic acid encoding a germline antibody V region and can be
screened for the expression of the germline V region. Those myeloma
cells with highest level of proteolytic light chain expression can
be subsequently fused with B cells that produce an antibody with
desired target protein specificity. The fusion cells will produce
two types of antibodies: one is a heterologous antibody containing
an endogenous antibody chain (either heavy or light) operably
joined to the recombinant germline V region (either heavy or
light), and the other is the same antibody that the parental B
cells would secrete (e.g. both endogenous heavy and light chains).
The operably joined heterologous heavy and light chains can be
isolated by conventional methods such as chromatography and
identification can be confirmed by target protein binding assays,
assays identifying a unique tag of the germline polypeptide, or
endopeptidase activity assays described in other sections of this
disclosure. In some cases, where the heterologous antibody is the
predominant type in quantity among the two types of antibodies,
such isolation may not be needed.
[0402] The hybridoma cells may be seeded and grown in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, parental myeloma
cells. For example, if the parental myeloma cells lack the enzyme
hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT),
the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which
substances prevent the growth of HGPRT-deficient cells.
[0403] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, myeloma cell lines may be murine myeloma
lines, such as those derived from MOPC-21 and MPC-11 mouse tumors
available from the Salk Institute Cell Distribution Center, San
Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the
American Type Culture Collection, Rockville, Md. USA. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker,
Inc., New York, 1987)).
[0404] Culture medium in which hybridoma cells are growing is
assayed for production of humanized antibodies comprising an
ultralong CDR3. For example, the binding specificity of monoclonal
antibodies produced by hybridoma cells may be determined by
immunoprecipitation or by an in vitro binding assay, such as an
enzyme-linked immunoadsorbent assay (ELISA).
[0405] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107:220 (1980).
[0406] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0407] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0408] The humanized antibodies comprising an ultralong CDR3 as
disclosed herein may be made by using combinatorial libraries to
screen for synthetic antibody clones with the desired activity or
activities. For example, synthetic antibody clones are selected by
screening phage libraries containing phage that display various
fragments of antibody variable regions (e.g., scFv or Fab) fused to
phage coat protein. Such phage libraries may be panned, for
example, by affinity chromatography against the desired antigen.
Clones expressing antibody fragments capable of binding to the
desired antigen may be adsorbed to the antigen and thus separated
from the non-binding clones in the library. The binding clones may
then be eluted from the antigen, and can be further enriched by
additional cycles of antigen adsorption/elution. Any of the
humanized antibodies comprising an ultralong CDR3 as disclosed
herein may be obtained by designing a suitable antigen screening
procedure to select for the phage clone of interest followed by
construction of a full length antibody comprising an ultralong CDR3
clone using the VH and VL (e.g., from scFv or Fab) sequences from
the phage clone of interest and suitable constant region (Fc)
sequences described in Kabat et al., Sequences of Proteins of
Immunological Interest, Fifth Edition, NIH Publication 91-3242,
Bethesda Md. (1991), vols. 1-3.
[0409] The antigen-binding domain of an antibody is formed from two
variable (V) regions, one each from the light (VL) and heavy (VH)
chains, that both present three hypervariable loops or
complementarity-determining regions (CDRs). Variable domains may be
displayed functionally on phage, either as single-chain Fv (scFv,
also referred to as single-chain antibody (SCA)) fragments, in
which VH and VL are covalently linked through a short, flexible
peptide, or as Fab fragments, in which they are each fused to a
constant domain and interact non-covalently, as described in Winter
et al., Ann. Rev. Immunol., 12: 433-455 (1994). scFv or SCA
encoding phage clones and Fab encoding phage clones may be
separately or collectively referred to as "Fv phage clones" or "Fv
clones".
[0410] Repertoires of VH and VL genes may be separately cloned by
polymerase chain reaction (PCR) and recombined randomly in phage
libraries, which can then be searched for antigen-binding clones as
described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
Libraries from immunized sources provide high-affinity antibodies
to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire may be cloned to
provide a single source of human antibodies to a wide range of
non-self and also self antigens without any immunization as
described by Griffiths et al., EMBO J. 12: 725-734 (1993). Finally,
naive libraries can also be made synthetically by cloning the
unrearranged V-gene segments from stem cells, and using PCR primers
containing random sequence to encode the highly variable CDR3
regions and to accomplish rearrangement in vitro as described by
Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
[0411] Filamentous phage is used to display antibody fragments by
fusion to the minor coat protein pill. Protein pill may include
truncated forms of pill. The antibody fragments can be displayed as
single chain Fv fragments, in which VH and VL domains are connected
on the same polypeptide chain by a flexible polypeptide spacer,
(e.g., as described by Marks et al., J. Mol. Biol., 222: 581-597
(1991)), or as Fab fragments, in which one chain is fused to pill
(e.g., a truncated pill) and the other is secreted into the
bacterial host cell periplasm where assembly of a Fab-coat protein
structure which becomes displayed on the phage surface by
displacing some of the wild type coat proteins, (e.g., as described
in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991)).
[0412] Nucleic acid encoding antibody variable gene segments
(including VH and VL segments) are recovered from the cells of
interest and and may be amplified or copies made by recombinant DNA
techniques (e.g., Kunkel mutagenesis). For example, in the case of
rearranged VH and VL gene libraries, the desired DNA may be
obtained by isolating genomic DNA or mRNA from lymphocytes followed
by polymerase chain reaction (PCR) with primers matching the 5' and
3' ends of rearranged VH and VL genes as described in Orlandi et
al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby
making diverse V gene repertoires for expression. The V genes may
be amplified from cDNA and genomic DNA, with back primers at the 5'
end of the exon encoding the mature V-domain and forward primers
based within the J-segment as described in Orlandi et al. (1989)
and in Ward et al., Nature, 341: 544-546 (1989). For amplifying
from cDNA, back primers can also be based in the leader exon as
described in Jones et al., Biotechnol., 9: 88-89 (1991), and
forward primers within the constant region as described in Sastry
et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To
enhance or maximize complementarity, degeneracy may be incorporated
in the primers as described in Orlandi et al. (1989) or Sastry et
al. (1989). Library diversity may be enhanced or maximized by using
PCR primers targeted to each V-gene family in order to amplify
available VH and VL arrangements present in the immune cell nucleic
acid sample, for example, as described in the method of Marks et
al., J. Mol. Biol., 222: 581-597 (1991) or as described in the
method of Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993).
For cloning of the amplified DNA into expression vectors, rare
restriction may can be introduced within the PCR primer as a tag at
one end as described in Orlandi et al. (1989), or by further PCR
amplification with a tagged primer as described in Clackson et al.,
Nature, 352: 624-628 (1991).
[0413] Repertoires of synthetically rearranged V genes may be
derived in vitro from V gene segments. Most of the human VH-gene
segments have been cloned and sequenced (e.g., reported in
Tomlinson et al., J. Mol. Biol., 227: 776-798 (1992)), and mapped
(e.g., reported in Matsuda et al., Nature Genet., 3: 88-94 (1993);
these cloned segments (including all the major conformations of the
H1 and H2 loop) may be used to generate diverse VH gene repertoires
with PCR primers encoding H3 loops of diverse sequence and length
as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388
(1992). VH repertoires may also be made with all the sequence
diversity focused in a long H3 loop of a single length as described
in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992).
Human V.kappa. and V.lamda.. segments have been cloned and
sequenced (reported in Williams and Winter, Eur. J. Immunol., 23:
1456-1461 (1993)) and can be used to make synthetic light chain
repertoires. Synthetic V gene repertoires, based on a range of VH
and VL folds, and L3 and H3 lengths, will encode antibodies of
considerable structural diversity. Following amplification of
V-gene encoding DNAs, germline V-gene segments can be rearranged in
vitro according to the methods of Hoogenboom and Winter, J. Mol.
Biol., 227: 381-388 (1992).
[0414] Repertoires of antibody fragments may be constructed by
combining VH and VL gene repertoires together in several ways. Each
repertoire may be created in different vectors, and the vectors
recombined in vitro, for example, as described in Hogrefe et al.,
Gene, 128: 119-126 (1993), or in vivo by combinatorial infection,
for example, the loxP system described in Waterhouse et al., Nucl.
Acids Res., 21: 2265-2266 (1993). The in vivo recombination
approach exploits the two-chain nature of Fab fragments to overcome
the limit on library size imposed by E. coli transformation
efficiency. Naive VH and VL repertoires are cloned separately, one
into a phagemid and the other into a phage vector. The two
libraries are then combined by phage infection of
phagemid-containing bacteria so that each cell contains a different
combination and the library size is limited only by the number of
cells present (about 10.sup.12 clones). Both vectors contain in
vivo recombination signals so that the VH and VL genes are
recombined onto a single replicon and are co-packaged into phage
virions. These large libraries may provide large numbers of diverse
antibodies of good affinity (K.sub.d.sup.-1 of about 10.sup.-8
M).
[0415] Alternatively, the repertoires may be cloned sequentially
into the same vector, for example, as described in Barbas et al.,
Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled
together by PCR and then cloned, for example, as described in
Clackson et al., Nature, 352: 624-628 (1991). PCR assembly may also
be used to join VH and VL DNAs with DNA encoding a flexible peptide
spacer to form single chain Fv (scFv) repertoires. In yet another
technique, "in cell PCR assembly" may be used to combine VH and VL
genes within lymphocytes by PCR and then clone repertoires of
linked genes as described in Embleton et al., Nucl. Acids Res., 20:
3831-3837 (1992).
[0416] The antibodies produced by naive libraries (either natural
or synthetic) can be of moderate affinity (K.sub.d.sup.-1 of about
10.sup.6 to 10.sup.7M.sup.-1), but affinity maturation may also be
mimicked in vitro by constructing and reselecting from secondary
libraries as described in Winter et al. (1994), supra. For example,
mutation can be introduced at random in vitro by using error-prone
polymerase (reported in Leung et al., Technique, 1: 11-15 (1989))
in the method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992)
or in the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89:
3576-3580 (1992). Additionally, affinity maturation may be
performed by randomly mutating one or more CDRs, for example, using
PCR with primers carrying random sequence spanning the CDR of
interest, in selected individual Fv clones and screening for higher
affinity clones. WO 9607754 described a method for inducing
mutagenesis in a complementarity determining region of an
immunoglobulin light chain to create a library of light chain
genes. Another effective approach is to recombine the VH or VL
domains selected by phage display with repertoires of naturally
occurring V domain variants obtained from unimmunized donors and
screen for higher affinity in several rounds of chain reshuffling
as described in Marks et al., Biotechnol., 10: 779-783 (1992). This
technique allows the production of antibodies and antibody
fragments with affinities in the 10.sup.-9 M range.
[0417] The phage library samples are contacted with an immobilized
protein under conditions suitable for binding of at least a portion
of the phage particles with the adsorbent. Normally, the
conditions, including pH, ionic strength, temperature and the like
are selected to mimic physiological conditions. The phages bound to
the solid phase are washed and then eluted by acid, e.g., as
described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:
7978-7982 (1991), or by alkali, (e.g., as described in Marks et
al., J. Mol. Biol., 222: 581-597 (1991)), or by antigen
competition, (e.g., in a procedure similar to the antigen
competition method of Clackson et al., Nature, 352: 624-628
(1991)). Phages may be enriched 20-1,000-fold in a single round of
selection. Moreover, the enriched phages may be grown in bacterial
culture and subjected to further rounds of selection.
[0418] The efficiency of selection depends on many factors,
including the kinetics of dissociation during washing, and whether
multiple antibody fragments on a single phage can simultaneously
engage with antigen. Antibodies with fast dissociation kinetics
(and weak binding affinities) may be retained by use of short
washes, multivalent phage display and high coating density of
antigen in solid phase. The high density not only stabilizes the
phage through multivalent interactions, but favors rebinding of
phage that has dissociated. The selection of antibodies with slow
dissociation kinetics (and good binding affinities) may be promoted
by use of long washes and monovalent phage display as described in
Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a
low coating density of antigen as described in Marks et al.,
Biotechnol., 10: 779-783 (1992).
[0419] DNA encoding the hybridoma-derived monoclonal antibodies or
phage display Fv clones disclosed herein is readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide primers designed to specifically amplify the heavy
and light chain coding regions of interest from hybridoma or phage
DNA template). Once isolated, the DNA can be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of the desired monoclonal antibodies in the
recombinant host cells. Recombinant expression in bacteria of
antibody-encoding DNA has been described by Better et al., U.S.
Pat. No. 6,204,023 (see also, e.g., Skerra et al., Curr. Opinion in
Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151
(1992)).
[0420] DNA encoding Fv clones as disclosed herein may be combined
with known DNA sequences encoding heavy chain and/or light chain
constant regions (e.g., the appropriate DNA sequences can be
obtained from Kabat et al., supra) to form clones encoding full or
partial length heavy and/or light chains. It will be appreciated
that constant regions of any isotype can be used for this purpose,
including IgG, IgM, IgA, IgD, and IgE constant regions, and that
such constant regions may be obtained from any human or animal
species. A Fv clone derived from the variable domain DNA of one
animal (such as human) species and then fused to constant region
DNA of another animal species to form coding sequence(s) for
"hybrid", full length heavy chain and/or light chain is included in
the definition of "chimeric" and "hybrid" antibody as used herein.
In a preferred Fv clone embodiment, aFv clone derived from human
variable DNA is fused to human constant region DNA to form coding
sequence(s) for all human, full or partial length heavy and/or
light chains.
[0421] DNA encoding a humanized antibody comprising an ultralong
CDR3 derived from a hybridoma disclosed herein may also be
modified, for example, by substituting the coding sequence for
human heavy- and light-chain constant domains in place of
homologous murine sequences derived from the hybridoma clone (e.g.,
as in the method of Morrison et al., Proc. Natl. Acad. Sci. USA,
81: 6851-6855 (1984)). DNA encoding a hybridoma or Fv clone-derived
antibody or fragment can be further modified by covalently joining
to the immunoglobulin coding sequence all or part of the coding
sequence for a non-immunoglobulin polypeptide. In this manner,
"chimeric" or "hybrid" antibodies are prepared that have the
binding specificity of the Fv clone or hybridoma clone-derived
antibodies disclosed herein.
Antibody Genes and Proteins
[0422] The present disclosure provides antibody genes and proteins
including, for example, humanized antibody genes or proteins that
comprise an ultralong CDR3 sequence and/or a CDR3 scaffold. The
present disclosure additionally provides VH, DH, and JH sequences
useful in the preparation of ultralong CDR3 sequences. Such
sequences may comprise motifs (e.g., cysteine motifs) as described
herein including those as described in the many embodiments
disclosed herein. In some embodiments, the antibodies disclosed
herein may selectively or specifically bind to an epitope of a
target protein. In some embodiments, the antibody may be an
antagonist (e.g., blocking) antibody or an agonist antibody.
[0423] The variable region of the heavy and light chains are
encoded by multiple germline gene segments separated by non-coding
regions, or introns, and often are present on different
chromosomes. For example, the genes for the human immunoglobulin
heavy chain region contains approximately 65 variable (VH) genes,
27 Diversity (DH) genes, and 6 Joining (JH) genes. The human kappa
(.kappa.) and lambda (.lamda.) light chains are also each encoded
by a similar number of VL and JL gene segments, but do not include
any D gene segments. Exemplary VH, DH, JH and VL (V.kappa. or
V.lamda.) and JL (J.kappa. or J.lamda.) germline gene segments are
set forth in WO 2010/054007.
[0424] During B cell differentiation germline DNA is rearranged
whereby one DH and one JH gene segment of the heavy chain locus are
recombined, which is followed by the joining of one VH gene segment
forming a rearranged VDJ gene that encodes a VH chain. The
rearrangement occurs only on a single heavy chain allele by the
process of allelic exclusion. Allelic exclusion is regulated by
in-frame or "productive" recombination of the VDJ segments, which
occurs in only about one-third of VDJ recombinations of the
variable heavy chain. When such productive recombination events
first occur in a cell, this result in production of a .mu. heavy
chain that gets expressed on the surface of a pre-B cell and
transmits a signal to shut off further heavy chain recombination,
thereby preventing expression of the allelic heavy chain locus. The
surface-expressed p heavy chain also acts to activate the kappa
(.kappa.) locus for rearrangement. The lambda (.lamda.) locus is
only activated for rearrangement if the .kappa. recombination is
unproductive on both loci. The light chain rearrangement events are
similar to the heavy chain, except that only the VL and JL segments
are recombined. Before primary transcription of each, the
corresponding constant chain gene is added. Subsequent
transcription and RNA splicing leads to mRNA that is translated
into an intact light chain or heavy chain.
[0425] The variable regions of antibodies confer antigen binding
and specificity due to recombination events of individual germline
V, D and J segments, whereby the resulting recombined nucleic acid
sequences encoding the variable region domains differ among
antibodies and confer antigen-specificity to a particular antibody.
The variation, however, is limited to three complementarity
determining regions (CDR1, CDR2, and CDR3) found within the
N-terminal domain of the heavy (H) and (L) chain variable regions.
The CDRs are interspersed with regions that are more conserved,
termed "framework regions" (FR). The extent of the framework region
and CDRs has been precisely defined (see e.g., Kabat, E. A. et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol.
Biol. 196:901-917). Each VH and VL is typically composed of three
CDRs and four FRs arranged from the amino terminus to carboxy
terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3
and FR4. Sequence variability among VL and VH domains is generally
limited to the CDRs, which are the regions that form the antigen
binding site. For example, for the heavy chain, generally, VH genes
encode the N-terminal three framework regions, the first two
complete CDRs and the first part of the third CDR), the DH gene
encodes the central portion of the third CDR, and the JH gene
encodes the last part of the third CDR and the fourth framework
region. For the light chain, the VL genes encode the first CDR and
second CDR. The third CDR (CDRL3) is formed by the joining of the
VL and JL gene segments. Hence, CDRs 1 and 2 are exclusively
encoded by germline V gene segment sequences. The VH and VL chain
CDR3s form the center of the Ag-binding site, with CDRs 1 and 2
form the outside boundaries; the FRs support the scaffold by
orienting the H and L CDRs. On average, an antigen binding site
typically requires at least four of the CDRs make contact with the
antigen's epitope, with CDR3 of both the heavy and light chain
being the most variable and contributing the most specificity to
antigen binding (see, e.g., Janis Kuby, Immunology, Third Edition,
New York, W.H. Freeman and Company, 1998, pp. 115-118). CDRH3,
which includes all of the D gene segment, is the most diverse
component of the Ab-binding site, and typically plays a critical
role in defining the specificity of the Ab. In addition to sequence
variation, there is variation in the length of the CDRs between the
heavy and light chains.
[0426] The constant regions, on the other hand, are encoded by
sequences that are more conserved among antibodies. These domains
confer functional properties to antibodies, for example, the
ability to interact with cells of the immune system and serum
proteins in order to cause clearance of infectious agents.
Different classes of antibodies, for example IgM, IgD, IgG, IgE and
IgA, have different constant regions, allowing them to serve
distinct effector functions.
[0427] These natural recombination events of V, D, and J, can
provide nearly 2.times.10.sup.7 different antibodies with both high
affinity and specificity. Additional diversity is introduced by
nucleotide insertions and deletions in the joining segments and
also by somatic hypermutation of V regions. The result is that
there are approximately 10.sup.10 antibodies present in an
individual with differing antigen specificities.
[0428] Antibodies include bovine antibody BLVH12 (e.g., heavy chain
variable region set forth in SEQ ID NO: 482, and light chain
variable region set forth in SEQ ID NO: 483); and bovine antibody
BLV5B8 (e.g., heavy chain variable region set forth in SEQ ID NO:
484, and light chain variable region set forth in SEQ ID NO:
485)
Antibody Fragments
[0429] The present disclosure encompasses antibody fragments. In
certain circumstances there are advantages of using antibody
fragments, rather than whole antibodies. The smaller size of the
fragments allows for rapid clearance, and may lead to improved
access to solid tumors. Antibody fragments include, but are not
limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments,
and other fragments described below. For a review of certain
antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003).
For a review of scFv fragments, see, e.g., Pluckthun, in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see
also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab')2 fragments comprising salvage receptor
binding epitope residues and having increased in vivo half-life,
see U.S. Pat. No. 5,869,046.
[0430] Diabodies are antibody fragments with two antigen binding
sites that may be bivalent or bispecific. See, for example, EP
404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003);
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993). Triabodies and tetrabodies are also described in Hudson et
al., Nat. Med. 9: 129134 (2003).
[0431] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516). Antibody fragments can be made by various
techniques, including but not limited to proteolytic digestion of
an intact antibody as well as production by recombinant host cells
(e.g. E. coli or phage), as described herein.
[0432] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments (see, e.g., U.S. Pat. No. 6,204,023).
Antibody fragments can be isolated from antibody phage libraries as
discussed above. Alternatively, Fab'-SH fragments can be directly
recovered from E. coli and chemically coupled to form F(ab').sub.2
fragments (see, e.g., Carter et al., Bio/Technology 10: 163-167
(1992)). According to another approach, F(ab').sub.2 fragments can
be isolated directly from recombinant host cell culture. Fab and
F(ab').sub.2 fragment with increased in vivo half-life comprising a
salvage receptor binding epitope residues (see, e.g., in U.S. Pat.
No. 5,869,046). Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. In other
embodiments, the antibody of choice is a single chain Fv fragment
(scFv or single chain antibody (SCA)). See WO 93/16185; U.S. Pat.
Nos. 5,571,894; and 5,587,458. Fv and sFv are the only species with
intact combining sites that are devoid of constant regions; thus,
they are suitable for reduced nonspecific binding during in vivo
use. sFv fusion proteins may be constructed to yield fusion of an
effector protein at either the amino or the carboxy terminus of an
sFv. See Antibody Engineering, ed. Borrebaeck, Supra. The antibody
fragment may also be a "linear antibody", for example, as described
in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be
monospecific or bispecific.
Humanized Antibodies
[0433] The present disclosure provides humanized antibodies
comprising an ultralong CDR3. Humanized antibodies may include
human engineered antibodies (see, e.g., Studnicka et al. (1994)
Protein Eng. 7(6) 805-814; and U.S. Pat. No. 5,766,886). Various
methods for humanizing non-human antibodies are known in the art.
For example, a humanized antibody can have one or more amino acid
residues introduced into it from a source which is human or
non-human. Humanization may be performed following the method of
Studnicka (see, e.g., Studnicka et al. (1994) Protein Eng. 7(6)
805-814; and U.S. Pat. No. 5,766,886), including the preparation of
modified antibody variable domains. Humanization may alternatively
be performed following the method of Winter and co-workers (Jones
et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature
332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by
substituting hypervariable region sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized" or
"human engineered" antibodies are chimeric antibodies, including
wherein substantially less than an intact human variable domain has
been substituted by or incorporated into the corresponding sequence
from a non-human species. For example, humanized antibodies may be
human antibodies in which some hypervariable region residues and
possibly some FR residues are substituted by residues from
analogous sites in rodent antibodies. Alternatively, humanized or
human engineered antibodies may be non-human (e.g, rodent)
antibodies in which some residues are substituted by residues from
analogious sites in human antibodies (see, e.g., Studnicka et al.
(1994) Protein Eng. 7(6) 805-814; and U.S. Pat. No. 5,766,886).
[0434] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is important to
reduce antigenicity. For example, to the so-called "best-fit"
method, the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework for the humanized
antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al.
(1987) J. Mol. Biol. 196:901). Another method uses a particular
framework derived from the consensus sequence of all human
antibodies of a particular subgroup of light or heavy chains. The
same framework may be used for several different humanized
antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA,
89:4285; Presta et al. (1993) J. Immunol., 151:2623).
[0435] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to one
method, humanized antibodies are prepared by a process of analysis
of the parental sequences and various conceptual humanized products
using three-dimensional models of the parental and humanized
sequences. Three-dimensional immunoglobulin models are commonly
available and are familiar to those skilled in the art. Computer
programs are available which illustrate and display probable
three-dimensional conformational structures of selected candidate
immunoglobulin sequences. Inspection of these displays permits
analysis of the likely role of the residues in the functioning of
the candidate immunoglobulin sequence, e.g., the analysis of
residues that influence the ability of the candidate immunoglobulin
to bind its antigen. In this way, FR residues can be selected and
combined from the recipient and import sequences so that the
desired antibody characteristic, such as increased affinity for the
target antigen(s), is achieved. In general, the hypervariable
region residues are directly and most substantially involved in
influencing antigen binding.
[0436] In some embodiments, the humanized antibodies comprising an
ultralong CDR3 may be deimmunized. Methods of deimmunizing an
antibody or protein are well known in the art. The immunogenicity
of therapeutic proteins such as antibodies is thought to result
from the presence of T-cell epitopes which can bind MHC class II
molecules and generate a proliferative and cytokine response in
CD4+ helper T-cells. These CD4+ helper cells then collaborate with
B-cells to generate an antibody response against the therapeutic
protein. Removal of the T-cell epitopes are thought to be key steps
in deimmunizing a recombinant protein. T-cell epitopes can be
predicted by in silico algorithms that identify residues required
for binding MHC. Alternatively, epitopes can be identified directly
by utilizing peripheral blood mononuclear cells from panels of
human donors and measuring their response against the therapeutic
protein when incubated with antigen presenting cells. Such in
silico and in vitro systems are well known in the art [Jones T D,
Crompton L J, Carr F J, Baker M P. Methods Mol Biol. 2009;
525:405-23, Deimmunization of monoclonal antibodies; and Baker M,
and Jones T D. The identification and removal of immunogenicity in
therapeutic proteins. Curr. Opin. Drug Discovery Dev. 2007; (2007);
10(2): 219-227]. When peptides are identified that bind MHC II or
otherwise stimulate CD4+ cell activation, the residues of the
peptide can be mutated one by one and tested for T-cell activation
until a mutation is found which disrupts MHC II binding and T-cell
activation. Such mutations, when found in an individual peptide,
can be encoded directly in the recombinant therapeutic protein.
Incubation of the whole protein with antigen presenting cells will
not induce a significant CD4+ response, indicating successful
deimmunization.
Bispecific Antibodies
[0437] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. For example, one of the binding
specificities may be for a first antigen and the other may be for
any other antigen. Exemplary bispecific antibodies may bind to two
different epitopes of the same protein. Bispecific antibodies may
also be used to localize cytotoxic agents to cells which express a
particular protein. These antibodies possess a binding arm specific
for the particular protein and an arm which binds the cytotoxic
agent (e.g., saporin, anti-interferon-.alpha., vinca alkaloid,
ricin A chain, methotrexate or radioactive isotope hapten).
Bispecific antibodies may be prepared as full length antibodies or
antibody fragments (e.g., F(ab').sub.2 bispecific antibodies).
[0438] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy chain-light chain pairs, where the two heavy chains have
different specificities (Milstein and Cuello, Nature, 305: 537
(1983)). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of 10 different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule, which is usually done by affinity chromatography steps,
is rather cumbersome, and the product yields are low. Similar
procedures are disclosed in WO 93/08829, and in Traunecker et al.,
EMBO J., 10: 3655 (1991).
[0439] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first heavy-chain constant
region (CH1), containing the site necessary for light chain
binding, present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for flexibility in adjusting the mutual proportions of the
three polypeptide fragments in embodiments when unequal ratios of
the three polypeptide chains used in the construction provide the
optimum yields. It is, however, possible to insert the coding
sequences for two or all three polypeptide chains in one expression
vector when the expression of at least two polypeptide chains in
equal ratios results in high yields or when the ratios are not of
particular significance.
[0440] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. This asymmetric structure may
facilitate the separation of the desired bispecific compound from
unwanted immunoglobulin chain combinations, as the presence of an
immunoglobulin light chain in only one half of the bispecific
molecule provides for a facile way of separation. This approach is
disclosed in WO 94/04690. For further details of generating
bispecific antibodies see, for example, Suresh et al., Methods in
Enzymology, 121:210 (1986).
[0441] According to another approach, the interface between a pair
of antibody molecules may can be engineered to maximize the
percentage of heterodimers which are recovered from recombinant
cell culture. The preferred interface comprises at least a part of
the CH3 domain of an antibody constant domain. In this method, one
or more small amino acid side chains from the interface of the
first antibody molecule are replaced with larger side chains (e.g.,
tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g., alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0442] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate may be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/00373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0443] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies may be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced may be used as agents for the
selective immobilization of enzymes.
[0444] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
HER2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0445] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. See, e.g., Kostelny et al., J.
Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from
the Fos and Jun proteins were linked to the Fab' portions of two
different antibodies by gene fusion. The antibody homodimers were
reduced at the hinge region to form monomers and then re-oxidized
to form the antibody heterodimers. This method can also be utilized
for the production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (VH) connected to a light-chain
variable domain (VL) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
VH and VL domains of one fragment are forced to pair with the
complementary VL and VH domains of another fragment, thereby
forming two antigen-binding sites. Another strategy for making
bispecific antibody fragments by the use of single-chain Fv (sFv)
dimers has also been reported. See, Gruber et al., J. Immunol.,
152:5368 (1994).
[0446] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. See, e.g.,
Tutt et al. J. Immunol. 147: 60 (1991).
Multivalent Antibodies
[0447] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The antibodies of the
present disclosure may be multivalent antibodies (which are other
than of the IgM class) with three or more antigen binding sites
(e.g., tetravalent antibodies), which may be produced by
recombinant expression of nucleic acid encoding the polypeptide
chains of the antibody. The multivalent antibody may comprise a
dimerization domain and three or more antigen binding sites. A
preferred dimerization domain may comprise (or consist of) an Fc
region or a hinge region. In this scenario, the antibody will
comprise an Fc region and three or more antigen binding sites
amino-terminal to the Fe region. A preferred multivalent antibody
may comprise (or consist of) three to about eight, but preferably
four, antigen binding sites. The multivalent antibody comprises at
least one polypeptide chain (and preferably two polypeptide
chains), wherein the polypeptide chain(s) comprise two or more
variable domains. For instance, the polypeptide chain(s) may
comprise VD1-(X1)n-VD2-(X.sub.2)n-Fc, wherein VD1 is a first
variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino
acid or polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region
chain; or VH-CH1-VH-CH1-Fc region chain. A multivalent antibody may
preferably further comprises at least two (and preferably four)
light chain variable domain polypeptides. A multivalent antibody
may, for instance, comprise from about two to about eight light
chain variable domain polypeptides. The light chain variable domain
polypeptides may comprise a light chain variable domain and,
optionally, further comprise a CL domain.
Antibody Variants
[0448] In some embodiments, amino acid sequence modification(s) of
the humanized antibodies comprising an ultralong CDR3 as described
herein are contemplated. For example, it may be desirable to
improve the binding affinity and/or other biological properties of
the antibody. Amino acid sequence variants of the antibody are
prepared by introducing appropriate nucleotide changes into the
antibody nucleic acid, or by peptide synthesis. Such modifications
include, for example, deletions from, and/or insertions into and/or
substitutions of, residues within the amino acid sequences of the
antibody. Any combination of deletion, insertion, and substitution
is made to arrive at the final construct, provided that the final
construct possesses the desired characteristics. The amino acid
alterations may be introduced in the subject antibody amino acid
sequence at the time that sequence is made.
[0449] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
immunoglobulins are screened for the desired activity.
[0450] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody to an
enzyme (e.g., for ADEPT) or a polypeptide which increases the serum
half-life of the antibody.
[0451] Glycosylation of polypeptides is typically either N-linked
or O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0452] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0453] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. For example, antibodies with a
mature carbohydrate structure that lacks fucose attached to an Fc
region of the antibody have been described (see, e.g., US
2003/0157108, US 2004/0093621. Antibodies with a bisecting
N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc
region of the antibody have been described (see, e.g., WO
2003/011878, and U.S. Pat. No. 6,602,684). Antibodies with at least
one galactose residue in the oligosaccharide attached to an Fc
region of the antibody WO 1997/30087; see, also, WO 1998/58964 and
WO 1999/22764 concerning antibodies with altered carbohydrate
attached to the Fc region thereof). Antigen-binding molecules with
modified glycosylation have been described (see, e.g., WO 99/54342,
U.S. Pat. Nos. 6,602,684 and 7,517,670, and US 2004/0072290; see
also, e.g., U.S. Pat. Nos. 7,214,775 and 7,682,610).
[0454] The preferred glycosylation variant herein comprises an Fc
region, wherein a carbohydrate structure attached to the Fc region
lacks fucose. Such variants have improved ADCC function.
Optionally, the Fc region further comprises one or more amino acid
substitutions therein which further improve ADCC, for example,
substitutions at positions 298, 333, and/or 334 of the Fc region
(Eu numbering of residues). Examples of publications related to
"defucosylated" or "fucose-deficient" antibodies include: US
2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614 (now
U.S. Pat. No. 6,946,292) US 2002/0164328 (now U.S. Pat. No.
7,064,191); US 2004/0093621; US 2004/0132140; US 2004/0110704; US
2004/0110282 (now U.S. Pat. No. 7,749,753); US 2004/0109865; WO
2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778;
WO2005/053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004);
Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of
cell lines producing defucosylated antibodies include Lec13 CHO
cells deficient in protein fucosylation (Ripka et al. Arch.
Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US
2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,
especially at Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells
(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).
[0455] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by a different residue. The sites of
greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR alterations are also contemplated.
Conservative substitutions are shown in Table 2 under the heading
of "preferred substitutions". If such substitutions result in a
change in biological activity, then more substantial changes,
denominated "exemplary substitutions", or as further described
below in reference to amino acid classes, may be introduced and the
products screened.
TABLE-US-00003 Original Preferred Residue Exemplary Substitutions
Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys
Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)
Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala
Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe;
Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys
(K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu;
Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val;
Ser Ser Trp (V\/) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val
(V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0456] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties: (1)
hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral
hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: asp, glu; (4)
basic: his, lys, arg; (5) residues that influence chain
orientation: gly, pro; and (6) aromatic: trp, tyr, phe.
[0457] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0458] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g., a
humanized or human antibody). Generally, the resulting variant(s)
selected for further development will have improved biological
properties relative to the parent antibody from which they are
generated. A convenient way for generating such substitutional
variants involves affinity maturation using phage display. Briefly,
several hypervariable region sites (e.g., 6-7 sites) are mutated to
generate all possible amino acid substitutions at each site. The
antibodies thus generated are displayed from filamentous phage
particles as fusions to the gene III product of M13 packaged within
each particle. The phage-displayed variants are then screened for
their biological activity (e.g., binding affinity) as herein
disclosed. In order to identify candidate hypervariable region
sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or additionally,
it may be beneficial to analyze a crystal structure of the
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues are candidates for substitution according to the
techniques elaborated herein. Once such variants are generated, the
panel of variants is subjected to screening as described herein and
antibodies with superior properties in one or more relevant assays
may be selected for further development.
[0459] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
[0460] It may be desirable to introduce one or more amino acid
modifications in an Fc region of the immunoglobulin polypeptides
disclosed herein, thereby generating a Fc region variant. The Fc
region variant may comprise a human Fc region sequence (e.g., a
human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g., a substitution) at one or more amino acid
positions including that of a hinge cysteine.
[0461] In accordance with this description and the teachings of the
art, it is contemplated that in some embodiments, an antibody used
in methods disclosed herein may comprise one or more alterations as
compared to the wild type counterpart antibody, e.g., in the Fc
region. These antibodies would nonetheless retain substantially the
same characteristics required for therapeutic utility as compared
to their wild type counterpart. For example, it is thought that
certain alterations can be made in the Fc region that would result
in altered (e.g., either improved or diminished) Clq binding and/or
Complement Dependent Cytotoxicity (CDC), e.g., as described in
WO99/51642. See also Duncan & Winter Nature 322:738-40 (1988);
U.S. Pat. Nos. 5,648,260; 5,624,821; and WO94/29351 concerning
other examples of Fc region variants. WO00/42072 and WO 2004/056312
describe antibody variants with improved or diminished binding to
FcRs. See, also, Shields et al. J. Biol. Chem. 9(2): 6591-6604
(2001). Antibodies with increased half lives and improved binding
to the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934 (Hinton et al.). These antibodies
comprise an Fc reg on with one or more substitutions therein which
improve binding of the Fc region to FcRn. Polypeptide variants with
altered Fc region amino acid sequences and increased or decreased
Clq binding capability are described in U.S. Pat. No. 6,194,551,
WO99/51642. See, also, Idusogie et al. J. Immunol. 164:4178-4184
(2000).
[0462] In certain embodiments, the present disclosure contemplates
an antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting
examples of in vitro assays to assess ADCC activity of a molecule
of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.
Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063
(1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA
82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., Exp.
Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays
methods may be employed (see, for example, ACTITM non-radioactive
cytotoxicity assay for flow cytometry (CellTecl1r1ology, Inc.
Mountain View, Calif.; and CytoTox 96.RTM. non-radioactive
cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells
for such assays include peripheral blood mononuclear cells (PBMC)
and Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. Proc.
Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also
be carried out to confirm that the antibody is unable to bind Clq
and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA
in WO 2006/029879 and WO 2005/100402. To assess complement
activation, a CDC assay may be performed (see, for example,
Gazzano-Santoro et al., Immunol. Methods 202:163 (1996); Cragg, M.
S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S, and M. J.
Glennie, Blood 103:27382743 (2004)). FcRn binding and in vivo
clearance/half life determinations can also be performed using
methods known in the art (see, e.g., Petkova, S. B. et al., Int'l
Immunol. 18(12):1759-1769 (2006)).
[0463] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0464] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., Biol. Chem. 9(2): 6591-6604
(2001).)
[0465] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0466] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) Clq
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. Immunol. 164: 41784184 (2000).
[0467] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., Immunol.
117:587 (1976) and Kim et al., Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
[0468] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning other
examples of Fc region variants.
Antibody Derivatives
[0469] The humanized antibodies comprising an ultralong CDR3 as
disclosed herein may be further modified to contain additional
nonproteinaceous moieties that are known in the art and readily
available. Preferably, the moieties suitable for derivatization of
the antibody are water soluble polymers. Non-limiting examples of
water soluble polymers include, but are not limited to,
polyethylene glycol (PEG), copolymers of ethylene glycol/propylene
glycol, carboxymethylcellulose, dextran, polyvinyl alcohol,
polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymers are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
Vectors, Host Cells and Recombinant Methods
[0470] For recombinant production of an antibody or fragment
thereof as disclosed herein, the nucleic acid encoding it is
isolated and inserted into a replicable vector for further cloning
(amplification of the DNA) or for expression. DNA encoding the
antibody is readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the heavy and light
chains of the antibody). In an exemplary embodiment, nucleic acid
encoding a humanized antibody comprising an ultralong CDR3, a
variable region comprising an ultralong CDR3, or an ultralong CDR3,
is isolated and inserted into a replicable vector for further
cloning (amplification of the DNA) or for expression. Many vectors
are available. The choice of vector depends in part on the host
cell to be used. Generally, preferred host cells are of either
prokaryotic or eukaryotic (generally mammalian) origin. It will be
appreciated that constant regions of any isotype can be used for
this purpose, including IgG, IgM, IgA, IgD, and IgE constant
regions, and that such constant regions can be obtained from any
human or animal species.
[0471] Expression vectors containing regulatory elements from
eukaryotic viruses are typically used in eukaryotic expression
vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors
derived from Epstein-Barr virus. Other exemplary eukaryotic vectors
include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE,
and any other vector allowing expression of proteins under the
direction of the CMV promoter, SV40 early promoter, SV40 later
promoter, metallothionein promoter, murine mammary tumor virus
promoter, Rous sarcoma virus promoter, polyhedrin promoter, or
other promoters shown effective for expression in eukaryotic
cells.
[0472] Some expression systems have markers that provide gene
amplification such as thymidine kinase and dihydrofolate reductase.
Alternatively, high yield expression systems not involving gene
amplification are also suitable, such as using a baculovirus vector
in insect cells, with a nucleic acid sequence encoding a partially
human ultralong CDR3 antibody chain under the direction of the
polyhedrin promoter or other strong baculovirus promoters.
[0473] a. Generating Antibodies Using Prokaryotic or Eukaryotic
Host Cells:
[0474] i. Vector Construction
[0475] Polynucleotide sequences encoding polypeptide components of
the antibodies disclosed herein can be obtained using standard
recombinant techniques. Desired polynucleotide sequences may be
isolated and sequenced from antibody producing cells such as
hybridoma cells. Alternatively, polynucleotides can be synthesized
using nucleotide synthesizer or PCR techniques. Once obtained,
sequences encoding the polypeptides are inserted into a recombinant
vector capable of replicating and expressing heterologous
polynucleotides in prokaryotic hosts. Many vectors that are
available and known in the art can be used for the purpose of the
present disclosure. Selection of an appropriate vector will depend
mainly on the size of the nucleic acids to be inserted into the
vector and the particular host cell to be transformed with the
vector. Each vector contains various components, depending on its
function (amplification or expression of heterologous
polynucleotide, or both) and its compatibility with the particular
host cell in which it resides. The vector components generally
include, but are not limited to: an origin of replication, a
selection marker gene, a promoter, a ribosome binding site (RBS), a
signal sequence, the heterologous nucleic acid insert and a
transcription termination sequence. Additionally, V regions
comprising an ultralong CDR3 may optionally be fused to a C-region
to produce an antibody comprising constant regions.
[0476] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species. pBR322 contains genes encoding
ampicillin (Amp) and tetracycline (Tet) resistance and thus
provides easy means for identifying transformed cells. pBR322, its
derivatives, or other microbial plasmids or bacteriophage may also
contain, or be modified to contain, promoters which can be used by
the microbial organism for expression of endogenous proteins.
Examples of pBR322 derivatives used for expression of particular
antibodies have been described (see, e.g., U.S. Pat. No.
5,648,237).
[0477] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, bacteriophage such as .lamda.GEM.TM.-11 may be utilized in
making a recombinant vector which can be used to transform
susceptible host cells such as E. coli LE392.
[0478] The expression vectors disclosed herein may comprise two or
more promoter-cistron pairs, encoding each of the polypeptide
components. A promoter is an untranslated regulatory sequence
located upstream (5') to a cistron that modulates its expression.
Prokaryotic promoters typically fall into two classes, inducible
and constitutive. Inducible promoter is a promoter that initiates
increased levels of transcription of the cistron under its control
in response to changes in the culture condition, e.g., the presence
or absence of a nutrient or a change in temperature.
[0479] A large number of promoters recognized by a variety of
potential host cells are well known. The selected promoter can be
operably linked to cistron DNA encoding the light or heavy chain by
removing the promoter from the source DNA via restriction enzyme
digestion and inserting the isolated promoter sequence into the
vector disclosed herein. Both the native promoter sequence and many
heterologous promoters may be used to direct amplification and/or
expression of the target genes. In some embodiments, heterologous
promoters are utilized, as they generally permit greater
transcription and higher yields of expressed target gene as
compared to the native target polypeptide promoter.
[0480] Promoters suitable for use with prokaryotic hosts include:
an ara B promoter, a PhoA promoter, .beta.-galactamase and lactose
promoter systems, a tryptophan (trp) promoter system and hybrid
promoters such as the tac or the trc promoter. However, other
promoters that are functional in bacteria (such as other known
bacterial or phage promoters) are suitable as well. Their
nucleotide sequences have been published, thereby enabling a
skilled worker operably to ligate them to cistrons encoding the
target light and heavy chains (e.g., Siebenlist et al. (1980) Cell
20: 269) using linkers or adaptors to supply any required
restriction sites.
[0481] Suitable bacterial promoters are well known in the art and
fully described in scientific literature such as Sambrook and
Russell, supra, and Ausubel et al, supra. Bacterial expression
systems for expressing antibody chains of the recombinant catalytic
polypeptide are available in, e.g., E. coli, Bacillus sp., and
Salmonella (Palva et al., Gene, 22:229-235 (1983); Mosbach et al.,
Nature, 302:543-545 (1983)).
[0482] In one aspect disclosed herein, each cistron within the
recombinant vector comprises a secretion signal sequence component
that directs translocation of the expressed polypeptides across a
membrane. In general, the signal sequence may be a component of the
vector, or it may be a part of the target polypeptide DNA that is
inserted into the vector. The signal sequence should be one that is
recognized and processed (e.g., cleaved by a signal peptidase) by
the host cell. For prokaryotic host cells that do not recognize and
process the signal sequences native to the heterologous
polypeptides, the signal sequence is substituted by a prokaryotic
signal sequence selected, for example PelB, OmpA, alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II
(STII) leaders, LamB, PhoE, and MBP. In one embodiment disclosed
herein, the signal sequences used in both cistrons of the
expression system are STII signal sequences or variants
thereof.
[0483] In another aspect, the production of the immunoglobulins
according to the disclosure can occur in the cytoplasm of the host
cell, and therefore does not require the presence of secretion
signal sequences within each cistron. In that regard,
immunoglobulin light and heavy chains are expressed, folded and
assembled to form functional immunoglobulins within the cytoplasm.
Certain host strains (e.g., the E. coli trxB-strains) provide
cytoplasm conditions that are favorable for disulfide bond
formation, thereby permitting proper folding and assembly of
expressed protein subunits (see e.g., Proba and Pluckthun Gene,
159:203 (1995)).
[0484] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. In one embodiment, the host cell is eukaryotic,
e.g. a Chinese Hamster Ovary (CHO) cell, Human Embryonic Kidney
(HEK) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell). For
example, antibodies may be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed. For
expression of antibody fragments and polypeptides in bacteria, see,
e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also
Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,
Humana Press, Totowa, N.J., 2003), pp. 245-254, describing
expression of antibody fragments in E. coli.) After expression, the
antibody may be isolated from the bacterial cell paste in a soluble
fraction and can be further purified. In addition to prokaryotes,
eukaryotic microbes such as filamentous fungi or yeast are suitable
cloning or expression hosts for antibody-encoding vectors,
including fungi and yeast strains whose glycosylation pathways have
been "humanized," resulting in the production of an antibody with a
partially or fully human glycosylation pattern. See Gemgross, Nat.
Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech.
24:210-215 (2006). Suitable host cells for the expression of
glycosylated antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells. These
examples are illustrative rather than limiting. Methods for
constructing derivatives of any of the above-mentioned bacteria
having defined genotypes are known in the art and described in, for
example, Bass et al., Proteins, 8:309-314 (1990). It is generally
necessary to select the appropriate bacteria taking into
consideration replicability of the replicon in the cells of a
bacterium. For example, E. coli, Serratia, or Salmonella species
can be suitably used as the host when well known plasmids such as
pBR322, pBR325, pACYC177, or pKN410 are used to supply the
replicon. Typically the host cell should secrete minimal amounts of
proteolytic enzymes, and additional protease inhibitors may
desirably be incorporated in the cell culture.
[0485] Plant cell cultures can also be utilized as hosts. See, e.g.
U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125, 978, and
6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants). Vertebrate cells may also be used
as hosts. For example, mammalian cell lines that are adapted to
grow in suspension may be useful. Other examples of useful
mammalian host cell lines are monkey kidney CV1 line transformed by
SV40 (COS-7); human embryonic kidney line (293 or 293 cells as
described, e.g., in Graham et al., Gen VII'0l. 36:59 (1977)); baby
hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as
described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1); African green monkey kidney cells (V
ERO-76); human cervical carcinoma cells (HELA); canine kidney cells
(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138);
human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TR1
cells, as described, e.g., in Mather et al., Annals NI'. Acad. Sci.
383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful
mammalian host cell lines include Chinese hamster ovary (CHO)
cells, including DHFR' CHO cells (Urlaub et al., Proc. Natl. Acad.
Sci. USA 77:4216 (1980)); and myeloma cell lines such as YO, NSO
and Sp2/0. For a review of certain mammalian host cell lines
suitable for antibody production, see, e.g., Yazaki and Wu, Methods
in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,
Totowa, N.].), pp. 255-268 (2003).
[0486] In one such embodiment, a host cell comprises (e.g., has
been transformed with): (1) a vector comprising a nucleic acid that
encodes an amino acid sequence comprising the VL of the antibody
and an amino acid sequence comprising the VH of the antibody, or
(2) a first vector comprising a nucleic acid that encodes an amino
acid sequence comprising the VL of the antibody and a second vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VH of the antibody.
[0487] ii. Antibody Production
[0488] For recombinant production of a partially human ultralong
CDR3 antibody, nucleic acid encoding a humanized antibody
comprising an ultralong CDR3 is inserted into one or more
expression vectors for further cloning and/or expression in a host
cell. Such nucleic acid may be readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of the antibody). Host cells are transformed with such
expression vectors and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0489] Transformation means introducing DNA into the prokaryotic
host so that the DNA is replicable, either as an extrachromosomal
element or by chromosomal integrant. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is
electroporation.
[0490] Prokaryotic cells used to produce the polypeptides disclosed
herein are grown in media known in the art and suitable for culture
of the selected host cells. Examples of suitable media include
luria broth (LB) plus necessary nutrient supplements. In some
embodiments, the media also contains a selection agent, chosen
based on the construction of the expression vector, to selectively
permit growth of prokaryotic cells containing the expression
vector. For example, ampicillin is added to media for growth of
cells expressing ampicillin resistant gene.
[0491] Any necessary supplements besides carbon, nitrogen, and
inorganic phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source. Optionally
the culture medium may contain one or more reducing agents selected
from the group consisting of glutathione, cysteine, cystamine,
thioglycollate, dithioerythritol and dithiothreitol.
[0492] The prokaryotic host cells are cultured at suitable
temperatures. For E. coli growth, for example, the preferred
temperature ranges from about 20.degree. C. to about 39.degree. C.,
more preferably from about 25.degree. C. to about 37.degree. C.,
even more preferably at about 30.degree. C. The pH of the medium
may be any pH ranging from about 5 to about 9, depending mainly on
the host organism. For E. coli, the pH is preferably from about 6.8
to about 7.4, and more preferably about 7.0.
[0493] If an inducible promoter is used in the expression vector
disclosed herein, protein expression is induced under conditions
suitable for the activation of the promoter. For example, an ara B
or phoA promoter may be used for controlling transcription of the
polypeptides. A variety of inducers may be used, according to the
vector construct employed, as is known in the art.
[0494] The expressed polypeptides of the present disclosure are
secreted into and recovered from the periplasm of the host cells or
transported into the culture media. Protein recovery from the
periplasm typically involves disrupting the microorganism,
generally by such means as osmotic shock, sonication or lysis. Once
cells are disrupted, cell debris or whole cells may be removed by
centrifugation or filtration. The proteins may be further purified,
for example, by affinity resin chromatography. Alternatively,
proteins that are transported into the culture media may be
isolated therein. Cells may be removed from the culture and the
culture supernatant being filtered and concentrated for further
purification of the proteins produced. The expressed polypeptides
can be further isolated and identified using commonly known methods
such as polyacrylamide gel electrophoresis (PAGE) and Western blot
assay.
[0495] Antibody production may be conducted in large quantity by a
fermentation process. Various large-scale fed-batch fermentation
procedures are available for production of recombinant proteins.
Large-scale fermentations have at least 1000 liters of capacity,
preferably about 1,000 to 100,000 liters of capacity. These
fermentors use agitator impellers to distribute oxygen and
nutrients, especially glucose (a preferred carbon/energy source).
Small scale fermentation refers generally to fermentation in a
fermentor that is no more than approximately 100 liters in
volumetric capacity, and can range from about 1 liter to about 100
liters.
[0496] In a fermentation process, induction of protein expression
is typically initiated after the cells have been grown under
suitable conditions to a desired density, e.g., an OD550 of about
180-220, at which stage the cells are in the early stationary
phase. A variety of inducers may be used, according to the vector
construct employed, as is known in the art and described above.
Cells may be grown for shorter periods prior to induction. Cells
are usually induced for about 12-50 hours, although longer or
shorter induction time may be used.
[0497] To improve the production yield and quality of the
polypeptides disclosed herein, various fermentation conditions can
be modified. For example, to improve the proper assembly and
folding of the secreted antibody polypeptides, additional vectors
overexpressing chaperone proteins, such as Dsb proteins (DsbA,
DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl
cis,trans-isomerase with chaperone activity) may be used to
co-transform the host prokaryotic cells. The chaperone proteins
have been demonstrated to facilitate the proper folding and
solubility of heterologous proteins produced in bacterial host
cells. (see e.g., Chen et al. (1999) J Bio Chem 274:19601-19605;
U.S. Pat. Nos. 6,083,715; 6,027,888; Bothmann and Pluckthun (2000)
J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol.
Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol.
39:199-210).
[0498] To minimize proteolysis of expressed heterologous proteins
(especially those that are proteolytically sensitive), certain host
strains deficient for proteolytic enzymes can be used for the
present disclosure. For example, host cell strains may be modified
to effect genetic mutation(s) in the genes encoding known bacterial
proteases such as Protease III, OmpT, DegP, Tsp, Protease I,
Protease Mi, Protease V, Protease VI and combinations thereof. Some
E. coli protease-deficient strains are available (see, e.g., Joly
et al. (1998), supra; U.S. Pat. Nos. 5,264,365; 5,508,192; Hara et
al., Microbial Drug Resistance, 2:63-72 (1996)).
[0499] E. coli strains deficient for proteolytic enzymes and
transformed with plasmids overexpressing one or more chaperone
proteins may be used as host cells in the expression systems
disclosed herein.
[0500] iii. Antibody Purification
[0501] Standard protein purification methods known in the art can
be employed. The following procedures are exemplary of suitable
purification procedures: fractionation on immunoaffinity or
ion-exchange columns, ethanol precipitation, reverse phase HPLC,
chromatography on silica or on a cation-exchange resin such as
DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation,
and gel filtration using, for example, Sephadex G-75.
[0502] In one aspect, Protein A immobilized on a solid phase is
used for immunoaffinity purification of the full length antibody
products disclosed herein. Protein A is a 41 kD cell wall protein
from Staphylococcus aureus which binds with a high affinity to the
Fc region of antibodies (see, e.g., Lindmark et al (1983) J.
Immunol. Meth. 62:1-13). The solid phase to which Protein A is
immobilized is preferably a column comprising a glass or silica
surface, more preferably a controlled pore glass column or a
silicic acid column. In some applications, the column has been
coated with a reagent, such as glycerol, in an attempt to prevent
nonspecific adherence of contaminants.
[0503] As the first step of purification, the preparation derived
from the cell culture as described above is applied onto the
Protein A immobilized solid phase to allow specific binding of the
antibody of interest to Protein A. The solid phase is then washed
to remove contaminants non-specifically bound to the solid phase.
Finally the antibody of interest is recovered from the solid phase
by elution.
[0504] b. Generating Antibodies Using Eukaryotic Host Cells:
[0505] The vector components generally include, but are not limited
to, one or more of the following: a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence.
[0506] (i) Signal Sequence Component
[0507] A vector for use in a eukaryotic host cell may also contain
a signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide of
interest. The heterologous signal sequence selected preferably is
one that is recognized and processed (e.g., cleaved by a signal
peptidase) by the host cell. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available.
[0508] The DNA for such precursor region is ligated in reading
frame to DNA encoding the antibody.
[0509] (ii) Origin of Replication
[0510] Generally, an origin of replication component is not needed
for mammalian expression vectors. For example, the SV40 origin may
be used only because it contains the early promoter.
[0511] (iii) Selection Gene Component
[0512] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, where relevant, or (c) supply
critical nutrients not available from complex media.
[0513] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0514] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the antibody nucleic acid, such as DHFR, thymidine
kinase, metallothionein-I and -II, preferably primate
metallothionein genes, adenosine deaminase, ornithine
decarboxylase, etc.
[0515] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell when wild-type DHFR is
employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity (e.g., ATCC CRL-9096).
[0516] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody, wild-type DHFR protein, and another
selectable marker such as aminoglycoside 3'-phosphotransferase
(APH) can be selected by cell growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See
U.S. Pat. No. 4,965,199.
[0517] (iv) Promoter Component
[0518] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the antibody polypeptide nucleic acid. Promoter sequences are known
for eukaryotes. Virtually alleukaryotic genes have an AT-rich
region located approximately 25 to 30 bases upstream from the site
where transcription is initiated. Another sequence found 70 to 80
bases upstream from the start of transcription of many genes is a
CNCAAT region where N may be any nucleotide. At the 3' end of most
eukaryotic genes is an AATAAA sequence that may be the signal for
addition of the poly A tail to the 3' end of the coding sequence.
All of these sequences are suitably inserted into eukaryotic
expression vectors.
[0519] Antibody polypeptide transcription from vectors in mammalian
host cells is controlled, for example, by promoters obtained from
the genomes of viruses such as polyoma virus, fowlpox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, from
heat-shock promoters, provided such promoters are compatible with
the host cell systems.
[0520] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. Alternatively, the Rous
Sarcoma Virus long terminal repeat can be used as the promoter.
[0521] (v) Enhancer Element Component
[0522] Transcription of DNA encoding the antibody polypeptide of
this disclosure by higher eukaryotes is often increased by
inserting an enhancer sequence into the vector. Many enhancer
sequences are now known from mammalian genes (globin, elastase,
albumin, .alpha.-fetoprotein, and insulin). An enhancer from a
eukaryotic cell virus may also be used. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the antibody
polypeptide-encoding sequence, but is preferably located at a site
5' from the promoter.
[0523] (vi) Transcription Termination Component
[0524] Expression vectors used in eukaryotic host cells will
typically also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are
commonly available from the 5' and, occasionally 3', untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding an antibody. One useful
transcription termination component is the bovine growth hormone
polyadenylation region. See WO94/11026 and the expression vector
disclosed therein.
[0525] (vii) Selection and Transformation of Host Cells
[0526] Suitable host cells for cloning or expressing the DNA in the
vectors herein include higher eukaryote cells described herein,
including vertebrate host cells. Propagation of vertebrate cells in
culture (tissue culture) has become a routine procedure. Examples
of useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO,
Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse
sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980); monkey
kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells
(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA,
ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat
liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC
CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor
(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y.
Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human
hepatoma line (Hep G2).
[0527] Any of the well-known procedures for introducing foreign
nucleotide sequences into host cells may be used. These include the
use of calcium phosphate transfection, polybrene, protoplast
fusion, electroporation, biolistics, liposomes, microinjection,
plasma vectors, viral vectors and any of the other well known
methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or
other foreign genetic material into a host cell (see, e.g.,
Sambrook and Russell, supra). It is only necessary that the
particular genetic engineering procedure used be capable of
successfully introducing at least both genes into the host cell
capable of expressing germline antibody polypeptide.
[0528] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0529] (viii) Culturing the Host Cells
[0530] The host cells used to produce an antibody of this
disclosure may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium ((DMEM), Sigma) are suitable for culturing the host cells.
In addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Reissue 30,985 may be used
as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0531] (ix) Purification of Antibody
[0532] When using recombinant techniques, the antibody can be
produced intracellularly, or directly secreted into the medium. If
the antibody is produced intracellularly, as a first step, the
particulate debris, either host cells or lysed fragments, are
removed, for example, by centrifugation or ultrafiltration. Where
the antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon.RTM. ultrafiltration unit. A
protease inhibitor such as PMSF may be included in any of the
foregoing steps to inhibit proteolysis and antibiotics may be
included to prevent the growth of adventitious contaminants.
[0533] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (cuss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM.
resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0534] Soluble forms of antibody or fragment present either in the
cytoplasm or released from the periplasmic space may be further
purified using methods known in the art, for example Fab fragments
are released from the bacterial periplasmic space by osmotic shock
techniques.
[0535] If inclusion bodies comprising an antibody or fragment have
formed, they can often bind to the inner and/or outer cellular
membranes and thus will be found primarily in the pellet material
after centrifugation. The pellet material can then be treated at pH
extremes or with chaotropic agent such as a detergent, guanidine,
guanidine derivatives, urea, or urea derivatives in the presence of
a reducing agent such as dithiothreitol at alkaline pH or tris
carboxyethyl phosphine at acid pH to release, break apart, and
solubilize the inclusion bodies. The soluble antibody or fragment
can then be analyzed using gel electrophoresis, immunoprecipitation
or the like. If it is desired to isolate a solublized antibody or
antigen binding fragment isolation may be accomplished using
standard methods such as those set forth below and in Marston et
al. (Meth. Enz., 182:264-275 (1990)).
[0536] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25 M salt).
[0537] In some cases, an antibody or fragment may not be
biologically active upon isolation. Various methods for "refolding"
or converting a polypeptide to its tertiary structure and
generating disulfide linkages, can be used to restore biological
activity. Such methods include exposing the solubilized polypeptide
to a pH usually above 7 and in the presence of a particular
concentration of a chaotrope. The selection of chaotrope is very
similar to the choices used for inclusion body solubilization, but
usually the chaotrope is used at a lower concentration and is not
necessarily the same as chaotropes used for the solubilization. In
most cases the refolding/oxidation solution will also contain a
reducing agent or the reducing agent plus its oxidized form in a
specific ratio to generate a particular redox potential allowing
for disulfide shuffling to occur in the formation of the protein's
cysteine bridge(s). Some of the commonly used redox couples include
cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric
chloride, dithiothreitol (DTT)/dithiane DTT, and
2-mercaptoethanol(bME)/di-thio-b(ME). In many instances, a
cosolvent may be used to increase the efficiency of the refolding,
and common reagents used for this purpose include glycerol,
polyethylene glycol of various molecular weights, arginine and the
like.
Immunoconjugates
[0538] The disclosure also provides immunoconjugates
(interchangeably termed "antibody-drug conjugates" or "ADC"),
comprising any of the humanized antibodies comprising an ultralong
CDR3 as described herein conjugated to a cytotoxic agent such as a
chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin
(e.g., an enzymatically active toxin of bacterial, fungal, plant,
or animal origin, or fragments thereof), or a radioactive isotope
(e.g., a radioconjugate).
[0539] The use of antibody-drug conjugates for the local delivery
of cytotoxic or cytostatic agents. For example, drugs to kill or
inhibit tumor cells in the treatment of cancer (Syrigos and
Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and
Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No.
4,975,278) allows targeted delivery of the drug moiety to tumors,
and intracellular accumulation therein, where systemic
administration of these unconjugated drug agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated (Baldwin et al., (1986) Lancet
pp. (Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal
Antibodies '84: Biological And Clinical Applications, A. Pinchera
et al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity
is sought thereby. Both polyclonal antibodies and monoclonal
antibodies have been reported as useful in these strategies
(Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87).
Drugs used in these methods include daunomycin, doxorubicin,
methotrexate, and vindesine (Rowland et al., (1986) Supra). Toxins
used in antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer
Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.
Chem. Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate
Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996)
Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode
et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.
53:3336-3342). The toxins may effect their cytotoxic and cytostatic
effects by mechanisms including tubulin binding, DNA binding, or
topoisomerase inhibition. Some cytotoxic drugs tend to be inactive
or less active when conjugated to large antibodies or protein
receptor ligands.
[0540] ZEVALIN.RTM. (ibritumomab tiuxetan, Biogen/Idec) is an
antibody-radioisotope conjugate composed of a murine IgG1 kappa
monoclonal antibody directed against the CD20 antigen found on the
surface of normal and malignant B lymphocytes and .sup.111In or
.sup.90Y radioisotope bound by a thiourea linker-chelator (Wiseman
et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al
(2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol.
20(10):2453-63; Witzig et al (2002) J. Clin. Oncol.
20(15):3262-69). Although ZEVALIN has activity against B-cell
non-Hodgkin's Lymphoma (NHL), administration results in severe and
prolonged cytopenias in most patients. MYLOTARG.TM. (gemtuzumab
ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate
composed of a hu CD33 antibody linked to calicheamicin, was
approved in 2000 for the treatment of acute myeloid leukemia by
injection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos.
4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116;
5,767,285; 5,773,001). Cantuzumab mertansine (Immunogen, Inc.), an
antibody drug conjugate composed of the huC242 antibody linked via
the disulfide linker SPP to the maytansinoid drug moiety, DM1, is
advancing into Phase II trials for the treatment of cancers that
express CanAg, such as colon, pancreatic, gastric, and others.
MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an
antibody drug conjugate composed of the anti-prostate specific
membrane antigen (PSMA) monoclonal antibody linked to the
maytansinoid drug moiety, DM1, is under development for the
potential treatment of prostate tumors. The auristatin peptides,
auristatin E (AE) and monomethylauristatin (MMAE), synthetic
analogs of dolastatin, were conjugated to chimeric monoclonal
antibodies cBR96 (specific to Lewis Y on carcinomas) and cAC10
(specific to CD30 on hematological malignancies) (Doronina et al
(2003) Nature Biotechnology 21(7):778-784) and are under
therapeutic development.
[0541] Chemotherapeutic agents useful in the generation of
immunoconjugates are described herein. Enzymatically active toxins
and fragments thereof that can be used include diphtheria A chain,
nonbinding active fragments of diphtheria toxin, exotoxin A chain
(from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
Momordica charantia inhibitor, curcin, crotin, Sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. See, e.g., WO 93/21232
published Oct. 28, 1993. A variety of radionuclides are available
for the production of radioconjugated antibodies. Examples include
.sup.212Bi, .sup.131I, .sup.131In, .sup.90Y, and .sup.186Re.
Conjugates of the antibody and cytotoxic agent are made using a
variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin may be prepared as described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0542] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, dolastatins,
aurostatins, a trichothecene, and CC1065, and the derivatives of
these toxins that have toxin activity, are also contemplated
herein.
[0543] a. Maytansine and Maytansinoids
[0544] In some embodiments, the immunoconjugate comprises a
humanized antibody (full length or fragments) comprising an
ultralong CDR3 as disclosed herein conjugated to one or more
maytansinoid molecules.
[0545] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.
[0546] Maytansinoid drug moieties are attractive drug moieties in
antibody drug conjugates because they are: (i) relatively
accessible to prepare by fermentation or chemical modification,
derivatization of fermentation products, (ii) amenable to
derivatization with functional groups suitable for conjugation
through the non-disulfide linkers to antibodies, (iii) stable in
plasma, and (iv) effective against a variety of tumor cell
lines.
[0547] Immunoconjugates containing maytansinoids, methods of making
same, and their therapeutic use are disclosed, for example, in U.S.
Pat. Nos. 5,208,020, 5,416,064 and EP 0 425 235. Liu et al., Proc.
Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates
comprising a maytansinoid designated DM1 linked to the monoclonal
antibody C242 directed against human colorectal cancer. The
conjugate was found to be highly cytotoxic towards cultured colon
cancer cells, and showed antitumor activity in an in vivo tumor
growth assay. Chari et al., Cancer Research 52:127-131 (1992)
describe immunoconjugates in which a maytansinoid was conjugated
via a disulfide linker to the murine antibody A7 binding to an
antigen on human colon cancer cell lines, or to another murine
monoclonal antibody TA.1 that binds the HER-2/neu oncogene. The
cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro
on the human breast cancer cell line SK-BR-3, which expresses
3.times.10.sup.5 HER-2 surface antigens per cell. The drug
conjugate achieved a degree of cytotoxicity similar to the free
maytansinoid drug, which could be increased by increasing the
number of maytansinoid molecules per antibody molecule. The
A7-maytansinoid conjugate showed low systemic cytotoxicity in
mice.
[0548] Antibody-maytansinoid conjugates are prepared by chemically
linking an antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. See, e.g., U.S. Pat. No.
5,208,020. An average of 3-4 maytansinoid molecules conjugated per
antibody molecule has shown efficacy in enhancing cytotoxicity of
target cells without negatively affecting the function or
solubility of the antibody, although even one molecule of
toxin/antibody would be expected to enhance cytotoxicity over the
use of naked antibody. Maytansinoids are well known in the art and
can be synthesized by known techniques or isolated from natural
sources. Suitable maytansinoids are disclosed, for example, in U.S.
Pat. No. 5,208,020 and in the other patents and nonpatent
publications referred to hereinabove. Preferred maytansinoids are
maytansinol and maytansinol analogues modified in the aromatic ring
or at other positions of the maytansinol molecule, such as various
maytansinol esters.
[0549] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. Nos. 5,208,020, 6,441,163, or EP Patent 0
425 235, Chari et al., Cancer Research 52:127-131 (1992).
Antibody-maytansinoid conjugates comprising the linker component
SMCC may be prepared. The linking groups include disulfide groups,
thioether groups, acid labile groups, photolabile groups, peptidase
labile groups, or esterase labile groups, as disclosed in the
above-identified patents, disulfide and thioether groups being
preferred. Additional linking groups are described and exemplified
herein.
[0550] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 (1978)) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0551] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
[0552] b. Auristatins and Dolastatins
[0553] In some embodiments, the immunoconjugate comprises an
antibody disclosed herein conjugated to dolastatins or dolostatin
peptidic analogs and derivatives, the auristatins (U.S. Pat. Nos.
5,635,483; 5,780,588). Dolastatins and auristatins have been shown
to interfere with microtubule dynamics, GTP hydrolysis, and nuclear
and cellular division (Woyke et al (2001) Antimicrob. Agents and
Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No.
5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob.
Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug
moiety may be attached to the antibody through the N (amino)
terminus or the C (carboxyl) terminus of the peptidic drug moiety
(WO 02/088172).
[0554] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, (see, e.g.,
U.S. Pat. No. 7,498,298).
[0555] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see, e.g., E.
Schroder and K. Lubke, "The Peptides", volume 1, pp 76-136, 1965,
Academic Press) that is well known in the field of peptide
chemistry. The auristatin/dolastatin drug moieties may be prepared
according to the methods of: U.S. Pat. Nos. 5,635,483; 5,780,588;
Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al.
(1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al.
Synthesis, 1996, 719-725; and Pettit et al. (1996) J. Chem. Soc.
Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol
21(7):778-784; U.S. Pat. No. 7,498,289, (disclosing, linkers and
methods of preparing monomethylvaline compounds such as MMAE and
MMAF conjugated to linkers).
[0556] c. Calicheamicin
[0557] In other embodiments, the immunoconjugate comprises an
antibody disclosed herein conjugated to one or more calicheamicin
molecules. The calicheamicin family of antibiotics are capable of
producing double-stranded DNA breaks at sub-picomolar
concentrations. For the preparation of conjugates of the
calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,
5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296.
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma..sub.1', .alpha..sub.2',
.alpha..sub.3', N-acetyl-.gamma..sub.1', PSAG and .theta.'.sub.1
(see, e.g., Hinman et al., Cancer Research 53:3336-3342 (1993),
Lode et al., Cancer Research 58:2925-2928 (1998) and the
aforementioned U.S. patents). Another anti-tumor drug that the
antibody can be conjugated is QFA which is an antifolate. Both
calicheamicin and QFA have intracellular sites of action and do not
readily cross the plasma membrane. Therefore, cellular uptake of
these agents through antibody mediated internalization greatly
enhances their cytotoxic effects.
[0558] d. Other Cytotoxic Agents
[0559] Other antitumor agents that can be conjugated to the
antibodies disclosed herein include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known
collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0560] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor,
curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0561] The present disclosure further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0562] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
antibodies. Examples include At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu. When the conjugate is
used for detection, it may comprise a radioactive atom for
scintigraphic studies, for example tc.sup.99m or I.sup.123, or a
spin label for nuclear magnetic resonance (NMR) imaging (also known
as magnetic resonance imaging, mri), such as iodine-123 again,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
[0563] The radiolabels or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods.
[0564] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat.
No. 5,208,020) may be used.
[0565] The compounds disclosed herein expressly contemplate, but
are not limited to, ADC prepared with cross-linker reagents: BMPS,
EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB,
SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,
U.S.A.). See pages 467-498, 2003-2004 Applications Handbook and
Catalog.
[0566] e. Preparation of Antibody Drug Conjugates
[0567] In the antibody drug conjugates (ADC) disclosed herein, an
antibody (Ab) is conjugated to one or more drug moieties (D), e.g.,
about 1 to about 20 drug moieties per antibody, through a linker
(L). An ADC of Formula I [Ab-(L-D).sub.p] may be prepared by
several routes, employing organic chemistry reactions, conditions,
and reagents known to those skilled in the art, including: (1)
reaction of a nucleophilic group of an antibody with a bivalent
linker reagent, to form Ab-L, via a covalent bond, followed by
reaction with a drug moiety D; and (2) reaction of a nucleophilic
group of a drug moiety with a bivalent linker reagent, to form D-L,
via a covalent bond, followed by reaction with the nucleophilic
group of an antibody. Additional methods for preparing ADC are
described herein.
[0568] The linker may be composed of one or more linker components.
Exemplary linker components include 6-maleimidocaproyl ("MC"),
maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"),
alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl
("PAB"), N-Succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"),
N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate
("SMCC"), and N-Succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB").
Additional linker components are known in the art and some are
disclosed herein (see, e.g., U.S. Pat. No. 7,498,298).
[0569] In some embodiments, the linker may comprise amino acid
residues. Exemplary amino acid linker components include a
dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
Exemplary dipeptides include: valine-citrulline (vc or val-cit),
alanine-phenylalanine (af or ala-phe). Exemplary tripeptides
include: glycine-valine-citrulline (gly-val-cit) and
glycine-glycine-glycine (gly-gly-gly). Amino acid residues which
comprise an amino acid linker component include those occurring
naturally, as well as minor amino acids and non-naturally occurring
amino acid analogs, such as citrulline. Amino acid linker
components can be designed and optimized in their selectivity for
enzymatic cleavage by a particular enzymes, for example, a
tumor-associated protease, cathepsin B, C and D, or a plasmin
protease.
[0570] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g., lysine, (iii) side chain thiol groups, e.g.,
cysteine, and (iv) sugar hydroxyl or amino groups where the
antibody is glycosylated. Amine, thiol, and hydroxyl groups are
nucleophilic and capable of reacting to form covalent bonds with
electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups. Certain antibodies have reducible interchain
disulfides, e.g., cysteine bridges. Antibodies may be made reactive
for conjugation with linker reagents by treatment with a reducing
agent such as DTT (dithiothreitol). Each cysteine bridge will thus
form, theoretically, two reactive thiol nucleophiles. Additional
nucleophilic groups can be introduced into antibodies through the
reaction of lysines with 2-iminothiolane (Traut's reagent)
resulting in conversion of an amine into a thiol. Reactive thiol
groups may be introduced into the antibody (or fragment thereof) by
introducing one, two, three, four, or more cysteine residues (e.g.,
preparing mutant antibodies comprising one or more non-native
cysteine amino acid residues).
[0571] Antibody drug conjugates disclosed herein may also be
produced by modification of the antibody to introduce electrophilic
moieties, which can react with nucleophilic substituents on the
linker reagent or drug. The sugars of glycosylated antibodies may
be oxidized, e.g., with periodate oxidizing reagents, to form
aldehyde or ketone groups which may react with the amine group of
linker reagents or drug moieties. The resulting imine Schiff base
groups may form a stable linkage, or may be reduced, e.g., by
borohydride reagents to form stable amine linkages. In one
embodiment, reaction of the carbohydrate portion of a glycosylated
antibody with either glactose oxidase or sodium meta-periodate may
yield carbonyl (aldehyde and ketone) groups in the protein that can
react with appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment, proteins containing N-terminal
serine or threonine residues can react with sodium meta-periodate,
resulting in production of an aldehyde in place of the first amino
acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146;
U.S. Pat. No. 5,362,852). Such aldehyde can be reacted with a drug
moiety or linker nucleophile.
[0572] Likewise, nucleophilic groups on a drug moiety include, but
are not limited to: amine, thiol, hydroxyl, hydrazide, oxime,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide groups capable of reacting to form covalent bonds
with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups.
[0573] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g., by recombinant techniques or
peptide synthesis. The length of DNA may comprise respective
regions encoding the two portions of the conjugate either adjacent
one another or separated by a region encoding a linker peptide
which does not destroy the desired properties of the conjugate.
[0574] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
Engineered Hybridomas
[0575] Hybridoma cells can be generated by fusing B cells producing
a desired antibody with an immortalized cell line, usually a
myeloma cell line, so that the resulting fusion cells will be an
immortalized cell line that secrets a particular antibody. By the
same principle, myeloma cells can be first transfected with a
nucleic acid encoding a germline antibody V region and can be
screened for the expression of the germline V region. Those myeloma
cells with highest level of proteolytic light chain expression can
be subsequently fused with B cells that produce an antibody with
desired target protein specificity. The fusion cells will produce
two types of antibodies: one is a heterologous antibody containing
an endogenous antibody chain (either heavy or light) operably
joined to the recombinant germline V region (either heavy or
light), and the other is the same antibody that the parental B
cells would secrete (e.g. both endogenous heavy and light chains).
The operably joined heterologous heavy and light chains can be
isolated by conventional methods such as chromatography and
identification can be confirmed by target protein binding assays,
assays identifying a unique tag of the germline polypeptide, or
endopeptidase activity assays described in other sections of this
disclosure. In some cases, where the heterologous antibody is the
predominant type in quantity among the two types of antibodies,
such isolation may not be needed. Hybridomas. Including bovine
hybridomas, may be a source of bovine antibody gene sequences,
including ultralong CDR3 sequences.
Transgenic Mammals
[0576] A nucleic acid sequence encoding a germline antibody
polypeptide of the present disclosure can be introduced into a
non-human mammal to generate a transgenic animal that expresses the
germline antibody polypeptide. Unlike the transgenic animal models
more commonly seen, the transgene expressed by the transgenic
mammals of the present disclosure need not replace at least one
allele of the endogenous coding sequence responsible for the
variable regions of antibody chains following somatic
recombination. Due to allelic exclusion, the presence of an
exogenous, post-somatic rearrangement version of the germline V
region DNA will inhibit the endogenous alleles of pre-somatic
rearrangement V minigenes from undergoing somatic rearrangement and
contributing to the makeup of antibody chains this mammal may
produce. Thus, when exposed to a particular antigen, the mammal
will generate heterologous antibodies comprising one endogenously
rearranged antibody chain, and one transgenic gene which was
rearranged a priori. Such heterologous antibodies are invaluable in
research and in treating certain conditions in live subjects. On
the other hand, a method that directs the integration of the
transgene to the locus of an endogenous allele will fully serve the
purpose of practicing the present disclosure as well.
[0577] The general methods of generating transgenic animals have
been well established and frequently practiced. For reviews and
protocols for generating transgenic animals and related methods for
genetic manipulations, see, e.g., Mansour et al., Nature
336:348-352 (1988); Capecchi et al., Trends Genet. 5:70-76 (1989);
Capecchi, Science 244:1288-1292 (1989); Capecchi et al., Current
Communications in Molecular Biology, pp 45-52, Capecchi, M. R.
(ed.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989);
Frohman et al., Cell 56: 145-147 (1989); Brinster et al., Proc.
Natl. Acad. Sci. USA 82:4438-4442 (1985); Evans et. al., Nature
292:154-156 (1981); Bradley et al., Nature 309:255-258 (1984);
Gossler et al., Proc. Natl. Acad. Sci. USA 83:9065-9069 (1986);
Robertson et al., Nature 322:445-448 (1986); Jaenisch Science
240:1468-1474 (1988); and Siedel, G. E., Jr., "Critical review of
embryo transfer procedures with cattle" in Fertilization and
Embryonic Development in Vitro, page 323, L. Mastroianni, Jr. and
J. D. Biggers, ed., Plenum Press, New York, N.Y. (1981).
[0578] An exemplary transgenic animal of the present disclosure is
mouse, whereas a number of other transgenic animals can also be
produced using the same general method. These animals include, but
are not limited to: rabbits, sheep, cattle, and pigs (Jaenisch
Science 240:1468-1474 (1988); Hammer et al., J. Animal. Sci. 63:269
(1986); Hammer et al. Nature 315:680 (1985); Wagner et al.,
Theriogenology 21:29 (1984)).
Pharmaceutical Compositions
[0579] Humanized antibodies comprising an ultralong CDR3, antibody
fragments, nucleic acids, or vectors disclosed herein can be
formulated in compositions, especially pharmaceutical compositions.
Such compositions with humanized antibodies comprising an ultralong
CDR3 comprise a therapeutically or prophylactically effective
amount of a humanized antibodies comprising an ultralong CDR3,
antibody fragment, nucleic acid, or vector disclosed herein in
admixture with a suitable carrier, e.g., a pharmaceutically
acceptable agent. Typically, humanized antibodies comprising an
ultralong CDR3, antibody fragments, nucleic acids, or vectors
disclosed herein are sufficiently purified for administration
before formulation in a pharmaceutical composition.
[0580] Pharmaceutically acceptable agents for use in the present
pharmaceutical compositions include carriers, excipients, diluents,
antioxidants, preservatives, coloring, flavoring and diluting
agents, emulsifying agents, suspending agents, solvents, fillers,
bulking agents, buffers, delivery vehicles, tonicity agents,
cosolvents, wetting agents, complexing agents, buffering agents,
antimicrobials, and surfactants.
[0581] Neutral buffered saline or saline mixed with serum albumin
are exemplary appropriate carriers. The pharmaceutical compositions
may include antioxidants such as ascorbic acid; low molecular
weight polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
Tween, pluronics, or polyethylene glycol (PEG). Also by way of
example, suitable tonicity enhancing agents include alkali metal
halides (preferably sodium or potassium chloride), mannitol,
sorbitol, and the like. Suitable preservatives include benzalkonium
chloride, thimerosal, phenethyl alcohol, methylparaben,
propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen
peroxide also may be used as preservative. Suitable cosolvents
include glycerin, propylene glycol, and PEG. Suitable complexing
agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or
hydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting
agents include sorbitan esters, polysorbates such as polysorbate
80, tromethamine, lecithin, cholesterol, tyloxapal, and the like.
The buffers may be conventional buffers such as acetate, borate,
citrate, phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be
about pH 4-5.5, and Tris buffer can be about pH 7-8.5. Additional
pharmaceutical agents are set forth in Remington's Pharmaceutical
Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing
Company, 1990.
[0582] The composition may be in liquid form or in a lyophilized or
freeze-dried form and may include one or more lyoprotectants,
excipients, surfactants, high molecular weight structural additives
and/or bulking agents (see, for example, U.S. Pat. Nos. 6,685,940,
6,566,329, and 6,372,716). In one embodiment, a lyoprotectant is
included, which is a non-reducing sugar such as sucrose, lactose or
trehalose. The amount of lyoprotectant generally included is such
that, upon reconstitution, the resulting formulation will be
isotonic, although hypertonic or slightly hypotonic formulations
also may be suitable. In addition, the amount of lyoprotectant
should be sufficient to prevent an unacceptable amount of
degradation and/or aggregation of the protein upon lyophilization.
Exemplary lyoprotectant concentrations for sugars (e.g., sucrose,
lactose, trehalose) in the pre-lyophilized formulation are from
about 10 mM to about 400 mM. In another embodiment, a surfactant is
included, such as for example, nonionic surfactants and ionic
surfactants such as polysorbates (e.g., polysorbate 20, polysorbate
80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol) phenyl
ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodium laurel
sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or
stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or
stearyl-sarcosine; linoleyl, myristyl-, or cetyl-betaine;
lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,
myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine
(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl ofeyl-taurate; and the MONAQUAT.TM.. series (Mona
Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.,
Pluronics, PF68 etc). Exemplary amounts of surfactant that may be
present in the pre-lyophilized formulation are from about
0.001-0.5%. High molecular weight structural additives (e.g.,
fillers, binders) may include for example, acacia, albumin, alginic
acid, calcium phosphate (dibasic), cellulose,
carboxymethylcellulose, carboxymethylcellulose sodium,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,
dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium
sulfate, amylose, glycine, bentonite, maltose, sorbitol,
ethylcellulose, disodium hydrogen phosphate, disodium phosphate,
disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar
gum, liquid glucose, compressible sugar, magnesium aluminum
silicate, maltodextrin, polyethylene oxide, polymethacrylates,
povidone, sodium alginate, tragacanth microcrystalline cellulose,
starch, and zein. Exemplary concentrations of high molecular weight
structural additives are from 0.1% to 10% by weight. In other
embodiments, a bulking agent (e.g., mannitol, glycine) may be
included.
[0583] Compositions may be suitable for parenteral administration.
Exemplary compositions are suitable for injection or infusion into
an animal by any route available to the skilled worker, such as
intraarticular, subcutaneous, intravenous, intramuscular,
intraperitoneal, intracerebral (intraparenchymal),
intracerebroventricular, intramuscular, intraocular, intraarterial,
or intralesional routes. A parenteral formulation typically will be
a sterile, pyrogen-free, isotonic aqueous solution, optionally
containing pharmaceutically acceptable preservatives.
[0584] Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringers' dextrose,
dextrose and sodium chloride, lactated Ringers, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers, such as those based on Ringer's dextrose,
and the like. Preservatives and other additives may also be
present, such as, for example, anti-microbials, anti-oxidants,
chelating agents, inert gases and the like. See generally,
Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.
[0585] Pharmaceutical compositions described herein may be
formulated for controlled or sustained delivery in a manner that
provides local concentration of the product (e.g., bolus, depot
effect) and/or increased stability or half-life in a particular
local environment. The compositions can include the formulation of
humanized antibodies comprising an ultralong CDR3, antibody
fragments, nucleic acids, or vectors disclosed herein with
particulate preparations of polymeric compounds such as polylactic
acid, polyglycolic acid, etc., as well as agents such as a
biodegradable matrix, injectable microspheres, microcapsular
particles, microcapsules, bioerodible particles beads, liposomes,
and implantable delivery devices that provide for the controlled or
sustained release of the active agent which then can be delivered
as a depot injection. Techniques for formulating such sustained- or
controlled-delivery means are known and a variety of polymers have
been developed and used for the controlled release and delivery of
drugs. Such polymers are typically biodegradable and biocompatible.
Polymer hydrogels, including those formed by complexation of
enantiomeric polymer or polypeptide segments, and hydrogels with
temperature or pH sensitive properties, may be desirable for
providing drug depot effect because of the mild and aqueous
conditions involved in trapping bioactive protein agents (e.g.,
humanized antibodies comprising an ultralong CDR3). See, for
example, the description of controlled release porous polymeric
microparticles for the delivery of pharmaceutical compositions in
WO 93/15722.
[0586] Suitable materials for this purpose include polylactides
(see, e.g., U.S. Pat. No. 3,773,919), polymers of
poly-(a-hydroxycarboxylic acids), such as
poly-D-(-)-3-hydroxybutyric acid (EP 133,988A), copolymers of
L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,
Biopolymers, 22: 547-556 (1983)), poly(2-hydroxyethyl-methacrylate)
(Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981), and
Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate, or
poly-D(-)-3-hydroxybutyric acid. Other biodegradable polymers
include poly(lactones), poly(acetals), poly(orthoesters), and
poly(orthocarbonates). Sustained-release compositions also may
include liposomes, which can be prepared by any of several methods
known in the art (see, e.g., Eppstein et al., Proc. Natl. Acad.
Sci. USA, 82: 3688-92 (1985)). The carrier itself, or its
degradation products, should be nontoxic in the target tissue and
should not further aggravate the condition. This can be determined
by routine screening in animal models of the target disorder or, if
such models are unavailable, in normal animals.
[0587] Microencapsulation of recombinant proteins for sustained
release has been performed successfully with human growth hormone
(rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson
et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther.,
27:1221-1223 (1993); Hora et al., Bio/Technology. 8:755-758 (1990);
Cleland, "Design and Production of Single Immunization Vaccines
Using Polylactide Polyglycolide Microsphere Systems," in Vaccine
Design: The Subunit and Adjuvant Approach, Powell and Newman, eds,
(Plenum Press: New York, 1995), pp. 439-462; WO 97/03692, WO
96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010. The
sustained-release formulations of these proteins were developed
using poly-lactic-coglycolic acid (PLGA) polymer due to its
biocompatibility and wide range of biodegradable properties. The
degradation products of PLGA, lactic and glycolic acids can be
cleared quickly within the human body. Moreover, the degradability
of this polymer can be depending on its molecular weight and
composition. Lewis, "Controlled release of bioactive agents from
lactide/glycolide polymer," in: M. Chasin and R. Langer (Eds.),
Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New
York, 1990), pp. 1-41. Additional examples of sustained release
compositions include, for example, EP 58,481A, U.S. Pat. No.
3,887,699, EP 158,277A, Canadian Patent No. 1176565, U. Sidman et
al., Biopolymers 22, 547 [1983], R. Langer et al., Chem. Tech. 12,
98 [1982], Sinha et al., J. Control. Release 90, 261 [2003], Zhu et
al., Nat. Biotechnol. 18, 24 [2000], and Dai et al., Colloids Surf
B Biointerfaces 41, 117 [2005].
[0588] Bioadhesive polymers are also contemplated for use in or
with compositions of the present disclosure. Bioadhesives are
synthetic and naturally occurring materials able to adhere to
biological substrates for extended time periods. For example,
Carbopol and polycarbophil are both synthetic cross-linked
derivatives of poly(acrylic acid). Bioadhesive delivery systems
based on naturally occurring substances include for example
hyaluronic acid, also known as hyaluronan. Hyaluronic acid is a
naturally occurring mucopolysaccharide consisting of residues of
D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acid is found
in the extracellular tissue matrix of vertebrates, including in
connective tissues, as well as in synovial fluid and in the
vitreous and aqueous humor of the eye. Esterified derivatives of
hyaluronic acid have been used to produce microspheres for use in
delivery that are biocompatible and biodegradable (see, for
example, Cortivo et al., Biomaterials (1991) 12:727-730; EP
517,565; WO 96/29998; Illum et al., J. Controlled Rel. (1994)
29:133-141). Exemplary hyaluronic acid containing compositions of
the present disclosure comprise a hyaluronic acid ester polymer in
an amount of approximately 0.1% to about 40% (w/w) of a humanized
antibody comprising an ultralong CDR3 to hyaluronic acid
polymer.
[0589] Both biodegradable and non-biodegradable polymeric matrices
may be used to deliver compositions of the present disclosure, and
such polymeric matrices may comprise natural or synthetic polymers.
Biodegradable matrices are preferred. The period of time over which
release occurs is based on selection of the polymer. Typically,
release over a period ranging from between a few hours and three to
twelve months is most desirable. Exemplary synthetic polymers which
may be used to form the biodegradable delivery system include:
polymers of lactic acid and glycolic acid, polyamides,
polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene
oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl
ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone,
polyglycolides, polysiloxanes, polyanhydrides, polyurethanes and
co-polymers thereof, poly(butic acid), poly(valeric acid), alkyl
cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, nitro celluloses, polymers of acrylic and methacrylic
esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose,
cellulose acetate, cellulose propionate, cellulose acetate
butyrate, cellulose acetate phthalate, carboxylethyl cellulose,
cellulose triacetate, cellulose sulphate sodium salt, poly(methyl
methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),
poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl
acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.
Exemplary natural polymers include alginate and other
polysaccharides including dextran and cellulose, collagen, chemical
derivatives thereof (substitutions, additions of chemical groups,
for example, alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art), albumin
and other hydrophilic proteins, zein and other prolamines and
hydrophobic proteins, copolymers and mixtures thereof. In general,
these materials degrade either by enzymatic hydrolysis or exposure
to water in vivo, by surface or bulk erosion. The polymer
optionally is in the form of a hydrogel (see, for example, WO
04/009664, WO 05/087201, Sawhney, et al., Macromolecules, 1993, 26,
581-587) that can absorb up to about 90% of its weight in water and
further, optionally is cross-linked with multivalent ions or other
polymers.
[0590] Delivery systems also include non-polymer systems that are
lipids including sterols such as cholesterol, cholesterol esters
and fatty acids or neutral fats such as mono-di- and
tri-glycerides; hydrogel release systems; silastic systems; peptide
based systems; wax coatings; compressed tablets using conventional
binders and excipients; partially fused implants; and the like.
Specific examples include, but are not limited to: (a) erosional
systems in which the product is contained in a form within a matrix
such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189 and
5,736,152 and (b) diffusional systems in which a product permeates
at a controlled rate from a polymer such as described in U.S. Pat.
Nos. 3,854,480, 5,133,974 and 5,407,686. Liposomes containing the
product may be prepared by methods known methods, such as for
example (DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA,
82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:
4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP
142,641; JP 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and
EP 102,324).
[0591] Alternatively or additionally, the compositions may be
administered locally via implantation into the affected area of a
membrane, sponge, or other appropriate material on to which a
humanized antibody comprising an ultralong CDR3, antibody fragment,
nucleic acid, or vector disclosed herein has been absorbed or
encapsulated. Where an implantation device is used, the device may
be implanted into any suitable tissue or organ, and delivery of a
humanized antibody comprising an ultralong CDR3 antibody fragment,
nucleic acid, or vector disclosed herein can be directly through
the device via bolus, or via continuous administration, or via
catheter using continuous infusion.
[0592] A pharmaceutical composition comprising a humanized antibody
comprising an ultralong CDR3, antibody fragment, nucleic acid, or
vector disclosed herein may be formulated for inhalation, such as
for example, as a dry powder. Inhalation solutions also may be
formulated in a liquefied propellant for aerosol delivery. In yet
another formulation, solutions may be nebulized. Additional
pharmaceutical composition for pulmonary administration include,
those described, for example, in WO 94/20069, which discloses
pulmonary delivery of chemically modified proteins. For pulmonary
delivery, the particle size should be suitable for delivery to the
distal lung. For example, the particle size may be from 1 .mu.m to
5 .mu.m; however, larger particles may be used, for example, if
each particle is fairly porous.
[0593] Certain formulations containing humanized antibodies
comprising an ultralong CDR3, antibody fragments, nucleic acids, or
vectors disclosed herein may be administered orally. Formulations
administered in this fashion may be formulated with or without
those carriers customarily used in the compounding of solid dosage
forms such as tablets and capsules. For example, a capsule can be
designed to release the active portion of the formulation at the
point in the gastrointestinal tract when bioavailability is
maximized and pre-systemic degradation is minimized. Additional
agents may be included to facilitate absorption of a selective
binding agent. Diluents, flavorings, low melting point waxes,
vegetable oils, lubricants, suspending agents, tablet
disintegrating agents, and binders also can be employed.
[0594] Another preparation may involve an effective quantity of a
humanized antibody comprising an ultralong CDR3, antibody fragment,
nucleic acid, or vector disclosed herein in a mixture with
non-toxic excipients which are suitable for the manufacture of
tablets. By dissolving the tablets in sterile water, or another
appropriate vehicle, solutions may be prepared in unit dose form.
Suitable excipients include, but are not limited to, inert
diluents, such as calcium carbonate, sodium carbonate or
bicarbonate, lactose, or calcium phosphate; or binding agents, such
as starch, gelatin, or acacia; or lubricating agents such as
magnesium stearate, stearic acid, or talc.
[0595] Suitable and/or preferred pharmaceutical formulations may be
determined in view of the present disclosure and general knowledge
of formulation technology, depending upon the intended route of
administration, delivery format, and desired dosage. Regardless of
the manner of administration, an effective dose may be calculated
according to patient body weight, body surface area, or organ size.
Further refinement of the calculations for determining the
appropriate dosage for treatment involving each of the formulations
described herein are routinely made in the art and is within the
ambit of tasks routinely performed in the art. Appropriate dosages
may be ascertained through use of appropriate dose-response
data.
[0596] In some embodiments, humanized antibodies comprising an
ultralong CDR3 or fragments thereof are provided with a modified Fc
region where a naturally-occurring Fc region is modified to
increase the half-life of the antibody or fragment in a biological
environment, for example, the serum half-life or a half-life
measured by an in vitro assay. Methods for altering the original
form of a Fc region of an IgG also are described in U.S. Pat. No.
6,998,253.
[0597] In certain embodiments, it may be desirable to modify the
antibody or fragment in order to increase its serum half-life, for
example, adding molecules such as PEG or other water soluble
polymers, including polysaccharide polymers, to antibody fragments
to increase the half-life. This may also be achieved, for example,
by incorporation of a salvage receptor binding epitope into the
antibody fragment (e.g., by mutation of the appropriate region in
the antibody fragment or by incorporating the epitope into a
peptide tag that is then fused to the antibody fragment at either
end or in the middle, e.g., by DNA or peptide synthesis) (see,
International Publication No. WO96/32478). Salvage receptor binding
epitope refers to an epitope of the Fc region of an IgG molecule
(e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
[0598] A salvage receptor binding epitope may include a region
wherein any one or more amino acid residues from one or two loops
of a Fc domain are transferred to an analogous position of the
antibody fragment. Even more preferably, three or more residues
from one or two loops of the Fc domain are transferred. Still more
preferred, the epitope is taken from the CH2 domain of the Fc
region (e.g., of an IgG) and transferred to the CH1, CH3, or VH
region, or more than one such region, of the antibody.
Alternatively, the epitope is taken from the CH2 domain of the Fc
region and transferred to the C.sub.L region or V.sub.L region, or
both, of the antibody fragment. See also WO 97/34631 and WO
96/32478 which describe Fc variants and their interaction with the
salvage receptor.
[0599] Mutation of residues within Fc receptor binding sites may
result in altered effector function, such as altered ADCC or CDC
activity, or altered half-life. Potential mutations include
insertion, deletion or substitution of one or more residues,
including substitution with alanine, a conservative substitution, a
non-conservative substitution, or replacement with a corresponding
amino acid residue at the same position from a different IgG
subclass (e.g., replacing an IgG1 residue with a corresponding IgG2
residue at that position). For example, it has been reported that
mutating the serine at amino acid position 241 in IgG4 to proline
(found at that position in IgG1 and IgG2) led to the production of
a homogeneous antibody, as well as extending serum half-life and
improving tissue distribution compared to the original chimeric
IgG4. (Angal et al., Mol. Immunol. 30:105-8, 1993).
Kits/Articles of Manufacture
[0600] As an additional aspect, the present disclosure includes
kits which comprise one or more compounds or compositions packaged
in a manner which facilitates their use to practice methods of the
present disclosure. In one embodiment, such a kit includes a
compound or composition described herein (e.g., a composition
comprising a humanized antibody comprising an ultralong CDR3 alone
or in combination with a second agent), packaged in a container
with a label affixed to the container or a package insert that
describes use of the compound or composition in practicing the
method. Suitable containers include, for example, bottles, vials,
syringes, etc. The containers may be formed from a variety of
materials such as glass or plastic. The container may have a
sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). The article of manufacture may
comprise (a) a first container with a composition contained
therein, wherein the composition comprises a humanized antibody
comprising an ultralong CDR3 as disclosed herein; and (b) a second
container with a composition contained therein, wherein the
composition comprises a further therapeutic agent. The article of
manufacture in this embodiment disclosed herein may further
comprise a package insert indicating that the first and second
compositions can be used to treat a particular condition.
Alternatively, or additionally, the article of manufacture may
further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes. Preferably, the
compound or composition is packaged in a unit dosage form. The kit
may further include a device suitable for administering the
composition according to a specific route of administration or for
practicing a screening assay. Preferably, the kit contains a label
that describes use of the humanized antibody comprising an
ultralong CDR3 composition.
[0601] The following are examples of the methods and compositions
of the disclosure. It is understood that various other embodiments
may be practiced, given the general description provided above.
EXAMPLES
Example 1. Generation of a Humanized Antibody with an Ultralong
CDR3 Sequence
[0602] At least 7 antibody heavy chain variable region sequences
are publicly available that comprise an ultralong CDR3 of bovine
origin. These sequences are shown in the alignment in FIG. 1. As
shown in FIG. 1, the ultralong CDR3s present within these variable
region sequences range in length from 56 to 61 amino acids.
Strikingly, the sequence alignment indicates that the amino acid
residues positioned in the N-terminal ends of the CDR3s are
particularly conserved. Following the first cysteine residue in
each of these variable region sequences, an amino acid sequence
pattern of "TTVHQ" (SEQ ID NO: 153) and variants thereof are found.
This is unusual in that most heavy chain variable regions of most
species end with the amino acid sequence "CAK" or "CAR". This
unusual sequence motif (e.g., "TTVHQ" (SEQ ID NO: 153)) may be
characteristic of the structure of an ultralong CDR3.
[0603] Additionally, the Bos taurus genome was searched and an
undescribed heavy chain variable region DNA sequence in the
antibody locus was found that encodes a "CTTVHQ" motif (e.g., SEQ
ID NO: 1). This variable region sequence is designated herein as
VH-UL (SEQ ID NO: 29). This sequence motif, discovered for the
first time in searching the cow genome, is important in antibodies
that comprise an ultralong CDR3.
[0604] To produce a humanized antibody comprising an ultralong
CDR3, human variable regions for acceptor human frameworks were
identified that are homologous to the bovine-derived VH-UL sequence
(SEQ ID NO: 29). Several members of the human VH4 family were
identified as being homologous to the VH-UL sequence, however none
of VH4 family members contained a "CTTVHQ" motif (SEQ ID NO: 185).
The sequences VH4-34*02 (SEQ ID NO: 33), VH4-39 (SEQ ID NO: 31),
and VH4-59*03 (SEQ ID NO: 32) are the most homologous to the VH-UL
sequence (see, e.g., FIG. 4B).
[0605] In a first exemplary method to produce a humanized antibody
comprising an ultralong CDR3, a VH4-39 human acceptor framework
(SEQ ID NO: 31) is used. The nucleotides encoding the last two
amino acid residues of VH4-39 (e.g., "AR") are removed from the
VH4-39 human acceptor framework, and nucleotides encoding a "TTVHQ"
motif (SEQ ID NO: 153) are added to the 3' end of the DNA sequence
encoding the VH4-39 human acceptor framework after the nucleotides
that encode the second cysteine residue of the VH4-39 human
acceptor framework (e.g., after the nucleotides that code for amino
acid position 97 of SEQ ID NO: 31). Next, the DNA encoding a
portion of the ultralong CDR3 from BLV1H12 (e.g., the portion of
the ultralong CDR3 beginning at the first glutamic acid residue (E)
in BLV1H12;
ETKKYQSCPDGYRERSDCSNRPACGTSDCCRVSVFGNCLTTLPVSYSYTYNYEWHVD) is fused
to the 3' end of the polynucleotide encoding the "TTVHQ" motif (SEQ
ID NO: 153). Finally, DNA encoding a partial human JH4 region,
beginning with the conserved tryptophan residue common to J region
sequences, is added to the 3' end of the portion of the ultralong
CDR3 sequence derived from BLV1H12. The following antibody gene
encoding a partially human antibody comprising an ultralong CDR3 is
derived:
TABLE-US-00004 (SEQ ID NO: 377)
cagctgcagctgcaggagtcgggcccaggactggtgaagccttcggagacc
ctgtccctcacctgcactgtctctggtggctccatcagcagtagtagttac
tactggggctggatccgccagcccccagggaaggggctggagtggattggg
agtatctattatagtgggagcacctactacaacccgtccctcaagagtcga
gtcaccatatccgtagacacgtccaagaaccagttctccctgaagctgagc
tctgtgaccgccgcagacacggctgtgtattactgtactactgtgcaccag
GAAACAAAAAAATACCAAAGTTGTCCTGATGGGTATAGAGAACGTTCGGAT
TGTAGTAACAGACCTGCTTGTGGTACTAGTGATTGTTGTCGTGTTAGTGTT
TTTGGTAATTGTCTTACTACTCTTCCTGTGAGTTATAGTTATACTTACAAT
TACGAATGGCACGTCGATGTCTGGGGCCAGGGAACCCTGGTCACCGTCTCC TCAG Regular
Font--derived from human VH4-39 Underlined--derived from VH-UL BOLD
BLACK--ultralong CDR3, derived from cow BLVH12 ITALICS--derived
from human JH4
[0606] The amino acid sequence as translated from this partially
human variable region gene is:
TABLE-US-00005 cagctgcagctgcaggagtcgggcccaggactggtgaagccttcggagacc
Q L Q L Q E S G P G L V K P S E T
ctgtccctcacctgcactgtctctggtggctccatcagcagtagtagttac L S L T C T V S
G G S I S S S S Y
tactggggctggatccgccagcccccagggaaggggctggagtggattggg Y W G W I R Q P
P G K G L E W I G
agtatctattatagtgggagcacctactacaacccgtccctcaagagtcga S I Y Y S G S T
Y Y N P S L K S R
gtcaccatatccgtagacacgtccaagaaccagttctccctgaagctgagc V T I S V D T S
K N Q F S L K L S
tctgtgaccgccgcagacacggctgtgtattactgtactactgtgcaccag S V T A A D T A
V Y Y C T T V H Q
gaaacaaaaaaataccaaagttgtcctgatgggtatagagaacgttcggat E T K K Y Q S C
P D G Y R E R S D
tgtagtaacagacctgcttgtggtactagtgattgttgtcgtgttagtgtt C S N R P A C G
T S D C C R V S V
tttggtaattgtcttactactcttcctgtgagttatagttatacttacaat F G N C L T T L
P V S Y S Y T Y N
tacgaatggcacgtcgatgtctggggccagggaaccctggtcaccgtctcc Y E W H V D V W
G Q G T L V T V S tcag (SEQ ID NO: 377) S (SEQ ID NO: 378)
[0607] In an embodiment, this humanized VH sequence is
recombinantly fused in-frame with a human heavy chain constant
region and paired with a light chain for recombinant antibody
production.
[0608] In another exemplary method to produce a humanized antibody
comprising an ultralong CDR3, an ultralong CDR3 derived from BLV5B8
(SEQ ID NO: 7) is incorporated into the VH4-34 human acceptor
framework (e.g., 4-34*02 (SEQ ID NO: 33); or 4-34*09 (SEQ ID NO:
34), along with a portion of the human JH2 region to produce a
humanized antibody comprising an ultralong CDR3. Next, the
nucleotides encoding the last two amino acid residues of the VH4-34
human acceptor framework (e.g., "AR") are removed from the VH4-34
human acceptor framework, and nucleotides encoding a "TTVHQ" motif
are added to the 3' end of the VH4-34 human acceptor framework
after the nucleotides that code for the second conserved cysteine
residue in the VH4-34 human acceptor framework (e.g., after the
nucleotides that code for amino acid position 95 of SEQ ID NOS: 33
or 34). Finally, DNA encoding a partial human JH2 region, beginning
with the conserved tryptophan residue common to J region sequences,
is added to the 3' end of the portion of the ultralong CDR3
sequence derived from BLV5B8. Thus, the following sequence encoding
a humanized antibody variable region comprising an ultralong CDR3
is produced:
TABLE-US-00006 (SEQ ID NO: 379)
caggtgcagctacaacagtggggcgcaggactgttgaagccttcggagacc
ctgtccctcacctgcgctgtctatggtgggtccttcagtggttactactgg
agctggatccgccagcccccagggaaggggctggagtggattggggaaatc
aatcatagtggaagcaccaactacaacccgtccctcaagagtcgagtcacc
atatcagtagacacgtccaagaaccagttctccctgaagctgagctctgtg
accgccgcggacacggctgtgtattactgtActactgtgcaccagGAAACC
AGAAAAACCTGTTCTGATGGTTATATGGCTGTAGATAGTTGTGGTCGTGGT
CAGAGTGATGGTTGTGTCAATGATTGCAATTGTTGTTATTATGGTTGGCGG
AACTGTCGCAGGCAGCCTGCAATTCAAAGTTACGAATTTCACGTCGATGCC
TGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAG Regular Font--derived from human
VH4-39 Underlined--derived from VH-UL BOLD BLACK--ultralong CDR3,
derived from cow BLVH12 ITALICS--derived from human JH2
[0609] The amino acid sequence as translated from this partially
human variable region gene is:
TABLE-US-00007 caggtgcagctacaacagtggggcgcaggactgttgaagccttcggagacc
Q V Q L Q Q W G A G L L K P S E T
ctgtccctcacctgcgctgtctatggtgggtccttcagtggttactactgg L S L T C A V Y
G G S F S G Y Y W
agctggatccgccagcccccagggaaggggctggagtggattggggaaatc S W I R Q P P G
K G L E W I G E I
aatcatagtggaagcaccaactacaacccgtccctcaagagtcgagtcacc N H S G S T N Y
N P S L K S R V T
atatcagtagacacgtccaagaaccagttctccctgaagctgagctctgtg I S V D T S K N
Q F S L K L S S V
accgccgcggacacggctgtgtattactgtactactgtgcaccaggaaacc T A A D T A V Y
Y C T T V H Q E T
agaaaaacctgttctgatggttatatggctgtagatagttgtggtcgtggt R K T C S D G Y
M A V D S C G R G
cagagtgatggttgtgtcaatgattgcaattgttgttattatggttggcgg Q S D G C V N D
C N C C Y Y G W R
aactgtcgcaggcagcctgcaattcaaagttacgaatttcacgtcgatgcc N C R R Q P A I
Q S Y E F H V D A tggggccgtggcaccctggtcActgtctcctcag (SEQ ID NO:
379) W G R G T L V T V S S (SEQ ID NO: 380)
[0610] In an embodiment, this humanized VH sequence is then
recombinantly fused in-frame with a human heavy chain constant
region and paired with a light chain for recombinant antibody
production.
[0611] Thus, any heavy chain variable region can be paired with a
light chain variable region to produce a recombinant antibody.
Bovine antibodies containing ultralong VH CDR3s typically pair with
a restricted set of lambda light chains. Several human VL sequences
can be used to pair with the sequences above, including VL1-47,
VL1-40, VL1-51, VL2-18, which are homologous to the lambda region
derived from Bos taurus.
Example 2: Generation of Libraries of Polynucleotides Encoding
Antibodies Comprising an Ultralong CDR3
[0612] Libraries of polynucleotides encoding antibodies that
comprise an ultralong CDR3 may be constructed by any method known
in the art. Such polynucleotide libraries may be present within a
plurality of vectors (e.g., a library of vectors) including, for
example, vectors present within a plurality of host cells (e.g., a
library of host cells). The libraries may present in any known
format including, in a spatially addressed format (see, e.g., WO
11/056997; and Mao et al. (2010) Nat Biotech 28:1195-1202).
[0613] In an exemplary method, bovine spleen and lymph nodes were
obtained from Animal Technologies (Tyler, Tex.), or from Texas
A&M University. Total RNA was isolated from bovine tissues from
three different cows (MID1, MID10, and MID 11) using TRIzol reagent
(Invitrogen, Carlsbad, Calif., USA) followed by on column digestion
of DNA using the RNeasy Mini Kit (Qiagen, Valencia, Calif., USA).
Alternatively, cDNA may be obtained from the lymph nodes of a
bovine immunized with an antigen (e.g., BVDV). Next, RNA quantity
and quality were assessed with Nanodrop (Thermal Scientific), Qubit
RNA and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, Calif.,
USA), following the manufacturer's protocols. Total RNA was used as
a template for cDNA synthesis catalyzed by Superscript II
(Invitrogen).
[0614] The library of amplified antibody variable regions were then
subjected to deep sequencing. Briefly, bar-coded primers (Table 1)
for each of the three cows (MID1, MID10, and MID11) were used to
amplify VH from bovine spleen cDNA.
TABLE-US-00008 TABLE 1 Bar-coded primers for deep sequencing Primer
Iso- # type Primers MID1 IgG CCTATCCCCTGTGTGCCTTGGCAGTCTCAGACGAG FW
TGCGTTTGAGCGACAAGGCTGTAGGCTG (SEQ ID NO: 381) MID1 IgG
CCATCTCATCCCTGCGTGTCTCCGACTCAGACGAG RV TGCGTCTTTCGGGGCTGTGGTGGAGGC
(SEQ ID NO: 382) MID10 IgM CCTATCCCCTGTGTGCCTTGGCAGTCTCAGTCTCT FW
ATGCGTTGAGCGACAAGGCTGTAGGCTG (SEQ ID NO: 383) MID10 IgM
CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTCT RV
ATGCGAGTGAAGACTCTCGGGTGTGATTCAC (SEQ ID NO: 384) MID11 IgM
CCTATCCCCTGTGTGCCTTGGCAGTCTCAGTGATA FW CGTCTTTGAGCGACAAGGCTGTAGGCTG
(SEQ ID NO: 385) MID11 IgM CCATCTCATCCCTGCGTGTCTCCGACTCAGTGATA RV
CGTCTAGTGAAGACTCTCGGGTGTGATTCAC (SEQ ID NO: 386)
[0615] Next, the amplicons of V.sub.H were purified from 2% agarose
gels and deep sequenced according to Roche 454 GS FLX instructions.
Multiple alignments were performed with the MUSCLE algorithm (Edgar
(2004) Nucleic Acids Research 32:1792-1797). MUSCLE was executed to
generate multiple long CDR H3 nucleotide alignments with relatively
high gap open (-20.0) and gap extend (-10.0) penalties due to the
large amount of heterogeneity observed in the sequences. Local
alignment was executed using the Smith-Waterman algorithm with the
following settings, match score=2.0, mismatch penalty=-1.0, gap
opening penalty=-2.0, and gap extension penalty=-0.5. CDR H3s were
defined by the third residue following the conserved cysteine in
framework 3 to the residue immediately preceding the conserved
tryptophan in framework 4. V.sub.HBUL was identified by BLAST
searching the bovine genome (assembly Btau_4.6.1) with multiple
ultralong V.sub.H sequences identified by deep sequencing. The deep
sequencing identified a total of 11,728 ultralong CDR3 sequences
with having a length between 44 and 69 amino acid residues. The
results of the deep sequencing are summarized in Table 2 below.
TABLE-US-00009 TABLE 2 Summary of deep sequencing results from
bovine spleen Cow#1 Cow#1 Cow#2 Source (Bar code) (MID1) (MID10)
(MID11) Ig Class IgG IgM IgM CDR H3 length range 44-66 44-68 44-69
Number of unique cysteine patterns 655 449 847 Total number of
unique long CDR H3 5633 1639 4456 sequences
[0616] The results of the deep sequencing also revealed that
ultralong CDR3 comprise a cysteine motif (e.g., a pattern of
cysteine residues) that comprises between 3 and 12 cysteine
residues. Representative examples of cysteine patterns are shown
for the deep sequencing run for three different cows (MID1, MID10,
and MID11) as well as their abundance in the run (Tables 3-5). The
cysteines in the ultralong CDR3 regions are symbolized as "C". The
amino acids between two cysteines are symbolized as "X.sub.n".
Exemplary sequences comprising cysteine motifs identified from the
deep sequencing are presented in FIGS. 2A-C.
TABLE-US-00010 TABLE 3 Cysteine patterns identified in ultralong
CDR3s from MID1 Cysteine pattern (MID1) SEQ ID NO Abundance (%)
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C 41 10.44%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C 42 8.11% CX.sub.11CXCX.sub.5C
43 5.22% CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C 44 2.56%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C 45 1.47%
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C 46 1.19%
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C 47 1.08%
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C 48 1.05%
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C 49 0.91%
CX.sub.2CX.sub.8CX.sub.8C 50 0.91%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C 51 0.59%
CX.sub.10CX.sub.5CX.sub.5C 52 0.57%
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C 53 0.50%
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C 54 0.43%
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C 55 0.41%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C 56 0.36%
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C 57 0.32%
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C 58 0.32%
CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C 59 0.30%
CX.sub.16CX.sub.5CXC 60 0.23%
TABLE-US-00011 TABLE 4 Cysteine patterns identified in ultralong
CDR3s from MID10 Cysteine pattern (MID10) SEQ ID NO Abundance (%)
CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C 59 2.87%
CX.sub.10CX.sub.5CX.sub.5C 52 0.73%
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C 57 0.67%
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C 61 0.61%
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C 62 0.55%
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C 63 0.43%
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C 64 0.37%
CX.sub.10CXCX.sub.6CX.sub.4CXC 65 0.31%
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C 66 0.31%
CX.sub.14CX.sub.2CX.sub.3CXCXC 67 0.31% CX.sub.15CX.sub.5CXC 68
0.31% CX.sub.4CX.sub.6CX.sub.6CX.sub.2CX.sub.11C 69 0.31%
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C 70 0.31%
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.6C 71 0.31%
CX.sub.10CX.sub.6CX.sub.5C 72 0.24%
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C 73 0.24%
CX.sub.7CX.sub.5CXCX.sub.2C 74 0.24% CX.sub.10CXCX.sub.6C 75 0.18%
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C 76 0.18%
CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C 77 0.18%
TABLE-US-00012 TABLE 5 Cysteine patterns identified in ultralong
CDR3s from MID11 Cysteine pattern (MID11) SEQ ID NO Abundance (%)
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C 58 1.19%
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C 59 0.96%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C 60 0.92%
CX.sub.16CX.sub.5CXCXCX.sub.14C 61 0.70%
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C 62 0.52%
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C 63 0.49%
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C 64 0.47%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C 65 0.45%
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C 66 0.45%
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C 67 0.43%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C 68 0.36%
CX.sub.10CX.sub.6CX.sub.5C 72 0.31%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C 69 0.29%
CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C 70 0.29%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C 71 0.25%
CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C 72 0.25%
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C 73 0.22%
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C 74 0.20%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C 45 0.20%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C 75 0.20%
[0617] Bovine V.sub.H regions were amplified from cDNA prepared in
example 9 using primers 5'-TTGAGCGACAAGGCTGTAGGCTG-3' (SEQ ID NO:
387) and 5'-CTTTCGGGGCTGTGGTGG-AGGC-3' (SEQ ID NO: 388) producing a
library of antibody variable region cDNA biased for ultralong CDRs.
Next, the mixture of V.sub.H regions was assembled by overlap PCR
with bovine C.sub.H1 and human IgG Fc. Briefly, EcoRI and NheI
sites were incorporated for ligation into pFUSE expression vector,
to afford a full-length heavy chain library ready for expression in
mammalian cells. The ligation product was transformed into E. coli
and 500 single E. coli transformants were picked. Each transformant
was then grown overnight in a separate vessel and DNA from each
colony was extracted using Qiagen minprep kits (Qiagen, Inc.) and
sequenced by BATJ, Inc. (San Diego, Calif.) using the oligo
5'-AGATCCAAGCTGTGACCGGC-3' (SEQ ID NO: 389). Sequences were
analyzed using VectorNTI (Invitrogen, Inc. Carlsbad, Calif.).
Duplicative sequences, sequences with no insert, and sequences
encoding a CDR shorter than 35 residues were excluded. 132 clones
containing unique long CDR heavy chain sequences were selected.
Each heavy chain in the 132 member library was then co-transfected
in parallel with pFUSE expression vector encoding the invariant
bovine light chain BLV1H12 (SEQ ID NO: 412) into 293T cells, to
generate a small spatially addressed library (Mao et al. (2010) Nat
Biotech 28:1195-1202). 130,000 293T cells per well were plated in
24 well plates and grown overnight in 500 ul DMEM media
(Invitrogen) with 10% FBS (Invitrogen), and
Penicillin/streptomycin/glutamine (Invitrogen) at 37.degree. C. and
5% CO.sub.2. 0.5 .mu.g of Hc-encoding pFuse vector and 0.5 .mu.g of
Lc-encoding pFuse vector were added to 25 .mu.l of optimem
(Invitrogen). 1 .mu.l of Lipofectamine 2000 or 293Fectin
transfection reagent (Invitrogen) was added to 25 .mu.l of optimem,
and incubated 5 minutes. Next, the DNA-optimem mix and transfection
reagent-optimem mix were combined and incubated 15 minutes, added
to 293T cells, and allowed to incubate on cells 4-6 hours. Then
media was aspirated from wells and replaced with fresh media, and
cells were allowed to grow and secrete IgG into the media for 4
days. Cell-culture supernatants containing IgG were harvested in 96
well format for further testing. The chimeric antibodies were
quantified by sandwich ELISA detecting human F.sub.c and screened
for binding to BVDV by ELISA.
[0618] Antibodies were then secreted into culture media and
harvested in a 96 well format to generate a small spatially
addressed library for further testing including, screening for
binding to BVDV by ELISA. For example, an ELISA was conducted to
screen the antibody library for binding to BVDV. Briefly, killed
BVDV (0.2 .mu.g) in 100 .mu.L DPBS was coated on 96-well MaxiSorp
ELISA plates (Nunc) for 1 hour at 37.degree. C. Next, the plates
were blocked with 200 .mu.L 3% BSA solution in DPBST, Dulbecco's
phosphate buffered saline, 0.25% Tween 20) for 1 hour at 37.degree.
C. Samples were then incubated with 3% BSA in DPBST for 1 hour at
37.degree. C. Subsequently, wells were washed 5 times with 200
.mu.L DPBST. Next, Goat Anti-Human IgG (Fc)-HRP conjugated antibody
(KPL Inc.) was added at a 1:1,000 dilution in blocking solution and
incubated for 1 hour at 37.degree. C. Wells were then washed 10
times with 200 .mu.L DPBST. A 100 .mu.L working solution of
QuantaBlu (Pierce) was added to each well and incubated for 5
minutes at room temperature before plates were read in a SpectraMax
M5 plate reader at ex325/em420 nm. Several candidate binders were
identified. Clone H12 has a 63-residue CDR3 with 6 cysteine
residues and was able to strongly bind BVDV in a dose dependent
fashion.
[0619] Additionally, binding of the chimeric recombinant antibodies
to BVDV antigens was evaluated by immunocytometric analysis of
transfected human embryonic kidney (HEK) 293A cells (Invitrogen),
as previously described (see, e.g., Njongmeta et al. (2012) Vaccine
30:1624-1635). Briefly, HEK 293A monolayers grown in 6-well tissue
culture plates were transfected with 2 .mu.g/well of plasmid
(pCDNA3.3, Invitrogen) encoding BVDV antigens (N.sup.pro, E2, or
non-structural proteins NS2-3) using Lipofectamine 2000 reagent
(Invitrogen), and incubated for 48 hr at 37.degree. C. with 5%
CO.sub.2. The monolayers were fixed with ice-cold 100% methanol for
10 minutes, rinsed with PBS, and after blocking for 1 hour with PBS
containing 5% fetal bovine serum (blocking buffer), the monolayers
were incubated at room temperature for 1 hr with 10 .mu.g/ml of a
mouse anti-FLAG M2-alkaline phosphatase (AP)-conjugate (Sigma) in
blocking buffer or 10 .mu.g/ml of the chimeric recombinant
antibodies (H12 or B8). Monolayers transfected with empty vector
were similarly reacted to serve as negative controls and, following
washes in blocking buffer, the monolayers probed with the chimeric
recombinant antibodies were incubated with a 1/200 dilution of
AP-conjugated goat anti-Human IgG (Fc specific) mAb (Sigma) in
blocking buffer for 1 hr. Following washes in blocking buffer, the
AP activity in all the wells was detected using Fast Red AS-MX
substrate (Sigma). Stained cells were visualized and photographed
using an IS70 inverted optical microscope (Olympus, Japan) equipped
with a camera. H12 strongly binds HEK293A cells transfected with
the NS2-3 non-structural proteins of BVDV but weakly bound to
untransfected cells while B8 had weak binding to both HEK293A cells
transfected with the NS2-3 non-structural proteins of BVDV and
untransfected cells.
Example 3: Construction of Vector Comprising an Antibody with an
Ultralong CDR3
[0620] Any vector known in the art may be used, or may be modified
to be used, for cloning and/or expression of a nucleotide sequence
encoding a heavy chain variable region that comprises an ultralong
CDR3. Such vectors may optionally comprise, or be modified to
comprise, a nucleotide sequence encoding the Fc portion of a human
immunoglobulin (e.g., IgG) linked to the nucleotide sequence
encoding the heavy chain variable region comprising an ultralong
CDR3. Additionally, the nucleotide sequence encoding the heavy
chain variable region comprising the ultralong CDR3 may be modified
according to known methods such that the ultralong CDR3 can accept
a nucleotide sequence encoding a non-bovine (e.g., non-antibody or
human) sequence including, for example, by unidirectional cloning
with restriction enzymes. Any vector known in the art may also be
used, or may be modified to be used, for cloning and/or expression
of a nucleotide sequence encoding a light chain variable
region.
[0621] In an exemplary method, a vector may be modified by
recombinant techniques to comprise a nucleotide sequence encoding a
heavy chain variable region having an ultralong CDR3 linked to a
nucleotide sequence encoding an IgG Fc. Briefly, a nucleotide
sequence encoding CH1-CH2-CH3 of human IgG1 (SEQ ID NO: 390) was
amplified by PCR and assembled via strand overlap elongation. Next,
a compatible cohesive-end strategy was employed to replace the
human Fc sequence encoded in vector pFUSE-hIgG2-Fc2 (SEQ ID NO:
457, InVivogen, San Diego Calif.) with CH1-CH2-CH3 of human IgG1,
which destroyed the existing 3' NheI site in the pFUSE-hIgG2-Fc2
vector and generated an NheI site at the 5' end of SEQ ID NO: 390.
The modified pFUSE-hIgG2-Fc2 vector ("HC pFuse", SEQ ID NO: 458)
allows insertion of VH fragments between the signal sequence and
the nucleotide sequence encoding CH1-CH2-CH3 of human IgG1 using
the existing EcoRI site and the newly introduced NheI site. For
example, the VH region from bovine antibody BLV1H12 (SEQ ID NO:
392) was amplified by PCR and subcloned in-frame between the signal
sequence and nucleotide sequence encoding CH1-CH2-CH3 of human IgG1
in the HC pFuse vector using EcoRI and NheI restriction enzymes
generating SEQ ID NO: 393.
[0622] Alternatively, nucleotide sequences encoding non-bovine
sequences were inserted into the nucleotide sequence encoding the
CDR3 of BLV1H12 heavy chain. Briefly, a pair of BsaI sites were
introduced by PCR strand overlap extension into the nucleotide
sequence encoding the CDR3 of BLV1H12 (SEQ ID NO: 395). The
modified nucleotide sequence incorporating the BsaI sites within
the CDR3 encoding sequence of BLV1H12 was then subcloned in-frame
between the signal sequence and the nucleotide sequence encoding
CH1-CH2-CH3 of human IgG1 in the HC pFuse vector described above
using EcoRI and NheI restriction enzymes. Next, non-antibody
sequences including cDNA encoding for interleukin 8 (IL-8, SEQ ID
NO: 475), interleukin 21 (IL-21, SEQ ID NO: 480) and
CXCL12/SDF-1alpha (SEQ ID NO: 479) (Origene) and oligoprimers
encoding the peptide hormone somatostain (SEQ ID NO: 477), the
venom peptides ProTx-II (SEQ ID NO: 481) and chlorotoxin (SEQ ID
NO: 478), and the synthetic conotoxin peptide ziconotide (SEQ ID
NO: 476) (IDTDNA) were modified by PCR amplification using
oligoprimers with BsaI flanks to comprise BsaI flanks to produce
non-bovine sequences with BsaI flanks for insertion into the CDR3
of BLV1H12. Next, the BsaI flanks in the modified non-antibody
sequences were cut with BsaI and ligated, in frame, with the
BLV1H12 BsaI digested vector, thereby inserting the nucleotide
sequence encoding the non-antibody sequence into the BLV1H12 heavy
chain variable region and replacing a portion (e.g., cysteine-rich
portion) of the ultralong CDR3, and in-frame with the sequence
encoding BVL1H12 variable region and CH1-CH2-CH3.
[0623] Similarly, a vector was modified by recombinant techniques
to comprise a nucleotide sequence encoding a light chain. Briefly,
the BLV1H12 light chain sequence (SEQ ID NO: 412) was amplified by
PCR to introduce EcoRI and NheI ends. The BLV1H12 light chain
variable region sequence was then digested with EcoRI and NheI and
subcloned into pFUSE-hIgG2-Fc2, deleting the portion of
pFUSE-hIgG2-Fc2 encoding Fc, and replacing it with only light chain
sequence (SEQ ID NO: 459).
[0624] In addition, overlap PCR was used to amplify and join
sequence from BLV1H12 light chain variable region and human lambda
light chain constant region (SEQ ID NO: 474). This was amplified by
PCR to introduce EcoRI and NheI ends. The hybrid BLV1H12 with human
lambda sequence was then digested with EcoRI and NheI and subcloned
into pFUSE-hIgG2-Fc2, deleting the portion of pFUSE-hIgG2-Fc2
encoding Fc, and replacing it with only light chain sequence.
Example 4: Expression of Polynucleotides Coding for Antibodies that
Comprise an Ultralong CDR3
[0625] Polynucleotides coding for antibodies comprising an
ultralong CDR3 may be expressed including, transiently expressed,
in a host cell by any method known in the art.
[0626] In an exemplary method, the vectors comprising the heavy and
light chains generated in Example 2 may be transfected into either
293T cells or Freestyle.TM. 293-F cells.
[0627] For example, 130,000 293T cells per well were plated in 24
well plates and grown overnight in 500 .mu.l DMEM media
(Invitrogen) with 10% FBS (Invitrogen), and
penicillin/streptomycin/glutamine (Invitrogen) at 37.degree. C. and
5% CO.sub.2. Next, 0.5 .mu.g of Heavy chain-encoding pFuse vector
(e.g., generated in Example 2) and 0.5 .mu.g of Light
chain-encoding pFuse vector (e.g., generated in Example 2) were
added to 25 .mu.l of Opti-MEM (Invitrogen). Subsequently, 1 .mu.l
of Lipofectamine 2000 or 293Fectin transfection reagent
(Invitrogen) was added to 25 .mu.l of Opti-MEM, and incubated 5
minutes. Then the dna-Opti-MEM solution and transfection
reagent-Opti-MEM solution were combined and incubated for 15
minutes, added to 293T cells, and allowed to incubate on cells for
4-6 hours. Then media was aspirated from wells and replaced with
fresh media, and the cells were allowed to grow and secrete IgG
into the media for 2-6 days.
[0628] Additionally, for example, 1.times.10.sup.6 293 Freestyle
suspension cells/ml were grown overnight in Freestyle.TM.293
Expression Medium (Invitrogen), and
penicillin/streptomycin/glutamine (Invitrogen) at 37.degree. C. and
5% CO.sub.2. Next, for each milliliter of cells, a 30 .mu.l
solution was made in buffer PBS comprising 0.5 .mu.g Heavy
chain-encoding pFuse DNA and 0.5 .mu.g of Lc-encoding pFuse.
Additionally, for each milliliter of cells, a solution comprising 1
.mu.l of 293Fectin transfection reagent was added to 30 .mu.l PBS
and incubated 5 minutes. Subsequently, the DNA and lipofectamine
solutions were combined and incubated 15 minutes before adding the
mixture to the cells. The cells were then allowed to grow and
secrete IgG into media for 2-6 days.
[0629] After the growth period for the 293T cells or Freestyle.TM.
293-F suspension cells, media was harvested, and IgG secreted into
the supernatant was evaluated by sandwich ELISA. Briefly,
Fc-specific anti Human IgG (Cat 12136, Sigma-Aldrich) was diluted
1:1000 in PBS and coated onto maxisorp plates (Nunc). Next, plates
were blocked with 2% BSA in TBST, washed, and IgG secreted
supernatants were incubated for 1 hour. After washing with TBST,
the appropriate HRP conjugated anti-Light chain antibody is diluted
1:1000 in TBST and incubated 1 hour (anti kappa-HRP, Cat #A-7164,
Sigma-Aldrich; anti-lambda-HRP, Cat #2070-05, Southern Biotech).
After washing with TBST, HRP was detected with TMB (Cat
#TMBS-1000-01, BioFX), and neutralized with 0.6M H.sub.2SO.sub.4.
Subsequently, absorbance (A450) was measured, and compared to a
standard curve of known IgG concentration to determine the
concentration of human antibody in cell culture supernatant.
[0630] The yield of antibody from supernatants from cells
transfected with heavy chain constructs encoding BLV1H12 with human
CH1-CH2-CH3 and a replacement of at least a portion of CDR3
(generated in Example 3) and a light chain construct encoding
BLV1H12 LV-human lambda LC was determined by ELISA, and normalized
to the yield of BLV1H12-IL8 (Table 6). The ELISA indicated that
some sequences (e.g., zinconotide) that were used to replace a
portion of CDR3 have a minor impact on expression of the antibody,
while some sequences (e.g., somatostatin, chlorotoxin, SDF1(alpha),
1L21) that were used to replace a portion of CDR3 had a modest
impact on expression of the antibody, and other sequences (ProTxII)
had an even greater impact on expression of the antibody, as
compared to BLV1H12-IL8 (the highest expressor) (Table 6).
TABLE-US-00013 TABLE 6 Expression yield of BLV1H12 with
replacements to Long CDR3 Normalized Yield (% Heavy Chain V region
Light Chain of highest expressor) BLV1H12-IL8 BLV1H12-HuLambdaC 100
BLV1H12-Ziconotide BLV1H12-HuLambdaC 94.7 BLV1H12-Somatostatin
BLV1H12-HuLambdaC 46.5 BLV1H12-Chlorotoxin BLV1H12-HuLambdaC 39.7
BLV1H12-SDF1(alpha) BLV1H12-HuLambdaC 38.9 BLV1H12-IL21
BLV1H12-HuLambdaC 32.3 BLV1H12-ProTxll BLV1H12-HuLambdaC 2.1
Example 5: Evaluation of Human Germline Sequences Comprising an
Ultralong CDR3 with a Non-Bovine Sequence
[0631] Human germline sequences that comprise an ultralong CDR3 may
be evaluated by any method known in the art to identify those human
germline sequences that permit expression of an ultralong CDR3 that
comprises a non-bovine sequence.
[0632] In an exemplary method, overlap PCR was used to insert BsaI
sites into human germline variable regions VH1-24, VH1-46, VH1-69,
VH3-23, VH4-4, and VH4-34. Next, PCR was used to introduce EcoRI
and NheI sites for sub-cloning in between the signal sequence and
CH1-CH2-CH3 region of the HC pFuse vector comprising BLV1H12
generated in Example 2. Subsequently, the IL-8-GSG cassette (SEQ ID
NO: 399) was introduced into the BsaI sites of each human germline
construct, giving VH1-24+CDR3-IL8 (SEQ ID NO: 425), VH1-46+CDR3-IL8
(SEQ ID NO: 426), VH1-69+CDR3-IL8 (SEQ ID NO: 427), VH3-23+CDR3-IL8
(SEQ ID NO: 428), VH4-4+CDR3-IL8 (SEQ ID NO: 429), and
VH4-34+CDR3-IL8 (SEQ ID NO: 430). Next, each pFuse vector encoding
a human germline heavy chain and CDR3-IL8 construct was
co-transfected with pFuse vector encoding BLV1H12 LV (SEQ ID NO:
474), and allowed to express and secrete these IgGs into the media
as described in Example 4. ELISA was then used to determine IgG
yield as described in Example 4. Yields of the constructs were
determined, and normalized to the highest expressing construct.
Surprisingly, the CDR3 from cow is not readily transposable to any
human heavy chain variable region. VH4-4 had the highest yield,
despite VH4-34 having the closest sequence homology to the BLV1H12
sequence (Table 7).
TABLE-US-00014 TABLE 7 Expression Yield of Humanized Heavy Chains
with Ultralong CDR3s (removed last column of values, with IgG nM,
only showing normalized values. Heavy Chain Normalized Yield (% of
Variable Region: highest expressor) BLV1H12 + CDR3IL8 100 BLV1H12
58.4 VH1-24 BLVCDR3IL8 4.4 VH1-46 BLVCDR3IL8 2.4 VH1-69 BLVCDR3IL8
1.4 VH3-23 BLVCDR3IL8 14.7 VH4-4 BLVCDR3IL8 22.5 VH4-34 BLVCDR3IL8
5.4
Example 6: Evaluation of Antibodies that Comprise an Ultralong CDR3
with a Non-Bovine Sequence
[0633] Antibodies comprising an ultralong CDR3 with a non-bovine
sequence (e.g., a replacement of at least a portion of the CDR3
with a non-bovine sequence) including, humanized antibodies, may be
evaluated by any method known in the art for binding of the
non-bovine sequence to its binding partner including, for example,
flow cytometry.
[0634] In an exemplary method, BLV1H12 IgG comprising a CDR3 with
an IL-8 insert (e.g., the IL-8 sequence replaced a portion of the
CDR3) was evaluated for binding to CXCR1 expressing cells by flow
cytometry. Briefly, a cell line expressing functionally validated
CXCR1 derived from U2OS cells was obtained from DiscoveRx and
cultured per manufacturer's instructions (Cat #93-0226C3, DiscoveRx
Corporation, Freemont, Calif.). The parental cell line U2OS was
obtained from ATCC and cultured under the same conditions as the
CXCR1 cells. Cell culture supernatants were then tested for binding
to cells by flow cytometry. The adherent U2OS or CXCR1-U2OS cells
were dissociated with Accutase (Innovative Cell Technologies, Inc.,
San Diego, Calif.), neutralized with an equal volume of media
containing 10% serum, centrifuged at 1000 g, and resuspended in PBS
with 2% BSA. Next, cells were dispensed into microtiter plates to
achieve between 30,000 to 300,000 cells per well, centrifuged
again, and resuspended in cell culture supernatant containing
expressed IgG, or a dilution of IgG-containing cell culture
supernatant. A fluorescent-conjugated anti-Human Fc antibody was
used to detect binding of the expressed antibody to cells.
Subsequently, cell fluorescence was measured by flow cytometry
(e.g., FACS), and median Arbitrary Fluorescence Units (AFU) were
calculated for each combination of antibody and cell type tested,
revealing the extent of IgG binding to those cells. The ratio of
median fluorescence (IgG binding) of CXCR1-U2OS cells versus U2OS
parental cells shows that the BLV1H12 IgG comprising a CDR3 with an
IL-8 insert has specificity for CXCR1 (Table 8).
TABLE-US-00015 TABLE 8 Binding of BLV1H12 frameworks to CXCR1 U2OS
cells ULcowV (Median ULcowV + IL8 (Median Arbitrary Fluorescence
Arbitrary Fluorescence Units (AFU)) Units (AFU)) Parental U2OS 4 76
CXCR1-U2OS 4 707
[0635] Additionally, the human germline CDR3-IL8 IgGs described in
Example 5 were evaluated for binding to CXCR1 expressing cells by
flow cytometry as described above. IgG binding to CXCR1-U2OS cells
is indicated by higher fluorescence values, but non-specific
binding to U2OS parental cells is also detectable. For a given IgG
with CDR3-IL-8, specific binding to CXCR1 is revealed by the ratio
of fluorescence on CXCR1-U2OS cells to the fluorescence on parental
U2OS cells (Table 9). While the BLV1H12 supported strong and
specific binding to CXCR1 (Table 8), the IL-8 in human germline
IgGs showed either weaker binding to CXCR1, or non-specific
interaction as seen in strong binding to both CXCR1-U2OS cells as
well as the parental control cells. The most specific interaction
to CXCR1 of IL-8 within a human germline IgG was seen with
VH4-34.
TABLE-US-00016 TABLE 9 Binding of IgGs with CDR3-1L8 to CXCR1 U2OS
cells Parental CXCR1- Ratio of CXCR1/ IgG with HC V region: U2OS
U2OS Parental BLV1H12 5 9 1.9 VH1-24 + CDR3-IL8 5 23 4.3 VH1-46 +
CDR3-IL8 5 6 1.1 VH1-69 + CDR3-I8 20 78 4.0 VH3-23 + CDR3-IL8 534
1443 2.7 VH4-4 + CDR3-IL8 1007 3079 3.1 VH4-34 + CDR3-IL8 70 648
9.3
[0636] In another exemplary method, IL-8 activation of the CXCR1
receptor was tested using the CXCR1-U2OS DiscoveRx cells described
above (Cat #93-0226C3, DiscoveRx). The DiscoveRx cells are
engineered such that upon activation by ligand, the resultant
beta-arrestin recruitment to the GPCR also causes activation of a
luminescent reporter enzyme present cell line. Lysis reagents and
luminescence substrate are included in the PathHunter Detection Kit
(Cat #93-001, DiscoveRx) which was used following manufacturer's
instructions. U2OS-CXCR1 cells were plated at 15,000 cells per well
and serum starved overnight in EMEM media without serum. Next,
media was removed and cells were incubated 1 hour at 37.degree. C.
with 80 .mu.l of 1:1 EMEM and PBS, containing dilutions of either
IL-8 or IgG with IL-8 insertion in CDR3, or control antibody. After
1 hour, 40 .mu.l of PathHunter Detection reagent mix was added.
Subsequently, after an hour at room temperature the luminescence in
each well was measured using a luminescence plate reader. Higher
luminescence signal reveals more activation of the CXCR1 receptor
at the tested concentrations of IL-8 or IgG. IgGs were purified by
scaling up the transfection method of Example 4, and purification
of the IgG from media using protein A sepharose following
manufacturer's instructions (Cat #17-1279-03 GE Healthcare) and
dialysis post-elution into PBS. Protein yield was determined by
A280 and calculated molar extinction coefficient. Increasing
concentrations of BLV1H12-IL8 IgG activated CXCR1 somewhat
similarly to the activation observed with IL-8, whereas increasing
concentration of BLV1H12 IgG had no effect on CXCR1 activation
(Table 10).
TABLE-US-00017 TABLE 10 Activation of CXCR1 by IL-8 as soluble
cytokine or as CDR3 replacement IL-8 BLV1H12-IL8 IgG IgG_BVL1H12
IgG nM (average RLU) (average RLU) (average RLU) 500.00 5720 6641
484 166.67 6770 6207 525 55.56 7365 5509 511 18.52 7295 5071 529
6.17 5869 4428 517 2.06 4110 3645 495 0.69 1939 2651 516 0.23 940
1515 455 0.08 504 822 461 0.01 440 435 460
[0637] Additionally, antibodies in which IL-8 was grafted into the
VH4-4 human germline variable region were tested for activation of
CXCR1. IgG with VH4-4+CDR3-IL8 (SEQ ID NO: 429) was compared to
BLV1H12+CDR3-IL8, and IgGVH4-4 CDR3 BsaI (lacking the IL-8 insert)
(SEQ ID NO: 431). A single concentration of soluble IL-8 was tested
as a positive control for assay function at 31.5 nM and gave
activation of 55525 average RLU. (Table 11). Although the IgG with
insertion of IL-8 into the human VH4-4 variable region did activate
CXCR1, it was not as potent in equal dose to the IgG with BLV1H12
sequence.
TABLE-US-00018 TABLE 11 Activation of CXCR1 by IgGs with CDR3 IL-8
BLV1H12 + VH4-4 + VH4-4 + IgG CDR3-IL8 CDR3-IL8 CDR3-Bsal (nM)
(average RLU) (average RLU) (average RLU) 261.500 56349 36481 7545
87.167 52128 29623 7139 29.056 47621 24889 7104 9.685 39280 15545
7174 3.228 34544 10233 7034 1.076 23700 8012 7184 0.359 13278 7338
6687 0.120 8867 7513 7331 0.040 7736 7323 6973 0.013 7329 6956 7048
0.004 7240 7067 7387 0.001 7437 7142 7500
Example 7: Modification of Humanized Antibodies Comprising an
Ultralong CDR3
[0638] Humanized antibodies generated in Example 5 may be modified
to comprise one or more amino acid substitutions in heavy chain
CDR1 and/or CDR2. Such amino acid substitutions may be introduced
into a human germline CDR1 and CDR2 at positions that are
hypothesized to interact with CDR3.
[0639] In an exemplary method, certain residues in VH4-34 CDR1
and/or CDR2 were substituted with corresponding CDR1 and/or CDR2
residues from BLV1H12. Briefly, the pFuse vector encoding heavy
chain VH4-34 CDR3-IL8 was modified by overlap PCR to replace all of
CDR1 with sequence from BLV1H12 (VH4-34+CDR3-IL8_CDR1 Cow, SEQ ID
NO: 432) or all of CDR2 with sequence from BLV1H12
(VH4-34+CDR3-IL8_CDR2 Cow, SEQ ID NO: 433). Additionally, overlap
PCR was used to introduce point mutants G31D and Y32K into CDR1
(VH4-34+CDR3-IL8_CDR1 G31D, Y32K, SEQ ID NO: 434) or point mutant
E50S into CDR2 (VH4-34+CDR3-IL8_CDR2 E505, SEQ ID NO: 435). These
heavy chain constructs were paired with BLV1H12 light chain and
were expressed as described in Example 4. Yield and CXCR1 binding
were determined as described in Example 6 (Table 12).
TABLE-US-00019 TABLE 12 CDR1 and 2 modification of VH4-34 CDR3-IL8
IgG IgG FACS CXCR1/ Yield Parental Heavy chain normalized Ratio
BLV1H12 100 1.9 VH4-34 + CDR3-IL8 20 9.3 VH4-34 + CDR3-IL8 + Cow
CDR1 29 2.1 VH4-34 + CDR3-IL8 + Cow CDR2 9 8.0 VH4-34 + CDR3-IL8 +
E31D, Y32K 19 8.1 VH4-34 + CDR3-IL8 + E50S 27 4.0
Example 8: Pairing of Heavy Chain Comprising an Ultralong CDR3 with
a Human Light Chain
[0640] Antibodies that comprise an ultralong CDR3 including,
antibodies that comprise an ultralong CDR3 where at least a portion
of the CDR3 has been replaced by a non-bovine sequence, may be
paired with a human light chain.
[0641] In an exemplary method, BLV1H12+CDR3-IL8 flexibility to pair
productively with human germline light chains was explored by
co-transfecting pFuse vector encoding heavy chain BLV1H12+CDR3-IL8
with pFuse vectors encoding each of several human germline light
chains (e.g., SEQ ID NOs: 445-456), or BLV1H12 light chain (SEQ ID
NO: 412). These human germline light chain sequences were
synthesized (Genscript, Inc) and amplified by PCR as in Example 3
for subcloning via EcoRI and NheI into the pFUSE LC vector.
BLV1H12+CDR3-1L8 encoding pFUSE vector was cotransfected with pFUSE
vectors encoding light chains as in Example 3. Secreted IgGs were
then tested by FACS as in Example 5 for specific binding to CXCR1
cells (Table 13). Human germ line light chains did not readily
support the function of IL-8 when expressed as BLV1H12+CDR3.
TABLE-US-00020 TABLE 13 Evaluation of BLV1H12 + CDR3-IL8 when
paired with germline human light chains Light chain FACS
CXCR1/Parental Ratio A20J1 0.8 A27J3 0.9 L6J1 0.8 L25J1 1.3 V1-2J7
0.8 V1-7J1 0.6 V1-11J2 0.8 V1-13J5 2.9 V1-16J6 0.6 V2-13J2 0.9
V2-14J4 1.0 V2-15J7 1.1 V2-17J2 2.0 V3-4J1 1.2 V5-4J2 0.8 BLV1H12
5.0
[0642] Alternatively, using the BLV1H12 light chain variable region
as a guide, the human germline sequences identified with closest
homology were V1-47 and V1-51. These light chain sequences were
synthesized (SEQ ID NO: 455, 456) (IDTDNA, Inc.) with the desired
EcoRI and NheI restriction sites for subcloning into the pFuse
vector. Subsequently, pFuse vector encoding V1-47 or V1-51 were
paired with pFuse vectors encoding VH4-34+CDR3-IL8, the
VH4-34+CDR3-IL8 with the CDR1 or CDR2 modifications described in
Example 6 (SEQ ID NOs: 432-435), as well as vectors encoding the
combinations of both CDR1 and CDR2 modifications (e.g.,
VH4-34+CDR3-IL8_CDR1 Cow_CDR2 Cow, SEQ ID NO: 436;
VH4-34+CDR3-IL8_CDR1 Cow_CDR2 E505, SEQ ID NO: 437;
VH4-34+CDR3-1L8_CDR1 G31D,Y32K_CDR2 Cow, SEQ ID NO: 438;
VH4-34+CDR3-IL8_CDR1 G31D,Y32K_CDR2 E505, SEQ ID NO: 439). These
IgGs were expressed, yield was determined by ELISA, and CXCR1
binding specificity was measured by flow cytometry as described
above (Table 14).
TABLE-US-00021 TABLE 14 Expression yield and CXCR1 specificity for
VH4-34 CDR3-IL8 IgGs having CDR1 and CDR2 modifications and paired
with Lc V1-47 or V1-51 CXCR1/Parental HC VH4-34 CDR3-IL8+ nM IgG
Ratio Hc-CDR1 Hc-CDR2 V1-47 V1-51 V1-47 V1-51 G31D Y32K VH4-34 1
2.5 3.6 3 Cow VH4-34 3 8.5 5.1 2.4 VH4-34 VH4-34 1.5 -0.4 2.6 2.9
G31D Y32K E50S 2 4.6 1.4 3 Cow E50S -- 22.8 2.6 3.1 VH4-34 E50S 3.3
3.3 3.3 1.8 G31D Y32K Cow -0.1 2.3 2 2.6 Cow Cow 0.7 4.9 4.6 3.1
VH4-34 Cow 0.6 0.6 2.5 3.9
Example 9: Modification of Human Light Chain and Pairing with Heavy
Chain Comprising an Ultralong CDR3
[0643] Human germline light chains including, light chains that may
be paired with a heavy chain comprising an ultralong CDR3, may be
modified by any method known in the art. Such modifications may
include the substitution of certain amino acid residues in the
human light chain to those residues at corresponding positions in a
bovine light chain sequence. The modified light chains may improve
the yield of the antibody comprising the ultralong CDR3 and/or
increase its binding specificity.
[0644] In an exemplary method, variants of V1-51 were made by
overlap PCR and subcloned into pFuse vector for expression as
described in Example 4. The engineered variants of V1-51 had: i)
substitutions 129V and N32G introduced in CDR1 (SEQ ID NO: 440),
ii) residues DNN (amino acids 51-53) in CDR2 changed to GDT (SEQ ID
NO: 441), iii) residues DNNKRP (SEQ ID NO: 471) in and near CDR2
changed to GDTSRA (SEQ ID NO: 472), or iv) the 14 residues at the
N-terminus were made identical to the first 14 resides of BLV1H12
light chain with point mutations S2A, T5N, PBS, A12G, A13S, and
P14L (SEQ ID NO: 443) or v) combining the changes set forth in ii)
and iv) (SEQ ID NO: 444). These V1-51 variants were paired with
heavy chains encoding VH4-34+CDR3-IL8 (SEQ ID NO: 430),
VH4-34+CDR3-IL8_CDR1 Cow (SEQ ID NO: 432), VH4-34+CDR3-IL8_CDR2 Cow
(SEQ ID NO: 433), or VH4-34+CDR3-IL8_CDR1 Cow_CDR2 Cow, (SEQ ID NO:
436). Transfections and ELISA were carried out as described above,
and flow cytometry was used to evaluate CXCR1 binding specificity
as previously described, with the modification that cells were
resuspended in 2% BSA in PBS plus 2 ug/ml Heparin sulfate, and IgGs
were normalized to 10 nM during incubation on cells. Only certain
combinations of heavy and light chains supported detectable
expression (Table 15). Those combinations that expressed heavy and
light chains were tested for CXCR1 binding specificity (Table
16).
TABLE-US-00022 TABLE 15 Expression yield (nM IgG) for VH4-34 +
CDR3-IL8 variants and V1-51 variants VH4- 34 VH4- VH4- CDR3- 34 34
IL8 VH4- CDR3- CDR3- CDR1- 34 IL8 IL8 Cow & CDR3- CDR1- CDR2-
CDR2- IL8 Cow Cow Cow Hc CDR1: Human Cow Human Cow CDR2: Human
Human Cow Cow V1-51 1 5 0 13 V1-51 CDR1 -1 5 0 3 I29V, N32G V1-51
CDR2 -1 23 -1 5 DNN to GDT V1-51 CDR2 -1 4 1 23 DNNKRP (SEQ ID NO:
471) to GDTSRA (SEQ ID NO: 472) V1-51 0 38 -1 22 S2A, T5N, P8S,
A12G, A13S, P14L V1-51 0 37 0 1 S2A, T5N, P8S, A12G, A13S, P14L
& DNN to GDT
TABLE-US-00023 TABLE 16 CXCR1 specificity for VH4-34 + CDR3-IL8
variants and V1-51 variants VH4- 34 VH4- VH4- CDR3- 34 34 IL8 VH4-
CDR3- CDR3- CDR1- 34 IL8 IL8 Cow & CDR3- CDR1- CDR2- CDR2- IL8
Cow Cow Cow Hc CDR1: Human Cow Human Cow CDR2: Human Human Cow Cow
V1-51 nt 7.2 nt 11.1 V1-51 CDR1 nt 8.9 nt 11.8 I29V, N32G V1-51
CDR2 nt 5.6 nt 16.7 DNN to GDT V1-51 CDR2 nt 8.0 nt 6.9 DNNKRP (SEQ
ID NO: 471) to GDTSRA (SEQ ID NO: 472) V1-51 nt 4.7 nt 12.2 S2A,
T5N, P8S, A12G, A13S, P14L V1-51 nt 6.8 nt S2A, T5N, P8S, A12G,
A13S, P14L & DNN to GDT
Example 10: Preparation of Antibody Libraries with Ultralong CDR3
Sequences
[0645] Libraries may be generated that comprise an ultralong CDR3
including, for example libraries that comprise at least a portion
of an ultralong CDR3, within an antibody framework (e.g., a heavy
chain framework). Such libraries may comprise a diversity of
ultralong CDR3 sequences, a diversity of one or more residues that
are positioned between one or more cysteine residues in the
cysteine domain of the ultralong CDR3, or a diversity of non-bovine
peptides that may be inserted (e.g., replace a portion of) in the
ultralong CDR3. The antibody framework may be derived from a bovine
sequence such as VH-UL, a human germline sequence, or a modified
human germline sequence such as described in Example 7. Heavy
chains with diverse ultralong CDR3 may be paired with light chains
of bovine, human, or modified composition (see, e.g., Example 9)
for expression of a library of antibody or antibody fragments
comprising a diverse ultralong CDR3.
[0646] In an exemplary method, a library is generated in which the
library comprises a diversity of ultralong CDR3 sequences (e.g.,
the diversity in the library resides in that the library contains a
plurality of diverse ultralong CDR3 sequences). For example, a
plurality of diverse ultralong CDR3 sequences is obtained from cDNA
extracted from the spleen and/or lymph nodes from either immunized
or non-immunized cows. Alternatively, a plurality of diverse
ultralong CDR3 sequences is captured as information from cDNA by
sequencing technology, such as described in Example 2. cDNA-derived
cow sequences are then amplified from the isolated cDNA or
synthesized from sequencing of cDNA, and diverse CDR3 sequences are
inserted into an antibody framework for expression of IgGs with
ultralong CDR3 sequences, producing a library of IgGs with CDR3s
derived from cows. The IgG library can exist in any format,
including as a spatially addressed array see, e.g., WO 11/056997;
and Mao et al. (2010) Nat Biotech 28:1195-1202).
[0647] In another exemplary method, a library is generated in which
members of the library comprise an ultralong CDR3 with a certain
cysteine domain, or multiple cysteine domains, wherein the members
are diverse in one or more residues that are positioned between one
or more cysteine residues in the cysteine domain. For example, in
the sequence CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 41),
a library is generated whereby any X residue is changed, or
combination of X residues are changed, to produce diversity within
CDR3. Such diversity is introduced by degenerate nucleotide
synthesis, error prone PCR, gene synthesis, or any other method
known in the art to make changes in nucleotide sequences. These
ultralong CDR3 loops are then integrated with sequence encoding the
antibody framework. Heavy chain sequences are then paired with
human or modified human light chain sequences for co-transfection
and expression to comprise an antibody library engineered from
bovine CDR3s.
[0648] In another exemplary method, a library is generated in which
members of the library comprise an ultralong CDR3 with diversity in
a non-bovine sequence inserted into (e.g., replace a portion of)
the ultralong CDR3. Ultralong CDR3s tolerate large non-bovine
sequence insertions (e.g., cytokines, peptide hormones, signaling
domains, and constituent proteins of arthropod toxins or reptile
venoms) including, for example, cysteine rich insertions, within an
antibody framework, and support the independent function of the
peptide encoded by the non-bovine sequence, as demonstrated in
Example 4 and Example 6. Sequences encoding non-bovine sequences
are amplified by PCR from cDNA or synthesized, and incorporated
with or without linker sequences, into an antibody sequence such
that they are expressed within, in place of, or replace at least a
portion of, an ultralong CDR3. Exemplary methods to insert a
non-bovine sequence into a vector for expression are set forth in
Example 3 above. Heavy chain sequences are then paired with human
or modified human light chain for co-transfection and expression.
For example, a non-bovine sequence is inserted into HC pFUSE,
encoding VH4-34*02 with CDR1 and CDR3 modifications from BLV1H12.
Briefly, the portion of the HC pFuse vector comprising a BsaI
cassette (see, Example 3; bolded in the sequence below) which is
flanked on both sides by a portion of an ultralong CDR3 (underlined
sequence below) is replaced by a non-bovine sequence.
TABLE-US-00024 (SEQ ID NO: 472)
caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagacg
ctgtccctcacctgcacagcaagcgggttttcactgagcgacaaggcagtg
ggatggattcgccagcccccagggaaggggctggagtggattggggaaatc
aatcatagtggaagcaccaactacaacccgtccctcaagagtcgagtcacc
atatcagtagacacgtccaagaaccagttctccctgaagctgagctctgtg
accgccgcggacacggctgtgtattactgtacctctgtgcaccaggaaact
aagaaataccagagcgagacctactatggttcgggtctctcttatacctac
aattatgaatggcatgtggatgtctggggacagggcctgctggtgacagtc
tctagtgctagc
[0649] After replacement of the BsaI cassette with the non-bovine
sequence the non-bovine sequence (referred to as insert in bold
text in the sequence below) is positioned such that it is flanked
on both sides by a portion of the ultralong CDR3.
TABLE-US-00025 (SEQ ID NO: 473)
qvqlqqwgagllkpsetlsltctasgfslsdkavgwirqppgkglewigei
nhsgstnynpslksrvtisvdtsknqfslklssvtaadtavyyctsvhqet kkyqs- insert
-sytynyewhvdvwgqgllvtvssas
[0650] Heavy chain sequences are then paired with human or modified
human light chain for co-transfection and expression.
[0651] Embodiments of the present subject matter described above,
some of which are set forth below, may be beneficial alone or in
combination, with one or more other embodiments. Such embodiments
include:
Embodiment 1
[0652] A humanized antibody or binding fragment thereof comprising
an ultralong CDR3. In some embodiments, the humanized antibody or
binding fragment thereof comprises one or more human variable
region framework sequences.
Embodiment 2
[0653] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 is 35 amino acids in
length or longer, 40 amino acids in length or longer, 45 amino
acids in length or longer, 50 amino acids in length or longer, 55
amino acids in length or longer, or 60 amino acids in length or
longer.
Embodiment 3
[0654] The humanized antibody or binding fragment thereof of
Embodiment 2, wherein the ultralong CDR3 is 35 amino acids in
length or longer.
Embodiment 4
[0655] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises 3 or more
cysteine residues, 4 or more cysteine residues, 5 or more cysteine
residues, 6 or more cysteine residues, 7 or more cysteine residues,
8 or more cysteine residues, 9 or more cysteine residues, 10 or
more cysteine residues, 11 or more cysteine residues, or 12 or more
cysteine residues.
Embodiment 5
[0656] The humanized antibody or binding fragment thereof of
Embodiment 4, wherein the ultralong CDR3 comprises 3 or more
cysteine residues.
Embodiment 6
[0657] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the antibodies or binding fragments thereof
comprise a cysteine motif.
Embodiment 7
[0658] The humanized antibody or binding fragment thereof of
Embodiment 6, wherein the cysteine motif is selected from the group
consisting of: CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO:
41), CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95).
Embodiment 8
[0659] The humanized antibody or binding fragment thereof of
Embodiment 6, wherein the cysteine motif is selected from the group
consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152).
Embodiment 9
[0660] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises 2 to 6 disulfide
bonds.
Embodiment 10
[0661] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises SEQ ID NO: 40 or
a derivative thereof.
Embodiment 11
[0662] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises amino acid
residues 3-6 of any of one SEQ ID NO: 1-4.
Embodiment 12
[0663] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises a non-human DH
or a derivative thereof.
Embodiment 13
[0664] The humanized antibody or binding fragment thereof of
Embodiment 12, wherein the non-human DH is SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11, or SEQ ID NO: 12.
Embodiment 14
[0665] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises a JH sequence or
a derivative thereof.
Embodiment 15
[0666] The humanized antibody or binding fragment thereof of
Embodiment 14, wherein the JH sequence is SEQ ID NO: 13, SEQ ID NO:
14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
Embodiment 16
[0667] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises: a non-human VH
sequence or a derivative thereof; a non-human DH sequence or a
derivative thereof; and/or a JH sequence or derivative thereof.
Embodiment 17
[0668] The humanized antibody or binding fragment thereof of
Embodiment 16, wherein the ultralong CDR3 comprises an additional
amino acid sequence comprising two to six amino acid residues or
more positioned between the VH sequence and the DH sequence.
Embodiment 18
[0669] The humanized antibody or binding fragment thereof of
Embodiment 17, wherein the additional amino acid sequence is
selected from the group consisting of: IR, IF, SEQ ID NO: 18, SEQ
ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
Embodiment 19
[0670] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises a sequence
derived from or based on SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:
28.
Embodiment 20
[0671] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises a non-bovine
sequence or a non-antibody sequence.
Embodiment 21
[0672] The humanized antibody or binding fragment thereof of
Embodiment 20, wherein the non-antibody sequence is a synthetic
sequence.
Embodiment 22
[0673] The humanized antibody or binding fragment thereof of
Embodiment 20, wherein the non-antibody sequence is a cytokine
sequence, a lymphokine sequence, a chemokine sequence, a growth
factor sequence, a hormone sequence, or a toxin sequence.
Embodiment 23
[0674] The humanized antibody or binding fragment thereof of
Embodiment 20, wherein the non-antibody sequence is an IL-8
sequence, an IL-21 sequence, an SDF-1 (alpha) sequence, a
somatostatin sequence, a chlorotoxin sequence, a Pro-TxII sequence,
or a ziconotide sequence.
Embodiment 24
[0675] The humanized antibody or binding fragment thereof of
Embodiment 20, wherein the non-antibody sequence is any one of SEQ
ID NOS: 475-481.
Embodiment 25
[0676] The humanized antibody or binding fragment thereof of
Embodiment 20, wherein the non-antibody sequence replaces at least
a portion of the ultralong CDR3.
Embodiment 26
[0677] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q).
Embodiment 27
[0678] The humanized antibody or binding fragment thereof of
Embodiment 26, wherein the X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif is TTVHQ (SEQ ID NO: 153), TSVHQ (SEQ ID NO: 154), SSVTQ (SEQ
ID NO: 155), STVHQ (SEQ ID NO: 156), ATVRQ (SEQ ID NO: 157), TTVYQ
(SEQ ID NO: 158), SPVHQ (SEQ ID NO: 159), ATVYQ (SEQ ID NO: 160),
TAVYQ (SEQ ID NO: 161), TNVHQ (SEQ ID NO: 162), ATVHQ (SEQ ID NO:
163), STVYQ (SEQ ID NO: 164), TIVHQ (SEQ ID NO: 165), AIVYQ (SEQ ID
NO: 166), TTVFQ (SEQ ID NO: 167), AAVFQ (SEQ ID NO: 168), GTVHQ
(SEQ ID NO: 169), ASVHQ (SEQ ID NO: 170), TAVFQ (SEQ ID NO: 171),
ATVFQ (SEQ ID NO: 172), AAAHQ (SEQ ID NO: 173), VVVYQ (SEQ ID NO:
174), GTVFQ (SEQ ID NO: 175), TAVHQ (SEQ ID NO: 176), ITVHQ (SEQ ID
NO: 177), ITAHQ (SEQ ID NO: 178), VTVHQ (SEQ ID NO: 179); AAVHQ
(SEQ ID NO: 180), GTVYQ (SEQ ID NO: 181), TTVLQ (SEQ ID NO: 182),
TTTHQ (SEQ ID NO: 183), or TTDYQ (SEQ ID NO: 184).
Embodiment 28
[0679] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif.
Embodiment 29
[0680] The humanized antibody or binding fragment thereof of
Embodiment 28, wherein the CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif is CTTVHQ (SEQ ID NO: 185), CTSVHQ (SEQ ID NO: 186), CSSVTQ
(SEQ ID NO: 187), CSTVHQ (SEQ ID NO: 188), CATVRQ (SEQ ID NO: 189),
CTTVYQ (SEQ ID NO: 190), CSPVHQ (SEQ ID NO: 191), CATVYQ (SEQ ID
NO: 192), CTAVYQ (SEQ ID NO: 193), CTNVHQ (SEQ ID NO: 194), CATVHQ
(SEQ ID NO: 195), CSTVYQ (SEQ ID NO: 196), CTIVHQ (SEQ ID NO: 197),
CAIVYQ (SEQ ID NO: 198), CTTVFQ (SEQ ID NO: 199), CAAVFQ (SEQ ID
NO: 200), CGTVHQ (SEQ ID NO: 201), CASVHQ (SEQ ID NO: 202), CTAVFQ
(SEQ ID NO: 203), CATVFQ (SEQ ID NO: 204), CAAAHQ (SEQ ID NO: 205),
CVVVYQ (SEQ ID NO: 206), CGTVFQ (SEQ ID NO: 207), CTAVHQ (SEQ ID
NO: 208), CITVHQ (SEQ ID NO: 209), CITAHQ (SEQ ID NO: 210), CVTVHQ
(SEQ ID NO: 211); CAAVHQ (SEQ ID NO: 212), CGTVYQ (SEQ ID NO: 213),
CTTVLQ (SEQ ID NO: 214), CTTTHQ (SEQ ID NO: 215), or CTTDYQ (SEQ ID
NO: 216).
Embodiment 30
[0681] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises a
(X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is any amino acid
residue, X.sup.b is an aromatic amino acid selected from the group
consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and
histidine (H), and wherein z is 1-4.
Embodiment 31
[0682] The humanized antibody or binding fragment thereof of
Embodiment 30, wherein the (X.sup.aX.sup.b).sub.z motif is CYTYNYEF
(SEQ ID NO: 217), HYTYTYDF (SEQ ID NO: 218), HYTYTYEW (SEQ ID NO:
219), KHRYTYEW (SEQ ID NO: 220), NYIYKYSF (SEQ ID NO: 221),
PYIYTYQF (SEQ ID NO: 222), SFTYTYEW (SEQ ID NO: 223), SYIYIYQW (SEQ
ID NO: 224), SYNYTYSW (SEQ ID NO: 225), SYSYSYEY (SEQ ID NO: 226),
SYTYNYDF (SEQ ID NO: 227), SYTYNYEW (SEQ ID NO: 228), SYTYNYQF (SEQ
ID NO: 229), SYVWTHNF (SEQ ID NO: 230), TYKYVYEW (SEQ ID NO: 231),
TYTYTYEF (SEQ ID NO: 232), TYTYTYEW (SEQ ID NO: 233), VFTYTYEF (SEQ
ID NO: 234), AYTYEW (SEQ ID NO: 235), DYIYTY (SEQ ID NO: 236),
IHSYEF (SEQ ID NO: 237), SFTYEF (SEQ ID NO: 238), SHSYEF (SEQ ID
NO: 239), THTYEF (SEQ ID NO: 240), TWTYEF (SEQ ID NO: 241), TYNYEW
(SEQ ID NO: 242), TYSYEF (SEQ ID NO: 243), TYSYEH (SEQ ID NO: 244),
TYTYDF (SEQ ID NO: 245), TYTYEF (SEQ ID NO: 246), TYTYEW (SEQ ID
NO: 247), AYEF (SEQ ID NO: 248), AYSF (SEQ ID NO: 249), AYSY (SEQ
ID NO: 250), CYSF (SEQ ID NO: 251), DYTY (SEQ ID NO: 252), KYEH
(SEQ ID NO: 253), KYEW (SEQ ID NO: 254), MYEF (SEQ ID NO: 255),
NWIY (SEQ ID NO: 256), NYDY (SEQ ID NO: 257), NYQW (SEQ ID NO:
258), NYSF (SEQ ID NO: 259), PYEW (SEQ ID NO: 260), RYNW (SEQ ID
NO: 261), RYTY (SEQ ID NO: 262), SYEF (SEQ ID NO: 263), SYEH (SEQ
ID NO: 264), SYEW (SEQ ID NO: 265), SYKW (SEQ ID NO: 266), SYTY
(SEQ ID NO: 267), TYDF (SEQ ID NO: 268), TYEF (SEQ ID NO: 269),
TYEW (SEQ ID NO: 270), TYQW (SEQ ID NO: 271), TYTY (SEQ ID NO:
272), or VYEW (SEQ ID NO: 273).
Embodiment 32
[0683] The humanized antibody or binding fragment thereof of
Embodiment 30, wherein the (X.sup.aX.sup.b).sub.z motif is
YXYXYX.
Embodiment 33
[0684] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n motif, wherein X.sub.1
is threonine (T), glycine (G), alanine (A), serine (S), or valine
(V), wherein X.sub.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sub.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sub.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), wherein X.sub.5 is
glutamine (Q), and wherein n is 27-54.
Embodiment 34
[0685] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises
X.sub.n(X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is any amino
acid residue, X.sup.b is an aromatic amino acid selected from the
group consisting of: tyrosine (Y), phenylalanine (F), tryptophan
(W), and histidine (H), wherein n is 27-54, and wherein z is
1-4.
Embodiment 35
[0686] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n(X.sup.aX.sup.b).sub.z
motif, wherein X.sup.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sup.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sup.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sup.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), and wherein X.sup.5 is glutamine (Q), wherein
X.sup.a is any amino acid residue, X.sup.b is an aromatic amino
acid selected from the group consisting of: tyrosine (Y),
phenylalanine (F), tryptophan (W), and histidine (H), wherein n is
27-54, and wherein z is 1-4.
Embodiment 36
[0687] The humanized antibody or binding fragment thereof of
Embodiment 35, wherein the X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif is TTVHQ (SEQ ID NO: 153) or TSVHQ (SEQ ID NO: 154), and
wherein the (X.sup.aX.sup.b).sub.z motif is YXYXYX.
Embodiment 37
[0688] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises: a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q), a cysteine motif selected from the group
consisting of: CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO:
41), CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95); and a (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
Embodiment 38
[0689] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 comprises: a
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q); a cysteine motif selected from the group
consisting of: wherein the cysteine motif is selected from the
group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152); and a (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
Embodiment 39
[0690] The humanized antibody or binding fragment thereof of
Embodiment 20, wherein the ultralong CDR3 comprises an additional
sequence that is a linker.
Embodiment 40
[0691] The humanized antibody or binding fragment thereof of
Embodiment 39, wherein the linker is linked to a C-terminus, a
N-terminus, or both C-terminus and N-terminus of the non-antibody
sequence.
Embodiment 41
[0692] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the ultralong CDR3 is a ruminant CDR3.
Embodiment 42
[0693] The humanized antibody or binding fragment thereof of
Embodiment 41, wherein the ruminant is a cow.
Embodiment 43
[0694] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the humanized antibody or binding fragment
thereof comprises a human heavy chain germline sequence that is a
VH4 family member germline sequence.
Embodiment 44
[0695] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the humanized antibody or binding fragment
thereof comprises a VH4-34 germline sequence.
Embodiment 45
[0696] The humanized antibody or binding fragment thereof of
Embodiment 44, wherein the humanized antibody or binding fragment
thereof comprises a human heavy chain germline sequence that is
derived from the VH4-34 germline sequence.
Embodiment 46
[0697] The humanized antibody or binding fragment thereof of
Embodiment 45, wherein the VH4-34 germline sequence comprises a
CDR1 comprising a Gly31Asp and Tyr32Lys substitution based on Kabat
numbering.
Embodiment 47
[0698] The humanized antibody or binding fragment thereof of
Embodiment 45, wherein the VH4-34 germline sequence comprises a
CDR2 comprising a Glu50Ser substitution based on Kabat
numbering.
Embodiment 48
[0699] The humanized antibody or binding fragment thereof of
Embodiment 45, wherein the VH4-34 germline sequence comprises a
CDR1 comprising Ala23Thr, Val24Ala, Tyr25Ser, Gly27Phe, Phe29Leu,
Gly31Asp, Tyr32Lys, Tyr33Ala, Trp34Val, and Ser35Gly substitutions,
and a CDR2 comprising Ile48Leu, Glu50Ser, Asn52Asp, His53Thr,
Ser54Gly, Ser56Asn, and Asn58Gly substitutions, based on Kabat
numbering.
Embodiment 49
[0700] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the humanized antibody or binding fragment
thereof comprises the amino acid sequence set forth in SEQ ID NO:
461, 462, 463, 464, 465, 466, 467, 468, or 469.
Embodiment 50
[0701] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the humanized antibody or binding fragment
thereof comprises the amino acid sequence set forth in SEQ ID NO:
470.
Embodiment 51
[0702] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the humanized antibody or binding fragment
thereof comprises the amino acid sequence set forth in SEQ ID NO:
461, 462, 463, 464, 465, 466, 467, 468, or 469; and the amino acid
sequence set forth in SEQ ID NO: 470.
Embodiment 52
[0703] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the humanized antibody or binding fragment
thereof comprises the amino acid sequence set forth in SEQ ID NO:
461, 462, 463, 464, 465, 466, 467, 468, or 469; the amino acid
sequence set forth in SEQ ID NO: 470; and a peptide sequence,
wherein the peptide sequence is positioned between SEQ ID NO: 461,
462, 463, 464, 465, 466, 467, 468, or 469, and SEQ ID NO: 470.
Embodiment 53
[0704] The humanized antibody or binding fragment thereof of
Embodiment 52, wherein the peptide sequence is a bovine sequence, a
non-bovine sequence, an antibody sequence, or a non-antibody
sequence.
Embodiment 54
[0705] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the humanized antibody or binding fragment
thereof comprises a light chain variable region sequence that is a
lambda light chain variable region sequence.
Embodiment 55
[0706] The humanized antibody or binding fragment thereof of
Embodiment 54, wherein the lambda light chain variable region
sequence is a bovine lambda light chain variable region
sequence.
Embodiment 56
[0707] The humanized antibody or binding fragment thereof of
Embodiment 54, wherein the lambda light chain variable region
sequence is a human lambda light chain variable region
sequence.
Embodiment 57
[0708] The humanized antibody or binding fragment thereof of
Embodiment 56, wherein the human lambda light chain variable region
sequence is a VL1-51 germline sequence.
Embodiment 58
[0709] The humanized antibody or binding fragment thereof of
Embodiment 56, wherein the human lambda light chain variable region
sequence is derived from a VL1-51 germline sequence.
Embodiment 59
[0710] The humanized antibody or binding fragment thereof of
Embodiment 58, wherein the VL1-51 germline sequence comprises a
CDR1 comprising Ile29Val and Asn32Gly substitution based on Kabat
numbering.
Embodiment 60
[0711] The humanized antibody or binding fragment thereof of
Embodiment 58, wherein the VL1-51 germline sequence comprises a
CDR2 comprising a substitution of DNN to GDT.
Embodiment 61
[0712] The humanized antibody or binding fragment thereof of
Embodiment 58, wherein the VL1-51 germline sequence comprises a
CDR2 comprising a substitution of DNNKRP (SEQ ID NO: 471) to GDTSRA
(SEQ ID NO: 472).
Embodiment 62
[0713] The humanized antibody or binding fragment thereof of
Embodiment 58, wherein the VL1-51 germline sequence comprises a
S2A, T5N, P8S, A12G, A13S, and P14L substitution based on Kabat
numbering.
Embodiment 63
[0714] The humanized antibody or binding fragment thereof of
Embodiment 58, wherein the VL1-51 germline sequence comprises a
S2A, T5N, P8S, A12G, A13S, and P14L substitution based on Kabat
numbering, and a CDR2 comprising a substitution of DNN to GDT.
Embodiment 64
[0715] The humanized antibody or binding fragment thereof of
Embodiment 1, wherein the humanized antibody or binding fragment
thereof comprising the amino acid sequence set forth in SEQ ID NO:
440, 441, 442, 443, or 444.
Embodiment 65
[0716] A polynucleotide encoding the humanized antibody or binding
fragment thereof of any one of Embodiments 1-64.
Embodiment 66
[0717] A polynucleotide encoding a humanized antibody or binding
fragment thereof that comprises an ultralong CDR3.
Embodiment 67
[0718] A polynucleotide encoding a humanized variable region that
comprises an ultralong CDR3.
Embodiment 68
[0719] A vector comprising the polynucleotide of any one of
Embodiments 65-67.
Embodiment 69
[0720] A host cell comprising the vector of Embodiment 68.
Embodiment 70
[0721] A nucleic acid library comprising a plurality of
polynucleotides comprising sequences coding for humanized
antibodies or binding fragments thereof, wherein the antibodies or
binding fragments thereof comprise an ultralong CDR3.
Embodiment 71
[0722] A library of humanized antibodies or binding fragments
thereof, wherein the antibodies or binding fragments thereof
comprise an ultralong CDR3. In some embodiments, the humanized
antibodies or binding fragments thereof in the library comprise one
or more human variable region framework sequences.
Embodiment 72
[0723] The library of Embodiment 71, wherein the ultralong CDR3 is
35 amino acids in length or longer, 40 amino acids in length or
longer, 45 amino acids in length or longer, 50 amino acids in
length or longer, 55 amino acids in length or longer, or 60 amino
acids in length or longer.
Embodiment 73
[0724] The library of Embodiment 72, wherein the ultralong CDR3 is
35 amino acids in length or longer.
Embodiment 74
[0725] The library of Embodiment 71, wherein the ultralong CDR3
comprises 3 or more cysteine residues, 4 or more cysteine residues,
5 or more cysteine residues, 6 or more cysteine residues, 7 or more
cysteine residues, 8 or more cysteine residues, 9 or more cysteine
residues, 10 or more cysteine residues, 11 or more cysteine
residues, or 12 or more cysteine residues.
Embodiment 75
[0726] The library of Embodiment 74, wherein the ultralong CDR3
comprises 3 or more cysteine residues.
Embodiment 76
[0727] The library of Embodiment 71, wherein the antibodies or
binding fragments thereof comprise a cysteine motif.
Embodiment 77
[0728] The library of Embodiment 76, wherein the cysteine motif is
selected from the group consisting of:
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 41),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95).
Embodiment 78
[0729] The library of Embodiment 76, wherein the cysteine motif is
selected from the group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152).
Embodiment 79
[0730] The library of Embodiment 71, wherein the ultralong CDR3
comprises 2 to 6 disulfide bonds.
Embodiment 80
[0731] The library of Embodiment 71, wherein the ultralong CDR3
comprises SEQ ID NO: 40 or a derivative thereof.
Embodiment 81
[0732] The library of Embodiment 71, wherein the ultralong CDR3
comprises amino acid residues 3-6 of any of one SEQ ID NO: 1-4.
Embodiment 82
[0733] The library of Embodiment 71, wherein the ultralong CDR3
comprises a non-human DH or a derivative thereof.
Embodiment 83
[0734] The library of Embodiment 82, wherein the non-human DH is
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12
Embodiment 84
[0735] The library of Embodiment 71, wherein the ultralong CDR3
comprises a JH sequence or a derivative thereof.
Embodiment 85
[0736] The library of Embodiment 84, wherein the JH sequence is SEQ
ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID
NO: 17.
Embodiment 86
[0737] The library of Embodiment 71, wherein the ultralong CDR3
comprises: a non-human VH sequence or a derivative thereof; a
non-human DH sequence or a derivative thereof; and/or a JH sequence
or derivative thereof.
Embodiment 87
[0738] The library of Embodiment 86, wherein the ultralong
[0739] CDR3 comprises an additional amino acid sequence comprising
two to six amino acid residues or more positioned between the VH
sequence and the DH sequence.
Embodiment 88
[0740] The library of Embodiment 87, wherein the additional amino
acid sequence is selected from the group consisting of: IR, IF, SEQ
ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
Embodiment 89
[0741] The library of Embodiment 88, wherein the ultralong CDR3
comprises a sequence derived from or based on SEQ ID NO: 22, SEQ ID
NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,
or SEQ ID NO: 28.
Embodiment 90
[0742] The library of Embodiment 71, wherein the ultralong CDR3
comprises a non-bovine sequence or a non-antibody sequence.
Embodiment 91
[0743] The library of Embodiment 90, wherein the non-antibody
sequence is a synthetic sequence.
Embodiment 92
[0744] The library of Embodiment 90, wherein the non-antibody
sequence is a cytokine sequence, a lymphokine sequence, a chemokine
sequence, a growth factor sequence, a hormone sequence, or a toxin
sequence.
Embodiment 93
[0745] The library of Embodiment 90, wherein the non-antibody
sequence is an IL-8 sequence, an IL-21 sequence, an SDF-1 (alpha)
sequence, a somatostatin sequence, a chlorotoxin sequence, a
Pro-TxII sequence, or a ziconotide sequence.
Embodiment 94
[0746] The library of Embodiment 90, wherein the non-antibody
sequence is any one of SEQ ID NOS: 475-481.
Embodiment 95
[0747] The library of Embodiment 71, wherein the ultralong CDR3
comprises a X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein
X.sup.1 is threonine (T), glycine (G), alanine (A), serine (S), or
valine (V), wherein X.sup.2 is serine (S), threonine (T), proline
(P), isoleucine (I), alanine (A), valine (V), or asparagine (N),
wherein X.sup.3 is valine (V), alanine (A), threonine (T), or
aspartic acid (D), wherein X.sup.4 is histidine (H), threonine (T),
arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and
wherein X.sup.5 is glutamine (Q).
Embodiment 96
[0748] The library of Embodiment 95, wherein the
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif is TTVHQ (SEQ ID NO:
153), TSVHQ (SEQ ID NO: 154), SSVTQ (SEQ ID NO: 155), STVHQ (SEQ ID
NO: 156), ATVRQ (SEQ ID NO: 157), TTVYQ (SEQ ID NO: 158), SPVHQ
(SEQ ID NO: 159), ATVYQ (SEQ ID NO: 160), TAVYQ (SEQ ID NO: 161),
TNVHQ (SEQ ID NO: 162), ATVHQ (SEQ ID NO: 163), STVYQ (SEQ ID NO:
164), TIVHQ (SEQ ID NO: 165), AIVYQ (SEQ ID NO: 166), TTVFQ (SEQ ID
NO: 167), AAVFQ (SEQ ID NO: 168), GTVHQ (SEQ ID NO: 169), ASVHQ
(SEQ ID NO: 170), TAVFQ (SEQ ID NO: 171), ATVFQ (SEQ ID NO: 172),
AAAHQ (SEQ ID NO: 173), VVVYQ (SEQ ID NO: 174), GTVFQ (SEQ ID NO:
175), TAVHQ (SEQ ID NO: 176), ITVHQ (SEQ ID NO: 177), ITAHQ (SEQ ID
NO: 178), VTVHQ (SEQ ID NO: 179); AAVHQ (SEQ ID NO: 180), GTVYQ
(SEQ ID NO: 181), TTVLQ (SEQ ID NO: 182), TTTHQ (SEQ ID NO: 183),
or TTDYQ (SEQ ID NO: 184).
Embodiment 97
[0749] The library of Embodiment 95, wherein the ultralong CDR3
comprises a CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif.
Embodiment 98
[0750] The library of Embodiment 97, wherein the
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif is CTTVHQ (SEQ ID NO:
185), CTSVHQ (SEQ ID NO: 186), CSSVTQ (SEQ ID NO: 187), CSTVHQ (SEQ
ID NO: 188), CATVRQ (SEQ ID NO: 189), CTTVYQ (SEQ ID NO: 190),
CSPVHQ (SEQ ID NO: 191), CATVYQ (SEQ ID NO: 192), CTAVYQ (SEQ ID
NO: 193), CTNVHQ (SEQ ID NO: 194), CATVHQ (SEQ ID NO: 195), CSTVYQ
(SEQ ID NO: 196), CTIVHQ (SEQ ID NO: 197), CAIVYQ (SEQ ID NO: 198),
CTTVFQ (SEQ ID NO: 199), CAAVFQ (SEQ ID NO: 200), CGTVHQ (SEQ ID
NO: 201), CASVHQ (SEQ ID NO: 202), CTAVFQ (SEQ ID NO: 203), CATVFQ
(SEQ ID NO: 204), CAAAHQ (SEQ ID NO: 205), CVVVYQ (SEQ ID NO: 206),
CGTVFQ (SEQ ID NO: 207), CTAVHQ (SEQ ID NO: 208), CITVHQ (SEQ ID
NO: 209), CITAHQ (SEQ ID NO: 210), CVTVHQ (SEQ ID NO: 211); CAAVHQ
(SEQ ID NO: 212), CGTVYQ (SEQ ID NO: 213), CTTVLQ (SEQ ID NO: 214),
CTTTHQ (SEQ ID NO: 215), or CTTDYQ (SEQ ID NO: 216).
Embodiment 99
[0751] The humanized antibody or binding fragment thereof of
Embodiment 71, wherein the ultralong CDR3 comprises a
(X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is any amino acid
residue, X.sup.b is an aromatic amino acid selected from the group
consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and
histidine (H), and wherein z is 1-4.
Embodiment 100
[0752] The humanized antibody or binding fragment thereof of
Embodiment 99, wherein the (X.sup.aX.sup.b).sub.z motif is CYTYNYEF
(SEQ ID NO: 217), HYTYTYDF (SEQ ID NO: 218), HYTYTYEW (SEQ ID NO:
219), KHRYTYEW (SEQ ID NO: 220), NYIYKYSF (SEQ ID NO: 221),
PYIYTYQF (SEQ ID NO: 222), SFTYTYEW (SEQ ID NO: 223), SYIYIYQW (SEQ
ID NO: 224), SYNYTYSW (SEQ ID NO: 225), SYSYSYEY (SEQ ID NO: 226),
SYTYNYDF (SEQ ID NO: 227), SYTYNYEW (SEQ ID NO: 228), SYTYNYQF (SEQ
ID NO: 229), SYVWTHNF (SEQ ID NO: 230), TYKYVYEW (SEQ ID NO: 231),
TYTYTYEF (SEQ ID NO: 232), TYTYTYEW (SEQ ID NO: 233), VFTYTYEF (SEQ
ID NO: 234), AYTYEW (SEQ ID NO: 235), DYIYTY (SEQ ID NO: 236),
IHSYEF (SEQ ID NO: 237), SFTYEF (SEQ ID NO: 238), SHSYEF (SEQ ID
NO: 239), THTYEF (SEQ ID NO: 240), TWTYEF (SEQ ID NO: 241), TYNYEW
(SEQ ID NO: 242), TYSYEF (SEQ ID NO: 243), TYSYEH (SEQ ID NO: 244),
TYTYDF (SEQ ID NO: 245), TYTYEF (SEQ ID NO: 246), TYTYEW (SEQ ID
NO: 247), AYEF (SEQ ID NO: 248), AYSF (SEQ ID NO: 249), AYSY (SEQ
ID NO: 250), CYSF (SEQ ID NO: 251), DYTY (SEQ ID NO: 252), KYEH
(SEQ ID NO: 253), KYEW (SEQ ID NO: 254), MYEF (SEQ ID NO: 255),
NWIY (SEQ ID NO: 256), NYDY (SEQ ID NO: 257), NYQW (SEQ ID NO:
258), NYSF (SEQ ID NO: 259), PYEW (SEQ ID NO: 260), RYNW (SEQ ID
NO: 261), RYTY (SEQ ID NO: 262), SYEF (SEQ ID NO: 263), SYEH (SEQ
ID NO: 264), SYEW (SEQ ID NO: 265), SYKW (SEQ ID NO: 266), SYTY
(SEQ ID NO: 267), TYDF (SEQ ID NO: 268), TYEF (SEQ ID NO: 269),
TYEW (SEQ ID NO: 270), TYQW (SEQ ID NO: 271), TYTY (SEQ ID NO:
272), or VYEW (SEQ ID NO: 273).
Embodiment 101
[0753] The humanized antibody or binding fragment thereof of
Embodiment 99, wherein the (X.sup.aX.sup.b).sub.z motif is
YXYXYX.
Embodiment 102
[0754] The library of Embodiment 71, wherein the ultralong CDR3
comprises a X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n motif,
wherein X.sup.1 is threonine (T), glycine (G), alanine (A), serine
(S), or valine (V), wherein X.sup.2 is serine (S), threonine (T),
proline (P), isoleucine (I), alanine (A), valine (V), or asparagine
(N), wherein X.sup.3 is valine (V), alanine (A), threonine (T), or
aspartic acid (D), wherein X.sup.4 is histidine (H), threonine (T),
arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and
wherein X.sup.5 is glutamine (Q), and wherein n is 27-54.
Embodiment 103
[0755] The library of Embodiment 71, wherein the ultralong CDR3
comprises X.sub.n(X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), wherein n is 27-54, and wherein
z is 1-4.
Embodiment 104
[0756] The library of Embodiment 71, wherein the ultralong CDR3
comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n(X.sup.aX.sup.b).sub.z
motif, wherein X.sub.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sub.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sub.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sub.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), wherein X.sub.5 is glutamine (Q), X.sup.a is any
amino acid residue, X.sup.b is an aromatic amino acid selected from
the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), wherein n is 27-54, and wherein
z is 1-4.
Embodiment 105
[0757] The library of Embodiment 104, wherein the
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif is TTVHQ (SEQ ID NO: 153)
or TSVHQ (SEQ ID NO: 154), and wherein the (X.sup.aX.sup.b).sub.z
motif is YXYXYX.
Embodiment 106
[0758] The library of Embodiment 71, wherein the ultralong CDR3
comprises: a CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein
X.sup.1 is threonine (T), glycine (G), alanine (A), serine (S), or
valine (V), wherein X.sup.2 is serine (S), threonine (T), proline
(P), isoleucine (I), alanine (A), valine (V), or asparagine (N),
wherein X.sup.3 is valine (V), alanine (A), threonine (T), or
aspartic acid (D), wherein X.sup.4 is histidine (H), threonine (T),
arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and
wherein X.sup.5 is glutamine (Q); a cysteine motif selected from
the group consisting of: CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ
ID NO: 41), CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95); and a (X.sup.aX.sup.b).sub.z motif, X.sup.a is any
amino acid residue, X.sup.b is an aromatic amino acid selected from
the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
Embodiment 107
[0759] The library of Embodiment 71, wherein the ultralong
[0760] CDR3 comprises: a CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif, wherein X.sup.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sup.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sup.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sup.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), and wherein X.sup.5 is glutamine (Q); a cysteine
motif selected from the group consisting of: wherein the cysteine
motif is selected from the group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152); and a (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
Embodiment 108
[0761] The library of Embodiment 90, wherein the ultralong CDR3
comprises an additional sequence that is a linker.
Embodiment 109
[0762] The library of Embodiment 108, wherein the linker is linked
to a C-terminus, a N-terminus, or both C-terminus and N-terminus of
the non-antibody sequence.
Embodiment 110
[0763] The library of Embodiment 71, wherein the ultralong CDR3 is
a ruminant CDR3.
Embodiment 111
[0764] The library of Embodiment 110, wherein the ruminant is a
cow.
Embodiment 112
[0765] The library of Embodiment 71, wherein the humanized antibody
or binding fragment thereof comprises a human heavy chain germline
sequence that is a VH4 family member germline sequence.
Embodiment 113
[0766] The library of Embodiment 71, wherein the humanized antibody
or binding fragment thereof comprises a VH4-34 germline
sequence.
Embodiment 114
[0767] The library of Embodiment 71, wherein the humanized antibody
or binding fragment thereof comprises a human heavy chain germline
sequence that is derived from VH4-34.
Embodiment 115
[0768] The library of Embodiment 114, wherein the VH4-34 germline
sequence comprises a CDR1 comprising a Gly31Asp and Tyr32Lys
substitution based on Kabat numbering.
Embodiment 116
[0769] The library of Embodiment 114, wherein the VH4-34 germline
sequence comprises a CDR2 comprising a Glu50Ser substitution based
on Kabat numbering.
Embodiment 117
[0770] The library of Embodiment 114, wherein the VH4-34 germline
sequence comprises a CDR1 comprising Ala23Thr, Val24Ala, Tyr25Ser,
Gly27Phe, Phe29Leu, Gly31Asp, Tyr32Lys, Tyr33Ala, Trp34Val, and
Ser35Gly substitutions, and a CDR2 comprising Ile48Leu, Glu50Ser,
Asn52Asp, His53Thr, Ser54Gly, Ser56Asn, and Asn58Gly substitutions,
based on Kabat numbering.
Embodiment 118
[0771] The library of Embodiment 71, wherein the humanized antibody
or binding fragment thereof comprises the amino acid sequence set
forth in SEQ ID NO: 470.
Embodiment 119
[0772] The library of Embodiment 71, wherein the humanized antibody
or binding fragment thereof comprises the amino acid sequence set
forth in SEQ ID NO: 461, 462, 463, 464, 465, 466, 467, 468, or 469;
and the amino acid sequence set forth in SEQ ID NO: 470.
Embodiment 120
[0773] The library of Embodiment 71, wherein the humanized antibody
or binding fragment thereof comprises the amino acid sequence set
forth in SEQ ID NO: 461, 462, 463, 464, 465, 466, 467, 468, or 469;
the amino acid sequence set forth in SEQ ID NO: 470; and a peptide
sequence, wherein the peptide sequence is positioned between SEQ ID
NO: 461, 462, 463, 464, 465, 466, 467, 468, or 469, and SEQ ID NO:
470.
Embodiment 121
[0774] The library of Embodiment 120, wherein the peptide sequence
is a bovine sequence, a non-bovine sequence, an antibody sequence,
or a non-antibody sequence.
Embodiment 122
[0775] The library of Embodiment 71, wherein the humanized antibody
or binding fragment thereof comprises a light chain variable region
sequence that is a lambda light chain variable region sequence.
Embodiment 123
[0776] The library of Embodiment 122, wherein the lambda light
chain variable region sequence is a bovine lambda light chain
variable region sequence.
Embodiment 124
[0777] The library of Embodiment 122, wherein the lambda light
chain variable region sequence is a human lambda light chain
variable region sequence.
Embodiment 125
[0778] The library of Embodiment 124, wherein the human lambda
light chain variable region sequence is VL1-51.
Embodiment 126
[0779] The library of Embodiment 124, wherein the human lambda
light chain variable region sequence is derived from VL1-51.
Embodiment 127
[0780] The library of Embodiment 126, wherein the VL1-51 germline
sequence comprises a CDR1 comprising Ile29Val and Asn32Gly
substitution based on Kabat numbering.
Embodiment 128
[0781] The library of Embodiment 126, wherein the VL1-51 germline
sequence comprises a CDR2 comprising a substitution of DNN to
GDT.
Embodiment 129
[0782] The library of Embodiment 126, wherein the VL1-51 germline
sequence comprises a CDR2 comprising a substitution of DNNKRP (SEQ
ID NO: 471) to GDTSRA (SEQ ID NO: 472).
Embodiment 130
[0783] The library of Embodiment 126, wherein the VL1-51 germline
sequence comprises a S2A, T5N, P8S, A12G, A13S, and P14L
substitution based on Kabat numbering.
Embodiment 131
[0784] The library of Embodiment 126, wherein the VL1-51 germline
sequence comprises a S2A, T5N, P8S, A12G, A13S, and P14L
substitution based on Kabat numbering, and a CDR2 comprising a
substitution of DNN to GDT.
Embodiment 132
[0785] The library of Embodiment 71, wherein the humanized antibody
or binding fragment thereof comprising the amino acid sequence set
forth in SEQ ID NO: 440, 441, 442, 443, or 444.
Embodiment 133
[0786] The library of any one of Embodiments 71-132, wherein the
humanized antibodies or binding fragments thereof are present in a
spatially addressed format.
Embodiment 134
[0787] A method of humanizing an antibody variable region
comprising the step of genetically combining a nucleic acid
sequence encoding a non-human ultralong CDR3 with a nucleic acid
sequence encoding a human variable region framework (FR)
sequence.
Embodiment 135
[0788] A method of generating a library of humanized antibodies
that comprises an ultralong CDR3, the method comprising: combining
a nucleic acid sequence encoding an ultralong CDR3 with a nucleic
acid sequence encoding a human variable region framework (FR)
sequence to produce nucleic acids encoding for humanized antibodies
that comprises an ultralong CDR3; and expressing the nucleic acids
encoding for humanized antibodies that comprises an ultralong CDR3
to generate a library of humanized antibodies that comprises an
ultralong CDR3.
Embodiment 136
[0789] A method of generating a library of humanized antibodies or
binding fragments thereof comprising an ultralong CDR3 that
comprises a non-antibody sequence, the method comprising: combining
a nucleic acid sequence encoding an ultralong CDR3, a nucleic acid
sequence encoding a human variable region framework (FR) sequence,
and a nucleic acid sequence encoding a non-antibody sequence to
produce nucleic acids encoding humanized antibodies or binding
fragments thereof comprising an ultralong CDR3 and a non-antibody
sequence, and expressing the nucleic acids encoding humanized
antibodies or binding fragments thereof comprising an ultralong
CDR3 and a non-antibody sequence to generate a library of humanized
antibodies or binding fragments thereof comprising an ultralong
CDR3 and a non-antibody sequence.
Embodiment 137
[0790] A library of humanized antibodies or binding fragments
thereof comprising an ultralong CDR3 that comprises a non-antibody
sequence.
Embodiment 138
[0791] A method of generating a library of humanized antibodies or
binding fragments thereof comprising an ultralong CDR3 that
comprises a cysteine motif, the method comprising: combining a
human variable region framework (FR) sequence, and a nucleic acid
sequence encoding an ultralong CDR3 and a cysteine motif;
introducing one or more nucleotide changes to the nucleic acid
sequence encoding one or more amino acid residues that are
positioned between one or more cysteine residues in the cysteine
motif for nucleotides encoding different amino acid residues to
produce nucleic acids encoding humanized antibodies or binding
fragments thereof comprising an ultralong CDR3 and a cysteine motif
with one or more nucleotide changes introduced between one or more
cysteine residues in the cysteine domain; and expressing the
nucleic acids encoding humanized antibodies or binding fragments
thereof comprising an ultralong CDR3 and a cysteine motif with one
or more nucleotide changes introduced between one or more cysteine
residues in the cysteine domain to generate a library of humanized
antibodies or binding fragments thereof comprising an ultralong
CDR3 and a cysteine motif with one or more amino acid changes
introduced between one or more cysteine residues in the cysteine
domain.
Embodiment 139
[0792] A library of humanized antibodies or binding fragments
thereof comprising an ultralong CDR3 that comprises a cysteine
motif, wherein the antibodies or binding fragments comprise one or
more substitutions of amino acid residues that are positioned
between cysteine residues in the cysteine motif.
Embodiment 140
[0793] A method of generating a library of humanized antibodies or
binding fragments thereof comprising a bovine ultralong CDR3, the
method comprising: combining a nucleic acid sequence encoding a
human variable region framework (FR) sequence and a nucleic acid
encoding a bovine ultralong CDR3, and expressing the nucleic acids
encoding a human variable region framework (FR) sequence and a
nucleic acid encoding a bovine ultralong CDR3 to generate a library
of humanized antibodies or binding fragments thereof comprising a
bovine ultralong CDR3.
Embodiment 141
[0794] A library of humanized antibodies or binding fragments
thereof comprising a bovine ultralong CDR3.
Embodiment 142
[0795] A CDR3 scaffold comprising a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif, wherein X.sup.1 is
threonine (T), glycine (G), alanine (A), serine (S), or valine (V),
wherein X.sup.2 is serine (S), threonine (T), proline (P),
isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein
X.sup.3 is valine (V), alanine (A), threonine (T), or aspartic acid
(D), wherein X.sup.4 is histidine (H), threonine (T), arginine (R),
tyrosine (Y), phenylalanine (F), or leucine (L), and wherein
X.sup.5 is glutamine (Q).
Embodiment 143
[0796] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold is 35 amino acids in length or longer, 40 amino acids in
length or longer, 45 amino acids in length or longer, 50 amino
acids in length or longer, 55 amino acids in length or longer, or
60 amino acids in length or longer.
Embodiment 144
[0797] The CDR3 scaffold of Embodiment 143, wherein the CDR3
scaffold is 35 amino acids in length or longer.
Embodiment 145
[0798] The CDR3 scaffold of Embodiment 143, wherein the CDR3
scaffold comprises 3 or more cysteine residues, 4 or more cysteine
residues, 5 or more cysteine residues, 6 or more cysteine residues,
7 or more cysteine residues, 8 or more cysteine residues, 9 or more
cysteine residues, 10 or more cysteine residues, 11 or more
cysteine residues, or 12 or more cysteine residues.
Embodiment 146
[0799] The CDR3 scaffold of Embodiment 145, wherein the CDR3
scaffold comprises 3 or more cysteine residues.
Embodiment 147
[0800] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises a cysteine motif.
Embodiment 148
[0801] The CDR3 scaffold of Embodiment 147, wherein the cysteine
motif is selected from the group consisting of:
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 41),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95).
Embodiment 149
[0802] The CDR3 scaffold of Embodiment 147, wherein the cysteine
motif is selected from the group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152).
Embodiment 150
[0803] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises 2 to 6 disulfide bonds.
Embodiment 151
[0804] The CDR3 scaffold of Embodiment 142, wherein the
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif in the CDR3 scaffold is
TTVHQ (SEQ ID NO: 153), TSVHQ (SEQ ID NO: 154), SSVTQ (SEQ ID NO:
155), STVHQ (SEQ ID NO: 156), ATVRQ (SEQ ID NO: 157), TTVYQ (SEQ ID
NO: 158), SPVHQ (SEQ ID NO: 159), ATVYQ (SEQ ID NO: 160), TAVYQ
(SEQ ID NO: 161), TNVHQ (SEQ ID NO: 162), ATVHQ (SEQ ID NO: 163),
STVYQ (SEQ ID NO: 164), TIVHQ (SEQ ID NO: 165), AIVYQ (SEQ ID NO:
166), TTVFQ (SEQ ID NO: 167), AAVFQ (SEQ ID NO: 168), GTVHQ (SEQ ID
NO: 169), ASVHQ (SEQ ID NO: 170), TAVFQ (SEQ ID NO: 171), ATVFQ
(SEQ ID NO: 172), AAAHQ (SEQ ID NO: 173), VVVYQ (SEQ ID NO: 174),
GTVFQ (SEQ ID NO: 175), TAVHQ (SEQ ID NO: 176), ITVHQ (SEQ ID NO:
177), ITAHQ (SEQ ID NO: 178), VTVHQ (SEQ ID NO: 179); AAVHQ (SEQ ID
NO: 180), GTVYQ (SEQ ID NO: 181), TTVLQ (SEQ ID NO: 182), TTTHQ
(SEQ ID NO: 183), or TTDYQ (SEQ ID NO: 184).
Embodiment 152
[0805] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises a CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5
motif.
Embodiment 153
[0806] The CDR3 scaffold of Embodiment 152, wherein the
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif in the CDR3 scaffold is
CTTVHQ (SEQ ID NO: 185), CTSVHQ (SEQ ID NO: 186), CSSVTQ (SEQ ID
NO: 187), CSTVHQ (SEQ ID NO: 188), CATVRQ (SEQ ID NO: 189), CTTVYQ
(SEQ ID NO: 190), CSPVHQ (SEQ ID NO: 191), CATVYQ (SEQ ID NO: 192),
CTAVYQ (SEQ ID NO: 193), CTNVHQ (SEQ ID NO: 194), CATVHQ (SEQ ID
NO: 195), CSTVYQ (SEQ ID NO: 196), CTIVHQ (SEQ ID NO: 197), CAIVYQ
(SEQ ID NO: 198), CTTVFQ (SEQ ID NO: 199), CAAVFQ (SEQ ID NO: 200),
CGTVHQ (SEQ ID NO: 201), CASVHQ (SEQ ID NO: 202), CTAVFQ (SEQ ID
NO: 203), CATVFQ (SEQ ID NO: 204), CAAAHQ (SEQ ID NO: 205), CVVVYQ
(SEQ ID NO: 206), CGTVFQ (SEQ ID NO: 207), CTAVHQ (SEQ ID NO: 208),
CITVHQ (SEQ ID NO: 209), CITAHQ (SEQ ID NO: 210), CVTVHQ (SEQ ID
NO: 211); CAAVHQ (SEQ ID NO: 212), CGTVYQ (SEQ ID NO: 213), CTTVLQ
(SEQ ID NO: 214), CTTTHQ (SEQ ID NO: 215), or CTTDYQ (SEQ ID NO:
216).
Embodiment 154
[0807] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises a (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a
is any amino acid residue, X.sup.b is an aromatic amino acid
selected from the group consisting of: tyrosine (Y), phenylalanine
(F), tryptophan (W), and histidine (H), and wherein z is 1-4.
Embodiment 155
[0808] The CDR3 scaffold of Embodiment 154, wherein the
(X.sup.aX.sup.b).sub.z motif in the CDR3 scaffold is CYTYNYEF (SEQ
ID NO: 217), HYTYTYDF (SEQ ID NO: 218), HYTYTYEW (SEQ ID NO: 219),
KHRYTYEW (SEQ ID NO: 220), NYIYKYSF (SEQ ID NO: 221), PYIYTYQF (SEQ
ID NO: 222), SFTYTYEW (SEQ ID NO: 223), SYIYIYQW (SEQ ID NO: 224),
SYNYTYSW (SEQ ID NO: 225), SYSYSYEY (SEQ ID NO: 226), SYTYNYDF (SEQ
ID NO: 227), SYTYNYEW (SEQ ID NO: 228), SYTYNYQF (SEQ ID NO: 229),
SYVWTHNF (SEQ ID NO: 230), TYKYVYEW (SEQ ID NO: 231), TYTYTYEF (SEQ
ID NO: 232), TYTYTYEW (SEQ ID NO: 233), VFTYTYEF (SEQ ID NO: 234),
AYTYEW (SEQ ID NO: 235), DYIYTY (SEQ ID NO: 236), IHSYEF (SEQ ID
NO: 237), SFTYEF (SEQ ID NO: 238), SHSYEF (SEQ ID NO: 239), THTYEF
(SEQ ID NO: 240), TWTYEF (SEQ ID NO: 241), TYNYEW (SEQ ID NO: 242),
TYSYEF (SEQ ID NO: 243), TYSYEH (SEQ ID NO: 244), TYTYDF (SEQ ID
NO: 245), TYTYEF (SEQ ID NO: 246), TYTYEW (SEQ ID NO: 247), AYEF
(SEQ ID NO: 248), AYSF (SEQ ID NO: 249), AYSY (SEQ ID NO: 250),
CYSF (SEQ ID NO: 251), DYTY (SEQ ID NO: 252), KYEH (SEQ ID NO:
253), KYEW (SEQ ID NO: 254), MYEF (SEQ ID NO: 255), NWIY (SEQ ID
NO: 256), NYDY (SEQ ID NO: 257), NYQW (SEQ ID NO: 258), NYSF (SEQ
ID NO: 259), PYEW (SEQ ID NO: 260), RYNW (SEQ ID NO: 261), RYTY
(SEQ ID NO: 262), SYEF (SEQ ID NO: 263), SYEH (SEQ ID NO: 264),
SYEW (SEQ ID NO: 265), SYKW (SEQ ID NO: 266), SYTY (SEQ ID NO:
267), TYDF (SEQ ID NO: 268), TYEF (SEQ ID NO: 269), TYEW (SEQ ID
NO: 270), TYQW (SEQ ID NO: 271), TYTY (SEQ ID NO: 272), or VYEW
(SEQ ID NO: 273).
Embodiment 156
[0809] The CDR3 scaffold of Embodiment 154, wherein the
(X.sup.aX.sup.b).sub.z motif in the CDR3 scaffold is YXYXYX.
Embodiment 157
[0810] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises a X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n
motif, wherein X.sup.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sup.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sup.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sup.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), and wherein X.sup.5 is glutamine (Q), and wherein n
is 27-54.
Embodiment 158
[0811] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises a X.sub.n(X.sup.aX.sup.b).sub.z motif, wherein
X.sup.a is any amino acid residue, X.sup.b is an aromatic amino
acid selected from the group consisting of: tyrosine (Y),
phenylalanine (F), tryptophan (W), and histidine (H), wherein n is
27-54, and wherein z is 1-4.
Embodiment 159
[0812] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sub.n(X.sup.aX.sup.b).sub.z
motif, wherein X.sub.1 is threonine (T), glycine (G), alanine (A),
serine (S), or valine (V), wherein X.sub.2 is serine (S), threonine
(T), proline (P), isoleucine (I), alanine (A), valine (V), or
asparagine (N), wherein X.sub.3 is valine (V), alanine (A),
threonine (T), or aspartic acid (D), wherein X.sub.4 is histidine
(H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F),
or leucine (L), wherein X.sub.5 is glutamine (Q), X.sup.a is any
amino acid residue, X.sup.b is an aromatic amino acid selected from
the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), wherein n is 27-54, and wherein
z is 1-4.
Embodiment 160
[0813] The CDR3 scaffold of Embodiment 159, wherein the
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif in the CDR3 scaffold is
TTVHQ (SEQ ID NO: 153) or TSVHQ (SEQ ID NO: 154), and wherein the
(X.sup.aX.sup.b).sub.z motif is YXYXYX.
Embodiment 161
[0814] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises: a CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif,
wherein X.sup.1 is threonine (T), glycine (G), alanine (A), serine
(S), or valine (V), wherein X.sup.2 is serine (S), threonine (T),
proline (P), isoleucine (I), alanine (A), valine (V), or asparagine
(N), wherein X.sup.3 is valine (V), alanine (A), threonine (T), or
aspartic acid (D), wherein X.sup.4 is histidine (H), threonine (T),
arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and
wherein X.sup.5 is glutamine (Q); a cysteine motif selected from
the group consisting of: CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C (SEQ
ID NO: 41), CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C (SEQ ID NO: 42),
CX.sub.11CXCX.sub.5C (SEQ ID NO: 43),
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C (SEQ ID NO: 44),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2C (SEQ ID NO: 45),
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C (SEQ ID NO: 46),
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C (SEQ ID NO: 47),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C (SEQ ID NO: 48),
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C (SEQ ID NO: 49),
CX.sub.13CX.sub.8CX.sub.8C (SEQ ID NO: 50),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C (SEQ ID NO: 51),
CX.sub.10CX.sub.5CX.sub.5C (SEQ ID NO: 52),
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C (SEQ ID NO: 53),
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C (SEQ ID NO: 54),
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C (SEQ ID NO: 55),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C (SEQ ID NO: 56),
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C (SEQ ID NO: 57),
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C (SEQ ID NO:
58), CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C (SEQ ID NO:
59), CX.sub.16CX.sub.5CXC (SEQ ID NO: 60),
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C (SEQ ID NO: 61),
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C (SEQ ID NO: 62),
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO: 63),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C (SEQ ID NO: 64),
CX.sub.10CXCX.sub.6CX.sub.4CXC (SEQ ID NO: 65),
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C (SEQ ID NO: 66),
CX.sub.14CX.sub.2CX.sub.3CXCXC (SEQ ID NO: 67),
CX.sub.15CX.sub.5CXC (SEQ ID NO: 68),
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C (SEQ ID NO: 69),
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C (SEQ ID NO: 70),
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C (SEQ ID NO: 71),
CX.sub.10CX.sub.6CX.sub.5C (SEQ ID NO: 72),
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C (SEQ ID NO: 73),
CX.sub.7CX.sub.5CXCX.sub.2C (SEQ ID NO: 74), CX.sub.10CXCX.sub.6C
(SEQ ID NO: 75),
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C (SEQ ID NO:
76), CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 77),
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C (SEQ ID NO: 78),
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C (SEQ ID NO: 79),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C (SEQ ID NO: 80),
CX.sub.16CX.sub.5CXCXCX.sub.14C (SEQ ID NO: 81),
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C (SEQ ID NO: 82),
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 83),
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C (SEQ ID NO: 84),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C (SEQ ID NO: 85),
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 86),
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C (SEQ ID NO: 87),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C (SEQ ID NO: 88),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C (SEQ ID NO:
89), CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C (SEQ ID NO: 90),
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C (SEQ ID NO:
91), CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C (SEQ ID NO: 92),
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C (SEQ ID NO: 93),
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C (SEQ ID NO:
94), and CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C (SEQ
ID NO: 95); and a (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
Embodiment 162
[0815] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises: a CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif,
wherein X.sup.1 is threonine (T), glycine (G), alanine (A), serine
(S), or valine (V), wherein X.sup.2 is serine (S), threonine (T),
proline (P), isoleucine (I), alanine (A), valine (V), or asparagine
(N), wherein X.sup.3 is valine (V), alanine (A), threonine (T), or
aspartic acid (D), wherein X.sup.4 is histidine (H), threonine (T),
arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and
wherein X.sup.5 is glutamine (Q); a cysteine motif selected from
the group consisting of: wherein the cysteine motif is selected
from the group consisting of:
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC (SEQ ID
NO: 96), CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC
(SEQ ID NO: 97), CX.sub.7CXCX.sub.5CX.sub.4CCCX.sub.4CX.sub.6CXC
(SEQ ID NO: 98), CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C
(SEQ ID NO: 99),
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC (SEQ ID NO:
100), CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C (SEQ
ID NO: 101), CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C (SEQ ID
NO: 102), CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C
(SEQ ID NO: 103), CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C
(SEQ ID NO: 104),
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C (SEQ ID
NO: 105), CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC
(SEQ ID NO: 106),
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC (SEQ ID NO:
107), CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C (SEQ ID
NO: 108), CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC
(SEQ ID NO: 109),
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C (SEQ
ID NO: 110), CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C (SEQ ID
NO: 111), CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C
(SEQ ID NO: 112), CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C
(SEQ ID NO: 113), CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C
(SEQ ID NO: 114), CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 115), CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC
(SEQ ID NO: 116), CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C
(SEQ ID NO: 117), CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC (SEQ ID
NO: 118), CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C (SEQ ID
NO: 119), CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C
(SEQ ID NO: 120), CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C (SEQ
ID NO: 121), CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C (SEQ ID NO:
122), CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C (SEQ ID
NO: 123), CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C (SEQ ID NO:
124), CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C (SEQ ID NO:
125), CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C (SEQ ID
NO: 126), CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C (SEQ ID NO:
127), CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C (SEQ ID NO: 128),
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C (SEQ ID NO:
129), CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C (SEQ ID NO:
130), CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C (SEQ ID NO:
131), CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C (SEQ ID
NO: 132), CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C (SEQ ID NO: 133),
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C (SEQ ID NO: 134),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C (SEQ ID NO: 135),
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C (SEQ ID NO: 136),
CX.sub.9CX.sub.6CX.sub.4CCXC (SEQ ID NO: 137),
CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 (SEQ ID NO: 138),
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C (SEQ ID NO: 139),
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C (SEQ ID NO: 140),
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC (SEQ ID NO: 141),
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C (SEQ ID NO: 142),
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C (SEQ ID NO: 143),
CX.sub.7CXCX.sub.3CXCX.sub.6C (SEQ ID NO: 144),
CX.sub.7CXCX.sub.4CXCX.sub.4C (SEQ ID NO: 145),
CX.sub.9CX.sub.5CX.sub.4C (SEQ ID NO: 146),
CX.sub.3CX.sub.6CX.sub.8C (SEQ ID NO: 147), CX.sub.10CXCX.sub.4C
(SEQ ID NO: 148), CX.sub.10CCX.sub.4C (SEQ ID NO: 149), CX.sub.15C
(SEQ ID NO: 150), CX.sub.10C (SEQ ID NO: 151), and CX.sub.9C (SEQ
ID NO: 152); and a (X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is
any amino acid residue, X.sup.b is an aromatic amino acid selected
from the group consisting of: tyrosine (Y), phenylalanine (F),
tryptophan (W), and histidine (H), and wherein z is 1-4.
Embodiment 163
[0816] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises SEQ ID NO: 40 or a derivative thereof.
Embodiment 164
[0817] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises amino acid residues 3-6 of any of one SEQ ID NO:
1-4.
Embodiment 165
[0818] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises a non-human DH or a derivative thereof.
Embodiment 166
[0819] The CDR3 scaffold of Embodiment 165, wherein the non-human
DH is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12
Embodiment 167
[0820] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises a JH sequence or a derivative thereof.
Embodiment 168
[0821] The CDR3 scaffold of Embodiment 167, wherein the JH sequence
is SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or
SEQ ID NO: 17.
Embodiment 169
[0822] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises: a non-human VH sequence or a derivative
thereof; a non-human DH sequence or a derivative thereof; and/or a
JH sequence or derivative thereof.
Embodiment 170
[0823] The CDR3 scaffold of Embodiment 169, wherein the CDR3
scaffold comprises an additional amino acid sequence comprising two
to six amino acid residues or more positioned between the VH
sequence and the DH sequence.
Embodiment 171
[0824] The CDR3 scaffold of Embodiment 170, wherein the additional
amino acid sequence is selected from the group consisting of: IR,
IF, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO:
21.
Embodiment 172
[0825] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold comprises a sequence derived from or based on SEQ ID NO:
22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ
ID NO: 27, or SEQ ID NO:
[0826] 28.
Embodiment 173
[0827] The CDR3 scaffold of Embodiment 142, wherein the CDR3
scaffold is a ruminant CDR3 scaffold.
Embodiment 174
[0828] The CDR3 scaffold of Embodiment 173, wherein the ruminant is
a cow.
Embodiment 175
[0829] A library comprising the CDR3 scaffold of any one of
Embodiments 142-174.
Embodiment 176
[0830] A polynucleotide encoding the CDR3 scaffold of any one of
Embodiments 142-174.
Embodiment 177
[0831] A vector comprising the polynucleotide of Embodiment
176.
Embodiment 178
[0832] A host cell comprising the vector of Embodiment 177.
Embodiment 179
[0833] The host cell of Embodiment 178, wherein the cell is a
bacteria, virus, or bacteriophage.
[0834] For the disclosure herein, the very least, and not as an
attempt to limit the application of the doctrine of equivalents to
the scope of the claims, each numerical parameter should at least
be construed in light of the number of reported significant digits
and by applying ordinary rounding techniques. Notwithstanding that
the numerical ranges and parameters setting forth the broad scope
of the disclosure are approximations, the numerical values set
forth in the specific examples are reported as precisely as
possible. Any numerical value, however, inherently contains certain
errors necessarily resulting from the standard deviation found in
their respective testing measurements.
[0835] The terms "a," "an," "the" and similar referents used in the
context of describing the exemplary embodiments (especially in the
context of the following claims) are to be construed to cover both
the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Recitation of ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein is intended
merely to better illuminate the exemplary embodiments and does not
pose a limitation on the scope of the exemplary embodiments
otherwise claimed. No language in the specification should be
construed as indicating any non-claimed element essential to the
practice of the exemplary embodiments.
[0836] Groupings of alternative elements or embodiments disclosed
herein are not to be construed as limitations. Each group member
can be referred to and claimed individually or in any combination
with other members of the group or other elements found herein. It
is anticipated that one or more members of a group can be included
in, or deleted from, a group for reasons of convenience and/or
patentability. When any such inclusion or deletion occurs, the
specification is deemed to contain the group as modified thus
fulfilling the written description of all Markush groups used in
the appended claims.
[0837] Certain embodiments are described herein, including the best
mode known to the inventors for carrying out the exemplary
embodiments. Of course, variations on these described embodiments
will become apparent to those of ordinary skill in the art upon
reading the foregoing description. The inventor expects skilled
artisans to employ such variations as appropriate, and the
inventors intend for the embodiments to be practiced otherwise than
specifically described herein. Accordingly, this disclosure
includes all modifications and equivalents of the subject matter
recited in the claims appended hereto as permitted by applicable
law. Moreover, any combination of the above-described elements in
all possible variations thereof is encompassed by the disclosure
unless otherwise indicated herein or otherwise clearly contradicted
by context.
[0838] Furthermore, numerous references have been made to patents
and printed publications. Each of the above-cited references is
individually incorporated herein by reference in their
entirety.
[0839] Specific embodiments disclosed herein can be further limited
in the claims using consisting of or and consisting essentially of
language. When used in the claims, whether as filed or added per
amendment, the transition term "consisting of" excludes any
element, step, or ingredient not specified in the claims. The
transition term "consisting essentially of" limits the scope of a
claim to the specified materials or steps and those that do not
materially affect the basic and novel characteristic(s). Exemplary
embodiments so claimed are inherently or expressly described and
enabled herein.
[0840] In closing, it is to be understood that the exemplary
embodiments disclosed herein are illustrative of the principles of
the present disclosure. Other modifications that can be employed
are within the scope of the disclosure. Thus, by way of example,
but not of limitation, alternative configurations of the present
exemplary embodiments can be utilized in accordance with the
teachings herein. Accordingly, the present exemplary embodiments
are not limited to that precisely as shown and described.
Sequence CWU 1
1
48516PRTArtificial sequenceSynthesized VH sequence (germline,
BLV5B8, BLV8C11, BF4E9, and F18) 1Cys Thr Thr Val His Gln1
526PRTArtificial sequenceSynthesized VH sequence (BLV1H12) 2Cys Thr
Ser Val His Gln1 536PRTArtificial sequenceSynthesized VH sequence
(BLV5D3) 3Cys Ser Ser Val Thr Gln1 546PRTArtificial
sequenceSynthesized VH sequence (BF1H1) 4Cys Thr Thr Val His Pro1
5548PRTArtificial sequenceSynthesized Germline sequence derived
from DH2 gene 5Ser Cys Pro Asp Gly Tyr Ser Tyr Gly Tyr Gly Cys Gly
Tyr Gly Tyr1 5 10 15Gly Cys Ser Gly Tyr Asp Cys Tyr Gly Tyr Gly Gly
Tyr Gly Gly Tyr 20 25 30Gly Gly Tyr Gly Tyr Ser Ser Tyr Ser Tyr Ser
Tyr Thr Tyr Glu Tyr 35 40 45648PRTArtificial sequenceSynthesized
Sequence derived from DH gene (BLV1H12) 6Ser Cys Pro Asp Gly Tyr
Arg Glu Arg Ser Asp Cys Ser Asn Arg Pro1 5 10 15Ala Cys Gly Thr Ser
Asp Cys Cys Arg Val Ser Val Phe Gly Asn Cys 20 25 30Leu Thr Thr Leu
Pro Val Ser Tyr Ser Tyr Thr Tyr Asn Tyr Glu Trp 35 40
45744PRTArtificial sequenceSynthesized Sequence derived from DH
gene (BLV5B8) 7Cys Ser Asp Gly Tyr Ile Ala Val Asp Ser Cys Gly Arg
Gly Gln Ser1 5 10 15Asp Gly Cys Val Asn Asp Cys Asn Ser Cys Tyr Tyr
Gly Trp Arg Asn 20 25 30Cys Arg Arg Gln Pro Ala Ile His Ser Tyr Glu
Phe 35 40844PRTArtificial sequenceSynthesized Sequence derived from
DH gene (BLV5D3) 8Ser Cys Pro Asp Gly Cys Ser Asp Gly Asp Gly Cys
Val Asp Gly Cys1 5 10 15Cys Cys Ser Ala Tyr Arg Cys Tyr Thr Pro Gly
Val Arg Asp Leu Ser 20 25 30Cys Thr Ser Tyr Ser Ile Thr Tyr Thr Tyr
Glu Trp 35 40945PRTArtificial sequenceSynthesized Sequence derived
from DH gene (BLV8C11) 9Cys Cys Ser Asp Ala Tyr Arg Tyr Asp Ser Gly
Cys Gly Ser Gly Cys1 5 10 15Asp Cys Cys Gly Ala Asp Cys Tyr Val Phe
Gly Ala Cys Thr Phe Gly 20 25 30Leu Asp Ser Ser Tyr Ser Tyr Ile Tyr
Ile Tyr Gln Trp 35 40 451047PRTArtificial sequenceSynthesized
Sequence derived from DH gene (BF4E9) 10Cys Pro Asp Gly Tyr Ser Tyr
Gly Tyr Gly Cys Gly Tyr Gly Tyr Gly1 5 10 15Cys Ser Gly Tyr Asp Cys
Tyr Gly Tyr Gly Gly Tyr Gly Tyr Gly Gly 20 25 30Tyr Gly Gly Tyr Ser
Ser Tyr Ser Tyr Ser Tyr Ser Tyr Glu Tyr 35 40 451144PRTArtificial
sequenceSynthesized Sequence derived from DH gene (BF1H1) 11Ser Pro
Asp Gly Tyr Ser Tyr Gly Tyr Gly Cys Gly Tyr Gly Tyr Gly1 5 10 15Cys
Ser Gly Tyr Asp Cys Tyr Gly Tyr Gly Gly Tyr Gly Tyr Gly Gly 20 25
30Tyr Gly Gly Tyr Ser Ser Tyr Ser Tyr Ser Tyr Ser 35
401242PRTArtificial sequenceSynthesized Sequence derived from DH
gene (F18) 12Cys Pro Asp Gly Tyr Gly Tyr Gly Tyr Gly Cys Gly Tyr
Gly Ser Tyr1 5 10 15Gly Tyr Ser Gly Tyr Asp Cys Tyr Gly Tyr Gly Gly
Tyr Gly Gly Tyr 20 25 30Gly Gly Tyr Gly Gly Tyr Ser Ser Tyr Ser 35
401315PRTArtificial sequenceSynthesized Germline sequence derived
from JH1 gene 13Tyr Val Asp Ala Trp Gly Gln Gly Leu Leu Val Thr Val
Ser Ser1 5 10 151415PRTArtificial sequenceSynthesized Sequence
derived from JH gene (BLV1H12) 14His Val Asp Val Trp Gly Gln Gly
Leu Leu Val Thr Val Ser Ser1 5 10 151515PRTArtificial
sequenceSynthesized Sequence derived from JH gene (BLV5B8) 15His
Val Asp Ala Trp Gly Arg Gly Leu Leu Val Thr Val Ser Ser1 5 10
151615PRTArtificial sequenceSynthesized Sequence derived from JH
gene (BLV5D3) 16Asn Val Asp Ala Trp Gly Arg Gly Leu Leu Val Thr Val
Ser Ser1 5 10 151715PRTArtificial sequenceSynthesized Sequence
derived from JH gene (BLV8C11 or BF4E9) 17Tyr Gly Asp Ala Trp Gly
Gln Gly Leu Leu Val Thr Val Ser Ser1 5 10 15186PRTArtificial
sequenceSynthesized Sequence derived from CDR3 (BLV1H12) 18Glu Thr
Lys Lys Tyr Gln1 5195PRTArtificial sequenceSynthesized Sequence
derived from CDR3 (BLV5B8) 19Glu Thr Arg Lys Thr1 5206PRTArtificial
sequenceSynthesized Sequence derived from CDR3 (BLV5D3) 20Arg Thr
His Val Ser Arg1 5216PRTArtificial sequenceSynthesized Sequence
derived from CDR3 (BLV8C11) 21Lys Thr Thr Arg Lys Thr1
52261PRTArtificial sequenceSynthesized ultralong CDR3 sequence
(BLV1H12) 22Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Cys Pro Asp
Gly Tyr1 5 10 15Arg Glu Arg Ser Asp Cys Ser Asn Arg Pro Ala Cys Gly
Thr Ser Asp 20 25 30Cys Cys Arg Val Ser Val Phe Gly Asn Cys Leu Thr
Thr Leu Pro Val 35 40 45Ser Tyr Ser Tyr Thr Tyr Asn Tyr Glu Trp His
Val Asp 50 55 602356PRTArtificial sequenceSynthesized ultralong
CDR3 sequence (BLV5B8) 23Thr Val His Gln Glu Thr Arg Lys Thr Cys
Ser Asp Gly Tyr Ile Ala1 5 10 15Val Asp Ser Cys Gly Arg Gly Gln Ser
Asp Gly Cys Val Asn Asp Cys 20 25 30Asn Ser Cys Tyr Tyr Gly Trp Arg
Asn Cys Arg Arg Gln Pro Ala Ile 35 40 45His Ser Tyr Glu Phe His Val
Asp 50 552457PRTArtificial sequenceSynthesized ultralong CDR3
sequence (BLV5D3) 24Ser Val Thr Gln Arg Thr His Val Ser Arg Ser Cys
Pro Asp Gly Cys1 5 10 15Ser Asp Gly Asp Gly Cys Val Asp Gly Cys Cys
Cys Ser Ala Tyr Arg 20 25 30Cys Tyr Thr Pro Gly Val Arg Asp Leu Ser
Cys Thr Ser Tyr Ser Ile 35 40 45Thr Tyr Thr Tyr Glu Trp Asn Val Asp
50 552558PRTArtificial sequenceSynthesized ultralong CDR3 sequence
(BLV8C11) 25Thr Val His Gln Lys Thr Thr Arg Lys Thr Cys Cys Ser Asp
Ala Tyr1 5 10 15Arg Tyr Asp Ser Gly Cys Gly Ser Gly Cys Asp Cys Cys
Gly Ala Asp 20 25 30Cys Tyr Val Phe Gly Ala Cys Thr Phe Gly Leu Asp
Ser Ser Tyr Ser 35 40 45Tyr Ile Tyr Ile Tyr Gln Trp Tyr Val Asp 50
552656PRTArtificial sequenceSynthesized ultralong CDR3 sequence
(BF4E9) 26Thr Val His Gln Ile Phe Cys Pro Asp Gly Tyr Ser Tyr Gly
Tyr Gly1 5 10 15Cys Gly Tyr Gly Tyr Gly Cys Ser Gly Tyr Asp Cys Tyr
Gly Tyr Gly 20 25 30Gly Tyr Gly Tyr Gly Gly Tyr Gly Gly Tyr Ser Ser
Tyr Ser Tyr Ser 35 40 45Tyr Ser Tyr Glu Tyr Tyr Gly Asp 50
552748PRTArtificial sequenceSynthesized ultralong CDR3 sequence
(BF1H1) 27Thr Val His Pro Ser Pro Asp Gly Tyr Ser Tyr Gly Tyr Gly
Cys Gly1 5 10 15Tyr Gly Tyr Gly Cys Ser Gly Tyr Asp Cys Tyr Gly Tyr
Gly Gly Tyr 20 25 30Gly Tyr Gly Gly Tyr Gly Gly Tyr Ser Ser Tyr Ser
Tyr Ser Tyr Ser 35 40 452848PRTArtificial sequenceSynthesized
ultralong CDR3 sequence (F18) 28Thr Val His Gln Ile Arg Cys Pro Asp
Gly Tyr Gly Tyr Gly Tyr Gly1 5 10 15Cys Gly Tyr Gly Ser Tyr Gly Tyr
Ser Gly Tyr Asp Cys Tyr Gly Tyr 20 25 30Gly Gly Tyr Gly Gly Tyr Gly
Gly Tyr Gly Gly Tyr Ser Ser Tyr Ser 35 40 4529100PRTArtificial
sequenceSynthesized exemplary heavy chain variable region sequence
suitable for modification or use with an ultralong CDR3 sequence
(VH-UL) 29Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro
Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu
Ser Asp Lys 20 25 30Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala
Leu Glu Trp Leu 35 40 45Gly Gly Ile Asp Thr Gly Gly Ser Thr Gly Tyr
Asn Pro Gly Leu Lys 50 55 60Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser
Lys Ser Gln Val Ser Leu65 70 75 80Ser Val Ser Ser Val Thr Thr Glu
Asp Ser Ala Thr Tyr Tyr Cys Thr 85 90 95Thr Val His Gln
1003097PRTArtificial sequenceSynthesized exemplary light chain
variable region sequence suitable for modification or use with an
ultralong CDR3 sequence (VL1X) 30Gln Ala Val Leu Thr Gln Pro Ser
Ser Val Ser Gly Ser Leu Gly Gln1 5 10 15Arg Val Ser Ile Thr Cys Ser
Gly Ser Ser Ser Asn Val Gly Asn Gly 20 25 30Tyr Val Ser Trp Tyr Gln
Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu 35 40 45Ile Tyr Gly Asp Thr
Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Arg Ser
Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Ala Glu
Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu Asp Ser Ser 85 90
95Ser3199PRTArtificial sequenceSynthesized exemplary heavy chain
variable region sequence suitable for modification or use with an
ultralong CDR 3 sequence (VH4-39) 31Gln Leu Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30Ser Tyr Tyr Trp Gly Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45Trp Ile Gly Ser Ile
Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe65 70 75 80Ser Leu
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95Cys
Ala Arg3296PRTArtificial sequenceSynthesized Human germline heavy
chain variable region sequence 4-59*03 32Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30Tyr Trp Ser Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile
Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 953397PRTArtificial sequenceSynthesized Human germline heavy
chain variable region sequence 4-34*02 33Gln Val Gln Leu Gln Gln
Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile
Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg3497PRTArtificial sequenceSynthesized Human germline
heavy chain variable region sequence 4-34*09 34Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu
Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg35110PRTArtificial sequenceSynthsized Bovine light chain
variable region sequence BLV1H12 35Gln Ala Val Leu Asn Gln Pro Ser
Ser Val Ser Gly Ser Leu Gly Gln1 5 10 15Arg Val Ser Ile Thr Cys Ser
Gly Ser Ser Ser Asn Val Gly Asn Gly 20 25 30Tyr Val Ser Trp Tyr Gln
Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu 35 40 45Ile Tyr Gly Asp Thr
Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Arg Ser
Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Ala Glu
Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu Asp Ser Ser 85 90 95Ser
Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu 100 105
1103698PRTArtificial sequenceSynthesized Human germline light chain
variable region sequence VL1-47 36Gln Ser Val Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser
Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Tyr Val Tyr Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Arg Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser
Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg65 70 75 80Ser Glu
Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Ser
Gly3799PRTArtificial sequenceSynthesized Human germline light chain
variable region sequence VL1-40*1 37Gln Ser Val Leu Thr Gln Pro Pro
Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr
Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Asp Val His Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr Gly Asn
Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys
Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75 80Gln Ala
Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95Leu
Ser Gly3898PRTArtificial sequenceSynthesized Human germline light
chain variable region sequence VL1-51*01 38Gln Ser Val Leu Thr Gln
Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5 10 15Lys Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30Tyr Val Ser Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Asp
Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser
Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln65 70 75
80Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu
85 90 95Ser Ala3999PRTArtificial sequenceSynthesized Human germline
light chain variable region sequence VL2-18*02 39Gln Ser Ala Leu
Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Val Thr
Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30Asn Arg
Val Ser Trp Tyr Gln Gln Pro Pro Gly Thr Ala Pro Lys Leu 35 40 45Met
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55
60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65
70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser
Ser 85 90 95Ser Thr Phe404PRTArtificial sequenceSynthesized
optional sequence in ultralong CDR3MISC_FEATURE(1)..(1)X is
threonine or serineMISC_FEATURE(3)..(3)X is histidine or
threonineMISC_FEATURE(4)..(4)X is glutamine or proline 40Xaa Val
Xaa
Xaa14128PRTArtificial sequenceSynthesized cysteine motif
1misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(17)Xaa can be any naturally occurring amino
acidmisc_feature(19)..(19)Xaa can be any naturally occurring amino
acidmisc_feature(21)..(27)Xaa can be any naturally occurring amino
acid 41Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 20
254243PRTArtificial sequenceSynthesized cysteine motif
2misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(26)Xaa can be any naturally occurring amino
acidmisc_feature(28)..(42)Xaa can be any naturally occurring amino
acid 42Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35
404321PRTArtificial sequenceSynthesized cysteine motif
3misc_feature(2)..(12)Xaa can be any naturally occurring amino
acidmisc_feature(14)..(14)Xaa can be any naturally occurring amino
acidmisc_feature(16)..(20)Xaa can be any naturally occurring amino
acid 43Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys
Xaa1 5 10 15Xaa Xaa Xaa Xaa Cys 204435PRTArtificial
sequenceSynthesized cysteine motif 4misc_feature(2)..(12)Xaa can be
any naturally occurring amino acidmisc_feature(14)..(18)Xaa can be
any naturally occurring amino acidmisc_feature(20)..(24)Xaa can be
any naturally occurring amino acidmisc_feature(26)..(26)Xaa can be
any naturally occurring amino acidmisc_feature(28)..(34)Xaa can be
any naturally occurring amino acid 44Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Cys
354541PRTArtificial sequenceSynthesized cysteine motif
5misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(26)Xaa can be any naturally occurring amino
acidmisc_feature(28)..(40)Xaa can be any naturally occurring amino
acid 45Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35
404634PRTArtificial sequenceSynthesized cysteine motif
6misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(17)Xaa can be any naturally occurring amino
acidmisc_feature(19)..(19)Xaa can be any naturally occurring amino
acidmisc_feature(21)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(33)Xaa can be any naturally occurring amino
acid 46Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Cys4736PRTArtificial sequenceSynthesized
cysteine motif 7misc_feature(2)..(11)Xaa can be any naturally
occurring amino acidmisc_feature(13)..(18)Xaa can be any naturally
occurring amino acidmisc_feature(20)..(25)Xaa can be any naturally
occurring amino acidmisc_feature(27)..(27)Xaa can be any naturally
occurring amino acidmisc_feature(29)..(35)Xaa can be any naturally
occurring amino acid 47Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys
Xaa Cys Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Cys 354836PRTArtificial
sequenceSynthesized cysteine motif 8misc_feature(2)..(11)Xaa can be
any naturally occurring amino acidmisc_feature(13)..(16)Xaa can be
any naturally occurring amino acidmisc_feature(18)..(24)Xaa can be
any naturally occurring amino acidmisc_feature(26)..(26)Xaa can be
any naturally occurring amino acidmisc_feature(28)..(35)Xaa can be
any naturally occurring amino acid 48Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Cys
354935PRTArtificial sequenceSynthesized cysteine motif
9misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(26)Xaa can be any naturally occurring amino
acidmisc_feature(28)..(34)Xaa can be any naturally occurring amino
acid 49Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Cys 355033PRTArtificial sequenceSynthesized
cysteine motif 10misc_feature(2)..(14)Xaa can be any naturally
occurring amino acidmisc_feature(16)..(23)Xaa can be any naturally
occurring amino acidmisc_feature(25)..(32)Xaa can be any naturally
occurring amino acid 50Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Cys5135PRTArtificial
sequenceSynthesized cysteine motif 11misc_feature(2)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(18)Xaa can
be any naturally occurring amino acidmisc_feature(20)..(24)Xaa can
be any naturally occurring amino acidmisc_feature(26)..(26)Xaa can
be any naturally occurring amino acidmisc_feature(28)..(34)Xaa can
be any naturally occurring amino acid 51Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Cys
355224PRTArtificial sequenceSynthesized cysteine motif
12misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(17)Xaa can be any naturally occurring amino
acidmisc_feature(19)..(23)Xaa can be any naturally occurring amino
acid 52Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys 205335PRTArtificial
sequenceSynthesized cysteine motif 13misc_feature(2)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(17)Xaa can
be any naturally occurring amino acidmisc_feature(19)..(24)Xaa can
be any naturally occurring amino acidmisc_feature(26)..(26)Xaa can
be any naturally occurring amino acidmisc_feature(28)..(34)Xaa can
be any naturally occurring amino acid 53Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Cys
355443PRTArtificial sequenceSynthesized cysteine motif
14misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(32)Xaa can be any naturally occurring amino
acidmisc_feature(34)..(42)Xaa can be any naturally occurring amino
acid 54Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35
405535PRTArtificial sequenceSynthesized cysteine motif
15misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(26)Xaa can be any naturally occurring amino
acidmisc_feature(28)..(34)Xaa can be any naturally occurring amino
acid 55Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Cys 355637PRTArtificial sequenceSynthesized
cysteine motif 16misc_feature(2)..(11)Xaa can be any naturally
occurring amino acidmisc_feature(13)..(18)Xaa can be any naturally
occurring amino acidmisc_feature(20)..(24)Xaa can be any naturally
occurring amino acidmisc_feature(26)..(26)Xaa can be any naturally
occurring amino acidmisc_feature(28)..(36)Xaa can be any naturally
occurring amino acid 56Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa
Cys Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Cys
355737PRTArtificial sequenceSynthesized cysteine motif
17misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(13)Xaa can be any naturally occurring amino
acidmisc_feature(15)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(36)Xaa can be any naturally occurring amino
acid 57Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Cys 355845PRTArtificial
sequenceSynthesized cysteine motif 18misc_feature(2)..(8)Xaa can be
any naturally occurring amino acidmisc_feature(10)..(12)Xaa can be
any naturally occurring amino acidmisc_feature(14)..(19)Xaa can be
any naturally occurring amino acidmisc_feature(21)..(25)Xaa can be
any naturally occurring amino acidmisc_feature(27)..(27)Xaa can be
any naturally occurring amino acidmisc_feature(29)..(33)Xaa can be
any naturally occurring amino acidmisc_feature(35)..(44)Xaa can be
any naturally occurring amino acid 58Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 20 25 30Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 455934PRTArtificial
sequenceSynthesized cysteine motif 19misc_feature(2)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(13)Xaa can
be any naturally occurring amino acidmisc_feature(15)..(18)Xaa can
be any naturally occurring amino acidmisc_feature(20)..(24)Xaa can
be any naturally occurring amino acidmisc_feature(26)..(26)Xaa can
be any naturally occurring amino acidmisc_feature(28)..(29)Xaa can
be any naturally occurring amino acidmisc_feature(31)..(33)Xaa can
be any naturally occurring amino acid 59Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys Xaa Xaa Cys Xaa Xaa 20 25 30Xaa
Cys6026PRTArtificial sequenceSynthesized cysteine motif
20misc_feature(2)..(17)Xaa can be any naturally occurring amino
acidmisc_feature(19)..(23)Xaa can be any naturally occurring amino
acidmisc_feature(25)..(25)Xaa can be any naturally occurring amino
acid 60Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 20
256126PRTArtificial sequenceSynthesized cysteine motif
21misc_feature(2)..(7)Xaa can be any naturally occurring amino
acidmisc_feature(9)..(12)Xaa can be any naturally occurring amino
acidmisc_feature(14)..(14)Xaa can be any naturally occurring amino
acidmisc_feature(16)..(19)Xaa can be any naturally occurring amino
acidmisc_feature(21)..(25)Xaa can be any naturally occurring amino
acid 61Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Cys
Xaa1 5 10 15Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys 20
256235PRTArtificial sequenceSynthesized cysteine motif
22misc_feature(2)..(12)Xaa can be any naturally occurring amino
acidmisc_feature(14)..(17)Xaa can be any naturally occurring amino
acidmisc_feature(19)..(23)Xaa can be any naturally occurring amino
acidmisc_feature(25)..(30)Xaa can be any naturally occurring amino
acidmisc_feature(32)..(34)Xaa can be any naturally occurring amino
acid 62Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa 20 25 30Xaa Xaa Cys 356326PRTArtificial sequenceSynthesized
cysteine motif 23misc_feature(2)..(9)Xaa can be any naturally
occurring amino acidmisc_feature(11)..(12)Xaa can be any naturally
occurring amino acidmisc_feature(14)..(19)Xaa can be any naturally
occurring amino acidmisc_feature(21)..(25)Xaa can be any naturally
occurring amino acid 63Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Cys Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys
20 256437PRTArtificial sequenceSynthesized cysteine motif
24misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(17)Xaa can be any naturally occurring amino
acidmisc_feature(19)..(23)Xaa can be any naturally occurring amino
acidmisc_feature(25)..(25)Xaa can be any naturally occurring amino
acidmisc_feature(27)..(36)Xaa can be any naturally occurring amino
acid 64Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Cys 356528PRTArtificial
sequenceSynthesized cysteine motif 25misc_feature(2)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(13)Xaa can
be any naturally occurring amino acidmisc_feature(15)..(20)Xaa can
be any naturally occurring amino acidmisc_feature(22)..(25)Xaa can
be any naturally occurring amino acidmisc_feature(27)..(27)Xaa can
be any naturally occurring amino acid 65Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Cys Xaa Cys 20 256629PRTArtificial sequenceSynthesized
cysteine motif 26misc_feature(2)..(11)Xaa can be any naturally
occurring amino acidmisc_feature(13)..(17)Xaa can be any naturally
occurring amino acidmisc_feature(19)..(23)Xaa can be any naturally
occurring amino acidmisc_feature(25)..(25)Xaa can be any naturally
occurring amino acidmisc_feature(27)..(28)Xaa can be any naturally
occurring amino acid 66Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys
Xaa Xaa Cys 20 256727PRTArtificial sequenceSynthesized cysteine
motif 27misc_feature(2)..(15)Xaa can be any naturally occurring
amino acidmisc_feature(17)..(18)Xaa can be any naturally occurring
amino acidmisc_feature(20)..(22)Xaa can be any naturally occurring
amino acidmisc_feature(24)..(24)Xaa can be any naturally occurring
amino acidmisc_feature(26)..(26)Xaa can be any naturally occurring
amino acid 67Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Cys Xaa Cys 20
256825PRTArtificial sequenceSynthesized cysteine motif
28misc_feature(2)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(22)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(24)Xaa can be any naturally occurring amino
acid 68Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 20
256938PRTArtificial sequenceSynthesized cysteine motif
29misc_feature(2)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(12)Xaa can be any naturally occurring amino
acidmisc_feature(14)..(22)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(25)Xaa can be any naturally occurring amino
acidmisc_feature(27)..(37)Xaa can be any naturally occurring amino
acid 69Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Cys 357038PRTArtificial
sequenceSynthesized cysteine motif 30misc_feature(2)..(7)Xaa can be
any naturally occurring amino acidmisc_feature(9)..(12)Xaa can be
any naturally occurring amino acidmisc_feature(14)..(18)Xaa can be
any naturally occurring amino acidmisc_feature(20)..(24)Xaa can be
any naturally occurring amino acidmisc_feature(26)..(37)Xaa can be
any naturally occurring amino acid 70Cys Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa
Cys 357138PRTArtificial sequenceSynthesized cysteine motif
31misc_feature(2)..(8)Xaa can be any naturally occurring amino
acidmisc_feature(10)..(12)Xaa can be any naturally occurring amino
acidmisc_feature(14)..(14)Xaa can be any naturally occurring amino
acidmisc_feature(16)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(23)..(27)Xaa can be any naturally occurring amino
acidmisc_feature(29)..(37)Xaa can be any naturally occurring amino
acid 71Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Cys
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Cys 357225PRTArtificial
sequenceSynthesized cysteine motif 32misc_feature(2)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(18)Xaa can
be any naturally occurring amino acidmisc_feature(20)..(24)Xaa can
be any naturally occurring amino acid 72Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys 20 257335PRTArtificial sequenceSynthesized cysteine
motif 33misc_feature(2)..(8)Xaa can be any naturally occurring
amino acidmisc_feature(10)..(12)Xaa can be any naturally occurring
amino acidmisc_feature(14)..(18)Xaa can be any naturally occurring
amino acidmisc_feature(20)..(24)Xaa can be any naturally occurring
amino acidmisc_feature(26)..(34)Xaa can be any naturally occurring
amino acid 73Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys
Xaa Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Cys 357420PRTArtificial
sequenceSynthesized cysteine motif 34misc_feature(2)..(8)Xaa can be
any naturally occurring amino acidmisc_feature(10)..(14)Xaa can be
any naturally occurring amino acidmisc_feature(16)..(16)Xaa can be
any naturally occurring amino acidmisc_feature(18)..(19)Xaa can be
any naturally occurring amino acid 74Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa1 5 10 15Cys Xaa Xaa Cys
207521PRTArtificial sequenceSynthesized cysteine motif
35misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(13)Xaa can be any naturally occurring amino
acidmisc_feature(15)..(20)Xaa can be any naturally occurring amino
acid 75Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa
Xaa1 5 10 15Xaa Xaa Xaa Xaa Cys 207643PRTArtificial
sequenceSynthesized cysteine motif 36misc_feature(2)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(15)Xaa can
be any naturally occurring amino acidmisc_feature(17)..(19)Xaa can
be any naturally occurring amino acidmisc_feature(21)..(25)Xaa can
be any naturally occurring amino acidmisc_feature(27)..(33)Xaa can
be any naturally occurring amino acidmisc_feature(35)..(35)Xaa can
be any naturally occurring amino acidmisc_feature(37)..(42)Xaa can
be any naturally occurring amino acid 76Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys1 5 10 15Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Cys Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Cys 35 407739PRTArtificial sequenceSynthesized
cysteine motif 37misc_feature(2)..(11)Xaa can be any naturally
occurring amino acidmisc_feature(13)..(16)Xaa can be any naturally
occurring amino acidmisc_feature(18)..(22)Xaa can be any naturally
occurring amino acidmisc_feature(24)..(35)Xaa can be any naturally
occurring amino acidmisc_feature(37)..(38)Xaa can be any naturally
occurring amino acid 77Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Cys Xaa Xaa Cys
357843PRTArtificial sequenceSynthesized cysteine motif
38misc_feature(2)..(13)Xaa can be any naturally occurring amino
acidmisc_feature(15)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(26)Xaa can be any naturally occurring amino
acidmisc_feature(28)..(28)Xaa can be any naturally occurring amino
acidmisc_feature(30)..(38)Xaa can be any naturally occurring amino
acidmisc_feature(40)..(42)Xaa can be any naturally occurring amino
acid 78Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Cys Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 35
407941PRTArtificial sequenceSynthesized cysteine motif
39misc_feature(2)..(13)Xaa can be any naturally occurring amino
acidmisc_feature(15)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(37)Xaa can be any naturally occurring amino
acidmisc_feature(39)..(40)Xaa can be any naturally occurring amino
acid 79Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 35
408039PRTArtificial sequenceSynthesized cysteine motif
40misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(26)Xaa can be any naturally occurring amino
acidmisc_feature(28)..(38)Xaa can be any naturally occurring amino
acid 80Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Cys 358143PRTArtificial
sequenceSynthesized cysteine motif 41misc_feature(2)..(17)Xaa can
be any naturally occurring amino acidmisc_feature(19)..(23)Xaa can
be any naturally occurring amino acidmisc_feature(25)..(25)Xaa can
be any naturally occurring amino acidmisc_feature(27)..(27)Xaa can
be any naturally occurring amino acidmisc_feature(29)..(42)Xaa can
be any naturally occurring amino acid 81Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa
Xaa Cys Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys 35 408236PRTArtificial sequenceSynthesized
cysteine motif 42misc_feature(2)..(11)Xaa can be any naturally
occurring amino acidmisc_feature(13)..(17)Xaa can be any naturally
occurring amino acidmisc_feature(19)..(19)Xaa can be any naturally
occurring amino acidmisc_feature(21)..(28)Xaa can be any naturally
occurring amino acidmisc_feature(30)..(35)Xaa can be any naturally
occurring amino acid 82Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Cys 358337PRTArtificial
sequenceSynthesized cysteine motif 43misc_feature(2)..(13)Xaa can
be any naturally occurring amino acidmisc_feature(15)..(18)Xaa can
be any naturally occurring amino acidmisc_feature(20)..(24)Xaa can
be any naturally occurring amino acidmisc_feature(26)..(33)Xaa can
be any naturally occurring amino acidmisc_feature(35)..(36)Xaa can
be any naturally occurring amino acid 83Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Cys Xaa Xaa Cys
358437PRTArtificial sequenceSynthesized cysteine motif
44misc_feature(2)..(13)Xaa can be any naturally occurring amino
acidmisc_feature(15)..(19)Xaa can be any naturally occurring amino
acidmisc_feature(21)..(25)Xaa can be any naturally occurring amino
acidmisc_feature(27)..(27)Xaa can be any naturally occurring amino
acidmisc_feature(29)..(36)Xaa can be any naturally occurring amino
acid 84Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa1 5 10 15Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Cys 358544PRTArtificial
sequenceSynthesized cysteine motif 45misc_feature(2)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(18)Xaa can
be any naturally occurring amino acidmisc_feature(20)..(24)Xaa can
be any naturally occurring amino acidmisc_feature(26)..(26)Xaa can
be any naturally occurring amino acidmisc_feature(28)..(31)Xaa can
be any naturally occurring amino acidmisc_feature(33)..(33)Xaa can
be any naturally occurring amino acidmisc_feature(35)..(43)Xaa can
be any naturally occurring amino acid 85Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys 20 25 30Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 408636PRTArtificial
sequenceSynthesized cysteine motif 46misc_feature(2)..(12)Xaa can
be any naturally occurring amino acidmisc_feature(14)..(17)Xaa can
be any naturally occurring amino acidmisc_feature(19)..(23)Xaa can
be any naturally occurring amino acidmisc_feature(25)..(32)Xaa can
be any naturally occurring amino acidmisc_feature(34)..(35)Xaa can
be any naturally occurring amino acid 86Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Cys Xaa Xaa Cys
358737PRTArtificial sequenceSynthesized cysteine motif
47misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(33)Xaa can be any naturally occurring amino
acidmisc_feature(35)..(36)Xaa can be any naturally occurring amino
acid 87Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Cys Xaa Xaa Cys 358836PRTArtificial
sequenceSynthesized cysteine motif 48misc_feature(2)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(18)Xaa can
be any naturally occurring amino acidmisc_feature(20)..(24)Xaa can
be any naturally occurring amino acidmisc_feature(26)..(26)Xaa can
be any naturally occurring amino acidmisc_feature(28)..(35)Xaa can
be any naturally occurring amino acid 88Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Cys
358943PRTArtificial sequenceSynthesized cysteine motif
49misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(26)Xaa can be any naturally occurring amino
acidmisc_feature(28)..(30)Xaa can be any naturally occurring amino
acidmisc_feature(32)..(39)Xaa can be any naturally occurring amino
acidmisc_feature(41)..(42)Xaa can be any naturally occurring amino
acid 89Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa
Cys Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 35
409038PRTArtificial sequenceSynthesized cysteine motif
50misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(28)Xaa can be any naturally occurring amino
acidmisc_feature(30)..(37)Xaa can be any naturally occurring amino
acid 90Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Cys 359143PRTArtificial
sequenceSynthesized cysteine motif 51misc_feature(2)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(18)Xaa can
be any naturally occurring amino acidmisc_feature(20)..(24)Xaa can
be any naturally occurring amino acidmisc_feature(26)..(26)Xaa can
be any naturally occurring amino acidmisc_feature(28)..(29)Xaa can
be any naturally occurring amino acidmisc_feature(31)..(36)Xaa can
be any naturally occurring amino acidmisc_feature(38)..(42)Xaa can
be any naturally occurring amino acid 91Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys Xaa Xaa Cys Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Cys 35 409234PRTArtificial sequenceSynthesized
cysteine motif 52misc_feature(2)..(8)Xaa can be any naturally
occurring amino acidmisc_feature(10)..(15)Xaa can be any naturally
occurring amino acidmisc_feature(17)..(19)Xaa can be any naturally
occurring amino acidmisc_feature(21)..(23)Xaa can be any naturally
occurring amino acidmisc_feature(25)..(33)Xaa can be any naturally
occurring amino acid 92Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Cys1 5 10 15Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Cys9339PRTArtificial
sequenceSynthesized cysteine motif 53misc_feature(2)..(10)Xaa can
be any naturally occurring amino acidmisc_feature(12)..(19)Xaa can
be any naturally occurring amino acidmisc_feature(21)..(25)Xaa can
be any naturally occurring amino acidmisc_feature(27)..(32)Xaa can
be any naturally occurring amino acidmisc_feature(34)..(38)Xaa can
be any naturally occurring amino acid 93Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Cys Xaa Xaa Xaa Xaa
Xaa Cys 359446PRTArtificial sequenceSynthesized cysteine motif
54misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(14)Xaa can be any naturally occurring amino
acidmisc_feature(16)..(17)Xaa can be any naturally occurring amino
acidmisc_feature(19)..(25)Xaa can be any naturally occurring amino
acidmisc_feature(27)..(27)Xaa can be any naturally occurring amino
acidmisc_feature(29)..(39)Xaa can be any naturally occurring amino
acidmisc_feature(41)..(45)Xaa can be any naturally occurring amino
acid 94Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Cys Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Cys Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Cys 35 40 459544PRTArtificial
sequenceSynthesized cysteine motif 55misc_feature(2)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(18)Xaa can
be any naturally occurring amino acidmisc_feature(20)..(24)Xaa can
be any naturally occurring amino acidmisc_feature(26)..(26)Xaa can
be any naturally occurring amino acidmisc_feature(28)..(29)Xaa can
be any naturally occurring amino acidmisc_feature(31)..(38)Xaa can
be any naturally occurring amino acidmisc_feature(40)..(43)Xaa can
be any naturally occurring amino acid 95Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys Xaa Xaa Cys Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Cys 35 409642PRTArtificial
sequenceSynthesized cysteine motif 56misc_feature(3)..(5)Xaa can be
any naturally occurring amino acidmisc_feature(7)..(7)Xaa can be
any naturally occurring amino acidmisc_feature(9)..(11)Xaa can be
any naturally occurring amino acidmisc_feature(13)..(14)Xaa can be
any naturally occurring amino acidmisc_feature(17)..(17)Xaa can be
any naturally occurring amino acidmisc_feature(19)..(23)Xaa can be
any naturally occurring amino acidmisc_feature(25)..(33)Xaa can be
any naturally occurring amino acidmisc_feature(35)..(39)Xaa can be
any naturally occurring amino acidmisc_feature(41)..(41)Xaa can be
any naturally occurring amino acid 96Cys Cys Xaa Xaa Xaa Cys Xaa
Cys Xaa Xaa Xaa Cys Xaa Xaa Cys Cys1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys 35 409739PRTArtificial sequenceSynthesized
cysteine motif 57misc_feature(2)..(7)Xaa can be any naturally
occurring amino acidmisc_feature(9)..(10)Xaa can be any naturally
occurring amino acidmisc_feature(12)..(16)Xaa can be any naturally
occurring amino acidmisc_feature(18)..(21)Xaa can be any naturally
occurring amino acidmisc_feature(24)..(24)Xaa can be any naturally
occurring amino acidmisc_feature(26)..(29)Xaa can be any naturally
occurring amino acidmisc_feature(31)..(36)Xaa can be any naturally
occurring amino acidmisc_feature(38)..(38)Xaa can be any naturally
occurring amino acid 97Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Cys Xaa
Xaa Xaa Xaa Cys Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Cys Xaa Cys
359838PRTArtificial sequenceSynthesized cysteine motif
58misc_feature(2)..(8)Xaa can be any naturally occurring amino
acidmisc_feature(10)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(25)..(28)Xaa can be any naturally occurring amino
acidmisc_feature(30)..(35)Xaa can be any naturally occurring amino
acidmisc_feature(37)..(37)Xaa can be any naturally occurring amino
acid 98Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Cys Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Cys Xaa Cys 359936PRTArtificial
sequenceSynthesized cysteine motif 59misc_feature(2)..(10)Xaa can
be any naturally occurring amino acidmisc_feature(12)..(14)Xaa can
be any naturally occurring amino acidmisc_feature(16)..(16)Xaa can
be any naturally occurring amino acidmisc_feature(18)..(19)Xaa can
be any naturally occurring amino acidmisc_feature(21)..(21)Xaa can
be any naturally occurring amino acidmisc_feature(25)..(30)Xaa can
be any naturally occurring amino acidmisc_feature(32)..(35)Xaa can
be any naturally occurring amino acid 99Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa1 5 10 15Cys Xaa Xaa Cys Xaa Cys
Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 20 25 30Xaa Xaa Xaa Cys
3510039PRTArtificial sequenceSynthesized cysteine motif
60misc_feature(2)..(6)Xaa can be any naturally occurring amino
acidmisc_feature(8)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(12)Xaa can be any naturally occurring amino
acidmisc_feature(14)..(17)Xaa can be any naturally occurring amino
acidmisc_feature(19)..(22)Xaa can be any naturally occurring amino
acidmisc_feature(25)..(34)Xaa can be any naturally occurring amino
acidmisc_feature(36)..(37)Xaa can be any naturally occurring amino
acid 100Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa
Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Cys Xaa Xaa Cys Cys 3510138PRTArtificial
sequenceSynthesized cysteine motif 61misc_feature(2)..(6)Xaa can be
any naturally occurring amino acidmisc_feature(8)..(8)Xaa can be
any naturally occurring amino acidmisc_feature(10)..(10)Xaa can be
any naturally occurring amino acidmisc_feature(12)..(12)Xaa can be
any naturally occurring amino acidmisc_feature(14)..(16)Xaa can be
any naturally occurring amino acidmisc_feature(19)..(21)Xaa can be
any naturally occurring amino acidmisc_feature(23)..(26)Xaa can be
any naturally occurring amino acidmisc_feature(28)..(37)Xaa can be
any naturally occurring amino acid 101Cys Xaa Xaa Xaa Xaa Xaa Cys
Xaa Cys Xaa Cys Xaa Cys Xaa Xaa Xaa1 5 10 15Cys Cys Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa
Cys 3510237PRTArtificial sequenceSynthesized cysteine motif
62misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(14)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(25)..(29)Xaa can be any naturally occurring amino
acidmisc_feature(31)..(36)Xaa can be any naturally occurring amino
acid 102Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Cys Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Cys Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Cys 3510336PRTArtificial
sequenceSynthesized cysteine motif 63misc_feature(3)..(10)Xaa can
be any naturally occurring amino acidmisc_feature(12)..(16)Xaa can
be any naturally occurring amino acidmisc_feature(18)..(21)Xaa can
be any naturally occurring amino acidmisc_feature(23)..(25)Xaa can
be any naturally occurring amino acidmisc_feature(27)..(30)Xaa can
be any naturally occurring amino acidmisc_feature(33)..(33)Xaa can
be any naturally occurring amino acidmisc_feature(35)..(35)Xaa can
be any naturally occurring amino acid 103Cys Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Cys 20 25 30Xaa Cys Xaa Cys
3510441PRTArtificial sequenceSynthesized cysteine motif
64misc_feature(3)..(8)Xaa can be any naturally occurring amino
acidmisc_feature(11)..(15)Xaa can be any naturally occurring amino
acidmisc_feature(19)..(22)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(27)Xaa can be any naturally occurring amino
acidmisc_feature(29)..(40)Xaa can be any naturally occurring amino
acid 104Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa
Cys1 5 10 15Cys Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35
4010536PRTArtificial sequenceSynthesized cysteine motif
65misc_feature(2)..(7)Xaa can be any naturally occurring amino
acidmisc_feature(9)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(14)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(23)..(27)Xaa can be any naturally occurring amino
acidmisc_feature(29)..(31)Xaa can be any naturally occurring amino
acidmisc_feature(33)..(35)Xaa can be any naturally occurring amino
acid 105Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Cys
Cys1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Cys 20 25 30Xaa Xaa Xaa Cys 3510639PRTArtificial
sequenceSynthesized cysteine motif 66misc_feature(2)..(4)Xaa can be
any naturally occurring amino acidmisc_feature(6)..(10)Xaa can be
any naturally occurring amino acidmisc_feature(12)..(17)Xaa can be
any naturally occurring amino acidmisc_feature(19)..(22)Xaa can be
any naturally occurring amino acidmisc_feature(25)..(25)Xaa can be
any naturally occurring amino acidmisc_feature(27)..(31)Xaa can be
any naturally occurring amino acidmisc_feature(33)..(36)Xaa can be
any naturally occurring amino acidmisc_feature(38)..(38)Xaa can be
any naturally occurring amino acid 106Cys Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa
Cys Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys 20 25 30Xaa Xaa Xaa Xaa Cys
Xaa Cys 3510741PRTArtificial sequenceSynthesized cysteine motif
67misc_feature(2)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(23)..(23)Xaa can be any naturally occurring amino
acidmisc_feature(25)..(35)Xaa can be any naturally occurring amino
acidmisc_feature(37)..(38)Xaa can be any naturally occurring amino
acidmisc_feature(40)..(40)Xaa can be any naturally occurring amino
acid 107Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Cys 35
4010836PRTArtificial sequenceSynthesized cysteine motif
68misc_feature(2)..(6)Xaa can be any naturally occurring amino
acidmisc_feature(8)..(9)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(26)Xaa can be any naturally occurring amino
acidmisc_feature(29)..(35)Xaa can be any naturally occurring amino
acid 108Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Cys Cys Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Cys 3510938PRTArtificial
sequenceSynthesized cysteine motif 69misc_feature(2)..(6)Xaa can be
any naturally occurring amino acidmisc_feature(8)..(12)Xaa can be
any naturally occurring amino acidmisc_feature(14)..(16)Xaa can be
any naturally occurring amino acidmisc_feature(18)..(19)Xaa can be
any naturally occurring amino acidmisc_feature(21)..(21)Xaa can be
any naturally occurring amino acidmisc_feature(24)..(27)Xaa can be
any naturally occurring amino acidmisc_feature(29)..(35)Xaa can be
any naturally occurring amino acidmisc_feature(37)..(37)Xaa can be
any naturally occurring amino acid 109Cys Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5 10 15Cys Xaa Xaa Cys Xaa Cys
Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Cys Xaa
Cys 3511037PRTArtificial sequenceSynthesized cysteine motif
70misc_feature(2)..(4)Xaa can be any naturally occurring amino
acidmisc_feature(6)..(12)Xaa can be any naturally occurring amino
acidmisc_feature(14)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(27)Xaa can be any naturally occurring amino
acidmisc_feature(29)..(33)Xaa can be any naturally occurring amino
acidmisc_feature(35)..(36)Xaa can be any naturally occurring amino
acid 110Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Cys Xaa Xaa Cys Xaa Xaa
Xaa Xaa 20 25 30Xaa Cys Xaa Xaa Cys 3511138PRTArtificial
sequenceSynthesized cysteine motif 71misc_feature(2)..(10)Xaa can
be any naturally occurring amino acidmisc_feature(12)..(14)Xaa can
be any naturally occurring amino acidmisc_feature(16)..(16)Xaa can
be any naturally occurring amino acidmisc_feature(18)..(21)Xaa can
be any naturally occurring amino acidmisc_feature(24)..(28)Xaa can
be any naturally occurring amino acidmisc_feature(32)..(37)Xaa can
be any naturally occurring amino acid 111Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa
Cys Cys Xaa Xaa Xaa Xaa Xaa Cys Cys Cys Xaa 20 25 30Xaa Xaa Xaa Xaa
Xaa Cys 3511238PRTArtificial sequenceSynthesized cysteine motif
72misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(14)Xaa can be any naturally occurring amino
acidmisc_feature(16)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(19)Xaa can be any naturally occurring amino
acidmisc_feature(21)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(29)Xaa can be any naturally occurring amino
acidmisc_feature(31)..(33)Xaa can be any naturally occurring amino
acidmisc_feature(35)..(37)Xaa can be any naturally occurring amino
acid 112Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys
Xaa1 5 10 15Cys Xaa Xaa Cys Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa 20 25 30Xaa Cys Xaa Xaa Xaa Cys 3511333PRTArtificial
sequenceSynthesized cysteine motif 73misc_feature(2)..(9)Xaa can be
any naturally occurring amino acidmisc_feature(12)..(12)Xaa can be
any naturally occurring amino acidmisc_feature(14)..(16)Xaa can be
any naturally occurring amino acidmisc_feature(19)..(21)Xaa can be
any naturally occurring amino acidmisc_feature(23)..(23)Xaa can be
any naturally occurring amino acidmisc_feature(25)..(27)Xaa can be
any naturally occurring amino acidmisc_feature(29)..(32)Xaa can be
any naturally occurring amino acid 113Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Cys Xaa Cys Xaa Xaa Xaa1 5 10 15Cys Cys Xaa Xaa Xaa Cys
Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 20 25
30Cys11434PRTArtificial sequenceSynthesized cysteine motif
74misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(19)Xaa can be any naturally occurring amino
acidmisc_feature(21)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(29)Xaa can be any naturally occurring amino
acidmisc_feature(31)..(33)Xaa can be any naturally occurring amino
acid 114Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Cys Xaa Cys Cys Xaa Cys Xaa Xaa Xaa Xaa Cys
Xaa Xaa 20 25 30Xaa Cys11537PRTArtificial sequenceSynthesized
cysteine motif 75misc_feature(2)..(11)Xaa can be any naturally
occurring amino acidmisc_feature(13)..(13)Xaa can be any naturally
occurring amino acidmisc_feature(15)..(17)Xaa can be any naturally
occurring amino acidmisc_feature(19)..(20)Xaa can be any naturally
occurring amino acidmisc_feature(22)..(22)Xaa can be any naturally
occurring amino acidmisc_feature(25)..(28)Xaa can be any naturally
occurring amino acidmisc_feature(30)..(34)Xaa can be any naturally
occurring amino acidmisc_feature(36)..(36)Xaa can be any naturally
occurring amino acid 115Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Cys Xaa Xaa1 5 10 15Xaa Cys Xaa Xaa Cys Xaa Cys Cys Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa 20 25
30Xaa Xaa Cys Xaa Cys 3511636PRTArtificial sequenceSynthesized
cysteine motif 76misc_feature(2)..(10)Xaa can be any naturally
occurring amino acidmisc_feature(12)..(12)Xaa can be any naturally
occurring amino acidmisc_feature(14)..(16)Xaa can be any naturally
occurring amino acidmisc_feature(18)..(19)Xaa can be any naturally
occurring amino acidmisc_feature(21)..(21)Xaa can be any naturally
occurring amino acidmisc_feature(24)..(27)Xaa can be any naturally
occurring amino acidmisc_feature(29)..(33)Xaa can be any naturally
occurring amino acidmisc_feature(35)..(35)Xaa can be any naturally
occurring amino acid 116Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
Xaa Cys Xaa Xaa Xaa1 5 10 15Cys Xaa Xaa Cys Xaa Cys Cys Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa 20 25 30Xaa Cys Xaa Cys
3511734PRTArtificial sequenceSynthesized cysteine motif
77misc_feature(2)..(7)Xaa can be any naturally occurring amino
acidmisc_feature(10)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(30)Xaa can be any naturally occurring amino
acidmisc_feature(32)..(33)Xaa can be any naturally occurring amino
acid 117Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Cys Xaa Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa
Cys Xaa 20 25 30Xaa Cys11828PRTArtificial sequenceSynthesized
cysteine motif 78misc_feature(2)..(7)Xaa can be any naturally
occurring amino acidmisc_feature(10)..(10)Xaa can be any naturally
occurring amino acidmisc_feature(12)..(14)Xaa can be any naturally
occurring amino acidmisc_feature(16)..(16)Xaa can be any naturally
occurring amino acidmisc_feature(19)..(21)Xaa can be any naturally
occurring amino acidmisc_feature(23)..(26)Xaa can be any naturally
occurring amino acid 118Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Cys
Xaa Xaa Xaa Cys Xaa1 5 10 15Cys Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Cys Cys 20 2511936PRTArtificial sequenceSynthesized cysteine motif
79misc_feature(2)..(7)Xaa can be any naturally occurring amino
acidmisc_feature(10)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(14)Xaa can be any naturally occurring amino
acidmisc_feature(16)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(19)Xaa can be any naturally occurring amino
acidmisc_feature(21)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(23)..(26)Xaa can be any naturally occurring amino
acidmisc_feature(28)..(35)Xaa can be any naturally occurring amino
acid 119Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Cys Xaa Xaa Xaa Cys
Xaa1 5 10 15Cys Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Cys 3512029PRTArtificial
sequenceSynthesized cysteine motif 80misc_feature(2)..(5)Xaa can be
any naturally occurring amino acidmisc_feature(7)..(8)Xaa can be
any naturally occurring amino acidmisc_feature(11)..(13)Xaa can be
any naturally occurring amino acidmisc_feature(15)..(15)Xaa can be
any naturally occurring amino acidmisc_feature(17)..(20)Xaa can be
any naturally occurring amino acidmisc_feature(23)..(24)Xaa can be
any naturally occurring amino acidmisc_feature(26)..(28)Xaa can be
any naturally occurring amino acid 120Cys Xaa Xaa Xaa Xaa Cys Xaa
Xaa Cys Cys Xaa Xaa Xaa Cys Xaa Cys1 5 10 15Xaa Xaa Xaa Xaa Cys Cys
Xaa Xaa Cys Xaa Xaa Xaa Cys 20 2512132PRTArtificial
sequenceSynthesized cysteine motif 81misc_feature(2)..(4)Xaa can be
any naturally occurring amino acidmisc_feature(6)..(10)Xaa can be
any naturally occurring amino acidmisc_feature(12)..(14)Xaa can be
any naturally occurring amino acidmisc_feature(18)..(21)Xaa can be
any naturally occurring amino acidmisc_feature(23)..(31)Xaa can be
any naturally occurring amino acid 121Cys Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Cys1 5 10 15Cys Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 20 25 3012229PRTArtificial
sequenceSynthesized cysteine motif 82misc_feature(3)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(15)Xaa can
be any naturally occurring amino acidmisc_feature(17)..(17)Xaa can
be any naturally occurring amino acidmisc_feature(20)..(22)Xaa can
be any naturally occurring amino acidmisc_feature(24)..(28)Xaa can
be any naturally occurring amino acid 122Cys Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys1 5 10 15Xaa Cys Cys Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys 20 2512335PRTArtificial
sequenceSynthesized cysteine motif 83misc_feature(2)..(10)Xaa can
be any naturally occurring amino acidmisc_feature(12)..(13)Xaa can
be any naturally occurring amino acidmisc_feature(15)..(17)Xaa can
be any naturally occurring amino acidmisc_feature(19)..(22)Xaa can
be any naturally occurring amino acidmisc_feature(25)..(28)Xaa can
be any naturally occurring amino acidmisc_feature(30)..(34)Xaa can
be any naturally occurring amino acid 123Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa
Xaa Cys Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 20 25 30Xaa Xaa Cys
3512439PRTArtificial sequenceSynthesized cysteine motif
84misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(23)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(26)Xaa can be any naturally occurring amino
acidmisc_feature(28)..(34)Xaa can be any naturally occurring amino
acidmisc_feature(36)..(38)Xaa can be any naturally occurring amino
acid 124Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Cys Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Cys Xaa Xaa Xaa Cys 3512535PRTArtificial
sequenceSynthesized cysteine motif 85misc_feature(2)..(10)Xaa can
be any naturally occurring amino acidmisc_feature(12)..(14)Xaa can
be any naturally occurring amino acidmisc_feature(18)..(27)Xaa can
be any naturally occurring amino acidmisc_feature(29)..(30)Xaa can
be any naturally occurring amino acidmisc_feature(32)..(34)Xaa can
be any naturally occurring amino acid 125Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Cys1 5 10 15Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa 20 25 30Xaa Xaa Cys
3512641PRTArtificial sequenceSynthesized cysteine motif
86misc_feature(2)..(4)Xaa can be any naturally occurring amino
acidmisc_feature(6)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(33)Xaa can be any naturally occurring amino
acidmisc_feature(35)..(40)Xaa can be any naturally occurring amino
acid 126Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys 35
4012736PRTArtificial sequenceSynthesized cysteine motif
87misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(30)Xaa can be any naturally occurring amino
acidmisc_feature(32)..(35)Xaa can be any naturally occurring amino
acid 127Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa
Cys Xaa 20 25 30Xaa Xaa Xaa Cys 3512836PRTArtificial
sequenceSynthesized cysteine motif 88misc_feature(2)..(8)Xaa can be
any naturally occurring amino acidmisc_feature(10)..(10)Xaa can be
any naturally occurring amino acidmisc_feature(12)..(17)Xaa can be
any naturally occurring amino acidmisc_feature(19)..(22)Xaa can be
any naturally occurring amino acidmisc_feature(26)..(35)Xaa can be
any naturally occurring amino acid 128Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa
Cys Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Cys
3512932PRTArtificial sequenceSynthesized cysteine motif
89misc_feature(2)..(9)Xaa can be any naturally occurring amino
acidmisc_feature(11)..(12)Xaa can be any naturally occurring amino
acidmisc_feature(14)..(17)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(23)Xaa can be any naturally occurring amino
acidmisc_feature(25)..(27)Xaa can be any naturally occurring amino
acidmisc_feature(29)..(31)Xaa can be any naturally occurring amino
acid 129Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa
Xaa1 5 10 15Xaa Cys Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa
Xaa Cys 20 25 3013036PRTArtificial sequenceSynthesized cysteine
motif 90misc_feature(2)..(8)Xaa can be any naturally occurring
amino acidmisc_feature(10)..(14)Xaa can be any naturally occurring
amino acidmisc_feature(16)..(16)Xaa can be any naturally occurring
amino acidmisc_feature(18)..(21)Xaa can be any naturally occurring
amino acidmisc_feature(24)..(30)Xaa can be any naturally occurring
amino acidmisc_feature(32)..(35)Xaa can be any naturally occurring
amino acid 130Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Cys Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa 20 25 30Xaa Xaa Xaa Cys 3513136PRTArtificial
sequenceSynthesized cysteine motif 91misc_feature(2)..(12)Xaa can
be any naturally occurring amino acidmisc_feature(14)..(16)Xaa can
be any naturally occurring amino acidmisc_feature(18)..(21)Xaa can
be any naturally occurring amino acidmisc_feature(25)..(32)Xaa can
be any naturally occurring amino acidmisc_feature(34)..(35)Xaa can
be any naturally occurring amino acid 131Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa
Cys Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Cys Xaa Xaa Cys
3513241PRTArtificial sequenceSynthesized cysteine motif
92misc_feature(2)..(3)Xaa can be any naturally occurring amino
acidmisc_feature(5)..(7)Xaa can be any naturally occurring amino
acidmisc_feature(9)..(12)Xaa can be any naturally occurring amino
acidmisc_feature(15)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(24)Xaa can be any naturally occurring amino
acidmisc_feature(26)..(40)Xaa can be any naturally occurring amino
acid 132Cys Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Cys Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35
4013331PRTArtificial sequenceSynthesized cysteine motif
93misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(30)Xaa can be any naturally occurring amino
acid 133Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys 20 25 3013433PRTArtificial sequenceSynthesized cysteine motif
94misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(18)Xaa can be any naturally occurring amino
acidmisc_feature(20)..(22)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(25)Xaa can be any naturally occurring amino
acidmisc_feature(27)..(32)Xaa can be any naturally occurring amino
acid 134Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Cys13538PRTArtificial sequenceSynthesized cysteine
motif 95misc_feature(2)..(10)Xaa can be any naturally occurring
amino acidmisc_feature(12)..(16)Xaa can be any naturally occurring
amino acidmisc_feature(18)..(21)Xaa can be any naturally occurring
amino acidmisc_feature(24)..(37)Xaa can be any naturally occurring
amino acid 135Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Cys
3513632PRTArtificial sequenceSynthesized cysteine motif
96misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(31)Xaa can be any naturally occurring amino
acid 136Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Cys 20 25 3013726PRTArtificial sequenceSynthesized cysteine
motif 97misc_feature(2)..(10)Xaa can be any naturally occurring
amino acidmisc_feature(12)..(17)Xaa can be any naturally occurring
amino acidmisc_feature(19)..(22)Xaa can be any naturally occurring
amino acidmisc_feature(25)..(25)Xaa can be any naturally occurring
amino acid 137Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa1 5 10 15Xaa Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Cys 20
2513833PRTArtificial sequenceSynthesized cysteine motif
98misc_feature(2)..(6)Xaa can be any naturally occurring amino
acidmisc_feature(9)..(15)Xaa can be any naturally occurring amino
acidmisc_feature(17)..(20)Xaa can be any naturally occurring amino
acidmisc_feature(22)..(33)Xaa can be any naturally occurring amino
acid 138Cys Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys1 5 10 15Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa13927PRTArtificial sequenceSynthesized cysteine
motif 99misc_feature(2)..(11)Xaa can be any naturally occurring
amino acidmisc_feature(13)..(15)Xaa can be any naturally occurring
amino acidmisc_feature(17)..(20)Xaa can be any naturally occurring
amino acidmisc_feature(23)..(26)Xaa can be any naturally occurring
amino acid 139Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Cys1 5 10 15Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Cys 20
2514028PRTArtificial sequenceSynthesized cysteine motif
100misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(15)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(22)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(27)Xaa can be any naturally occurring amino
acid 140Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Cys1 5 10 15Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys 20
2514131PRTArtificial sequenceSynthesized cysteine motif
101misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(15)Xaa can be any naturally occurring amino
acidmisc_feature(17)..(20)Xaa can be any naturally occurring amino
acidmisc_feature(22)..(28)Xaa can be any naturally occurring amino
acidmisc_feature(30)..(30)Xaa can be any naturally occurring amino
acid 141Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Cys1 5 10 15Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Cys 20 25
3014227PRTArtificial sequenceSynthesized cysteine motif
102misc_feature(2)..(8)Xaa can be any naturally occurring amino
acidmisc_feature(10)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(19)Xaa can be any naturally occurring amino
acidmisc_feature(21)..(22)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(26)Xaa can be any naturally occurring amino
acid 142Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Cys 20
2514329PRTArtificial sequenceSynthesized cysteine motif
103misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(15)Xaa can be any naturally occurring amino
acidmisc_feature(17)..(20)Xaa can be any naturally occurring amino
acidmisc_feature(23)..(28)Xaa can be any naturally occurring amino
acid 143Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Cys1 5 10 15Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys 20
2514424PRTArtificial sequenceSynthesized cysteine motif
104misc_feature(2)..(8)Xaa can be any naturally occurring amino
acidmisc_feature(10)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(14)Xaa can be any naturally occurring amino
acidmisc_feature(16)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(23)Xaa can be any naturally occurring amino
acid 144Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys 2014523PRTArtificial
sequenceSynthesized cysteine motif 105misc_feature(2)..(8)Xaa can
be any naturally occurring amino acidmisc_feature(10)..(10)Xaa can
be any naturally occurring amino acidmisc_feature(12)..(15)Xaa can
be any naturally occurring amino acidmisc_feature(17)..(17)Xaa can
be any naturally occurring amino acidmisc_feature(19)..(22)Xaa can
be any naturally occurring amino acid 145Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys1 5 10 15Xaa Cys Xaa Xaa Xaa
Xaa Cys 2014622PRTArtificial sequenceSynthesized cysteine motif
106misc_feature(2)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(21)Xaa can be any naturally occurring amino
acid 146Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa1 5 10 15Cys Xaa Xaa Xaa Xaa Cys 2014721PRTArtificial
sequenceSynthesized cysteine motif 107misc_feature(2)..(4)Xaa can
be any naturally occurring amino acidmisc_feature(6)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(20)Xaa can
be any naturally occurring amino acid 147Cys Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Cys
2014819PRTArtificial sequenceSynthesized cysteine motif
108misc_feature(2)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(13)Xaa can be any naturally occurring amino
acidmisc_feature(15)..(18)Xaa can be any naturally occurring amino
acid 148Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa
Xaa1 5 10 15Xaa Xaa Cys14918PRTArtificial sequenceSynthesized
cysteine motif 109misc_feature(2)..(11)Xaa can be any naturally
occurring amino acidmisc_feature(14)..(17)Xaa can be any naturally
occurring amino acid 149Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Cys Xaa Xaa Xaa1 5 10 15Xaa Cys15017PRTArtificial
sequenceSynthesized cysteine motif 110misc_feature(2)..(16)Xaa can
be any naturally occurring amino acid 150Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10
15Cys15112PRTArtificial sequenceSynthesized cysteine motif
111misc_feature(2)..(11)Xaa can be any naturally occurring amino
acid 151Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys1 5
1015211PRTArtificial sequenceSynthesized cysteine motif
112misc_feature(2)..(10)Xaa can be any naturally occurring amino
acid 152Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys1 5
101535PRTArtificial sequenceSynthesized X1X2X3X4X5 motif 1 153Thr
Thr Val His Gln1 51545PRTArtificial sequenceSynthesized X1X2X3X4X5
motif 2 154Thr Ser Val His Gln1 51555PRTArtificial
sequenceSynthesized X1X2X3X4X5 motif 3 155Ser Ser Val Thr Gln1
51565PRTArtificial sequenceSynthesized X1X2X3X4X5 motif 4 156Ser
Thr Val His Gln1 51575PRTArtificial sequenceSynthesized X1X2X3X4X5
motif 5 157Ala Thr Val Arg Gln1 51585PRTArtificial
sequenceSynthesized X1X2X3X4X5 motif 6 158Thr Thr Val Tyr Gln1
51595PRTArtificial sequenceSynthesized X1X2X3X4X5 motif 7 159Ser
Pro Val His Gln1 51605PRTArtificial sequenceSynthesized X1X2X3X4X5
motif 8 160Ala Thr Val Tyr Gln1 51615PRTArtificial
sequenceSynthesized X1X2X3X4X5 motif 9 161Thr Ala Val Tyr Gln1
51625PRTArtificial sequenceSynthesized X1X2X3X4X5 motif 10 162Thr
Asn Val His Gln1 51635PRTArtificial sequenceSynthesized X1X2X3X4X5
motif 11 163Ala Thr Val His Gln1 51645PRTArtificial
sequenceSynthesized X1X2X3X4X5 motif 12 164Ser Thr Val Tyr Gln1
51655PRTArtificial sequenceSynthesized X1X2X3X4X5 motif 13 165Thr
Ile Val His Gln1 51665PRTArtificial sequenceSynthesized X1X2X3X4X5
motif 14 166Ala Ile Val Tyr Gln1 51675PRTArtificial
sequenceSynthesized X1X2X3X4X5 motif 15 167Thr Thr Val Phe Gln1
51685PRTArtificial sequenceSynthesized X1X2X3X4X5 motif 16 168Ala
Ala Val Phe Gln1 51695PRTArtificial sequenceSynthesized X1X2X3X4X5
motif 17 169Gly Thr Val His Gln1 51705PRTArtificial
sequenceSynthesized X1X2X3X4X5 motif 18 170Ala Ser Val His Gln1
51715PRTArtificial sequenceSynthesized X1X2X3X4X5 motif 19 171Thr
Ala Val Phe Gln1 51725PRTArtificial sequenceSynthesized X1X2X3X4X5
motif 20 172Ala Thr Val Phe Gln1 51735PRTArtificial
sequenceSynthesized X1X2X3X4X5 motif 21 173Ala Ala Ala His Gln1
51745PRTArtificial sequenceSynthesized X1X2X3X4X5 motif 22 174Val
Val Val Tyr Gln1 51755PRTArtificial sequenceSynthesized X1X2X3X4X5
motif 23 175Gly Thr Val Phe Gln1 51765PRTArtificial
sequenceSynthesized X1X2X3X4X5 motif 24 176Thr Ala Val His Gln1
51775PRTArtificial sequenceSynthesized X1X2X3X4X5 motif 25 177Ile
Thr Val His Gln1 51785PRTArtificial sequenceSynthesized X1X2X3X4X5
motif 26 178Ile Thr Ala His Gln1 51795PRTArtificial
sequenceSynthesized X1X2X3X4X5 motif 27 179Val Thr Val His Gln1
51805PRTArtificial sequenceSynthesized X1X2X3X4X5 motif 28 180Ala
Ala Val His Gln1 51815PRTArtificial sequenceSynthesized X1X2X3X4X5
motif 29 181Gly Thr Val Tyr Gln1 51825PRTArtificial
sequenceSynthesized X1X2X3X4X5 motif 30 182Thr Thr Val Leu Gln1
51835PRTArtificial sequenceSynthesized X1X2X3X4X5 motif 31 183Thr
Thr Thr His Gln1 51845PRTArtificial sequenceSynthesized X1X2X3X4X5
motif 32 184Thr Thr Asp Tyr Gln1 51856PRTArtificial
sequenceSynthesized CX1X2X3X4X5 motif 1 185Cys Thr Thr Val His Gln1
51866PRTArtificial sequenceSynthesized CX1X2X3X4X5 motif 2 186Cys
Thr Ser Val His Gln1 51876PRTArtificial sequenceSynthesized
CX1X2X3X4X5 motif 3 187Cys Ser Ser Val Thr Gln1 51886PRTArtificial
sequenceSynthesized CX1X2X3X4X5 motif 4 188Cys Ser Thr Val His Gln1
51896PRTArtificial sequenceSynthesized CX1X2X3X4X5 motif 5 189Cys
Ala Thr Val Arg Gln1 51906PRTArtificial sequenceSynthesized
CX1X2X3X4X5 motif 6 190Cys Thr Thr Val Tyr Gln1 51916PRTArtificial
sequenceSynthesized CX1X2X3X4X5 motif 7 191Cys Ser Pro Val His Gln1
51926PRTArtificial sequenceSynthesized CX1X2X3X4X5 motif 8 192Cys
Ala Thr Val Tyr Gln1 51936PRTArtificial sequenceSynthesized
CX1X2X3X4X5 motif 9 193Cys Thr Ala Val Tyr Gln1 51946PRTArtificial
sequenceSynthesized CX1X2X3X4X5 motif 10 194Cys Thr Asn Val His
Gln1 51956PRTArtificial sequenceSynthesized CX1X2X3X4X5 motif 11
195Cys Ala Thr Val His Gln1 51966PRTArtificial sequenceSynthesized
CX1X2X3X4X5 motif 12 196Cys Ser Thr Val Tyr Gln1 51976PRTArtificial
sequenceSynthesized CX1X2X3X4X5 motif 13 197Cys Thr Ile Val His
Gln1 51986PRTArtificial sequenceSynthesized CX1X2X3X4X5 motif 14
198Cys Ala Ile Val Tyr Gln1 51996PRTArtificial sequenceSynthesized
CX1X2X3X4X5 motif 15 199Cys Thr Thr Val Phe Gln1 52006PRTArtificial
sequenceSynthesized CX1X2X3X4X5 motif 16 200Cys Ala Ala Val Phe
Gln1 52016PRTArtificial sequenceSynthesized CX1X2X3X4X5 motif 17
201Cys Gly Thr Val His Gln1 52026PRTArtificial sequenceSynthesized
CX1X2X3X4X5 motif 18 202Cys Ala Ser Val His Gln1 52036PRTArtificial
sequenceSynthesized CX1X2X3X4X5 motif 19 203Cys Thr Ala Val Phe
Gln1 52046PRTArtificial sequenceSynthesized CX1X2X3X4X5 motif 20
204Cys Ala Thr Val Phe Gln1 52056PRTArtificial sequenceSynthesized
CX1X2X3X4X5 motif 21 205Cys Ala Ala Ala His Gln1 52066PRTArtificial
sequenceSynthesized CX1X2X3X4X5 motif 22 206Cys Val Val Val Tyr
Gln1 52076PRTArtificial sequenceSynthesized CX1X2X3X4X5 motif 23
207Cys Gly Thr Val Phe Gln1 52086PRTArtificial sequenceSynthesized
CX1X2X3X4X5 motif 24 208Cys Thr Ala Val His Gln1 52096PRTArtificial
sequenceSynthesized CX1X2X3X4X5 motif 25 209Cys Ile Thr Val His
Gln1 52106PRTArtificial sequenceSynthesized CX1X2X3X4X5 motif 26
210Cys Ile Thr Ala His Gln1 52116PRTArtificial sequenceSynthesized
CX1X2X3X4X5 motif 27 211Cys Val Thr Val His Gln1 52126PRTArtificial
sequenceSynthesized CX1X2X3X4X5 motif 28 212Cys Ala Ala Val His
Gln1 52136PRTArtificial sequenceSynthesized CX1X2X3X4X5 motif 29
213Cys Gly Thr Val Tyr Gln1 52146PRTArtificial sequenceSynthesized
CX1X2X3X4X5 motif 30 214Cys Thr Thr Val Leu Gln1 52156PRTArtificial
sequenceSynthesized CX1X2X3X4X5 motif 31 215Cys Thr Thr Thr His
Gln1 52166PRTArtificial sequenceSynthesized CX1X2X3X4X5 motif 32
216Cys Thr Thr Asp Tyr Gln1 52178PRTArtificial sequenceSynthesized
XAXBZ motif 1 217Cys Tyr Thr Tyr Asn Tyr Glu Phe1
52188PRTArtificial sequenceSynthesized XAXBZ motif 2 218His Tyr Thr
Tyr Thr Tyr Asp Phe1 52198PRTArtificial sequenceSynthesized XAXBZ
motif 3 219His Tyr Thr Tyr Thr Tyr Glu Trp1 52208PRTArtificial
sequenceSynthesized XAXBZ motif 4 220Lys His Arg Tyr Thr Tyr Glu
Trp1 52218PRTArtificial sequenceSynthesized XAXBZ motif 5 221Asn
Tyr Ile Tyr Lys Tyr Ser Phe1 52228PRTArtificial sequenceSynthesized
XAXBZ motif 6 222Pro Tyr Ile Tyr Thr Tyr Gln Phe1
52238PRTArtificial sequenceSynthesized XAXBZ motif 7 223Ser Phe Thr
Tyr Thr Tyr Glu Trp1 52248PRTArtificial sequenceSynthesized XAXBZ
motif 8 224Ser Tyr Ile Tyr Ile Tyr Gln Trp1 52258PRTArtificial
sequenceSynthesized XAXBZ motif 9 225Ser Tyr Asn Tyr Thr Tyr Ser
Trp1 52268PRTArtificial sequenceSynthesized XAXBZ motif 10 226Ser
Tyr Ser Tyr Ser Tyr Glu Tyr1 52278PRTArtificial sequenceSynthesized
XAXBZ motif 11 227Ser Tyr Thr Tyr Asn Tyr Asp Phe1
52288PRTArtificial sequenceSynthesized XAXBZ motif 12 228Ser Tyr
Thr Tyr Asn Tyr Glu Trp1 52298PRTArtificial sequenceSynthesized
XAXBZ motif 13 229Ser Tyr Thr Tyr Asn Tyr Gln Phe1
52308PRTArtificial sequenceSynthesized XAXBZ motif 14 230Ser Tyr
Val Trp Thr His Asn Phe1 52318PRTArtificial sequenceSynthesized
XAXBZ motif 15 231Thr Tyr Lys Tyr Val Tyr Glu Trp1
52328PRTArtificial sequenceSynthesized XAXBZ motif 16 232Thr Tyr
Thr Tyr Thr Tyr Glu Phe1 52338PRTArtificial sequenceSynthesized
XAXBZ motif 17 233Thr Tyr Thr Tyr Thr Tyr Glu Trp1
52348PRTArtificial sequenceSynthesized XAXBZ motif 18 234Val Phe
Thr Tyr Thr Tyr Glu Phe1 52356PRTArtificial sequenceSynthesized
XAXBZ motif 19 235Ala Tyr Thr Tyr Glu Trp1 52366PRTArtificial
sequenceSynthesized XAXBZ motif 20 236Asp Tyr Ile Tyr Thr Tyr1
52376PRTArtificial sequenceSynthesized XAXBZ motif 21 237Ile His
Ser Tyr Glu Phe1 52386PRTArtificial sequenceSynthesized XAXBZ motif
22 238Ser Phe Thr Tyr Glu Phe1 52396PRTArtificial
sequenceSynthesized XAXBZ motif 23 239Ser His Ser Tyr Glu Phe1
52406PRTArtificial sequenceSynthesized XAXBZ motif 24 240Thr His
Thr Tyr Glu Phe1 52416PRTArtificial sequenceSynthesized XAXBZ motif
25 241Thr Trp Thr Tyr Glu Phe1 52426PRTArtificial
sequenceSynthesized XAXBZ motif 26 242Thr Tyr Asn Tyr Glu Trp1
52436PRTArtificial sequenceSynthesized XAXBZ motif 27 243Thr Tyr
Ser Tyr Glu Phe1 52446PRTArtificial sequenceSynthesized XAXBZ motif
28 244Thr Tyr Ser Tyr Glu His1 52456PRTArtificial
sequenceSynthesized XAXBZ motif 29 245Thr Tyr Thr Tyr Asp Phe1
52466PRTArtificial sequenceSynthesized XAXBZ motif 30 246Thr Tyr
Thr Tyr Glu Phe1 52476PRTArtificial sequenceSynthesized XAXBZ motif
31 247Thr Tyr Thr Tyr Glu Trp1 52484PRTArtificial
sequenceSynthesized XAXBZ motif 32 248Ala Tyr Glu
Phe12494PRTArtificial sequenceSynthesized XAXBZ motif 33 249Ala Tyr
Ser Phe12504PRTArtificial sequenceSynthesized XAXBZ motif 34 250Ala
Tyr Ser Tyr12514PRTArtificial sequenceSynthesized XAXBZ motif 35
251Cys Tyr Ser Phe12524PRTArtificial sequenceSynthesized XAXBZ
motif 36 252Asp Tyr Thr Tyr12534PRTArtificial sequenceSynthesized
XAXBZ motif 37 253Lys Tyr Glu His12544PRTArtificial
sequenceSynthesized XAXBZ motif 38 254Lys Tyr Glu
Trp12554PRTArtificial sequenceSynthesized XAXBZ motif 39 255Met Tyr
Glu Phe12564PRTArtificial sequenceSynthesized XAXBZ motif 40 256Asn
Trp Ile Tyr12574PRTArtificial sequenceSynthesized XAXBZ motif 41
257Asn Tyr Asp Tyr12584PRTArtificial sequenceSynthesized XAXBZ
motif 42 258Asn Tyr Gln Trp12594PRTArtificial sequenceSynthesized
XAXBZ motif 43 259Asn Tyr Ser Phe12604PRTArtificial
sequenceSynthesized XAXBZ motif 44 260Pro Tyr Glu
Trp12614PRTArtificial sequenceSynthesized XAXBZ motif 45 261Arg Tyr
Asn Trp12624PRTArtificial sequenceSynthesized XAXBZ motif 46 262Arg
Tyr Thr Tyr12634PRTArtificial sequenceSynthesized XAXBZ motif 47
263Ser Tyr Glu Phe12644PRTArtificial sequenceSynthesized XAXBZ
motif 48 264Ser Tyr Glu His12654PRTArtificial sequenceSynthesized
XAXBZ motif 49 265Ser Tyr Glu Trp12664PRTArtificial
sequenceSynthesized XAXBZ motif 50 266Ser Tyr Lys
Trp12674PRTArtificial sequenceSynthesized XAXBZ motif 51 267Ser Tyr
Thr Tyr12684PRTArtificial sequenceSynthesized XAXBZ motif 52 268Thr
Tyr Asp Phe12694PRTArtificial sequenceSynthesized XAXBZ motif 53
269Thr Tyr Glu Phe12704PRTArtificial sequenceSynthesized XAXBZ
motif 54 270Thr Tyr Glu Trp12714PRTArtificial sequenceSynthesized
XAXBZ motif 55 271Thr Tyr Gln Trp12724PRTArtificial
sequenceSynthesized XAXBZ motif 56 272Thr Tyr Thr
Tyr12734PRTArtificial sequenceSynthesized XAXBZ motif 57 273Val Tyr
Glu Trp127448PRTArtificial sequenceSynthesized DH2 Germline
sequence 274Ser Cys Pro Asp Gly Tyr Ser Tyr Gly Tyr Gly Cys Gly Tyr
Gly Tyr1 5 10 15Gly Cys Ser Gly Tyr Asp Cys Tyr Gly Tyr Gly Gly Tyr
Gly Gly Tyr 20 25 30Gly Gly Tyr Gly Tyr Ser Ser Tyr Ser Tyr Ser Tyr
Thr Tyr Glu Tyr 35 40 4527515PRTArtificial sequenceSynthesized JH1
Germline sequence 275Tyr Val Asp Ala Trp Gly Gln Gly Leu Leu Val
Thr Val Ser Ser1 5 10 1527665PRTArtificial sequenceSynthesized
BLV1H12 276Cys Thr Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Cys
Pro Asp1 5 10 15Gly Tyr Arg Glu Arg Ser Asp Cys Ser Asn Arg Pro Ala
Cys Gly Thr 20 25 30Ser Asp Cys Cys Arg Val Ser Val Phe Gly Asn Cys
Leu Thr Thr Leu 35 40 45Pro Val Ser Tyr Ser Tyr Thr Tyr Asn Tyr Glu
Trp His Val Asp Val 50 55 60Trp6527760PRTArtificial
sequenceSynthesized BLV5B8 277Cys Thr Thr Val His Gln Glu Thr Arg
Lys Thr Cys Ser Asp Gly Tyr1 5 10 15Ile Ala Val Asp Ser Cys Gly Arg
Gly Gln Ser Asp Gly Cys Val Asn 20 25 30Asp Cys Asn Ser Cys Tyr Tyr
Gly Trp Arg Asn Cys Arg Arg Gln Pro 35 40 45Ala Ile His Ser Tyr Glu
Phe His Val Asp Ala Trp 50 55 6027861PRTArtificial
sequenceSynthesized
BLV5D3 278Cys Ser Ser Val Thr Gln Arg Thr His Val Ser Arg Ser Cys
Pro Asp1 5 10 15Gly Cys Ser Asp Gly Asp Gly Cys Val Asp Gly Cys Cys
Cys Ser Ala 20 25 30Tyr Arg Cys Tyr Thr Pro Gly Val Arg Asp Leu Ser
Cys Thr Ser Tyr 35 40 45Ser Ile Thr Tyr Thr Tyr Glu Trp Asn Val Asp
Ala Trp 50 55 6027962PRTArtificial sequenceSynthesized BLV8C11
279Cys Thr Thr Val His Gln Lys Thr Thr Arg Lys Thr Cys Cys Ser Asp1
5 10 15Ala Tyr Arg Tyr Asp Ser Gly Cys Gly Ser Gly Cys Asp Cys Cys
Gly 20 25 30Ala Asp Cys Tyr Val Phe Gly Ala Cys Thr Phe Gly Leu Asp
Ser Ser 35 40 45Tyr Ser Tyr Ile Tyr Ile Tyr Gln Trp Tyr Val Asp Ala
Trp 50 55 6028060PRTArtificial sequenceSynthesized BF4E9 280Cys Thr
Thr Val His Gln Ile Phe Cys Pro Asp Gly Tyr Ser Tyr Gly1 5 10 15Tyr
Gly Cys Gly Tyr Gly Tyr Gly Cys Ser Gly Tyr Asp Cys Tyr Gly 20 25
30Tyr Gly Gly Tyr Gly Tyr Gly Gly Tyr Gly Gly Tyr Ser Ser Tyr Ser
35 40 45Tyr Ser Tyr Ser Tyr Glu Tyr Tyr Gly Asp Ala Trp 50 55
6028170PRTArtificial sequenceSynthesized B-L1 281Cys Ser Thr Val
His Gln Lys Thr Arg Thr Thr Gln Gly Asn Thr Cys1 5 10 15Pro Asp Gly
Tyr Thr Leu Lys Asp Asp Cys Pro Arg Cys Arg Gly Gly 20 25 30Cys Asp
Gly Tyr Asp Cys Cys Trp Gly Asp Ala Cys Arg Ser Ser Gly 35 40 45Leu
Cys Trp Gly His Asn Pro Leu Val Thr Glu Thr Tyr Thr Tyr Glu 50 55
60Phe Tyr Ile Asp Ala Trp65 7028263PRTArtificial
sequenceSynthesized B-L2 282Cys Ala Thr Val Arg Gln Thr Thr Leu Arg
Asp Cys Pro Gly Gly Tyr1 5 10 15Thr Glu Asp Arg Ser Cys Val Asn Thr
Tyr Ser Cys Gly Ala Asp Asp 20 25 30Cys Cys Gly Arg Gly Asp Val Gly
Tyr Pro Ala Leu Tyr Gly Tyr Arg 35 40 45Cys Ala Ala His Ile Gln Arg
Tyr Asn Trp His Ala Asp Ala Trp 50 55 6028371PRTArtificial
sequenceSynthesized UL1 283Cys Ser Thr Val His Gln Lys Thr Arg Thr
Thr Gln Gly Glu Tyr Leu1 5 10 15Ser Leu Met Val Thr Leu Leu Lys Asp
Asp Cys Pro Arg Cys Arg Gly 20 25 30Gly Cys Asp Gly Tyr Asp Cys Cys
Trp Gly Asp Ala Cys Arg Ser Ser 35 40 45Gly Leu Cys Trp Gly His Asn
Pro Leu Val Thr Glu Thr Tyr Thr Tyr 50 55 60Glu Phe Tyr Ile Asp Ala
Trp65 7028471PRTArtificial sequenceSynthesized UL2 284Cys Ser Thr
Val His Gln Lys Thr Arg Thr Thr Gln Gly Asn Asn Leu1 5 10 15Ser Leu
Met Val Thr Leu Leu Lys Asp Asp Cys Pro Arg Cys Arg Gly 20 25 30Gly
Cys Asp Gly Tyr Asp Cys Cys Trp Gly Asp Ala Cys Arg Ser Ser 35 40
45Gly Leu Cys Trp Gly His Asn Pro Leu Val Thr Glu Thr Tyr Thr Tyr
50 55 60Glu Phe Tyr Ile Asp Ala Trp65 7028570PRTArtificial
sequenceSynthesized UL3 285Cys Ser Thr Val His Gln Lys Thr Arg Thr
Thr Gln Gly Asn Thr Cys1 5 10 15Pro Asp Gly Tyr Thr Leu Lys Asp Asp
Cys Pro Arg Cys Arg Gly Gly 20 25 30Cys Asp Gly Tyr Asp Cys Cys Trp
Gly Asp Ala Cys Arg Ser Ser Gly 35 40 45Leu Cys Trp Gly His Asn Pro
Leu Val Thr Glu Thr Tyr Thr Tyr Glu 50 55 60Phe Tyr Ile Asp Ala
Trp65 7028670PRTArtificial sequenceSynthesized UL4 286Cys Ser Thr
Val His Gln Lys Thr Arg Thr Thr Gln Gly Asn Thr Cys1 5 10 15Pro Asp
Gly Tyr Thr Phe Lys Asp Asp Cys Pro Arg Cys Arg Gly Gly 20 25 30Cys
Asp Gly Tyr Asp Cys Cys Trp Gly Asp Ala Cys Arg Ser Ser Gly 35 40
45Leu Cys Trp Gly His Asn Pro Leu Val Thr Glu Thr Tyr Thr Tyr Glu
50 55 60Phe Tyr Ile Asp Ala Trp65 7028770PRTArtificial
sequenceSynthesized UL5 287Cys Thr Thr Val His Gln Lys Thr Arg Thr
Thr Gln Gly Asn Thr Cys1 5 10 15Pro Asp Gly Tyr Thr Leu Lys Asp Asp
Cys Pro Arg Cys Arg Gly Gly 20 25 30Cys Asp Gly Tyr Asp Cys Cys Trp
Gly Asp Ala Cys Arg Ser Ser Gly 35 40 45Leu Cys Trp Gly His Asn Pro
Leu Val Thr Glu Thr Tyr Thr Tyr Glu 50 55 60Phe Tyr Ile Asp Ala
Trp65 7028870PRTArtificial sequenceSynthesized UL6 288Cys Ser Thr
Val His Gln Lys Thr Arg Thr Thr Gln Gly Asn Thr Cys1 5 10 15Pro Asp
Gly Tyr Thr Leu Lys Asn Asp Cys Pro Arg Cys Arg Gly Gly 20 25 30Cys
Asp Gly Tyr Asp Cys Cys Trp Gly Asp Ala Cys Arg Ser Ser Gly 35 40
45Leu Cys Trp Gly His Asn Pro Leu Val Thr Glu Thr Tyr Thr Tyr Glu
50 55 60Phe Tyr Ile Asp Ala Trp65 7028970PRTArtificial
sequenceSynthesized UL7 289Cys Thr Thr Val Tyr Gln Lys Thr Arg Thr
Thr Gln Gly Asn Thr Cys1 5 10 15Pro Asp Gly Tyr Thr Leu Lys Asp Asp
Cys Pro Arg Cys Arg Gly Gly 20 25 30Cys Asp Gly Tyr Asp Cys Cys Trp
Gly Asp Ala Cys Arg Ser Ser Gly 35 40 45Leu Cys Trp Gly His Asn Pro
Leu Val Thr Glu Thr Tyr Thr Tyr Glu 50 55 60Phe Tyr Ile Asp Ala
Trp65 7029070PRTArtificial sequenceSynthesized UL8 290Cys Ser Thr
Val His Gln Lys Pro Gly Gln His Lys Gly Ile Leu Val1 5 10 15Leu Met
Val Thr Leu Leu Lys Asp Asp Cys Pro Arg Cys Arg Gly Gly 20 25 30Cys
Asp Gly Tyr Asp Cys Cys Trp Gly Asp Ala Cys Arg Ser Ser Gly 35 40
45Leu Cys Trp Gly His Asn Pro Leu Val Thr Glu Thr Tyr Thr Tyr Glu
50 55 60Phe Tyr Ile Asp Ala Trp65 7029170PRTArtificial
sequenceSynthesized UL9 291Cys Ser Thr Val His Gln Lys Thr Arg Thr
Thr Gln Gly Ile Leu Val1 5 10 15Leu Met Val Thr Leu Leu Lys Asp Asp
Cys Pro Arg Cys Arg Gly Gly 20 25 30Cys Asp Gly Tyr Asp Cys Cys Trp
Gly Asp Ala Cys Arg Ser Ser Gly 35 40 45Leu Cys Trp Gly His Asn Pro
Leu Val Thr Glu Thr Tyr Thr Tyr Glu 50 55 60Phe Tyr Ile Asp Ala
Trp65 7029263PRTArtificial sequenceSynthesized UL10 292Cys Ser Pro
Val His Gln Glu Ile Arg Lys Cys Cys Pro Ala Gly Cys1 5 10 15Gln Cys
Gly Arg Ser Cys Gly Ala Cys Cys Gly Cys Ala Gly Asp Glu 20 25 30Phe
Cys Gly Ile Asn Val Tyr Gly Tyr Val Thr Cys Gly Gly Tyr Arg 35 40
45Thr Cys Ser Cys Ile Asp Thr Tyr Asp Phe Tyr Val Asp Ala Trp 50 55
6029363PRTArtificial sequenceSynthesized UL11 293Cys Ser Pro Val
His Gln Gln Thr Arg Lys Cys Cys Pro Ala Gly Cys1 5 10 15Gln Cys Gly
Arg Ser Cys Gly Ala Cys Cys Gly Cys Ala Gly Asp Glu 20 25 30Phe Cys
Gly Ile Asn Val Tyr Gly Tyr Ile Thr Cys Gly Gly Tyr Arg 35 40 45Thr
Cys Ser Cys Ile Asp Thr Tyr Asp Phe Tyr Val Glu Ala Trp 50 55
6029468PRTArtificial sequenceSynthesized UL12 294Cys Ala Thr Val
Tyr Gln Lys Thr Asn Gln Ser Lys Asn Cys Pro Glu1 5 10 15Gly Ser Ala
Trp Cys Arg Ser Cys Asp Gly Gly Ala Gly Cys Ala Asp 20 25 30Tyr Glu
Cys Cys Arg Cys Gly Trp Ser Gly Cys Ser Trp Arg Asn Gly 35 40 45Ala
Cys Glu Cys Ser Ser Leu Ser Ser Ser Tyr Thr Tyr Glu Leu His 50 55
60Val Asp Ala Trp6529565PRTArtificial sequenceSynthesized UL13
295Cys Ser Thr Val His Gln Thr Thr His Gln Ile His Thr Cys Pro Asn1
5 10 15Gly Trp Thr Gly Gly Cys Val Cys Ser Ser Arg Phe Asn Cys Arg
Gly 20 25 30Asn Asn Cys Cys Cys Arg Thr Ala Tyr Cys Ser Val Asp Arg
Tyr Val 35 40 45Cys Ala Cys Pro Thr Val Thr Tyr Thr Tyr Glu Phe Asn
Val Asp Ser 50 55 60Trp6529665PRTArtificial sequenceSynthesized
UL14 296Cys Thr Ala Val Tyr Gln Lys Thr Thr Ser Ile Arg Ser Cys Pro
Gly1 5 10 15Gly Thr Thr Leu Arg Asn Gly Cys Arg Ser Ala Cys Gly Cys
Asn Asp 20 25 30Cys Asp Cys Cys Cys Gly Ser Ser Trp Asp Ile Cys Tyr
Met Ser Lys 35 40 45Cys Thr Ser Ala Pro Glu Thr Tyr Thr Tyr Glu Leu
His Ile Asp Ala 50 55 60Trp6529765PRTArtificial sequenceSynthesized
UL15 297Cys Thr Asn Val His Gln Lys Thr Lys Lys Thr Cys Pro Asp Asp
Tyr1 5 10 15Thr Cys Gly Val Ser Cys Ser Cys Ser Ser Ser Gly Cys Ala
Asp Tyr 20 25 30Gly Cys Cys Ser Tyr Ile Thr Tyr Gly Val Pro Gly Asp
Cys Gly Gly 35 40 45Cys Cys Ser Tyr Lys His Arg Tyr Thr Tyr Glu Trp
Asn Val Asp Ala 50 55 60Trp6529863PRTArtificial sequenceSynthesized
UL16 298Cys Thr Thr Val His Gln Lys Thr Lys Lys Leu Cys Pro Asn Gly
Arg1 5 10 15Thr Cys Gly Cys Gly Cys Asp Cys Gly Ser Gly Cys Cys Thr
Ser Tyr 20 25 30Cys Asp Ser Phe Gly Cys Trp Gly Gly Arg Asp Thr Phe
Gly Ser Ser 35 40 45Cys Thr Ser Ala Thr Tyr Thr Tyr Glu Trp Gly Val
Asp Ala Trp 50 55 6029964PRTArtificial sequenceSynthesized UL17
299Cys Ala Thr Val His Gln His Thr Asn Lys Lys Arg Cys Pro Asp Gly1
5 10 15Tyr Glu Phe Ser Ala Gly Cys Cys Cys Gly Glu Gly Cys Ser Gly
Ser 20 25 30Asp Cys Cys Cys Asn Ser Arg Leu Arg Cys Ser Trp Tyr Glu
Ile Tyr 35 40 45Cys Ser Val Ser Pro Ser Asp Thr Tyr Glu Phe His Val
Asp Ala Trp 50 55 6030064PRTArtificial sequenceSynthesized UL18
300Cys Thr Thr Val His Gln His Thr Asn Lys Lys Arg Cys Pro Asp Gly1
5 10 15Tyr Arg Phe Ser Ala Ala Cys Cys Cys Gly Glu Gly Cys Ser Gly
Asn 20 25 30Glu Cys Cys Cys Asn Thr Arg Leu Arg Cys Ser Trp Tyr Glu
Ile Tyr 35 40 45Cys Ser Val Ser Pro Ser Asp Thr Tyr Glu Phe His Val
Asp Ala Trp 50 55 6030164PRTArtificial sequenceSynthesized UL19
301Cys Thr Thr Val His Gln His Thr Asn Gln Asn Arg Cys Pro Thr Gly1
5 10 15Tyr Lys His Ser Ala Gly Cys Cys Cys Gly Val Gly Cys Ser Gly
Asn 20 25 30Asp Cys Cys Cys Asn Ser Arg Leu Arg Cys Ser Trp Tyr Glu
Thr Tyr 35 40 45Cys Ser Leu Ser Pro Thr Asp Met Tyr Glu Phe Tyr Val
Asp Ala Trp 50 55 6030264PRTArtificial sequenceSynthesized UL20
302Cys Ser Thr Val His Gln His Thr Asn Gln Asn Arg Cys Pro Ala Gly1
5 10 15Tyr Lys His Ser Ala Gly Cys Cys Cys Gly Val Gly Cys Ser Gly
Asn 20 25 30Asp Cys Cys Cys Asn Ser Arg Leu Arg Cys Ser Trp Tyr Glu
Thr Tyr 35 40 45Cys Ser Leu Ser Pro Thr Asp Met Tyr Glu Phe Tyr Val
Asp Ala Trp 50 55 6030363PRTArtificial sequenceSynthesized UL21
303Cys Thr Thr Val His Gln Lys Thr Asn Glu Arg Cys Cys Arg Val Val1
5 10 15Ser Asp Asp Gly Glu Cys Gly Asp Gly Asn Ser Cys His Arg Trp
Leu 20 25 30Cys Ser Asp Tyr Cys Tyr Ser Gly Asp Cys Cys Ala Cys Gly
Cys Arg 35 40 45Ala Tyr His Tyr Thr Tyr Thr Tyr Glu Trp Asn Ile Asp
Ala Trp 50 55 6030463PRTArtificial sequenceSynthesized UL22 304Cys
Thr Thr Val His Gln Lys Thr Asn Glu Arg Cys Cys Arg Val Val1 5 10
15Ser Asp Asp Gly Glu Cys Gly Asp Gly Asn Ser Cys His Arg Trp Leu
20 25 30Cys Ser Asp Tyr Cys Tyr Ser Gly Asp Cys Cys Ala Cys Gly Cys
Arg 35 40 45Ala Tyr His Tyr Thr Tyr Thr Tyr Asp Phe Arg Ile Asp Val
Trp 50 55 6030564PRTArtificial sequenceSynthesized UL23 305Cys Thr
Thr Val His Gln Lys Thr Asn Arg Glu Arg Cys Cys Pro Asp1 5 10 15Gly
Tyr Tyr Tyr Cys Cys Arg Ser Val Ser Asp Cys Cys Cys Ser Thr 20 25
30Arg Ala Cys Val Gly Asp Ser Cys Gly Trp Thr Asp Phe Gly Ser Thr
35 40 45His Asn Val Asp Cys Ser Phe Thr Tyr Glu Phe His Val Asp Ala
Trp 50 55 6030663PRTArtificial sequenceSynthesized UL24 306Cys Thr
Thr Val His Gln Gln Thr Arg Lys Ser Cys Pro Asp Gly Tyr1 5 10 15Thr
Tyr Cys His Asp Cys Gly Tyr Gly Cys Cys Cys Gly Ala Ser Phe 20 25
30Cys Arg Asp Tyr Gly Gly Cys Gly Ser Leu Cys Gly Arg Tyr Cys Thr
35 40 45Ser Phe Asp Tyr Ile Tyr Thr Tyr Glu Asn Tyr Val Glu Thr Trp
50 55 6030764PRTArtificial sequenceSynthesized UL25 307Cys Thr Thr
Val His Gln Glu Thr Lys Lys Asn Cys Pro Asp Asn Cys1 5 10 15Tyr Tyr
Glu Asn Ser Cys Gly Asp Tyr Gly Ser Gly Cys Asn Gly Gly 20 25 30Asp
Cys Cys Arg Cys Gly Thr Trp Leu Thr Cys Ser Val Ser Gly Cys 35 40
45Thr Cys Ile Arg Ala Thr Asn Thr Tyr Gln Trp Tyr Val Asn Ala Trp
50 55 6030864PRTArtificial sequenceSynthesized UL26 308Cys Thr Thr
Val His Gln Ser Thr Asn Lys Lys Ser Cys Pro Asp Arg1 5 10 15Val Cys
Trp Ala Val Gly Cys Cys Phe Gly Glu Asp Cys Thr Ser Ser 20 25 30Asp
Cys Thr Cys Tyr Ala Ser Pro Gly Asn Pro Tyr Arg His Asp Cys 35 40
45Gly Asn Cys Asp Cys Arg Ser Ser Tyr Glu His His Val Asp Ala Trp
50 55 6030963PRTArtificial sequenceSynthesized UL27 309Cys Thr Thr
Val Arg Gln Glu Thr Leu Ile Arg Cys Arg Asp Gly Pro1 5 10 15Ser Cys
Ala Ala Cys Cys Arg Ser Gly Arg Arg Cys Ser Gly Tyr Gly 20 25 30Cys
Cys Thr Asp Gly Cys Cys Ser Asp Asn Asp Tyr Ala Asp Cys Ile 35 40
45Arg Gly Glu Phe Val Asp Val Tyr Glu Trp Asn Val Asp Ala Trp 50 55
6031063PRTArtificial sequenceSynthesized UL28 310Cys Ser Thr Val
Tyr Gln Lys Thr Arg Thr Thr Cys Pro Asp Gly Tyr1 5 10 15Thr Cys Gly
Asp Gly Ala Arg Cys Glu Lys Ala Cys Arg Gly Cys Asp 20 25 30Cys Cys
Arg Thr Thr Val Cys Asp Thr Val Trp Ser Ser Tyr Cys Ser 35 40 45Cys
Tyr Ser Phe Thr Asp Ser Tyr Glu Phe Tyr Val Asp Ala Trp 50 55
6031166PRTArtificial sequenceSynthesized UL29 311Cys Ala Thr Val
Tyr Gln Lys Thr Asn Arg Glu Met Ser Cys Pro Asp1 5 10 15Gly Cys Arg
Ile His Asn Ala Arg Leu Cys Leu Ser Gly Cys Ser Gly 20 25 30Ser Asp
Cys Cys Ser Cys Gly Asp Cys Val Ser Asp Ala Arg Cys Tyr 35 40 45Asn
Cys Arg Ser Ala Val Phe Thr Tyr Thr Tyr Glu Phe His Val Asp 50 55
60Ala Trp6531263PRTArtificial sequenceSynthesized UL30 312Cys Thr
Ile Val His Gln Glu Thr Lys Arg Ser Cys Pro Asp Gly Tyr1 5 10 15Asn
Thr Gly Thr Arg Cys Phe Gly Ser Cys Gly Cys Ile Gly Ser Asn 20 25
30Cys Cys Arg Ser Thr Thr Ser Cys Cys Cys Ala Gly Ile Tyr Ser Gln
35 40 45Cys Thr Thr Ser Thr Leu Thr Tyr Glu Trp His Ala Asp Val Trp
50 55
6031363PRTArtificial sequenceSynthesized UL31 313Cys Ala Ile Val
Tyr Gln Arg Thr Arg Gln Arg Cys Pro Asp Gly Tyr1 5 10 15Asn Thr Gly
Thr Arg Cys Phe Gly Thr Cys Gly Cys Asn Gly Ser Asn 20 25 30Cys Cys
Arg Phe Thr Thr Ser Cys Cys Cys Ala Gly Val Tyr Ser Gln 35 40 45Cys
Thr Thr Ser Thr Leu Thr Tyr Glu Trp His Ala Asp Val Trp 50 55
6031463PRTArtificial sequenceSynthesized UL32 314Cys Thr Thr Val
His Gln Lys Thr Glu Thr Arg Cys Pro Asp Gly Tyr1 5 10 15Ser Ser Thr
Asn Gly Cys Asp Ala Arg Cys Gly Cys Ser Asp Cys Asp 20 25 30Cys Cys
Asn Val Gly Arg Trp Gly Cys Pro Leu Ile Cys Ser Arg Asn 35 40 45Cys
Arg Ser Phe Thr Tyr Thr Tyr Glu Trp Tyr Ala Asp Ala Trp 50 55
6031563PRTArtificial sequenceSynthesized UL33 315Cys Thr Thr Val
His Gln Lys Thr Asn Lys Lys Glu Ser Cys Pro Asp1 5 10 15Gly Tyr Thr
Met Asn Glu Cys Cys Gly Cys Gly Tyr Gly Cys Cys Arg 20 25 30Gly Gly
Cys Val Cys Ser Ala Tyr Cys Ser Arg Pro Asn Cys Trp Arg 35 40 45Glu
Leu Thr Tyr Thr Tyr Thr Tyr Glu Phe Tyr Val Asp Thr Trp 50 55
6031663PRTArtificial sequenceSynthesized UL34 316Cys Thr Thr Val
Tyr Gln Lys Ser Arg Lys Glu Ser Ser Cys Pro Asn1 5 10 15Gly Trp Ile
Tyr Gly Lys Asp Cys Cys Ser Trp Ser Tyr Cys Thr Asp 20 25 30Cys Asp
Cys Cys Leu Cys Gly Asp Leu His Cys Tyr Asp Gly Cys Ser 35 40 45Ser
Phe Gly Val Thr Trp Thr Tyr Glu Phe His Val Asp Ala Trp 50 55
6031762PRTArtificial sequenceSynthesized UL35 317Cys Thr Thr Val
Phe Gln Glu Thr Arg Lys Ser Cys Pro Thr Gly Phe1 5 10 15Tyr Val Asp
Gly Ser Thr Cys Gly Cys Ala Thr Tyr Cys Arg Thr Cys 20 25 30Asp Cys
Cys Gly Gly Tyr Arg Cys Ser Gly Gly Gly Ser Cys Ala Cys 35 40 45Ser
Ser Tyr Thr Tyr Asn Tyr Asp Phe His Val Asp Ala Trp 50 55
6031861PRTArtificial sequenceSynthesized UL36 318Cys Ala Ala Val
Phe Gln Glu Thr Arg Thr Asn Cys Pro Ser Gly Tyr1 5 10 15Gly Asn Ala
Phe Ser Cys Gly Cys Pro Ile Ala Cys Arg Asp Cys Asp 20 25 30Cys Cys
Gly Gly Tyr Trp Cys Ser Gly Gly Ala Asp Cys His Cys Val 35 40 45Ser
Tyr Asn Tyr Thr Tyr Ser Trp His Val Asp Ala Trp 50 55
6031962PRTArtificial sequenceSynthesized UL37 319Cys Ala Thr Val
Tyr Gln Lys Thr Glu Lys His Cys Pro Leu Phe His1 5 10 15Ser Ile Cys
Cys His Cys Gly Glu Gly Val Gly Cys Ser Gly Gly Asp 20 25 30Cys Cys
Gly Cys Glu Arg Arg Ser Gly Cys Val Val Cys Thr Met Arg 35 40 45Asn
Ser Tyr Thr Tyr Asn Tyr Gln Phe His Val Asp Ala Trp 50 55
6032063PRTArtificial sequenceSynthesized UL38 320Cys Gly Thr Val
His Gln Lys Thr Lys Glu Leu Cys Pro Asp Asp Ser1 5 10 15Thr Tyr Cys
Cys Gly Cys Val Ser Gly Cys Ala Cys Cys Thr Tyr Gly 20 25 30Cys Asp
Gly Val Gly Cys Cys Arg Val Ser Leu Trp Thr Thr Tyr Ile 35 40 45Lys
Asp Ile Val Gly Val Ser Tyr Glu Trp His Val Asp Ala Trp 50 55
6032161PRTArtificial sequenceSynthesized UL39 321Cys Ala Ser Val
His Gln His Thr Glu Pro Thr Cys Pro Ala Gly Tyr1 5 10 15Thr Tyr Cys
Cys Gly Cys Leu Tyr Lys Cys Asn Cys Gly Asp Cys Gly 20 25 30Cys Tyr
Asn Val Gly Cys Gly Ser Gly Trp Leu Gly Lys Ala Cys Gly 35 40 45Asp
Tyr Arg Glu Thr Tyr Glu Trp Tyr Val Asp Ala Trp 50 55
6032261PRTArtificial sequenceSynthesized UL40 322Cys Ala Ser Val
His Gln His Thr Glu Pro Thr Cys Pro Ala Gly Tyr1 5 10 15Thr Tyr Cys
Cys Gly Cys Leu Tyr Lys Cys Asn Cys Gly Asp Cys Gly 20 25 30Cys Tyr
Asn Ala Gly Cys Gly Ser Gly Trp Leu Gly Lys Ala Cys Gly 35 40 45Asp
Tyr Arg Glu Thr Tyr Glu Trp Tyr Val Asp Ala Trp 50 55
6032361PRTArtificial sequenceSynthesized UL41 323Cys Thr Thr Val
Phe Gln Glu Thr Arg Lys Ser Cys Pro Ser Gly Phe1 5 10 15Arg Asp Arg
Asp Ala Cys Gly Cys Ala Val Thr Cys Arg Asn Cys Asp 20 25 30Cys Cys
Gly Gly Gly Pro Cys Asn Gly Gly Gly Ser Cys Arg Cys Asn 35 40 45Asn
Tyr Ile Tyr Lys Tyr Ser Phe His Val Asp Ala Trp 50 55
6032461PRTArtificial sequenceSynthesized UL42 324Cys Thr Ala Val
Phe Gln Glu Thr Arg Lys Asp Cys Pro Ser Gly Tyr1 5 10 15Gly Ser Ala
Phe Thr Cys Gly Cys Leu Ala Ala Cys His Gly Cys Asp 20 25 30Cys Cys
Gly Gly Gly Trp Cys Ser Gly Gly Gly Asp Cys Arg Cys Arg 35 40 45Ser
Tyr Ser Thr Ala Tyr Ser Phe His Ile Asp Ala Trp 50 55
6032561PRTArtificial sequenceSynthesized UL43 325Cys Ala Thr Val
Phe Gln Glu Thr Arg Lys Ser Cys Pro Ser Gly Tyr1 5 10 15Ala Asp Arg
Phe Thr Cys Asp Cys Val Tyr Tyr Cys Gln Thr Cys Asp 20 25 30Cys Cys
Gly Gly Asn Arg Cys Ser Gly Gly Gly Pro Cys Arg Cys Ser 35 40 45Ser
Tyr Ser Ile Asn Tyr Ser Phe His Val Asp Thr Trp 50 55
6032658PRTArtificial sequenceSynthesized UL44 326Cys Ala Ala Ala
His Gln Glu Thr Lys Lys Ser Cys Pro Asp Gly Thr1 5 10 15Cys Arg Gln
Cys Cys Gly Gly Val Cys Arg Cys His Ala Ser Gly Cys 20 25 30Cys Tyr
Trp Cys Thr Thr Gly Cys Val Gly Arg Ala Leu Ser Glu Ser 35 40 45His
Ser Tyr Glu Phe His Val Asp Thr Trp 50 5532770PRTArtificial
sequenceSynthesized UL45 327Cys Ser Thr Val His Gln Lys Thr Arg Thr
Thr Gln Gly Asn Thr Cys1 5 10 15Pro Asp Gly Tyr Thr Leu Lys Asp Asp
Cys Pro Arg Cys Arg Gly Gly 20 25 30Cys Asp Gly Tyr Asp Cys Cys Trp
Gly Asp Ala Cys Arg Ser Ser Gly 35 40 45Leu Cys Trp Gly His Asn Pro
Leu Val Thr Glu Thr Tyr Thr Tyr Glu 50 55 60Phe Tyr Ile Asp Ala
Trp65 7032868PRTArtificial sequenceSynthesized UL46 328Cys Val Val
Val Tyr Gln Lys Thr Asn Ser Gln Lys Ser Cys Pro Arg1 5 10 15Gly Tyr
Thr Glu Arg Glu Thr Cys Asn Arg Arg Tyr Gly Trp Gly Cys 20 25 30Gly
Arg Tyr Asp Cys Cys Asp Cys Asp Arg Trp Val Ser Gly Asn Cys 35 40
45Ala Asn Ile Cys Thr Asp Tyr Thr Asp Thr His Thr Tyr Glu Phe His
50 55 60Ala Asp Ala Trp6532964PRTArtificial sequenceSynthesized
UL47 329Cys Gly Thr Val Phe Gln Gln Thr His Lys Val Arg Asp Cys Pro
Asp1 5 10 15Gly Phe Thr Ala Ala Pro Arg Cys Gly Gly Glu Cys Cys Cys
Ser Asn 20 25 30Val Asn Ser Arg Ser Gly Gly Trp Cys Arg Tyr Cys Gly
Arg Asp Cys 35 40 45Thr Ala Pro Thr Glu Thr Ser Thr Tyr Glu Phe His
Val Asp Ala Trp 50 55 6033063PRTArtificial sequenceSynthesized UL48
330Cys Thr Ala Val Tyr Gln Arg Thr Gly Gln Lys Cys Pro Glu Gly Cys1
5 10 15Glu Ser Arg Asn Thr Cys Leu Tyr Ser Arg Asn Cys Gly Asp Tyr
Thr 20 25 30Cys Cys Gly Gly Ser Arg Ala Ser Gly Ser Gly Ala Cys Gly
Trp Asn 35 40 45Ser Val Asp Cys Lys Asn Lys Tyr Glu His His Val Asp
Ala Trp 50 55 6033163PRTArtificial sequenceSynthesized UL49 331Cys
Thr Thr Val Tyr Gln Lys Thr Lys Gln Asn Cys Pro Asp Gly Tyr1 5 10
15Asp Phe Arg Asp Thr Cys Gly Ser Gln Ser Tyr Cys Ser Gly Tyr Asp
20 25 30Cys Cys Arg Cys Ser Arg Phe Gly Gly Cys Ser Ile Gly Thr Cys
Ile 35 40 45Ser Tyr Ser Asp Ala Tyr Thr Tyr Glu Trp Tyr Val Asp Ala
Trp 50 55 6033263PRTArtificial sequenceSynthesized UL50 332Cys Thr
Thr Val His Gln Gln Thr His Glu Lys Arg Ser Cys Pro Glu1 5 10 15Ser
Tyr Ser Tyr Ser Cys Ser Cys Ala Ser Gly Val Val Gly Cys Gly 20 25
30Pro Asp Asp Cys Cys Cys Thr Tyr Arg Ile Ser Ile Arg Gly Tyr Thr
35 40 45Cys Ser Ser Leu Ser Asn Ser Tyr Glu Trp Tyr Val Asp Ala Trp
50 55 6033361PRTArtificial sequenceSynthesized UL51 333Cys Thr Ala
Val His Gln Gln Thr Lys Arg Lys Ser Gly Cys Pro Asp1 5 10 15Gly Tyr
Ser Asp Glu Ser Cys Ser Tyr Cys Gly Ser Ser Trp Cys Cys 20 25 30Pro
Val Tyr Trp Cys Gly Ser Pro Cys Ser Tyr Arg Cys Leu Arg His 35 40
45Thr Asp Thr Tyr Ser Tyr Glu His His Val Asp Ala Trp 50 55
6033460PRTArtificial sequenceSynthesized UL52 334Cys Ala Thr Val
Tyr Gln Glu Thr Lys Arg Thr Cys Ala Gly Gly His1 5 10 15Ser Val Glu
Cys Asp Ser Pro Tyr Asp Cys Asn Cys Arg Gly Gly Asp 20 25 30Cys Cys
Arg Ser Pro Ile Phe Asn Asp Cys Trp Ala Ala Ser Cys Ser 35 40 45Ala
Thr Lys Thr Tyr Glu Trp His Val Glu Ser Trp 50 55
6033558PRTArtificial sequenceSynthesized UL53 335Cys Ile Thr Val
His Gln Glu Thr Gln Lys Ser Cys Pro Asp Asp Tyr1 5 10 15Thr Tyr Tyr
Gly Asp Gly Thr Cys Ala Tyr Val Cys Ser Ile Asp Lys 20 25 30Cys Cys
Cys Gly Arg Thr Trp Leu Ser Ser Gly Cys Leu Pro Cys Arg 35 40 45Tyr
Thr Tyr Asn Leu His Val Asp Ala Trp 50 5533658PRTArtificial
sequenceSynthesized UL54 336Cys Ile Thr Val His Gln Glu Thr Gln Lys
Ser Cys Pro Asp Asp Tyr1 5 10 15Thr Ser Tyr Gly Asp Ala Thr Cys Ala
Tyr Val Cys Ser Thr Asp Glu 20 25 30Cys Cys Cys Gly Arg Thr Trp Leu
Ser Ala Gly Cys Arg Pro Cys Arg 35 40 45Tyr Thr Tyr Asn Leu His Val
Asp Ala Trp 50 5533758PRTArtificial sequenceSynthesized UL55 337Cys
Ile Thr Val His Gln Glu Thr Gln Lys Ser Cys Phe Asp Asp Tyr1 5 10
15Thr Tyr Tyr Gly Asp Ala Ser Cys Ala Tyr Val Cys Ser Thr Asp Glu
20 25 30Cys Cys Cys Gly Arg Thr Trp Leu Ser Ala Gly Cys Arg Pro Cys
Arg 35 40 45Tyr Thr Tyr Asn Leu His Val Asp Ala Trp 50
5533858PRTArtificial sequenceSynthesized UL56 338Cys Ile Thr Ala
His Gln Glu Thr Gln Lys Ser Cys Ser Asp Asp Tyr1 5 10 15Thr Tyr Tyr
Gly Asp Ala Thr Cys Ala Tyr Val Cys Ser Thr Asp Glu 20 25 30Cys Cys
Cys Gly Arg Thr Trp Leu Ser Ala Gly Cys Arg Pro Cys Arg 35 40 45Tyr
Thr Tyr Asn Leu His Val Asp Ala Trp 50 5533958PRTArtificial
sequenceSynthesized UL57 339Cys Ile Thr Val His Gln Glu Thr Gln Lys
Ser Cys Pro Asp Asp Tyr1 5 10 15Thr Tyr Tyr Gly Asp Gly Thr Cys Ala
Tyr Val Cys Ser Ile Asp Asn 20 25 30Cys Cys Cys Gly Arg Thr Trp Leu
Ser Ser Gly Cys Leu Pro Cys Arg 35 40 45Tyr Thr Tyr Asn Leu His Val
Asp Ala Trp 50 5534061PRTArtificial sequenceSynthesized UL58 340Cys
Val Thr Val His Gln Gln Thr His Ala Thr Arg Arg Cys Pro Asp1 5 10
15Gly Tyr Gly Asp Ser Tyr Ala Cys Lys Ser Asn Tyr Gly Cys Ser Ala
20 25 30Glu Gly Cys Cys Arg Trp Gly Pro Gly Ser Gly Ala Cys Thr Gly
Ala 35 40 45Ile Tyr Thr Ser Pro Tyr Glu Trp Tyr Val Asp Ala Trp 50
55 6034160PRTArtificial sequenceSynthesized UL59 341Cys Ala Ala Val
His Gln Arg Thr Glu Gly Gln Gln Ser Cys Pro Asp1 5 10 15Gly Tyr Leu
Glu Thr Arg Val Cys Pro Tyr Arg Met Tyr Arg Cys Ile 20 25 30Gly Trp
Asp Cys Cys Arg Cys Ser Asp Gly Ser Arg Asp Asn Tyr Ile 35 40 45Met
Thr Tyr Ser Tyr Glu Phe His Val Asp Val Trp 50 55
6034259PRTArtificial sequenceSynthesized UL60 342Cys Thr Thr Val
Tyr Gln Glu Thr Lys Thr Lys Ser Gly Cys Pro Asp1 5 10 15Gly Tyr Ser
Cys Cys Tyr Asn Gly Arg Ser Arg Ser Cys Arg Pro Asn 20 25 30Asp Cys
Ser Thr Tyr Gly Glu Val Arg Ser Leu Ser Arg Ser Cys Tyr 35 40 45Thr
Tyr Asn Tyr Glu Phe Tyr Val Asp Ala Trp 50 5534358PRTArtificial
sequenceSynthesized UL61 343Cys Gly Thr Val Tyr Gln His Thr Lys Glu
Ile Lys Thr Cys Pro Asp1 5 10 15Gly Tyr Ser Asp Val Phe Thr Tyr Cys
Pro Val Thr Cys Pro Gly Trp 20 25 30Asp Cys Cys Arg Arg Asn Asp Cys
Gly Arg Thr Arg Tyr Thr Val Ala 35 40 45Tyr Ser Tyr Ala Leu His Val
Asp Val Trp 50 5534457PRTArtificial sequenceSynthesized UL62 344Cys
Thr Thr Val Leu Gln Glu Thr His Gln Gln Arg Gly Cys Pro Ala1 5 10
15Gly Tyr Gln Val Val Asp Gly Cys Pro Tyr Gly Asp Cys Cys Arg Thr
20 25 30Ser Tyr Val Cys Gly Pro Leu Thr Cys Thr Ser Asn Thr Ala Thr
Arg 35 40 45Asn Tyr Gln Trp Tyr Val Asp Ala Trp 50
5534556PRTArtificial sequenceSynthesized UL63 345Cys Ser Thr Val
Tyr Gln Lys Thr Glu Lys Lys Cys Pro Asp Gly Tyr1 5 10 15Thr Asp Arg
Arg Asp Glu Cys Pro Asn Thr Cys Lys Asn Phe Asp Cys 20 25 30Glu Asn
Glu Gly Gly Leu Arg Cys Leu Cys Ser Ala Tyr Ile Ser Ala 35 40 45Tyr
Glu Phe His Val Asp Ala Trp 50 5534655PRTArtificial
sequenceSynthesized UL64 346Cys Thr Thr Thr His Gln Arg Thr Gln Lys
Ser Cys Pro Asp Tyr Ala1 5 10 15Ser Tyr Asp Cys Gly Ser Pro Asp Asp
Glu Glu Cys Ser Ser Cys Arg 20 25 30Ser Cys Thr Arg Trp Cys Ala Pro
Thr Ala Pro Tyr Ile Tyr Thr Tyr 35 40 45Gln Phe Tyr Ile Asp Ala Trp
50 5534754PRTArtificial sequenceSynthesized UL65 347Cys Thr Thr Val
His Gln Gln Thr Asn Lys Arg Cys Pro Thr Gly Tyr1 5 10 15Asn Ser Gly
Thr Leu Cys Asn Met Ile Gly Cys Ser Gly Asp Glu Cys 20 25 30Cys Asn
Tyr Gly Arg Val Glu Cys Thr Ser Tyr Val Trp Thr His Asn 35 40 45Phe
Tyr Val Asp Ala Trp 5034853PRTArtificial sequenceSynthesized UL66
348Cys Thr Thr Val His Gln Glu Thr Gln Arg Thr Ser Cys Pro Ser Gly1
5 10 15Trp Thr Tyr Thr Cys Asn Cys Arg Asn Gly Cys Gly Cys Tyr Arg
Pro 20 25 30Ser Gln Leu Cys Gly Ala Tyr Val Ala Val Thr His Thr Tyr
Glu Phe 35 40 45His Val Asp Ala Trp 5034953PRTArtificial
sequenceSynthesized UL67 349Cys Ala Thr Val His Gln Lys Asp Lys His
Cys Pro Ala Gly Tyr Arg1 5 10 15Ser Gly Thr Leu Cys Arg Met Ile Gly
Cys Thr Gly Asp Asp Cys Cys 20 25 30Asn Tyr Asp Arg Val Glu Cys Thr
Asn Tyr Asp Tyr Thr Asn Asn Phe 35 40 45Tyr Val Asp Ala Trp
5035052PRTArtificial sequenceSynthesized UL68 350Cys Thr Ala Val
His Gln Gln Thr Thr Glu Lys Gly Lys Thr Cys Pro1 5
10 15Pro Arg Ser Arg Asp Met Gly Thr Arg Cys Arg Asp Asp Arg Tyr
Tyr 20 25 30Pro Trp Arg Tyr Ser Asp Tyr Thr Tyr Thr Tyr Thr Tyr Glu
Trp His 35 40 45Val Asp Ala Trp 5035151PRTArtificial
sequenceSynthesized UL69 351Cys Thr Ser Val His Gln Lys Thr Asp Val
Thr Cys Pro Ser Gly Ala1 5 10 15Thr Tyr Arg Cys Asp Cys Gly Gly Arg
Gly Cys Gly Cys Tyr Asp Pro 20 25 30Trp Cys Ser Thr Thr Tyr Arg Gly
Thr Tyr Thr Tyr Asp Phe His Val 35 40 45Glu Thr Trp
5035250PRTArtificial sequenceSynthesized UL70 352Cys Gly Thr Val
His Gln Glu Thr His Thr Gln Arg Thr Cys Pro Asp1 5 10 15Ala Cys Asp
Val Thr Gly Asp Asn Cys Lys Val Arg Arg Asn Gly Asp 20 25 30Trp Cys
Gly Arg Ala Ser Lys Thr Asp Thr Tyr Asp Phe Tyr Val Asp 35 40 45Ala
Trp 5035349PRTArtificial sequenceSynthesized UL71 353Cys Thr Thr
Asp Tyr Gln Lys Thr Glu Lys Ser Cys Pro Glu Asn Tyr1 5 10 15Tyr Ala
Glu Thr Gly Tyr Cys Met Cys Gly Ser Trp Arg Cys Gly Tyr 20 25 30Gly
Ser Thr Thr Ser Leu Ile Val Ser Tyr Lys Trp Tyr Val Asp Ala 35 40
45Trp35449PRTArtificial sequenceSynthesized UL72 354Cys Thr Thr Val
His Gln Lys Thr Asn Gln Lys Trp Gly Cys Pro Asp1 5 10 15Gly Tyr Val
His Met Ser Gly Ser Cys Cys Arg Gly Ser Ile Cys Thr 20 25 30Asn Gly
Leu Phe Arg Asn Thr Tyr Thr Tyr Glu Phe Asn Val Glu Ala 35 40
45Trp35548PRTArtificial sequenceSynthesized UL73 355Cys Thr Thr Val
Tyr Gln Glu Thr Arg Thr Asn Cys Pro Asp Gly Tyr1 5 10 15Asn Tyr Arg
Ser Gly Asp Cys Arg Arg Trp Asn His Trp Leu Gly Glu 20 25 30Gln Arg
Val Ser Pro Thr Tyr Asn Tyr Glu Trp Tyr Val Asp Ser Trp 35 40
4535647PRTArtificial sequenceSynthesized UL74 356Cys Thr Thr Val
Tyr Gln Lys Thr Thr Thr Thr Lys Ser Cys Pro Gly1 5 10 15Gly Phe Asp
Asn Gly Arg Arg Cys Ile Met Gly Leu Gly Asp Leu Arg 20 25 30Asp Tyr
Thr Tyr Phe Asn Lys Tyr Glu Trp Tyr Val Glu Thr Trp 35 40
4535746PRTArtificial sequenceSynthesized UL75 357Cys Ser Thr Val
His Gln Lys Thr Glu Gln Arg Cys Leu Asp Gly Tyr1 5 10 15Asp Asp Arg
Gly Ala Tyr Cys Tyr Asp Ser Val Arg Gly Leu Met Ser 20 25 30Trp Thr
Tyr Lys Tyr Val Tyr Glu Trp Arg Val Asp Thr Trp 35 40
4535845PRTArtificial sequenceSynthesized UL76 358Cys Thr Asn Val
His Gln Met Thr Ile Lys Thr Cys Pro Asp Gly Gly1 5 10 15Ser Tyr Gly
Trp Tyr Trp Pro Tyr Gly Tyr Gly Cys Asn Gly Gly Val 20 25 30Ser Ala
Thr Tyr Thr Tyr Glu Phe Tyr Val Asp Ala Trp 35 40
4535944PRTArtificial sequenceSynthesized UL77 359Cys Thr Thr Val
Tyr Gln Lys Thr Glu Ser Val Arg Ser Cys Pro Asp1 5 10 15Gly Ser Met
Asp Gly Trp Arg Cys Arg Leu Gly Thr Met Asn Trp Ile 20 25 30Tyr Ser
Asn Thr Tyr Glu Phe Tyr Val Asp Ala Trp 35 4036075PRTArtificial
sequenceSynthesized BLV1H12 360Cys Thr Ser Val His Gln Glu Thr Lys
Lys Tyr Gln Ser Cys Pro Asp1 5 10 15Gly Tyr Arg Glu Arg Ser Asp Cys
Ser Asn Arg Pro Ala Cys Gly Thr 20 25 30Ser Asp Cys Cys Arg Val Ser
Val Phe Gly Asn Cys Leu Thr Thr Leu 35 40 45Pro Val Ser Tyr Ser Tyr
Thr Tyr Asn Tyr Glu Trp His Val Asp Val 50 55 60Trp Gly Gln Gly Leu
Leu Val Thr Val Ser Ser65 70 7536170PRTArtificial
sequenceSynthesized BLV5B8 361Cys Thr Thr Val His Gln Glu Thr Arg
Lys Thr Cys Ser Asp Gly Tyr1 5 10 15Ile Ala Val Asp Ser Cys Gly Arg
Gly Gln Ser Asp Gly Cys Val Asn 20 25 30Asp Cys Asn Ser Cys Tyr Tyr
Gly Trp Arg Asn Cys Arg Arg Gln Pro 35 40 45Ala Ile His Ser Tyr Glu
Phe His Val Asp Ala Trp Gly Arg Gly Leu 50 55 60Leu Val Thr Val Ser
Ser65 7036271PRTArtificial sequenceSynthesized BLV5D3 362Cys Ser
Ser Val Thr Gln Arg Thr His Val Ser Arg Ser Cys Pro Asp1 5 10 15Gly
Cys Ser Asp Gly Asp Gly Cys Val Asp Gly Cys Cys Cys Ser Ala 20 25
30Tyr Arg Cys Tyr Thr Pro Gly Val Arg Asp Leu Ser Cys Thr Ser Tyr
35 40 45Ser Ile Thr Tyr Thr Tyr Glu Trp Asn Val Asp Ala Trp Gly Arg
Gly 50 55 60Leu Leu Val Thr Val Ser Ser65 7036372PRTArtificial
sequenceSynthesized BLV8C11 363Cys Thr Thr Val His Gln Lys Thr Thr
Arg Lys Thr Cys Cys Ser Asp1 5 10 15Ala Tyr Arg Tyr Asp Ser Gly Cys
Gly Ser Gly Cys Asp Cys Cys Gly 20 25 30Ala Asp Cys Tyr Val Phe Gly
Ala Cys Thr Phe Gly Leu Asp Ser Ser 35 40 45Tyr Ser Tyr Ile Tyr Ile
Tyr Gln Trp Tyr Val Asp Ala Trp Gly Gln 50 55 60Gly Leu Leu Val Thr
Val Ser Ser65 7036470PRTArtificial sequenceSynthesized BF4E9 364Cys
Thr Thr Val His Gln Ile Phe Cys Pro Asp Gly Tyr Ser Tyr Gly1 5 10
15Tyr Gly Cys Gly Tyr Gly Tyr Gly Cys Ser Gly Tyr Asp Cys Tyr Gly
20 25 30Tyr Gly Gly Tyr Gly Tyr Gly Gly Tyr Gly Gly Tyr Ser Ser Tyr
Ser 35 40 45Tyr Ser Tyr Ser Tyr Glu Tyr Tyr Gly Asp Ala Trp Gly Gln
Gly Leu 50 55 60Leu Val Thr Val Ser Ser65 7036550PRTArtificial
sequenceSynthesized BF1H1 365Cys Thr Thr Val His Pro Ser Pro Asp
Gly Tyr Ser Tyr Gly Tyr Gly1 5 10 15Cys Gly Tyr Gly Tyr Gly Cys Ser
Gly Tyr Asp Cys Tyr Gly Tyr Gly 20 25 30Gly Tyr Gly Tyr Gly Gly Tyr
Gly Gly Tyr Ser Ser Tyr Ser Tyr Ser 35 40 45Tyr Ser
5036650PRTArtificial sequenceSynthesized F18 366Cys Thr Thr Val His
Gln Ile Arg Cys Pro Asp Gly Tyr Gly Tyr Gly1 5 10 15Tyr Gly Cys Gly
Tyr Gly Ser Tyr Gly Tyr Ser Gly Tyr Asp Cys Tyr 20 25 30Gly Tyr Gly
Gly Tyr Gly Gly Tyr Gly Gly Tyr Gly Gly Tyr Ser Ser 35 40 45Tyr Ser
50367300DNAArtificial sequenceSynthesized Bovine VH-UL
367caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac
cctctcgctc 60acctgcacgg cctctggatt ctcattgagc gacaaggctg taggctgggt
ccgccaggct 120ccagggaagg cgctggagtg gctcggtggt atagacactg
gtggaagcac aggctataac 180ccaggcctga aatcccggct cagcatcacc
aaggacaact ccaagagcca agtctctctg 240tcagtgagca gcgtgacaac
tgaggactcg gccacatact actgtactac tgtgcaccag 300368311DNAArtificial
sequenceSynthesized 4-39 368cagctgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc
agtagtagtt actactgggg ctggatccgc 120cagcccccag ggaaggggct
ggagtggatt gggagtatct attatagtgg gagcacctac 180tacaacccgt
ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc
240tccctgaagc tgagctctgt gaccgccgca gacacggctg tgtattactg
tgcgagacac 300acagtgaggg g 311369288DNAArtificial
sequenceSynthesized 4-59*03 369caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
agttactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg
gattgggtat atctattaca gtgggagcac caactacaac 180ccctccctca
agagtcgagt caccatatca gtagacacgt ccaagaacca attctccctg
240aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcg
288370291DNAArtificial sequenceSynthesized 4-34*09 370caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60acctgcgctg
tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc
120ccagggaagg gactggagtg gattggggaa atcaatcata gtggaagcac
caactacaac 180ccgtccctca agagtcgagt taccatatca gtagacacgt
ctaagaacca gttctccctg 240aagctgagct ctgtgactgc cgcggacacg
gccgtgtatt actgtgcgag a 291371293DNAArtificial sequenceSynthesized
4-34*02 371caggtgcagc tacaacagtg gggcgcagga ctgttgaagc cttcggagac
cctgtccctc 60acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat
ccgccagccc 120ccagggaagg ggctggagtg gattggggaa atcaatcata
gtggaagcac caactacaac 180ccgtccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc
cgcggacacg gctgtgtatt actgtgcgag agg 293372330DNAArtificial
sequenceSynthesized BLV1H12 VL 372caggctgtgc tgaatcagcc atcatccgtg
tccgggtccc tgggccagag ggtctccatc 60acctgctctg gaagcagcag caatgttgga
aatggatatg tgagctggta ccaactgatc 120ccaggatcgg cccccagaac
cctcatctat ggtgacacca gtcgagcctc gggggtcccc 180gaccgattct
ccggctccag gtctgggaac acagccaccc tgaccatcag ctcgctccag
240gctgaggacg aggcagatta tttctgtgca tctgctgagg atagtagcag
taatgctgtt 300ttcggcagcg ggaccacact gaccgtcctg
330373296DNAArtificial sequenceSynthesized Vl1-47 373cagtctgtgc
tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg
gaagcagctc caacatcgga agtaattatg tatactggta ccagcagctc
120ccaggaacgg cccccaaact cctcatctat aggaataatc agcggccctc
aggggtccct 180gaccgattct ctggctccaa gtctggcacc tcagcctccc
tggccatcag tgggctccgg 240tccgaggatg aggctgatta ttactgtgca
gcatgggatg acagcctgag tggtcc 296374299DNAArtificial
sequenceSynthesized Vl1-40*1 374cagtctgtgc tgacgcagcc gccctcagtg
tctggggccc cagggcagag ggtcaccatc 60tcctgcactg ggagcagctc caacatcggg
gcaggttatg atgtacactg gtaccagcag 120cttccaggaa cagcccccaa
actcctcatc tatggtaaca gcaatcggcc ctcaggggtc 180cctgaccgat
tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc
240caggctgagg atgaggctga ttattactgc cagtcctatg acagcagcct gagtggttc
299375296DNAArtificial sequenceSynthesized Vl1-51 *01 375cagtctgtgt
tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg
gaagcagctc caacattggg aataattatg tatcctggta ccagcagctc
120ccaggaacag cccccaaact cctcatttat gacaataata agcgaccctc
agggattcct 180gaccgattct ctggctccaa gtctggcacg tcagccaccc
tgggcatcac cggactccag 240actggggacg aggccgatta ttactgcgga
acatgggata gcagcctgag tgctgg 296376297DNAArtificial
sequenceSynthesized Vl2-18*02 376cagtctgccc tgactcagcc tccctccgtg
tccgggtctc ctggacagtc agtcaccatc 60tcctgcactg gaaccagcag tgacgttggt
agttataacc gtgtctcctg gtaccagcag 120cccccaggca cagcccccaa
actcatgatt tatgaggtca gtaatcggcc ctcaggggtc 180cctgatcgct
tctctgggtc caagtctggc aacacggcct ccctgaccat ctctgggctc
240caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cactttc
297377514DNAArtificial sequenceSynthesized partially human antibody
comprising an ultralong CDR3 377cagctgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc
agtagtagtt actactgggg ctggatccgc 120cagcccccag ggaaggggct
ggagtggatt gggagtatct attatagtgg gagcacctac 180tacaacccgt
ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc
240tccctgaagc tgagctctgt gaccgccgca gacacggctg tgtattactg
tactactgtg 300caccaggaaa caaaaaaata ccaaagttgt cctgatgggt
atagagaacg ttcggattgt 360agtaacagac ctgcttgtgg tactagtgat
tgttgtcgtg ttagtgtttt tggtaattgt 420cttactactc ttcctgtgag
ttatagttat acttacaatt acgaatggca cgtcgatgtc 480tggggccagg
gaaccctggt caccgtctcc tcag 514378171PRTArtificial
sequenceSynthesized partially human antibody comprising an
ultralong CDR3 amino acid sequence 378Gln Leu Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30Ser Tyr Tyr Trp Gly
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45Trp Ile Gly Ser
Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60Leu Lys Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe65 70 75 80Ser
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90
95Cys Thr Thr Val His Gln Glu Thr Lys Lys Tyr Gln Ser Cys Pro Asp
100 105 110Gly Tyr Arg Glu Arg Ser Asp Cys Ser Asn Arg Pro Ala Cys
Gly Thr 115 120 125Ser Asp Cys Cys Arg Val Ser Val Phe Gly Asn Cys
Leu Thr Thr Leu 130 135 140Pro Val Ser Tyr Ser Tyr Thr Tyr Asn Tyr
Glu Trp His Val Asp Val145 150 155 160Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 165 170379493DNAArtificial sequenceSynthesized
partially human antibody V region comprised of an ultralong CDR3
379caggtgcagc tacaacagtg gggcgcagga ctgttgaagc cttcggagac
cctgtccctc 60acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat
ccgccagccc 120ccagggaagg ggctggagtg gattggggaa atcaatcata
gtggaagcac caactacaac 180ccgtccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc
cgcggacacg gctgtgtatt actgtactac tgtgcaccag 300gaaaccagaa
aaacctgttc tgatggttat atggctgtag atagttgtgg tcgtggtcag
360agtgatggtt gtgtcaatga ttgcaattgt tgttattatg gttggcggaa
ctgtcgcagg 420cagcctgcaa ttcaaagtta cgaatttcac gtcgatgcct
ggggccgtgg caccctggtc 480actgtctcct cag 493380164PRTArtificial
sequenceSynthesized partially human antibody V region comprised of
an ultralong CDR3 380Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu
Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly
Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn His Ser Gly Ser
Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val
Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95Thr Val His Gln
Glu Thr Arg Lys Thr Cys Ser Asp Gly Tyr Met Ala 100 105 110Val Asp
Ser Cys Gly Arg Gly Gln Ser Asp Gly Cys Val Asn Asp Cys 115 120
125Asn Cys Cys Tyr Tyr Gly Trp Arg Asn Cys Arg Arg Gln Pro Ala Ile
130 135 140Gln Ser Tyr Glu Phe His Val Asp Ala Trp Gly Arg Gly Thr
Leu Val145 150 155 160Thr Val Ser Ser38163DNAArtificial
sequenceSynthesized MID1 FW 381cctatcccct gtgtgccttg gcagtctcag
acgagtgcgt ttgagcgaca aggctgtagg 60ctg 6338262DNAArtificial
sequenceSynthesized MID1 RV 382ccatctcatc cctgcgtgtc tccgactcag
acgagtgcgt ctttcggggc tgtggtggag 60gc 6238363DNAArtificial
sequenceSynthesized MID10 FW 383cctatcccct gtgtgccttg gcagtctcag
tctctatgcg ttgagcgaca aggctgtagg 60ctg 6338466DNAArtificial
sequenceSynthesized MID10 RV 384ccatctcatc cctgcgtgtc tccgactcag
tctctatgcg agtgaagact ctcgggtgtg 60attcac 6638563DNAArtificial
sequenceSynthesized MID11 FW 385cctatcccct gtgtgccttg gcagtctcag
tgatacgtct ttgagcgaca aggctgtagg 60ctg 6338666DNAArtificial
sequenceSynthesized MID11 RV 386ccatctcatc cctgcgtgtc tccgactcag
tgatacgtct agtgaagact ctcgggtgtg 60attcac 6638723DNAArtificial
sequenceSynthesized primer 1 for bovine VH region 387ttgagcgaca
aggctgtagg ctg 2338822DNAArtificial sequenceSynthesized primer 2
for bovine VH region 388ctttcggggc tgtggtggag gc
2238920DNAArtificial sequenceSynthesized sequencing primer
389agatccaagc tgtgaccggc 203901002DNAArtificial sequenceSynthesized
CH1 CH2 CH3 of human IgG1CDS(1)..(1002) 390gct agc acc aag ggc cca
tcg gtc ttc ccc ctg gca ccc tcc tcc aag 48Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15agc
acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac 96Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30ttc ccc gag ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc
144Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac
tcc 192Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc
cag acc 240Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr65 70 75 80tac atc tgc aac gtg aat cac aag ccc agc aac acc
aag gtg gac aag 288Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95aaa gtt gag ccc aaa tct tgt gac aaa act cac
aca tgc cca ccg tgc 336Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110cca gca cct gaa ctc ctg ggg gga ccg
tca gtc ttc ctc ttc ccc cca 384Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125aaa ccc aag gac acc ctc atg
atc tcc cgg acc cct gag gtc aca tgc 432Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140gtg gtg gtg gac gtg
agc cac gaa gac cct gag gtc aag ttc aac tgg 480Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160tac gtg
gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag 528Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg
576Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtg
tcc aac 624His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc
aaa gcc aaa ggg 672Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220cag ccc cga gaa cca cag gtg tac acc ctg
ccc cca tcc cgg gat gag 720Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240ctg acc aag aac cag gtc agc
ctg acc tgc ctg gtc aaa ggc ttc tat 768Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255ccc agc gac atc gcc
gtg gag tgg gag agc aat ggg cag ccg gag aac 816Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270aac tac aag
acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc 864Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285ctc
tac agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac 912Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg
960Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320cag aag agc ctc tcc ctg tct ccg ggt aaa tga taa
tct aga 1002Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Arg 325
330391330PRTArtificial sequenceSynthetic Construct 391Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310
315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330392513DNAArtificial sequenceSynthesized BLV1H12 V region
392caggtccagc tgagagagag cggcccttca ctggtcaagc catcccagac
actgagcctg 60acatgcacag caagcgggtt ttcactgagc gacaaggcag tgggatgggt
ccgacaggca 120ccaggaaaag ccctggaatg gctgggcagc atcgataccg
gcgggaacac agggtacaat 180cccggactga agagcagact gtccattacc
aaggacaact ctaaaagtca ggtgtcactg 240agcgtgagct ccgtcaccac
agaggatagt gcaacttact attgcacctc tgtgcaccag 300gaaactaaga
aataccagag ctgtcctgac ggctatcggg agagatctga ttgcagtaat
360aggccagctt gtggcacatc cgactgctgt cgcgtgtctg tcttcgggaa
ctgcctgact 420accctgcctg tgtcctactc ttatacctac aattatgaat
ggcatgtgga tgtctgggga 480cagggcctgc tggtgacagt ctctagtgct agc
5133931563DNAArtificial sequenceSynthesized (Sig Seq- VRegion -
CH1CH2CH3) BLV1H12 V in human IgGCDS(1)..(1563) 393atg cgc agg atg
caa ctc ctg ttg ctg att gca cta agt ctt gca ctt 48Met Arg Arg Met
Gln Leu Leu Leu Leu Ile Ala Leu Ser Leu Ala Leu1 5 10 15gtc acg aat
tcg cag gtc cag ctg aga gag agc ggc cct tca ctg gtc 96Val Thr Asn
Ser Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val 20 25 30aag cca
tcc cag aca ctg agc ctg aca tgc aca gca agc ggg ttt tca 144Lys Pro
Ser Gln Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser 35 40 45ctg
agc gac aag gca gtg gga tgg gtc cga cag gca cca gga aaa gcc 192Leu
Ser Asp Lys Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala 50 55
60ctg gaa tgg ctg ggc agc atc gat acc ggc ggg aac aca ggg tac aat
240Leu Glu Trp Leu Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr
Asn65 70 75 80ccc gga ctg aag agc aga ctg tcc att acc aag gac aac
tct aaa agt 288Pro Gly Leu Lys Ser Arg Leu Ser Ile Thr Lys Asp Asn
Ser Lys Ser 85 90 95cag gtg tca ctg agc gtg agc tcc gtc acc aca gag
gat agt gca act 336Gln Val Ser Leu Ser Val Ser Ser Val Thr Thr Glu
Asp Ser Ala Thr 100 105 110tac tat tgc acc tct gtg cac cag gaa act
aag aaa tac cag agc tgt 384Tyr Tyr Cys Thr Ser Val His Gln Glu Thr
Lys Lys Tyr Gln Ser Cys 115 120 125cct gac ggc tat cgg gag aga tct
gat tgc agt aat agg cca gct tgt 432Pro Asp Gly Tyr Arg Glu Arg Ser
Asp Cys Ser Asn Arg Pro Ala Cys 130 135 140ggc aca tcc gac tgc tgt
cgc gtg tct gtc ttc ggg aac tgc ctg act 480Gly Thr Ser Asp Cys Cys
Arg Val Ser Val Phe Gly Asn Cys Leu Thr145 150 155 160acc ctg cct
gtg tcc tac tct tat acc tac aat tat gaa tgg cat gtg 528Thr Leu Pro
Val Ser Tyr Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val 165 170 175gat
gtc tgg gga cag ggc ctg ctg gtg aca gtc tct agt gct agc acc 576Asp
Val Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser Ala Ser Thr 180 185
190aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct
624Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
195 200 205ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc
ccc gag 672Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu 210 215 220ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc
agc ggc gtg cac 720Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His225 230 235 240acc ttc ccg gct gtc cta cag tcc tca
gga ctc tac tcc ctc agc agc 768Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser 245 250 255gtg gtg acc gtg ccc tcc agc
agc ttg ggc acc cag acc tac atc tgc 816Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys 260 265 270aac gtg aat cac aag
ccc agc aac acc aag gtg gac aag aaa gtt gag 864Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 275 280 285ccc aaa tct
tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct 912Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 290 295 300gaa
ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag 960Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys305 310
315 320gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg
gtg 1008Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val 325 330 335gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg
tac gtg gac 1056Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp 340 345 350ggc gtg gag gtg cat aat gcc aag aca aag ccg
cgg gag gag cag tac 1104Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr 355 360 365aac agc acg tac cgt gtg gtc agc gtc
ctc acc gtc ctg cac cag gac 1152Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 370 375 380tgg ctg aat ggc aag gag tac
aag tgc aag gtg tcc aac aaa gcc ctc 1200Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu385 390 395 400cca gcc ccc atc
gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 1248Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 405 410 415gaa cca
cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag 1296Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 420 425
430aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac
1344Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
435 440 445atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac
tac aag 1392Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 450 455 460acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc
ttc ctc tac agc 1440Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser465 470 475 480aag ctc acc gtg gac aag agc agg tgg
cag cag ggg aac gtc ttc tca 1488Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser 485 490 495tgc tcc gtg atg cat gag gct
ctg cac aac cac tac acg cag aag agc 1536Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser 500 505 510ctc tcc ctg tct ccg
ggt aaa tga taa 1563Leu Ser Leu Ser Pro Gly Lys
515394519PRTArtificial sequenceSynthetic Construct 394Met Arg Arg
Met Gln Leu Leu Leu Leu Ile Ala Leu Ser Leu Ala Leu1 5 10 15Val Thr
Asn Ser Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val 20 25 30Lys
Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser 35 40
45Leu Ser Asp Lys Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala
50 55 60Leu Glu Trp Leu Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr
Asn65 70 75 80Pro Gly Leu Lys Ser Arg Leu Ser Ile Thr Lys Asp Asn
Ser Lys Ser 85 90 95Gln Val Ser Leu Ser Val Ser Ser Val Thr Thr Glu
Asp Ser Ala Thr 100 105 110Tyr Tyr Cys Thr Ser Val His Gln Glu Thr
Lys Lys Tyr Gln Ser Cys 115 120 125Pro Asp Gly Tyr Arg Glu Arg Ser
Asp Cys Ser Asn Arg Pro Ala Cys 130 135 140Gly Thr Ser Asp Cys Cys
Arg Val Ser Val Phe Gly Asn Cys Leu Thr145 150 155 160Thr Leu Pro
Val Ser Tyr Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val 165 170 175Asp
Val Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser Ala Ser Thr 180 185
190Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
195 200 205Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu 210 215 220Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His225 230 235 240Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser 245 250 255Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys 260 265 270Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 275 280 285Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 290 295 300Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys305 310
315 320Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val 325 330 335Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp 340 345 350Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr 355 360 365Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 370 375 380Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu385 390 395 400Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 405 410 415Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 420 425
430Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
435 440 445Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 450 455 460Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser465 470 475 480Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser 485 490 495Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser 500 505 510Leu Ser Leu Ser Pro
Gly Lys 5153951470DNAArtificial sequenceSynthesized BLV1H12 V in
human IgG with BsaI cassette in CDR3CDS(1)..(1470) 395atg cgc agg
atg caa ctc ctg ttg ctg att gca cta agt ctt gca ctt 48Met Arg Arg
Met Gln Leu Leu Leu Leu Ile Ala Leu Ser Leu Ala Leu1 5 10 15gtc acg
aat tcg cag gtc cag ctg aga gag agc ggc cct tca ctg gtc 96Val Thr
Asn Ser Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val 20 25 30aag
cca tcc cag aca ctg agc ctg aca tgc aca gca agc ggg ttt tca 144Lys
Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser 35 40
45ctg agc gac aag gca gtg gga tgg gtc cga cag gca cca gga aaa gcc
192Leu Ser Asp Lys Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala
50 55 60ctg gaa tgg ctg ggc agc atc gat acc ggc ggg aac aca ggg tac
aat 240Leu Glu Trp Leu Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr
Asn65 70 75 80ccc gga ctg aag agc aga
ctg tcc att acc aag gac aac tct aaa agt 288Pro Gly Leu Lys Ser Arg
Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser 85 90 95cag gtg tca ctg agc
gtg agc tcc gtc acc aca gag gat agt gca act 336Gln Val Ser Leu Ser
Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr 100 105 110tac tat tgc
acc tct gtg cac cag gaa act aag aaa tac cag agc gag 384Tyr Tyr Cys
Thr Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Glu 115 120 125acc
tac tat ggt tcg ggt ctc tct tat acc tac aat tat gaa tgg cat 432Thr
Tyr Tyr Gly Ser Gly Leu Ser Tyr Thr Tyr Asn Tyr Glu Trp His 130 135
140gtg gat gtc tgg gga cag ggc ctg ctg gtg aca gtc tct agt gct agc
480Val Asp Val Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser Ala
Ser145 150 155 160acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc
tcc aag agc acc 528Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr 165 170 175tct ggg ggc aca gcg gcc ctg ggc tgc ctg
gtc aag gac tac ttc ccc 576Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro 180 185 190gag ccg gtg acg gtg tcg tgg aac
tca ggc gcc ctg acc agc ggc gtg 624Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val 195 200 205cac acc ttc ccg gct gtc
cta cag tcc tca gga ctc tac tcc ctc agc 672His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 210 215 220agc gtg gtg acc
gtg ccc tcc agc agc ttg ggc acc cag acc tac atc 720Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile225 230 235 240tgc
aac gtg aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt 768Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 245 250
255gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca
816Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
260 265 270cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca
aaa ccc 864Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro 275 280 285aag gac acc ctc atg atc tcc cgg acc cct gag gtc
aca tgc gtg gtg 912Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val 290 295 300gtg gac gtg agc cac gaa gac cct gag gtc
aag ttc aac tgg tac gtg 960Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val305 310 315 320gac ggc gtg gag gtg cat aat
gcc aag aca aag ccg cgg gag gag cag 1008Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln 325 330 335tac aac agc acg tac
cgt gtg gtc agc gtc ctc acc gtc ctg cac cag 1056Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln 340 345 350gac tgg ctg
aat ggc aag gag tac aag tgc aag gtg tcc aac aaa gcc 1104Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 355 360 365ctc
cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc 1152Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 370 375
380cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc
1200Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr385 390 395 400aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc
ttc tat ccc agc 1248Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser 405 410 415gac atc gcc gtg gag tgg gag agc aat ggg
cag ccg gag aac aac tac 1296Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr 420 425 430aag acc acg cct ccc gtg ctg gac
tcc gac ggc tcc ttc ttc ctc tac 1344Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr 435 440 445agc aag ctc acc gtg gac
aag agc agg tgg cag cag ggg aac gtc ttc 1392Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 450 455 460tca tgc tcc gtg
atg cat gag gct ctg cac aac cac tac acg cag aag 1440Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys465 470 475 480agc
ctc tcc ctg tct ccg ggt aaa tga taa 1470Ser Leu Ser Leu Ser Pro Gly
Lys 485396488PRTArtificial sequenceSynthetic Construct 396Met Arg
Arg Met Gln Leu Leu Leu Leu Ile Ala Leu Ser Leu Ala Leu1 5 10 15Val
Thr Asn Ser Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val 20 25
30Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser
35 40 45Leu Ser Asp Lys Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys
Ala 50 55 60Leu Glu Trp Leu Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly
Tyr Asn65 70 75 80Pro Gly Leu Lys Ser Arg Leu Ser Ile Thr Lys Asp
Asn Ser Lys Ser 85 90 95Gln Val Ser Leu Ser Val Ser Ser Val Thr Thr
Glu Asp Ser Ala Thr 100 105 110Tyr Tyr Cys Thr Ser Val His Gln Glu
Thr Lys Lys Tyr Gln Ser Glu 115 120 125Thr Tyr Tyr Gly Ser Gly Leu
Ser Tyr Thr Tyr Asn Tyr Glu Trp His 130 135 140Val Asp Val Trp Gly
Gln Gly Leu Leu Val Thr Val Ser Ser Ala Ser145 150 155 160Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 165 170
175Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
180 185 190Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val 195 200 205His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser 210 215 220Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile225 230 235 240Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val 245 250 255Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 260 265 270Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 275 280 285Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 290 295
300Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val305 310 315 320Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln 325 330 335Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln 340 345 350Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala 355 360 365Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 370 375 380Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr385 390 395 400Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 405 410
415Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
420 425 430Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr 435 440 445Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe 450 455 460Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys465 470 475 480Ser Leu Ser Leu Ser Pro Gly
Lys 485397258DNAArtificial sequenceSynthesized IL-8 26-99
InsertCDS(1)..(258) 397aat tcg ggt ctc aag agc cca agg agt gct aaa
gaa ctt aga tgt cag 48Asn Ser Gly Leu Lys Ser Pro Arg Ser Ala Lys
Glu Leu Arg Cys Gln1 5 10 15tgc ata aag aca tac tcc aaa cct ttc cac
ccc aag ttc atc aag gag 96Cys Ile Lys Thr Tyr Ser Lys Pro Phe His
Pro Lys Phe Ile Lys Glu 20 25 30ctg aga gtg att gag agt gga cca cac
tgc gcc aac aca gag att att 144Leu Arg Val Ile Glu Ser Gly Pro His
Cys Ala Asn Thr Glu Ile Ile 35 40 45gta aag ctt tct gat ggg aga gag
ctc tgc ctg gac ccc aag gaa aac 192Val Lys Leu Ser Asp Gly Arg Glu
Leu Cys Leu Asp Pro Lys Glu Asn 50 55 60tgg gtg cag agg gtc gtg gag
aag ttc ttg aag agg gct gag aac tca 240Trp Val Gln Arg Val Val Glu
Lys Phe Leu Lys Arg Ala Glu Asn Ser65 70 75 80tct tat gag acc agc
taa 258Ser Tyr Glu Thr Ser 8539885PRTArtificial sequenceSynthetic
Construct 398Asn Ser Gly Leu Lys Ser Pro Arg Ser Ala Lys Glu Leu
Arg Cys Gln1 5 10 15Cys Ile Lys Thr Tyr Ser Lys Pro Phe His Pro Lys
Phe Ile Lys Glu 20 25 30Leu Arg Val Ile Glu Ser Gly Pro His Cys Ala
Asn Thr Glu Ile Ile 35 40 45Val Lys Leu Ser Asp Gly Arg Glu Leu Cys
Leu Asp Pro Lys Glu Asn 50 55 60Trp Val Gln Arg Val Val Glu Lys Phe
Leu Lys Arg Ala Glu Asn Ser65 70 75 80Ser Tyr Glu Thr Ser
85399267DNAArtificial sequenceSynthesized IL8 26-99 + GlySerGly
Linker InsertCDS(1)..(267) 399aat tcg ggt ctc aag agc cca agg agt
gct aaa gaa ctt aga tgt cag 48Asn Ser Gly Leu Lys Ser Pro Arg Ser
Ala Lys Glu Leu Arg Cys Gln1 5 10 15tgc ata aag aca tac tcc aaa cct
ttc cac ccc aag ttc atc aag gag 96Cys Ile Lys Thr Tyr Ser Lys Pro
Phe His Pro Lys Phe Ile Lys Glu 20 25 30ctg aga gtg att gag agt gga
cca cac tgc gcc aac aca gag att att 144Leu Arg Val Ile Glu Ser Gly
Pro His Cys Ala Asn Thr Glu Ile Ile 35 40 45gta aag ctt tct gat ggg
aga gag ctc tgc ctg gac ccc aag gaa aac 192Val Lys Leu Ser Asp Gly
Arg Glu Leu Cys Leu Asp Pro Lys Glu Asn 50 55 60tgg gtg cag agg gtc
gtg gag aag ttc ttg aag agg gct gag aac tca 240Trp Val Gln Arg Val
Val Glu Lys Phe Leu Lys Arg Ala Glu Asn Ser65 70 75 80ggc agc ggt
tct tat gag acc agc taa 267Gly Ser Gly Ser Tyr Glu Thr Ser
8540088PRTArtificial sequenceSynthetic Construct 400Asn Ser Gly Leu
Lys Ser Pro Arg Ser Ala Lys Glu Leu Arg Cys Gln1 5 10 15Cys Ile Lys
Thr Tyr Ser Lys Pro Phe His Pro Lys Phe Ile Lys Glu 20 25 30Leu Arg
Val Ile Glu Ser Gly Pro His Cys Ala Asn Thr Glu Ile Ile 35 40 45Val
Lys Leu Ser Asp Gly Arg Glu Leu Cys Leu Asp Pro Lys Glu Asn 50 55
60Trp Val Gln Arg Val Val Glu Lys Phe Leu Lys Arg Ala Glu Asn Ser65
70 75 80Gly Ser Gly Ser Tyr Glu Thr Ser 85401435DNAArtificial
sequenceSynthesized IL-21 InsertCDS(1)..(435) 401aat tcg ggt ctc
aag agc caa ggt caa gat cgc cac atg atc aga atg 48Asn Ser Gly Leu
Lys Ser Gln Gly Gln Asp Arg His Met Ile Arg Met1 5 10 15cgt cag ctc
ata gat att gtt gat cag ctg aag aac tac gtg aac gac 96Arg Gln Leu
Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp 20 25 30ttg gtc
cct gaa ttt ctg cca gct ccc gaa gat gta gag aca aac tgt 144Leu Val
Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys 35 40 45gag
tgg tca gcc ttc tcc tgc ttt cag aag gcc caa cta aag tca gca 192Glu
Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala 50 55
60aat acc ggc aac aac gag agg ata atc aat gta tca atc aaa aag ctg
240Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys
Leu65 70 75 80aag agg aag cca cct tcc aca aat gca ggg aga cgg cag
aaa cac cgc 288Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln
Lys His Arg 85 90 95ctg aca tgc cct tca tgt gat tct tac gag aag aag
cca ccc aaa gag 336Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys
Pro Pro Lys Glu 100 105 110ttc cta gag cgg ttc aag tca ctt ctc gac
aag atg att gat cag cat 384Phe Leu Glu Arg Phe Lys Ser Leu Leu Asp
Lys Met Ile Asp Gln His 115 120 125ctg tcc tct cgc aca cac gga agt
gaa gat tcc tct tat gag acc agc 432Leu Ser Ser Arg Thr His Gly Ser
Glu Asp Ser Ser Tyr Glu Thr Ser 130 135 140taa
435402144PRTArtificial sequenceSynthetic Construct 402Asn Ser Gly
Leu Lys Ser Gln Gly Gln Asp Arg His Met Ile Arg Met1 5 10 15Arg Gln
Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp 20 25 30Leu
Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys 35 40
45Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala
50 55 60Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys
Leu65 70 75 80Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln
Lys His Arg 85 90 95Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys
Pro Pro Lys Glu 100 105 110Phe Leu Glu Arg Phe Lys Ser Leu Leu Asp
Lys Met Ile Asp Gln His 115 120 125Leu Ser Ser Arg Thr His Gly Ser
Glu Asp Ser Ser Tyr Glu Thr Ser 130 135 140403435DNAArtificial
sequenceSynthesized IL-21 (Q116D, H120D) InsertCDS(1)..(435) 403aat
tcg ggt ctc aag agc caa ggt caa gat cgc cac atg atc aga atg 48Asn
Ser Gly Leu Lys Ser Gln Gly Gln Asp Arg His Met Ile Arg Met1 5 10
15cgt cag ctc ata gat att gtt gat cag ctg aag aac tac gtg aac gac
96Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp
20 25 30ttg gtc cct gaa ttt ctg cca gct ccc gaa gat gta gag aca aac
tgt 144Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn
Cys 35 40 45gag tgg tca gcc ttc tcc tgc ttt cag aag gcc caa cta aag
tca gca 192Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys
Ser Ala 50 55 60aat acc ggc aac aac gag agg ata atc aat gta tca atc
aaa aag ctg 240Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile
Lys Lys Leu65 70 75 80aag agg aag cca cct tcc aca aat gca ggg aga
cgg cag aaa cac cgc 288Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg
Arg Gln Lys His Arg 85 90 95ctg aca tgc cct tca tgt gat tct tac gag
aag aag cca ccc aaa gag 336Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu
Lys Lys Pro Pro Lys Glu 100 105 110ttc cta gag cgg ttc aag tca ctt
ctc gac aag atg att gat cag cat 384Phe Leu Glu Arg Phe Lys Ser Leu
Leu Asp Lys Met Ile Asp Gln His 115 120 125ctg tcc tct cgc aca cac
gga agt gaa gat tcc tct tat gag acc agc 432Leu Ser Ser Arg Thr His
Gly Ser Glu Asp Ser Ser Tyr Glu Thr Ser 130 135 140taa
435404144PRTArtificial sequenceSynthetic Construct 404Asn Ser Gly
Leu Lys Ser Gln Gly Gln Asp Arg His Met Ile Arg Met1 5 10 15Arg Gln
Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp 20 25 30Leu
Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys 35 40
45Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala
50 55 60Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys
Leu65 70 75 80Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln
Lys His Arg 85 90 95Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys
Pro Pro Lys Glu 100 105 110Phe Leu Glu Arg Phe Lys Ser Leu Leu Asp
Lys Met Ile Asp Gln His 115 120 125Leu Ser Ser Arg Thr His Gly Ser
Glu Asp Ser Ser Tyr Glu Thr Ser 130 135 140405249DNAArtificial
sequenceSynthesized SDF-1alpha-GSG Insert 405aattcgggtc tcaagagcaa
gcccgtcagc ctgagctaca gatgcccatg ccgattcttc 60gaaagccatg ttgccagagc
caacgtcaag catctcaaaa ttctcaacac tccaaactgt
120gcccttcaga ttgtagcccg gctgaagaac aacaacagac aagtgtgcat
tgacccgaag 180ctaaagtgga ttcaggagta cctggagaaa gctttaaaca
agggcagcgg ttcttatgag 240accagctaa 24940678DNAArtificial
sequenceSynthesized Somatostatin-14CDS(1)..(78) 406aat tcg ggt ctc
aag agc gct ggc tgc aag aat ttc ttc tgg aag act 48Asn Ser Gly Leu
Lys Ser Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr1 5 10 15ttc aca tcc
tgt tct tat gag acc agc taa 78Phe Thr Ser Cys Ser Tyr Glu Thr Ser
20 2540725PRTArtificial sequenceSynthetic Construct 407Asn Ser Gly
Leu Lys Ser Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr1 5 10 15Phe Thr
Ser Cys Ser Tyr Glu Thr Ser 20 25408126DNAArtificial
sequenceSynthesized ProTx-II InsertCDS(1)..(126) 408aat tcg ggt ctc
aag agc tat tgc cag aag tgg atg tgg acc tgc gat 48Asn Ser Gly Leu
Lys Ser Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp1 5 10 15agc gaa cgg
aaa tgt tgc gaa ggc atg gtg tgc cgc ctg tgg tgc aag 96Ser Glu Arg
Lys Cys Cys Glu Gly Met Val Cys Arg Leu Trp Cys Lys 20 25 30aag aaa
ctc tgg tct tat gag acc agc taa 126Lys Lys Leu Trp Ser Tyr Glu Thr
Ser 35 4040941PRTArtificial sequenceSynthetic Construct 409Asn Ser
Gly Leu Lys Ser Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp1 5 10 15Ser
Glu Arg Lys Cys Cys Glu Gly Met Val Cys Arg Leu Trp Cys Lys 20 25
30Lys Lys Leu Trp Ser Tyr Glu Thr Ser 35 40410138DNAArtificial
sequenceSynthesized Chlorotoxin Insert 410aattcgggtc tcaagagcat
gtgtatgccc tgcttcacga ccgatcacca gatggcgcgc 60aaatgcgatg actgttgcgg
cggtaaaggt cgcggaaagt gctatggccc gcagtgtctg 120tcttatgaga ccagctaa
138411111DNAArtificial sequenceSynthesized Ziconotide Insert
411aattcgggtc tcaagagctg caagggcaaa ggtgcgaaat gcagccgcct
gatgtatgat 60tgctgtaccg ggtcctgccg cagtggcaag tgctcttatg agaccagcta
a 111412672DNAArtificial sequenceSynthesized BLV1H12 Light
ChainCDS(1)..(672) 412tca cga att cgc agg ccg tcc tga acc agc caa
gca gcg tct ccg ggt 48Ser Arg Ile Arg Arg Pro Ser Thr Ser Gln Ala
Ala Ser Pro Gly1 5 10 15ctc tgg ggc agc ggg tct caa tca cct gta gcg
ggt ctt cct cca atg 96Leu Trp Gly Ser Gly Ser Gln Ser Pro Val Ala
Gly Leu Pro Pro Met 20 25 30tcg gca acg gct acg tgt ctt ggt atc agc
tga tcc ctg gca gtg ccc 144Ser Ala Thr Ala Thr Cys Leu Gly Ile Ser
Ser Leu Ala Val Pro 35 40 45cac gaa ccc tga tct acg gcg aca cat cca
gag ctt ctg ggg tcc ccg 192His Glu Pro Ser Thr Ala Thr His Pro Glu
Leu Leu Gly Ser Pro 50 55 60atc ggt tct cag gga gca gat ccg gaa aca
cag cta ctc tga cca tca 240Ile Gly Ser Gln Gly Ala Asp Pro Glu Thr
Gln Leu Leu Pro Ser65 70 75gct ccc tgc agg ctg agg acg aag cag att
att tct gcg cat ctg ccg 288Ala Pro Cys Arg Leu Arg Thr Lys Gln Ile
Ile Ser Ala His Leu Pro 80 85 90agg act cta gtt caa atg ccg tgt ttg
gaa gcg gca cca cac tga cag 336Arg Thr Leu Val Gln Met Pro Cys Leu
Glu Ala Ala Pro His Gln 95 100 105tcc tgg ggc agc cca aga gtc ccc
ctt cag tga ctc tgt tcc cac cct 384Ser Trp Gly Ser Pro Arg Val Pro
Leu Gln Leu Cys Ser His Pro 110 115 120cta ccg agg aac tga acg gaa
aca agg cca cac tgg tgt gtc tga tca 432Leu Pro Arg Asn Thr Glu Thr
Arg Pro His Trp Cys Val Ser 125 130 135gcg act ttt acc ctg gat ccg
tca ctg tgg tct gga agg cag atg gca 480Ala Thr Phe Thr Leu Asp Pro
Ser Leu Trp Ser Gly Arg Gln Met Ala140 145 150gca caa tta cta gga
acg tgg aaa cta ccc gcg cct cca agc agt cta 528Ala Gln Leu Leu Gly
Thr Trp Lys Leu Pro Ala Pro Pro Ser Ser Leu 155 160 165ata gta aat
acg ccg cca gct cct atc tga gcc tga cct cta gtg att 576Ile Val Asn
Thr Pro Pro Ala Pro Ile Ala Pro Leu Val Ile 170 175 180gga agt cca
aag ggt cat ata gct gcg aag tga ccc atg aag gct caa 624Gly Ser Pro
Lys Gly His Ile Ala Ala Lys Pro Met Lys Ala Gln 185 190 195ccg tga
cta aga ctg tga aac cat ccg agt gct cct agg cta gct ggc 672Pro Leu
Arg Leu Asn His Pro Ser Ala Pro Arg Leu Ala Gly 200 205
2104137PRTArtificial sequenceSynthetic Construct 413Ser Arg Ile Arg
Arg Pro Ser1 541434PRTArtificial sequenceSynthetic Construct 414Thr
Ser Gln Ala Ala Ser Pro Gly Leu Trp Gly Ser Gly Ser Gln Ser1 5 10
15Pro Val Ala Gly Leu Pro Pro Met Ser Ala Thr Ala Thr Cys Leu Gly
20 25 30Ile Ser4158PRTArtificial sequenceSynthetic Construct 415Ser
Leu Ala Val Pro His Glu Pro1 541625PRTArtificial sequenceSynthetic
Construct 416Ser Thr Ala Thr His Pro Glu Leu Leu Gly Ser Pro Ile
Gly Ser Gln1 5 10 15Gly Ala Asp Pro Glu Thr Gln Leu Leu 20
2541732PRTArtificial sequenceSynthetic Construct 417Pro Ser Ala Pro
Cys Arg Leu Arg Thr Lys Gln Ile Ile Ser Ala His1 5 10 15Leu Pro Arg
Thr Leu Val Gln Met Pro Cys Leu Glu Ala Ala Pro His 20 25
3041811PRTArtificial sequenceSynthetic Construct 418Gln Ser Trp Gly
Ser Pro Arg Val Pro Leu Gln1 5 104199PRTArtificial
sequenceSynthetic Construct 419Leu Cys Ser His Pro Leu Pro Arg Asn1
54209PRTArtificial sequenceSynthetic Construct 420Thr Glu Thr Arg
Pro His Trp Cys Val1 542142PRTArtificial sequenceSynthetic
Construct 421Ser Ala Thr Phe Thr Leu Asp Pro Ser Leu Trp Ser Gly
Arg Gln Met1 5 10 15Ala Ala Gln Leu Leu Gly Thr Trp Lys Leu Pro Ala
Pro Pro Ser Ser 20 25 30Leu Ile Val Asn Thr Pro Pro Ala Pro Ile 35
4042214PRTArtificial sequenceSynthetic Construct 422Pro Leu Val Ile
Gly Ser Pro Lys Gly His Ile Ala Ala Lys1 5 104236PRTArtificial
sequenceSynthetic Construct 423Pro Met Lys Ala Gln Pro1
542410PRTArtificial sequenceSynthetic Construct 424Asn His Pro Ser
Ala Pro Arg Leu Ala Gly1 5 10425641DNAArtificial
sequenceSynthesized VH1-24+CDR3-IL8 425tcacgaattc gcaggtccag
ctggtacagt ctggggctga ggtgaagaag cctggggcct 60cagtgaaggt gtcctgcaag
gtttccggat acaccctcac tgaattatcc atgcactggg 120tgcgacaggc
tcctggaaaa gggcttgagt ggatgggagg ttttgatcct gaagatggtg
180aaacaatcta cgcacagaag ttccagggca gagtcaccat gaccgaggac
acatctacag 240acacagccta catggagctg agcagcctga gatctgagga
cacggccgtg tattactgca 300cctctgtgca ccaggaaact aagaaatacc
agagcccaag gagtgctaaa gaacttagat 360gtcagtgcat aaagacatac
tccaaacctt tccaccccaa gttcatcaag gagctgagag 420tgattgagag
tggaccacac tgcgccaaca cagagattat tgtaaagctt tctgatggga
480gagagctctg cctggacccc aaggaaaact gggtgcagag ggtcgtggag
aagttcttga 540agagggctga gaactcaggc agcggttctt atacctacaa
ttatgaatgg catgtggatg 600tctggggaca gggcctgctg gtgacagtct
ctagtgctag c 641426641DNAArtificial sequenceSynthesized
VH1-46+CDR3-IL8 426tcacgaattc gcaggtgcag ctggtgcagt ctggggctga
ggtgaagaag cctggggcct 60cagtgaaggt ttcctgcaag gcatctggat acaccttcac
cagctactat atgcactggg 120tgcgacaggc ccctggacaa gggcttgagt
ggatgggaat aatcaaccct agtggtggta 180gcacaagcta cgcacagaag
ttccagggca gagtcaccat gaccagggac acgtccacga 240gcacagtcta
catggagctg agcagcctga gatctgagga cacggccgtg tattactgca
300cctctgtgca ccaggaaact aagaaatacc agagcccaag gagtgctaaa
gaacttagat 360gtcagtgcat aaagacatac tccaaacctt tccaccccaa
gttcatcaag gagctgagag 420tgattgagag tggaccacac tgcgccaaca
cagagattat tgtaaagctt tctgatggga 480gagagctctg cctggacccc
aaggaaaact gggtgcagag ggtcgtggag aagttcttga 540agagggctga
gaactcaggc agcggttctt atacctacaa ttatgaatgg catgtggatg
600tctggggaca gggcctgctg gtgacagtct ctagtgctag c
641427641DNAArtificial sequenceSynthesized VH1-69+CDR3-IL8
427tcacgaattc gcaggtgcag ctggtgcagt ctggggctga ggtgaagaag
cctgggtcct 60cggtgaaggt gtcctgcaag gcttctggag gcaccttcag cagctatgct
atcagctggg 120tgcgacaggc ccctggacaa gggcttgagt ggatgggagg
gatcatccct atctttggta 180cagcaaacta cgcacagaag ttccagggca
gagtcacgat taccgcggac aaatccacga 240gcacagccta catggagctg
agcagcctga gatctgagga cacggccgtg tattactgca 300cctctgtgca
ccaggaaact aagaaatacc agagcccaag gagtgctaaa gaacttagat
360gtcagtgcat aaagacatac tccaaacctt tccaccccaa gttcatcaag
gagctgagag 420tgattgagag tggaccacac tgcgccaaca cagagattat
tgtaaagctt tctgatggga 480gagagctctg cctggacccc aaggaaaact
gggtgcagag ggtcgtggag aagttcttga 540agagggctga gaactcaggc
agcggttctt atacctacaa ttatgaatgg catgtggatg 600tctggggaca
gggcctgctg gtgacagtct ctagtgctag c 641428641DNAArtificial
sequenceSynthesized VH3-23+CDR3-IL8 428tcacgaattc ggaggtgcag
ctgttggagt ctgggggagg cttggtacag cctggggggt 60ccctgagact ctcctgtgca
gcctctggat tcacctttag cagctatgcc atgagctggg 120tccgccaggc
tccagggaag gggctggagt gggtgagcgc aattagtggt agtggcggta
180gcacatacta cgcagactcc gtgaagggcc ggttcaccat ctcacgtgac
aattccaaga 240acacgctgta tctgcaaatg aacagcctga gagccgagga
cacggccgtg tattactgca 300cctctgtgca ccaggaaact aagaaatacc
agagcccaag gagtgctaaa gaacttagat 360gtcagtgcat aaagacatac
tccaaacctt tccaccccaa gttcatcaag gagctgagag 420tgattgagag
tggaccacac tgcgccaaca cagagattat tgtaaagctt tctgatggga
480gagagctctg cctggacccc aaggaaaact gggtgcagag ggtcgtggag
aagttcttga 540agagggctga gaactcaggc agcggttctt atacctacaa
ttatgaatgg catgtggatg 600tctggggaca gggcctgctg gtgacagtct
ctagtgctag c 641429638DNAArtificial sequenceSynthesized
VH4-4+CDR3-IL8 429tcacgaattc gcaggtgcag ctgcaggagt cgggcccagg
actggtgaag ccttcggaga 60cgctgtccct cacctgcact gtctctggtg gctccatcag
tagttactac tggagctgga 120ttcggcagcc cgccgggaag ggactggagt
ggattgggcg tatctatacc agtgggagca 180ccaactacaa cccctccctc
aagagtcgag tcaccatgtc agtagacacg tccaagaacc 240agttctccct
gaagctgagc tctgtgaccg ccgcggacac ggccgtgtat tactgcacct
300ctgtgcacca ggaaactaag aaataccaga gcccaaggag tgctaaagaa
cttagatgtc 360agtgcataaa gacatactcc aaacctttcc accccaagtt
catcaaggag ctgagagtga 420ttgagagtgg accacactgc gccaacacag
agattattgt aaagctttct gatgggagag 480agctctgcct ggaccccaag
gaaaactggg tgcagagggt cgtggagaag ttcttgaaga 540gggctgagaa
ctcaggcagc ggttcttata cctacaatta tgaatggcat gtggatgtct
600ggggacaggg cctgctggtg acagtctcta gtgctagc 638430638DNAArtificial
sequenceSynthesized VH4-34+CDR3-IL8 430tcacgaattc gcaggtgcag
ctacagcagt ggggcgcagg actgttgaag ccttcggaga 60cgctgtccct cacctgcgct
gtctatggtg ggtccttcag tggttactac tggagctgga 120ttcgccagcc
cccagggaag gggctggagt ggattgggga aatcaatcat agtggaagca
180ccaactacaa cccgtccctc aagagtcgag tcaccatatc agtagacacg
tccaagaacc 240agttctccct gaagctgagc tctgtgaccg ccgcggacac
ggctgtgtat tactgtacct 300ctgtgcacca ggaaactaag aaataccaga
gcccaaggag tgctaaagaa cttagatgtc 360agtgcataaa gacatactcc
aaacctttcc accccaagtt catcaaggag ctgagagtga 420ttgagagtgg
accacactgc gccaacacag agattattgt aaagctttct gatgggagag
480agctctgcct ggaccccaag gaaaactggg tgcagagggt cgtggagaag
ttcttgaaga 540gggctgagaa ctcaggcagc ggttcttata cctacaatta
tgaatggcat gtggatgtct 600ggggacaggg cctgctggtg acagtctcta gtgctagc
638431431DNAArtificial sequenceSynthesized IgGVH4-4 CDR3 BsaI
(lacking the IL-8 insert) 431tcacgaattc gcaggtgcag ctgcaggagt
cgggcccagg actggtgaag ccttcggaga 60cgctgtccct cacctgcact gtctctggtg
gctccatcag tagttactac tggagctgga 120ttcggcagcc cgccgggaag
ggactggagt ggattgggcg tatctatacc agtgggagca 180ccaactacaa
cccctccctc aagagtcgag tcaccatgtc agtagacacg tccaagaacc
240agttctccct gaagctgagc tctgtgaccg ccgcggacac ggccgtgtat
tactgcacct 300ctgtgcacca ggaaactaag aaataccaga gcgagaccta
ctatggttcg ggtctctctt 360atacctacaa ttatgaatgg catgtggatg
tctggggaca gggcctgctg gtgacagtct 420ctagtgctag c
431432638DNAArtificial sequenceSynthesized VH4-34+CDR3-IL8_CDR1 Cow
432tcacgaattc gcaggtgcag ctacagcagt ggggcgcagg actgttgaag
ccttcggaga 60cgctgtccct cacctgcaca gcaagcgggt tttcactgag cgacaaggca
gtgggatgga 120ttcgccagcc cccagggaag gggctggagt ggattgggga
aatcaatcat agtggaagca 180ccaactacaa cccgtccctc aagagtcgag
tcaccatatc agtagacacg tccaagaacc 240agttctccct gaagctgagc
tctgtgaccg ccgcggacac ggctgtgtat tactgtacct 300ctgtgcacca
ggaaactaag aaataccaga gcccaaggag tgctaaagaa cttagatgtc
360agtgcataaa gacatactcc aaacctttcc accccaagtt catcaaggag
ctgagagtga 420ttgagagtgg accacactgc gccaacacag agattattgt
aaagctttct gatgggagag 480agctctgcct ggaccccaag gaaaactggg
tgcagagggt cgtggagaag ttcttgaaga 540gggctgagaa ctcaggcagc
ggttcttata cctacaatta tgaatggcat gtggatgtct 600ggggacaggg
cctgctggtg acagtctcta gtgctagc 638433638DNAArtificial
sequenceSynthesized VH4-34+CDR3-IL8_CDR2 Cow 433tcacgaattc
gcaggtgcag ctacagcagt ggggcgcagg actgttgaag ccttcggaga 60cgctgtccct
cacctgcgct gtctatggtg ggtccttcag tggttactac tggagctgga
120ttcgccagcc cccagggaag gggctggagt ggctgggcag catcgatacc
ggcgggaaca 180cagggtacaa cccgtccctc aagagtcgag tcaccatatc
agtagacacg tccaagaacc 240agttctccct gaagctgagc tctgtgaccg
ccgcggacac ggctgtgtat tactgtacct 300ctgtgcacca ggaaactaag
aaataccaga gcccaaggag tgctaaagaa cttagatgtc 360agtgcataaa
gacatactcc aaacctttcc accccaagtt catcaaggag ctgagagtga
420ttgagagtgg accacactgc gccaacacag agattattgt aaagctttct
gatgggagag 480agctctgcct ggaccccaag gaaaactggg tgcagagggt
cgtggagaag ttcttgaaga 540gggctgagaa ctcaggcagc ggttcttata
cctacaatta tgaatggcat gtggatgtct 600ggggacaggg cctgctggtg
acagtctcta gtgctagc 638434638DNAArtificial sequenceSynthesized
VH4-34+CDR3-IL8_CDR1 G31D, Y32K 434tcacgaattc gcaggtgcag ctacagcagt
ggggcgcagg actgttgaag ccttcggaga 60cgctgtccct cacctgcgct gtctatggtg
ggtccttcag tgacaagtac tggagctgga 120ttcgccagcc cccagggaag
gggctggagt ggattgggga aatcaatcat agtggaagca 180ccaactacaa
cccgtccctc aagagtcgag tcaccatatc agtagacacg tccaagaacc
240agttctccct gaagctgagc tctgtgaccg ccgcggacac ggctgtgtat
tactgtacct 300ctgtgcacca ggaaactaag aaataccaga gcccaaggag
tgctaaagaa cttagatgtc 360agtgcataaa gacatactcc aaacctttcc
accccaagtt catcaaggag ctgagagtga 420ttgagagtgg accacactgc
gccaacacag agattattgt aaagctttct gatgggagag 480agctctgcct
ggaccccaag gaaaactggg tgcagagggt cgtggagaag ttcttgaaga
540gggctgagaa ctcaggcagc ggttcttata cctacaatta tgaatggcat
gtggatgtct 600ggggacaggg cctgctggtg acagtctcta gtgctagc
638435638DNAArtificial sequenceSynthesized VH4-34+CDR3-IL8_CDR2
E50S 435tcacgaattc gcaggtgcag ctacagcagt ggggcgcagg actgttgaag
ccttcggaga 60cgctgtccct cacctgcgct gtctatggtg ggtccttcag tggttactac
tggagctgga 120ttcgccagcc cccagggaag gggctggagt ggattgggag
catcaatcat agtggaagca 180ccaactacaa cccgtccctc aagagtcgag
tcaccatatc agtagacacg tccaagaacc 240agttctccct gaagctgagc
tctgtgaccg ccgcggacac ggctgtgtat tactgtacct 300ctgtgcacca
ggaaactaag aaataccaga gcccaaggag tgctaaagaa cttagatgtc
360agtgcataaa gacatactcc aaacctttcc accccaagtt catcaaggag
ctgagagtga 420ttgagagtgg accacactgc gccaacacag agattattgt
aaagctttct gatgggagag 480agctctgcct ggaccccaag gaaaactggg
tgcagagggt cgtggagaag ttcttgaaga 540gggctgagaa ctcaggcagc
ggttcttata cctacaatta tgaatggcat gtggatgtct 600ggggacaggg
cctgctggtg acagtctcta gtgctagc 638436638DNAArtificial
sequenceSynthesized VH4-34+CDR3-IL8_CDR1 Cow_CDR2 Cow 436tcacgaattc
gcaggtgcag ctacagcagt ggggcgcagg actgttgaag ccttcggaga 60cgctgtccct
cacctgcaca gcaagcgggt tttcactgag cgacaaggca gtgggatgga
120ttcgccagcc cccagggaag gggctggagt ggctgggcag catcgatacc
ggcgggaaca 180cagggtacaa cccgtccctc aagagtcgag tcaccatatc
agtagacacg tccaagaacc 240agttctccct gaagctgagc tctgtgaccg
ccgcggacac ggctgtgtat tactgtacct 300ctgtgcacca ggaaactaag
aaataccaga gcccaaggag tgctaaagaa cttagatgtc 360agtgcataaa
gacatactcc aaacctttcc accccaagtt catcaaggag ctgagagtga
420ttgagagtgg accacactgc gccaacacag agattattgt aaagctttct
gatgggagag 480agctctgcct ggaccccaag gaaaactggg tgcagagggt
cgtggagaag ttcttgaaga 540gggctgagaa ctcaggcagc ggttcttata
cctacaatta tgaatggcat gtggatgtct 600ggggacaggg cctgctggtg
acagtctcta gtgctagc 638437638DNAArtificial sequenceSynthesized
VH4-34+CDR3-IL8_CDR1 Cow_CDR2 E50S 437tcacgaattc gcaggtgcag
ctacagcagt ggggcgcagg actgttgaag ccttcggaga 60cgctgtccct cacctgcaca
gcaagcgggt tttcactgag cgacaaggca gtgggatgga 120ttcgccagcc
cccagggaag gggctggagt ggattgggag catcaatcat agtggaagca
180ccaactacaa cccgtccctc aagagtcgag tcaccatatc agtagacacg
tccaagaacc 240agttctccct gaagctgagc tctgtgaccg ccgcggacac
ggctgtgtat tactgtacct 300ctgtgcacca ggaaactaag aaataccaga
gcccaaggag tgctaaagaa cttagatgtc 360agtgcataaa gacatactcc
aaacctttcc accccaagtt catcaaggag ctgagagtga 420ttgagagtgg
accacactgc gccaacacag agattattgt aaagctttct gatgggagag
480agctctgcct ggaccccaag gaaaactggg tgcagagggt cgtggagaag
ttcttgaaga
540gggctgagaa ctcaggcagc ggttcttata cctacaatta tgaatggcat
gtggatgtct 600ggggacaggg cctgctggtg acagtctcta gtgctagc
638438638DNAArtificial sequenceSynthesized VH4-34+CDR3-IL8_CDR1
G31D, Y32K_CDR2 Cow 438tcacgaattc gcaggtgcag ctacagcagt ggggcgcagg
actgttgaag ccttcggaga 60cgctgtccct cacctgcgct gtctatggtg ggtccttcag
tgacaagtac tggagctgga 120ttcgccagcc cccagggaag gggctggagt
ggctgggcag catcgatacc ggcgggaaca 180cagggtacaa cccgtccctc
aagagtcgag tcaccatatc agtagacacg tccaagaacc 240agttctccct
gaagctgagc tctgtgaccg ccgcggacac ggctgtgtat tactgtacct
300ctgtgcacca ggaaactaag aaataccaga gcccaaggag tgctaaagaa
cttagatgtc 360agtgcataaa gacatactcc aaacctttcc accccaagtt
catcaaggag ctgagagtga 420ttgagagtgg accacactgc gccaacacag
agattattgt aaagctttct gatgggagag 480agctctgcct ggaccccaag
gaaaactggg tgcagagggt cgtggagaag ttcttgaaga 540gggctgagaa
ctcaggcagc ggttcttata cctacaatta tgaatggcat gtggatgtct
600ggggacaggg cctgctggtg acagtctcta gtgctagc 638439638DNAArtificial
sequenceSynthesized VH4-34+CDR3-IL8_CDR1 G31D, Y32K_CDR2 E50S
439tcacgaattc gcaggtgcag ctacagcagt ggggcgcagg actgttgaag
ccttcggaga 60cgctgtccct cacctgcgct gtctatggtg ggtccttcag tgacaagtac
tggagctgga 120ttcgccagcc cccagggaag gggctggagt ggattgggag
catcaatcat agtggaagca 180ccaactacaa cccgtccctc aagagtcgag
tcaccatatc agtagacacg tccaagaacc 240agttctccct gaagctgagc
tctgtgaccg ccgcggacac ggctgtgtat tactgtacct 300ctgtgcacca
ggaaactaag aaataccaga gcccaaggag tgctaaagaa cttagatgtc
360agtgcataaa gacatactcc aaacctttcc accccaagtt catcaaggag
ctgagagtga 420ttgagagtgg accacactgc gccaacacag agattattgt
aaagctttct gatgggagag 480agctctgcct ggaccccaag gaaaactggg
tgcagagggt cgtggagaag ttcttgaaga 540gggctgagaa ctcaggcagc
ggttcttata cctacaatta tgaatggcat gtggatgtct 600ggggacaggg
cctgctggtg acagtctcta gtgctagc 638440671DNAArtificial
sequenceSynthesized V1-51, I29V, N32G 440tcacgaattc gcagtccgtg
ctgacccaac ccccgtcagt gtctgctgcc cccgggcaga 60aggtgactat cagctgctct
ggctcatcct ccaatgtcgg caacggctac gtcagctggt 120accagcagct
gcctggaaca gctcctaaac tgctcattta tgacaataac aagcgcccat
180ccggaatccc tgaccgattc agcggaagca aatcagggac ctctgcaact
ctgggaatca 240ctgggcttca gacaggagat gaggcagatt actattgcgc
ctctgcagag gacagctcca 300gcaatgccgt gttcgggtct ggtaccactc
ttacagtcct aggtcagccc aaggctgccc 360cctcggtcac tctgttcccg
ccctcctctg aggagcttca agccaacaag gccacactgg 420tgtgtctcat
aagtgacttc tacccgggag ccgtgacagt ggcctggaag gcagatagca
480gccccgtcaa ggcgggagtg gaaacaacca caccctccaa acaaagcaac
aacaagtacg 540cggccagcag ctatctgagc ctgacgcctg agcagtggaa
gtcccacaga agctacagct 600gccaggtcac gcatgaaggg agcaccgtgg
agaagacagt ggcccctaca gaatgttcat 660aatgagctag c
671441671DNAArtificial sequenceSynthesized V1-51, DNN (aa51-53)
changed to GDT 441tcacgaattc gcagtccgtg ctgacccaac ccccgtcagt
gtctgctgcc cccgggcaga 60aggtgactat cagctgctct ggctcatcaa gcaacatcgg
gaataattac gtcagctggt 120accagcagct gcctggaaca gctcctaaac
tgctcattta tggcgacaca aagcgcccat 180ccggaatccc tgaccgattc
agcggaagca aatcagggac ctctgcaact ctgggaatca 240ctgggcttca
gacaggagat gaggcagatt actattgcgc ctctgcagag gacagctcca
300gcaatgccgt gttcgggtct ggtaccactc ttacagtcct aggtcagccc
aaggctgccc 360cctcggtcac tctgttcccg ccctcctctg aggagcttca
agccaacaag gccacactgg 420tgtgtctcat aagtgacttc tacccgggag
ccgtgacagt ggcctggaag gcagatagca 480gccccgtcaa ggcgggagtg
gaaacaacca caccctccaa acaaagcaac aacaagtacg 540cggccagcag
ctatctgagc ctgacgcctg agcagtggaa gtcccacaga agctacagct
600gccaggtcac gcatgaaggg agcaccgtgg agaagacagt ggcccctaca
gaatgttcat 660aatgagctag c 671442671DNAArtificial
sequenceSynthesized V1-51, DNNKRP (aa51-56) changed to GDTSRA
442tcacgaattc gcagtccgtg ctgacccaac ccccgtcagt gtctgctgcc
cccgggcaga 60aggtgactat cagctgctct ggctcatcaa gcaacatcgg gaataattac
gtcagctggt 120accagcagct gcctggaaca gctcctaaac tgctcattta
tggcgacaca tccagagctt 180ccggaatccc tgaccgattc agcggaagca
aatcagggac ctctgcaact ctgggaatca 240ctgggcttca gacaggagat
gaggcagatt actattgcgc ctctgcagag gacagctcca 300gcaatgccgt
gttcgggtct ggtaccactc ttacagtcct aggtcagccc aaggctgccc
360cctcggtcac tctgttcccg ccctcctctg aggagcttca agccaacaag
gccacactgg 420tgtgtctcat aagtgacttc tacccgggag ccgtgacagt
ggcctggaag gcagatagca 480gccccgtcaa ggcgggagtg gaaacaacca
caccctccaa acaaagcaac aacaagtacg 540cggccagcag ctatctgagc
ctgacgcctg agcagtggaa gtcccacaga agctacagct 600gccaggtcac
gcatgaaggg agcaccgtgg agaagacagt ggcccctaca gaatgttcat
660aatgagctag c 671443671DNAArtificial sequenceSynthesized V1-51,
S2A,T5N,P8S,A12G,A13S, and P14L 443tcacgaattc gcaggccgtc ctgaaccagc
caagcagcgt ctccgggtct ctggggcaga 60aggtgactat cagctgctct ggctcatcaa
gcaacatcgg gaataattac gtcagctggt 120accagcagct gcctggaaca
gctcctaaac tgctcattta tgacaataac aagcgcccat 180ccggaatccc
tgaccgattc agcggaagca aatcagggac ctctgcaact ctgggaatca
240ctgggcttca gacaggagat gaggcagatt actattgcgc ctctgcagag
gacagctcca 300gcaatgccgt gttcgggtct ggtaccactc ttacagtcct
aggtcagccc aaggctgccc 360cctcggtcac tctgttcccg ccctcctctg
aggagcttca agccaacaag gccacactgg 420tgtgtctcat aagtgacttc
tacccgggag ccgtgacagt ggcctggaag gcagatagca 480gccccgtcaa
ggcgggagtg gaaacaacca caccctccaa acaaagcaac aacaagtacg
540cggccagcag ctatctgagc ctgacgcctg agcagtggaa gtcccacaga
agctacagct 600gccaggtcac gcatgaaggg agcaccgtgg agaagacagt
ggcccctaca gaatgttcat 660aatgagctag c 671444671DNAArtificial
sequenceSynthesized V1-51, S2A,T5N,P8S,A12G,A13S, P14L, and DNN
(aa51-53) changed to GDT 444tcacgaattc gcaggccgtc ctgaaccagc
caagcagcgt ctccgggtct ctggggcaga 60aggtgactat cagctgctct ggctcatcaa
gcaacatcgg gaataattac gtcagctggt 120accagcagct gcctggaaca
gctcctaaac tgctcattta tggcgacaca aagcgcccat 180ccggaatccc
tgaccgattc agcggaagca aatcagggac ctctgcaact ctgggaatca
240ctgggcttca gacaggagat gaggcagatt actattgcgc ctctgcagag
gacagctcca 300gcaatgccgt gttcgggtct ggtaccactc ttacagtcct
aggtcagccc aaggctgccc 360cctcggtcac tctgttcccg ccctcctctg
aggagcttca agccaacaag gccacactgg 420tgtgtctcat aagtgacttc
tacccgggag ccgtgacagt ggcctggaag gcagatagca 480gccccgtcaa
ggcgggagtg gaaacaacca caccctccaa acaaagcaac aacaagtacg
540cggccagcag ctatctgagc ctgacgcctg agcagtggaa gtcccacaga
agctacagct 600gccaggtcac gcatgaaggg agcaccgtgg agaagacagt
ggcccctaca gaatgttcat 660aatgagctag c 671445657DNAArtificial
sequenceSynthesized A20J1 445gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcgagtca gggcattagc
aattatttag cctggtatca gcagaaacca 120gggaaagttc ctaagctcct
gatctatgct gcatccactt tgcaatcagg ggtcccatct 180cggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct
240gaagatgttg caacttatta ctgtcaaaag tataacagtg cccctccgtg
gacgttcggc 300caagggacca aggtggaaat caaacgtacg gtggctgcac
catctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaact
gcctctgttg tgtgcctgct gaataacttc 420tatcccagag aggccaaagt
acagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagtg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg
540acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt
cacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag
agtgttaatg agctagc 657446657DNAArtificial sequenceSynthesized L6J1
446gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca
acagaaacct 120ggccaggctc ccaggctcct catctatgat gcatccaaca
gggccactgg catcccagcc 180aggttcagtg gcagtgggtc tgggacagac
ttcactctca ccatcagcag cctagagcct 240gaagattttg cagtttatta
ctgtcagcag cgtagcaact ggcctccgtg gacgttcggc 300caagggacca
aggtggaaat caaacgtacg gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgttaatg agctagc
657447660DNAArtificial sequenceSynthesized L25J1 447gaaatagtga
tgacgcagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agcagctact tatcctggta ccagcagaaa
120cctgggcagg ctcccaggct cctcatctat ggtgcatcca ccagggccac
tggcatccca 180gccaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagcctgcag 240cctgaagatt ttgcagttta ttactgtcag
caggattata acttacctcc gtggacgttc 300ggccaaggga ccaaggtgga
aatcaaacgt acggtggctg caccatctgt cttcatcttc 360ccgccatctg
atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac
420ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca
atcgggtaac 480tcccaggaga gtgtcacaga gcaggacagc aaggacagca
cctacagcct cagcagcacc 540ctgacgctga gcaaagcaga ctacgagaaa
cacaaagtct acgcctgcga agtcacccat 600cagggcctga gctcgcccgt
cacaaagagc ttcaacaggg gagagtgtta atgagctagc 660448663DNAArtificial
sequenceSynthesized V1-2J7 448cagtctgccc tgactcagcc tccctccgcg
tccgggtctc ctggacagtc agtcaccatc 60tcctgcactg gaaccagcag tgacgttggt
ggttataact atgtctcctg gtaccaacag 120cacccaggca aagcccccaa
actcatgatt tatgaggtca gtaagcggcc ctcaggggtc 180cctgatcgct
tctctggctc caagtctggc aacacggcct ccctgaccgt ctctgggctc
240caggctgagg atgaggctga ttattactgc agctcatatg caggcagcaa
caatttcgct 300gtgttcggag gaggcaccca gctgaccgtc ctaggtcagc
ccaaggctgc cccctcggtc 360actctgttcc cgccctcctc tgaggagctt
caagccaaca aggccacact ggtgtgtctc 420ataagtgact tctacccggg
agccgtgaca gtggcctgga aggcagatag cagccccgtc 480aaggcgggag
tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc
540agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag
ctgccaggtc 600acgcatgaag ggagcaccgt ggagaagaca gtggccccta
cagaatgttc ataatgagct 660agc 663449663DNAArtificial
sequenceSynthesized V1-7J1 449cagtctgccc tgactcagcc tgcctccgtg
tctggctccc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag tgatgttggg
agttataacc ttgtctcctg gtaccaacag 120cacccaggca aagcccccaa
actcatgatt tatgagggca gtaagcggcc ctcaggggtt 180tctaatcgct
tctctggctc caagtctggc aacacggcct ccctgacaat ctctgggctc
240caggctgagg acgaggctga ttattactgc tgctcatatg caggtagtag
cactttctat 300gtcttcggaa ctgggaccaa ggtcaccgtc ctaggtcagc
ccaaggctgc cccctcggtc 360actctgttcc cgccctcctc tgaggagctt
caagccaaca aggccacact ggtgtgtctc 420ataagtgact tctacccggg
agccgtgaca gtggcctgga aggcagatag cagccccgtc 480aaggcgggag
tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc
540agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag
ctgccaggtc 600acgcatgaag ggagcaccgt ggagaagaca gtggccccta
cagaatgttc ataatgagct 660agc 663450663DNAArtificial
sequenceSynthesized V1-11J2 450cagtctgtgc tgactcagcc accctcggtg
tctgaagccc ccaggcagag ggtcaccatc 60tcctgttctg gaagcagctc caacatcgga
aataatgctg taaactggta ccagcagctc 120ccaggaaagg ctcccaaact
cctcatctat tatgatgatc tgctgccctc aggggtctct 180gaccgattct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag
240tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa
tggtcctgtg 300gtattcggcg gagggaccaa gctgaccgtc ctaggtcagc
ccaaggctgc cccctcggtc 360actctgttcc cgccctcctc tgaggagctt
caagccaaca aggccacact ggtgtgtctc 420ataagtgact tctacccggg
agccgtgaca gtggcctgga aggcagatag cagccccgtc 480aaggcgggag
tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc
540agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag
ctgccaggtc 600acgcatgaag ggagcaccgt ggagaagaca gtggccccta
cagaatgttc ataatgagct 660agc 663451336DNAArtificial
sequenceSynthesized V1-16J6 451ccagtctgtg ctgactcagc caccctcagc
gtctgggacc cccgggcaga gggtcaccat 60ctcttgttct ggaagcagct ccaacatcgg
aagtaatact gtaaactggt accagcagct 120cccaggaacg gcccccaaac
tcctcatcta tagtaataat cagcggccct caggggtccc 180tgaccgattc
tctggctcca agtctggcac ctcagcctcc ctggccatca gtgggctcca
240gtctgaggat gaggctgatt attactgtgc agcatgggat gacagcctga
atggtcctaa 300tgtgttcggc agtggcacca aggtgaccgt cctagg
336452657DNAArtificial sequenceSynthesized V2-17J2 452tcctatgagc
tgacacagcc accctcggtg tcagtgtccc caggacagac ggccaggatc 60acctgctctg
gagatgcatt gccaaagcaa tatgcttatt ggtaccagca gaagccaggc
120caggcccctg tgctggtgat atataaagac agtgagaggc cctcaggcat
ccctgagcga 180ttctctggct ccagctcagg gacaacagtc acgttgacca
tcagtggagt ccaggcagaa 240gatgaggctg actattactg tcaatcagca
gacagcagtg gtacttatcc tgtggtattc 300ggcggaggga ccaagctgac
cgtcctaggt cagcccaagg ctgccccctc ggtcactctg 360ttcccgccct
cctctgagga gcttcaagcc aacaaggcca cactggtgtg tctcataagt
420gacttctacc cgggagccgt gacagtggcc tggaaggcag atagcagccc
cgtcaaggcg 480ggagtggaaa caaccacacc ctccaaacaa agcaacaaca
agtacgcggc cagcagctat 540ctgagcctga cgcctgagca gtggaagtcc
cacagaagct acagctgcca ggtcacgcat 600gaagggagca ccgtggagaa
gacagtggcc cctacagaat gttcataatg agctagc 657453663DNAArtificial
sequenceSynthesized V3-4J1 453cagactgtgg tgacccagga gccatcgttc
tcagtgtccc ctggagggac agtcacactc 60acttgtggct tgagctctgg ctcagtctct
actagttact accccagctg gtaccagcag 120accccaggcc aggctccacg
cacgctcatc tacagcacaa acactcgctc ttctggggtc 180cctgatcgct
tctctggctc catccttggg aacaaagctg ccctcaccat cacgggggcc
240caggcagatg atgaatctga ttattactgt gtgctgtata tgggtagtgg
catttcttat 300gtcttcggaa ctgggaccaa ggtcaccgtc ctaggtcagc
ccaaggctgc cccctcggtc 360actctgttcc cgccctcctc tgaggagctt
caagccaaca aggccacact ggtgtgtctc 420ataagtgact tctacccggg
agccgtgaca gtggcctgga aggcagatag cagccccgtc 480aaggcgggag
tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc
540agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag
ctgccaggtc 600acgcatgaag ggagcaccgt ggagaagaca gtggccccta
cagaatgttc ataatgagct 660agc 663454666DNAArtificial
sequenceSynthesized V5-4J2 454cagcctgtgc tgactcaatc atcctctgcc
tctgcttccc tgggctcctc ggtcaagctc 60acctgcactc tgagcagtgg gcacagtagc
tacatcatcg catggcatca gcagcagcca 120gggaaggccc ctcggtactt
gatgaagctt gaaggtagtg gaagctacaa caaggggagc 180ggagttcctg
atcgcttctc aggctccagc tctggggctg accgctacct caccatctcc
240aacctccagt ttgaggatga ggctgattat tactgtgaga cctgggacag
taacactcat 300gtggtattcg gcggagggac caagctgacc gtcctaggtc
agcccaaggc tgccccctcg 360gtcactctgt tcccgccctc ctctgaggag
cttcaagcca acaaggccac actggtgtgt 420ctcataagtg acttctaccc
gggagccgtg acagtggcct ggaaggcaga tagcagcccc 480gtcaaggcgg
gagtggagac caccacaccc tccaaacaaa gcaacaacaa gtacgcggcc
540agcagctatc tgagcctgac gcctgagcag tggaagtccc acagaagcta
cagctgccag 600gtcacgcatg aagggagcac cgtggagaag acagtggccc
ctacagaatg ttcataatga 660gctagc 666455660DNAArtificial
sequenceSynthesized V1-47 455caatcagttt tgacccagcc accctccgca
tccggcaccc ccgggcaacg cgttacaata 60agctgtagcg gcagctcatc taatattggc
agcaactacg tttattggta ccagcagctt 120ccagggaccg cccccaaatt
gcttatctac cggaataatc agaggccttc cggggtgcca 180gataggttct
ctgggagtaa atctggcact agcgcaagtc tggctatcag cgggctccgg
240tctgaggatg aagccgacta ttattgcgcg agcgctgagg actcatcttc
taatgctgtg 300tttggctccg gtaccacact caccgtccta ggtcagccca
aggctgcccc ctcggtcact 360ctgttcccgc cctcctctga ggagcttcaa
gccaacaagg ccacactggt gtgtctcata 420agtgacttct acccgggagc
cgtgacagtg gcctggaagg cagatagcag ccccgtcaag 480gcgggagtgg
aaacaaccac accctccaaa caaagcaaca acaagtacgc ggccagcagc
540tatctgagcc tgacgcctga gcagtggaag tcccacagaa gctacagctg
ccaggtcacg 600catgaaggga gcaccgtgga gaagacagtg gcccctacag
aatgttcata atgagctagc 660456660DNAArtificial sequenceSynthesized
V1-51 456cagtccgtgc tgacccaacc cccgtcagtg tctgctgccc ccgggcagaa
ggtgactatc 60agctgctctg gctcatcaag caacatcggg aataattacg tcagctggta
ccagcagctg 120cctggaacag ctcctaaact gctcatttat gacaataaca
agcgcccatc cggaatccct 180gaccgattca gcggaagcaa atcagggacc
tctgcaactc tgggaatcac tgggcttcag 240acaggagatg aggcagatta
ctattgcgcc tctgcagagg acagctccag caatgccgtg 300ttcgggtctg
gtaccactct tacagtccta ggtcagccca aggctgcccc ctcggtcact
360ctgttcccgc cctcctctga ggagcttcaa gccaacaagg ccacactggt
gtgtctcata 420agtgacttct acccgggagc cgtgacagtg gcctggaagg
cagatagcag ccccgtcaag 480gcgggagtgg aaacaaccac accctccaaa
caaagcaaca acaagtacgc ggccagcagc 540tatctgagcc tgacgcctga
gcagtggaag tcccacagaa gctacagctg ccaggtcacg 600catgaaggga
gcaccgtgga gaagacagtg gcccctacag aatgttcata atgagctagc
6604574188DNAArtificial sequenceSynthesized vector pFUSE-hIgG2-Fc2
457ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc
acagtccccg 60agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg
cgcggggtaa 120actgggaaag tgatgtcgtg tactggctcc gcctttttcc
cgagggtggg ggagaaccgt 180atataagtgc agtagtcgcc gtgaacgttc
tttttcgcaa cgggtttgcc gccagaacac 240agctgaagct tcgaggggct
cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300gccatccacg
ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg
360cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt
ccggcgctcc 420cttggagcct acctagactc agccggctct ccacgctttg
cctgaccctg cttgctcaac 480tctacgtctt tgtttcgttt tctgttctgc
gccgttacag atccaagctg tgaccggcgc 540ctacctgaga tcaccggcga
aggagggcca ccatgtacag gatgcaactc ctgtcttgca 600ttgcactaag
tcttgcactt gtcacgaatt cgatatcggc catggttaga tctgtggagt
660gcccaccttg cccagcacca cctgtggcag gaccttcagt cttcctcttc
cccccaaaac 720ccaaggacac cctgatgatc tccagaaccc ctgaggtcac
gtgcgtggtg gtggacgtga 780gccacgaaga ccccgaggtc cagttcaact
ggtacgtgga cggcatggag gtgcataatg 840ccaagacaaa gccacgggag
gagcagttca acagcacgtt
ccgtgtggtc agcgtcctca 900ccgtcgtgca ccaggactgg ctgaacggca
aggagtacaa gtgcaaggtc tccaacaaag 960gcctcccagc ccccatcgag
aaaaccatct ccaaaaccaa agggcagccc cgagaaccac 1020aggtgtacac
cctgccccca tcccgggagg agatgaccaa gaaccaggtc agcctgacct
1080gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc
aatgggcagc 1140cggagaacaa ctacaagacc acacctccca tgctggactc
cgacggctcc ttcttcctct 1200acagcaagct caccgtggac aagagcaggt
ggcagcaggg gaacgtcttc tcatgctccg 1260tgatgcatga ggctctgcac
aaccactaca cacagaagag cctctccctg tctccgggta 1320aatgagtgcc
acggctagct ggccagacat gataagatac attgatgagt ttggacaaac
1380cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
ctattgcttt 1440atttgtaacc attataagct gcaataaaca agttaacaac
aacaattgca ttcattttat 1500gtttcaggtt cagggggagg tgtgggaggt
tttttaaagc aagtaaaacc tctacaaatg 1560tggtatggaa ttaattctaa
aatacagcat agcaaaactt taacctccaa atcaagcctc 1620tacttgaatc
cttttctgag ggatgaataa ggcataggca tcaggggctg ttgccaatgt
1680gcattagctg tttgcagcct caccttcttt catggagttt aagatatagt
gtattttccc 1740aaggtttgaa ctagctcttc atttctttat gttttaaatg
cactgacctc ccacattccc 1800tttttagtaa aatattcaga aataatttaa
atacatcatt gcaatgaaaa taaatgtttt 1860ttattaggca gaatccagat
gctcaaggcc cttcataata tcccccagtt tagtagttgg 1920acttagggaa
caaaggaacc tttaatagaa attggacagc aagaaagcga gcttctagct
1980tatcctcagt cctgctcctc tgccacaaag tgcacgcagt tgccggccgg
gtcgcgcagg 2040gcgaactccc gcccccacgg ctgctcgccg atctcggtca
tggccggccc ggaggcgtcc 2100cggaagttcg tggacacgac ctccgaccac
tcggcgtaca gctcgtccag gccgcgcacc 2160cacacccagg ccagggtgtt
gtccggcacc acctggtcct ggaccgcgct gatgaacagg 2220gtcacgtcgt
cccggaccac accggcgaag tcgtcctcca cgaagtcccg ggagaacccg
2280agccggtcgg tccagaactc gaccgctccg gcgacgtcgc gcgcggtgag
caccggaacg 2340gcactggtca acttggccat gatggctcct cctgtcagga
gaggaaagag aagaaggtta 2400gtacaattgc tatagtgagt tgtattatac
tatgcagata tactatgcca atgattaatt 2460gtcaaactag ggctgcaggg
ttcatagtgc cacttttcct gcactgcccc atctcctgcc 2520caccctttcc
caggcataga cagtcagtga cttaccaaac tcacaggagg gagaaggcag
2580aagcttgaga cagacccgcg ggaccgccga actgcgaggg gacgtggcta
gggcggcttc 2640ttttatggtg cgccggccct cggaggcagg gcgctcgggg
aggcctagcg gccaatctgc 2700ggtggcagga ggcggggccg aaggccgtgc
ctgaccaatc cggagcacat aggagtctca 2760gccccccgcc ccaaagcaag
gggaagtcac gcgcctgtag cgccagcgtg ttgtgaaatg 2820ggggcttggg
ggggttgggg ccctgactag tcaaaacaaa ctcccattga cgtcaatggg
2880gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga
tgtactgcca 2940aaaccgcatc atcatggtaa tagcgatgac taatacgtag
atgtactgcc aagtaggaaa 3000gtcccataag gtcatgtact gggcataatg
ccaggcgggc catttaccgt cattgacgtc 3060aatagggggc gtacttggca
tatgatacac ttgatgtact gccaagtggg cagtttaccg 3120taaatactcc
acccattgac gtcaatggaa agtccctatt ggcgttacta tgggaacata
3180cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg
ggccatttac 3240cgtaagttat gtaacgcctg caggttaatt aagaacatgt
gagcaaaagg ccagcaaaag 3300gccaggaacc gtaaaaaggc cgcgttgctg
gcgtttttcc ataggctccg cccccctgac 3360gagcatcaca aaaatcgacg
ctcaagtcag aggtggcgaa acccgacagg actataaaga 3420taccaggcgt
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
3480accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
tagctcacgc 3540tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
tgggctgtgt gcacgaaccc 3600cccgttcagc ccgaccgctg cgccttatcc
ggtaactatc gtcttgagtc caacccggta 3660agacacgact tatcgccact
ggcagcagcc actggtaaca ggattagcag agcgaggtat 3720gtaggcggtg
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
3780gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
tggtagctct 3840tgatccggca aacaaaccac cgctggtagc ggtggttttt
ttgtttgcaa gcagcagatt 3900acgcgcagaa aaaaaggatc tcaagaagat
cctttgatct tttctacggg gtctgacgct 3960cagtggaacg aaaactcacg
ttaagggatt ttggtcatgg ctagttaatt aacatttaaa 4020tcagcggccg
caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg
4080aatcgtaact aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa
ctagcaaaat 4140aggctgtccc cagtgcaagt gcaggtgcca gaacatttct ctatcgaa
41884584495DNAArtificial sequenceSynthesized vector HC pFuse
458ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc
acagtccccg 60agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg
cgcggggtaa 120actgggaaag tgatgtcgtg tactggctcc gcctttttcc
cgagggtggg ggagaaccgt 180atataagtgc agtagtcgcc gtgaacgttc
tttttcgcaa cgggtttgcc gccagaacac 240agctgaagct tcgaggggct
cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300gccatccacg
ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg
360cgtccgccgt ctaggtaagt ttaaagctca ggtccagacc gggcctttgt
ccggcgctcc 420cttggagcct acctagactc agccggctct ccacgctttg
cctgaccctg cttgctcaac 480tctacgtctt tgtttcgttt tctgttctgc
gccgttacag atccaagctg tgaccggcgc 540ctacctgaga tcaccggcga
aggagggcca ccatgcgcag gatgcaactc ctgttgctga 600ttgcactaag
tcttgcactt gtcacgaatt cgccatggct cctcagctag caccaagggc
660ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac
agcggccctg 720ggctgcctgg tcaaggacta cttccccgag ccggtgacgg
tgtcgtggaa ctcaggcgcc 780ctgaccagcg gcgtgcacac cttcccggct
gtcctacagt cctcaggact ctactccctc 840agcagcgtgg tgaccgtgcc
ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 900aatcacaagc
ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa
960actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc
agtcttcctc 1020ttccccccaa aacccaagga caccctcatg atctcccgga
cccctgaggt cacatgcgtg 1080gtggtggacg tgagccacga agaccctgag
gtcaagttca actggtacgt ggacggcgtg 1140gaggtgcata atgccaagac
aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 1200gtcagcgtcc
tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag
1260gtgtccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc
caaagggcag 1320ccccgagaac cacaggtgta caccctgccc ccatcccggg
atgagctgac caagaaccag 1380gtcagcctga cctgcctggt caaaggcttc
tatcccagcg acatcgccgt ggagtgggag 1440agcaatgggc agccggagaa
caactacaag accacgcctc ccgtgctgga ctccgacggc 1500tccttcttcc
tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc
1560ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa
gagcctctcc 1620ctgtctccgg gtaaatgata atctagtggc cagacatgat
aagatacatt gatgagtttg 1680gacaaaccac aactagaatg cagtgaaaaa
aatgctttat ttgtgaaatt tgtgatgcta 1740ttgctttatt tgtaaccatt
ataagctgca ataaacaagt taacaacaac aattgcattc 1800attttatgtt
tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct
1860acaaatgtgg tatggaatta attctaaaat acagcatagc aaaactttaa
cctccaaatc 1920aagcctctac ttgaatcctt ttctgaggga tgaataaggc
ataggcatca ggggctgttg 1980ccaatgtgca ttagctgttt gcagcctcac
cttctttcat ggagtttaag atatagtgta 2040ttttcccaag gtttgaacta
gctcttcatt tctttatgtt ttaaatgcac tgacctccca 2100cattcccttt
ttagtaaaat attcagaaat aatttaaata catcattgca atgaaaataa
2160atgtttttta ttaggcagaa tccagatgct caaggccctt cataatatcc
cccagtttag 2220tagttggact tagggaacaa aggaaccttt aatagaaatt
ggacagcaag aaagcgagct 2280tctagcttat cctcagtcct gctcctctgc
cacaaagtgc acgcagttgc cggccgggtc 2340gcgcagggcg aactcccgcc
cccacggctg ctcgccgatc tcggtcatgg ccggcccgga 2400ggcgtcccgg
aagttcgtgg acacgacctc cgaccactcg gcgtacagct cgtccaggcc
2460gcgcacccac acccaggcca gggtgttgtc cggcaccacc tggtcctgga
ccgcgctgat 2520gaacagggtc acgtcgtccc ggaccacacc ggcgaagtcg
tcctccacga agtcccggga 2580gaacccgagc cggtcggtcc agaactcgac
cgctccggcg acgtcgcgcg cggtgagcac 2640cggaacggca ctggtcaact
tggccatgat ggctcctcct gtcaggagag gaaagagaag 2700aaggttagta
caattgctat agtgagttgt attatactat gcagatatac tatgccaatg
2760attaattgtc aaactagggc tgcagggttc atagtgccac ttttcctgca
ctgccccatc 2820tcctgcccac cctttcccag gcatagacag tcagtgactt
accaaactca caggagggag 2880aaggcagaag cttgagacag acccgcggga
ccgccgaact gcgaggggac gtggctaggg 2940cggcttcttt tatggtgcgc
cggccctcgg aggcagggcg ctcggggagg cctagcggcc 3000aatctgcggt
ggcaggaggc ggggccgaag gccgtgcctg accaatccgg agcacatagg
3060agtctcagcc ccccgcccca aagcaagggg aagtcacgcg cctgtagcgc
cagcgtgttg 3120tgaaatgggg gcttgggggg gttggggccc tgactagtca
aaacaaactc ccattgacgt 3180caatggggtg gagacttgga aatccccgtg
agtcaaaccg ctatccacgc ccattgatgt 3240actgccaaaa ccgcatcatc
atggtaatag cgatgactaa tacgtagatg tactgccaag 3300taggaaagtc
ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
3360tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc
aagtgggcag 3420tttaccgtaa atactccacc cattgacgtc aatggaaagt
ccctattggc gttactatgg 3480gaacatacgt cattattgac gtcaatgggc
gggggtcgtt gggcggtcag ccaggcgggc 3540catttaccgt aagttatgta
acgcctgcag gttaattaag aacatgtgag caaaaggcca 3600gcaaaaggcc
aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
3660ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
cgacaggact 3720ataaagatac caggcgtttc cccctggaag ctccctcgtg
cgctctcctg ttccgaccct 3780gccgcttacc ggatacctgt ccgcctttct
cccttcggga agcgtggcgc tttctcatag 3840ctcacgctgt aggtatctca
gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 3900cgaacccccc
gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
3960cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
ttagcagagc 4020gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
cctaactacg gctacactag 4080aagaacagta tttggtatct gcgctctgct
gaagccagtt accttcggaa aaagagttgg 4140tagctcttga tccggcaaac
aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 4200gcagattacg
cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
4260tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatggcta
gttaattaac 4320atttaaatca gcggccgcaa taaaatatct ttattttcat
tacatctgtg tgttggtttt 4380ttgtgtgaat cgtaactaac atacgctctc
catcaaaaca aaacgaaaca aaacaaacta 4440gcaaaatagg ctgtccccag
tgcaagtgca ggtgccagaa catttctcta tcgaa 44954593822DNAArtificial
sequenceSynthesized vector LC-pFuse 459ggatctgcga tcgctccggt
gcccgtcagt gggcagagcg cacatcgccc acagtccccg 60agaagttggg gggaggggtc
ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa 120actgggaaag
tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
180atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc
gccagaacac 240agctgaagct tcgaggggct cgcatctctc cttcacgcgc
ccgccgccct acctgaggcc 300gccatccacg ccggttgagt cgcgttctgc
cgcctcccgc ctgtggtgcc tcctgaactg 360cgtccgccgt ctaggtaagt
ttaaagctca ggtccagacc gggcctttgt ccggcgctcc 420cttggagcct
acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac
480tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg
tgaccggcgc 540ctacctgaga tcaccggcga aggagggcca ccatgcgcag
gatgcaactc ctgttgctga 600ttgcactaag tcttgcactt gtcacgaatt
cgccatggcc ctaggtcagc ccaaggctgc 660cccctcggtc actctgttcc
cgccctcctc tgaggagctt caagccaaca aggccacact 720ggtgtgtctc
ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag
780cagccccgtc aaggcgggag tggaaacaac cacaccctcc aaacaaagca
acaacaagta 840cgcggccagc agctatctga gcctgacgcc tgagcagtgg
aagtcccaca gaagctacag 900ctgccaggtc acgcatgaag ggagcaccgt
ggagaagaca gtggccccta cagaatgttc 960ataatgagct agctggccag
acatgataag atacattgat gagtttggac aaaccacaac 1020tagaatgcag
tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
1080aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
ttatgtttca 1140ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
aacctctaca aatgtggtat 1200ggaattaatt ctaaaataca gcatagcaaa
actttaacct ccaaatcaag cctctacttg 1260aatccttttc tgagggatga
ataaggcata ggcatcaggg gctgttgcca atgtgcatta 1320gctgtttgca
gcctcacctt ctttcatgga gtttaagata tagtgtattt tcccaaggtt
1380tgaactagct cttcatttct ttatgtttta aatgcactga cctcccacat
tcccttttta 1440gtaaaatatt cagaaataat ttaaatacat cattgcaatg
aaaataaatg ttttttatta 1500ggcagaatcc agatgctcaa ggcccttcat
aatatccccc agtttagtag ttggacttag 1560ggaacaaagg aacctttaat
agaaattgga cagcaagaaa gcgagcttct agcttatcct 1620cagtcctgct
cctctgccac aaagtgcacg cagttgccgg ccgggtcgcg cagggcgaac
1680tcccgccccc acggctgctc gccgatctcg gtcatggccg gcccggaggc
gtcccggaag 1740ttcgtggaca cgacctccga ccactcggcg tacagctcgt
ccaggccgcg cacccacacc 1800caggccaggg tgttgtccgg caccacctgg
tcctggaccg cgctgatgaa cagggtcacg 1860tcgtcccgga ccacaccggc
gaagtcgtcc tccacgaagt cccgggagaa cccgagccgg 1920tcggtccaga
actcgaccgc tccggcgacg tcgcgcgcgg tgagcaccgg aacggcactg
1980gtcaacttgg ccatgatggc tcctcctgtc aggagaggaa agagaagaag
gttagtacaa 2040ttgctatagt gagttgtatt atactatgca gatatactat
gccaatgatt aattgtcaaa 2100ctagggctgc agggttcata gtgccacttt
tcctgcactg ccccatctcc tgcccaccct 2160ttcccaggca tagacagtca
gtgacttacc aaactcacag gagggagaag gcagaagctt 2220gagacagacc
cgcgggaccg ccgaactgcg aggggacgtg gctagggcgg cttcttttat
2280ggtgcgccgg ccctcggagg cagggcgctc ggggaggcct agcggccaat
ctgcggtggc 2340aggaggcggg gccgaaggcc gtgcctgacc aatccggagc
acataggagt ctcagccccc 2400cgccccaaag caaggggaag tcacgcgcct
gtagcgccag cgtgttgtga aatgggggct 2460tgggggggtt ggggccctga
ctagtcaaaa caaactccca ttgacgtcaa tggggtggag 2520acttggaaat
ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg
2580catcatcatg gtaatagcga tgactaatac gtagatgtac tgccaagtag
gaaagtccca 2640taaggtcatg tactgggcat aatgccaggc gggccattta
ccgtcattga cgtcaatagg 2700gggcgtactt ggcatatgat acacttgatg
tactgccaag tgggcagttt accgtaaata 2760ctccacccat tgacgtcaat
ggaaagtccc tattggcgtt actatgggaa catacgtcat 2820tattgacgtc
aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
2880ttatgtaacg cctgcaggtt aattaagaac atgtgagcaa aaggccagca
aaaggccagg 2940aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
tccgcccccc tgacgagcat 3000cacaaaaatc gacgctcaag tcagaggtgg
cgaaacccga caggactata aagataccag 3060gcgtttcccc ctggaagctc
cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 3120tacctgtccg
cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
3180tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
accccccgtt 3240cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
agtccaaccc ggtaagacac 3300gacttatcgc cactggcagc agccactggt
aacaggatta gcagagcgag gtatgtaggc 3360ggtgctacag agttcttgaa
gtggtggcct aactacggct acactagaag aacagtattt 3420ggtatctgcg
ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
3480ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
gattacgcgc 3540agaaaaaaag gatctcaaga agatcctttg atcttttcta
cggggtctga cgctcagtgg 3600aacgaaaact cacgttaagg gattttggtc
atggctagtt aattaacatt taaatcagcg 3660gccgcaataa aatatcttta
ttttcattac atctgtgtgt tggttttttg tgtgaatcgt 3720aactaacata
cgctctccat caaaacaaaa cgaaacaaaa caaactagca aaataggctg
3780tccccagtgc aagtgcaggt gccagaacat ttctctatcg aa
3822460107PRTArtificial sequenceSynthesized VH4-34+CDR3 460Gln Val
Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr
Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25
30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Thr 85 90 95Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser
100 105461107PRTArtificial sequenceSynthesized VH4-34+CDR3_CDR1 Cow
461Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp
Lys 20 25 30Ala Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro
Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys Thr 85 90 95Ser Val His Gln Glu Thr Lys Lys Tyr
Gln Ser 100 105462107PRTArtificial sequenceSynthesized
VH4-34+CDR3_CDR2 Cow 462Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu
Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly
Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Ser Ile Asp Thr Gly Gly Asn
Thr Gly Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val
Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95Ser Val His Gln
Glu Thr Lys Lys Tyr Gln Ser 100 105463107PRTArtificial
sequenceSynthesized VH4-34+CDR3_CDR1 G31D, Y32K 463Gln Val Gln Leu
Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Lys 20 25 30Tyr Trp
Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly
Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Thr 85 90 95Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser 100
105464107PRTArtificial sequenceSynthesized VH4-34+CDR3_CDR2 E50S
464Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly
Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Ser Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro
Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln
Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Thr 85 90 95Ser Val His Gln Glu Thr Lys Lys Tyr Gln
Ser 100 105465107PRTArtificial sequenceSynthesized VH4-34+CDR3_CDR1
Cow_CDR2 Cow 465Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser
Leu Ser Asp Lys 20 25 30Ala Val Gly Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Leu 35 40 45Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly
Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95Ser Val His Gln Glu Thr
Lys Lys Tyr Gln Ser 100 105466107PRTArtificial sequenceSynthesized
VH4-34+CDR3_CDR1 Cow_CDR2 E50S 466Gln Val Gln Leu Gln Gln Trp Gly
Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr
Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30Ala Val Gly Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Ser Ile Asn His
Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95Ser
Val His Gln Glu Thr Lys Lys Tyr Gln Ser 100 105467107PRTArtificial
sequenceSynthesized VH4-34+CDR3_CDR1 G31D,Y32K_CDR2 Cow 467Gln Val
Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr
Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Lys 20 25
30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Thr 85 90 95Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser
100 105468107PRTArtificial sequenceSynthesized VH4-34+CDR3_CDR1
G31D,Y32K_CDR2 E50S 468Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu
Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly
Gly Ser Phe Ser Asp Lys 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Ser Ile Asn His Ser Gly Ser
Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val
Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95Ser Val His Gln
Glu Thr Lys Lys Tyr Gln Ser 100 10546925PRTArtificial
sequenceSynthesized J region of antibody comprising UL CDR3 469Ser
Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val Trp Gly Gln Gly1 5 10
15Leu Leu Val Thr Val Ser Ser Ala Ser 20 254706PRTArtificial
sequenceSynthesized VL1-51 CDR2 substituted amino acid resides
470Asp Asn Asn Lys Arg Pro1 54716PRTArtificial sequenceSynthesized
VL1-51 CDR2 substituted amino acids 471Gly Asp Thr Ser Arg Ala1
5472420DNAArtificial sequenceSynthesized HC-pFuse vector with BsaI
cassette 472caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac
gctgtccctc 60acctgcacag caagcgggtt ttcactgagc gacaaggcag tgggatggat
tcgccagccc 120ccagggaagg ggctggagtg gattggggaa atcaatcata
gtggaagcac caactacaac 180ccgtccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc
cgcggacacg gctgtgtatt actgtacctc tgtgcaccag 300gaaactaaga
aataccagag cgagacctac tatggttcgg gtctctctta tacctacaat
360tatgaatggc atgtggatgt ctggggacag ggcctgctgg tgacagtctc
tagtgctagc 420473133PRTArtificial sequenceSynthesized Hc-pFuse
vector with non-bovine sequence insertMISC_FEATURE(107)..(107)X is
an insert of a non-bovine sequencemisc_feature(108)..(108)Xaa can
be any naturally occurring amino acid 473Gln Val Gln Leu Gln Gln
Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30Ala Val Gly Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile
Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr
85 90 95Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Xaa Ser Tyr Thr
Tyr 100 105 110Asn Tyr Glu Trp His Val Asp Val Trp Gly Gln Gly Leu
Leu Val Thr 115 120 125Val Ser Ser Ala Ser 130474662DNAArtificial
sequenceSynthesized BLV1H12 LV-human lambda LC 474tcacgaattc
gcaggccgtc ctgaaccagc caagcagcgt ctccgggtct ctggggcagc 60gggtctcaat
cacctgtagc gggtcttcct ccaatgtcgg caacggctac gtgtcttggt
120atcagctgat ccctggcagt gccccacgaa ccctgatcta cggcgacaca
tccagagctt 180ctggggtccc cgatcggttc tcagggagca gatccggaaa
cacagctact ctgaccatca 240gctccctgca ggctgaggac gaagcagatt
atttctgcgc atctgccgag gactctagtt 300caaatgccgt gtttggaagc
ggcaccacac tgacagtcct aggtcagccc aaggctgccc 360cctcggtcac
tctgttcccg ccctcctctg aggagcttca agccaacaag gccacactgg
420tgtgtctcat aagtgacttc tacccgggag ccgtgacagt ggcctggaag
gcagatagca 480gccccgtcaa ggcgggagtg gagaccacca caccctccaa
acaaagcaac aacaagtacg 540cggccagcag ctatctgagc ctgacgcctg
agcagtggaa gtcccacaga agctacagct 600gccaggtcac gcatgaaggg
agcaccgtgg agaagacagt ggcccctaca gaatgttcat 660aa
66247574PRTArtificial sequenceSynthesized IL-8 sequence 475Pro Arg
Ser Ala Lys Glu Leu Arg Cys Gln Cys Ile Lys Thr Tyr Ser1 5 10 15Lys
Pro Phe His Pro Lys Phe Ile Lys Glu Leu Arg Val Ile Glu Ser 20 25
30Gly Pro His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly
35 40 45Arg Glu Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln Arg Val
Val 50 55 60Glu Lys Phe Leu Lys Arg Ala Glu Asn Ser65
7047625PRTArtificial sequenceSynthesized ziconotide sequence 476Cys
Lys Gly Lys Gly Ala Lys Cys Ser Arg Leu Met Tyr Asp Cys Cys1 5 10
15Thr Gly Ser Cys Arg Ser Gly Lys Cys 20 2547715PRTArtificial
sequenceSynthesized somatostatin sequence 477Ala Gly Cys Lys Asn
Phe Phe Trp Lys Thr Phe Thr Ser Cys Gly1 5 10 1547834PRTArtificial
sequenceSynthesized chlorotoxin sequence 478Met Cys Met Pro Cys Phe
Thr Thr Asp His Gln Met Ala Arg Lys Cys1 5 10 15Asp Asp Cys Cys Gly
Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30Cys
Leu47968PRTArtificial sequenceSynthesized SDF1(alpha) sequence
479Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser1
5 10 15His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr
Pro 20 25 30Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn
Arg Gln 35 40 45Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr
Leu Glu Lys 50 55 60Ala Leu Asn Lys65480133PRTArtificial
sequenceSynthesized IL-21 sequence 480Gln Gly Gln Asp Arg His Met
Ile Arg Met Arg Gln Leu Ile Asp Ile1 5 10 15Val Asp Gln Leu Lys Asn
Tyr Val Asn Asp Leu Val Pro Glu Phe Leu 20 25 30Pro Ala Pro Glu Asp
Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser 35 40 45Cys Phe Gln Lys
Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu 50 55 60Arg Ile Ile
Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser65 70 75 80Thr
Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys 85 90
95Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys
100 105 110Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg
Thr His 115 120 125Gly Ser Glu Asp Ser 13048130PRTArtificial
sequenceSynthesized ProTxII sequence 481Tyr Cys Gln Lys Trp Met Trp
Thr Cys Asp Ser Glu Arg Lys Cys Cys1 5 10 15Glu Gly Met Val Cys Arg
Leu Trp Cys Lys Lys Lys Leu Trp 20 25 30482274PRTArtificial
sequenceSynthesized Hc BLV1H12 482Gln Val Gln Leu Arg Glu Ser Gly
Pro Ser Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr
Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30Ala Val Gly Trp Val Arg
Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45Gly Ser Ile Asp Thr
Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55 60Ser Arg Leu Ser
Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu65 70 75 80Ser Val
Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr 85 90 95Ser
Val His Gln Glu Thr Lys Lys Tyr Gln Ser Cys Pro Asp Gly Tyr 100 105
110Arg Glu Arg Ser Asp Cys Ser Asn Arg Pro Ala Cys Gly Thr Ser Asp
115 120 125Cys Cys Arg Val Ser Val Phe Gly Asn Cys Leu Thr Thr Leu
Pro Val 130 135 140Ser Tyr Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val
Asp Val Trp Gly145 150 155 160Gln Gly Leu Leu Val Thr Val Ser Ser
Ala Ser Thr Thr Ala Pro Lys 165 170 175Val Tyr Pro Leu Ser Ser Cys
Cys Gly Asp Lys Ser Ser Ser Thr Val 180 185 190Thr Leu Gly Cys Leu
Val Ser Ser Tyr Met Pro Glu Pro Val Thr Val 195 200 205Thr Trp Asn
Ser Gly Ala Leu Lys Ser Gly Val His Thr Phe Pro Ala 210 215 220Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val225 230
235 240Pro Gly Ser Thr Ser Gly Gln Thr Phe Thr Cys Asn Val Ala His
Pro 245 250 255Ala Ser Ser Thr Lys Val Asp Lys Ala Val Glu Pro Lys
Ser Cys Asp 260 265 270Gly Ser483216PRTArtificial
sequenceSynthesized Lc BLV1H12 483Gln Ala Val Leu Asn Gln Pro Ser
Ser Val Ser Gly Ser Leu Gly Gln1 5 10 15Arg Val Ser Ile Thr Cys Ser
Gly Ser Ser Ser Asn Val Gly Asn Gly 20 25 30Tyr Val Ser Trp Tyr Gln
Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu 35 40 45Ile Tyr Gly Asp Thr
Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Arg Ser
Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Ala Glu
Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu Asp Ser Ser 85 90 95Ser
Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu Gly Gln 100 105
110Pro Lys Ser Pro Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu Glu
115 120 125Leu Asn Gly Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
Phe Tyr 130 135 140Pro Gly Ser Val Thr Val Val Trp Lys Ala Asp Gly
Ser Thr Ile Thr145 150 155 160Arg Asn Val Glu Thr Thr Arg Ala Ser
Lys Gln Ser Asn Ser Lys Tyr 165 170 175Ala Ala Ser Ser Tyr Leu Ser
Leu Thr Ser Ser Asp Trp Lys Ser Lys 180 185 190Gly Ser Tyr Ser Cys
Glu Val Thr His Glu Gly Ser Thr Val Thr Lys 195 200 205Thr Val Lys
Pro Ser Glu Cys Ser 210 215484269PRTArtificial sequenceSynthesized
Hc BLV5B8 484Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Gln
Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser
Leu Ser Asp Lys 20 25 30Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys
Ala Leu Glu Trp Leu 35 40 45Gly Ser Ile Asp Thr Gly Gly Ser Thr Gly
Tyr Asn Pro Gly Leu Lys 50 55 60Ser Arg Leu Ser Ile Thr Lys Asp Asn
Ser Lys Ser Gln Val Ser Leu65 70 75 80Ser Val Ser Ser Val Thr Thr
Glu Asp Ser Ala Thr Tyr Tyr Cys Thr 85 90 95Thr Val His Gln Glu Thr
Arg Lys Thr Cys Ser Asp Gly Tyr Ile Ala 100 105 110Val Asp Ser Cys
Gly Arg Gly Gln Ser Asp Gly Cys Val Asn Asp Cys 115 120 125Asn Ser
Cys Tyr Tyr Gly Trp Arg Asn Cys Arg Arg Gln Pro Ala Ile 130 135
140His Ser Tyr Glu Phe His Val Asp Ala Trp Gly Arg Gly Leu Leu
Val145 150 155 160Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Lys Val
Tyr Pro Leu Ser 165 170 175Ser Cys Cys Gly Asp Lys Ser Ser Ser Thr
Val Thr Leu Gly Cys Leu 180 185 190Val Ser Ser Tyr Met Pro Glu Pro
Val Thr Val Thr Trp Asn Ser Gly 195 200 205Ala Leu Lys Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser 210 215 220Gly Leu Tyr Ser
Leu Ser Ser Met Val Thr Val Pro Gly Ser Thr Ser225 230 235 240Gly
Gln Thr Phe Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys 245 250
255Val Asp Lys Ala Val Glu Pro Lys Ser Cys Asp Gly Ser 260
265485216PRTArtificial sequenceSynthesized Lc BLV5B8 485Gln Ala Val
Leu Asn Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln1 5 10 15Arg Val
Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Gly 20 25 30Tyr
Val Ser Trp Tyr Gln Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu 35 40
45Ile Tyr Gly Asp Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu
Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu
Asp Ser Ser 85 90 95Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr
Val Leu Gly Gln 100 105 110Pro Lys Ser Pro Pro Ser Val Thr Leu Phe
Pro Pro Ser Thr Glu Glu 115 120 125Leu Asn Gly Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140Pro Gly Ser Val Thr Val
Val Trp Lys Ala Asp Gly Ser Thr Ile Thr145 150 155 160Arg Asn Val
Glu Thr Thr Arg Ala Ser Lys Gln Ser Asn Ser Lys Tyr 165 170 175Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Ser Ser Asp Trp Lys Ser Lys 180 185
190Gly Ser Tyr Ser Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys
195 200 205Thr Val Lys Pro Ser Glu Cys Ser 210 215
* * * * *