U.S. patent application number 13/737913 was filed with the patent office on 2014-02-20 for ultralong complementarity determining regions and uses thereof.
This patent application is currently assigned to THE SCRIPPS RESEARCH INSTITUTE. The applicant listed for this patent is THE SCRIPPS RESEARCH INSTITUTE. Invention is credited to Omar A. Bazirgan, Dan Ekiert, Hongyuan Helen Mao, Peter Schultz, Vaughn Smider, Feng Wang, Ian Wilson, Yong Zhang.
Application Number | 20140050720 13/737913 |
Document ID | / |
Family ID | 48782074 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140050720 |
Kind Code |
A1 |
Smider; Vaughn ; et
al. |
February 20, 2014 |
ULTRALONG COMPLEMENTARITY DETERMINING REGIONS AND USES THEREOF
Abstract
Disclosed herein are immunoglobulin constructs comprising at
least one immunoglobulin domain or fragment thereof; and a
therapeutic polypeptide or derivative or variant thereof attached
to or inserted into said immunoglobulin domain. Also provided are
immunoglobulin constructs comprising a mammalian immunoglobulin
heavy chain comprising at least a portion of a knob domain in the
complementarity-determining region 3 (CDR3H) or fragment thereof;
and a therapeutic polypeptide attached to or inserted into said
knob domain of the CDR3H. Also provided are immunoglobulin
constructs comprising a mammalian immunoglobulin heavy chain
comprising at least a portion of a stalk domain in the
complementarity-determining region 3 (CDR3H) or fragment thereof;
and a therapeutic polypeptide attached to or inserted into said
stalk domain of the CDR3H. Also described herein are methods and
compositions comprising the immunoglobulin constructs described
herein for treatment and prevention of a disease or condition in a
subject.
Inventors: |
Smider; Vaughn; (San Diego,
CA) ; Bazirgan; Omar A.; (San Diego, CA) ;
Ekiert; Dan; (San Francisco, CA) ; Mao; Hongyuan
Helen; (San Diego, CA) ; Schultz; Peter; (La
Jolla, CA) ; Wang; Feng; (Encinitas, CA) ;
Wilson; Ian; (La Jolla, CA) ; Zhang; Yong;
(San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE SCRIPPS RESEARCH INSTITUTE; |
|
|
US |
|
|
Assignee: |
THE SCRIPPS RESEARCH
INSTITUTE
La Jolla
CA
|
Family ID: |
48782074 |
Appl. No.: |
13/737913 |
Filed: |
January 9, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61584680 |
Jan 9, 2012 |
|
|
|
61671629 |
Jul 13, 2012 |
|
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Current U.S.
Class: |
424/133.1 ;
435/252.33; 435/254.21; 435/254.23; 435/320.1; 435/328; 435/69.6;
506/16; 506/18; 530/387.3; 536/23.53 |
Current CPC
Class: |
C07K 16/00 20130101;
C07K 16/46 20130101; C07K 2317/565 20130101; C07K 2317/50 20130101;
A61P 35/00 20180101; C07K 2317/73 20130101; C07K 2317/94 20130101;
C07K 2318/00 20130101; C07K 2317/55 20130101; C07K 2319/00
20130101; C07K 14/53 20130101; C07K 2319/30 20130101; C07K 2319/55
20130101; C07K 2317/20 20130101; C07K 16/10 20130101; C07K 2317/569
20130101; C07K 2319/50 20130101; C07K 2317/76 20130101 |
Class at
Publication: |
424/133.1 ;
506/18; 506/16; 530/387.3; 536/23.53; 435/320.1; 435/69.6;
435/252.33; 435/254.21; 435/254.23; 435/328 |
International
Class: |
C07K 16/10 20060101
C07K016/10; C07K 16/46 20060101 C07K016/46 |
Claims
1. A library of antibodies or binding fragments thereof, wherein
the antibodies or binding fragments thereof comprise an ultralong
CDR3.
2. A library of polynucleotides encoding for antibodies or binding
fragments thereof, wherein the encoded antibodies or binding
fragments thereof comprise an ultralong CDR3.
3-47. (canceled)
48. A recombinant antibody or fragment thereof, wherein at least a
portion of the recombinant antibody or fragment thereof is based on
or derived from at least a portion of an ultralong CDR3.
49. An antibody or fragment thereof comprising: (a) a first
antibody sequence, wherein at least a portion of the first antibody
sequence is derived from at least a portion of an ultralong CDR3;
(b) a non-antibody sequence; and (c) optionally, a second antibody
sequence, wherein at least a portion of the second antibody
sequence is derived from at least a portion of an ultralong
CDR3.
50-158. (canceled)
159. A library of antibodies or binding fragments thereof, wherein
the antibodies or binding fragments thereof comprise an ultralong
CDR3.
160. A library of antibodies or binding fragments thereof, wherein
the antibodies or binding fragments thereof comprise the antibody
or binding fragment of claim 48.
161. A nucleic acid library comprising a plurality of
polynucleotides comprising sequences coding for antibodies or
binding fragments thereof, wherein the antibodies or binding
fragments thereof comprise an ultralong CDR3.
162. A nucleic acid library comprising a plurality of
polynucleotides comprising sequences coding for antibodies or
binding fragments thereof, wherein the antibodies or binding
fragments thereof comprise the antibody or binding fragment of
claim 48.
163-165. (canceled)
166. A polynucleotide comprising a nucleic acid sequence that
encodes the antibody or binding fragment thereof of claim 48.
167. A vector comprising a polynucleotide, wherein the
polynucleotide comprises a nucleic acid sequence that encodes the
antibody or binding fragment thereof of claim 48.
168. A host cell comprising a polynucleotide, wherein the
polynucleotide comprises a nucleic acid sequence that encodes the
antibody or binding fragment thereof of claim 48.
169. A method of producing an antibody or binding fragment thereof
comprising an ultralong CDR3 or fragment thereof comprising
culturing the host cell of claim 168 under conditions wherein the
polynucleotide sequence is expressed and the antibody or binding
fragment thereof comprising an ultralong CDR3 or fragment thereof
is produced.
170. (canceled)
171. A pharmaceutical composition comprising an antibody or
fragment thereof of claim 48.
172. A pharmaceutical composition comprising (a) an antibody or
fragment thereof comprising sequence based on or derived from at
least a portion of an ultralong CDR3; and (b) a pharmaceutically
acceptable excipient.
173-174. (canceled)
175. A method of treating a disease or condition in a subject in
need thereof comprising administering to the mammal a
therapeutically effective amount of the antibody of claim 48.
176-188. (canceled)
189. A library of antibodies or binding fragments thereof, wherein
the antibodies or binding fragments thereof comprise the antibody
or binding fragment of claim 49.
190. A nucleic acid library comprising a plurality of
polynucleotides comprising sequences coding for antibodies or
binding fragments thereof, wherein the antibodies or binding
fragments thereof comprise the antibody or binding fragment of
claim 49.
191. A polynucleotide comprising a nucleic acid sequence that
encodes the antibody or binding fragment thereof of claim 49.
192. A vector comprising a polynucleotide, wherein the
polynucleotide comprises a nucleic acid sequence that encodes the
antibody or binding fragment thereof of claim 49.
193. A host cell comprising a polynucleotide, wherein the
polynucleotide comprises a nucleic acid sequence that encodes the
antibody or binding fragment thereof claim 49.
194. A method of producing an antibody or binding fragment thereof
comprising an ultralong CDR3 or fragment thereof comprising
culturing the host cell of claim 193 under conditions wherein the
polynucleotide sequence is expressed and the antibody or binding
fragment thereof comprising an ultralong CDR3 or fragment thereof
is produced.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/584,680 filed Jan. 9, 2012, and U.S. Provisional
Application No. 61/671,629, filed Jul. 13, 2012, both of which are
incorporated by reference herein in their entirety.
FIELD
[0002] Described herein are immunoglobulin constructs comprising at
least a portion of an ultralong CDR3, methods of making such
constructs, pharmaceutical compositions and medicaments comprising
such constructs, and methods of using such constructs and
compositions to prevent, inhibit, and/or treat a disease or
condition in a subject.
BACKGROUND
[0003] 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.
[0004] 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
[0005] The present disclosure provides antibodies that comprise an
utralong CDR3 including, libraries that comprise an ultralong CDR3,
and uses thereof.
[0006] The present disclosure also provides a library of antibodies
or binding fragments thereof, wherein the antibodies or binding
fragments thereof comprise an ultralong CDR3.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises 3 or more
cysteine residues.
[0011] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
comprise a cysteine motif.
[0012] In some embodiments of each or any of the above or below
mentioned embodiments, the cysteine motif is selected from the
group consisting of SEQ ID NOS: 45-156. In some embodiments of each
or any of the above or below mentioned embodiments, the cysteine
motif is selected from the group consisting of SEQ ID NOS: 45-99.
In some embodiments of each or any of the above or below mentioned
embodiments, the cysteine motif is selected from the group
consisting of SEQ ID NOS: 45-135. In some embodiments of each or
any of the above or below mentioned embodiments, the cysteine motif
is selected from the group consisting of SEQ ID NOS: 100-135. In
some embodiments of each or any of the above or below mentioned
embodiments, the cysteine motif is selected from the group
consisting of SEQ ID NOS: 136-156.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a non-bovine
sequence.
[0018] In some embodiments of each or any of the above or below
mentioned embodiments, the non-bovine sequence is a non-antibody or
a human sequence.
[0019] In some embodiments of each or any of the above or below
mentioned embodiments, the non-bovine sequence replaces at least a
portion of the ultralong CDR3.
[0020] In some embodiments of each or any of the above or below
mentioned embodiments, the non-bovine sequence is a hormone,
lymphokine, interleukin, chemokine, cytokine, toxin, or combination
thereof.
[0021] In some embodiments of each or any of the above or below
mentioned embodiments, the non-bovine sequence is a cytokine.
[0022] In some embodiments of each or any of the above or below
mentioned embodiments, the cytokine is granulocyte
colony-stimulating factor (G-CSF).
[0023] 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.
[0024] 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.
[0025] In some embodiments of each or any of the above or below
mentioned embodiments, the linker is (GGGGS).sub.n(SEQ ID NO: 339),
where n is an integer between 0 and 5. Alternatively, or
additionally, the linker is (GSG).sub.n (SEQ ID NO: 342), GGGSGGGGS
(SEQ ID NO: 337) or GGGGSGGGS (SEQ ID NO: 338)
[0026] 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.
[0027] 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: 159) or TSVHQ (SEQ ID NO: 160).
[0028] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 further 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.
[0029] 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.
[0030] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
X.sup.1X.sup.2.sub.X.sup.3.sub.X.sup.4.sub.X.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.
[0031] 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: 159) or TSVHQ (SEQ ID NO: 160), and
wherein the (X.sup.aX.sup.b).sub.z motif is YXYXYX.
[0032] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises TSVHQETKKYQ
(SEQ ID NO. 157).
[0033] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises VHQETKKYQ (SEQ
ID NO: 158).
[0034] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises CTTVHQX.sub.n
(SEQ ID NO. 223).
[0035] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises CTSVHQX.sub.n
(SEQ ID NO. 223), wherein n is 1-8.
[0036] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises TTVHQ (SEQ ID
NO. 159).
[0037] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises TSVHQ (SEQ ID
NO. 160).
[0038] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises VHQ.
[0039] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises KKQ.
[0040] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises VYQ.
[0041] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises CX.sup.1
X.sup.2 X.sup.3 X.sup.4Q (SEQ ID NO: 228), wherein X.sup.1 is T, S,
A, or G, wherein X.sup.2 is T, S, A, P, or I, wherein X.sup.3 is V
or K, and wherein X.sup.4 is H, K, or Y.
[0042] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises CX.sup.1
X.sup.2VHQ (SEQ ID NO: 230), wherein X.sup.1 is T, S, A, or G, and
wherein X.sup.2 is T, S, A, P, or I.
[0043] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises CX.sup.1
X.sup.2VX.sup.3Q (SEQ ID NO: 232), wherein X.sup.1 is T, S, A, or
G, wherein X.sup.2 is T, S, A, P, or I, and wherein X.sup.3 is H,
Y, or K.
[0044] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises
CX.sup.1X.sup.2KKQ (SEQ ID NO: 234), wherein X.sup.1 is T, S, A, or
G, and wherein X.sup.2 is T, S, A, P, or I.
[0045] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises YTYNYEW (SEQ ID
NO: 235).
[0046] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises
YX.sup.1YX.sup.2 (SEQ ID NO: 296), wherein X.sup.2 is E or D.
[0047] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises
YX.sup.1YX.sup.2 Y (SEQ ID NO: 297).
[0048] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises YEX, wherein X
is H, W, N, F, I or Y.
[0049] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises YDX, wherein X
is H, W, N, F, I or Y.
[0050] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises XYE, wherein X
is T, S, N or I.
[0051] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises XYD, wherein X
is T, S, N or I.
[0052] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises
Y(E/D)X.sup.1X.sub.nW (SEQ ID NOS: 304-305), wherein X.sup.1 is H,
W, N, F, I or Y, and wherein n is 1-4.
[0053] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
are chimeric, human engineered, or humanized.
[0054] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 is a ruminant CDR3.
[0055] In some embodiments of each or any of the above or below
mentioned embodiments, the ruminant is a cow.
[0056] The present disclosure also provides a library of
polynucleotides encoding for antibodies or binding fragments
thereof, wherein the encoded antibodies or binding fragments
thereof comprise an ultralong CDR3.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises 3 or more
cysteine residues.
[0061] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
comprise a cysteine motif.
[0062] In some embodiments of each or any of the above or below
mentioned embodiments, the cysteine motif is selected from the
group consisting of SEQ ID NOS: 45-156. In some embodiments of each
or any of the above or below mentioned embodiments, the cysteine
motif is selected from the group consisting of SEQ ID NOS: 45-99.
In some embodiments of each or any of the above or below mentioned
embodiments, the cysteine motif is selected from the group
consisting of SEQ ID NOS: 45-135. In some embodiments of each or
any of the above or below mentioned embodiments, the cysteine motif
is selected from the group consisting of SEQ ID NOS: 100-135. In
some embodiments of each or any of the above or below mentioned
embodiments, the cysteine motif is selected from the group
consisting of SEQ ID NOS: 136-156.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a non-bovine
sequence.
[0068] In some embodiments of each or any of the above or below
mentioned embodiments, the non-bovine sequence is a non-antibody or
a human sequence.
[0069] In some embodiments of each or any of the above or below
mentioned embodiments, the non-bovine sequence replaces at least a
portion of the ultralong CDR3.
[0070] In some embodiments of each or any of the above or below
mentioned embodiments, the non-antibody sequence is a hormone,
lymphokine, interleukin, chemokine, cytokine, toxin, or combination
thereof.
[0071] In some embodiments of each or any of the above or below
mentioned embodiments, the non-bovine sequence is a cytokine.
[0072] In some embodiments of each or any of the above or below
mentioned embodiments, the cytokine is granulocyte
colony-stimulating factor (G-CSF).
[0073] 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.
[0074] 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.
[0075] In some embodiments of each or any of the above or below
mentioned embodiments, the linker is (GGGGS).sub.n (SEQ ID NO:
339), where n is an integer between 0 and 5. Alternatively, the
linker is (GSG).sub.n (SEQ ID NO: 342), GGGSGGGGS (SEQ ID NO: 337)
or GGGGSGGGS (SEQ ID NO: 338).
[0076] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises a
X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.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.sub.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.sub.5 is glutamine (Q).
[0077] 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:159) or TSVHQ (SEQ ID NO: 160).
[0078] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 further comprises a
(X.sup.aX.sup.b).sub.z motif, wherein X.sup.a is an aromatic amino
acid selected from the group consisting of: tyrosine (Y),
phenylalanine (F), tryptophan (W), and histidine (H), wherein
X.sup.b is any amino acid residue, and wherein z is 1-4.
[0079] 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.
[0080] 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.6X.sup.7).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.
[0081] 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: 159) or TSVHQ (SEQ ID NO: 160), and
wherein the (X.sup.a-X.sup.b).sub.z motif is YXYXYX.
[0082] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises TSVHQETKKYQ
(SEQ ID NO. 157).
[0083] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises VHQETKKYQ (SEQ
ID NO: 158).
[0084] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises CTTVHQXn (SEQ
ID NO. 223).
[0085] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises CTSVHQXn (SEQ
ID NO. 224), wherein n is 1-8.
[0086] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises TTVHQ (SEQ ID
NO. 159).
[0087] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises TSVHQ (SEQ ID
NO. 160).
[0088] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises VHQ.
[0089] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises KKQ.
[0090] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises VYQ.
[0091] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises CX.sup.1
X.sup.2 X.sup.3 X.sup.4Q, wherein X.sup.1 is T, S, A, or G, wherein
X.sup.2 is T, S, A, P, or I, wherein X.sup.3 is V or K, and wherein
X.sup.4 is H, K, or Y.
[0092] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises CX.sup.1
X.sup.2VHQ, wherein X.sup.1 is T, S, A, or G, and wherein X.sup.2
is T, S, A, P, or I.
[0093] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises CX.sup.1
X.sup.2VX.sup.3Q, wherein X.sup.1 is T, S, A, or G, wherein X.sup.2
is T, S, A, P, or I, and wherein X.sup.3 is H, Y, or K.
[0094] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises CX.sup.1
X.sup.2KKQ, wherein X.sup.1 is T, S, A, or G, and wherein X.sup.2
is T, S, A, P, or I.
[0095] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises YTYNYEW (SEQ ID
NO: 235).
[0096] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises
YX.sup.1YX.sup.2, wherein X.sup.2 is E or D.
[0097] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises
YX.sup.1YX.sup.2 Y.
[0098] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises YEX, wherein X
is H, W, N, F, I or Y.
[0099] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises YDX, wherein X
is H, W, N, F, I or Y.
[0100] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises XYE, wherein X
is T, S, N or I.
[0101] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises XYD, wherein X
is T, S, N or I.
[0102] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises
Y(E/D)X.sup.1X.sub.nW, wherein X.sup.1 is H, W, N, F, I or Y, and
wherein n is 1-4.
[0103] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
are chimeric, human engineered, or humanized.
[0104] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 is a ruminant CDR3.
[0105] In some embodiments of each or any of the above or below
mentioned embodiments, the ruminant is a cow.
[0106] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
are present in a spatially addressed format.
[0107] In some embodiments of each or any of the above or below
mentioned embodiments, the polynucleotides coding for the
antibodies or binding fragments thereof are present in a spatially
addressed format.
[0108] The present disclosure also provides a library of vectors
comprising any of the library of polynucleotides disclosed
herein.
[0109] The present disclosure also provides a library of host cells
comprising the any library of vectors disclosed herein.
[0110] In some embodiments of each or any of the above or below
mentioned embodiments, the cell is a bacteria, virus, or
bacteriophage.
[0111] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
are displayed on the cell surface.
[0112] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
are secreted from the cell.
[0113] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
are present in a spatially addressed format.
[0114] The present disclosure also provides an antibody or binding
fragment thereof comprising an ultralong CDR3, wherein the
ultralong CDR3 comprises a non-bovine sequence.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 comprises 3 or more
cysteine residues.
[0119] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
comprise a cysteine motif.
[0120] In some embodiments of each or any of the above or below
mentioned embodiments, the cysteine motif is selected from the
group consisting of SEQ ID NOS: 45-156. In some embodiments of each
or any of the above or below mentioned embodiments, the cysteine
motif is selected from the group consisting of SEQ ID NOS: 45-99.
In some embodiments of each or any of the above or below mentioned
embodiments, the cysteine motif is selected from the group
consisting of SEQ ID NOS: 45-135. In some embodiments of each or
any of the above or below mentioned embodiments, the cysteine motif
is selected from the group consisting of SEQ ID NOS: 100-135. In
some embodiments of each or any of the above or below mentioned
embodiments, the cysteine motif is selected from the group
consisting of SEQ ID NOS: 136-156.
[0121] In some embodiments of each or any of the above or below
mentioned embodiments, the non-bovine sequence is a non-antibody
sequence or a human sequence.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] In some embodiments of each or any of the above or below
mentioned embodiments, the non-bovine sequence replaces at least a
portion of the ultralong CDR3.
[0127] In some embodiments of each or any of the above or below
mentioned embodiments, the non-bovine sequence is a hormone,
lymphokine, interleukin, chemokine, cytokine, toxin, or combination
thereof.
[0128] In some embodiments of each or any of the above or below
mentioned embodiments, the non-bovine sequence is a cytokine.
[0129] In some embodiments of each or any of the above or below
mentioned embodiments, the cytokine is granulocyte
colony-stimulating factor (G-CSF).
[0130] In some embodiments of each or any of the above or below
mentioned embodiments, the antibodies or binding fragments thereof
are chimeric, human engineered, or humanized.
[0131] In some embodiments of each or any of the above or below
mentioned embodiments, the ultralong CDR3 is a ruminant CDR3.
[0132] In some embodiments of each or any of the above or below
mentioned embodiments, the ruminant is a cow.
[0133] The present disclosure also provides a polynucleotide
encoding for any antibody or binding fragment thereof disclosed
herein.
[0134] The present disclosure also provides a vector comprising any
polynucleotide encoding for the antibody or binding fragment
thereof disclosed herein.
[0135] The present disclosure also provides a host cell comprising
any vector disclosed herein.
[0136] In some embodiments of each or any of the above or below
mentioned embodiments, the cell is a bacteria, virus, or
bacteriophage.
[0137] In some embodiments of each or any of the above or below
mentioned embodiments, the antibody or binding fragment thereof is
displayed on the surface of the cell.
[0138] In some embodiments of each or any of the above or below
mentioned embodiments, the antibody or binding fragment thereof is
secreted from the cell.
[0139] The present disclosure also provides a method of producing
an antibody or binding fragment thereof comprising an ultralong
CDR3 or fragment thereof comprising culturing a host cell
comprising a polynucleotide encoding any of the antibodies
disclosed herein under conditions wherein the polynucleotide
sequence is expressed and the antibody or binding fragment thereof
comprising an ultralong CDR3 or fragment thereof is produced.
[0140] In some embodiments of each or any of the above or below
mentioned embodiments, the methods further comprise recovering the
antibody or binding fragment thereof comprising an ultralong CDR3
or fragment thereof from the host cell culture.
[0141] The present disclosure also provides a pharmaceutical
composition comprising any antibody or binding fragment thereof
disclosed herein.
[0142] The present disclosure also provides a method of treating a
mammal in need thereof comprising administering to the mammal an
amount of any antibody disclosed herein.
[0143] In some embodiments of each or any of the above or below
mentioned embodiments, the mammal is a human.
[0144] In some embodiments is a recombinant antibody or fragment
thereof, wherein at least a portion of the recombinant antibody or
fragment thereof is based on or derived from at least a portion of
an ultralong CDR3.
[0145] In some embodiments is an antibody or fragment thereof
comprising at least a portion of an ultralong CDR3 sequence and at
least a portion of a non-bovine sequence.
[0146] In some embodiments is an antibody or fragment thereof
comprising (a) a first antibody sequence, wherein at least a
portion of the first antibody sequence is derived from at least a
portion of an ultralong CDR3; (b) a non-antibody sequence; and (c)
optionally, a second antibody sequence, wherein at least a portion
of the second antibody sequence is derived from at least a portion
of an ultralong CDR3.
[0147] The antibodies disclosed herein may be a chimeric, human
engineered, or humanized antibody. The antibodies disclosed herein
may be a bovine engineered, bovinized, or fully bovine antibody.
The antibodies disclosed herein may comprise a Fab, a scFv, dsFv,
diabody, (dsFv).sub.2, minibody, flex minibody or bi-specific
fragment. The antibodies disclosed herein may be an isolated
antibody.
[0148] The antibodies disclosed herein may further comprise a
non-antibody sequence. The non-antibody sequence may be derived
from a mammal. The mammal may be a bovine, human, or non-bovine
mammal. The antibodies disclosed herein may comprise a non-antibody
sequence derived from a non-bovine animal. The non-bovine animal
may be a scorpion. The non-bovine animal may be a lizard. The
lizard may be a gila monster. The non-antibody sequence may be a
derived from a growth factor. The growth factor may be a GCSF,
GMCSF or FGF21. The GCSF may be a bovine GCSF. Alternatively, the
GCSF may be a human GCSF. The GMCSF and/or the FGF21 may be from a
human. The non-antibody sequence may be a derived from a cytokine.
The cytokine may be a beta-interferon. The non-antibody sequence
may be a derived from a hormone. The hormone may be an exendin-4,
GLP-1, somatostatin, or erythropoietin. The GLP-1 and/or
erythropoietin may be from a human. The non-antibody sequence may
be a derived from a toxin. The toxin may be a Moka1, VM-24,
ziconotide, chlorotoxin, or protoxin2 (ProTxII). The non-antibody
sequence may be IL8, ziconotide, somatostatin, chlorotoxin,
SDF1(alpha), or IL21. The non-antibody sequence may comprise an
amino acid sequence based on or derived from any of SEQ ID NOS:
317-332. The non-antibody sequence may replace at least a portion
of the ultralong CDR3. The non-antibody sequence may be inserted
into the sequence of the ultralong CDR3. The non-antibody sequence
may be conjugated to at least a portion of the antibody (e.g.,
ultralong CDR3, variable region, heavy chain, light chain). The
non-antibody sequence may be attached to the ultralong CDR3,
linker, cleavage site, non-bovine sequence, non-ultralong CDR3
antibody sequence, or combination thereof. The non-antibody
sequence may be adjacent to the ultralong CDR3, linker, cleavage
site, non-bovine sequence, non-ultralong CDR3 antibody sequence, or
combination thereof.
[0149] The antibodies disclosed herein may comprise an ultralong
CDR3 may be based on or derived from a cow ultralong CDR3. At least
a portion of the antibodies disclosed herein may be from a mammal.
At least a portion of the first antibody sequence and/or at least a
portion of the second antibody sequence of the antibodies disclosed
herein may be from a mammal. The mammal may be a bovine, human or
non-bovine mammal.
[0150] The antibodies disclosed herein may comprise 3 or more amino
acids in length. The antibodies disclosed herein may comprise a
sequence that may be based on or derived from an ultralong CDR3
disclosed herein. The antibodies disclosed herein may comprise 1 or
more amino acid residues based on or derived from a stalk domain of
the ultralong CDR3. The antibodies disclosed herein may comprise 1
or more amino acid residues based on or derived from a knob domain
of the ultralong CDR3.
[0151] At least a portion of the antibodies disclosed herein may be
based on or derived from at least a portion of an ultralong CDR3
disclosed herein. The portion of the antibody based on or derived
from at least a portion of the ultralong CDR3 may be 20 or fewer
amino acids in length. The portion of the antibody based on or
derived from at least a portion of the ultralong CDR3 may be 3 or
more amino acids in length
[0152] The antibodies disclosed herein may comprise 1 or more
conserved motifs derived from a stalk domain of the ultralong CDR3.
The 1 or more conserved motifs derived from the stalk domain of the
ultralong CDR3 may comprise any of the stalk domain conserved
motifs disclosed herein.
[0153] The portion of the ultralong CDR3s disclosed herein may
comprise at least a portion of a stalk domain of the ultralong
CDR3, at least a portion of the knob domain of the ultralong CDR3,
or a combination thereof.
[0154] The antibodies disclosed herein may comprise a sequence
selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS:
333-336. The antibodies disclosed herein may comprise a sequence
that may be 50% or more homologous to a sequence selected from any
one of SEQ ID NOS: 157-224 and 235-295.
[0155] A portion of any of the antibodies disclosed herein may be
based on or derived from at least a portion of a single ultralong
CDR3 sequence. A portion of the antibodies disclosed herein may be
based on or derived from at least a portion of two or more
different ultralong CDR3 sequences.
[0156] In any of the embodiments disclosed herein, the portion of
the ultralong CDR3 is based on or derived from a BLV1H12 ultralong
CDR3 sequence. The portion of the ultralong CDR3 may be based on or
derived from a sequence that may be 50% or more homologous to a
BLV1H12 ultralong CDR3 sequence. The portion of the ultralong CDR3
may be based on or derived from a BLV5B8, BLVCV1, BLV5D3, BLV8C11,
BF1H1, or F18 ultralong CDR3 sequence. The portion of the ultralong
CDR3 may be based on or derived from a sequence that may be 50% or
more homologous to a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18
ultralong CDR3 sequence.
[0157] The antibodies disclosed herein may comprise a first and/or
second antibody sequence that comprises 3 or more amino acids in
length. A portion of the first antibody sequence derived from at
least a portion of the ultralong CDR3 and/or the portion of the
second antibody sequence derived from at least a portion of the
ultralong CDR3 may be 20 or fewer amino acids in length. A portion
of the first antibody sequence derived from at least a portion of
the ultralong CDR3 and/or the portion of the second antibody
sequence derived from at least a portion of the ultralong CDR3 may
be 3 or more amino acids in length.
[0158] In any of the embodiments disclosed herein, the first and/or
second antibody sequences comprise one or more amino acid residues
based on or derived from a stalk domain of the ultralong CDR3. The
first and/or second antibody sequences may comprise one or more
amino acid residues based on or derived from a knob domain of the
ultralong CDR3. The one or more amino acid residues derived from
the knob domain of the ultralong CDR3 may be a serine and/or
cysteine residue. The first and/or second antibody sequences may
comprise one or more conserved motifs derived from a stalk domain
of the ultralong CDR3. The one or more conserved motifs derived
from the stalk domain of the ultralong CDR3 may comprise a sequence
selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS:
333-336.
[0159] In any of the embodiments disclosed herein, the portion of
the first antibody sequence derived from at least a portion of the
ultralong CDR3 and/or the portion of the second antibody sequence
derived from at least a portion of the ultralong CDR3 comprises a
sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID
NOS: 333-336. The portion of the first antibody sequence derived
from at least a portion of the ultralong CDR3 and/or the portion of
the second antibody sequence derived from at least a portion of the
ultralong CDR3 may comprise a sequence that may be 50% or more
homologous to a sequence selected from any one of SEQ ID NOS:
157-224 and 235-295. The portion of the first antibody sequence
derived from at least a portion of the ultralong CDR3 may comprise
a sequence selected from any one of SEQ ID NOS: 157-234. The
portion of the first antibody sequence derived from at least a
portion of the ultralong CDR3 may comprise a sequence that may be
50% or more homologous to a sequence selected from any one of SEQ
ID NOS: 157-224.
[0160] In any of the embodiments disclosed herein, the portion of
the second antibody sequence derived from at least a portion of the
ultralong CDR3 comprises a sequence selected from any one of SEQ ID
NOS: 235-307 and SEQ ID NOS: 333-336. The portion of the second
antibody sequence derived from at least a portion of the ultralong
CDR3 may comprise a sequence that may be 50% or more homologous to
a sequence selected from any one of SEQ ID NOS: 235-295. The
portion of the first antibody sequence derived from at least a
portion of the ultralong CDR3 and the portion of the second
antibody sequence derived from at least a portion of the ultralong
CDR3 may be derived from the same ultralong CDR3 sequence.
[0161] In any of the embodiments disclosed herein, the portion of
the first antibody sequence derived from at least a portion of the
ultralong CDR3 and the portion of the second antibody sequence
derived from at least a portion of the ultralong CDR3 is derived
from two or more different ultralong CDR3 sequences. The portions
of the ultralong CDR3 of the first and/or second antibody sequences
may be based on or derived from a BLV1H12 ultralong CDR3 sequence.
The portions of the ultralong CDR3 of the first and/or second
antibody sequences may be based on or derived from a sequence that
may be 50% or more homologous to a BLV1H12 ultralong CDR3 sequence.
The portions of the ultralong CDR3 of the first and/or second
antibody sequences may be based on or derived from a BLV5B8,
BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralong CDR3 sequence. The
portions of the ultralong CDR3 of the first and/or second antibody
sequences may be based on or derived from a sequence that may be
50% or more homologous to a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1,
or F18 ultralong CDR3 sequence.
[0162] In any of the embodiments disclosed herein, the ultralong
CDR3 is based on or derived from an ultralong CDR3 that may be 35
or more amino acids in length. The ultralong CDR3 may be based on
or derived from an ultralong CDR3 comprising 3 or more cysteine
residues.
[0163] In any of the embodiments disclosed herein, the ultralong
CDR3 is based on or derived from an ultralong CDR3 may comprise one
or more cysteine motifs. The one or more cysteine motifs may be
selected from the group consisting of SEQ ID NOS: 45-156. In some
embodiments of each or any of the above or below mentioned
embodiments, the cysteine motif is selected from the group
consisting of SEQ ID NOS: 45-99. In some embodiments of each or any
of the above or below mentioned embodiments, the cysteine motif is
selected from the group consisting of SEQ ID NOS: 45-135. In some
embodiments of each or any of the above or below mentioned
embodiments, the cysteine motif is selected from the group
consisting of SEQ ID NOS: 100-135. In some embodiments of each or
any of the above or below mentioned embodiments, the cysteine motif
is selected from the group consisting of SEQ ID NOS: 136-156.
[0164] The antibodies disclosed herein may be based on or derived
from an ultralong CDR3 that may be 35 or more amino acids in
length. The antibodies disclosed herein may be based on or derived
from an ultralong CDR3 comprising 3 or more cysteine residues. The
antibodies disclosed herein may be based on or derived from an
ultralong CDR3 may comprise 1 or more cysteine motifs.
[0165] The antibodies disclosed herein may comprise an ultralong
CDR3 that is 35 or more amino acids in length. The antibodies
disclosed herein may comprise an ultralong CDR3 comprising 3 or
more cysteine residues. The antibodies disclosed herein may
comprise an ultralong CDR3 comprising one or more cysteine
motifs.
[0166] In any of the embodiments disclosed herein, the ultralong
CDR3 may be a heavy chain CDR3. The ultralong CDR3 may comprise an
amino acid sequence derived from or based on a non-human DH
sequence. The ultralong CDR3 may comprise an amino acid sequence
derived from or based on a JH sequence. The ultralong CDR3 may
comprise an amino acid sequence derived from or based on a
non-human VH sequence; an amino acid sequence derived from or based
on a non-human DH sequence; and/or an amino acid sequence derived
from or based on a JH sequence. The ultralong CDR3 may comprise an
additional amino acid sequence comprising at least about two amino
acid residues positioned between the VH derived amino acid sequence
and the DH derived amino acid sequence.
[0167] Any of the antibodies disclosed herein may comprise a
sequence based on or derived from a sequence selected from SEQ ID
NOS: 24-44, the antibody or binding fragment thereof encoded by a
DNA sequence based on or derived from the DNA of SEQ ID NOS: 2-22.
Any of the antibodies disclosed herein may comprise a sequence
based on or derived from a sequence selected from SEQ ID NO: 23,
the antibody or binding fragment thereof encoded by a DNA sequence
based on or derived from the DNA of SEQ ID NO: 1.
[0168] Any of the ultralong CDR3s disclosed herein may comprise a
sequence based on or derived from a sequence selected from SEQ ID
NOS: 24-44. Any of the antibodies disclosed herein may comprise a
sequence based on or derived from a sequence selected from SEQ ID
NO: 23. Any of the ultralong CDR3s disclosed herein may be encoded
by a DNA sequence that may be derived from or based on SEQ ID NOS:
2-22. Any of the antibodies disclosed herein may comprise a portion
encoded by a DNA sequence that may be derived from or based on SEQ
ID NO: 1.
[0169] Any of the antibodies disclosed herein may comprise one or
more linkers. Any of the antibodies disclosed herein may comprise a
first linker sequence. Any of the antibodies disclosed herein may
comprise a second linker sequence. The first and second linker
sequences may comprise the same sequence. The first and second
linker sequences may comprise different sequences. The first and/or
second linker sequences may be the same length. The first and/or
second linker sequences may be different lengths. The first and/or
second linker sequences may be 3 or more amino acids in length.
[0170] The first and/or second linker sequence may attach the
non-antibody sequence to the portion based on or derived from the
portion of the ultralong CDR3. The first and/or second linker
sequences may attach the non-antibody sequence to the first
antibody sequence. The first and/or second linker sequences may
attach the non-antibody sequence to the second antibody sequence.
The first and/or second linker sequences may be adjacent to a
non-antibody sequence, a portion of an ultralong CDR3 sequence, a
cleavage site sequence, a non-bovine sequence, an antibody
sequence, or a combination thereof.
[0171] The first and/or second linker sequences may comprise one or
more glycine residues. The first and/or second linker sequences may
comprise two or more consecutive glycine residues. The first and/or
second linker sequences may comprise one or more serine residues.
The first and/or second linker sequence may comprise one or more
polar amino acid residues. The one or more polar amino acid
residues may be selected from serine, threonine, asparagine, or
glutamine. The polar amino acid residues may comprise uncharged
side chains. The first and/or second linker sequences may comprise
the sequence (GGGGS).sub.n, wherein n=1 to 5; the sequence
GGGSGGGGS; the sequence GGGGSGGGS; the sequence of (GSG)n, wherein
n is greater than or equal to one; or a combination thereof.
[0172] Any of the antibodies disclosed herein may comprise one or
more cleavage sites. The one or more cleavage sites may comprise a
recognition site for a protease. The protease may be a Factor Xa or
thrombin. The one or more cleavage sites may comprise an amino acid
sequence of IEGR.
[0173] The one or more cleavage site may be between a first
antibody sequence and the non-antibody sequence. The one or more
cleavage sites may be between a second antibody sequence and the
non-antibody sequence. The one or more cleavage sites may be
between the one or more linkers and the non-antibody sequence. The
one or more cleavage sites may be between a first antibody sequence
and the one or more linkers. The one or more cleavage sites may be
between a second antibody sequence and the one or more linkers. The
one or more cleavage sites may be adjacent to a non-antibody
sequence, a portion of an ultralong CDR3 sequence, a linker
sequence, an antibody sequence, or a combination thereof.
[0174] In some embodiments is library of antibodies or binding
fragments thereof, wherein the antibodies or binding fragments
thereof may comprise an ultralong CDR3.
[0175] In some embodiments is library of antibodies or binding
fragments thereof, wherein the antibodies or binding fragments
thereof may comprise any of the antibodies disclosed herein.
[0176] In some embodiments is nucleic acid library comprising a
plurality of polynucleotides comprising sequences coding for
antibodies or binding fragments thereof, wherein the antibodies or
binding fragments thereof may comprise an ultralong CDR3.
[0177] In some embodiments is nucleic acid library comprising a
plurality of polynucleotides comprising sequences coding for
antibodies or binding fragments thereof, wherein the antibodies or
binding fragments thereof may comprise any of the antibodies
disclosed herein.
[0178] In some embodiments is polynucleotide comprising a nucleic
acid sequence that encodes a variable region, wherein the variable
region may comprise an ultralong CDR3.
[0179] In some embodiments is vector comprising a polynucleotide,
wherein the polynucleotide comprises a nucleic acid sequence that
encodes a variable region, wherein the variable region may comprise
an ultralong CDR3.
[0180] In some embodiments is host cell comprising a
polynucleotide, wherein the polynucleotide comprises a nucleic acid
sequence that encodes a variable region, wherein the variable
region may comprise an ultralong CDR3.
[0181] In some embodiments is polynucleotide comprising a nucleic
acid sequence that encodes the antibody or binding fragment thereof
of any of the antibodies disclosed herein.
[0182] In some embodiments is vector comprising a polynucleotide,
wherein the polynucleotide comprises a nucleic acid sequence that
encodes the antibody or binding fragment thereof of any of the
antibodies disclosed herein.
[0183] In some embodiments is host cell comprising a
polynucleotide, wherein the polynucleotide comprises a nucleic acid
sequence that encodes the antibody or binding fragment thereof of
any of the antibodies disclosed herein.
[0184] In some embodiments is method of producing an antibody or
binding fragment thereof comprising an ultralong CDR3 or fragment
thereof comprising culturing a host cell comprising a
polynucleotide, wherein the polynucleotide may comprise a nucleic
acid sequence that encodes the antibody or binding fragment thereof
of any of the antibodies disclosed herein under conditions wherein
the polynucleotide sequence may be expressed and the antibody or
binding fragment thereof comprising an ultralong CDR3 or fragment
thereof may be produced. The method may comprise recovering the
antibody or binding fragment thereof comprising the ultralong CDR3
or fragment thereof from the host cell culture.
[0185] In some embodiments is pharmaceutical composition comprising
any of the antibodies disclosed herein. The pharmaceutical
compostion may comprise two or more antibodies, wherein at least
one of the two or more antibodies comprises at least a portion of
an ultralong CDR3.
[0186] In some embodiments is pharmaceutical composition comprising
(a) an antibody or fragment thereof comprising sequence based on or
derived from at least a portion of an ultralong CDR3; and (b) a
pharmaceutically acceptable excipient.
[0187] In some embodiments is method of treating a disease or
condition in a subject in need thereof comprising administering to
the mammal a therapeutically effective amount of any of the
antibodies disclosed herein. In some instances, the antibodies
disclosed herein comprise an ultralong CDR3 sequence and a
non-antibody sequence. In some instances, the non-antibody sequence
is selected from the group comprising Moka1, Vm24, human GLP-1,
Exendin-4, beta-interferon, human EPO, human FGF21, human GMCSF,
human interferon-beta, bovine GCSF, human GCSF, be IL8, ziconotide,
somatostatin, chlorotoxin, SDF1(alpha), IL21 and a derivative or
variant thereof. The non-antibody sequence may comprise an amino
acid sequence based on or derived from any of SEQ ID NOS:
317-332.
[0188] The disease or condition may be selected from the group
comprising autoimmune disease, heteroimmune disease or condition,
inflammatory disease, pathogenic infection, thromboembolic
disorder, respiratory disease or condition, metabolic disease,
central nervous system (CNS) disorder, bone disease, cancer, blood
disorder, obesity, diabetes, osteoporosis, anemia, or pain.
[0189] The disease or condition would benefit from the modulation
of an ion channel. The ion channel may be selected from the group
comprising a potassium ion channel, sodium ion channel, or acid
sensing ion channel. The ion channel may be selected from the group
comprising Kv1.3 ion channel, Nav1.7 ion channel and acid sensing
ion channel (ASIC).
[0190] The disease or condition would benefit from the modulation
of a receptor. The receptor may be selected from the group
comprising GLP1R, GCGR, EPO receptor, FGFR, FGF21R, CSFR, GMCSFR,
IL8R, IL21R and GCSFR.
[0191] The disease or condition may be mastitis.
[0192] The subject may be a mammal. The mammal may be a bovine or
human.
[0193] In some embodiments are crystals based on or derived from
the antibodies disclosed herein. The crystals may have a space
group P2.sub.12.sub.12.sub.1. In some instances, the crystal has
the unit cell dimensions of "a" between about 40 to 80 angstroms,
between 45 to about 75 angstroms, or between about 50 to about 75
angstroms; "b" between about 40 to 140 angstroms, between about 50
to about 130 angstroms, between about 55 to about 130 angstroms;
and "c" between 100 to about 350 angstroms, between 120 to about
340 angstroms, or between about 125 to about 330 angstroms. The
crystal may comprise a bovine antibody or portion thereof. The
crystal may comprise a Fab fragment based on or derived from a
bovine antibody. The crystal may be an isolated crystal.
[0194] In some embodiments, is an isolated crystal comprising a
bovine antibody Fab fragment comprising SEQ ID NO: 24 and SEQ ID
NO: 23, wherein the crystal has a space group
P2.sub.12.sub.12.sub.1 and unit cell dimensions of a=71.4
angstroms, b=127.6 angstroms and c=127.9 angstroms.
[0195] In some embodiments, is an isolated crystal comprising a
bovine antibody Fab fragment comprising SEQ ID NO: 340 and SEQ ID
NO: 341, wherein the crystal has a space group
P2.sub.12.sub.12.sub.1 and unit cell dimensions of a=54.6
angstroms, b=53.7 angstroms and c=330.5 angstroms.
[0196] In any or all of the above or below disclosure (e.g.,
antibodies, uses, or methods) or embodiments utilizing an antibody
comprising an ultralong CDR3, any antibody comprising an ultralong
CDR3 may be used including, for example, any of the above mentioned
antibodies comprising an ultralong CDR3.
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. 1A-C present the identification of a new structural
domain in bovine antibodies. FIG. 1A shows a comparison of CDR H3
length amongst murine, human, and bovine repertoires. An ultralong
subset of over 60 amino acids is uniquely found in bovine heavy
chains. FIG. 1B shows sequences of representative CDR H3 from
murine (mu), human (hu), or bovine sequences from the literature
along with six bovine sequences (B-S1 to B-S4, and B-L1 and B-L2)
from our deep sequencing results. The conserved cysteine of
framework 3 and tryptophan of framework 4 that define CDR H3
boundaries in all antibody variable regions are shaded in grey for
reference. The lengths of the CDR H3s are indicated at the right.
The murine antibodies include D44.1, an anti-HEL antibody, 93F3, an
aldolase, and OKT3, a therapeutic antibody targeting human CD3.
This antibody is unusual in having a free cysteine in CDR H3. The
human antibodies include Yvo, a cryoglobulin, CR6261, an
anti-influenza A hemaglutinin, and PG9, an anti-HIV antibody which
has one of the longest human CDR H3s. The bovine antibodies
represent the ultralong sequences in the literature, and short
sequences for comparison. BLV5B8 and BLV1H12 (indicated in bold)
were used in our structure determinations. Relatively conserved
TTVHQ and CPDG motifs are in bold. FIG. 1C shows crystal structures
of BLV1H12 (left) and BLV5B8 (middle) Fabs compared to the 93F3 Fab
with a "normal" CDR H3 (right). A superlong, two .beta.-stranded
stalk protrudes from each bovine V.sub.H immunoglobulin domain and
terminates in an unusual three disulfide-linked knob domain.
[0199] FIG. 2A-C present the structural diversity in ultralong
bovine antibodies. FIG. 2A shows a comparison of the structure of
the two knobs showing differences in disulfide patterns. Close up
views of the knobs of BLV1H12 (left) and BLV5H8 (right) are shown,
in addition to a two-dimensional representation of the knob and its
disulfide pattern. Disulfides are in numbered and circled. The
sequences of the knob regions are shown below, with cysteines
conserved with the D.sub.H2 germline gene segment underlined. The
disulfide pattern is indicated above each sequence. FIG. 2B shows
an overlay of the variable regions of BLV1H12 and BLV5B8 shows
structural homology in the variable regions except the upper part
of the stalk and knob, which are significantly divergent. FIG. 2C
shows surface and charge density representation of BLV1H12 (left)
and BLV5B8 (right) showing different shapes and charge in the knob
region.
[0200] FIG. 3A-C present the genetic basis for ultralong antibody
formation. FIG. 3A shows the identification of V.sub.HBUL, a
germline variable region used in ultralong antibodies. The leader
sequence is in light grey, coding sequence is indicated with the
amino acid translation above, the intron is in italics, and the
unique TTVHQ extension, which forms a portion of the ascending
strand of the stalk is in bold. The recombination signal sequence
heptamer and nonamer are underlined. FIG. 3B shows the V.sub.HBUL
region is found on chromosome 21. Partial cattle metaphase spread
(left) and enlarged chromosome 21 (top right) showing the location
of V.sub.HBUL region in BTA21q24 by two-color FISH with BAC clones
318H2 and 14-74H6. International nomenclature for BTA21 is depicted
at the bottom. FIG. 3C shows a schematic of the bovine
immunoglobulin loci depicting V.sub.HBUL, D.sub.H2, and
V.sub..lamda.1x, which are preferentially used in ultralong
antibodies. The process of V(D)J recombination assembles the gene
segments to form functional ultralong heavy and light chain genes.
(bottom left) The V-D-J regions mapped onto the BLV1H12 Fab
structure. V.sub.HBUL is unique in encoding CDR H1 and CDR5H2
residues that interact with the stalk, as well as a TTVHQ motif
that initiates the ascending .beta.-strand. Similarly, the
V.sub..lamda.1x light chain encodes CDR L1 and CDR L2 residues that
interact with the stalk. Arrows indicate areas of potential
junctional diversity. Relatively long V-D insertions are indicated
in purple. It is unclear whether this sequence results from
N-additions, gene conversion, or another mechanism. (bottom right)
A detailed depiction of the interactions of CDR H1, H2, L1, and L2
with the stalk of BLV1H12, as well as the location of the YxYxY
motif of the descending strand.
[0201] FIG. 4A-C depict deep sequence diversity of bovine ultralong
V.sub.H CDR H3s. FIG. 4A shows the distribution of the number of
cysteines in bovine ultralong CDR H3s of IgM and IgG. FIG. 4B shows
the length distribution of ultralong CDR H3s. Note that clonal
sequences selected during an immune response can bias the
proportion at any given length. FIG. 4C shows representative
sequences of ultralong bovine V.sub.H CDR H3s. The terminal portion
of the V.sub.HBUL region is shown, along with junctional diversity
at the V-D joint, D.sub.H2 and J.sub.H (top). The sequences of
BLVH12 and BLV5B8 are shown for comparison, followed by 20
ultralong CDR H3 sequences (bottom). Cysteines conserved with
D.sub.H2 are underlined. The conserved cysteine and tryptophan that
define the CDR H3 boundaries in all antibody variable regions are
highlighted in grey for reference. The CPDG motif is underlined in
grey and the region of the descending strand encoding a YxYxY motif
is underlined in grey.
[0202] FIG. 5A-C show that cysteine mutations contribute to
ultralong CDR H3 diversity. FIG. 5A shows that the consensus of
ultralong CDR H3 deep sequences aligns with D.sub.H2. A consensus
sequence for three deep sequencing runs (from two cows) were
determined, and aligned with one another and with D.sub.H2. The
consensus aligns well except for some areas of
insertions/deletions. Thus, either a single D.sub.H gene, or highly
related genes, produce the diversity of sequences in ultralong CDR
H3 antibodies. FIG. 5B shows that the D.sub.H2 gene region analysis
showing residues that can readily mutate to cysteine, including SH
hotspots. The nucleotide sequence is above and translated
amino-acid sequence below. RGYW hotspots, which are recognized by
AID for SH and/or gene conversion, are boxed. Nucleotides at
positions 3, 15, 19, 21, 25, 27, 31, 33, 39, 43, 45, 49, 51, 57,
60, 64, 73, 75, 79, 81, 84, 88, 90, 93, 97, 102, 106, 112, 117,
121, 123, 127, 129, 133, 139, and 145 in (B) can be altered in a
single mutation to a cysteine-encoding codon. FIG. 5C shows
affinity maturation groups show mutation to and from cysteine.
Several groups of clonally related sequences were identified and
analyzed for somatic hypermutation. Three groups are shown as
examples (labeled 1 to 3 on the left). Sequence differences from
cysteine are highlighted in grey. The number of times each sequence
is represented in the cluster is shown at the right.
[0203] FIG. 6A-D show bovine antibodies with ultralong CDR H3s bind
antigen. FIG. 6A shows immunization experimental scheme for
identifying antigen-specific, ultralong CDR H3 antibodies. Heavy
chain variable region mRNA was isolated, amplified by RT PCR, and
paired with the invariant light chain to produce a small library of
IgG produced in HEK293 cells. FIG. 6B shows ELISA of 132 ultralong
CDR H3 antibodies against BVDV (left), and binding activity of the
"hits" B8, B9, and H12 in a titration assay (right). FIG. 6C shows
the sequences of B8, H9, and H12 in comparison to BLV1H12 and the
germline D.sub.H2 region. Lengths (L) of the CDR H3 are indicated
at the right. Cysteines conserved with D.sub.H2 are underlined.
FIG. 6D shows that H12 binds NS2-3 on cells. A flag-tagged BVDV
NS2-3 protein construct was transfected into HEK293A cells and
stained with anti-Flag as a positive control (left), the H12
antibody (middle), and B8 (right). Binding assays with
untransfected cells are shown on the bottom.
[0204] FIG. 7A-B depict a model for ultralong CDR H3
diversification into novel minifolds. FIG. 7A shows a schematic of
the D.sub.H2 knob with 4 cysteines is shown on the left, with SH
and/or gene conversion leading to a multitude of new cysteine
patterns and new loops on the right. FIG. 7B shows mechanisms for
generating antibody diversity. In humans and mice (left),
combinatorial diversity through V(D)J recombination and
V.sub.H-V.sub.L pairing creates a multitude of different binding
sites, which are further optimized following antigen exposure by
somatic hypermutation. In cows (right), combinatorial diversity is
severely limited; however, somatic mutation to and from cysteines
can reshape the "knob" region, creating substantial structural
diversity in ultralong CDR H3s. These antibodies may be further
optimized through SH and may bind unique targets such as pores or
channels.
[0205] FIG. 8A-J depict schemes showing attachment of bovine G-CSF
onto the knob domain of a heavy chain region of bovine BLV1H12
antibody to design an immunoglobulin construct described herein.
FIG. 8A shows a ribbon diagram of a heavy chain region and light
chain region of bovine BLV1H12 antibody. The boxed region
highlights the extended region of the ultralong CDR3 comprising the
stalk and knob domain. FIG. 8B shows a ribbon diagram of a growth
factor (e.g., bovine G-CSF) inserted into or replacing a portion of
the knob domain of a heavy chain region of a bovine BLV1H12
antibody. FIG. 8C shows a ribbon diagram of a toxin (e.g., Moka1,
VM-24) inserted into or replacing a portion of the knob domain of a
heavy chain region of a bovine BLV1H12 antibody. FIG. 8D shows a
ribbon diagram of a receptor ligand (e.g., GLP-1, exendin-4)
inserted into or replacing a portion of the knob domain of a heavy
chain region of a bovine BLV1H12 antibody. FIG. 8E shows a ribbon
diagram of a hormone (e.g., erythropoietin) inserted into or
replacing a portion of the knob domain of a heavy chain region of a
bovine BLV1H12 antibody. FIG. 8F shows a cartoon depicting a heavy
chain region and light chain region of bovine BLV1H12 antibody.
FIG. 8G shows a cartoon of a growth factor (e.g., bovine G-CSF)
inserted into or replacing a portion of the knob domain of a heavy
chain region of a bovine BLV1H12 antibody. FIG. 8H shows a cartoon
of a toxin (e.g., Moka1, VM-24) inserted into or replacing a
portion of the knob domain of a heavy chain region of a bovine
BLV1H12 antibody. FIG. 8I shows a cartoon of a receptor ligand
(e.g., GLP-1, exendin-4) inserted into or replacing a portion of
the knob domain of a heavy chain region of a bovine BLV1H12
antibody. The left panel shows both ends of the receptor ligand
inserted into or replacing a portion of the knob domain; the right
panel shows the N-terminus of the receptor ligand released from the
knob domain after treatment with a protease. FIG. 8J shows a
cartoon of a hormone (e.g., erythropoietin) inserted into or
replacing a portion of the knob domain of a heavy chain region of a
bovine BLV1H12 antibody. Said attachment of the growth factor,
toxin, receptor ligand, and hormone can be by means of a
polypeptide linker of sequence GGGGS (Ab-peptide L1) or GGGSGGGGS
and GGGGSGGGS (Ab-peptide L2). Another construct (Ab-peptide L0) in
which attachment is not by means of a linker is not shown in this
cartoon.
[0206] FIG. 9A-9E present illustrative mouse NFS-60 cell
proliferative activity for the Ab-bGCSF fusion proteins. FIG. 9A-9B
depict proliferative activity of bovine G-CSF and human G-CSF
respectively. FIG. 9C depicts the lack of proliferative activity
for bovine BLV1H12 antibody in the absence of G-CSF. FIG. 9D
depicts proliferative activity of bovine G-CSF inserted into or
replacing a portion of the knob domain in the absence of a linker
(Ab-bGCSF L0). FIG. 9E depicts proliferative activity of bovine
G-CSF inserted into or replacing a portion of the knob domain by
means of a polypeptide linker of sequence GGGGS (Ab-bGCSF L1).
[0207] FIG. 10A-10E present human granulocyte progenitor cell
proliferative activities of the Ab-GCSF fusion proteins. FIG.
10A-10B depict proliferative activity of bovine G-CSF and human
G-CSF respectively. FIG. 10C depicts the lack of proliferative
activity for bovine BLV1H12 antibody (Ab) in the absence of G-CSF.
FIG. 10D depicts proliferative activity of bovine G-CSF inserted
into or replacing a portion of the knob domain in the absence of a
linker (Ab-bGCSF L0). FIG. 10E depicts proliferative activity of
bovine G-CSF inserted into or replacing a portion of the knob
domain, said attachment by means of a polypeptide linker of
sequence GGGGS (Ab-bGCSF L1).
[0208] FIG. 11A-11B depict pharmacokinetics of Ab-bGCSF fusion
proteins in mice.
[0209] FIG. 12A-12B provide Proliferative activities of Ab-bGCSF
fusion proteins on mice neutrophils that are blood stained and
counted at the 10th day post-injection.
[0210] FIG. 13A-13C display expression and purification of Ab-bGCSF
fusion proteins in Pichia pastoris. FIG. 13A shows a map of Pichia
expression vector for an immunoglobulin construct provided herein.
FIG. 13B provides a western blot post-induction of expression of
the immunoglobulin constructs Ab-bGCSF L0 and Ab-bGCSF L1. FIG. 13C
provides SDS-PAGE gel of purified Ab-bGCSF L0 and Ab-bGCSF L1
expressed in Pichia at a yield of .about.70 .mu.g per 100 mL
culture.
[0211] FIG. 14A-14C show vectors for expression of the
immunoglobulin constructs described herein in free style HEK 293
cells. FIG. 14A provides a vector of Ab-bGCSF L0 heavy chain for
expression in free style HEK 293 cells. FIG. 14B provides a vector
of Ab-bGCSF L1 heavy chain for expression in free style HEK 293
cells. FIG. 14C provides a vector of Ab-bGCSF light chain for
expression in free style HEK 293 cells.
[0212] FIG. 15A shows SDS-PAGE gel of purified antibody fusions.
FIG. 15A SDS-PAGE gel of purified Ab-bGCSF L0 and Ab-bGCSF L1
proteins from HEK 293 cells. FIG. 15B provides a SDS PAGE of the
immunoglobulin constructs Ab-Protoxin2 comprising a GGGGS linker
attached to both ends of protoxin2.
[0213] FIG. 16A-16B provide SDS PAGE and activities of the
immunoglobulin constructs Ab-Moka1 L0 (no linker) and Ab-Moka1 L1
(linker). FIG. 16A provides a SDS PAGE of the immunoglobulin fusion
proteins Ab-Moka1 L0 and Ab-Moka1 L1. FIG. 16B provides
BLV1H12-Moka1 fusion proteins inhibitory activities on T cells
activation
[0214] FIG. 17 provides BLV1H12-Moka1 fusion proteins inhibitory
activities on human peripheral blood mononuclear cells (PBMCs)
[0215] FIG. 18A-18C provide SDS PAGE and activities of the
immunoglobulin constructs Ab-VM24 L1 (GGGGS linker) and Ab-VM24 L2
(GGGSGGGGS and GGGGSGGGS linkers). FIG. 18A provides a SDS PAGE of
the immunoglobulin fusion proteins Ab-VM24 L1 and Ab-VM24 L2. FIG.
18B provides BLV1H12-VM24 L1 fusion protein inhibitory activities
on T cells activation. FIG. 18C provides BLV1H12-VM24 L2 fusion
protein inhibitory activities on T cells activation.
[0216] FIG. 19 provides a SDS PAGE of the immunoglobulin constructs
Ab-GLP-1 and Ab-Exendin-4.
[0217] FIG. 20 provides activity of Ab-GLP-1 and Ab-Ex4 on HEK293
cells expressing GLP-1 receptor.
[0218] FIG. 21 provides proliferative activity of Ab-hEPO fusion
proteins on TF1 cells.
[0219] FIG. 22A-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.
Moving from left to right, the first segment is derived from the
V.sub.H germline and is represented throughout the disclosure as a
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5 motif. The second segment is a
string of spacer amino acid residues designated throughout the
disclosure as X.sub.n residues. The third segment is a string of
amino acid residues derived from the germline D.sub.H2 region and
the fourth segment is a string of amino acid residues derived from
the germline J.sub.H1 region.
DETAILED DESCRIPTION
[0220] Disclosed herien are antibodies and fragments thereof.
Generally, the antibodies and fragments thereof comprise at least a
portion of an ultralong CDR3. The portion of the ultralong CDR3 may
be derived from or based on an ultralong CDR3 sequence. The portion
of the ultralong CDR3 may be derived from or based on a stalk
domain of an ultralong CDR3 sequence. Alternatively, or
additionally, the portion of the ultralong CDR3 may be derived from
or based on a knob domain of an ultralong CDR3 sequence. The
antibodies and fragments thereof may further comprise one or more
therapeutic polypeptides. The therapeutic polypeptides may be
inserted into the portion of the ultralong CDR3. The therapeutic
polypeptides may replace one or more amino acid residues in the
amino acid sequence of the portion of the ultralong CDR3. The
therapeutic polypeptides may replace one or more nucleotides in the
nucleic acid sequence of the portion of the ultralong CDR3.
Alternatively, the therapeutic polypeptides may be conjugated or
attached to the portion of the ultralong CDR3. The antibodies and
fragments disclosed herein may further comprise one or more
linkers. Additionally, the antibodies and fragments disclosed
herein further comprise a cleavage site. A portion of the
antibodies and fragments disclosed herien may be based on or
derived from an antibody sequence from a different animal or specie
from with the ultralong CDR3 is derived. For example, the ultralong
CDR3 may be derived from or based on a bovine antibody sequence and
the additional and another portion of the antibody sequence may be
derived from or based on a non-bovine antibody sequence. Further
details of the antibodies and fragments thereof are described
herein.
Ultralong CDR3Proteins
[0221] The present disclosure provides antibodies or immunoglobulin
constructs comprising ultralong CDR3 sequences or portions
thereof.
[0222] In an embodiment, the present disclosure provides an
antibody comprising an ultralong CDR3. The ultralong CDR3 may be 35
amino acids in length or more (e.g., 40 or more, 45 or more, 50 or
more, 55 or more, 60 or more). The ultralong CDR3 may comprise at
least a portion of a knob domain of a CDR3, at least a portion of a
stalk domain of a CDR3, or a combination thereof. The portion of
the knob domain of the CDR3 may comprise one or more conserved
motifs derived from the knob domain of the ultralong CDR3. The
portion of the stalk domain of the CDR3 may comprise one or more
conserved motis derived from the stalk domain of the ultralong
CDR3. Such an 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. The antibody may comprise one or more cysteine motifs. The
antibody may comprise a non-antibody sequence within the ultralong
CDR3. Alternatively, or additionally, the antibody comprises a
non-bovine sequence. The non-bovine sequence can be linked to the
ultralong CDR3 sequence. The antibody may further comprise a
linker. The linker can comprise an amino acid sequence of
(GGGGS).sub.n wherein n=1 to 5. Alternatively, the linker comprises
an amino acids sequence of (GSG).sub.n, GGGSGGGGS or GGGGSGGGS. The
antibody may comprise a non-bovine sequence within the ultralong
CDR3. The antibody may further comprise an antibody sequence,
wherein the antibody sequence does not comprise an ultralong CDR3
sequence. The antibody may further comprise an antibody sequence,
wherein the amino acid sequence identitity of the antibody peptide
sequence to the ultralong CDR3 peptide sequence is about 40% or
less (e.g., about 35% or less, about 30% or less, about 25% or
less, about 20% or less, about 15% or less, 10% or less, about 5%
or less, about 3% or less, about 1% or less). The antibody may
comprise a cytotoxic agent or therapeutic polypeptide. The
cytotoxic agent or therapeutic polypeptide may be conjugated to the
ultralong CDR3. The antibody may bind to a target. The target may
be a protein target. The protein target may be a transmembrane
protein target. The antibody may comprise at least a portion of a
BLV1H12 and/or BLVCV1 antibody. Alternatively, or additionally, the
antibody comprises at least a portion of a BLV5D3, BLV8C11, BF1H1,
BLV5B8 and/or F18 antibody. The antibody may comprise at least a
portion of a human antibody. The antibody may be a chimeric,
recombinant, engineered, synthetic, humanized, fully human, or
human engineered antibody. The antibody may comprise antibody
sequences from two or more different antibodies. The two or more
different antibodies may be from the same species. For example, the
specie may be a bovine specie, human specie, or murine specie. The
two or more different antibodies may be from the same type of
animal. For example the two or more different antibodies may be
from a cow. The two or more different antibodies may be from a
human. Alternatively, the two or more different antibodies are from
different species. For example, the two or more different
antibodies are from a human specie and bovine specie. In another
example, the two or more diffent antibodies are from a bovine
specie and a non-bovine specie. In another example, the two or more
different antibodies are from a human specie and a non-human
specie.
[0223] In another embodiment, the present disclosure provides an
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). The antibody may
comprise at least a portion of a BLV1H12 and/or BLVCV1 antibody.
Alternatively, or additionally, the antibody comprises at least a
portion of a BLV5D3, BLV8C11, BF1H1, BLV5B8 and/or F18 antibody.
Alternatively, the ultralong CDR3 sequence may be derived from a
camelid or shark CDR3 sequence.
[0224] In another embodiment, the present disclosure provides an
antibody comprising an ultralong CDR3, wherein the CDR3 comprises 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 derived
from a therapeutic polypeptide. The non-antibody sequence may be of
human or non-human origin. The non-antibody sequence may comprise a
synthetic sequence. The non-antibody sequence 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 antibody. The CDR3 may be 35 amino acids in
length or more. The ultralong CDR3 may comprise at least a portion
of a knob domain of a CDR3, at least a portion of a stalk domain of
a CDR3, or a combination thereof. The portion of the knob domain of
the CDR3 may comprise one or more conserved motifs derived from the
knob domain of the ultralong CDR3. The portion of the stalk domain
of the CDR3 may comprise one or more conserved motis derived from
the stalk domain of the ultralong CDR3. Alternatively, or
additionally, the antibody comprises at least 3 cysteine residues
or more. The antibody can comprise one or more cysteine motifs. The
antibody may comprise at least a portion of a BLV1H12 and/or BLVCV1
antibody. Alternatively, or additionally, the antibody comprises at
least a portion of a BLV5D3, BLV8C11, BF1H1, BLV5B8 and/or F18
antibody.
[0225] In another embodiment, the present disclosure provides an
antibody comprising an ultralong CDR3 and a non-bovine sequence.
The ultralong CDR3 can be derived from a ruminant. The ruminant can
be a bovine. The non-bovine sequence can be derived from or based
on a non-bovine mammal sequence. For example, the non-bovine
sequence can be derived from or based on a human, mouse, rat,
sheep, dog, and/or goat sequence. The ultralong CDR3 sequence can
comprise the non-bovine sequence. Alternatively, the non-bovine
sequence is linked to the ultralong CDR3 sequence. The non-bovine
sequence can be derived from or based on at least a portion of an
antibody sequence. The antibody sequence can encode a variable
region, constant region or a combination thereof. The CDR3 may be
35 amino acids in length or more. The ultralong CDR3 may comprise
at least a portion of a knob domain of a CDR3, at least a portion
of a stalk domain of a CDR3, or a combination thereof. The portion
of the knob domain of the CDR3 may comprise one or more conserved
motifs derived from the knob domain of the ultralong CDR3. The
portion of the stalk domain of the CDR3 may comprise one or more
conserved motis derived from the stalk domain of the ultralong
CDR3. Alternatively, or additionally, the antibody comprises at
least 3 cysteine residues or more. The antibody can comprise one or
more cysteine motifs.
[0226] In another embodiment, the present disclosure provides an
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.
[0227] In another embodiment, the present disclosure provides for
an 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.
[0228] In another embodiment, the present disclosure provides an
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.
[0229] In another embodiment, the present disclosure provides an
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 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.
[0230] In another embodiment, the present disclosure provides an
antibody comprising an ultralong CDR3, wherein the 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.
The CDR3 may be 35 amino acids in length or more. The ultralong
CDR3 may comprise at least a portion of a knob domain of a CDR3, at
least a portion of a stalk domain of a CDR3, or a combination
thereof. The portion of the knob domain of the CDR3 may comprise
one or more conserved motifs derived from the knob domain of the
ultralong CDR3. The portion of the stalk domain of the CDR3 may
comprise one or more conserved motis derived from the stalk domain
of the ultralong CDR3. Alternatively, or additionally, the antibody
comprises at least 3 cysteine residues or more. The antibody can
comprise one or more cysteine motifs.
[0231] Provided herein is an immunoglobulin construct comprising a
mammalian immunoglobulin heavy chain comprising at least a portion
of complementarity-determining region 3 (CDR3H); and a therapeutic
polypeptide, wherein the therapeutic polypeptide is inserted into
or replaces at least a portion of the CDR3H. The immunoglobulin
construct may comprise one or more linkers. The one or more linkers
can connect the therapeutic polypeptide to the heavy chain. In some
embodiments, the linker comprises an amino acid sequence of
(GGGGS)n wherein n=1 to 5. Alternatively, or additionally, the
linker comprises an amino acid acid sequence of (GSG)n (SEQ ID NO:
342), GGGSGGGGS (SEQ ID NO: 337) or GGGGSGGGS. In some embodiments
provided are immunoglobulin constructs described herein, wherein
the therapeutic polypeptide is selected from a hormone, a
lymphokine, an interleukin, a chemokines, a cytokine and
combinations thereof. In certain embodiments, the therapeutic
polypeptide is a cytokine. In some embodiments, the therapeutic
polypeptide is a colony stimulating factor polypeptide. In certain
embodiments, the colony stimulating factor is macrophage
colony-stimulating factor (M-C SF), Granulocyte-macrophage
colony-stimulating factor (GM-CSF), Granulocyte colony-stimulating
factor (G-CSF) or fragment, or variant thereof. In an embodiment,
the colony stimulating factor is mammalian G-CSF or derivative or
variant thereof. In a certain embodiment, the colony stimulating
factor is bovine G-CSF or derivative or variant thereof. In other
instances, the therapeutic polypeptide is Moka1, Vm24, human GLP-1,
Exendin-4, human EPO, human FGF21, human GMCSF, or human
interferon-beta. Provided herein are immunoglobulin constructs
comprising a mammalian immunoglobulin heavy chain comprising a knob
domain in the complementarity-determining region 3 (CDR3H) or
fragment thereof; and a therapeutic polypeptide attached to said
knob domain of the CDR3H, wherein said mammalian immunoglobulin is
a bovine immunoglobulin. In some embodiments, the bovine
immunoglobulin is a BLV1H12 antibody. In some embodiments of the
immunoglobulin constructs described herein, at least a portion of
the knob domain is replaced by the therapeutic polypeptide. The
knob domain of the CDR3H may comprise one or more conserved motifs
derived from the knob domain of the ultralong CDR3H. The
immunoglobulin construct may further comprise at least a portion of
a stalk domain in the CDR3H. The portion of the stalk domain of the
CDRH3 may comprise one or more conserved motis derived from the
stalk domain of the ultralong CDR33H.
[0232] Further provided herein are antibodies or fragments thereof
comprising a stalk domain in the complementarity-determining region
3 (CDR3H) or fragment thereof; and a therapeutic polypeptide. In
some instances, the complementarity-determining region 3 (CDR3H) is
derived from a bovine ultralong CDR3H. The therapeutic polypeptide
can be any of the therapeutic polypeptides disclosed herein. For
example, the therapeutic polypeptide is Moka1, Vm24, GLP-1,
Exendin-4, human EPO, human FGF21, human GMCSF, or human
interferon-beta. The therapeutic polypeptide can be attached to the
stalk domain. In some instances, the antibody or fragment thereof
further comprises a linker. The linker can attach the therapeutic
polypeptide to the stalk domain. Alternatively, or additionally,
the antibody or fragment thereof further comprises at least a
portion of a knob domain in the CDR3H. In some instances, the
linker attaches the therapeutic polypeptide to the knob domain. In
some instances, the knob domain is attached to the stalk domain.
The portion of the knob domain of the CDR3 may comprise one or more
conserved motifs derived from the knob domain of the ultralong
CDR3. The stalk domain of the CDR3 may comprise one or more
conserved motis derived from the stalk domain of the ultralong
CDR3.
[0233] In some instances, an antibody or fragment thereof is
provided herein. The antibody or fragment thereof can comprise at
least one immunoglobulin domain or fragment thereof; and a
therapeutic polypeptide or derivative or variant thereof. The
therapeutic polypeptide, or derivative or variant thereof can be
attached to the immunoglobulin domain. In some instances, the
therapeutic polypeptide is Moka1, Vm24, GLP-1, Exendin-4, human
EPO, human FGF21, human GMCSF, human interferon-beta, or derivative
or variant thereof. In some embodiments, the immunoglobulin domain
is an immunoglobulin A, an immunoglobulin D, an immunoglobulin E,
an immunoglobulin G, or an immunoglobulin M. The immunoglobulin
domain can be an immunoglobulin heavy chain region or fragment
thereof. In some instances, the immunoglobulin domain is from a
mammalian antibody. Alternatively, the immunoglobulin domain is
from a chimeric antibody. In some instances, the immunoglobulin
domain is from an engineered antibody or recombinant antibody. In
other instances, the immunoglobulin domain is from a humanized,
human engineered or fully human antibody. In certain embodiments,
the mammalian antibody is a bovine antibody. In other instances,
the mammalin antibody is a human antibody. In other instances, the
mammalian antibody is a murine antibody. In some instances, the
immunoglobulin domain is a heavy chain region comprising a knob
domain in the complementarity-determining region 3 (CDR3H) or
fragment thereof. The therapeutic polypeptide can be attached to
the knob domain. Alternatively, or additionally, the immunoglobulin
domain is a heavy chain region comprising a stalk domain in the
complementarity-determining region 3 (CDR3H) or fragment thereof.
In some instances, the therapeutic polypeptide is attached to the
stalk domain. In some instances, the antibody or fragment thereof
further comprises a linker. The linker can attach the therapeutic
polypeptide to the immunoglobulin domain or fragment thereof. The
knob domain of the CDR3 may comprise one or more conserved motifs
derived from the knob domain of the ultralong CDR3. The stalk
domain of the CDR3 may comprise one or more conserved motis derived
from the stalk domain of the ultralong CDR3.
[0234] Provided herein is an immunoglobulin construct comprising at
least one immunoglobulin domain or fragment thereof; and a G-CSF
polypeptide or derivative or variant thereof attached to said
immunoglobulin domain. In some embodiments, the immunoglobulin
domain is an immunoglobulin A, an immunoglobulin D, an
immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. In
some embodiments, the immunoglobulin domain is an immunoglobulin
heavy chain region or fragment thereof. In an embodiment, the
immunoglobulin domain is from a mammalian or chimeric antibody. In
other instances, the immunoglobulin domain is from a humanized,
human engineered or fully human antibody. In certain embodiments,
the mammalian antibody is a bovine antibody. In some instances, the
mammalian antibody is a human antibody. In other instances, the
mammalian antibody is a murine antibody. In an embodiment, the
immunoglobulin domain is a heavy chain region comprising a knob
domain in the complementarity-determining region 3 (CDR3H) or
fragment thereof. In an embodiment, the G-CSF polypeptide is
attached to the knob domain. The immunoglobulin domain may be a
heavy chain region comprising a stalk domain in the
complementarity-determining region 3 (CDR3H) or fragment thereof.
The G-CSF polypeptide may be attached to the stalk domain. The knob
domain of the CDR3 may comprise one or more conserved motifs
derived from the knob domain of the ultralong CDR3. The stalk
domain of the CDR3 may comprise one or more conserved motis derived
from the stalk domain of the ultralong CDR3.
[0235] In certain embodiments, provided is an immunoglobulin
construct comprising at least one immunoglobulin domain or fragment
thereof; and a G-CSF polypeptide or derivative or variant thereof
attached to said immunoglobulin domain, wherein said G-CSF
polypeptide is a bovine G-CSF polypeptide or derivative or variant
thereof. In certain embodiments provided herein is a pharmaceutical
composition comprising an immunoglobulin construct provided herein,
and a pharmaceutically acceptable carrier. In certain embodiments
is provided a method of preventing or treating a disease in a
mammal in need thereof comprising administering a pharmaceutical
composition described herein to said mammal. The immunoglobulin
domain may be a heavy chain region comprising a knob domain in the
complementarity-determining region 3 (CDR3H) or fragment thereof.
The G-CSF polypeptide may be attached to the knob domain. The
immunoglobulin domain may be a heavy chain region comprising a
stalk domain in the complementarity-determining region 3 (CDR3H) or
fragment thereof. The G-CSF polypeptide may be attached to the
stalk domain. The knob domain of the CDR3 may comprise one or more
conserved motifs derived from the knob domain of the ultralong
CDR3. The stalk domain of the CDR3 may comprise one or more
conserved motis derived from the stalk domain of the ultralong
CDR3.
[0236] In some embodiments is an antibody or fragment thereof
comprising: (a) a first antibody sequence, wherein at least a
portion of the first antibody sequence is derived from at least a
portion of an ultralong CDR3; and (b) a non-antibody sequence. The
antibody or fragment thereof may further comprise a second antibody
sequence, wherein at least a portion of the second antibody
sequence is derived from at least a portion of an ultralong CDR3.
The ultralong CDR3 from which the first antibody sequence and/or
second antibody sequence may be derived from a ruminant. The
ruminant can be a cow. At least a portion of the first antibody
sequence and/or at least a portion of the second antibody sequence
can be derived from a mammal. The mammal may be a bovine.
Alternatively, the mammal is a non-bovine mammal, such as a human.
The first and/or second antibody sequences may be 3 or more amino
acids in length. The amino acids may be consecutive amino acids.
Alternatively, the amino acids are non-consecutive amino acids. The
first and/or second antibody sequences may comprise a bovine
antibody sequence comprising 3 or more amino acids in length. The
bovine antibody may be a BLVH12, BLV5B8, BLVCV1, BLV5D3, BLV8C11,
BF1H1, or F18 antibody. The first and/or second antibody sequences
may comprise a human antibody sequence comprising 3 or moreore
amino acids in length. The portion of the first antibody sequence
derived from at least a portion of the ultralong CDR3 and/or the
portion of the second antibody sequence derived from at least a
portion of the ultralong CDR3 can be 20 or fewer amino acids in
length. The portion of the first antibody sequence derived from at
least a portion of the ultralong CDR3 and/or the portion of the
second antibody sequence derived from at least a portion of the
ultralong CDR3 may be 3 or more amino acids in length. The first
and/or second antibody sequences can comprise 1 or more, 2 or more,
3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9
or more, 10 or more, 15 or more, 20 or more, 30 or more, or 40 or
more amino acid residues derived from a knob domain of the
ultralong CDR3. The 1 or more amino acid residues derived from the
knob domain of the ultralong CDR3 may be a serine and/or cysteine
residue. The first and/or second antibody sequences may comprise 1
or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7
or more, 8 or more, 9 or more, or 10 or more amino acid residues
derived from a stalk domain of the ultralong CDR3. The first and/or
second antibody sequences may comprise 1 or more, 2 or more, 3 or
more, 4 or more, or 5 or more conserved motifs derived from a stalk
domain of the ultralong CDR3. The one or more conserved motifs
derived from the stalk domain of the ultralong CDR3 may comprise a
sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID
NOS: 333-336. The portion of the first antibody sequence derived
from at least a portion of the ultralong CDR3 and/or the portion of
the second antibody sequence derived from at least a portion of the
ultralong CDR3 may comprise a sequence selected from any one of SEQ
ID NOS: 157-307 and SEQ ID NOS: 333-336. The portion of the first
antibody sequence derived from at least a portion of the ultralong
CDR3 and/or the portion of the second antibody sequence derived
from at least a portion of the ultralong CDR3 may comprise a
sequence that is 50% or more homologous to a sequence selected from
any one of SEQ ID NOS: 157-224 and 235-295. The portion of the
first antibody sequence derived from at least a portion of the
ultralong CDR3 may comprise a sequence selected from any one of SEQ
ID NOS: 157-234. The portion of the first antibody sequence derived
from at least a portion of the ultralong CDR3 may comprise a
sequence that is 50% or more homologous to a sequence selected from
any one of SEQ ID NOS: 157-224. The portion of the first antibody
sequence derived from at least a portion of the ultralong CDR3 may
comprise a sequence that is 50% or more homologous to a sequence
selected from any one of SEQ ID NOS: 225-227. The portion of the
second antibody sequence derived from at least a portion of the
ultralong CDR3 may comprise a sequence selected from any one of SEQ
ID NOS: 235-307 and SEQ ID NOS: 333-336. The portion of the second
antibody sequence derived from at least a portion of the ultralong
CDR3 may comprise a sequence that may be 50% or more homologous to
a sequence selected from any one of SEQ ID NOS: 235-295. The
portion of the first antibody sequence derived from at least a
portion of the ultralong CDR3 and the portion of the second
antibody sequence derived from at least a portion of the ultralong
CDR3 may be derived from the same ultralong CDR3 sequence. The
portion of the first antibody sequence derived from at least a
portion of the ultralong CDR3 and the portion of the second
antibody sequence derived from at least a portion of the ultralong
CDR3 may be derived from two or more different ultralong CDR3
sequences. The portions of the ultralong CDR3 of the first and/or
second antibody sequences may be based on or derived from a BLV1H12
ultralong CDR3 sequence. The portions of the ultralong CDR3 of the
first and/or second antibody sequences may be based on or derived
from a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralong
CDR3 sequence. The antibody may further comprise one or more linker
sequences.
[0237] The present disclosure also provides antibodies that
comprise a heavy chain polypeptide, wherein the heavy chain
polypeptide comprises at least a portion of an ultralong CDR3
sequence. The heavy chain polypeptide may comprise a polypeptide
sequence based on or derived from a polypeptide sequence of any one
of SEQ ID NOS: 24-44. The heavy chain polypeptide may comprise a
polypeptide sequence encoded by a DNA sequence based on or derived
from the DNA sequence of any one of SEQ ID NOS: 2-22. Also provided
are antibodies comprising a heavy chain polypeptide, wherein the
heavy chain polypeptide comprises an ultralong CDR3 sequence and
the heavy chain polypeptide sequences are substantially similar to
those polypeptide sequences provided by any one of SEQ ID NOS:
24-44. A heavy chain polypeptide sequence may be considered
substantially similar to a polypeptide sequence provided by any one
of SEQ ID NOS: 24-44 where the heavy chain polypeptide sequence
shares 60%, 70%, 80%, 90%, 95%, 99%, or more nucleic acid identity
to a nucleotide sequence provided by any one of SEQ ID NOS: 24-44.
The antibodies may further comprise a light chain polypeptide. The
light chain polypeptide may comprise a polypeptide sequence based
on or derived from a polypeptide sequence of SEQ ID NO: 23. The
light chain polypeptide may comprise a polypeptide sequence encoded
by a DNA sequence based on or derived from the DNA sequence of SEQ
ID NO: 1. Also provided are antibodies further comprising a light
chain polypeptide, wherein the light chain polypeptide comprises an
ultralong CDR3 sequence and the light chain polypeptide sequences
are substantially similar to those polypeptide sequences provided
by SEQ ID NO: 23. A light chain polypeptide sequence may be
considered substantially similar to a polypeptide sequence provided
by SEQ ID NO: 1 where the light chain polypeptide sequence shares
60%, 70%, 80%, 90%, 95%, 99%, or more nucleic acid identity to a
nucleotide sequence provided by any one of SEQ ID NO: 1. The
antibody may have therapeutic activity in an animal. The antibody
can have therapeutic activity in infectious disease in a subject.
The antibody may comprise a monoclonal antibody, polyclonal
antibody, chimeric antibody, recombinant antibody, engineered
antibody, or synthetic antibody. The antibody may comprise a
mammalian antibody. The antibody may comprise a bovine antibody.
The antibody may comprise a G-CSF polypeptide, or derivative or
variant thereof. The antibody may comprise a mammalian G-CSF
polypeptide, or derivative or variant thereof. The antibody may
comprise a bovine G-CSF, or derivative or variant thereof. In some
embodiments, a pharmaceutical composition of therapeutic
formulation comprises an antibody described herein and a
pharmaceutically acceptable carrier. In certain embodiments, the
antibody is used in a method of treating a subject in need thereof,
with a therapeutically effective amount of the antibody or a
pharmaceutical composition described herein. In some embodiments, a
nucleic acid molecule or a complement thereof encodes a therapeutic
immunoglobulin described herein.
Genetic Sequences
[0238] The present disclosure provides genetic sequences (e.g.,
genes, nucleic acids, polynucleotides) encoding antibodies
comprising ultralong CDR3 sequences or portions thereof. The
present disclosure provides genetic sequences (e.g., genes, nucleic
acids, polynucleotides) encoding antibodies comprising the knob
domain and/or knob domain of ultralong CDR3 sequences. In another
embodiment, the present disclosure provides genetic sequences
encoding an antibody or immunoglobulin construct described
herein.
[0239] The present disclosure also provides genetic sequences
(e.g., genes, nucleic acids, polynucleotides) encoding an ultralong
CDR3 or portion thereof. The present disclosure also provides
genetic sequences (e.g., genes, nucleic acids, polynucleotides)
encoding the knob domain and/or knob domain of an ultralong
CDR3.
[0240] In an embodiment, the present disclosure provides genetic
sequences encoding an antibody comprising an ultralong CDR3. The
ultralong CDR3 may be 35 amino acids in length or more (e.g., 40 or
more, 45 or more, 50 or more, 55 or more, 60 or more). The
ultralong CDR3 may comprise at least a portion of a knob domain of
a CDR3, at least a portion of a stalk domain of a CDR3, or a
combination thereof. Such an 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. The antibody may comprise one or more
cysteine motifs. The antibody may comprise a non-antibody sequence
within the ultralong CDR3. Alternatively, or additionally, the
antibody comprises a non-bovine sequence. The antibody may further
comprise an antibody sequence. The antibody may comprise a
cytotoxic agent or therapeutic polypeptide. The cytotoxic agent or
therapeutic polypeptide may be conjugated to the ultralong CDR3.
The antibody may bind to a target. The target may be a protein
target, such as a transmembrane protein target.
[0241] In another embodiment, the present disclosure provides
genetic sequences encoding an 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). Alternatively, the
ultralong CDR3 sequence may be derived from a camelid or shark CDR3
sequence.
[0242] In another embodiment, the present disclosure provides
genetic sequences encoding an antibody comprising an ultralong
CDR3, wherein the CDR3 comprises 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 sequence
may be derived from a therapeutic polypeptide. The non-antibody
protein sequence may be of human or non-human origin. The
non-antibody sequence may comprise a synthetic sequence. The
non-antibody sequencemay 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 antibody. The CDR3 may be 35 amino acids in
length or more. The ultralong CDR3 may comprise at least a portion
of a knob domain of a CDR3, at least a portion of a stalk domain of
a CDR3, or a combination thereof. Alternatively, or additionally,
the antibody comprises at least 3 cysteine residues or more. The
antibody can comprise one or more cysteine motifs.
[0243] In another embodiment, the present disclosure provides
genetic sequences encoding an antibody comprising an ultralong CDR3
and a non-bovine sequence. The ultralong CDR3 can be derived from a
ruminant. The ruminant can be a bovine. The non-bovine sequence can
be derived from or based on a non-bovine mammal sequence. For
example, the non-bovine sequence can be derived from or based on a
human, mouse, rat, sheep, dog, and/or goat sequence. The non-bovine
sequence can be within the ultralong CDR3. Alternatively, the
non-bovine sequence is linked or attached to the ultralong CDR3
sequence. The non-bovine sequence can be derived from or based on
at least a portion of an antibody sequence. The antibody sequence
can encode a variable region, constant region or a combination
thereof. The CDR3 may be 35 amino acids in length or more. The
ultralong CDR3 may comprise at least a portion of a knob domain of
a CDR3, at least a portion of a stalk domain of a CDR3, or a
combination thereof. Alternatively, or additionally, the antibody
comprises at least 3 cysteine residues or more. The antibody can
comprise one or more cysteine motifs.
[0244] In another embodiment, the present disclosure provides
genetic sequences encoding an 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.
[0245] In another embodiment, the present disclosure provides
genetic sequences encoding an 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.
[0246] In another embodiment, the present disclosure provides
genetic sequences encoding an 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.
[0247] In another embodiment, the present disclosure provides
genetic sequences encoding an 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 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.
[0248] In another embodiment, the present disclosure provides
genetic sequences encoding an antibody comprising an ultralong
CDR3, wherein the 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. The CDR3 may be 35 amino acids in
length or more. The ultralong CDR3 may comprise at least a portion
of a knob domain of a CDR3, at least a portion of a stalk domain of
a CDR3, or a combination thereof. Alternatively, or additionally,
the antibody comprises at least 3 cysteine residues or more. The
antibody can comprise one or more cysteine motifs.
[0249] In another embodiment, the present disclosure provides
genetic sequences encoding an antibody or fragment thereof
comprising: (a) a first antibody sequence, wherein at least a
portion of the first antibody sequence is derived from at least a
portion of an ultralong CDR3; and (b) a non-antibody sequence. The
antibody or fragment thereof may further comprise a second antibody
sequence, wherein at least a portion of the second antibody
sequence is derived from at least a portion of an ultralong CDR3.
The ultralong CDR3 from which the first antibody sequence and/or
second antibody sequence may be derived from a ruminant. The
ruminant can be a cow. At least a portion of the first antibody
sequence and/or at least a portion of the second antibody sequence
can be derived from a mammal. The mammal may be a bovine.
Alternatively, the mammal is a non-bovine mammal, such as a human.
The first and/or second antibody sequences may be 3 or more amino
acids in length. The amino acids may be consecutive amino acids.
Alternatively, the amino acids are non-consecutive amino acids. The
first and/or second antibody sequences may comprise a bovine
antibody sequence comprising 3 or more amino acids in length. The
bovine antibody may be a BLVH12, BLV5B8, BLVCV1, BLV5D3, BLV8C11,
BF1H1, or F18 antibody. The first and/or second antibody sequences
may comprise a human antibody sequence comprising 3 or more, 4 or
more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or
more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more,
60 or more, or 70 or more amino acids in length. The portion of the
first antibody sequence derived from at least a portion of the
ultralong CDR3 and/or the portion of the second antibody sequence
derived from at least a portion of the ultralong CDR3 can be 20 or
fewer amino acids in length. The portion of the first antibody
sequence derived from at least a portion of the ultralong CDR3
and/or the portion of the second antibody sequence derived from at
least a portion of the ultralong CDR3 may be 3 or more amino, 4 or
more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or
more, 15 or more, or 20 or more acids in length. The first and/or
second antibody sequences can comprise 1 or more, 2 or more, 3 or
more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or
more, 10 or more, 15 or more, 20 or more, 30 or more, or 40 or more
amino acid residues derived from a knob domain of the ultralong
CDR3. The 1 or more amino acid residues derived from the knob
domain of the ultralong CDR3 may be a serine and/or cysteine
residue. The first and/or second antibody sequences may comprise 1
or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7
or more, 8 or more, 9 or more, or 10 or more amino acid residues
derived from a stalk domain of the ultralong CDR3. The first and/or
second antibody sequences may comprise 1 or more, 2 or more, 3 or
more, 4 or more, or 5 or more conserved motifs derived from a stalk
domain of the ultralong CDR3. The one or more conserved motifs
derived from the stalk domain of the ultralong CDR3 may comprise a
sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID
NOS: 333-336. The portion of the first antibody sequence derived
from at least a portion of the ultralong CDR3 and/or the portion of
the second antibody sequence derived from at least a portion of the
ultralong CDR3 may comprise a sequence selected from any one of SEQ
ID NOS: 157-307 and SEQ ID NOS: 333-336. The portion of the first
antibody sequence derived from at least a portion of the ultralong
CDR3 and/or the portion of the second antibody sequence derived
from at least a portion of the ultralong CDR3 may comprise a
sequence that is 50% or more homologous to a sequence selected from
any one of SEQ ID NOS: 157-224 and 235-295. The portion of the
first antibody sequence derived from at least a portion of the
ultralong CDR3 may comprise a sequence selected from any one of SEQ
ID NOS: 157-234. The portion of the first antibody sequence derived
from at least a portion of the ultralong CDR3 may comprise a
sequence that is 50% or more homologous to a sequence selected from
any one of SEQ ID NOS: 157-224. The portion of the first antibody
sequence derived from at least a portion of the ultralong CDR3 may
comprise a sequence that is 50% or more homologous to a sequence
selected from any one of SEQ ID NOS: 225-227. The portion of the
second antibody sequence derived from at least a portion of the
ultralong CDR3 may comprise a sequence selected from any one of SEQ
ID NOS: 235-307 and SEQ ID NOS: 333-336. The portion of the second
antibody sequence derived from at least a portion of the ultralong
CDR3 may comprise a sequence that may be 50% or more homologous to
a sequence selected from any one of SEQ ID NOS: 235-295. The
portion of the first antibody sequence derived from at least a
portion of the ultralong CDR3 and the portion of the second
antibody sequence derived from at least a portion of the ultralong
CDR3 may be derived from the same ultralong CDR3 sequence. The
portion of the first antibody sequence derived from at least a
portion of the ultralong CDR3 and the portion of the second
antibody sequence derived from at least a portion of the ultralong
CDR3 may be derived from two or more different ultralong CDR3
sequences. The portions of the ultralong CDR3 of the first and/or
second antibody sequences may be based on or derived from a BLV1H12
ultralong CDR3 sequence. The portions of the ultralong CDR3 of the
first and/or second antibody sequences may be based on or derived
from a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralong
CDR3 sequence. The antibody may further comprise one or more linker
sequences.
[0250] The present disclosure also provides isolated genetic
sequences (e.g., genes, nucleic acids, polynucleotides,
oligonucleotides) such as genetic sequences encoding antibodies
that comprise an ultralong CDR sequence including, for example, a
CDR3 sequence as provided by any one of SEQ ID NOS: 2-22. Also
provided are ultralong CDR3 nucleic acid sequences that are
substantially similar to those CDR3 sequences provided by any one
of SEQ ID NOS: 2-22. A CDR3 sequence may be considered
substantially similar to a CDR3 sequence provided by any one of SEQ
ID NOS: 2-22 where the CDR3 sequence shares 80%, 85%, 90%, 95%,
99%, or more, nucleic acid sequence identity to a CDR3 sequence
provided by any one of SEQ ID NOS: 2-22 or hybridizes to any one of
SEQ ID NOS: 2-22 under stringent hybridization conditions.
[0251] The present disclosure also provides isolated genetic
sequences (e.g., genes, nucleic acids, polynucleotides,
oligonucleotides) such as genetic sequences encoding antibodies
that comprise a heavy chain polypeptide, wherein the heavy chain
polypeptide comprises at least a portion of an ultralong CDR3
sequence. The heavy chain polypeptide may comprise a polypeptide
sequence of any one of SEQ ID NOS: 24-44. The heavy chain
polypeptide may comprise a polypeptide sequence encoded by the DNA
of any one of SEQ ID NOS: 2-22. Also provided are isolated genetic
sequences (e.g., genes, nucleic acids, polynucleotides,
oligonucleotides) such as genetic sequences encoding antibodies
comprising a heavy chain polypeptide, wherein the heavy chain
polypeptide comprises an ultralong CDR3 sequence and the heavy
chain polypeptide sequences are substantially similar to those
polypeptide sequences provided by any one of SEQ ID NOS: 24-44. A
heavy chain polypeptide sequence may be considered substantially
similar to a polypeptide sequence provided by any one of SEQ ID
NOS: 24-44 where the heavy chain polypeptide sequence shares 60%,
70%, 80%, 90%, 95%, 99%, or more nucleic acid identity to a
nucleotide sequence provided by any one of SEQ ID NOS: 24-44 or
hybridizes to any one of SEQ ID NOS: 24-44 under stringent
hybridization conditions. The isolated genetic sequences (e.g.,
genes, nucleic acids, polynucleotides, oligonucleotides) such as
genetic sequences encoding antibodies may further comprise a light
chain polypeptide. The light chain polypeptide may comprise a
polypeptide sequence of SEQ ID NO: 23. The light chain polypeptide
may comprise a polypeptide sequence encoded by the DNA of SEQ ID
NO: 1. Also provided are isolated genetic sequences (e.g., genes,
nucleic acids, polynucleotides, oligonucleotides) such as genetic
sequences encoding antibodies further comprising a light chain
polypeptide, wherein the light chain polypeptide comprises an
ultralong CDR3 sequence and the light chain polypeptide sequences
are substantially similar to those polypeptide sequences provided
by SEQ ID NO: 23. A light chain polypeptide sequence may be
considered substantially similar to a polypeptide sequence provided
by SEQ ID NO: 1 where the light chain polypeptide sequence shares
60%, 70%, 80%, 90%, 95%, 99%, or more nucleic acid identity to a
nucleotide sequence provided by any one of SEQ ID NO: 1 or
hybridizes to SEQ ID NOS: 1 under stringent hybridization
conditions.
Libraries and Arrays
[0252] The present disclosure provides collections, libraries and
arrays of antibodies comprising ultralong CDR3 sequences. In some
embodiments, members of the collections, libraries, or arrays may
exhibit sequence diversity.
[0253] In an embodiment, the present disclosure provides a library
or an array of 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.
[0254] In another embodiment, the present disclosure provides for a
library or an array of 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.
[0255] In another embodiment, the present disclosure provides
libraries or arrays of 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.
[0256] In another embodiment, the present disclosure provides
libraries or arrays of antibodies comprising ultralong CDR3
sequences wherein at least two members of the library differ in the
human framework used to construct the antibody comprising an
ultralong CDR3.
[0257] In another embodiment, the present disclosure provides
libraries or arrays of 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 antibody.
[0258] In another embodiment, the present disclosure provides
libraries or arrays of antibodies comprising ultralong CDR3
sequences wherein at least two members of the library or the array
differ in having a non-bovine sequence. The non-bovine sequence can
be derived from or based on a non-bovine mammal sequence. For
example, the non-bovine sequence can be derived from or based on a
human, mouse, rat, sheep, dog, and/or goat sequence. The non-bovine
sequence can be within the ultralong CDR3. Alternatively, the
non-bovine sequence is linked or attached to the ultralong CDR3
sequence. The non-bovine sequence can be derived from or based on
at least a portion of an antibody sequence. The antibody sequence
can encode a variable region, constant region or a combination
thereof.
[0259] In another embodiment, the present disclosure provides
libraries or arrays of antibodies comprising ultralong CDR3
sequences wherein at least two members of the library or array
differ in having a cytoxic agent or therapeutic polypeptide that is
conjugated to the ultralong CDR3. The cytoxic agent or therapeutic
polypeptide may include, but is not limited to, 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). The cytotoxic agent or therapeutic polypeptide can
be encoded by a non-antibody sequence.
[0260] In another embodiment, the present disclosure provides
libraries or arrays of antibodies comprising ultralong CDR3
sequences wherein at least two members of the library or array
differ in binding to targets. The target can be a protein target.
The protein target can be a transmembrane protein target. Such
transmembrane targets may include, but are not limited to, GPCRs,
ion channels, transporters, and cell surface receptors.
[0261] 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
[0262] The present disclosure provides cells comprising genetic
sequences encoding antibodies comprising ultralong CDR3 sequences
or portions thereof. The present disclosure provides cells
comprising genetic sequences encoding antibodies comprising at
least a portion of a knob domain or at least a portion of a knob
domain of an ultralong CDR3 sequence.
[0263] The present disclosure provides cells comprising genetic
sequences (e.g., genes, nucleic acids, polynucleotides) encoding an
ultralong CDR3 or portion thereof. The present disclosure also
provides cells comprising genetic sequences (e.g., genes, nucleic
acids, polynucleotides) encoding the knob domain and/or knob domain
of an ultralong CDR3.
[0264] In an embodiment, the present disclosure provides cells
expressing an antibody comprising an ultralong CDR3. The cells may
be prokaryotic or eukaryotic, and an antibody comprising an
ultralong CDR3 may be expressed on the cell surface or secreted
into the media. When displayed on the cell surface an 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, an 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 an antibody comprising an ultralong
CDR3. Alternatively, a stable cell line or stable pools may be
created by transfecting or transducing genetic sequences encoding
an 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 antibodies
comprising ultralong CDR3 sequences include prokaryotic cells like
E. coli, eukaryotic cells like the yeasts Saccharomyces cerevisiae
and Pichia pastoris, insect cells (e.g., Sf9, Hi5), chinese hamster
ovary (CHO) cells, monkey cells like COS-1, or human cells like
HEK-293, HeLa, SP-1.
Library Methods
[0265] The present disclosure provides methods for making libraries
comprising 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.
[0266] The present disclosure also provides methods of screening
libraries of antibodies comprising ultralong CDR3 sequences.
DEFINITIONS
[0267] 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.
[0268] 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 at least a portion of a knob domain and/or knob domain. An
ultralong CDR3 may comprise a non-antibody sequence, including, for
example, a cytokine, chemokine, growth factor or hormone sequence.
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, or 8 or
more cysteine residues. An ultralong CDR3 may comprise one or more
cysteine motifs. An ultralong CDR3 may comprise an amino acid
sequence that is derived from or based on SEQ ID NOS: 23-44 or is
encoded by a DNA sequence that is derived from or based on SEQ ID
NOS: 2-22. A variable region that comprises an ultralong CDR3 may
include an amino acid sequence that is derived from or based on SEQ
ID NOS: 23-44 or is encoded by a DNA sequence that is derived from
or based on SEQ ID NOS: 2-22. Such a sequence may be derived from
or based on a bovine germline VH gene sequence. An ultralong CDR3
may comprise a sequence derived from or based on a non-human DH
gene sequence (see, e.g., Koti, et al. (2010) Mol. Immunol. 47:
2119-2128). An ultralong CDR3 may comprise a sequence derived from
or based on a JH sequence, (see e.g., Hosseini, et al. (2004) Int.
Immunol. 16: 843-852). In an embodiment, an ultralong CDR3 may
comprise a sequence derived from or based on a non-human VH
sequence and/or a sequence derived from or based on a non-human DH
sequence and/or a sequence derived from or based on a JH sequence,
and optionally an additional sequence comprising two to six amino
acids or more such as, for example, between the VH derived sequence
and the DH derived sequence. In another embodiment, an ultralong
CDR3 may comprise a sequence that is about 50% or more homologous
to a sequence derived from or based on SEQ ID NOS: 23-44. For
example, the ultralong CDR3 may comprise a sequence that is about
60%, 70%, 80%, 85%, 90%, 95%, 97% or more homologous to a sequence
derived from or based on SEQ ID NOS: 23-44. In another embodiment,
an ultralong CDR3 may comprise a sequence that aligns to 5 or more
amino acids to a sequence derived from or based SEQ ID NOS: 23-44.
For example, the ultralong CDR3 may comprise a sequence that aligns
to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more amino
acids to a sequence derived from or based SEQ ID NOS: 23-44. In
another embodiment, an ultralong CDR3 may comprise a sequence that
comprises 5 or more consecutive amino acids to a sequence derived
from or based SEQ ID NOS: 23-44. For example, the ultralong CDR3
may comprise a sequence that comprises 5, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60 or more consecutive amino acids to a sequence
derived from or based SEQ ID NOS: 23-44. In another embodiment, an
ultralong CDR3 may comprise a sequence that is about 50% or more
homologous to a DNA sequence that is derived from or based on a SEQ
ID NOS: 2-22. For example, the ultralong CDR3 may comprise a
sequence that is about 60%, 70%, 80%, 85%, 90%, 95%, 97% or more
homologous to a DNA sequence that is derived from or based on SEQ
ID NOS: 2-22. In another embodiment, an ultralong CDR3 may comprise
a sequence that aligns to 5 or more nucleic acids to a DNA sequence
that is derived from or based SEQ ID NOS: 2-22. For example, the
ultralong CDR3 may comprise a sequence that aligns to 5, 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 80, 100, 120, 140, 150, 175,
200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600 or more
nucleic acids to a DNA sequence that is derived from or based SEQ
ID NOS: 2-22. In another embodiment, an ultralong CDR3 may comprise
a sequence that comprises 5 or more consecutive nucleic acids to a
DNA sequence that is derived from or based SEQ ID NOS: 2-22. For
example, the ultralong CDR3 may comprise a sequence that comprises
5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 80, 100, 120, 140,
150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600 or
more consecutive amino acids to a DNA sequence that is derived from
or based SEQ ID NOS: 2-22. In another embodiment, an ultralong CDR3
may comprise a sequence that is about 50% or more homologous to a
sequence derived from or based on a knob domain sequence. For
example, the ultralong CDR3 may comprise a sequence that is about
60%, 70%, 80%, 85%, 90%, 95%, 97% or more homologous to a sequence
derived from or based on a knob domain sequence. In another
embodiment, an ultralong CDR3 may comprise a sequence that aligns
to 5 or more amino acids to a sequence derived from or based a knob
domain sequence. For example, the ultralong CDR3 may comprise a
sequence that aligns to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60 or more amino acids to a sequence derived from or based a knob
domain sequence In another embodiment, an ultralong CDR3 may
comprise a sequence that comprises 5 or more consecutive amino
acids to a sequence derived from or based a knob domain sequence
For example, the ultralong CDR3 may comprise a sequence that
comprises 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more
consecutive amino acids to a sequence derived from or based a knob
domain sequence. In another embodiment, an ultralong CDR3 may
comprise a sequence that is about 50% or more homologous to a
sequence derived from or based on a knob domain sequence. For
example, the ultralong CDR3 may comprise a sequence that is about
60%, 70%, 80%, 85%, 90%, 95%, 97% or more homologous to a sequence
derived from or based on a knob domain sequence. In another
embodiment, an ultralong CDR3 may comprise a sequence that aligns
to 5 or more amino acids to a sequence derived from or based a knob
domain sequence For example, the ultralong CDR3 may comprise a
sequence that aligns to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60 or more amino acids to a sequence derived from or based a knob
domain sequence In another embodiment, an ultralong CDR3 may
comprise a sequence that comprises 5 or more consecutive amino
acids to a sequence derived from or based a stalk domain sequence
For example, the ultralong CDR3 may comprise a sequence that
comprises 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more
consecutive amino acids to a sequence derived from or based a stalk
domain sequence. The antibodies disclosed herein may comprise at
least a portion of an ultralong CDR3 derived from or based on a
sequence of any of the ultralong CDR3s disclosed herein. The
sequence of the ultralong CDR3 or a portion thereof may be modified
or altered to contain one or more non-bovine antibody-based
nucleotides and/or amino acids. The modifications and/or
alterations in the sequence of the ultralong CDR3 or portion
thereof may improve one or more features of the expressed antibody.
For example, the modifications and/or alterations may improve
expression, folding, half-life, activity and/or solubility of the
antibody.
[0269] 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.
[0270] "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.
[0271] "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.
[0272] 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.
[0273] 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.
[0274] 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.
[0275] "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.
[0276] "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.
[0277] 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 (CM5, Biacore Inc.) and
according to the supplier's instructions.
[0278] "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.
[0279] "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., mRNA, 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.
[0280] "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.
[0281] "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.
[0282] "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.
[0283] "Recombinant" when used with reference to a cell, nucleic
acid, protein, antibody 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 herein.
[0284] "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.
[0285] "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.
[0286] "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.
[0287] "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)).
[0288] "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.
[0289] "Antibody", "immunoglobulin" and "immunoglobulin construct"
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).
The term "antibody" can refer to a full length antibody or a
portion thereof. An antibody can refer to a peptide comprising at
least one antibody sequence. The antibody sequence can comprise 5
or more amino acids of an antibody sequence. For example the
antibody sequence can comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20 or more amino acids of an antibody sequence. The
5 or more amino acids may be consecutive amino acids of an antibody
sequence. Alternatively, the 5 or more amino acids are
non-consecutive amino acids of an antibody sequence. For example,
the 5 or more amino acids may comprise a conserved motif within the
antibody sequence. For example, the 5 or more amino acids may
comprise a conserved motif within an ultralong CDR3 sequence. An
antibody can be human, humanized, fully human and/or affinity
matured. An antibody can be a chimeric antibody. An antibody can be
a recombinant, engineered, or synthetic antibody. An antibody may
be a bovine, bovine engineered, fully bovine and/or affinity
matured. The bovine engineered antibody may comprise one or more
nucleotides or peptides derived from a bovine antibody sequence. A
fully bovine antibody may comprise replacing one or more
nucleotides or peptides from a non-bovine antibody sequence with
one or more nucleotides or peptides based on a bovine antibody
sequence. 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. In some instances, the antibodies provided herein
comprise at least one immunoglobulin domain from an avian antibody,
reptilian antibody, amphibian antibody, insect antibody, or
chimeric combinations thereof. The antibodies can comprise at least
one immunoglobulin domain from a chimeric antibody. The chimeric
antibody can be derived from two or more different species (e.g.,
mouse and human, bovine and human). The antibodies can comprise at
least one immunoglobulin domain from an engineered, recombinant or
synthetic antibody. In some instances, engineered, recombinant or
synthetic antibodies are created using antibody genes made in a
laboratory or taken from cells. The antibody genes can be derived
from one or more mammals. For example, the antibody genes are
derived from a human. The antibody genes may be derived from a
bovine. Alternatively, or additionally, the antibodies disclosed
herein comprise at least one immunoglobulin domain from a
humanized, human engineered or fully human antibody. The antibody
may comprise antibody sequences from two or more different
antibodies. The two or more different antibodies may be from the
same species. For example, the specie may be a bovine specie, human
specie, or murine specie. The two or more different antibodies may
be from the same type of animal. For example the two or more
different antibodies may be from a cow. The two or more different
antibodies may be from a human. Alternatively, the two or more
different antibodies are from different species. For example, the
two or more different antibodies are from a human specie and bovine
specie. In another example, the two or more diffent antibodies are
from a bovine specie and a non-bovine specie. In another example,
the two or more different antibodies are from a human specie and a
non-human specie. The two or more different antibodies may be from
different animals. For example, the two different animals are a
human and a cow. The different animals may be from the same specie.
For example, the different animals may be a cow and a water
buffalo.
[0290] "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.
[0291] 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.
[0292] "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.
[0293] 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.
[0294] 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.
[0295] 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.
[0296] "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 (e.g., (ds Fv).sub.2), Fd and Fd'
fragments Fab fragments, Fd fragments, scFv fragments, linear
antibodies, single-chain antibody molecules, minibodies, flex
minibodies, bispecific fragments, 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.
[0297] A "dsFv" refers to an Fv with an engineered intermolecular
disulfide bond, which stabilizes the VH-VL pair.
[0298] 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.
[0299] 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.
[0300] 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.
[0301] A "Fab' fragment" refers to a fragment containing one half
(one heavy chain and one light chain) of the F(ab')2 fragment.
[0302] A "Fd' fragment refers to a fragment of an antibody
containing one heavy chain portion of a F(ab')2 fragment.
[0303] A "Fv' fragment" refers to a fragment containing only the VH
and VL domains of an antibody molecule.
[0304] 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.
[0305] Diabodies are dimeric scFv; diabodies typically have shorter
peptide linkers than scFvs, and they preferentially dimerize.
[0306] "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).
[0307] "Hypervariable region", "HVR", or "HV", as well as
"complementary determining 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.
(See also, for example, FIG. 1 and bold, italicized text for Kabat
CDRs and underlined text for Chothia CDRs for 12.3 ICI
antibody).
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
[0308] 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 (see also, for example, FIG. 1 and bracketed text for
12.3 1C1 antibody).
[0309] 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.
[0310] "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.
[0311] "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 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)).
[0312] "Humanized" or "Human engineered" forms of non-human (e.g.,
murine, bovine) 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).
[0313] "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.
[0314] "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.
[0315] "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).
[0316] 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.
[0317] "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).
[0318] 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, an 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.
[0319] 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.
[0320] "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).
[0321] "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).
[0322] 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.
[0323] 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).
[0324] 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.
[0325] "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).
[0326] "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.
[0327] "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. Immuno19:457-92 (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)).
[0328] 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.
[0329] "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.
[0330] 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)).
[0331] "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.
[0332] "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.
[0333] "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.
[0334] "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.
[0335] 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.
[0336] "Disorder" or "disease" refers to any condition that would
benefit from treatment with a substance/molecule (e.g., an 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.
[0337] "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.
[0338] "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.
[0339] "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.
[0340] "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.
[0341] "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.
[0342] "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.
[0343] "Pharmaceutically acceptable vehicle" refers to a diluent,
adjuvant, excipient, or carrier with which at least one antibody of
the present disclosure is administered.
[0344] "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).
[0345] 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.
[0346] "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.
[0347] 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.
[0348] 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.
[0349] "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.
[0350] 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." 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.
[0351] "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
(K) and lambda (K), based on the amino acid sequence of its
constant domain.
[0352] "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.
[0353] 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.
[0354] 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.
[0355] 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.
[0356] "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.
[0357] "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.
[0358] "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).
[0359] A portion of an antibody includes sufficient amino acids to
form an antigen binding site.
[0360] 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.
[0361] 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.
[0362] 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..
[0363] 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.
[0364] "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.
[0365] "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.
[0366] 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.
[0367] 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.
[0368] 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).
[0369] 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.
[0370] 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-L1 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.
[0371] 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.
[0372] 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.
[0373] An "array" refers to a collection of elements, such as
antibodies, containing three or more members.
[0374] 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.
[0375] 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.
[0376] 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.
[0377] "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.
[0378] "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.
[0379] 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.
[0380] "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.
[0381] "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.
[0382] A "target protein" or "protein target" 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. Modulating the activity can
comprise increasing, decreasing, stimulating, or preventing the
activity or expression of the target protein. 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. In some
instances, the target protein is a transmembrane protein target.
Transmembrane protein targets include, but are not limited to,
GPCRs, ion channels, transporters, and cell surface receptors. Ion
channels may be potassium ion channels, sodium ion channels,
calcium ion channels, and voltage gated channels. In some
instances, the antibodies disclosed herein modulate a Kv1.3 ion
channel, Nav1.7 ion channel, or acid sensing ion channel (ASIC).
The antibodies disclosed herein may modulate cell surface receptors
such as GLP1R, GCGR, EPO receptor, FGFR, FGF21R, CSFR, GMCSFR, and
GCSFR. Additional target proteins include, but are not limited to,
cytokines, kinases, interferons, hormones, and growth factors. The
target proteins can be from a mammal or non-mammal. The target
proteins can be from a human. Alternatively, the target proteins
are from a bovine.
[0383] "Hit" refers to an antibody or portion thereof identified,
recognized or selected as having an activity in a screening
assay.
[0384] "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.
[0385] "High-throughput" refers to a large-scale method or process
that permits manipulation of large numbers of molecules or
compounds, generally tens to hundreds 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.
[0386] 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.
[0387] 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.
[0388] 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.
[0389] "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.
[0390] "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.
[0391] "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.
[0392] A "peptide" refers to a polypeptide that is from 2 to 40
amino acids in length.
[0393] 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.
[0394] 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).
[0395] "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
[0396] 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.
[0397] An "immunoconjugate" refers to an antibody conjugated to one
or more heterologous molecule(s), including but not limited to a
cytotoxic agent, non-antibody peptide or therapeutic polypeptide.
An immunoconjugate may include non-antibody sequences. The
non-antibody sequence can be conjugated to the antibody.
Alternatively, the non-antibody sequence can be within the antibody
sequence.
[0398] A "non-antibody peptide" refers to a peptide encoded by a
non-antibody antibody sequence. For example, a non-antibody peptide
may be a hormone, a lymphokine, an interleukin, a chemokines, a
cytokine or a peptide toxin.
[0399] As used herein, the terms "therapeutic polypeptide,"
"therapeutic peptides," and therapeutic immunoglobulin construct"
mean one or more peptides having demonstrated or potential use in
treating, preventing, or ameliorating one or more diseases,
disorders, or conditions in a subject in need thereof, as well as
related peptides. Therapeutic peptides include peptides found to
have use in treating, preventing, or ameliorating one or more
diseases, disorders, or conditions after the time of filing of this
application. Related peptides include fragments of therapeutic
peptides, therapeutic peptide variants, and therapeutic peptide
derivatives that retain some or all of the therapeutic activities
of the therapeutic peptide. As will be known to one of skill in the
art, as a general principle, modifications may be made to peptides
that do not alter, or only partially abrogate, the properties and
activities of those peptides. In some instances, modifications
result in an increase in therapeutic activities. The terms
"therapeutic polypeptide" or "therapeutic peptides" encompass
modifications to the therapeutic peptides defined and/or disclosed
herein. In certain embodiments, the therapeutic polypeptide is
selected from a hormone, a lymphokine, an interleukin, a
chemokines, a cytokine, a peptide toxin, and combinations thereof.
Therapeutic polypeptides can be peptides encoded by non-antibody
sequences.
[0400] A derivative or a variant of a polypeptide is said to share
"homology" or be "homologous" with the peptide if the amino acid
sequences of the derivative or variant has at least 50% identity
with the original peptide. In certain embodiments, the derivative
or variant is at least 75% the same as that of either the peptide
or a fragment of the peptide having the same number of amino acid
residues as the derivative. In certain embodiments, the derivative
or variant is at least 85% the same as that of either the peptide
or a fragment of the peptide having the same number of amino acid
residues as the derivative. In certain embodiments, the amino acid
sequence of the derivative is at least 90% the same as the peptide
or a fragment of the peptide having the same number of amino acid
residues as the derivative. In some embodiments, the amino acid
sequence of the derivative is at least 95% the same as the peptide
or a fragment of the peptide having the same number of amino acid
residues as the derivative. In certain embodiments, the derivative
or variant is at least 99% the same as that of either the peptide
or a fragment of the peptide having the same number of amino acid
residues as the derivative.
[0401] 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.
[0402] 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.
[0403] 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.
[0404] 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.
[0405] 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.
General Techniques
[0406] 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).
[0407] 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.
[0408] 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 Devanter 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).
[0409] 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.
Antibodies with Ultralong CDR3 Sequences
[0410] 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.
[0411] 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).
[0412] In an embodiment, bovine antibodies are identified and/or
produced. Multiple techniques exist to identify and/or produce
antibodies.
[0413] Antibodies of the present disclosure may be isolated by
screening including, high-throughput screening, of 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). Such screening may be iterative until a hit is
obtained.
[0414] 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.
[0415] Typically, a non-human antibody is humanized to reduce
immunogenicity 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.
[0416] 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.
[0417] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions 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 regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions 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)).
[0418] Antibodies with ultralong CDR3 sequences may also include
non-antibody sequences, such as cytokines, therapeutic polypeptides
or growth factors, into the CDR3 region. The resultant antibody can
be effective in treating or preventing a disease or condition. For
example, an antibody comprising an ultralong CDR3 inhibits tumor
metastasis. In some embodiments, the cytokine, therapeutic
polypeptide or growth factor may be shown to have an
antiproliferative effect on at least one cell population.
Alternatively, or additionally, the resultant antibody modulates
the expression or activity of a target (e.g., protein target,
transmembrane protein target). For example, an antibody comprising
an ultralong CDR3 inhibits or blocks an ion channel. The
non-antibody sequence may be a hormone, a lymphokine, an
interleukin, a chemokines, a cytokine, a peptide toxin, and
combinations thereof. Such cytokines, therapeutic polypeptides,
toxins, lymphokines, growth factors, or other hematopoietic factors
include Granulocyte colony-stimulating factor (G-CSF), macrophage
colony-stimulating factor (M-CSF), Granulocyte-macrophage
colony-stimulating factor (GM-CSF), Meg-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 (e.g., .alpha.-interferon,
.beta.-interferon, a .lamda.-interferon), TNF-alpha, TNF1, TNF2,
thrombopoietin, stem cell factor, and erythropoietin (EPO).
Additional growth factors for use in the 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 21
(FGF21)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. In some embodiments, the
therapeutic polypeptide is a mammalian G-CSF, a growth hormone, a
leptin, a .alpha.-interferon, a .beta.-interferon, a
.lamda.-interferon, a GM-CSF, a IL-11, a IL-10, a mokal (e.g.,
Moka, mokatoxin-1), or a VM-24. In some embodiments, the
therapeutic polypeptide is a glucagon-like peptide 1 (GLP-1),
exendin-4 (Ex-4), erythropoietin (EPO), fibroblast growth factor
(FGF21), IL8, ziconotide, somatostatin, chlorotoxin, SDF1(alpha),
IL21, or derivative or variant thereof. The G-CSF may be a bovine
G-CSF. The G-CSF, GM-CSF, EPO, FGF21, .beta.-interferon and GLP-1
may be from a human.
[0419] The non-antibody sequence may comprise an amino acid
sequence based on or derived from any of SEQ ID NOS: 317-332. The
non-antibody sequence may comprise an amino acid sequence that is
50%, 60%, 70% 80%, 90%, 95%, 97%, 99% identical to any of SEQ ID
NOS: 317-332. The non-antibody sequence may comprise an amino acid
sequence that comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, or more amino acid residues of SEQ ID NOS: 317-332.
The amino acid residues may be consecutive. Alternatively, the
amino acid residues are non-consecutive. The non-antibody sequence
may comprise at least a portion of any of SEQ ID NOS: 317-332.
[0420] The antibodies disclosed herein may comprise one or more
sequences based on or derived from a mammalian, avian, reptilian,
amphibian, fish, insect, bug, or arachnid sequence. Mammals
include, but are not limited to, cows, bison, buffalo, humans,
mice, dogs, cats, sheep, goats, or rabbits. Avians include, but are
not limited to, chicken, geese, doves, eagles, sparrows, and
pidgeons. Reptiles include, but are not limited to, lizards,
gators, snakes, and turtles. Amphibians include, but are not
limited to, frogs, salamanders, toads, and newts. Fish include, but
are not limited to, tuna, salmon, whales, and sharks. Insects,
bugs, and arachnids include, but are not limited to, flies,
mosquitos, spiders, and scorpions. The non-antibody sequence may be
based on or derived from a bovine or human sequence. Alternatively,
the non-antibody sequence is based on or derived from a lizard,
snail, snake or scorpion sequence. The lizard may be a gila
monster. The snail may be a cone snail.
[0421] In some embodiments, the non-antibody sequence is linked to
an end of an ultralong CDR3 sequence. For example, the non-antibody
sequence can be linked to the 5' end or 3' end of the ultralong
CDR3 nucleotide sequence. In another example, the non-antibody
sequence can be linked to the N-terminus or C-terminus of the
ultralong CDR3 peptide sequence.
[0422] In another embodiment, the non-antibody sequence is inserted
within an ultralong CDR3 sequence. For example, the non-antibody
sequence is inserted between the stalk domain of an ultralong CDR3
sequence. The non-antibody sequence can be inserted within the
stalk domain of an ultralong CDR3 sequence. In another example, the
non-antibody sequence is inserted between the stalk domain and the
knob domain of an ultralong CDR3 sequence. Alternatively, the
non-antibody sequence is inserted within the knob domain of an
ultralong CDR3 sequence.
[0423] In some embodiments, the non-antibody sequence replaces at
least a portion of an ultralong CDR3 sequence. The non-antibody
sequence can replace about 1 or more, 2 or more, 3 or more, 4 or
more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30
or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more
amino acids of the ultralong CDR3 peptide sequence. The
non-antibody sequence can replace about 10% or more, 20% or more,
30% or more, 40% or more, 50% or more, 55% or more, 60% or more,
65% or more, 70% or more, 75% or more, 80% or more, 85% or more,
90% or more, 95% or more of the ultralong CDR3 peptide sequence.
The non-antibody sequence can replace at least a portion of a knob
domain of an ultralong CDR3. The non-antibody sequence can replace
about 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30
or more, 35 or more, 40 or more, 45 or more amino acids of the knob
domain of an ultralong CDR3 peptide sequence. The non-antibody
sequence can replace about 10% or more, 20% or more, 30% or more,
40% or more, 50% or more, 55% or more, 60% or more, 65% or more,
70% or more, 75% or more, 80% or more, 85% or more, 90% or more,
95% or more of the knob domain of the ultralong CDR3 peptide
sequence. The non-antibody sequence can replace at least a portion
of a stalk domain of an ultralong CDR3. The non-antibody sequence
can replace about 1 or more, 2 or more, 3 or more, 4 or more, 5 or
more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or
more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more
amino acids of the stalk domain of an ultralong CDR3 peptide
sequence. The non-antibody sequence can replace about 10% or more,
20% or more, 30% or more, 40% or more, 50% or more, 55% or more,
60% or more, 65% or more, 70% or more, 75% or more, 80% or more,
85% or more, 90% or more, 95% or more of the stalk domain of the
ultralong CDR3 peptide sequence. The amino acids may be consecutive
amino acids. Alternatively, the amino acids are non-consecutive
amino acids. The ultralong CDR3 may comprise one or more conserved
motifs. The conserved motifs may be stalk domain conserved motifs
as disclosed herein. Alternatively, the conserved motifs may be
knob domain conserved motifs as disclosed herein.
[0424] In some embodiments, the non-antibody sequence replaces at
least a portion of an ultralong CDR3 sequence. The non-antibody
sequence can replace about 5 or more, 10 or more, 15 or more, 20 or
more, 25 or more, 30 or more, 40 or more, 50 or more, 55 or more,
60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or
more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or
more, 170 or more nucleotides of the ultralong CDR3 nucleotide
sequence. The non-antibody sequence can replace about 10% or more,
20% or more, 30% or more, 40% or more, 50% or more, 55% or more,
60% or more, 65% or more, 70% or more, 75% or more, 80% or more,
85% or more, 90% or more, 95% or more of the ultralong CDR3
nucleotide sequence. The non-antibody sequence can replace at least
a portion of a knob domain of an ultralong CDR3. The non-antibody
sequence can replace about 5 or more, 10 or more, 15 or more, 20 or
more, 25 or more, 30 or more, 40 or more, 50 or more, 55 or more,
60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or
more, 120 or more, 130 or more, 140 or more nucleotides of the knob
domain of an ultralong CDR3 nucleotide sequence. The non-antibody
sequence can replace about 10% or more, 20% or more, 30% or more,
40% or more, 50% or more, 55% or more, 60% or more, 65% or more,
70% or more, 75% or more, 80% or more, 85% or more, 90% or more,
95% or more of the knob domain of the ultralong CDR3 nucleotide
sequence. The non-antibody sequence can replace at least a portion
of a stalk domain of an ultralong CDR3. The non-antibody sequence
can replace about 5 or more, 10 or more, 15 or more, 20 or more, 25
or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or
more, 55 or more, 60 or more, 65 or more, 70 or more nucleotides of
the stalk domain of an ultralong CDR3 nucleotide sequence. The
non-antibody sequence can replace about 10% or more, 20% or more,
30% or more, 40% or more, 50% or more, 55% or more, 60% or more,
65% or more, 70% or more, 75% or more, 80% or more, 85% or more,
90% or more, 95% or more of the stalk domain of the ultralong CDR3
nucleotide sequence. The nucleotides may be consecutive
nucleotides. Alternatively, the nucleotides are non-consecutive
nucleotides. The ultralong CDR3 may comprise one or more conserved
motifs. The conserved motifs may be stalk domain conserved motifs
as disclosed herein. Alternatively, the conserved motifs may be
knob domain conserved motifs as disclosed herein.
[0425] An antibody comprising an ultralong CDR3 sequence and a
non-antibody sequence may further comprise one or more cleavage
sites between the ultralong CDR3 sequence and the non-antibody
sequence. The one or more cleavage sites may be in front of the
N-terminus of the non-antibody peptide sequence. For example, a
cleavage site is inserted at the N-terminus of the non-antibody
peptide sequence and at the C-terminus of the ultralong CDR3
peptide sequence. Alternatively, the one or more cleave sites are
behind the C-terminus of the non-antibody peptide sequence. For
example the cleavage site is inserted at the C-terminus of the
non-antibody peptide sequence and at the N-terminus of the
ultralong CDR3 peptide sequence. The one or more cleavage sites may
flank both the N-terminus and the C-terminus of the non-antibody
peptide sequence. The one or more cleavage sites may be upstream of
the non-antibody nucleotide sequence. For example, the one or more
cleavage sites may be at the 5' end of the non-antibody nucleotide
sequence and at the 3' end of the ultralong CDR3 nucleotide
sequence. The one or more cleavage sites may be downstream of the
non-antibody nucleotide sequence. For example, the one or more
cleavage sites may be at the 3' end of the non-antibody nucleotide
sequence and at the 5' end of the ultralong CDR3 nucleotide
sequence. The one or more cleavage sites may flank both the 5' end
and the 3' end of the non-antibody nucleotide sequence. The one or
more cleavage sites may directly flank the non-antibody sequence.
For example, there are zero nucleotides or amino acids between the
cleavage site sequence and the non-antibody sequence.
Alternatively, the one or more cleavage sites may indirectly flank
the non-antibody sequence. For example, there are one or more
nucleotides between the cleavage site nucleotide sequence and the
non-antibody nucleotide sequence. There may be 2 or more, 3 or
more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or
more, 10 or more, 12 or more, 15 or more, 20 or more nucleotides
between the cleavage site nucleotide sequence and the non-antibody
nucleotide sequence. In another example, there are one or more
amino acids between the cleavage site peptide sequence and the
non-antibody peptide sequence. There may be 2 or more, 3 or more, 4
or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10
or more, 12 or more, 15 or more, 20 or more amino acids between the
cleavage site peptide sequence and the non-antibody peptide
sequence. The cleavage site may be adjacent to the sequence based
on or derived from the ultralong CDR3 sequence, linker sequence,
non-antibody sequence, non-bovine sequence, or a combination
thereof. The cleavage site may be between the sequence based on or
derived from the ultralong CDR3 sequence and the linker sequence.
The cleavage site may be between the sequence based on or derived
from the ultralong CDR3 sequence and the non-antibody sequence. The
cleavage site may be between the linker sequence and the
non-antibody sequence. The cleavage site may be for a protease. The
protease may be a serine protease, threonine protease, cysteine
protease, aspartate protease, or metalloprotease. The protease may
include, but is not limited to, Factor Xa protease,
chymotrypsin-like protease, trypsin-like protease, elastase-like
protease, subtilisin-like protease, actinidain, bromelain,
calpains, caspases, cathepsins, Mir1-CP, papain, HIV-1 protease,
chymosin, renin, cathepsin D, pepsin, plasmepsin, nepenthesin,
metalloexopeptidases, and metalloendopeptidases. The cleavage site
may be a cleavage site for Factor Xa or thrombin. For example, the
cleavage site may comprise the amino acid sequence of IEGR.
Alternatively, the cleavage site is for a nuclease. The antibody
comprising the ultralong CDR3 sequence and non-antibody sequence
may be cleaved by one or more proteases. Cleavage of the antibody
by the one or more protease can result in release of one or more
ends of the non-antibody peptide from the ultralong CDR3 region of
the antibody. For example, cleavage of the antibody results in
release of the N-terminus of the non-antibody peptide from the
ultralong CDR3 region. Alternatively, cleavage of the antibody
results in release of the C-terminus of the non-antibody peptide
from the ultralong CDR3 region.
[0426] The non-antibody sequence may be linked to the ultralong
CDR3 sequence via one or more linkers. The non-antibody sequence
may be inserted with an ultralong CDR3 sequence. In some instances,
two or more linkers are used to link the non-antibody sequence to
the ultralong CDR3 sequence. The two or more linkers may comprise
the same sequence. Alternatively, the two or more linkers comprise
different sequences. The one or more linker sequences may be the
same length. The one or more linker sequences may be different
lengths. The one or more linker sequences may be 3 or more, 4 or
more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10
or more amino acids in length. The one or more linker sequences may
comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6
or more, 7 or more, 8 or more, 9 or more, or 10 or more glycine
residues. The one or more linker sequences may comprise 2 or more,
3 or more, 4 or more, or 5 or more consecutive glycine residues.
The one or more linker sequences may comprise 1 or more serine
residues. The one or more linker sequences may comprise 1 or more,
2 or more, 3 or more, 4 or more, or 5 or more polar amino acid
residues. The polar amino acid residues may be selected from
serine, threonine, asparagine, or glutamine. The polar amino acid
residues may comprise uncharged side chains. The linkers may be
attached to the N-terminal, C-terminal, or both N- and C-termini of
the non-antibody peptide sequence. The linkers may be attached to
the 5'-end, 3'-end, or both the 5'- and 3' ends of the non-antibody
nucleotide sequence. 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). Alternatively, the linker
comprises an amino acid sequence of (GGGGS). (SEQ ID NO: 339)
wherein n=1 to 5. The linker may comprise an amino acid sequence of
GGGSGGGGS (SEQ ID NO: 337) or GGGGSGGGS (SEQ ID NO: 338). 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 acids including 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.
[0427] The ultralong CDR3 may be based on or derived from a single
ultralong CDR3 sequence. Alternatively, the ultralong CDR3 is based
on or derived from two or more ultralong CDR3 sequences. The two or
more ultralong CDR3 sequences may be from the same animal.
Alternatively, the two or more ultralong CDR3 sequences are from
two or more different animals.
[0428] The ultralong CDR3 may comprise at least a portion of a
stalk domain of an ultralong CDR3. The antibodies disclosed herein
may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more,
6 or more 7 or more, 8 or more, 9 or more, or 10 or more amino
acids derived from or based on the stalk domain of the ultralong
CDR3. The antibodies disclosed herien may comprise 20 or fewer, 19
or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or
fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or
fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer,
3 or fewer, or 2 or fewer amino acids derived from or based the
stalk domain of the ultralong CDR3. The amino acids may be
consecutive amino acids. Alternatively, the amino acids are
non-consecutive amino acids. The antibodies disclosed herein may
comprise a sequence that is 50% or more, 60% or more, 70% or more,
80% or more, 85% or more, 90% or more, 95% or more, 97% or more,
99% or more, or 100% homologous the sequence of the stalk domain of
the ultralong CDR3. The ultralong CDR3 may comprise one or more
conserved motifs derived from or based on a stalk domain of the
ultralong CDR3. The antibodies disclosed herein may comprise 1 or
more, 2 or more, 3 or more, 4 or more, or 5 or more conserved
motifs derived from or based on the stalk domain of the ultralong
CDR3. The one or more conserved motifs derived from or based on the
stalk domain of the ultralong CDR3 may comprise a sequence selected
from any one of SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The
antibodies disclosed herein may comprise a sequence that is 50% or
more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or
more, 95% or more, 97% or more, 99% or more, or 100% homologous to
a sequence selected from any one of SEQ ID NOS: 157-224 and
235-295. The antibodies disclosed herein may comprise a sequence
that is 50% or more, 60% or more, 70% or more, 80% or more, 85% or
more, 90% or more, 95% or more, 97% or more, 99% or more, or 100%
homologous to a sequence selected from any one of SEQ ID NOS:
225-227.
[0429] The one or more conserved motifs derived from or based on
the stalk domain of the ultralong CDR3 may comprise a CT(T/S)VHQ
motif. Alternatively, the one or more conserved motifs derived from
or based on the stalk domain of the ultralong CDR3 comprise a
CT(T/S)VHQX.sub.n motif. In some instances, n is between 1 to 8,
between 1 to 7, between 1 to 6, between 1 to 5, between 1 to 4, or
between 1 to 3. The one or more conserved motifs derived from or
based on the stalk domain of the ultralong CDR3 may comprise a
CX.sup.1 X.sup.2 X.sup.3 X.sup.4Q motif. X.sup.1 may be a T, S, A,
or G residue. X.sup.2 may be a T, S, A, P, or I residue. X.sup.3
may be a V or K residue. X.sup.4 may be an H, K, or Y residue. The
one or more conserved motifs derived from or based on the stalk
domain of the ultralong CDR3 may comprise an X.sup.1 X.sup.2VHQ
motif. X.sup.1 may be a T, S, A, or G residue. X.sup.2 may be a T,
S, A, P or I residue. The one or more conserved motifs derived from
or based on the stalk domain of the ultralong CDR3 may comprise a
CX.sup.1 X.sup.2VHQ motif. X.sup.1 may be a T, S, A, or G residue.
X.sup.2 may be a T, S, A, P or I residue. The one or more conserved
motifs derived from or based on the stalk domain of the ultralong
CDR3 may comprise an X.sup.1 X.sup.2VX.sup.3Q motif. X.sup.1 may be
a T, S, A, or G residue. X.sup.2 may be a T, S, A, P or I residue.
X.sup.3 may be an H, Y or K residue. The one or more conserved
motifs derived from or based on the stalk domain of the ultralong
CDR3 may comprise a CX.sup.1 X.sup.2VX.sup.3Q motif. X.sup.1 may be
a T, S, A, or G residue. X.sup.2 may be a T, S, A, P or I residue.
X.sup.3 may be an H, Y or K residue. The one or more conserved
motifs derived from or based on the stalk domain of the ultralong
CDR3 may comprise an X.sup.1 X.sup.2KKQ motif. X.sup.1 may be a T,
S, A, or G residue. X.sup.2 may be a T, S, A, P or I residue. The
one or more conserved motifs derived from or based on the stalk
domain of the ultralong CDR3 may comprise a CX.sup.1 X.sup.2KKQ
motif. X.sup.1 may be a T, S, A, or G residue. X.sup.2 may be a T,
S, A, P or I residue.
[0430] The one or more conserved motifs derived from or based on
the stalk domain of the ultralong CDR3 may comprise an
YX.sup.1YX.sup.2 motif. X.sup.1 may be a T, S, N, or I residue.
X.sup.2 may be an E or D residue. The one or more conserved motifs
derived from or based on the stalk domain of the ultralong CDR3 may
comprise an YX.sup.1YX.sup.2Y motif. X.sup.1 may be an L, S, T, or
R residue. X.sup.2 may be a T, S, N or I residue. The one or more
conserved motifs derived from or based on the stalk domain of the
ultralong CDR3 may comprise an YX.sup.1YX.sup.2YX.sup.3 motif.
X.sup.1 may be an L, S, T, or R residue. X.sup.2 may be a T, S, N
or I residue. X.sup.3 may be an E or D residue. The one or more
conserved motifs derived from or based on the stalk domain of the
ultralong CDR3 may comprise an YX.sup.1YX.sup.2YX.sup.3X.sup.4
motif X.sup.1 may be an L, S, T, or R residue. X.sup.2 may be a T,
S, N or I residue. X.sup.3 may be an E or D residue. X.sup.4 may be
an H, W, N, F, I or Y residue. The one or more conserved motifs
derived from or based on the stalk domain of the ultralong CDR3 may
comprise an Y(E/D)X motif X may be an H, W, N, F, I or Y residue.
The one or more conserved motifs derived from or based on the stalk
domain of the ultralong CDR3 may comprise an XY(E/D) motif X may be
a T, S, N or I residue. The one or more conserved motifs derived
from or based on the stalk domain of the ultralong CDR3 may
comprise an Y(E/D)X.sup.1X.sub.nW motif X.sup.1 may be an H, W, N,
F, I or Y residue. In some instances, n is between 1 to 4, between
1 to 3, or between 1 to 2. The one or more conserved motifs derived
from or based on the stalk domain of the ultralong CDR3 may
comprise an Y(E/D)X.sup.1X.sup.2 X.sup.3X.sup.4X.sup.5W motif
X.sup.1 may be an H, W, N, F, I or Y residue. X.sup.2 may be an Y,
H, G, or N residue. X3 may be a V, I, or A residue. X.sup.4 may be
a D, N, T, or E residue. X.sup.5 may be an A, V, S, or T
residue.
[0431] The antibodies disclosed herein may comprise a first
conserved motif derived from or based on the stalk domain of the
ultralong CDR3 selected from any of SEQ ID NOS: 157-234 and a
second conserved motif derived from or based on the stalk domain of
the ultralong CDR3 selected from any of SEQ ID NOS: 235-307 and SEQ
ID NOS: 333-336. The antibodies disclosed herein may comprise a
first conserved motif derived from or based on the stalk domain of
the ultralong CDR3 selected from a group comprising
CT(T/S)VHQX.sub.n, CX.sup.1X.sup.2 X.sup.3X.sup.4Q,
X.sup.1X.sup.2VHQ, CX.sup.1X.sup.2 VHQ, X.sup.1X.sup.2VX.sup.3Q,
CX.sup.1X.sup.2VX.sup.3Q, X.sup.1X.sup.2KKQ, and CX.sup.1X.sup.2KKQ
and a second conserved motif derived from or based on the stalk
domain of the ultralong CDR3 selected from the group comprising
YX.sup.1YX.sup.2, YX.sup.1YX.sup.2Y, YX.sup.1YX.sup.2YX.sup.3,
YX.sup.1YX.sup.2YX.sup.3X.sup.4, Y(E/D)X, XY(E/D),
Y(E/D)X.sup.1X.sub.nW, and Y(E/D)X.sup.1X.sup.2
X.sup.3X.sup.4X.sup.5W.
[0432] The ultralong CDR3 may comprise at least a portion of a knob
domain of an ultralong CDR3. The antibodies disclosed herein may
comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6
or more 7 or more, 8 or more, 9 or more, or 10 or more amino acids
derived from or based on the knob domain of the ultralong CDR3. The
antibodies disclosed herien may comprise 20 or fewer, 19 or fewer,
18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13
or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or
fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer,
or 2 or fewer amino acids derived from or based the knob domain of
the ultralong CDR3. The amino acids may be consecutive amino acids.
Alternatively, the amino acids are non-consecutive amino acids. The
antibodies disclosed herein may comprise a sequence that is 50% or
more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or
more, 95% or more, 97% or more, 99% or more, or 100% homologous the
sequence of the knob domain of the ultralong CDR3. The ultralong
CDR3 may comprise one or more conserved motifs derived from or
based on a knob domain of the ultralong CDR3. The antibodies
disclosed herein may comprise 1 or more, 2 or more, 3 or more, 4 or
more, or 5 or more conserved motifs derived from or based on the
knob domain of the ultralong CDR3. The one or more conserved motifs
derived from or based on the knob domain may comprise a cysteine
motif as disclosed herein. Alternatively, or additionally, one or
more conserved motifs derived from or based on the knob domain
comprises a C(P/S)DG motif.
[0433] The antibodies disclosed herein may comprise a sequence
based on or derived from a mammal. The mammal may be a bovine.
Alternatively, the mammal is a non-bovine mammal, such as a human.
The antibody sequences may be 3 or more, 4 or more, 5 or more, 6 or
more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12
or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or
more, 40 or more 45 or more, 50 or more, 55 or more, 60 or more, 65
or more 70 or more, 80 or more, 90 or more, 100 or more, 110 or
more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or
more, 170 or more, 180 or more 190 or more, 200 or more, 220 or
more, 230 or more, 240 or more 250 or more 260 or more, 270 or
more, 280 or more, 290 or more or 300 or more amino acids in
length. The amino acids may be consecutive amino acids.
Alternatively, the amino acids are non-consecutive amino acids.
[0434] The antibody sequences may comprise a bovine antibody
sequence comprising 3 or more, 4 or more, 5 or more, 6 or more, 7
or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more,
15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or
more 45 or more, 50 or more, 55 or more, 60 or more, 65 or more 70
or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or
more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or
more, 180 or more 190 or more, 200 or more, 220 or more, 230 or
more, 240 or more 250 or more 260 or more, 270 or more, 280 or
more, 290 or more or 300 or more amino acids in length. The bovine
antibody may be a BLVH12, BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1,
or F18 antibody. The antibody sequences may comprise a human
antibody sequence comprising 3 or more, 4 or more, 5 or more, 6 or
more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12
or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or
more, 40 or more 45 or more, 50 or more, 55 or more, 60 or more, 65
or more 70 or more, 80 or more, 90 or more, 100 or more, 110 or
more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or
more, 170 or more, 180 or more 190 or more, 200 or more, 220 or
more, 230 or more, 240 or more 250 or more 260 or more, 270 or
more, 280 or more, 290 or more or 300 or more amino acids in
length. The amino acids may be consecutive amino acids.
Alternatively, the amino acids are non-consecutive amino acids.
[0435] The antibody sequence based on or derived from at least a
portion of the ultralong CDR3 can be 20 or fewer, 19 or fewer, 18
or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or
fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or
fewer, 7 or fewer, 6 or fewer, or 5 or fewer amino acids in length.
The antibody sequence based on or derived from at least a portion
of the ultralong CDR3 may be 3 or more, 4 or more, 5 or more, 6 or
more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12
or more, 13 or more, 14 or more, or 15 or more amino acids in
length. The amino acids may be consecutive amino acids.
Alternatively, the amino acids are non-consecutive amino acids.
[0436] The antibody sequence based on or derived from at least a
portion of the ultralong CDR3 can contain 1 or more, 2 or more, 3
or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9
or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or
more, 35 or more, 40 or more, 50 or more, 60 or more, 70 or more,
80 or more, 90 or more, or 100 or more nucleic acid modifications
or alterations in the nucleotide sequence of the ultralong CDR3
from which it is based on or derived from. The modifications and/or
alterations may comprise substitutions, deletions, and/or
insertions. Substitutions may comprise replacing one nucleic acid
with another nucleic acid. The nucleic acid may be a natural
nucleic acid or a non-natural nucleic acid.
[0437] The antibody sequence based on or derived from at least a
portion of the ultralong CDR3 can contain 1 or more, 2 or more, 3
or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9
or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or
more, 35 or more, 40 or more, 50 or more, or 60 or more amino acid
modifications or alterations in the peptide sequence of the
ultralong CDR3 from which it is based on or derived from. The
modifications and/or alterations may comprise substitutions,
deletions, and/or insertions. Substitutions may comprise replacing
one amino acid with another amino acid. The amino acids to be
substituted may contain one or more similar features to the amino
acid by which it is replaced. The features may include, but are not
limited to, size, polarity, hydrophobicity, acidity, side chain,
and bond formations. The amino acid may be a natural amino acid or
a non-natural amino acid.
[0438] In certain embodiments, the half-life of an antibody
described herein is greater than the half-life of the un-conjugated
therapeutic peptide or un-conjugated non-antibody peptide that is
incorporated in the antibody. In some embodiments, the half-life of
an antibody provided herein is greater than 4 hours when
administered to a subject. In certain embodiments, the half-life of
an antibody provided herein is greater than 4 hours, greater than 6
hours, greater than 12 hours, greater than 24 hours, greater than
36 hours, greater than 2 days, greater than 3 days, greater than 4
days, greater than 5 days, greater than 6 days, greater than 7
days, greater than 8 days, greater than 9 days, greater than 10
days, greater than 11 days, greater than 12 days, greater than 13
days, or greater than 14 days when administered to a subject. In
some instances, the subject is a mammal. In some embodiments, the
subject is a mouse or a bovine. In other instances, the subject is
a human. In certain embodiments, a pharmaceutical composition
comprising the antibody is administered to the subject once a day,
every two days, every three days, every 4 days, every 7 days, every
10 days, every 14 days, every 21 days, every 28 days, every 2
months, or every three months.
[0439] The antibodies may be modified or altered to reduce
immunogenicity. For example, the sequence of a partially bovine or
non-bovine antibody may be modified or altered to reduce
immunogenicity to humans. A non-human antibody may be humanized to
reduce immunogenicity 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.
[0440] The 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 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.
[0441] 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.
[0442] The 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.
[0443] 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.
[0444] 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.
[0445] 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 X.sup.63-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)).
[0446] Culture medium in which hybridoma cells are growing is
assayed for production of 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).
[0447] 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).
[0448] 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.
[0449] 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.
[0450] The 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 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.
[0451] 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".
[0452] 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).
[0453] Filamentous phage is used to display antibody fragments by
fusion to the minor coat protein pIII. Protein pIII may include
truncated forms of pIII. 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 pIII
(e.g., a truncated pIII) 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)).
[0454] Nucleic acid encoding antibody variable gene segments
(including VH and VL segments) are recovered from the cells of
interest 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).
[0455] 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).
[0456] 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).
[0457] 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).
[0458] 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.
[0459] 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.
[0460] 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).
[0461] 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)).
[0462] 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, a Fv 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.
[0463] DNA encoding an 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
[0464] The present disclosure provides antibody genes and proteins
including, for example, chimeric, recombinant, engineered,
synthetic, hybrid, bovine, fully bovine, bovinized, human, fully
human or humanized antibody genes or proteins that comprise an
ultralong CDR3 sequence. 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.
[0465] 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.
[0466] 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 .mu. heavy chain also acts to activate the kappa
(.kappa.) locus for rearrangement. The lambda (.lamda.) locus is
only activated for rearrangement if the K 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.
[0467] 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.
[0468] 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.
[0469] 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.
Antibody Fragments
[0470] 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, Fv', Fd, Fd', scFv,
hsFv fragments, and diabodies, 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.
[0471] 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).
[0472] 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.
[0473] 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
[0474] 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).
[0475] 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).
[0476] 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.
[0477] 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, Can 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.
Bovine Antibodies
[0478] The present disclosure provides for bovine antibodies
comprising an ultralong CDR3. The bovine antibodies may be
recombinant antibodies, engineered antibodies, synthetic
antibodies, bovinized antibodies, or fully bovine antibodies.
Bovinized antibodies may include bovine engineered antibodies.
Methods for producing a bovinized antibody may comprise introducing
one or more amino acid residues into it from a source which is a
bovine. In some instances, methods for producing a bovinized
antibody may comprise introducing one or more amino acid residues
into it from a source which is a non-bovine. Bovinization may be
performed by preparing a modified antibody variable domains.
Alternatively, bovinization may be performed by substituting
hypervariable region sequences for the corresponding sequences of a
bovine antibody. Accordingly, such "bovinized" or "bovine
engineered" antibodies are chimeric antibodies. Chimeric antibodies
may include antibodies wherein substantially less than an intact
bovine variable domain has been substituted by or incorporated into
the corresponding sequence from a non-bovine species. Bovinized or
bovine engineered antibodies may be bovine antibodies in which some
hypervariable region residues and constant region residues are
substituted by residues from analogous sites in non-bovine
antibodies. Alternatively, bovinized, bovine engineered or fully
bovine antibodies may be non-bovine (e.g, human) antibodies in
which some residues are substituted by residues from analogious
sites in bovine antibodies. For example, a bovine immunoglobuline
region can be used to replace a non-bovine (e.g., human, rodent)
immunoglobulin region to produce a fully bovine, bovinized or
bovine engineered antibody.
Bispecific Antibodies
[0479] 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).
[0480] 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).
[0481] 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.
[0482] 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).
[0483] 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 C.sub.H3 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.
[0484] 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.
[0485] 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.
[0486] 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.
[0487] 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).
[0488] 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
[0489] 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-(X2)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
[0490] In some embodiments, amino acid sequence modification(s) of
the 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 including, for example,
conservatively modified 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.
[0491] 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.
[0492] 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.
[0493] 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.
[0494] 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).
[0495] 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).
[0496] 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)).
[0497] 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 TABLE 1 Original Exemplary Preferred Residue
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; Leu Norleucine Leu (L) Norleucine; Ile; Val; Ile
Met; Ala; Phe 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 (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine
[0498] 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 amino acids 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.
[0499] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0500] 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.
[0501] 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.
[0502] 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.
[0503] 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. No. 5,648,260; U.S. Pat. No. 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).
[0504] 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'! 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, ACTI.TM. non-radioactive
cytotoxicity assay for flow cytometry (CellTecllrlology, 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). Clq 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)).
[0505] 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).
[0506] 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).)
[0507] 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).
[0508] 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).
[0509] 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).
[0510] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
Antibody Derivatives
[0511] The 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
[0512] 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 an 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.
[0513] 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.
[0514] 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.
[0515] a. Generating Antibodies Using Prokaryotic or Eukaryotic
Host Cells:
[0516] i. Vector Construction
[0517] 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.
[0518] 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).
[0519] 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.
[0520] 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.
[0521] 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.
[0522] 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.
[0523] 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)).
[0524] 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.
[0525] 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)).
[0526] 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.
[0527] 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'01. 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).
[0528] 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.
[0529] ii. Antibody Production
[0530] For recombinant production of a partially human ultralong
CDR3 antibody, nucleic acid encoding an 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.
[0531] 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.
[0532] 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.
[0533] 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.
[0534] 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.
[0535] 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.
[0536] 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.
[0537] 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.
[0538] 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.
[0539] 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. No. 6,083,715; U.S. Pat. No. 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).
[0540] 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. No. 5,264,365; U.S. Pat. No.
5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72
(1996)).
[0541] 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.
[0542] iii. Antibody Purification
[0543] 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.
[0544] 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 aureas 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.
[0545] 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.
[0546] b. Generating Antibodies Using Eukaryotic Host Cells:
[0547] 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.
[0548] (i) Signal Sequence Component
[0549] 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.
[0550] The DNA for such precursor region is ligated in reading
frame to DNA encoding the antibody.
[0551] (ii) Origin of Replication
[0552] 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.
[0553] (Iii) Selection Gene Component
[0554] 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.
[0555] 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.
[0556] 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.
[0557] 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).
[0558] 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.
[0559] (iv) Promoter Component
[0560] 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.
[0561] 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.
[0562] 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.
[0563] (v) Enhancer Element Component
[0564] 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.
[0565] (vi) Transcription Termination Component
[0566] 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.
[0567] (vii) Selection and Transformation of Host Cells
[0568] 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); TR1 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).
[0569] 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.
[0570] 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.
[0571] (viii) Culturing the Host Cells
[0572] 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. No. 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. Patent 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.
[0573] (ix) Purification of Antibody
[0574] 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.
[0575] 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 (Guss 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.
[0576] 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.
[0577] 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)).
[0578] 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).
[0579] 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
[0580] The disclosure also provides immunoconjugates
(interchangeably termed "antibody-drug conjugates" or "ADC"),
comprising any of the 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). Alternatively, the immunoconjugate
comprises any of the antibodies comprising an ultralong CDR3 as
described herein conjugated to a peptide. The peptide may be a
non-antibody peptide, therapeutic polypeptide, cytokine, hormone or
growth factor. The peptide may be encoded by a non-antibody
sequence.
[0581] The antibody-drug conjugates may be used 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.
[0582] 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.
[0583] 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.
[0584] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, dolastatins,
aurostatins, a trichothecene, and CC 1065, and the derivatives of
these toxins that have toxin activity, are also contemplated
herein.
[0585] a. Maytansine and Maytansinoids
[0586] In some embodiments, the immunoconjugate comprises an
antibody (full length or fragments) comprising an ultralong CDR3 as
disclosed herein conjugated to one or more maytansinoid
molecules.
[0587] 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.
[0588] 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.
[0589] 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.
[0590] 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.
[0591] 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.
[0592] 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.
[0593] 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.
[0594] b. Auristatins and Dolastatins
[0595] 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).
[0596] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, (see, e.g.,
U.S. Pat. No. 7,498,298).
[0597] 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. No. 5,635,483; U.S. Pat. No.
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).
[0598] c. Calicheamicin
[0599] 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.sup.I, .alpha..sub.2.sup.I,
.alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I, PSAG and
.theta..sup.I.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.
[0600] d. Other Cytotoxic Agents
[0601] 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).
[0602] 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.
[0603] 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).
[0604] 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.
[0605] 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.
[0606] 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.
[0607] 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.
[0608] e. Preparation of Antibody Drug Conjugates
[0609] 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.
[0610] 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).
[0611] 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 acids including 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.
[0612] 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).
[0613] 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.
[0614] 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.
[0615] 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.
[0616] 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
[0617] 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
[0618] 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.
[0619] 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).
[0620] 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
[0621] Antibodies comprising an ultralong CDR3, antibody fragments,
nucleic acids, or vectors disclosed herein can be formulated in
compositions, especially pharmaceutical compositions. Such
compositions with antibodies comprising an ultralong CDR3 comprise
a therapeutically or prophylactically effective amount of
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,
antibodies comprising an ultralong CDR3, antibody fragments,
nucleic acids, or vectors disclosed herein are sufficiently
purified for administration before formulation in a pharmaceutical
composition.
[0622] Pharmaceutically acceptable salts, excipients, or vehicles
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.
[0623] 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.
[0624] 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.
[0625] 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.
[0626] 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 Ringer's, 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.
[0627] 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
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.,
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.
[0628] 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.
[0629] 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].
[0630] 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 an antibody
comprising an ultralong CDR3 to hyaluronic acid polymer.
[0631] 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 multi-valent ions or other
polymers.
[0632] 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).
[0633] 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 an
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 an 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.
[0634] A pharmaceutical composition comprising an 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.
[0635] Certain formulations containing 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.
[0636] Another preparation may involve an effective quantity of an
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.
[0637] 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.
[0638] In some embodiments, 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.
[0639] 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.
[0640] 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.
[0641] 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).
[0642] In some embodiments is a pharmaceutical composition
comprising an antibody comprising an ultralong CDR3; and a
pharmaceutically acceptable carrier. The antibody may comprise a
therapeutic polypeptide, or derivative or variant thereof. The
therapeutic polypeptide may be a non-antibody sequence. The
therapeutic polypeptide, or derivative or variant thereof can be
within the ultralong CDR3. In some instances, the therapeutic
polypeptide is Moka1, Vm24, GLP-1, Exendin-4, human EPO, human
FGF21, human GMCSF, human interferon-beta, human GCSF, bovine GCSF
or derivative or variant thereof. Alternatively, the antibody is an
immunoconjugate as described herein. The antibody can comprise one
or more immunoglobulin domains. In some embodiments, the
immunoglobulin domain is an immunoglobulin A, an immunoglobulin D,
an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M.
The immunoglobulin domain can be an immunoglobulin heavy chain
region or fragment thereof. In some instances, the immunoglobulin
domain is from a mammalian antibody. Alternatively, the
immunoglobulin domain is from a chimeric antibody. In some
instances, the immunoglobulin domain is from an engineered antibody
or recombinant antibody. In other instances, the immunoglobulin
domain is from a humanized, human engineered or fully human
antibody. The mammalian antibody may be a bovine antibody. The
mammalin antibody may be a human antibody. In other instances, the
mammalian antibody is a murine antibody. The ultralong CDR3 can
comprise at least a portion of a knob domain in the CDR3. The
therapeutic polypeptide can be attached to the knob domain.
Alternatively, or additionally, the ultralong CDR3 comprises at
least a portion of a stalk domain in the CDR3. The therapeutic
polypeptide may be attached to the stalk domain. In some instances,
the antibody further comprises a linker. The linker can be within
the ultralong CDR3. The linker can attach the therapeutic
polypeptide to the immunoglobulin domain or fragment thereof. In
other instances, the linker attaches the therapeutic polypeptide to
the knob domain or stalk domain. In certain embodiments is a method
of preventing or treating a disease in a subject in need thereof
comprising administering this pharmaceutical composition to the
subject. In some embodiments, the pharmaceutical composition
comprising an immunoglobulin construct comprising a heavy chain
polypeptide comprising a sequence based on or derived from a
sequence selected from any one of SEQ ID NOS: 24-44 and the
polypeptide sequence encoded by the DNA any one of SEQ ID NOS:
2-22; and a light chain polypeptide comprising a sequence selected
from SEQ ID NO: 23 and a polypeptide sequence encoded by the DNA of
SEQ ID NO: 1; and a pharmaceutically acceptable carrier. In certain
embodiments is a method of preventing or treating a disease in a
mammal in need thereof comprising administering this
phartmaceutical composition to the mammal. In some embodiments, the
disease is an infectious disease such as mastitis. In certain
embodiments, the mammal in need is a dairy animal selected from a
list comprising cow, camel, donkey, goat, horse, reindeer, sheep,
water buffalo, moose and yak. In some embodiments, the mammal in
need is bovine.
[0643] In some embodiments, the pharmaceutical compositions
disclosed herein may be useful for providing prognostic or
providing diagnostic information.
Kits/Articles of Manufacture
[0644] 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 an 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
an 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 antibody comprising an ultralong CDR3
composition.
[0645] In certain embodiments, the composition comprising the
antibody is formulated in accordance with routine procedures as a
pharmaceutical composition adapted for intravenous administration
to mammals, such as humans, bovines, felines, canines, and murines.
Typically, compositions for intravenous administration are
solutions in sterile isotonic aqueous buffer. Where necessary, the
composition may also include a solubilizing agent and a local
anesthetic such as lignocaine to ease pain at the site of the
injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent. Where the composition is to be
administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water or saline.
Where the composition is administered by injection, an ampoule of
sterile water for injection or saline can be provided so that the
ingredients may be mixed prior to administration.
[0646] The amount of the composition described herein which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a Therapeutic protein can be determined by standard clinical
techniques. In addition, in vitro assays may optionally be employed
to help identify optimal dosage ranges. The precise dose to be
employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses are extrapolated from
dose-response curves derived from in vitro or animal model test
systems.
[0647] 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.
Methods of Treatment
[0648] Further disclosed herein are methods of preventing or
treating a disease or condition in a subject in need thereof
comprising administering a composition comprising one or more
antibodies comprising an ultralong CDR3 as disclosed herein to said
subject. The composition can further comprise a pharmaceutically
acceptable carrier. The subject may be a mammal. The mammal may be
a human. Alternatively, the mammal is a bovine. The antibody may
comprise a therapeutic polypeptide, or derivative or variant
thereof. The therapeutic polypeptide can be encoded by a
non-antibody sequence. The therapeutic polypeptide, or derivative
or variant thereof can be attached to the immunoglobulin domain.
The therapeutic polypeptide, or derivative or variant thereof may
be within the ultralong CDR3. Alternatively, the therapeutic
polypeptide, or derivative or variant thereof is conjugated to the
ultralong CDR3. In some instances, the therapeutic polypeptide is
Moka1, Vm24, human GLP-1, Exendin-4, human EPO, human FGF21, human
GMCSF, human interferon-beta, or derivative or variant thereof. The
antibody may be an immunoconjugate as described herein. The
antibody can comprise one or more immunoglobulin domains. The
immunoglobulin domain may be an immunoglobulin A, an immunoglobulin
D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin
M. The immunoglobulin domain can be an immunoglobulin heavy chain
region or fragment thereof. In some instances, the immunoglobulin
domain is from a mammalian antibody. Alternatively, the
immunoglobulin domain is from a chimeric antibody. The
immunoglobulin domain may be from an engineered antibody or
recombinant antibody. The immunoglobulin domain may be from a
humanized, human engineered or fully human antibody. The mammalian
antibody can be a bovine antibody. The mammalin antibody may be a
human antibody. In other instances, the mammalian antibody is a
murine antibody. The ultralong CDR3 may be 35 amino acids in length
or more. The ultralong CDR3 may comprise at least 3 cysteine
residues or more. The ultralong CDR3 may comprise one or more
cysteine motifs. The one or more cysteine motifs may be based on or
derived from SEQ ID NOS: 45-156. The one or more cysteine motifs
may be based on or derived from SEQ ID NOS: 45-99. The one or more
cysteine motifs may be based on or derived from SEQ ID NOS:
100-135. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 136-156. The ultralong CDR3 can comprise at least
a portion of a knob domain. The knob domain may comprise a
conserved motif within the knob domain of an ultralong CDR3. For
example, the knob domain may comprise a cysteine motif disclosed
herein. The therapeutic polypeptide can be attached to the knob
domain. Alternatively, or additionally, the ultralong CDR3
comprises at least a portion of a stalk domain. The stalk domain
may comprise a conserved motif within the stalk domain of an
ultralong CDR3. The conserved motif within the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 157-224. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 157-161. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 223-234. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 235-239. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 296-299. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from a first
sequence selected from the derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence basegroup comprising SEQ ID NOS:
157-234 and a second sequence selected from the group comprising
SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336. The antibodies
disclosed herein may comprise 2 or more, 3 or more, 4 or more, 5 or
more sequences based on or derived from SEQ ID NOS: 157-307 and SEQ
ID NOS: 333-336. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. For
example, the stalk domain may comprise a T(S/T)VHQ motif. The
therapeutic polypeptide can be attached to the stalk domain. In
some instances, the antibody, antibody fragment or immunoglobulin
construct further comprises a linker. The linker can attach the
therapeutic polypeptide to the immunoglobulin domain or fragment
thereof. In other instances, the linker attaches the therapeutic
polypeptide to the knob domain or stalk domain. In some instances,
the disease or condition is an autoimmune disease, heteroimmune
disease or condition, inflammatory disease, pathogenic infection,
thromboembolic disorder, respiratory disease or condition,
metabolic disease, central nervous system (CNS) disorder, bone
disease or cancer. In other instances, the disease or condition is
a blood disorder. In some instances, the disease or condition is
obesity, diabetes, osteoporosis, anemia, or pain.
[0649] In some embodiments is a method of preventing or treating a
disease or condition in a subject in need thereof comprising
administering to the subject a composition comprising: an
immunoglobulin construct comprising a heavy chain polypeptide
comprising a sequence that is substantially similar to a sequence
selected from SEQ ID NOS: 24-44; and a light chain polypeptide
comprising the sequence that is substantially similar to a sequence
of SEQ ID NO: 23. The heavy chain polypeptide sequence may share
50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more amino acid
sequence identity to a heavy chain sequence provided by any one of
SEQ ID NOS: 24-44. The light chain polypeptide sequence may share
50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more amino acid
sequence identity to a light chain sequence provided by SEQ ID NO:
23. In some instances, the disease or condition is an autoimmune
disease, heteroimmune disease or condition, inflammatory disease,
pathogenic infection, thromboembolic disorder, respiratory disease
or condition, metabolic disease, central nervous system (CNS)
disorder, bone disease or cancer. In other instances, the disease
or condition is a blood disorder. In some instances, the disease or
condition is obesity, diabetes, osteoporosis, anemia, or pain.
[0650] In an embodiment is provided a method of preventing or
treating a disease or condition in a subject in need thereof
comprising administering to the subject a composition comprising:
an immunoglobulin construct comprising a heavy chain polypeptide
comprising a polypeptide sequence encoded by a DNA sequence that is
substantially similar to a sequence selected from SEQ ID NOS: 2-22;
and a light chain polypeptide comprising a polypeptide sequence
encoded by a DNA sequence that is substantially similar to a
sequence of SEQ ID NO: 1. The heavy chain nucleotide sequence may
share 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more homology
to a heavy chain sequence provided by any one of SEQ ID NOS: 2-22.
The light chain nucleotide sequence may share 50%, 60%, 70%, 80%,
85%, 90%, 95%, 97%, 99%, or more homology to a light chain sequence
provided by SEQ ID NO: 1. In some instances, the disease or
condition is an autoimmune disease, heteroimmune disease or
condition, inflammatory disease, pathogenic infection,
thromboembolic disorder, respiratory disease or condition,
metabolic disease, central nervous system (CNS) disorder, bone
disease or cancer. In other instances, the disease or condition is
a blood disorder. In some instances, the disease or condition is
obesity, diabetes, osteoporosis, anemia, or pain.
[0651] Disclosed herein in some embodiments is a method of
preventing or treating an autoimmune disease in a subject in need
thereof comprising administering a composition comprising one or
more antibodies comprising an ultralong CDR3 as disclosed herein to
said subject. The composition can further comprise a
pharmaceutically acceptable carrier. The subject may be a mammal.
The mammal may be a human. Alternatively, the mammal is a bovine.
The antibody may comprise a therapeutic polypeptide, or derivative
or variant thereof. The therapeutic polypeptide can be encoded by a
non-antibody sequence. The therapeutic polypeptide, or derivative
or variant thereof can be attached to the immunoglobulin domain.
The therapeutic polypeptide, or derivative or variant thereof may
be within the ultralong CDR3. Alternatively, the therapeutic
polypeptide, or derivative or variant thereof is conjugated to the
ultralong CDR3. In some instances, the therapeutic polypeptide is
Moka1, VM-24 or beta-interferon or derivative or variant thereof.
The antibody may be an immunoconjugate as described herein. The
antibody can comprise one or more immunoglobulin domains. The
immunoglobulin domain may be an immunoglobulin A, an immunoglobulin
D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin
M. The immunoglobulin domain can be an immunoglobulin heavy chain
region or fragment thereof. In some instances, the immunoglobulin
domain is from a mammalian antibody. Alternatively, the
immunoglobulin domain is from a chimeric antibody. The
immunoglobulin domain may be from an engineered antibody or
recombinant antibody. The immunoglobulin domain may be from a
humanized, human engineered or fully human antibody. The mammalian
antibody can be a bovine antibody. The mammalin antibody may be a
human antibody. In other instances, the mammalian antibody is a
murine antibody. The ultralong CDR3 may be 35 amino acids in length
or more. The ultralong CDR3 may comprise at least 3 cysteine
residues or more. The ultralong CDR3 may comprise one or more
cysteine motifs. The one or more cysteine motifs may be based on or
derived from SEQ ID NOS: 45-156. The one or more cysteine motifs
may be based on or derived from SEQ ID NOS: 45-99. The one or more
cysteine motifs may be based on or derived from SEQ ID NOS:
100-135. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least a
portion of a knob domain. The knob domain may comprise a conserved
motif within the knob domain of an ultralong CDR3. For example, the
knob domain may comprise a cysteine motif disclosed herein. The
Moka1, VM-24, beta-interferon, or a derivative or variant thereof
can be attached to the knob domain. Alternatively, or additionally,
the ultralong CDR3 comprises at least a portion of a stalk domain.
The stalk domain may comprise a conserved motif within the stalk
domain of an ultralong CDR3. The conserved motif within the stalk
domain of the ultralong CDR3 may comprise a polypeptide sequence
based on or derived from SEQ ID NOS: 157-307 and SEQ ID NOS:
333-336. The conserved motif with the stalk domain of the ultralong
CDR3 may comprise a polypeptide sequence based on or derived from
SEQ ID NOS: 157-224. The conserved motif with the stalk domain of
the ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 157-161. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 223-234. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 235-239. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 296-299. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from a first
sequence selected from the derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence basegroup comprising SEQ ID NOS:
157-234 and a second sequence selected from the group comprising
SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336. The antibodies
disclosed herein may comprise 2 or more, 3 or more, 4 or more, 5 or
more sequences based on or derived from SEQ ID NOS: 157-307 and SEQ
ID NOS: 333-336. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. For
example, the stalk domain may comprise a T(S/T)VHQ motif. The
Moka1, VM-24, beta-interferon, or a derivative or variant thereof
can be attached to the stalk domain. In some instances, the
antibody, antibody fragment or immunoglobulin construct further
comprises a linker. The linker can attach Moka1, VM-24,
beta-interferon, or a derivative or variant thereof to the
immunoglobulin domain or fragment thereof. In other instances, the
linker attaches Moka1, VM-24, beta-interferon, or a derivative or
variant thereof to the knob domain or stalk domain. In some
instances, the autoimmune disease is a T-cell mediated autoimmune
disease. T-cell mediated autoimmune diseases include, but are not
limited to, multiple sclerosis, type-1 diabetes, and psoriasis. In
other instances, the autoimmune disease lupus, Sjogren's syndrome,
scleroderma, rheumatoid arthritis, dermatomyositis, Hasmimoto's
thyroiditis, Addison's disease, celiac disease, Crohn's disease,
pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune
hemolytic anemia, idiopathic thrombocytopenic purpura, myasthenia
gravis, Ord's thyroiditis, Graves' disease, Guillain-Barre
syndrome, acute disseminated encephalomyelitis,
opsoclonus-myoclonus syndrome, ankylosing spondylitisis,
antiphospholipid antibody syndrome, aplastic anemia, autoimmune
hepatitis, Goodpasture's syndrome, Reiter's syndrome, Takayasu's
arteritis, temporal arteritis, Wegener's granulomatosis, alopecia
universalis, Behcet's disease, chronic fatigue, dysautonomia,
endometriosis, interstitial cystitis, neuromyotonia, scleroderma,
and vulvodynia. Lupus can include, but is not limited to, acute
cutaneous lupus erythematosus, subacute cutaneous lupus
erythematosus, chronic cutaneous lupus erythematosus, discoid lupus
erythematosus, childhood discoid lupus erythematosus, generalized
discoid lupus erythematosus, localized discoid lupus erythematosus,
chilblain lupus erythematosus (hutchinson), lupus
erythematosus-lichen planus overlap syndrome, lupus erythematosus
panniculitis (lupus erythematosus profundus), tumid lupus
erythematosus, verrucous lupus erythematosus (hypertrophic lupus
erythematosus), complement deficiency syndromes, drug-induced lupus
erythematosus, neonatal lupus erythematosus, and systemic lupus
erythematosus.
[0652] Further disclosed herein is a method of preventing or
treating a disease or condition which would benefit from the
modulation of a potassium voltage-gated channel in a subject in
need thereof comprising administering a composition comprising one
or more antibodies comprising an ultralong CDR3 as disclosed herein
to said subject. The composition can further comprise a
pharmaceutically acceptable carrier. In some instances, the
potassium voltage-gated channel is a KCNA3 or K.sub.v1.3 channel.
The subject may be a mammal. The mammal may be a human.
Alternatively, the mammal is a bovine. The antibody may comprise a
therapeutic polypeptide, or derivative or variant thereof. The
therapeutic polypeptide can be encoded by a non-antibody sequence.
The therapeutic polypeptide, or derivative or variant thereof can
be attached to the immunoglobulin domain. The therapeutic
polypeptide, or derivative or variant thereof may be within the
ultralong CDR3. Alternatively, the therapeutic polypeptide, or
derivative or variant thereof is conjugated to the ultralong CDR3.
In some instances, the therapeutic polypeptide is Moka1, VM-24, or
derivative or variant thereof. The antibody may be an
immunoconjugate as described herein. The antibody can comprise one
or more immunoglobulin domains. The immunoglobulin domain may be an
immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an
immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain
can be an immunoglobulin heavy chain region or fragment thereof. In
some instances, the immunoglobulin domain is from a mammalian
antibody. Alternatively, the immunoglobulin domain is from a
chimeric antibody. The immunoglobulin domain may be from an
engineered antibody or recombinant antibody. The immunoglobulin
domain may be from a humanized, human engineered or fully human
antibody. The mammalian antibody can be a bovine antibody. The
mammalin antibody may be a human antibody. In other instances, the
mammalian antibody is a murine antibody. The ultralong CDR3 may be
35 amino acids in length or more. The ultralong CDR3 may comprise
at least 3 cysteine residues or more. The ultralong CDR3 may
comprise one or more cysteine motifs. The one or more cysteine
motifs may be based on or derived from SEQ ID NOS: 45-156. The one
or more cysteine motifs may be based on or derived from SEQ ID NOS:
45-99. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 100-135. The one or more cysteine motifs may be
based on or derived from SEQ ID NOS: 136-156. The ultralong CDR3
comprises at least a portion of a knob domain. The knob domain may
comprise a conserved motif within the knob domain of an ultralong
CDR3. For example, the knob domain may comprise a cysteine motif
disclosed herein. The Moka1, VM-24, or a derivative or variant
thereof can be attached to the knob domain. Alternatively, or
additionally, the ultralong CDR3 comprises at least a portion of a
stalk domain. The stalk domain may comprise a conserved motif
within the stalk domain of an ultralong CDR3. The conserved motif
within the stalk domain of the ultralong CDR3 may comprise a
polypeptide sequence based on or derived from SEQ ID NOS: 157-307
and SEQ ID NOS: 333-336. The conserved motif with the stalk domain
of the ultralong CDR3 may comprise a polypeptide sequence based on
or derived from SEQ ID NOS: 157-224. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 157-161. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 223-234. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 235-239. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 296-299. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 300-303. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from a first sequence selected from the derived from SEQ ID
NOS: 300-303. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence basegroup
comprising SEQ ID NOS: 157-234 and a second sequence selected from
the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
The antibodies disclosed herein may comprise 2 or more, 3 or more,
4 or more, 5 or more sequences based on or derived from SEQ ID NOS:
157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalk
domain of the ultralong CDR3 may comprise a polypeptide sequence
based on or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS:
333-336. For example, the stalk domain may comprise a T(S/T)VHQ
motif. The Moka1, VM-24, or a derivative or variant thereof can be
attached to the stalk domain. In some instances, the antibody,
antibody fragment or immunoglobulin construct further comprises a
linker. The linker can attach Moka1, VM-24, or a derivative or
variant thereof to the immunoglobulin domain or fragment thereof.
In other instances, the linker attaches Moka1, VM-24,
beta-interferon, or a derivative or variant thereof to the knob
domain or stalk domain. In some instances, the disease or condition
is an autoimmune disease. The autoimmune disease can be a T-cell
mediated autoimmune disease. In some instances, modulating a
potassium voltage-gated channel comprises inhibiting or blocking a
potassium voltage-gated channel. In some instances, the disease or
condition is episodic ataxia, seizure, or neuromyotonia.
[0653] Provided herein is a method of preventing or treating a
metabolic disease or condition in a subject in need thereof
comprising administering a composition comprising one or more
antibodies comprising an ultralong CDR3 as disclosed herein to said
subject. The composition can further comprise a pharmaceutically
acceptable carrier. The subject may be a mammal. The mammal may be
a human. Alternatively, the mammal is a bovine. The antibody may
comprise a therapeutic polypeptide, or derivative or variant
thereof. The therapeutic polypeptide can be encoded by a
non-antibody sequence. The therapeutic polypeptide, or derivative
or variant thereof can be attached to the immunoglobulin domain.
The therapeutic polypeptide, or derivative or variant thereof may
be within the ultralong CDR3. Alternatively, the therapeutic
polypeptide, or derivative or variant thereof is conjugated to the
ultralong CDR3. In some instances, the therapeutic polypeptide is
GLP-1, Exendin-4, FGF21, or derivative or variant thereof. The
GLP-1 may be a human GLP-1. In some instances, the FGF21 is a human
FGF21. The antibody may be an immunoconjugate as described herein.
The antibody can comprise one or more immunoglobulin domains. The
immunoglobulin domain may be an immunoglobulin A, an immunoglobulin
D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin
M. The immunoglobulin domain can be an immunoglobulin heavy chain
region or fragment thereof. In some instances, the immunoglobulin
domain is from a mammalian antibody. Alternatively, the
immunoglobulin domain is from a chimeric antibody. The
immunoglobulin domain may be from an engineered antibody or
recombinant antibody. The immunoglobulin domain may be from a
humanized, human engineered or fully human antibody. The mammalian
antibody can be a bovine antibody. The mammalin antibody may be a
human antibody. In other instances, the mammalian antibody is a
murine antibody. The ultralong CDR3 may be 35 amino acids in length
or more. The ultralong CDR3 may comprise at least 3 cysteine
residues or more. The ultralong CDR3 may comprise one or more
cysteine motifs. The one or more cysteine motifs may be based on or
derived from SEQ ID NOS: 45-156. The one or more cysteine motifs
may be based on or derived from SEQ ID NOS: 45-99. The one or more
cysteine motifs may be based on or derived from SEQ ID NOS:
100-135. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least a
portion of a knob domain. The knob domain may comprise a conserved
motif within the knob domain of an ultralong CDR3. For example, the
knob domain may comprise a cysteine motif disclosed herein. The
GLP-1, Exendin-4, FGF21, or a derivative or variant thereof can be
attached to the knob domain. Alternatively, or additionally, the
ultralong CDR3 comprises at least a portion of a stalk domain. The
stalk domain may comprise a conserved motif within the stalk domain
of an ultralong CDR3. The conserved motif within the stalk domain
of the ultralong CDR3 may comprise a polypeptide sequence based on
or derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 157-224. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 157-161. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 223-234. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 235-239. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 296-299. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from a first
sequence selected from the derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence basegroup comprising SEQ ID NOS:
157-234 and a second sequence selected from the group comprising
SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336. The antibodies
disclosed herein may comprise 2 or more, 3 or more, 4 or more, 5 or
more sequences based on or derived from SEQ ID NOS: 157-307 and SEQ
ID NOS: 333-336. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. For
example, the stalk domain may comprise a T(S/T)VHQ motif The GLP-1,
Exendin-4, FGF21, or a derivative or variant thereof can be
attached to the stalk domain. In some instances, the antibody,
antibody fragment or immunoglobulin construct further comprises a
linker. The linker can attach GLP-1, Exendin-4, FGF21, or a
derivative or variant thereof to the immunoglobulin domain or
fragment thereof. In other instances, the linker attaches GLP-1,
Exendin-4, FGF21, or a derivative or variant thereof to the knob
domain or stalk domain. Metabolic diseases and/or conditions can
include disorders of carbohydrate metabolism, amino acid
metabolism, organic acid metabolism (organic acidurias), fatty acid
oxidation and mitochondrial metabolism, porphyrin metabolism,
purine or pyrimidine metabolism, steroid metabolism, mitochondrial
function, peroxisomal function, urea cycle disorder, urea cycle
defects or lysosomal storage disorders. In some instances, the
metabolic disease or condition is diabetes. In other instances, the
metabolic disese or condition is glycogen storage disease,
phenylketonuria, maple syrup urine disease, glutaric acidemia type
1, Carbamoyl phosphate synthetase I deficiency, alcaptonuria,
Medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD),
acute intermittent porphyria, Lesch-Nyhan syndrome, lipoid
congenital adrenal hyperplasia, congenital adrenal hyperplasia,
Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or
Niemann Pick disease.
[0654] Provided herein is a method of preventing or treating a
central nervous system (CNS) disorder in a subject in need thereof
comprising administering a composition comprising one or more
antibodies comprising an ultralong CDR3 as disclosed herein to said
subject. The composition can further comprise a pharmaceutically
acceptable carrier. The subject may be a mammal. The mammal may be
a human. Alternatively, the mammal is a bovine. The antibody may
comprise a therapeutic polypeptide, or derivative or variant
thereof. The therapeutic polypeptide can be encoded by a
non-antibody sequence. The therapeutic polypeptide, or derivative
or variant thereof can be attached to the immunoglobulin domain.
The therapeutic polypeptide, or derivative or variant thereof may
be within the ultralong CDR3. Alternatively, the therapeutic
polypeptide, or derivative or variant thereof is conjugated to the
ultralong CDR3. In some instances, the therapeutic polypeptide is
GLP-1, Exendin-4, or derivative or variant thereof. The GLP-1 may
be a human GLP-1. The antibody may be an immunoconjugate as
described herein. The antibody can comprise one or more
immunoglobulin domains. The immunoglobulin domain may be an
immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an
immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain
can be an immunoglobulin heavy chain region or fragment thereof. In
some instances, the immunoglobulin domain is from a mammalian
antibody. Alternatively, the immunoglobulin domain is from a
chimeric antibody. The immunoglobulin domain may be from an
engineered antibody or recombinant antibody. The immunoglobulin
domain may be from a humanized, human engineered or fully human
antibody. The mammalian antibody can be a bovine antibody. The
mammalin antibody may be a human antibody. In other instances, the
mammalian antibody is a murine antibody. The ultralong CDR3 may be
35 amino acids in length or more. The ultralong CDR3 may comprise
at least 3 cysteine residues or more. The ultralong CDR3 may
comprise one or more cysteine motifs. The one or more cysteine
motifs may be based on or derived from SEQ ID NOS: 45-156. The one
or more cysteine motifs may be based on or derived from SEQ ID NOS:
45-99. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 100-135. The one or more cysteine motifs may be
based on or derived from SEQ ID NOS: 136-156. The ultralong CDR3
comprises at least a portion of a knob domain. The knob domain may
comprise a conserved motif within the knob domain of an ultralong
CDR3. For example, the knob domain may comprise a cysteine motif
disclosed herein. The GLP-1, Exendin-4, or a derivative or variant
thereof can be attached to the knob domain. Alternatively, or
additionally, the ultralong CDR3 comprises at least a portion of a
stalk domain. The stalk domain may comprise a conserved motif
within the stalk domain of an ultralong CDR3. The conserved motif
within the stalk domain of the ultralong CDR3 may comprise a
polypeptide sequence based on or derived from SEQ ID NOS: 157-307
and SEQ ID NOS: 333-336. The conserved motif with the stalk domain
of the ultralong CDR3 may comprise a polypeptide sequence based on
or derived from SEQ ID NOS: 157-224. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 157-161. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 223-234. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 235-239. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 296-299. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 300-303. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from a first sequence selected from the derived from SEQ ID
NOS: 300-303. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence basegroup
comprising SEQ ID NOS: 157-234 and a second sequence selected from
the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
The antibodies disclosed herein may comprise 2 or more, 3 or more,
4 or more, 5 or more sequences based on or derived from SEQ ID NOS:
157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalk
domain of the ultralong CDR3 may comprise a polypeptide sequence
based on or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS:
333-336. For example, the stalk domain may comprise a T(S/T)VHQ
motif. The GLP-1, Exendin-4, or a derivative or variant thereof can
be attached to the stalk domain. In some instances, the antibody,
antibody fragment or immunoglobulin construct further comprises a
linker. The linker can attach GLP-1, Exendin-4, or a derivative or
variant thereof to the immunoglobulin domain or fragment thereof.
In other instances, the linker attaches GLP-1, Exendin-4, or a
derivative or variant thereof to the knob domain or stalk domain.
In some instances, the CNS disorder is Alzheimer's disease (AD).
Additional CNS disorders include, but are not limited to,
encephalitis, meningitis, tropical spastic paraparesis, arachnoid
cysts, Huntington's disease, locked-in syndrome, Parkinson's
disease, Tourette's, and multiple sclerosis.
[0655] Provided herein is a method of preventing or treating a
disease or condition which benefits from a GLP-1R and/or glucagon
receptor (GCGR) agonist in a subject in need thereof comprising
administering a composition comprising one or more antibodies
comprising an ultralong CDR3 as disclosed herein to said subject.
The composition can further comprise a pharmaceutically acceptable
carrier. The subject may be a mammal. The mammal may be a human.
Alternatively, the mammal is a bovine. The antibody may comprise a
therapeutic polypeptide, or derivative or variant thereof. The
therapeutic polypeptide can be encoded by a non-antibody sequence.
The therapeutic polypeptide, or derivative or variant thereof can
be attached to the immunoglobulin domain. The therapeutic
polypeptide, or derivative or variant thereof may be within the
ultralong CDR3. Alternatively, the therapeutic polypeptide, or
derivative or variant thereof is conjugated to the ultralong CDR3.
In some instances, the therapeutic polypeptide is GLP-1, Exendin-4,
or derivative or variant thereof. The GLP-1 may be a human GLP-1.
The antibody may be an immunoconjugate as described herein. The
antibody can comprise one or more immunoglobulin domains. The
immunoglobulin domain may be an immunoglobulin A, an immunoglobulin
D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin
M. The immunoglobulin domain can be an immunoglobulin heavy chain
region or fragment thereof. In some instances, the immunoglobulin
domain is from a mammalian antibody. Alternatively, the
immunoglobulin domain is from a chimeric antibody. The
immunoglobulin domain may be from an engineered antibody or
recombinant antibody. The immunoglobulin domain may be from a
humanized, human engineered or fully human antibody. The mammalian
antibody can be a bovine antibody. The mammalin antibody may be a
human antibody. In other instances, the mammalian antibody is a
murine antibody. The ultralong CDR3 may be 35 amino acids in length
or more. The ultralong CDR3 may comprise at least 3 cysteine
residues or more. The ultralong CDR3 may comprise one or more
cysteine motifs. The one or more cysteine motifs may be based on or
derived from SEQ ID NOS: 45-156. The one or more cysteine motifs
may be based on or derived from SEQ ID NOS: 45-99. The one or more
cysteine motifs may be based on or derived from SEQ ID NOS:
100-135. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least a
portion of a knob domain. The knob domain may comprise a conserved
motif within the knob domain of an ultralong CDR3. For example, the
knob domain may comprise a cysteine motif disclosed herein. The
GLP-1, Exendin-4, or a derivative or variant thereof can be
attached to the knob domain. Alternatively, or additionally, the
ultralong CDR3 comprises at least a portion of a stalk domain. The
stalk domain may comprise a conserved motif within the stalk domain
of an ultralong CDR3. The conserved motif within the stalk domain
of the ultralong CDR3 may comprise a polypeptide sequence based on
or derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 157-224. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 157-161. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 223-234. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 235-239. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 296-299. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from a first
sequence selected from the derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence basegroup comprising SEQ ID NOS:
157-234 and a second sequence selected from the group comprising
SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336. The antibodies
disclosed herein may comprise 2 or more, 3 or more, 4 or more, 5 or
more sequences based on or derived from SEQ ID NOS: 157-307 and SEQ
ID NOS: 333-336. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. For
example, the stalk domain may comprise a T(S/T)VHQ motif. The
GLP-1, Exendin-4, or a derivative or variant thereof can be
attached to the stalk domain. In some instances, the antibody,
antibody fragment or immunoglobulin construct further comprises a
linker. The linker can attach GLP-1, Exendin-4, or a derivative or
variant thereof to the immunoglobulin domain or fragment thereof.
In other instances, the linker attaches GLP-1, Exendin-4, or a
derivative or variant thereof to the knob domain or stalk domain.
The disease or condition can be a metabolic disease or disorder. In
some instances, the disease or condition is diabetes. In other
instances, the disease or condition is obesity. Additional diseases
and/or conditions which benefit from a GLP-1R and/or GCGR agonist
include, but are not limited to, dyslipidemia, cardiovascular and
fatty liver diseases.
[0656] Provided herein is a method of preventing or treating a
blood disorder in a subject in need thereof comprising
administering a composition comprising one or more antibodies
comprising an ultralong CDR3 as disclosed herein to said subject.
The composition can further comprise a pharmaceutically acceptable
carrier. The subject may be a mammal. The mammal may be a human.
Alternatively, the mammal is a bovine. The antibody may comprise a
therapeutic polypeptide, or derivative or variant thereof. The
therapeutic polypeptide can be encoded by a non-antibody sequence.
The therapeutic polypeptide, or derivative or variant thereof can
be attached to the immunoglobulin domain. The therapeutic
polypeptide, or derivative or variant thereof may be within the
ultralong CDR3. Alternatively, the therapeutic polypeptide, or
derivative or variant thereof is conjugated to the ultralong CDR3.
In some instances, the therapeutic polypeptide is erythropoietin,
GMCSF, or derivative or variant thereof. The erythropoietin may be
a human erythropoietin. The GMCSF may be a human GMCSF. The
antibody may be an immunoconjugate as described herein. The
antibody can comprise one or more immunoglobulin domains. The
immunoglobulin domain may be an immunoglobulin A, an immunoglobulin
D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin
M. The immunoglobulin domain can be an immunoglobulin heavy chain
region or fragment thereof. In some instances, the immunoglobulin
domain is from a mammalian antibody. Alternatively, the
immunoglobulin domain is from a chimeric antibody. The
immunoglobulin domain may be from an engineered antibody or
recombinant antibody. The immunoglobulin domain may be from a
humanized, human engineered or fully human antibody. The mammalian
antibody can be a bovine antibody. The mammalin antibody may be a
human antibody. In other instances, the mammalian antibody is a
murine antibody. The ultralong CDR3 may be 35 amino acids in length
or more. The ultralong CDR3 may comprise at least 3 cysteine
residues or more. The ultralong CDR3 may comprise one or more
cysteine motifs. The one or more cysteine motifs may be based on or
derived from SEQ ID NOS: 45-156. The one or more cysteine motifs
may be based on or derived from SEQ ID NOS: 45-99. The one or more
cysteine motifs may be based on or derived from SEQ ID NOS:
100-135. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least a
portion of a knob domain. The knob domain may comprise a conserved
motif within the knob domain of an ultralong CDR3. For example, the
knob domain may comprise a cysteine motif disclosed herein. The
erythropoietin, GMCSF, or a derivative or variant thereof can be
attached to the knob domain. Alternatively, or additionally, the
ultralong CDR3 comprises at least a portion of a stalk domain. The
stalk domain may comprise a conserved motif within the stalk domain
of an ultralong CDR3. The conserved motif within the stalk domain
of the ultralong CDR3 may comprise a polypeptide sequence based on
or derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 157-224. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 157-161. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 223-234. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 235-239. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 296-299. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from a first
sequence selected from the derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence basegroup comprising SEQ ID NOS:
157-234 and a second sequence selected from the group comprising
SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336. The antibodies
disclosed herein may comprise 2 or more, 3 or more, 4 or more, 5 or
more sequences based on or derived from SEQ ID NOS: 157-307 and SEQ
ID NOS: 333-336. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. For
example, the stalk domain may comprise a T(S/T)VHQ motif. The
erythropoietin, GMCSF, or a derivative or variant thereof can be
attached to the stalk domain. In some instances, the antibody,
antibody fragment or immunoglobulin construct further comprises a
linker. The linker can attach erythropoietin, GMCSF, or a
derivative or variant thereof to the immunoglobulin domain or
fragment thereof. In other instances, the linker attaches
erythropoietin, GMCSF, or a derivative or variant thereof to the
knob domain or stalk domain. In some instances, the blood disorder
is anemia. Examples of anemia include, but are not limited to,
herditary xerocytosis, congenital dyserythropoietic anemia, Rh null
disease, infectious mononucleosis related anemia, drugs-related
anemia, aplastic anemia, microcytic anemia, macrocytic anemia,
normocytic anemia, hemolytic anemia, poikilocytic anemia,
spherocytic anemia, drepanocytic anemia, normochromic anemia,
hyperchromic anemia, hypochromic anemia, macrocytic-normochromic
anemia, microcytic-hypochromic anemia, normocytic-normochromic
anemia, iron-deficiency anemia, pernicious anemia,
folate-deficiency anemia, thalassemia, sideroblastic anemia,
posthemorrhagic anemia, sickle cell anemia, chronic anemia,
achrestic anemia, autoimmune haemolytic anemia, Cooley's anemia,
drug-induced immune haemolytic anemia, erythroblastic anemia,
hypoplastic anemia, Diamond-Blackfan anemia, Pearson's anemia,
transient anemia, Fanconi's anemia, Lederer's anemia, myelpathic
anemia, nutritional anemia, spur-cell anemia, Von Jaksh's anemia,
sideroblatic anemia, sideropenic anemia, alpha thalassemia, beta
thalassemia, hemoglobin h disease, acute acquired hemolytic anemia,
warm autoimmune hemolytic anemia, cold autoimmune hemolytic anemia,
primary cold autoimmune hemolytic anemia, secondary cold autoimmune
hemolytic anemia, secondary autoimmune hemolytic anemia, primary
autoimmune hemolytic anemia, x-linked sideroblastic anemia,
pyridoxine-responsive anemia, nutritional sideroblastic anemia,
pyridoxine deficiency-induced sideroblastic anemia, copper
deficiency-induced sideroblastic anemia, cycloserine-induced
sideroblastic anemia, chloramphenicol-induced sideroblastic anemia,
ethanol-induced sideroblastic anemia, isoniazid-induced
sideroblastic anemia, drug-induced sideroblastic anemia,
toxin-induced sideroblastic anemia, microcytic hyperchromic anemia,
macrocytic hyperchromic anemia, megalocytic-normochromic anemia,
drug-induced immune hemolytic anemia, non-hereditary spherocytic
anemia, inherited spherocytic anemia, and congenital spherocytic
anemia. In other instances, the blood disorder is malaria.
Alternatively, the blood disorder is lymphoma, leukemia, multiple
myeloma, or myelodysplastic syndrome. In some instances, the blood
disorder is neutropenia, Shwachmann-Daimond syndrome, Kostmann
syndrome, chronic granulomatous disease, leukocyte adhesion
deficiency, meyloperoxidase deficiency, or Chediak Higashi
syndrome.
[0657] Provided herein is a method of preventing or treating a
disease or disorder which benefitis from stimulating or increasing
white blood cell production in a subject in need thereof comprising
administering a composition comprising one or more antibodies
comprising an ultralong CDR3 as disclosed herein to said subject.
The composition can further comprise a pharmaceutically acceptable
carrier. The subject may be a mammal. The mammal may be a human.
Alternatively, the mammal is a bovine. The antibody may comprise a
therapeutic polypeptide, or derivative or variant thereof. The
therapeutic polypeptide can be encoded by a non-antibody sequence.
The therapeutic polypeptide, or derivative or variant thereof can
be attached to the immunoglobulin domain. The therapeutic
polypeptide, or derivative or variant thereof may be within the
ultralong CDR3. Alternatively, the therapeutic polypeptide, or
derivative or variant thereof is conjugated to the ultralong CDR3.
In some instances, the therapeutic polypeptide is GMCSF, or
derivative or variant thereof. The GMCSF may be a human GMCSF. The
antibody may be an immunoconjugate as described herein. The
antibody can comprise one or more immunoglobulin domains. The
immunoglobulin domain may be an immunoglobulin A, an immunoglobulin
D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin
M. The immunoglobulin domain can be an immunoglobulin heavy chain
region or fragment thereof. In some instances, the immunoglobulin
domain is from a mammalian antibody. Alternatively, the
immunoglobulin domain is from a chimeric antibody. The
immunoglobulin domain may be from an engineered antibody or
recombinant antibody. The immunoglobulin domain may be from a
humanized, human engineered or fully human antibody. The mammalian
antibody can be a bovine antibody. The mammalin antibody may be a
human antibody. In other instances, the mammalian antibody is a
murine antibody. The ultralong CDR3 may be 35 amino acids in length
or more. The ultralong CDR3 may comprise at least 3 cysteine
residues or more. The ultralong CDR3 may comprise one or more
cysteine motifs. The one or more cysteine motifs may be based on or
derived from SEQ ID NOS: 45-156. The one or more cysteine motifs
may be based on or derived from SEQ ID NOS: 45-99. The one or more
cysteine motifs may be based on or derived from SEQ ID NOS:
100-135. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least a
portion of a knob domain. The knob domain may comprise a conserved
motif within the knob domain of an ultralong CDR3. For example, the
knob domain may comprise a cysteine motif disclosed herein. The
GMCSF, or a derivative or variant thereof can be attached to the
knob domain. Alternatively, or additionally, the ultralong CDR3
comprises at least a portion of a stalk domain. The stalk domain
may comprise a conserved motif within the stalk domain of an
ultralong CDR3. The conserved motif within the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 157-224. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 157-161. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 223-234. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 235-239. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 296-299. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from a first
sequence selected from the derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence basegroup comprising SEQ ID NOS:
157-234 and a second sequence selected from the group comprising
SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336. The antibodies
disclosed herein may comprise 2 or more, 3 or more, 4 or more, 5 or
more sequences based on or derived from SEQ ID NOS: 157-307 and SEQ
ID NOS: 333-336. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. For
example, the stalk domain may comprise a T(S/T)VHQ motif. The
GMCSF, or a derivative or variant thereof can be attached to the
stalk domain. In some instances, the antibody, antibody fragment or
immunoglobulin construct further comprises a linker. The linker can
attach GMCSF, or a derivative or variant thereof to the
immunoglobulin domain or fragment thereof. In other instances, the
linker attaches GMCSF, or a derivative or variant thereof to the
knob domain or stalk domain. In some instances, the disese or
disorder is neutropenia, Shwachmann-Daimond syndrome, Kostmann
syndrome, chronic granulomatous disease, leukocyte adhesion
deficiency, meyloperoxidase deficiency, or Chediak Higashi
syndrome.
[0658] Provided herein is a method of preventing or treating a
disease or disorder which benefitis from stimulating or increasing
red blood cell production in a subject in need thereof comprising
administering a composition comprising one or more antibodies
comprising an ultralong CDR3 as disclosed herein to said subject.
The composition can further comprise a pharmaceutically acceptable
carrier. The subject may be a mammal. The mammal may be a human.
Alternatively, the mammal is a bovine. The antibody may comprise a
therapeutic polypeptide, or derivative or variant thereof. The
therapeutic polypeptide can be encoded by a non-antibody sequence.
The therapeutic polypeptide, or derivative or variant thereof can
be attached to the immunoglobulin domain. The therapeutic
polypeptide, or derivative or variant thereof may be within the
ultralong CDR3. Alternatively, the therapeutic polypeptide, or
derivative or variant thereof is conjugated to the ultralong CDR3.
In some instances, the therapeutic polypeptide is erythropoietin,
or derivative or variant thereof. The erythropoietin may be a human
erythropoietin. The antibody may be an immunoconjugate as described
herein. The antibody can comprise one or more immunoglobulin
domains. The immunoglobulin domain may be an immunoglobulin A, an
immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an
immunoglobulin M. The immunoglobulin domain can be an
immunoglobulin heavy chain region or fragment thereof. In some
instances, the immunoglobulin domain is from a mammalian antibody.
Alternatively, the immunoglobulin domain is from a chimeric
antibody. The immunoglobulin domain may be from an engineered
antibody or recombinant antibody. The immunoglobulin domain may be
from a humanized, human engineered or fully human antibody. The
mammalian antibody can be a bovine antibody. The mammalin antibody
may be a human antibody. In other instances, the mammalian antibody
is a murine antibody. The ultralong CDR3 may be 35 amino acids in
length or more. The ultralong CDR3 may comprise at least 3 cysteine
residues or more. The ultralong CDR3 may comprise one or more
cysteine motifs. The one or more cysteine motifs may be based on or
derived from SEQ ID NOS: 45-156. The one or more cysteine motifs
may be based on or derived from SEQ ID NOS: 45-99. The one or more
cysteine motifs may be based on or derived from SEQ ID NOS:
100-135. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least a
portion of a knob domain. The knob domain may comprise a conserved
motif within the knob domain of an ultralong CDR3. For example, the
knob domain may comprise a cysteine motif disclosed herein. The
erythropoietin, or a derivative or variant thereof can be attached
to the knob domain. Alternatively, or additionally, the ultralong
CDR3 comprises at least a portion of a stalk domain. The stalk
domain may comprise a conserved motif within the stalk domain of an
ultralong CDR3. The conserved motif within the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 157-224. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 157-161. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 223-234. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 235-239. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 296-299. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from a first
sequence selected from the derived from SEQ ID NOS: 300-303. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence basegroup comprising SEQ ID NOS:
157-234 and a second sequence selected from the group comprising
SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336. The antibodies
disclosed herein may comprise 2 or more, 3 or more, 4 or more, 5 or
more sequences based on or derived from SEQ ID NOS: 157-307 and SEQ
ID NOS: 333-336. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. For
example, the stalk domain may comprise a T(S/T)VHQ motif. The
erythropoietin, or a derivative or variant thereof can be attached
to the stalk domain. In some instances, the antibody, antibody
fragment or immunoglobulin construct further comprises a linker.
The linker can attach erythropoietin, or a derivative or variant
thereof to the immunoglobulin domain or fragment thereof. In other
instances, the linker attaches erythropoietin, or a derivative or
variant thereof to the knob domain or stalk domain. In some
instances, the disease or disorder is anemia.
[0659] Provided herein is a method of preventing or treating
obesity in a subject in need thereof comprising administering a
composition comprising one or more antibodies comprising an
ultralong CDR3 as disclosed herein to said subject. The composition
can further comprise a pharmaceutically acceptable carrier. The
subject may be a mammal. The mammal may be a human. Alternatively,
the mammal is a bovine. The antibody may comprise a therapeutic
polypeptide, or derivative or variant thereof. The therapeutic
polypeptide can be encoded by a non-antibody sequence. The
therapeutic polypeptide, or derivative or variant thereof can be
attached to the immunoglobulin domain. The therapeutic polypeptide,
or derivative or variant thereof may be within the ultralong CDR3.
Alternatively, the therapeutic polypeptide, or derivative or
variant thereof is conjugated to the ultralong CDR3. In some
instances, the therapeutic polypeptide is GLP-1, Exendin-4, FGF21,
or derivative or variant thereof. The GLP-1 may be a human GLP-1.
In some instances, the FGF21 is a human FGF21. The antibody may be
an immunoconjugate as described herein. The antibody can comprise
one or more immunoglobulin domains. The immunoglobulin domain may
be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E,
an immunoglobulin G, or an immunoglobulin M. The immunoglobulin
domain can be an immunoglobulin heavy chain region or fragment
thereof. In some instances, the immunoglobulin domain is from a
mammalian antibody. Alternatively, the immunoglobulin domain is
from a chimeric antibody. The immunoglobulin domain may be from an
engineered antibody or recombinant antibody. The immunoglobulin
domain may be from a humanized, human engineered or fully human
antibody. The mammalian antibody can be a bovine antibody. The
mammalin antibody may be a human antibody. In other instances, the
mammalian antibody is a murine antibody. The ultralong CDR3 may be
35 amino acids in length or more. The ultralong CDR3 may comprise
at least 3 cysteine residues or more. The ultralong CDR3 may
comprise one or more cysteine motifs. The one or more cysteine
motifs may be based on or derived from SEQ ID NOS: 45-156. The one
or more cysteine motifs may be based on or derived from SEQ ID NOS:
45-99. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 100-135. The one or more cysteine motifs may be
based on or derived from SEQ ID NOS: 136-156. The ultralong CDR3
comprises at least a portion of a knob domain. The knob domain may
comprise a conserved motif within the knob domain of an ultralong
CDR3. For example, the knob domain may comprise a cysteine motif
disclosed herein. The GLP-1, Exendin-4, FGF21, or a derivative or
variant thereof can be attached to the knob domain. Alternatively,
or additionally, the ultralong CDR3 comprises at least a portion of
a stalk domain. The stalk domain may comprise a conserved motif
within the stalk domain of an ultralong CDR3. The conserved motif
within the stalk domain of the ultralong CDR3 may comprise a
polypeptide sequence based on or derived from SEQ ID NOS: 157-307
and SEQ ID NOS: 333-336. The conserved motif with the stalk domain
of the ultralong CDR3 may comprise a polypeptide sequence based on
or derived from SEQ ID NOS: 157-224. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 157-161. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 223-234. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 235-239. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 296-299. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 300-303. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from a first sequence selected from the derived from SEQ ID
NOS: 300-303. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence basegroup
comprising SEQ ID NOS: 157-234 and a second sequence selected from
the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
The antibodies disclosed herein may comprise 2 or more, 3 or more,
4 or more, 5 or more sequences based on or derived from SEQ ID NOS:
157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalk
domain of the ultralong CDR3 may comprise a polypeptide sequence
based on or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS:
333-336. For example, the stalk domain may comprise a T(S/T)VHQ
motif. The GLP-1, Exendin-4, FGF21, or a derivative or variant
thereof can be attached to the stalk domain. In some instances, the
antibody, antibody fragment or immunoglobulin construct further
comprises a linker. The linker can attach GLP-1, Exendin-4, FGF21,
or a derivative or variant thereof to the immunoglobulin domain or
fragment thereof. In other instances, the linker attaches GLP-1,
Exendin-4, FGF21, or a derivative or variant thereof to the knob
domain or stalk domain.
[0660] Provided herein is a method of preventing or treating a pain
in a subject in need thereof comprising administering a composition
comprising one or more antibodies, antibody fragments, or
immunoglobulin constructs described herein to said subject. In some
instances, the subject is a mammal. In certain instances, the
mammal is a human. Alternatively, the mammal is a bovine. In some
instances, the one or more antibodies, antibody fragments, or
immunoglobulin constructs comprise a protoxin2 or a derivative or
variant thereof. Alternatively, or additionally, the one or more
antibodies, antibody fragments, or immunoglobulin constructs
comprise at least a portion of a CDR3H. The portion of the CDR3H
can be a stalk domain or knob domain in the CDR3H. In some
instances, the one or more antibodies, antibody fragments, or
immunoglobulin constructs further comprise a linker. The linker can
attach the protoxin2 or a derivative or variant thereof to the
portion of the CDR3H.
[0661] Provided herein is a method of preventing or treating a
disease or condition which benefits from modulating a sodium ion
channel in a subject in need thereof comprising administering a
composition comprising one or more antibodies, antibody fragments,
or immunoglobulin constructs described herein to said subject. In
some instances, the subject is a mammal. In certain instances, the
mammal is a human. Alternatively, the mammal is a bovine. In some
instances, the one or more antibodies, antibody fragments, or
immunoglobulin constructs comprise a protoxin2 or a derivative or
variant thereof. Alternatively, or additionally, the one or more
antibodies, antibody fragments, or immunoglobulin constructs
comprise at least a portion of a CDR3H. The portion of the CDR3H
can be a stalk domain or knob domain in the CDR3H. In some
instances, the one or more antibodies, antibody fragments, or
immunoglobulin constructs further comprise a linker. The linker can
attach the protoxin2 or a derivative or variant thereof to the
portion of the CDR3H. In some instances, the sodium ion channel is
a Na.sub.v channel. In some instances, the Na.sub.c channel is a
Na.sub.v1.7 channel. In some instances, modulating a sodium ion
channel comprises inhibiting or blocking a sodium ion channel. In
some instances, the disease or condition is Dravet Syndrome,
generalized epilepsy with febrile seizures plus (GEFS+),
paramyotonia congenital or erythromelalgia. In some instances, the
disease or condition is pain.
[0662] Provided herein is a method of preventing or treating a
disease or condition which benefits from modulating an acid sensing
ion channel (ASIC) in a subject in need thereof comprising
administering a composition comprising one or more antibodies,
antibody fragments, or immunoglobulin constructs described herein
to said subject. In some instances, the subject is a mammal. In
certain instances, the mammal is a human. Alternatively, the mammal
is a bovine. In some instances, the one or more antibodies,
antibody fragments, or immunoglobulin constructs comprise a
protoxin2 or a derivative or variant thereof. Alternatively, or
additionally, the one or more antibodies, antibody fragments, or
immunoglobulin constructs comprise at least a portion of a CDR3H.
The portion of the CDR3H can be a stalk domain or knob domain in
the CDR3H. In some instances, the one or more antibodies, antibody
fragments, or immunoglobulin constructs further comprise a linker.
The linker can attach the protoxin2 or a derivative or variant
thereof to the portion of the CDR3H. In some instances, modulating
an ASIC comprises inhibiting or blocking the ASIC. In some
instances, the disease or condition is a central nervous system
disorder. In other instances, the disease or condition is pain.
[0663] Provided herein is a method of preventing or treating a
pathogenic infection in a subject in need thereof comprising
administering a composition comprising one or more antibodies
comprising an ultralong CDR3 as disclosed herein to said subject.
The composition can further comprise a pharmaceutically acceptable
carrier. The subject may be a mammal. The mammal may be a human.
Alternatively, the mammal is a bovine. The antibody may comprise a
therapeutic polypeptide, or derivative or variant thereof. The
therapeutic polypeptide can be encoded by a non-antibody sequence.
The therapeutic polypeptide, or derivative or variant thereof can
be attached to the immunoglobulin domain. The therapeutic
polypeptide, or derivative or variant thereof may be within the
ultralong CDR3. Alternatively, the therapeutic polypeptide, or
derivative or variant thereof is conjugated to the ultralong CDR3.
In some instances, the therapeutic polypeptide is beta-interferon,
or derivative or variant thereof. The antibody may be an
immunoconjugate as described herein. The antibody can comprise one
or more immunoglobulin domains. The immunoglobulin domain may be an
immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an
immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain
can be an immunoglobulin heavy chain region or fragment thereof. In
some instances, the immunoglobulin domain is from a mammalian
antibody. Alternatively, the immunoglobulin domain is from a
chimeric antibody. The immunoglobulin domain may be from an
engineered antibody or recombinant antibody. The immunoglobulin
domain may be from a humanized, human engineered or fully human
antibody. The mammalian antibody can be a bovine antibody. The
mammalin antibody may be a human antibody. In other instances, the
mammalian antibody is a murine antibody. The ultralong CDR3 may be
35 amino acids in length or more. The ultralong CDR3 may comprise
at least 3 cysteine residues or more. The ultralong CDR3 may
comprise one or more cysteine motifs. The one or more cysteine
motifs may be based on or derived from SEQ ID NOS: 45-156. The one
or more cysteine motifs may be based on or derived from SEQ ID NOS:
45-99. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 100-135. The one or more cysteine motifs may be
based on or derived from SEQ ID NOS: 136-156. The ultralong CDR3
comprises at least a portion of a knob domain. The knob domain may
comprise a conserved motif within the knob domain of an ultralong
CDR3. For example, the knob domain may comprise a cysteine motif
disclosed herein. The beta-interferon, or a derivative or variant
thereof can be attached to the knob domain. Alternatively, or
additionally, the ultralong CDR3 comprises at least a portion of a
stalk domain. The stalk domain may comprise a conserved motif
within the stalk domain of an ultralong CDR3. The conserved motif
within the stalk domain of the ultralong CDR3 may comprise a
polypeptide sequence based on or derived from SEQ ID NOS: 157-307
and SEQ ID NOS: 333-336. The conserved motif with the stalk domain
of the ultralong CDR3 may comprise a polypeptide sequence based on
or derived from SEQ ID NOS: 157-224. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 157-161. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 223-234. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 235-239. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 296-299. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 300-303. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from a first sequence selected from the derived from SEQ ID
NOS: 300-303. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence basegroup
comprising SEQ ID NOS: 157-234 and a second sequence selected from
the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
The antibodies disclosed herein may comprise 2 or more, 3 or more,
4 or more, 5 or more sequences based on or derived from SEQ ID NOS:
157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalk
domain of the ultralong CDR3 may comprise a polypeptide sequence
based on or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS:
333-336. For example, the stalk domain may comprise a T(S/T)VHQ
motif. The beta-interferon, or a derivative or variant thereof can
be attached to the stalk domain. In some instances, the antibody,
antibody fragment or immunoglobulin construct further comprises a
linker. The linker can attach beta-interferon, or a derivative or
variant thereof to the immunoglobulin domain or fragment thereof.
In other instances, the linker attaches beta-interferon, or a
derivative or variant thereof to the knob domain or stalk domain.
In some instances, the pathogenic infection is a viral, bacterial,
fungal, or parasitic infection. In some instances, the viral
infection is a herpes virus.
[0664] Provided herein is a method of preventing or treating a
cancer in a subject in need thereof comprising administering a
composition comprising one or more antibodies comprising an
ultralong CDR3 as disclosed herein to said subject. The composition
can further comprise a pharmaceutically acceptable carrier. The
subject may be a mammal. The mammal may be a human. Alternatively,
the mammal is a bovine. The antibody may comprise a therapeutic
polypeptide, or derivative or variant thereof. The therapeutic
polypeptide can be encoded by a non-antibody sequence. The
therapeutic polypeptide, or derivative or variant thereof can be
attached to the immunoglobulin domain. The therapeutic polypeptide,
or derivative or variant thereof may be within the ultralong CDR3.
Alternatively, the therapeutic polypeptide, or derivative or
variant thereof is conjugated to the ultralong CDR3. In some
instances, the therapeutic polypeptide is beta-interferon, or
derivative or variant thereof. The antibody may be an
immunoconjugate as described herein. The antibody can comprise one
or more immunoglobulin domains. The immunoglobulin domain may be an
immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an
immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain
can be an immunoglobulin heavy chain region or fragment thereof. In
some instances, the immunoglobulin domain is from a mammalian
antibody. Alternatively, the immunoglobulin domain is from a
chimeric antibody. The immunoglobulin domain may be from an
engineered antibody or recombinant antibody. The immunoglobulin
domain may be from a humanized, human engineered or fully human
antibody. The mammalian antibody can be a bovine antibody. The
mammalin antibody may be a human antibody. In other instances, the
mammalian antibody is a murine antibody. The ultralong CDR3 may be
35 amino acids in length or more. The ultralong CDR3 may comprise
at least 3 cysteine residues or more. The ultralong CDR3 may
comprise one or more cysteine motifs. The one or more cysteine
motifs may be based on or derived from SEQ ID NOS: 45-156. The one
or more cysteine motifs may be based on or derived from SEQ ID NOS:
45-99. The one or more cysteine motifs may be based on or derived
from SEQ ID NOS: 100-135. The one or more cysteine motifs may be
based on or derived from SEQ ID NOS: 136-156. The ultralong CDR3
comprises at least a portion of a knob domain. The knob domain may
comprise a conserved motif within the knob domain of an ultralong
CDR3. For example, the knob domain may comprise a cysteine motif
disclosed herein. The beta-interferon, or a derivative or variant
thereof can be attached to the knob domain. Alternatively, or
additionally, the ultralong CDR3 comprises at least a portion of a
stalk domain. The stalk domain may comprise a conserved motif
within the stalk domain of an ultralong CDR3. The conserved motif
within the stalk domain of the ultralong CDR3 may comprise a
polypeptide sequence based on or derived from SEQ ID NOS: 157-307
and SEQ ID NOS: 333-336. The conserved motif with the stalk domain
of the ultralong CDR3 may comprise a polypeptide sequence based on
or derived from SEQ ID NOS: 157-224. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 157-161. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 223-234. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from SEQ ID NOS: 235-239. The conserved motif with the
stalk domain of the ultralong CDR3 may comprise a polypeptide
sequence based on or derived from SEQ ID NOS: 296-299. The
conserved motif with the stalk domain of the ultralong CDR3 may
comprise a polypeptide sequence based on or derived from SEQ ID
NOS: 300-303. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence based on or
derived from a first sequence selected from the derived from SEQ ID
NOS: 300-303. The conserved motif with the stalk domain of the
ultralong CDR3 may comprise a polypeptide sequence basegroup
comprising SEQ ID NOS: 157-234 and a second sequence selected from
the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
The antibodies disclosed herein may comprise 2 or more, 3 or more,
4 or more, 5 or more sequences based on or derived from SEQ ID NOS:
157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalk
domain of the ultralong CDR3 may comprise a polypeptide sequence
based on or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS:
333-336. For example, the stalk domain may comprise a T(S/T)VHQ
motif. The beta-interferon, or a derivative or variant thereof can
be attached to the stalk domain. In some instances, the antibody,
antibody fragment or immunoglobulin construct further comprises a
linker. The linker can attach beta-interferon, or a derivative or
variant thereof to the immunoglobulin domain or fragment thereof.
In other instances, the linker attaches beta-interferon, or a
derivative or variant thereof to the knob domain or stalk domain.
In some instances, the cancer is a hematological malignancy. The
hematological malignancy can be a leukemia or lymphoma. In some
instances, the hematological malignancy is a B-cell lymphoma,
T-cell lymphoma, follicular lymphoma, marginal zone lymphoma, hairy
cell leukemia, chronic myeloid leukemia, mantle cell lymphoma,
nodular lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma,
chronic lymphocytic leukemia, or small lymphocytic leukemia.
[0665] Provided herein is a method of preventing or treating a
disease in a mammal in need thereof comprising administering a
pharmaceutical composition described herein to said mammal. In some
embodiments, the disease is an infectious disease. In certain
embodiments, the infectious disease is mastitis. In some
embodiments, the infectious disease is a respiratory disease. In
certain embodiments, the respiratory disease is bovine respiratory
disease of shipping fever. In certain embodiments, the mammal in
need is a dairy animal selected from a list comprising cow, camel,
donkey, goat, horse, reindeer, sheep, water buffalo, moose and yak.
In some embodiments, the mammal in need is bovine.
[0666] Provided is a method of preventing or treating mastitis in a
dairy animal, comprising providing to said dairy animal an
effective amount of a composition comprising: an immunoglobulin
construct comprising a heavy chain polypeptide comprising a
sequence selected from SEQ ID NO: 25 and SEQ ID NO: 26; and a light
chain polypeptide comprising the sequence of SEQ ID NO: 23. In an
embodiment is provided a method of preventing or treating mastitis
in a dairy animal, comprising providing to said dairy animal an
effective amount of a composition comprising: an immunoglobulin
construct comprising a heavy chain polypeptide comprising a
polypeptide sequence encoded by the DNA selected from SEQ ID NO: 4
and SEQ ID NO: 5; and a light chain polypeptide comprising a
polypeptide sequence encoded by the DNA of SEQ ID NO: 1. In some
embodiments, the dairy animal is a cow or a water buffalo. Provided
are methods of treatment, inhibition and prevention by
administration to a subject of an effective amount of an antibody
or pharmaceutical composition described herein. The antibody may be
substantially purified (e.g., substantially free from substances
that limit its effect or produce undesired side-effects). The
subject can be an animal, including but not limited to animals such
as cows, pigs, sheep, goats, rabbits, horses, chickens, cats, dogs,
mice, etc. The subject can be a mammal. In some instances, the
subject is a human. Alternatively, the subject is a bovine.
[0667] Various delivery systems are known and can be used to
administer an antibody formulation described herein, e.g.,
encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the compound,
receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem.
262:4429-4432 (1987)), construction of a nucleic acid as part of a
retroviral or other vector, etc. Methods of introduction include
but are not limited to intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes.
The compounds or compositions may be administered by any convenient
route, for example by infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings (e.g., oral mucosa,
rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. Administration can
be systemic or local. In addition, in certain embodiments, it is
desirable to introduce the heteromultimer compositions described
herein into the central nervous system by any suitable route,
including intraventricular and intrathecal injection;
intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir.
[0668] In a specific embodiment, it is desirable to administer the
antibody, or compositions described herein locally to the area in
need of treatment; this may be achieved by, for example, and not by
way of limitation, local infusion, topical application, e.g., in
conjunction with a wound dressing after surgery, by injection, by
means of a catheter, by means of a suppository, or by means of an
implant, said implant being of a porous, non-porous, or gelatinous
material, including membranes, such as sialastic membranes, or
fibers. Preferably, when administering a protein, including an
antibody, of the invention, care must be taken to use materials to
which the protein does not absorb.
[0669] In another embodiment, the antibody or pharmaceutical
composition is delivered in a vesicle, in particular a liposome
(see Langer, Science 249:1527-1533 (1990); Treat et al., in
Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365
(1989); Lopez-Berestein, ibid., pp. 317-327; see generally
ibid.)
[0670] In yet another embodiment, the heteromultimers or
composition can be delivered in a controlled release system. In one
embodiment, a pump may be used (see Langer, supra; Sefton, CRC
Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery
88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In
another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,
New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev.
Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190
(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al.,
J. Neurosurg. 71:105 (1989)). In yet another embodiment, a
controlled release system can be placed in proximity of the
therapeutic target, e.g., the brain, thus requiring only a fraction
of the systemic dose (see, e.g., Goodson, in Medical Applications
of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other
controlled release systems are discussed in the review by Langer
(Science 249:1527-1533 (1990)).
[0671] In a specific embodiment comprising a nucleic acid encoding
a antibody described herein, the nucleic acid can be administered
in vivo to promote expression of its encoded protein, by
constructing it as part of an appropriate nucleic acid expression
vector and administering it so that it becomes intracellular, e.g.,
by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by
direct injection, or by use of microparticle bombardment (e.g., a
gene gun; Biolistic, Dupont), or coating with lipids or
cell-surface receptors or transfecting agents, or by administering
it in linkage to a homeobox-like peptide which is known to enter
the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA
88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be
introduced intracellularly and incorporated within host cell DNA
for expression, by homologous recombination.
[0672] In some embodiments are crystals based on or derived from
the antibodies disclosed herein. The crystals may have a space
group P2.sub.12.sub.12.sub.1. In some instances, the crystal has
the unit cell dimensions of "a" between about 40 to 80 angstroms,
between 45 to about 75 angstroms, or between about 50 to about 75
angstroms; "b" between about 40 to 140 angstroms, between about 50
to about 130 angstroms, between about 55 to about 130 angstroms;
and "c" between 100 to about 350 angstroms, between 120 to about
340 angstroms, or between about 125 to about 330 angstroms.
Alternatively, the crystal has the unit cell dimensions of "a"
greater than or equal to 40, 50, 60, 70, or 80 angstroms; "b"
greater than or equal to 40, 50, 60, 70, 80, 90, 100, 110, 120, or
130 angstroms; and "c" greater than or equal to 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
270, 280, 290, 300, 310, 320, 330, or 340 angstroms.
[0673] The crystal may comprise a bovine antibody or portion
thereof. The crystal may comprise a Fab fragment based on or
derived from a bovine antibody. The crystal may comprise a
non-antibody sequence, linker, cleave site, non-bovine sequence, or
a combination thereof. The crystal may be an isolated crystal. The
antibody may be based on or derived from the peptide sequence of
any of SEQ ID NOS: 1-44. The antibody may comprise at least a
portion of a heavy chain. The portion of the heavy chain may
comprise the peptide sequence of any of SEQ ID NOS: 24-44. The
antibody may comprise at least a portion of a heavy chain. The
portion of the heavy chain may be encoded by a DNA sequence based
on or derived from the DNA sequence of any of SEQ ID NOS: 2-22. The
antibody may comprise at least a portion of a light chain. The
portion of the light chain may comprise the peptide sequence of SEQ
ID NO: 23. The antibody may comprise at least a portion of a heavy
chain. The portion of the heavy chain may be encoded by a DNA
sequence based on or derived from the DNA sequence of SEQ ID NOS:
1.
[0674] In some embodiments, is an isolated crystal comprising a
bovine antibody Fab fragment comprising SEQ ID NO: 24 and SEQ ID
NO: 23, wherein the crystal has a space group
P2.sub.12.sub.12.sub.1 and unit cell dimensions of a=71.4
angstroms, b=127.6 angstroms and c=127.9 angstroms.
[0675] In some embodiments, is an isolated crystal comprising a
bovine antibody Fab fragment comprising SEQ ID NO: 340 and SEQ ID
NO: 341, wherein the crystal has a space group
P2.sub.12.sub.12.sub.1 and unit cell dimensions of a=54.6
angstroms, b=53.7 angstroms and c=330.5 angstroms.
Example 1
Purification and Crystallization of Antibodies Comprising an
Ultralong CDR3
[0676] An antibody that comprises an ultralong CDR3 including, for
example, an antibody generated by any of the examples described
herein, may be purified and subsequently crystallized to determine
the structure of the antibody.
[0677] A. Purification:
[0678] Genes encoding the heavy and light chain Fab regions of
BLV1H12 and BLV5B8 were generated by gene synthesis (GenScript,
Piscataway, N.J.). A DNA fragment derived from the promoter region
of pFastBacDual (Invitrogen) was fused to the gp67 and the honey
bee mellitin (HBM) signal peptides by overlap PCR, yielding a
fragment with head-to-head p10 and polyhedrin promoters upstream of
the HBM and gp67 signal peptides, respectively (i.e.,
HBM-p10-pPolyH-gp67). Bovine Fab heavy and light chain regions were
fused to the promoter-signal peptide cassette by overlap PCR (heavy
chain downstream of pPolyH-gp67 and light chain downstream of
p10-HBM), and ligated into the SfiI sites of pDCE361, a derivative
of pFastBacDual. Next, a His6-tag was introduced at the C-terminus
of the heavy chain to facilitate purification. The resulting
baculovirus transfer vectors were used to generate recombinant
bacmids using the Bac-to-Bac system (Invitrogen) and virus was
rescued by transfecting purified bacmid DNA into Sf9 cells using
Cellfectin II (Invitrogen). Both Fab proteins were produced by
infecting suspension cultures of Sf9 cells with recombinant
baculovirus at an MOI of 5-10 and incubating at 28.degree. C. with
shaking at 110 RPM. After 72 hours, the cultures were clarified by
two rounds of centrifugation at 2000 g and 10,000 g at 4.degree. C.
The supernatant, containing secreted, soluble Fab were then
concentrated and buffer exchanged into 1.times.PBS, pH 7.4. After
metal affinity chromatography using Ni-NTA resin, Fabs were
purified by protein G affinity chromatography (GE Healthcare),
cation exchange chromatography (MonoS, GE healthcare), and gel
filtration (Superdex200, GE Healthcare).
[0679] B. Crystallization and Structure Determination
[0680] Gel filtration fractions containing the bovine Fabs were
concentrated to .about.10 mg/mL in 10 mM Tris, pH 8.0 and 50 mM
NaCl. Initial crystallization trials were set up using the
automated Rigaku Crystalmation robotic system at the Joint Center
for Structural Genomics. Several hits were obtained for BLV1H12 and
BLV5B8, and crystals used for data collection were grown by the
sitting drop vapor diffusion method with a reservoir solution (100
.mu.L) containing 0.27 M potassium citrate and 22% PEG 3350
(BLV1H12) and 0.2 M di-sodium tartrate and 20% PEG 3350 (BLV5B8).
Drops consisting of 100 nL protein+100 mL precipitant were set up
at 20.degree. C., and crystals appeared within 3-7 days. The
resulting crystals were cryoprotected using well solution
supplemented with 15% ethylene glycol then flash cooled and stored
in liquid nitrogen until data collection.
[0681] Diffraction data were then collected on the GM/CA-CAT 23ID-D
beamline at the Advanced Photon Source at Argonne National
Laboratory (BLV1H12) and the 11-1 beamline at the Stanford
Synchrotron Radiation Lightsource for BLV5B8. Both datasets were
indexed in spacegroup P212121, integrated, scaled, and merged using
HKL2000 (BLV5B8; HKL Research) or XPREP (BLV1H12; Bruker). The
BLV1H12 structure was solved by molecular replacement to 1.88 .ANG.
resolution using Phaser (McCoy et al., 2007). Fab variable domains
from 1BVK and constant domains from 2FB4 were used as search models
and two complete BLV1H12 Fabs were found in the asymmetric unit.
The BLV5B8 dataset was also solved by molecular replacement (to
2.20 .ANG.), using the refined BLV1H12 coordinates as a model.
Rigid body refinement, simulated annealing and restrained
refinement (including TLS refinement, with one group for each Ig
domain and one for each CDR H3) were carried out in Phenix (Adams
et al. (2010) Acta Crystallogr D Biol Crystallogr 66:213-221).
Riding hydrogens were used during refinement and are included in
the final model.
[0682] Between rounds of refinement, the model was built and
adjusted using Coot. Waters were built automatically using the
"ordered solvent" modeling function in Phenix (Adams et al. (2010)
Acta Crystallogr D Biol Crystallogr 66:213-221). Structures were
validated using the JCSG QC Server (publicly available at
http://smb.slac.stanford.edu/jcsg/QC/), which includes Molprobity
(Chen et al. (2010) Acta Crystallogr D Biol Crystallogr 66:12-21)
Table 1
TABLE-US-00004 TABLE 1 Data collection and refinement statistics
Data collection BLV1H12 Fab BLV5B8 Fab Beamline APS 231D-D SSRL
11-1 Wavelength (.ANG.) 1.033 0.979 Space group
P2.sub.12.sub.12.sub.1 P2.sub.12.sub.12.sub.1 Unit cell parameters
a = 71.4, b = 127.6, c = 127.9, A = 54.6, b = 53.7, c = 330.5,
(.ANG., .degree.) .alpha. = .beta. = .gamma. = 90 .alpha. = .beta.
= .gamma. = 90 Resolution (.ANG.) 50-1.88 (1.92-1.88) 50-2.20
(2.28-2.20) Observations 638,900 313,175 Unique Reflections 96,353
49,527 Redundancy 6.2 (4.9) 6.3 (3.5) Completeness (%) 97.3 (98.2)
96.7 (75.4) <1/.alpha..sub.1> 14.7 (2.5) 17.8 (2.3)
R.sub.sym.sup.b 0.09 (0.76) 0.10 (0.45) Z.sub.a.sup.c 2 2
Refinement statistics Resolution (.ANG.) 50-1.88 50-2.20
Reflections (work) 89,254 46,900 Reflections (test) 4,704 2,441
R.sub.cryst(%).sup.d/R.sub.free(%).sup.c 20.8/23.9 2.07/24.8
Average B (.ANG..sup.2) 43.0 42.7 Wilson B (.ANG..sup.2) 32.5 32.0
Protein atoms 6,724 6,939 Carbohydrate atoms 0 0 Waters 501 474
Other 1 0 RMSD from ideal geometry Bond length (.ANG.) 0.014 0.003
Bond angles (.degree.) 1.12 0.79 Ramachandran statistics (%).sup.r
Favored 96.9 95.2 Outliers 0.1 0.5 PDB Code .sup.g wwww Xxxx
.sup.aNumbers in parentheses refer to the highest resolution shell.
.sup.bR.sub.sym = .SIGMA..sub.hkl.SIGMA..sub.i | I.sub.hkl, i -
<I.sub.hkl> |/.SIGMA..sub.hkl.SIGMA..sub.iI.sub.hkl, I and
R.sub.pim = .SIGMA..sub.hkl (1/(n - 1)).sup.1/2.SIGMA..sub.i |
I.sub.hkl, i - <I.sub.hkl> |
/.SIGMA..sub.hkl.SIGMA..sub.iI.sub.hkl, I, where I.sub.hkl, i is
the scaled intensity of the i.sup.th measurement of relection h, k,
l, <I.sub.hkl> is the average intensity for that reflection,
and n is the redundancy (Emsley et al. (2010) Acta Crystallogr D
Biol Crystallogr 66: 486-501). .sup.cZ.sub.a is the number of Fabs
per crystallographic asymmetric unit. .sup.dR.sub.cryst =
.SIGMA..sub.hkl | F.sub.o - F.sub.c |/.SIGMA..sub.hkl | F.sub.o |
.times. 100 .sup.eR.sub.free was calculated as for R.sub.cryst, but
on a test set comprising 5% of the data excluded from refinement.
.sup.fCalculated using Molprobity (Chen et al. (2010) Acta
Crystallogr D Biol Crystallogr 66: 12-21). .sup.g Coordinates and
structure factors will be deposited in the PDB prior to publication
and be available immediately on publication.
Example 2
Generation of Libraries of Polynucleotides Encoding Antibodies
Comprising an Ultralong CDR3
[0683] 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). 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).
[0684] The library of amplified antibody variable regions were then
subjected to deep sequencing. Briefly, bar-coded primers (Table 2)
for each of the three cows (MID1, MID10, and MID11) were used to
amplify V.sub.H from bovine spleen cDNA.
TABLE-US-00005 TABLE 2 Bar-coded primers for deep sequencing SEQ ID
Description NO: Isotype Primers MID1 FW 308 IgG
CCTATCCCCTGTGTGCCTTGGCAGTCTCAGAC GAGTGCGTTTGAGCGACAAGGCTGTAGGCTG
MID1 RV 309 IgG CCATCTCATCCCTGCGTGTCTCCGACTCAGAC
GAGTGCGTCTTTCGGGGCTGTGGTGGAGGC MID10 FW 310 IgM
CCTATCCCCTGTGTGCCTTGGCAGTCTCAGTC TCTATGCGTTGAGCGACAAGGCTGTAGGCTG
MID10 RV 311 IgM CCATCTCATCCCTGCGTGTCTCCGACTCAGTC
TCTATGCGAGTGAAGACTCTCGGGTGTGATT CAC MID11 FW 312 IgM
CCTATCCCCTGTGTGCCTTGGCAGTCTCAGTG ATACGTCTTTGAGCGACAAGGCTGTAGGCTG
MID11 RV 313 IgM CCATCTCATCCCTGCGTGTCTCCGACTCAGTG
ATACGTCTAGTGAAGACTCTCGGGTGTGATT CAC Primer A 314
TTGAGCGACAAGGCTGTAGGCTG Primer B 315 CTTTCGGGGCTGTGGTGG-AGGC Primer
C 316 AGATCCAAGCTGTGACCGGC
[0685] 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.sub.--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 3 below.
TABLE-US-00006 TABLE 3 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
[0686] 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 (Table 4-6; SEQ ID
NOS: 45-156). The cysteines in the ultralong CDR3 regions are
symbolized as "C". The amino acids between two cysteines are
symbolized as "X.sub.n". Additional exemplary cysteine motifs are
shown in the ultralong CDR3 sequences set forth in Table 23.
TABLE-US-00007 TABLE 4 Cysteine patterns identified in ultralong
CDR3s from MID1 Cysteine pattern (MID1) Abundance (%)
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C 10.44%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C 8.11% CX.sub.11CXCX.sub.5C
5.22% CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C 2.56%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C 1.47%
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C 1.19%
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C 1.08%
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C 1.05%
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C 0.91%
CX.sub.13CX.sub.8CX.sub.8C 0.91%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C 0.59%
CX.sub.10CX.sub.5CX.sub.5C 0.57%
CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C 0.50%
CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C 0.43%
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C 0.41%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C 0.36%
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C 0.32%
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C 0.32%
CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C 0.30%
CX.sub.16CX.sub.5CXC 0.23%
TABLE-US-00008 TABLE 5 Cysteine patterns identified in ultralong
CDR3s from MID10 Cysteine pattern (MID10) Abundance (%)
CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C 2.87%
CX.sub.10CX.sub.5CX.sub.5C 0.73%
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C 0.67%
CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C 0.61%
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C 0.55%
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C 0.43%
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C 0.37%
CX.sub.10CXCX.sub.6CX.sub.4CXC 0.31%
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C 0.31%
CX.sub.14CX.sub.2CX.sub.3CXCXC 0.31% CX.sub.15CX.sub.5CXC 0.31%
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C 0.31%
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C 0.31%
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C 0.31%
CX.sub.10CX.sub.6CX.sub.5C 0.24%
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C 0.24%
CX.sub.7CX.sub.5CXCX.sub.2C 0.24% CX.sub.10CXCX.sub.6C 0.18%
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C 0.18%
CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C 0.18%
TABLE-US-00009 TABLE 6 Cysteine patterns identified in ultralong
CDR3s from MID11 Cysteine pattern (MID11) Abundance (%)
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C 1.19%
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C 0.96%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C 0.92%
CX.sub.16CX.sub.5CXCXCX.sub.14C 0.70%
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C 0.52%
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C 0.49%
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C 0.47%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C 0.45%
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C 0.45%
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C 0.43%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C 0.36%
CX.sub.10CX.sub.6CX.sub.5C 0.31%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C 0.29%
CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C 0.29%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C 0.25%
CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C 0.25%
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C 0.22%
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C 0.20%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C 0.20%
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C 0.20%
[0687] Bovine V.sub.H regions were amplified from cDNA prepared in
example 9 using primers 5'-TTGAGCGACAAGGCTGTAGGCTG-3' (SEQ ID NO:
314) and 5'-CTTTCGGGGCTGTGGTGG-AGGC-3' (SEQ ID NO: 315) 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: 316). 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 (SEQ ID NO: N) 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 then 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.
[0688] 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 (FIG. 1B, left). Clone H12 has a 63-residue CDR3 with 6
cysteine residues and was able to strongly bind BVDV in a dose
dependent fashion (FIG. 1B, right; and 1C).
[0689] 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 hr 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 (FIG. 1D).
Example 3
Generation and Testing of Libraries of Antibodies Comprising an
Ultralong CDR3
[0690] A library of polynucleotides coding for antibodies that
comprise an ultralong CDR3 is generated by immunization of cattle
with whole killed bovine viral diarrhea virus (BVDV) (FIG. 1A).
Briefly, a four month-old Holstein steer was immunized by
intradermal inoculation of a mixture of heat killed BVDV-1 and
BVDV-2 (100 .mu.g of each). The inactivated virus mixture was
suspended in 500 .mu.l PBS and emulsified in 500 .mu.l Freund's
Complete Adjuvant by repeated passage through a double barrel
needle. Next, the immunogen was inoculated intradermally (200
.mu.l/injection) at the neck region using a 26.times.1.sup.1/2 G
needle. The steer was then boosted three times at monthly intervals
with the same amount of antigen but formulated in Freund's
Incomplete Adjuvant. Sero-conversion was tested by ELISA using
plates coated with the inactivated virus and by immunocytometric
analysis of MDBK cells infected with either BVDV-1 or BVDV-2. The
steer was then bled from the jugular vein and blood was collected
in heparin. Lymphocytes were purified through Lymphocyte Separation
Media (Mediatech) centrifugation and stored in RNAlater. A cDNA
library was then made from the plurality of lymphocyte RNA as
described in Example 2.
Example 4
Constructing Vectors of BLV1H12-bGCSF Fusion Proteins for
Expression in Mammalian Cells
[0691] A gene encoding bovine G-CSF (bGCSF) was synthesized by
Genscript (NJ, USA) and amplified by polymerase chain reaction
(PCR). To optimize the folding and stability of the immunoglobulin
constructs, flexible linkers of (GGGGS)n (n=0, 1) were added on
both ends of the bGCSF fragment. Subsequently, PCR fragments of
bGCSF with varied lengths of linkers were grafted into the
complementarity determining region 3 of the heavy chain (CDR3H) of
BLV1H12 antibody by exploiting overlap extension PCR, to replace
the `knob` domain as shown in the crystal structure of BLV1H12. The
expression vectors of BLV1H12-bGCSF fusion proteins were generated
by in-frame ligation of the amplified BLV1H12-bGCSF fusion genes to
the pFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly, the
gene encoding the light chain of BLV1H12 antibody was cloned into
the pFuse vector without hIgG1 Fc fragment. The obtained expression
vectors were confirmed by sequencing. FIGS. 8F, and 8G provide
depictions of the bovine G-CSF inserted into or replacing at least
a portion of the knob domain of a heavy chain region of bovine
BLV1H12 antibody. FIG. 14 shows vectors for expression of the
BLV1H12-bGCSF fusion protein. DNA sequences encoding the heavy and
light chain immunoglobulin constructs Ab-bGCSF L0 (n=0) and
Ab-bGCSF L1 (n=1) are shown in Table 19. Amino acid sequences
encoding the heavy and light chain immunoglobulin constructs
Ab-bGCSF L0 and Ab-bGCSF L1 are shown in Table 21.
Example 5
Expression and Purification of BLV1H12-bGCSF Fusion Antibodies
[0692] BLV1H12-bGCSF fusion antibodies were expressed through
transient transfections of free style HEK 293 cells with vectors
encoding BLV1H12-bGCSF fusion heavy chain and BLVH1H12 light chain.
Expressed BLV1H12-bGCSF fusion antibodies were secreted into the
culture medium and harvested at 48 hours and 96 hours after
transfection. The BLV1H12-bGCSF fusion antibodies were purified by
Protein A/G chromatography (Thermo Fisher Scientific, IL), and
analyzed by SDS-PAGE gel. FIG. 15A shows SDS-PAGE gel of purified
Ab-bGCSF L0 and Ab-bGCSF L1 fusion antibodies from HEK 293
cells.
Example 6
In Vitro Study of Proliferative Activities of the BLV1H12-bGCSF
Fusion Antibodies on Mouse NFS-60 Cells
[0693] Mouse NFS-60 cells were obtained from American Type Culture
Collection (ATCC), VA, and cultured in RPMI-1640 medium
supplemented with 10% fetal bovine serum (FBS), 0.05 mM
2-mercapoethanol and 62 ng/ml human macrophage colony stimulating
factor (M-CSF). For proliferation assay, mouse NFS-60 cells were
washed three times with RPMI-1640 medium and resuspended in
RPMI-1640 medium with 10% FBS and 0.05 mM 2-mercapoethanol at a
density of 1.5.times.10.sup.5 cells/ml. In 96-well plates, 100
.mu.l of cell suspension was added into each well, followed by the
addition of varied concentrations of bGCSF, BLV1H12 antibody, and
the immunoglobulin constructs described herein, Equal volume of PBS
buffer was added into the control wells. The plates were incubated
at 37.degree. C. in a 5% CO.sub.2 incubator for 72 hours. Cells
were then treated with AlamarBlue (Invitrogen) ( 1/10 volume of
cell suspension) for 4 hours at 37.degree. C. Fluorescence at 595
nm for each well was read to indicate the cell viability. As seen
in FIGS. 9A-9E and Tables 7-8, the immunoglobulin constructs
Ab-bGCSF L0 and Ab-bGCSF L1 display similar activity as the bovine
G-CSF and human G-CSF.
TABLE-US-00010 TABLE 7 bGCSF hGCSF Ab (ng/mL) F.I. (ng/mL) F.I.
(ng/mL) F.I. 0.09145 1213.67567 0.09145 1266.554 0.45725 1039.57933
0.27435 1360.925 0.27435 1478.287 1.37174 1009.55533 0.82305
1761.00533 0.82305 1752.216 4.11523 983.27867 2.46914 2100.51733
2.46914 2224.028 12.34568 971.84967 7.40741 2405.09667 7.40741
2587.222 37.03704 960.54933 22.22222 2646.24067 22.22222 2751.249
111.11111 991.83 66.66667 2812.098 66.66667 2815.37 333.33333
964.798 200 3087.144 200 2948.509
TABLE-US-00011 TABLE 8 Ab-bGCSF Ab-bGCSF L1 (ng/mL) F.I. (ng/mL)
F.I. 0.09876 1327.94667 0.09808 1412.387 0.29627 1435.92467 0.29424
1654.776 0.88881 1734.765 0.88272 2082.718 2.66642 2188.50333
2.64816 2550.674 7.99927 2680.08167 7.94449 2997.807 23.9978
2899.28333 23.83346 3277.812 71.9934 2920.56767 71.50039 3308.383
215.9802 3416.20433 214.50117 3868.075
Example 7
In Vitro Study of Proliferative Activities of BLV1H12-bGCSF Fusion
Antibodies on Human Granulocyte Progenitors
[0694] Human mPB CD34.sup.+ cells were purchased from AllCells.
Cells were resuspended in HSC expansion medium (StemSpan SFEM,
StemCell Technologies) supplemented with 1.times. antibiotics and
the following recombinant human cytokines thrombopoietin, IL6, Flt3
ligand, and stem cell factor (100 ng/mL, R & D Systems), then
plated in 96-well plates (1000 cells per well), with varied
concentrations of BLV1H12-bGCSF fusion antibodies. Cells were
cultured for 7 days at 37.degree. C. in a 5% CO.sub.2 incubator,
then analyzed by flow cytometry to measure cell number and
expression of CD45ra and CD41. Cells were stained in staining
medium (HBSS supplemented with 2% FBS and 2 mM EDTA) at 4.degree.
C. for 1 h with PECy7 anti-CD45ra and eFluor 450 anti-CD41
(eBiosciences), then washed with staining medium and analyzed.
Multicolor analysis for cell phenotyping was performed on a LSR II
flow cytometer (Becton Dickinson). FIGS. 10A-10E and Tables 9-10
show the human granulocyte progenitor cell proliferative activities
of the Ab-GCSF fusion antibodies.
TABLE-US-00012 TABLE 9 Number of Number of Number of bGCSF CD45RA-
hGCSF CD45RA- Ab CD45RA- (ng/mL) CD41- (ng/mL) CD41- (ng/mL) CD41-
0.55886 7354 0.05081 4175 0.28959 3666 1.67657 9776 0.15242 3651
0.86877 3839 5.02972 12700 0.45725 3671 2.60631 3852 15.08916 13200
1.37174 4299 23.45679 3519 45.26749 13700 4.11523 5900 70.37037
3541 135.80247 13300 12.34568 7784 211.11111 3606 407.40741 13700
37.03704 10500 5700 4100 1222.22222 14100 111.11111 12200
3666.66667 13100 333.33333 14100 11000 13800 1000 13900
TABLE-US-00013 TABLE 10 Number of Number of Ab-bGCSF CD45RA-
Ab-bGCSF L1 CD45RA- (ng/mL) CD41- (ng/mL) CD41- 0.14225 2601
0.30483 4070 0.42676 2253 0.91449 3601 1.28029 4155 2.74348 4303
3.84088 4399 8.23045 6404 11.52263 7262 24.69136 9122 34.5679 9902
74.07407 11200 103.7037 11500 222.22222 11000 311.11111 11500
666.66667 11400 933.33333 11900 2000 11400 2800 12100 6000
13200
Example 8
Pharmacokinetics of BLV1H12-bGCSF Fusion Antibodies in Mice
[0695] For PK study in mice, 70 .mu.g of the immunoglobulin
constructs described herein were injected into 3 BALB/c mice per
group. Blood samples were drawn from time 0 to 14 days with
extended intervals and analyzed by ELISA using anti-human IgG Fc
antibody with horseradish peroxidase (HRP) labeled (KPL) and
anti-6.times.His antibody with HRP labeled (Clontech). Data were
normalized by taking maximal concentration at the first time point
(30 min). As seen in FIGS. 11A-11B and Table 11, the half-life for
bovine G-CSF was significantly increased when provided in the form
of an immunoglobulin construct described herein.
TABLE-US-00014 TABLE 11 bGCSF Ab Ab-GCSF L1 Ab-GCSF L0 hour
Percentage day Percentage day Percentage day Percentage 0.5 100.00%
0.021 100.00% 0.021 100.00% 0.021 100.00% 1 79.52% 0.042 106.09%
0.042 102.12% 0.042 91.12% 3 56.81% 0.083 95.88% 0.083 104.03%
0.083 91.24% 6 14.94% 0.125 86.54% 0.125 101.71% 0.125 91.56% 24
4.08% 0.250 74.26% 0.250 93.90% 0.250 61.22% 48 0.00% 1.000 52.43%
1.000 59.26% 1.000 44.22% 72 0.00% 2.000 39.46% 2.000 56.66% 2.000
36.36% 3.000 36.97% 3.000 39.09% 3.000 27.38% 8.000 45.44% 8.000
22.21% 8.000 11.53% 10.000 40.98% 10.000 21.15% 10.000 12.44%
14.000 51.96% 14.000 21.41% 14.000 14.35%
Example 9
Neutrophils Counts in Mice
[0696] On the 10.sup.th day after injection of BLV1H12-bGCSF fusion
antibodies into mice for PK study, blood samples were drawn from
the mice and stained using the Diff Quick Staining Kit (Thermo
Fisher Scientific, IL). Neutrophils and white blood cells were
counted under microscope and the percentages of neutrophils were
analyzed. FIGS. 12A-12B and Table 12 show proliferative activities
of BLV1H12-bGCSF fusion antibodies on mice neutrophils that are
blood stained and counted at the 10th day post-injection.
TABLE-US-00015 TABLE 12 Percentage of Neutrophil N.C 16.07 Ab 18.81
Ab-GCSF L1 25.32 Ab-GCSF L0 25.05 bGCSF 16.48
Example 10
Construction of Vectors of BLV1H12-bGCSF Fusion Proteins for
Expression in Pichia pastoris
[0697] Gene fragments encoding BLV1H12-bGCSF chain heavy chain and
BLV1H12 light chain were amplified from the pFuse expression
vectors and subsequently ligated into the same pPICZ.alpha. vector
(Invitrogen). The expressions of the heavy and light chains were
under the control of AOX1 promoter.
Example 11
Expression and Purification of BLV1H12-bGCSF Fusion Antibodies in
Pichia
[0698] The Pichia GS190 cells were transformed with the
pPICZ.alpha. vectors encoding immunoglobulin construct heavy and
light chains by electroporation. Positive transformants were
selected based on zeocin resistance and confirmed by PCR. Pichia
cells with integrated BLV1H12-bGCSF fusion genes were grown in BMGY
medium till OD.sub.600=2.about.6. The cells were then transferred
into BMMY medium in 1/5 of its original volume for induction of the
proteins expression. For every 24 hours, a final concentration of
0.5% methanol was added into the medium to maintain the induction.
Medium containing the secreted immunoglobulin constructs were
harvested after 96-hour induction. The BLV1H12-bGCSF fusion
antibodies were purified by Protein A/G chromatography (Thermo
Fisher Scientific, IL) and analyzed by SDS-PAGE gel. FIGS. 13A-13C
show expression and purification of BLV1H12-bGCSF fusion antibodies
in Pichia pastoris.
Example 12
Constructing Vectors of BLV1H12-Moka1 Fusion Proteins for
Expression in Mammalian Cells
[0699] A gene encoding Moka1 was synthesized by Genscript or IDT,
and amplified by polymerase chain reaction (PCR). To optimize the
folding and stability of fusion proteins, flexible linkers of
(GGGGS)n (n=0, 1) were added on both ends of the Moka1 fragment.
Subsequently, PCR fragments of Moka1 with and without the linker
were grafted into the complementarity determining region 3 of the
heavy chain (CDR3H) of BLV1H12 antibody by exploiting overlap
extension PCR, to replace the `knob` domain as shown in the crystal
structure of BLV1H12. The expression vectors of BLV1H12-Moka1
fusion proteins were generated by in-frame ligation of the
amplified BLV1H12-Moka1 fusion genes to the pFuse-hIgG1-Fc backbone
vector (InvivoGen, CA). Similarly, the gene encoding the light
chain of BLV1H12 antibody was cloned into the pFuse vector without
hIgG1 Fc fragment. The obtained expression vectors were confirmed
by sequencing. FIGS. 8F and 8H provide depictions of a Moka1
peptide inserted into or replacing at least a portion of the knob
domain of a heavy chain region of bovine BLV1H12 antibody.
Example 13
Expression and Purification of BLV1H12 Ab-Moka1 Fusion
Antibodies
[0700] BLV1H12-Moka1 fusion antibodies were expressed through
transient transfections of free style HEK 293 cells with vectors
encoding BLV1H12-Moka1 fusion heavy chain and the BLV1H12 light
chain. BLV1H12-Moka1 fusion antibodies were secreted into the
culture medium and harvested at 48 hours and 96 hours after
transfection. The BLV1H12-Moka1 fusion antibodies were purified by
Protein A/G chromatography (Thermo Fisher Scientific, IL), and
analyzed by SDS-PAGE gel. FIG. 16A shows a SDS PAGE of the
immunoglobulin fusion antibodiess Ab-Moka1 L0 (n=0) and Ab-Moka1 L1
(n=1).
Example 14
In Vitro Study of BLV1H12-Moka1 Fusion Antibodies Inhibitory
Activities on Human Peripheral Blood Mononuclear Cells (PBMCs)/T
Cells Activation
[0701] Human PBMCs were isolated from fresh venous blood of healthy
donors through ficoll gradient centrifugation, followed by
resuspension in RPMI1640 medium with 10% FBS and plating in 96-well
plates at a density of 1.times.10.sup.6 cells/mL. Human T cells
were purified from the isolated PBMCs using T cell enrichment kit.
Purified PBMCs and T cells were pretreated for 1 h at 37.degree. C.
with 5% CO.sub.2 with various concentrations of purified
BLV1H12-Moka1 fusion antibodies and then activated by anti-CD3 and
CD28 antibodies. After 24 h treatment, supernatant was collected
for measurement of the levels of secreted TNF-.alpha. using ELISA
kit. FIG. 17 and Table 13 shows BLV1H12-Moka1 fusion antibodies
inhibitory activities on human peripheral blood mononuclear cells
(PBMCs). FIG. 16B and Table 14 shows BLV1H12-Moka1 fusion
antibodies inhibitory activities on T cells activation.
TABLE-US-00016 TABLE 13 Concentration Ab Ab-Moka L0 Ab-Moka L1 (nM)
F.I. F.I. F.I. 0 1966.657 1966.657 1966.657 2 2333.599333
1679.371333 1394.048 20 2186.372667 1441.220667 1294.799333 200
1981.540333 928.0533333 773.3666667 400 1732.831333 664.9696667
505.102 N.A. 183.3106667 183.3106667 183.3106667
TABLE-US-00017 TABLE 14 [Ab-Moka-L1] nM F.I. 0.3658 479.8675
1.09739 498.558 3.29218 452.342 9.87654 445.013 29.62963 467.268
88.88889 360.2535 266.66667 233.809 800 226.155
Example 15
Constructing Vectors of BLV1H12-VM24 Fusion Proteins for Expression
in Mammalian Cells
[0702] A gene encoding Vm24 was synthesized by Genscript or IDT,
and amplified by polymerase chain reaction (PCR). To optimize the
folding and stability of BLV1H12-VM24 fusion proteins, flexible
linkers of GGGGS or GGGSGGGGS were added on both ends of the Vm24
fragment. Subsequently, PCR fragments of VM24 with varied lengths
of linkers were grafted into the complementarity determining region
3 of the heavy chain (CDR3H) of BLV1H12 antibody by exploiting
overlap extension PCR, to replace the `knob` domain as shown in the
crystal structure of BLV1H12. The expression vectors of
BLV1H12-VM24 fusion proteins were generated by in-frame ligation of
the amplified BLV1H12-VM24 fusion genes to the pFuse-hIgG1-Fc
backbone vector (InvivoGen, CA). Similarly, the gene encoding the
light chain of BLV1H12 antibody was cloned into the pFuse vector
without hIgG1 Fc fragment. The obtained expression vectors were
confirmed by sequencing. FIGS. 8F, and 8H provide depictions of a
VM24 peptide inserted into or replacing at least a portion of the
knob domain of a heavy chain region of bovine BLV1H12 antibody.
Example 16
Expression and Purification of BLV1H12-VM24 Fusion Antibodies
[0703] BLV1H12-VM24 fusion antibodies were expressed through
transient transfections of free style HEK 293 cells with vectors
encoding BLV1H12-VM24 fusion heavy chain and the BLV1H12 light
chain. Expressed BLV1H12-VM24 fusion antibodies were secreted into
the culture medium and harvested every 48 hours for twice after
transfection. The BLV1H12-VM24 fusion antibodies were purified by
Protein A/G chromatography (Thermo Fisher Scientific, IL), and
analyzed by SDS-PAGE gel. FIG. 18A shows a SDS PAGE of the
immunoglobulin constructs Ab-VM24 L1 (linker=GGGGS) and Ab-VM24 L2
(first linker=GGGSGGGGS and second linker=GGGGSGGGS).
Example 17
In Vitro Study of BLV1H12-Vm24 Fusion Antibodies Inhibitory
Activities on T Cells Activation
[0704] Human T cells were purified from the isolated PBMCs using T
cell enrichment kit. Purified T cells were pretreated for 1 h at
37.degree. C. with 5% CO.sub.2 with various concentrations of
purified BLV1H12-VM24 fusion antibodies and then activated by
anti-CD3 and CD28 antibodies. After 24 h treatment, supernatant was
collected for measurement of the levels of secreted TNF-a using
ELISA kit. FIGS. 18B-C and Table 15 show the BLV1H12-VM24 fusion
antibodies inhibitory activities on T cells activation.
TABLE-US-00018 TABLE 15 [Ab-VM24 L1] [Ab-VM24-L2] nM F.I. nM F.I.
0.45725 3427.004 0.45725 3156.626 1.37174 3265.969 1.37174 3345.846
4.11523 3499.82 4.11523 3518.316 12.34568 3627.431 12.34568
3607.5755 37.03704 3575.4815 37.03704 3508.0475 111.11111 3085.439
111.11111 3220.2475 333.33333 1853.645 333.33333 2465.838 1000
990.818 1000 1306.0215
Example 18
Constructing Vectors of BLV1H12-Ex-4 Fusion Proteins for Expression
in Mammalian Cells
[0705] A gene encoding Exendin-4 (Ex-4) was synthesized by
Genscript or IDT, and amplified by polymerase chain reaction (PCR).
A cleavage site of Factor Xa was placed in front of the N-terminal
of Exendin-4. In addition to this protease cleavage site, GGGGS
linker followed with a cysteine were also added on both ends of the
Exendin-4 fragment. Subsequently, PCR fragments of Exendin-4 were
grafted into the complementarity determining region 3 of the heavy
chain (CDR3H) of BLV1H12 antibody by exploiting overlap extension
PCR, to replace the `knob` domain as shown in the crystal structure
of BLV1H12. The expression vectors of BLV1H12-Ex-4 clip fusion
proteins were generated by in-frame ligation of the amplified
BLV1H12-fusion genes to the pFuse-hIgG1-Fc backbone vector
(InvivoGen, CA). Similarly, the gene encoding the light chain of
BLV1H12 antibody was cloned into the pFuse vector without hIgG1 Fc
fragment. The obtained expression vectors were confirmed by
sequencing. FIG. 8F, and 8I show cartoons depicting Exendin-4
peptide inserted into or replacing at least a portion of the knob
domain of an immunoglobulin heavy chain region. FIG. 8I also
depicts the clipped version of the BLV1H12-Exendin-4 fusion
protein, wherein Exendin-4 has a free N-terminus.
Example 19
Expression and Purification of BLV1H12-Ex-4 Clip Fusion
Proteins
[0706] BLV1H12-Ex-4 fusion antibodies were expressed through
transient transfections of free style HEK 293 cells with vectors
encoding BLV1H12-Ex-4 fusion heavy chain and the BLV1H12 light
chain. Expressed BLV1H12-Ex-4 fusion antibodies were secreted into
the culture medium and harvested at 48 hours and 96 hours after
transfection. The BLV1H12-Ex-4 fusion antibodies were purified by
Protein A/G chromatography (Thermo Fisher Scientific, IL).
BLV1H12-Ex-4 fusion antibodies were further treated with Factor Xa
protease (GE Healthcare) following manufacture's protocol to
release N-terminal of Ex-4 peptides fused to the BLV1H12 antibody.
After treatment, BLV1H12-Ex-4 fusion antibodies were re-purified by
Protein A/G affinity column to remove protease and analyzed by
SDS-PAGE gel. FIG. 19 shows a western blot of expression of the
immunoglobulin construct Ab-Exendin-4.
Example 20
In Vitro Study of BLV1H12-Ex-4 Clip Fusion Antibodies Activation
Activities on GLP-1 Receptor (GLP-1R)
[0707] HEK293 cells expressing surface GLP-1R and cAMP responsive
luciferase reporter gene were seeded in 384 well plates at a
density of 5000 cells per well. After 24 h incubation at 37.degree.
C. with 5% CO.sub.2, cells were treated with various concentrations
of Exendin-4 peptides and BLV1H12-Ex-4 clip fusion antibodies and
incubated for another 24 h. Subsequently, luciferase assay was
performed using One-Glo luciferase reagent according manufacture's
instruction (Promega). FIG. 20 and Tables 16-17 show the activity
of Ab-Ex4 fusion antibodies on HEK293 cells expressing GLP-1
receptor.
TABLE-US-00019 TABLE 16 Ab- Ab-GLP1 GLP1(RN) Ab (nM) RLU (nM) RLU
Ab-Ex4 (nM) RLU (nM) RLU 1.26953 5200 1.26953 18600 1.26953 6120
1.26953 4740 2.53906 5000 2.53906 24500 2.53906 6360 2.53906 5800
5.07813 5600 5.07813 43200 5.07813 8500 5.07813 4400 10.15625 5500
10.15625 66560 10.15625 10420 10.15625 5200 20.3125 5380 20.3125
105040 20.3125 19340 20.3125 7780 40.625 4600 40.625 143780 40.625
27960 40.625 13600 81.25 6140 81.25 151060 81.25 54800 81.25 33760
162.5 5760 162.5 166640 162.5 90660 162.5 65800 325 5600 325 171400
325 117900 325 100920 650 5800 650 159780 650 134760 650 140640
1300 14000 1300 184960 1300 159660 1300 169060
TABLE-US-00020 TABLE 17 Ab-Ex4(RN) Ex4 (nM) RLU (nM) RLU 0.00248
24120 0.00248 62500 0.00496 26320 0.00496 71840 0.00992 28140
0.00992 72160 0.01984 33500 0.01984 71360 0.03967 34180 0.03967
69720 0.07935 48860 0.07935 72380 0.15869 63460 0.15869 77680
0.31738 80740 0.31738 87220 0.63477 117240 0.63477 93760 1.26953
128740 1.26953 134100 2.53906 153820 2.53906 128120 5.07813 163020
5.07813 138220 10.15625 169360 10.15625 158700 20.3125 161380
20.3125 165300 40.625 154920 40.625 175200 81.25 163700 162.5
163860 325 164160 650 155700 1300 168740
Example 21
Constructing Vectors of BLV1H12-GLP-1 Clip Fusion Proteins for
Expression in Mammalian Cells
[0708] A gene encoding GLP-1 was synthesized by Genscript or IDT,
and amplified by polymerase chain reaction (PCR). To optimize the
folding and stability of fusion proteins, flexible linkers of
(GGGGS)n (n=0, 1) were added on both ends of the Moka1 fragment.
Linkers of GGGGS or GGGSGGGGS were added on both ends of the GLP-1
fragment. A cleavage site of Factor Xa was placed in front of the
N-terminal of GLP-1. In addition to this protease cleavage site,
GGGGS linker followed with a cysteine were also added on both ends
of the GLP-1 fragment. Subsequently, PCR fragments of GLP-1 were
grafted into the complementarity determining region 3 of the heavy
chain (CDR3H) of BLV1H12 antibody by exploiting overlap extension
PCR, to replace the `knob` domain as shown in the crystal structure
of BLV1H12. The expression vectors of BLV1H12-GLP-1 clip fusion
proteins were generated by in-frame ligation of the amplified
BLV1H12-GLP-1 fusion genes to the pFuse-hIgG1-Fc backbone vector
(InvivoGen, CA). Similarly, the gene encoding the light chain of
BLV1H12 antibody was cloned into the pFuse vector without hIgG1 Fc
fragment. The obtained expression vectors were confirmed by
sequencing. FIGS. 8F and 8I show cartoons depicting a GLP1 peptide
inserted into or replacing at least a portion of the knob domain of
an immunoglobulin heavy chain region. FIG. 8I also shows shows a
clipped version of the BLV1H12-GLP-1 fusion protein, wherein GLP-1
has a free N-terminus.
Example 22
Expression and Purification of BLV1H12-GLP-1 Clip Fusion
Antibodies
[0709] BLV1H12-GLP-1 fusion antibodies were expressed through
transient transfections of free style HEK 293 cells with vectors
encoding BLV1H12-GLP-1 fusion heavy chain and the BLV1H12 light
chain. Expressed BLV1H12-GLP-1 fusion antibodies were secreted into
the culture medium and harvested at 48 hours and 96 hours after
transfection. The BLV1H12-GLP-1 fusion antibodies were purified by
Protein A/G chromatography (Thermo Fisher Scientific, IL).
BLV1H12-GLP-1 fusion antibodies were further treated with Factor Xa
protease (GE Healthcare) following manufacture's protocol to
release N-terminal of GLP-1 peptide fused to the BLV1H12 antibody.
After treatment, BLV1H12-GLP-1 clip fusion antibodies were
re-purified by Protein A/G affinity column to remove protease and
analyzed by SDS-PAGE gel. FIG. 19 provides a western blot of
expression of the immunoglobulin construct Ab-GLP-1.
Example 23
In Vitro Study of BLV1H12-GLP-1 Clip Fusion Antibodies Activation
Activities on GLP-1 Recepto (GLP-1R)
[0710] HEK293 cells expressing surface GLP-1R and cAMP responsive
luciferase reporter gene were seeded in 384 well plates at a
density of 5000 cells per well. After 24 h incubation at 37.degree.
C. with 5% CO.sub.2, cells were treated with various concentrations
of peptides and BLV1H12-GLP-1 clip fusion proteins and incubated
for another 24 h. Subsequently, luciferase assay was performed
using One-Glo luciferase reagent according manufacture's
instruction (Promega). FIG. 20 and Table 16 show the activity of
Ab-GLP-1 f fusion antibodies on HEK293 cells expressing GLP-1
receptor.
Example 24
Constructing Vectors of BLV1H12-hEPO Fusion Proteins for Expression
in Mammalian Cells
[0711] A gene encoding human EPO was synthesized by Genscript or
IDT, and amplified by polymerase chain reaction (PCR). To optimize
the folding and stability of fusion proteins, flexible linkers of
(GGGGS) were added on both ends of human EPO. Subsequently, PCR
fragments of hEPO were grafted into the complementarity determining
region 3 of the heavy chain (CDR3H) of BLV1H12 antibody by
exploiting overlap extension PCR, to replace the `knob` domain as
shown in the crystal structure of BLV1H12. The expression vectors
of BLV1H12-hEPO fusion proteins were generated by in-frame ligation
of the amplified BLV1H12-fusion genes to the pFuse-hIgG1-Fc
backbone vector (InvivoGen, CA). Similarly, the gene encoding the
light chain of BLV1H12 antibody was cloned into the pFuse vector
without hIgG1 Fc fragment. The obtained expression vectors were
confirmed by sequencing. FIGS. 8F and 8J show cartoons depicting
hEPO peptide attached to the knob domain of an immunoglobulin heavy
chain region.
Example 25
Expression and Purification of BLV1H12-hEPO Fusion Antibodies
[0712] BLV1H12-hEPO fusion antibodies were expressed through
transient transfections of free style HEK 293 cells with vectors
encoding BLV1H12-hEPO fusion heavy chain and the BLV1H12 light
chain. Expressed BLV1H12-hEPO fusion antibodies were secreted into
the culture medium and harvested at 48 hours and 96 hours after
transfection. The BLV1H12-hEPO fusion antibodies were purified by
Protein A/G chromatography (Thermo Fisher Scientific, IL), and
analyzed by SDS-PAGE gel.
Example 26
In Vitro Study of BLV1H12-hEPO Fusion Antibody Proliferative
Activities on TF-1 Cells
[0713] TF-1 cells were obtained from American Type Culture
Collection (ATCC), VA, and cultured in RPMI-1640 medium
supplemented with 10% fetal bovine serum (FBS), penicillin,
streptomycin and 2 ng/mL granulocyte-macrophage colony-stimulating
factor (GM-CSF). For proliferation assay, TF-1 cells were washed
three times with RPMI-1640 medium and resuspended in RPMI-1640
medium with 10% FBS and penicillin and streptomycin at a density of
1.5.times.10.sup.5 cells/ml. Cells were plated in 96-well plates
and treated with varied concentrations of BLV1H12-hEPO fusion
antibodies. After 72 h of incubation at 37.degree. C. with 5%
CO.sub.2, cells viabilities were measured using AlamarBlue
(Invitrogen) assay following manufacture's instruction. FIG. 21 and
Table 18 show the proliferative activity of Ab-hEPO fusion
antibodies on TF1 cells.
TABLE-US-00021 TABLE 18 hEPO Ab-hEPO Ab (nM) F.I. (nM) F.I. (nM)
F.I. 2.25E-05 4261.364 9.42E-05 4361.92 9.42E-05 4108.119 1.13E-04
4722.771 4.71E-04 4324.037 4.71E-04 4223.257 5.63E-04 4481.459
0.00236 4198.65 0.00236 4274.07 0.00282 5128.302 0.01178 4757.196
0.01178 4267.586 0.01408 5522.459 0.05888 5265.069 0.05888 3905.529
0.0704 7125.093 0.2944 6430.723 0.2944 4091.452 0.352 8629.194
1.472 8963.889 1.472 4109.106 1.76 9748.017 7.36 10330.18 7.36
4071.234 8.8 10392.97 36.8 10776.73 36.8 4100.011 44 9357.346 184
10330.44 184 4413.497
Example 27
Constructing Vectors of BLV1H12-hFGF21 Fusion Proteins for
Expression in Mammalian Cells
[0714] A gene encoding human FGF21 (hFGF21) was synthesized by
Genscript or IDT, and amplified by polymerase chain reaction (PCR).
To optimize the folding and stability of fusion proteins, flexible
linkers of (GGGGS) were added on both ends of human FGF21.
Subsequently, PCR fragments of hFGF21 were grafted into the
complementarity determining region 3 of the heavy chain (CDR3H) of
BLV1H12 antibody by exploiting overlap extension PCR, to replace
the `knob` domain as shown in the crystal structure of BLV1H12. The
expression vectors of BLV1H12-hFGF21 fusion proteins were generated
by in-frame ligation of the amplified BLV1H12-fusion genes to the
pFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly, the gene
encoding the light chain of BLV1H12 antibody was cloned into the
pFuse vector without hIgG1 Fc fragment. The obtained expression
vectors were confirmed by sequencing.
Example 28
Expression and Purification of BLV1H12-hFGF21 Fusion Antibody
[0715] BLV1H12-hFGF21 fusion antibodies were expressed through
transient transfections of free style HEK 293 cells with vectors
encoding BLV1H12-hFGF21 fusion heavy chain and the BLV1H12 light
chain. Expressed BLV1H12-hFGF21 fusion antibodies were secreted
into the culture medium and harvested every 48 hours for twice
after transfection. The BLV1H12-hFGF21 fusion antibodies were
purified by Protein A/G chromatography (Thermo Fisher Scientific,
IL), and analyzed by SDS-PAGE gel.
Example 29
Constructing Vectors of BLV1H12-hGMCSF Fusion Proteins for
Expression in Mammalian Cells
[0716] A gene encoding human GMCSF (hGMCSF) was synthesized by
Genscript or IDT, and amplified by polymerase chain reaction (PCR).
To optimize the folding and stability of fusion proteins, flexible
linkers of (GGGGS) were added on both ends of human GMCSF.
Subsequently, PCR fragments of hGMCSF were grafted into the
complementarity determining region 3 of the heavy chain (CDR3H) of
BLV1H12 antibody by exploiting overlap extension PCR, to replace
the `knob` domain as shown in the crystal structure of BLV1H12. The
expression vectors of BLV1H12-hGMCSF fusion proteins were generated
by in-frame ligation of the amplified BLV1H12-hGMCSF fusion genes
to the pFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly,
the gene encoding the light chain of BLV1H12 antibody was cloned
into the pFuse vector without hIgG1 Fc fragment. The obtained
expression vectors were confirmed by sequencing.
Example 30
Expression and Purification of BLV1H12-hGMCSF Fusion Antibodies
[0717] BLV1H12-hGMCSF fusion antibodies can be expressed through
transient transfections of free style HEK 293 cells with vectors
encoding BLV1H12-hGMCSF fusion heavy chain and the BLV1H12 light
chain. Expressed BLV1H12-hGMCSF fusion antibodies can be secreted
into the culture medium and harvested at 48 hours and 96 hours
after transfection. The BLV1H12-hGMCSF fusion antibodies can be
purified by Protein A/G chromatography (Thermo Fisher Scientific,
IL), and analyzed by SDS-PAGE gel.
Example 31
Constructing Vectors of BLV1H12-hIFN-b Proteins for Expression in
Mammalian Cells
[0718] A gene encoding human interferon-beta (hIFN-b) was
synthesized by Genscript or IDT, and amplified by polymerase chain
reaction (PCR). To optimize the folding and stability of fusion
proteins, flexible linkers of (GGGGS) were added on both ends of
human interferon-beta. Subsequently, PCR fragments of hIFN-b were
grafted into the complementarity determining region 3 of the heavy
chain (CDR3H) of BLV1H12 antibody by exploiting overlap extension
PCR, to replace the `knob` domain as shown in the crystal structure
of BLV1H12. The expression vectors of BLV1H12-hIFN-b fusion
proteins were generated by in-frame ligation of the amplified
BLV1H12-hIFN-b fusion genes to the pFuse-hIgG1-Fc backbone vector
(InvivoGen, CA). Similarly, the gene encoding the light chain of
BLV1H12 antibody was cloned into the pFuse vector without hIgG1 Fc
fragment. The obtained expression vectors were confirmed by
sequencing.
Example 32
Expression and Purification of BLV1H12-hIFN-b Fusion Antibodies
[0719] BLV1H12-hIFN-b fusion antibodies can be expressed through
transient transfections of free style HEK 293 cells with vectors
encoding BLV1H12-hIFN-b fusion heavy chain and the BLV1H12 light
chain. Expressed BLV1H12-hIFN-b fusion antibodies can be secreted
into the culture medium and harvested at 48 hours and 96 hours
after transfection. The BLV1H12-hIFN-b fusion antibodies can be
purified by Protein A/G chromatography (Thermo Fisher Scientific,
IL), and analyzed by SDS-PAGE gel.
Example 33
Constructing Vectors of BLV1H12-Fusion Proteins for Expression in
Mammalian Cells
[0720] Genes encoding various genes were synthesized by Genscript
(NJ, USA) and amplified by polymerase chain reaction (PCR). To
optimize the folding and stability of the immunoglobulin
constructs, one or more flexible linkers of (GGGGS)n (n=0, 1),
GGGSGGGGS, and/or GGGGSGGGS were added on both ends of the gene
fragment. Subsequently, PCR fragments of the genes with varied
lengths of linkers were grafted into the complementarity
determining region 3 of the heavy chain (CDR3H) of BLV1H12 antibody
by exploiting overlap extension PCR, to replace at least a portion
of the `knob` domain as shown in the crystal structure of BLV1H12
(FIGS. 8A-8J). The expression vectors of BLV1H12-fusion proteins
were generated by in-frame ligation of the amplified BLV1H12-fusion
genes to the pFuse-hIgG1-Fc backbone vector (InvivoGen, CA).
Similarly, the gene encoding the light chain of BLV1H12 antibody
was cloned into the pFuse vector without hIgG1 Fc fragment. The
obtained expression vectors were confirmed by sequencing.
[0721] Nucleic acid sequences of the BLV1H12-fusion proteins are
displayed in Table 19 (SEQ ID NOS: 1-15). Peptide sequences of the
BLVH12-fusion proteins are displayed in Table 21 (SEQ ID NOS:
23-37). As shown in the Table 19 and Table 21, the bovine heavy
chain sequence is in bold font; the human heavy chain sequence is
highlighted with a dashed underline; the non-antibody sequence is
in italicized font; the stalk domain is in bold font and
underlined; the knob domain is in bold font and double underlined;
the linker sequence is in italicized font and squiggly
underlined.
Example 34
Constructing Vectors of BLV1H12-IL8 Fusion Proteins for Expression
in Mammalian Cells
[0722] Gene encoding a human IL-8 sequence (see, e.g., SEQ ID NO:
317 corresponding to amino acids 26-99 of IL-8, designated IL8
herein) was amplified from cDNA (OriGene) by polymerase chain
reaction (PCR). In some constructs, a linker (e.g., GSG or repeats
of GSG) may be added on one or both ends of the IL8 fragment.
Subsequently, PCR fragments of IL8 with or without a linker were
grafted into the complementarity determining region 3 of the heavy
chain (CDR3H) of BLV1H12 antibody by PCR, to replace the `knob`
domain as shown in the crystal structure of BLV1H12. The expression
vectors of BLV1H12-IL8 fusion proteins were generated by in-frame
ligation of the amplified BLV1H12-IL8 fusion genes to CH1--CH2-CH3
in a pFuse-backbone vector (InvivoGen, CA). The BLV1H12-IL8 heavy
chain variable region sequence is shown as SEQ ID NO: 16
(nucleotide) and SEQ ID NO: 38 (amino acid). Similarly, the gene
encoding the light chain of BLV1H12 antibody was cloned into a
pFuse vector without hIgG1 Fc fragment. The obtained expression
vectors were confirmed by sequencing.
Example 35
Expression of BLV1H12-IL8 Fusion Proteins
[0723] BLV1H12-IL8 fusion proteins were expressed through transient
transfections of 293T cells or Freestyle.TM. 293-F cells with
vectors encoding BLV1H12-IL8 heavy and light chains. Expressed
fusion proteins were secreted into the culture medium and culture
supernatants obtained after 2 days of cell culture. The expression
of BLV1H12-IL8 fusion proteins in the supernatants was determined
by ELISA to be 37.2 nM
Example 36
In Vitro Study of Activities of the BLV1H12-IL8 Fusion Proteins on
CXCR1 Expressing Cells
[0724] 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 U20S was
obtained from ATCC and cultured under the same conditions as the
CXCR1 cells. Cell culture supernatants from Example 2 above were
then tested for binding to cells by flow cytometry. The adherent
U20S or CXCR1-U20S 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
expressedBLV1H12-IL8 fusion proteins to cells. Subsequently, cell
fluorescence was measured by flow cytometry (e.g., FACS), and
median Arbitrary Fluorescence Units (AFU) were calculated,
revealing the extent of IgG binding to those cells. The ratio of
median fluorescence (IgG binding) of CXCR1-U20S cells versus U20S
parental cells shows that the BLV1H12-IL8 fusion protein has
specificity for CXCR1 (Table 27).
TABLE-US-00022 TABLE 27 BLV1H12 (Median BLV1H12-IL8 (Median
Arbitrary Fluorescence Arbitrary Fluorescence Units (AFU)) Units
(AFU)) Parental U2OS 4 76 CXCR1-U2OS 4 707
Example 37
Constructing Vectors of BLV1H12-Ziconotide Fusion Proteins for
Expression in Mammalian Cells
[0725] Gene encoding a ziconotide sequence (see, e.g., SEQ ID NO:
318) was prepared from multiple oligonucleotides and then amplified
by polymerase chain reaction (PCR). In some constructs, a linker
(e.g., GSG or repeats of GSG) may be added on one or both ends of
the ziconotide fragment. Subsequently, PCR fragments of ziconotide
with or without a linker were grafted into the complementarity
determining region 3 of the heavy chain (CDR3H) of BLV1H12 antibody
by PCR, to replace the `knob` domain as shown in the crystal
structure of BLV1H12. The expression vectors of BLV1H12-ziconotide
fusion proteins were generated by in-frame ligation of the
amplified BLV1H12-ziconotide fusion genes to CH1-CH2-CH3 in a
pFuse-backbone vector (InvivoGen, CA). The BLV1H12-ziconotide heavy
chain variable region sequence is shown as SEQ ID NO: 17
(nucleotide) and SEQ ID NO: 39 (amino acid). Similarly, the gene
encoding the light chain of BLV1H12 antibody was cloned into a
pFuse vector without hIgG1 Fc fragment. The obtained expression
vectors were confirmed by sequencing.
Example 38
Expression of BLV1H12-Ziconotide Fusion Proteins
[0726] BLV1H12-ziconotide fusion proteins were expressed through
transient transfections of 293T cells or Freestyle.TM. 293-F cells
with vectors encoding BLV1H12-ziconotide heavy and light chains.
Expressed fusion proteins were secreted into the culture medium and
culture supernatants obtained after 2 days of cell culture. The
expression of BLV1H12-ziconotide fusion proteins in the
supernatants was determined by ELISA and normalized as compared to
the IL8 construct in Example 35 above to be 94.7% of the IL8
construct.
Example 39
Constructing Vectors of BLV1H12-Somatostatin Fusion Proteins for
Expression in Mammalian Cells
[0727] Gene encoding a somatostatin sequence (see, e.g., SEQ ID NO:
319) was prepared from multiple oligonucleotides and then amplified
by polymerase chain reaction (PCR). In some constructs, a linker
(e.g., GSG or repeats of GSG) may be added on one or both ends of
the somatostatin fragment. Subsequently, PCR fragments of
somatostatin with or without a linker were grafted into the
complementarity determining region 3 of the heavy chain (CDR3H) of
BLV1H12 antibody by PCR, to replace the `knob` domain as shown in
the crystal structure of BLV1H12. The expression vectors of
BLV1H12-somatostatin fusion proteins were generated by in-frame
ligation of the amplified BLV1H12-somatostatin fusion genes to
CH1-CH2-CH3 in a pFuse-backbone vector (InvivoGen, CA). The
BLV1H12-somatostatin heavy chain variable region sequence is shown
as SEQ ID NO: 18 (nucleotide) and SEQ ID NO: 40 (amino acid).
Similarly, the gene encoding the light chain of BLV1H12 antibody
was cloned into a pFuse vector without hIgG1 Fc fragment. The
obtained expression vectors were confirmed by sequencing.
Example 40
Expression of BLV1H12-Somatostatin Fusion Proteins
[0728] BLV1H12-somatostatin fusion proteins were expressed through
transient transfections of 293T cells or Freestyle.TM. 293-F cells
with vectors encoding BLV1H12-somatostatin heavy and light chains.
Expressed fusion proteins were secreted into the culture medium and
culture supernatants obtained after 2 days of cell culture. The
expression of BLV1H12-somatostatin fusion proteins in the
supernatants was determined by ELISA and normalized as compared to
the IL8 construct in Example 35 above to be 46.5% of the IL8
construct.
Example 41
Constructing Vectors of BLV1H12-Chlorotoxin Fusion Proteins for
Expression in Mammalian Cells
[0729] Gene encoding a chlorotoxin sequence (see, e.g., SEQ ID NO:
320) was prepared from multiple oligonucleotides and then amplified
by polymerase chain reaction (PCR). In some constructs, a linker
(e.g., GSG or repeats of GSG) may be added on one or both ends of
the chlorotoxin fragment. Subsequently, PCR fragments of
chlorotoxin with or without a linker were grafted into the
complementarity determining region 3 of the heavy chain (CDR3H) of
BLV1H12 antibody by PCR, to replace the `knob` domain as shown in
the crystal structure of BLV1H12. The expression vectors of
BLV1H12-chlorotoxin fusion proteins were generated by in-frame
ligation of the amplified BLV1H12-chlorotoxin fusion genes to
CH1-CH2-CH3 in a pFuse-backbone vector (InvivoGen, CA). The
BLV1H12-chlorotoxin heavy chain variable region sequence is shown
as SEQ ID NO: 19 (nucleotide) and SEQ ID NO: 41 (amino acid).
Similarly, the gene encoding the light chain of BLV1H12 antibody
was cloned into a pFuse vector without hIgG1 Fc fragment. The
obtained expression vectors were confirmed by sequencing.
Example 42
Expression of BLV1H12-Chlorotoxin Fusion Proteins
[0730] BLV1H12-chlorotoxin fusion proteins were expressed through
transient transfections of 293T cells or Freestyle.TM. 293-F cells
with vectors encoding BLV1H12-chlorotoxin heavy and light chains.
Expressed fusion proteins were secreted into the culture medium and
culture supernatants obtained after 2 days of cell culture. The
expression of BLV1H12-chlorotoxin fusion proteins in the
supernatants was determined by ELISA and normalized as compared to
the IL8 construct in Example 35 above to be 39.7% of the IL8
construct.
Example 43
Constructing Vectors of BLV1H12-SDF-1 (Alpha) Fusion Proteins for
Expression in Mammalian Cells
[0731] Gene encoding a SDF-1 alpha sequence (see, e.g., SEQ ID NO:
321) was amplified from cDNA (OriGene) by polymerase chain reaction
(PCR). In some constructs, a linker (e.g., GSG or repeats of GSG)
may be added on one or both ends of the SDF-1 (alpha) fragment.
Subsequently, PCR fragments of SDF-1 (alpha) with or without a
linker were grafted into the complementarity determining region 3
of the heavy chain (CDR3H) of BLV1H12 antibody by PCR, to replace
the `knob` domain as shown in the crystal structure of BLV1H12. The
expression vectors of BLV1H12-SDF-1 (alpha) fusion proteins were
generated by in-frame ligation of the amplified BLV1H12-SDF-1
(alpha) fusion genes to CH1-CH2-CH3 in a pFuse-backbone vector
(InvivoGen, CA). The BLV1H12-SDF-1 (alpha) heavy chain variable
region sequence is shown as SEQ ID NO: 20 (nucleotide) and SEQ ID
NO: 42 (amino acid). Similarly, the gene encoding the light chain
of BLV1H12 antibody was cloned into a pFuse vector without hIgG1 Fc
fragment. The obtained expression vectors were confirmed by
sequencing.
Example 44
Expression of BLV1H12-SDF-1 (Alpha) Fusion Proteins
[0732] BLV1H12-SDF-1 (alpha) fusion proteins were expressed through
transient transfections of 293T cells or Freestyle.TM. 293-F cells
with vectors encoding BLV1H12-SDF-1 (alpha) heavy and light chains.
Expressed fusion proteins were secreted into the culture medium and
culture supernatants obtained after 2 days of cell culture. The
expression of BLV1H12-SDF-1 (alpha) fusion proteins in the
supernatants was determined by ELISA and normalized as compared to
the IL8 construct in Example 35 above to be 38.9% of the IL8
construct.
Example 45
Constructing Vectors of BLV1H12-IL21 Fusion Proteins for Expression
in Mammalian Cells
[0733] Gene encoding an IL21 sequence (see, e.g., SEQ ID NO: 322)
was amplified from cDNA (OriGene) by polymerase chain reaction
(PCR). In some constructs, a linker (e.g., GSG or repeats of GSG)
may be added on one or both ends of the IL21 fragment.
Subsequently, PCR fragments of IL21 with or without a linker were
grafted into the complementarity determining region 3 of the heavy
chain (CDR3H) of BLV1H12 antibody by PCR, to replace the `knob`
domain as shown in the crystal structure of BLV1H12. The expression
vectors of BLV1H12-IL21 fusion proteins were generated by in-frame
ligation of the amplified BLV1H12-IL21 fusion genes to CH1-CH2-CH3
in a pFuse-backbone vector (InvivoGen, CA). The BLV1H12-IL21 heavy
chain variable region sequence is shown as SEQ ID NO: 21
(nucleotide) and SEQ ID NO: 43 (amino acid). Similarly, the gene
encoding the light chain of BLV1H12 antibody was cloned into a
pFuse vector without hIgG1 Fc fragment. The obtained expression
vectors were confirmed by sequencing.
Example 46
Expression of BLV1H12-IL21 Fusion Proteins
[0734] BLV1H12-IL21 fusion proteins were expressed through
transient transfections of 293T cells or Freestyle.TM. 293-F cells
with vectors encoding BLV1H12-IL21 heavy and light chains.
Expressed fusion proteins were secreted into the culture medium and
culture supernatants obtained after 2 days of cell culture. The
expression of BLV1H12-IL21 fusion proteins in the supernatants was
determined by ELISA and normalized as compared to the IL8 construct
in Example 35 above to be 32.3% of the IL8 construct.
Example 47
Constructing Vectors of BLV1H12-Protoxin2 Fusion Proteins for
Expression in Mammalian Cells
[0735] Gene encoding protoxin2 was synthesized by Genscript or IDT,
and amplified by polymerase chain reaction (PCR). To optimize the
folding and stability of fusion proteins, flexible linkers of GGGGS
were added on both ends of protoxin2 fragments. Subsequently, PCR
fragments of protoxin2 (called protoxin2-L1) were grafted into the
complementarity determining region 3 of the heavy chain (CDR3H) of
BLV1H12 antibody by exploiting overlap extension PCR, to replace
the `knob` domain as shown in the crystal structure of BLV1H12. The
expression vectors of BLV1H12-fusion proteins were generated by
in-frame ligation of the amplified BLV1H12-protoxin2-L1 fusion
genes (SEQ ID NO: 15) to the pFuse-hIgG1-Fc backbone vector
(InvivoGen, CA). Similarly, the gene encoding the light chain of
BLV1H12 antibody was cloned into the pFuse vector without hIgG1 Fc
fragment. The obtained expression vectors were confirmed by
sequencing.
Example 48
Expression and Purification of BLV1H12-Protoxin2-L1 Fusion
Proteins
[0736] BLV1H12-protoxin2-L1 fusion antibodies were expressed
through transient transfections of free style HEK 293 cells with
vectors encoding BLV1H12-protoxin2 fusion heavy chain and the
BLV1H12 light chain. Expressed BLV1H12-protoxin2-L1 fusion
antibodies were secreted into the culture medium and harvested at
48 hours and 96 hours after transfection. The BLV1H12-protoxin2-L1
fusion antibodies were purified by Protein A/G chromatography
(Thermo Fisher Scientific, IL), and analyzed by SDS-PAGE gel (FIG.
15B).
Example 49
Constructing Vectors of BLV1H12-ProTxII Fusion Proteins for
Expression in Mammalian Cells
[0737] Gene encoding a ProTxII sequence (see, e.g., SEQ ID NO: 323)
was prepared from multiple oligonucleotides and then amplified by
polymerase chain reaction (PCR). In some constructs, a linker
(e.g., GSG or repeats of GSG) may be added on one or both ends of
the ProTxII fragment. Subsequently, PCR fragments of ProTxII with
or without a linker were grafted into the complementarity
determining region 3 of the heavy chain (CDR3H) of BLV1H12 antibody
by PCR, to replace the `knob` domain as shown in the crystal
structure of BLV1H12. The expression vectors of BLV1H12-ProTxII
fusion proteins were generated by in-frame ligation of the
amplified BLV1H12-ProTxII fusion genes to CH1-CH2-CH3 in a
pFuse-backbone vector (InvivoGen, CA). The BLV1H12-ProTxII heavy
chain variable region sequence is shown as SEQ ID NO: 22
(nucleotide) and SEQ ID NO: 44 (amino acid). Similarly, the gene
encoding the light chain of BLV1H12 antibody was cloned into a
pFuse vector without hIgG1 Fc fragment. The obtained expression
vectors were confirmed by sequencing.
Example 50
Expression of BLV1H12-ProTxII Fusion Proteins
[0738] BLV1H12-ProTxII fusion proteins were expressed through
transient transfections of 293T cells or Freestyle.TM. 293-F cells
with vectors encoding BLV1H12-ProTxII heavy and light chains.
Expressed fusion proteins were secreted into the culture medium and
culture supernatants obtained after 2 days of cell culture. The
expression of BLV1H12-ProTxII fusion proteins in the supernatants
was determined by ELISA and normalized as compared to the IL8
construct in Example 35 above to be 2.1% of the IL8 construct.
[0739] 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.
TABLE-US-00023 TABLE 19 SEQ ID Description NO: Sequence Light Chain
1 CAGGCCGTCCTGAACCAGCCAAGCAGCGTCTCCGGGTCT
CTGGGGCAGCGGGTCTCAATCACCTGTAGCGGGTCTTCCT
CCAATGTCGGCAACGGCTACGTGTCTTGGTATCAGCTGAT
CCCTGGCAGTGCCCCACGAACCCTGATCTACGGCGACAC
ATCCAGAGCTTCTGGGGTCCCCGATCGGTTCTCAGGGAGC
AGATCCGGAAACACAGCTACTCTGACCATCAGCTCCCTG
CAGGCTGAGGACGAAGCAGATTATTTCTGCGCATCTGCC
GAGGACTCTAGTTCAAATGCCGTGTTTGGAAGCGGCACC
ACACTGACAGTCCTGGGGCAGCCCAAGAGTCCCCCTTCA
GTGACTCTGTTCCCACCCTCTACCGAGGAACTGAACGGA
AACAAGGCCACACTGGTGTGTCTGATCAGCGACTTTTACC
CTGGATCCGTCACTGTGGTCTGGAAGGCAGATGGCAGCA
CAATTACTAGGAACGTGGAAACTACCCGCGCCTCCAAGC
AGTCTAATAGTAAATACGCCGCCAGCTCCTATCTGAGCCT
GACCTCTAGTGATTGGAAGTCCAAAGGGTCATATAGCTG
CGAAGTGACCCATGAAGGCTCAACCGTGACTAAGACTGT GAAACCATCCGAGTGCTCC Heavy
Chain- 2 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA no insertion
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC
ACCAGGAAACTAAGAAATACCAGAGCTGTCCTGACGG
CTATCGGGAGAGATCTGATTGCAGTAATAGGCCAGCT
TGTGGCACATCCGACTGCTGTCGCGTGTCTGTCTTCG
GGAACTGCCTGACTACCCTGCCTGTGTCCTACTCTTAT
ACCTACAATTATGAATGGCATGTGGATGTCTGGGGAC AGGGCCTGCTGGTGACAGTCTCTAGT
IFN-beta 3 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ##STR00001##
AATTTTCAGTGTCAGAAGCTCCTGTGGCAATTGAATGGGAGG
CTTGAATACTGCCTCAAGGACAGGATGAACTTTGACATCCCT
GAGGAGATTAAGCAGCTGCAGCAGTTCCAGAAGGAGGACGC
CGCATTGACCATCTATGAGATGCTCCAGAACATCTTTGCTATT
TTCAGACAAGATTCATCTAGCACTGGCTGGAATGAGACTATTG
TTGAGAACCTCCTGGCTAATGTCTATCATCAGATAAACCATCT
GAAGACAGTCCTGGAAGAAAAACTGGAGAAAGAAGATTTCAC
CAGGGGAAAACTCATGAGCAGTCTGCACCTGAAAAGATATTA
TGGGAGGATTCTGCATTACCTGAAGGCCAAGGAGTACAGTCA
CTGTGCCTGGACCATAGTCAGAGTGGAAATCCTAAGGAACTT ##STR00002##
GGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCT
AGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGT
CAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGAC
ACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCT
GTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCG
GAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGG
CCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCA
GTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCC
TGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAA ##STR00003## bGCSF-L0 4
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC
ACCAGGAAACTAAGAAATACCAGAGCACCCCCCTTGGC
CCTGCCCGATCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTA
GAGCAAGTGAGGAAAATCCAGGCTGATGGCGCCGAGCTGCA
GGAGAGGCTGTGTGCCGCCCACAAGCTGTGCCACCCGGAG
GAGCTGATGCTGCTCAGGCACTCTCTGGGCATCCCCCAGGC
TCCCCTAAGCAGCTGCTCCAGCCAGTCCCTGCAGCTGACGA
GCTGCCTGAACCAACTACACGGCGGCCTCTTTCTCTACCAGG
GCCTCCTGCAGGCCCTGGCGGGCATCTCCCCAGAGCTGGCC
CCCACCTTGGACACACTGCAGCTGGACGTCACTGACTTTGCC
ACGAACATCTGGCTGCAGATGGAGGACCTGGGGGCGGCCCC
CGCTGTGCAGCCCACCCAGGGCGCCATGCCGACCTTCACTT
CAGCCTTCCAACGCAGAGCAGGAGGGGTCCTGGTTGCTTCC
CAGCTGCATCGTTTCCTGGAGCTGGCATACCGTGGCCTGCG
CTACCTTGCTGAGCCCTCTTATACCTACAATTATGAATG
GCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACA
GTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACC
CCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTAC
CGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCC
GAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGA
AAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTC
CTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTC
CCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATG
TGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGC ##STR00004## bGCSF-L1 5
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ##STR00005##
CTGCTCAAGTGCTTAGAGCAAGTGAGGAAAATCCAGGCTGAT
GGCGCCGAGCTGCAGGAGAGGCTGTGTGCCGCCCACAAGC
TGTGCCACCCGGAGGAGCTGATGCTGCTCAGGCACTCTCTG
GGCATCCCCCAGGCTCCCCTAAGCAGCTGCTCCAGCCAGTC
CCTGCAGCTGACGAGCTGCCTGAACCAACTACACGGCGGCC
TCTTTCTCTACCAGGGCCTCCTGCAGGCCCTGGCGGGCATCT
CCCCAGAGCTGGCCCCCACCTTGGACACACTGCAGCTGGAC
GTCACTGACTTTGCCACGAACATCTGGCTGCAGATGGAGGAC
CTGGGGGCGGCCCCCGCTGTGCAGCCCACCCAGGGCGCCA
TGCCGACCTTCACTTCAGCCTTCCAACGCAGAGCAGGAGGG
GTCCTGGTTGCTTCCCAGCTGCATCGTTTCCTGGAGCTGGCA ##STR00006##
TCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGC
TTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGC
TGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGG
GATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGAC
TGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTG
CACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGT
ATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTAC
TTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCT
GCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCA ##STR00007## GMCSF 6
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ##STR00008##
GGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTG
AACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTA
GAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGC
CTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGG
CAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCA
GCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCT
GTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCT
GAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCC ##STR00009##
TTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTG
CTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAA
AGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATC
CTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGC
TATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAG
GAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGT
GCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATG
GTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCA
CCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGT ##STR00010## hFGF21 7
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ##STR00011##
GGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCC
AGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACG
GTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCA
GCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGT
CAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCC
TGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCC
GGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCG
AAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCC
CCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCT
GCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCC
GGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGA
CCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCA ##STR00012##
TTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTG
CTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAA
AGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATC
CTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGC
TATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAG
GAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGT
GCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATG
GTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCA
CCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGT ##STR00013## Ex-4 8
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ##STR00014##
TGCTCTTATACCTACAATTATGAATGGCATGTGGATGT
CTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCT
TCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCT
GCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGG
ATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACT
GTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGC
ACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTA
TAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACT
TCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTG
CCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAA ##STR00015## hGLP-1 9
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ##STR00016##
TGCTCTTATACCTACAATTATGAATGGCATGTGGATGT
CTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCT
TCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCT
GCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGG
ATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACT
GTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGC
ACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTA
TAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACT
TCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTG
CCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAA ##STR00017## hEPO 10
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ##STR00018##
AGAGGTACCTCTTGGAGGCCAAGGAGGCCGAGAATATCACG
ACGGGCTGTGCTGAACACTGCAGCTTGAATGAGAATATCACT
GTCCCAGACACCAAAGTTAATTTCTATGCCTGGAAGAGGATG
GAGGTCGGGCAGCAGGCCGTAGAAGTCTGGCAGGGCCTGG
CCCTGCTGTCGGAAGCTGTCCTGCGGGGCCAGGCCCTGTTG
GTCAACTCTTCCCAGCCGTGGGAGCCCCTGCAGCTGCATGT
GGATAAAGCCGTCAGTGGCCTTCGCAGCCTCACCACTCTGCT
TCGGGCTCTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCAG
ATGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACA
CTTTCCGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGG
AAAGCTGAAGCTGTACACAGGGGAGGCCTGCAGGACAGGGG ##STR00019##
AATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGT
GACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTG
TACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTA
GTACCGTGACACTGGGATGCCTGGTCTCAAGCTATAT
GCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCC
CTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGC
AGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGAC
AGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGT
AATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACA ##STR00020## Moka-L0 11
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC
ACCAGGAAACTAAGAAATACCAGAGCATCAACGTGAAGT
GCAGCCTGCCCCAGCAGTGCATCAAGCCCTGCAAGGACGCC
GGCATGCGGTTCGGCAAGTGCATGAACAAGAAGTGCAGGTG
CTACAGCTCTTATACCTACAATTATGAATGGCATGTGG
ATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAG
TGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCA
AGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACAC
TGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGT
GACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGA
GTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCC
TGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAG
TACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCAT
CCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAAC ##STR00021## Moka-L1 12
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ##STR00022##
ATACCTACAATTATGAATGGCATGTGGATGTCTGGGG
ACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACA
ACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTG
GGGACAAATCCTCTAGTACCGTGACACTGGGATGCCT
GGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACC
TGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCT
TCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCT
GAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGG
CAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCT
CCACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTG ##STR00023## VM-24-L1 13
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ##STR00024##
TCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGC
TTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGC
TGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGG
GATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGAC
TGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTG
CACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGT
ATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTAC
TTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCT
GCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCA ##STR00025## VM-24-L2 14
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ##STR00026##
AATTATGAATGGCATGTGGATGTCTGGGGACAGGGCC
TGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACC
AAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAA
TCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAA
GCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTC
AGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCT
GTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAA
TGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTT
CACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAA ##STR00027## Protoxin2-L1 15
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA
AGCCATCCCAGACACTGAGCCTGACATGCACAGCAAG
CGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTC
CGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGC
AGCATCGATACCGGCGGGAACACAGGGTACAATCCCG
GACTGAAGAGCAGACTGTCCATTACCAAGGACAACTC
TAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC
ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ##STR00028##
TATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGC
TGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAA
GGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCC
TCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCT
ATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGG
AGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTG
CTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGG
TGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCAC
CTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTG ##STR00029## ##STR00030##
TABLE-US-00024 TABLE 20 SEQ ID Description NO: Sequence BLV1H12-IL8
16 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA
GCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCG
GGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGAC
AGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATC
GATACCGGCGGGAACACAGGGTACAATCCCGGACTGAA
GAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCA
GGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAG
TGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAA
GAAATACCAGAGCCCAAGGAGTGCTAAAGAACTTAGAT
GTCAGTGCATAAAGACATACTCCAAACCTTTCCACCCCA
AGTTCATCAAGGAGCTGAGAGTGATTGAGAGTGGACCA
CACTGCGCCAACACAGAGATTATTGTAAAGCTTTCTGAT
GGGAGAGAGCTCTGCCTGGACCCCAAGGAAAACTGGGT
GCAGAGGGTCGTGGAGAAGTTCTTGAAGAGGGCTGAGA
ACTCAGGCAGCGGTTCTTATACCTACAATTATGAATGGC
ATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTC BLV1H12- 17
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA Ziconotide
GCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCG
GGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGAC
AGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATC
GATACCGGCGGGAACACAGGGTACAATCCCGGACTGAA
GAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCA
GGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAG
TGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAA
GAAATACCAGAGCTGCAAGGGCAAAGGTGCGAAATGCA
GCCGCCTGATGTATGATTGCTGTACCGGGTCCTGCCGCA
GTGGCAAGTGCTCTTATACCTACAATTATGAATGGCATG
TGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTC BLV1H12- 18
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA Somatostatin
GCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCG
GGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGAC
AGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATC
GATACCGGCGGGAACACAGGGTACAATCCCGGACTGAA
GAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCA
GGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAG
TGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAA
GAAATACCAGAGCGCTGGCTGCAAGAATTTCTTCTGGAA
GACTTTCACATCCTGTGGTTCTTATACCTACAATTATGAA
TGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACA GTC BLV1H12- 19
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA Chlorotoxin
GCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCG
GGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGAC
AGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATC
GATACCGGCGGGAACACAGGGTACAATCCCGGACTGAA
GAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCA
GGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAG
TGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAA
GAAATACCAGAGCATGTGTATGCCCTGCTTCACGACCGA
TCACCAGATGGCGCGCAAATGCGATGACTGTTGCGGCGG
TAAAGGTCGCGGAAAGTGCTATGGCCCGCAGTGTCTGTC
TTATACCTACAATTATGAATGGCATGTGGATGTCTGGGG ACAGGGCCTGCTGGTGACAGTC
BLV1H12- 20 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA SDF1(alpha)
GCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCG
GGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGAC
AGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATC
GATACCGGCGGGAACACAGGGTACAATCCCGGACTGAA
GAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCA
GGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAG
TGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAA
GAAATACCAGAGCAAGCCCGTCAGCCTGAGCTACAGAT
GCCCATGCCGATTCTTCGAAAGCCATGTTGCCAGAGCCA
ACGTCAAGCATCTCAAAATTCTCAACACTCCAAACTGTG
CCCTTCAGATTGTAGCCCGGCTGAAGAACAACAACAGAC
AAGTGTGCATTGACCCGAAGCTAAAGTGGATTCAGGAGT
ACCTGGAGAAAGCTTTAAACAAGGGCAGCGGTTCTTATA
CCTACAATTATGAATGGCATGTGGATGTCTGGGGACAGG GCCTGCTGGTGACAGTC BLV1H12-
21 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA IL21
GCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCG
GGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGAC
AGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATC
GATACCGGCGGGAACACAGGGTACAATCCCGGACTGAA
GAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCA
GGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAG
TGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAA
GAAATACCAGAGCCAAGGTCAAGATCGCCACATGATCA
GAATGCGTCAGCTCATAGATATTGTTGATCAGCTGAAGA
ACTACGTGAACGACTTGGTCCCTGAATTTCTGCCAGCTC
CCGAAGATGTAGAGACAAACTGTGAGTGGTCAGCCTTCT
CCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACCG
GCAACAACGAGAGGATAATCAATGTATCAATCAAAAAG
CTGAAGAGGAAGCCACCTTCCACAAATGCAGGGAGACG
GCAGAAACACCGCCTGACATGCCCTTCATGTGATTCTTA
CGAGAAGAAGCCACCCAAAGAGTTCCTAGAGCGGTTCA
AGTCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCT
CTCGCACACACGGAAGTGAAGATTCCTCTTATACCTACA
ATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGC TGGTGACAGTC BLV1H12- 22
CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA ProTxII
GCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCG
GGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGAC
AGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATC
GATACCGGCGGGAACACAGGGTACAATCCCGGACTGAA
GAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCA
GGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAG
TGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAA
GAAATACCAGAGCTATTGCCAGAAGTGGATGTGGACCT
GCGATAGCGAACGGAAATGTTGCGAAGGCATGGTGTGC
CGCCTGTGGTGCAAGAAGAAACTCTGGTCTTATACCTAC
AATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTG CTGGTGACAGTC
TABLE-US-00025 TABLE 21 SEQ ID Name NO Sequence Light 23
QAVLNQPSSVSGSLGQRVSITCSGSSSNVGNGYVSWYQLIPGSAP Chain
RTLIYGDTSRASGVPDRFSGSRSGNTATLTISSLQAEDEADYFCAS
AEDSSSNAVFGSGTTLTVLGQPKSPPSVTLFPPSTEELNGNKATLV
CLISDFYPGSVTVVWKADGSTITRNVETTRASKQSNSKYAASSYL
SLTSSDWKSKGSYSCEVTHEGSTVTKTVKPSECS Heavy 24
QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG Chain
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV ##STR00031## IFN- 25
QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG beta
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYCTSVHQETKKYQS
MSYNLLGFLQRSSNFQC QKLLWQLNGRLEYCLKDRIVINFDIPEEIKQLQQFQKEDAALTIYEMLQ
NIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTR
GKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTG YLRN
SYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYP
LSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTF
PAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKA ##STR00032##
##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037##
bGCSF- 26 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L0
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV
TTEDSATYYCTSVHQETKKYQSTPLGPARSLPQSFLLKCLEQVRKI
QADGAELQERLCAAHKLCHPEELMLLRHSLGIPQAPLSSCSSQSLQLT
SCLNQLHGGLFLYQGLLQALAGISPELAPTLDTLQLDVTDFATNIWLQ
MEDLGAAPAVQPTQGAMPTFTSAFQRRAGGVLVASQLHRFLELAYRG
LRYLAEPSYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYPLSS
CCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPA
VLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVE ##STR00038##
##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043##
bGCSF- 27 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L1
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYCTSVHQETKKYQS
TPLGPARSLPQSFLLKCL EQVRKIQADGAELQERLCAAHKLCHPEELMLLRHSLGIPQAPLSSCSS
QSLQLTSCLNQLHGGLFLYQGLLQALAGISPELAPTLDTLQLDVTDFA
TNIWLQMEDLGAAPAVQPTQGAMPTFTSAFQRRAGGVLVASQLHRFL ELAYRGLRYLAEP
SYTYNYEWHVDVWGQGLLVTVSSAS
TTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGA
LKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPA ##STR00044##
##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049##
GMCSF 28 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYCTSVHQETKKYQS
APARSPSPSTQPWEHVN AIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQG
LRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFICENLKDF LLVIPFDCWEPVQE
SYTYNYEWHVDVWGQGLLVTVSSA
STTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSG
ALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHP ##STR00050##
##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055##
hFGF21 29 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYCTSVHQETKKYQS
HPIPDSSPLLQFGGQVR QRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQI
LGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAH
GLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPD
VGSSDPLSMVGPSQGRSPSYAS SYTYNYEWHVDVWGQGL
LVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVT
VTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFT ##STR00056##
##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061##
Ex-4 30 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYCTSVHQETKKYQSC
IEGRHGEGTFTSDLSK QMEEEAVRLFIEWLKNGGPSSGAPPPS CSYTYNYEWHVDV
WGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSY
MPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGS ##STR00062##
##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067##
hGLP-1 31 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV ##STR00068##
LEGQAAKEFIAWLVKGR CSYTYNYEWHVDVWGQGLLVT
VSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVT
WNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCN ##STR00069##
##STR00070## ##STR00071## ##STR00072## ##STR00073## ##STR00074##
hEPO 32 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYCTSVHQETKKYQS
APPRLICDSRVLERYLLE AKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRNIEVGQQAVEVWQ
GLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALG
AQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACR TGDR
SYTYNYEWHVDVWGQGLLVTVSSASTTAPKVYP
LSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTF
PAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKA ##STR00075##
##STR00076## ##STR00077## ##STR00078## ##STR00079## ##STR00080##
Moka- 33 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L0
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV
TTEDSATYYCTSVHQETKKYQSINVKCSLPQQCIICPCKDAGMRF
GKCMNKKCRCYSSYTYNYEWHVDVWGQGLLVTVSSASTTAPK
VYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGV
HTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKV ##STR00081##
##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086##
Moka- 34 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L1
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYCTSVHQETKKYQS
INVKCSLPQQCIKPCKDA GMRFGKCMNKKCRCYS SYTYNYEWHVDVWGQGLLVTV
SSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTW
NSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNV ##STR00087##
##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092##
VM-24- 35 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L1
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYCTSVHQETKKYQS
AAAISCVGSPECPPKCRA QGCKNGKCMNRKCKCYYC SYTYNYEWHVDVWGQGLLV
TVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVT
WNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCN ##STR00093##
##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098##
VM-24- 36 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L2
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYCTSVHQETKKYQS
AAAISCVGSPECPP KCRAQGCKNGKCMNRKCKCYYC SYTYNYEWHVDV
WGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSY
MPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGS ##STR00099##
##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104##
Protoxin 37 QVQLRESGPSLKPSQTLSLTCTASGFSLSDKAVGWVRQAPG 2-L1
KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYCTSVHQETKKYQS
YCQKWIVIWTCDSERKCC EGMVCRLWCKKKLWG SYTYNYEWHVDVWGQGLLVTVSS
ASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNS
GALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAH ##STR00105##
##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110##
For SEQ ID NOS: 24-37 bovine heavy chain sequence = bold human
heavy chain sequence = dashed underline non-antibody sequence =
italic Stalk = bold, underline; knob = bold, double underline;
linker = italic, squiggly underline
TABLE-US-00026 TABLE 22 SEQ ID Name NO SEQUENCE (bold font =
non-antibody sequence) BLV1H12-IL8 38
QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL
EWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSA
TYYCTSVHQETKKYQSPRSAKELRCQCIKTYSKPFHPKFIKELR
VIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRA
ENSGSGSYTYNYEWHVDVWGQGLLVTV BLV1H12- 39
QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL Ziconotide
EWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSA
TYYCTSVHQETKKYQSCKGKGAKCSRLMYDCCTGSCRSGKCS YTYNYEWHVDVWGQGLLVTV
BLV1H12- 40 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL
Somatostatin EWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSA
TYYCTSVHQETKKYQSAGCKNFFWKTFTSCGSYTYNYEWHVD VWGQGLLVTV BLV1H12- 41
QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL Chlorotoxin
EWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSA
TYYCTSVHQETKKYQSMCMPCFTTDHQMARKCDDCCGGKGR
GKCYGPQCLSYTYNYEWHVDVWGQGLLVTV BLV1H12- 42
QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL SDF1(alpha)
EWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSA
TYYCTSVHQETKKYQSKPVSLSYRCPCRFFESHVARANVKHLK
ILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKGS GSYTYNYEWHVDVWGQGLLVTV
BLV1H12-IL21 43 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL
EWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSA
TYYCTSVHQETKKYQSQGQDRHMIRMRQLIDIVDQLKNYVND
LVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINV
SIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFK
SLLQKMIHQHLSSRTHGSEDSSYTYNYEWHVDVWGQGLLVTV BLV1H12- 44
QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL ProTxII
EWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSA
TYYCTSVHQETKKYQSYCQKWMWTCDSERKCCEGMVCRLW
CKKKLWSYTYNYEWHVDVWGQGLLVTV BLV5B8 340
QVQLRESGPSLVQPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL VHCH1
EWLGSIDTGGSTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSA
TYYCTTVHQETRKTCSDGYIAVDSCGRGQSDGCVNDCNSCYYG
WRNCRRQPAIHSYEFHVDAWGRGLLVTVSSASTTAPKVYPLSSC
CGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQS
SGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSCDG S BLV5B8 341
QAVLNQPSSVSGSLGQRVSITCSGSSSNVGNGYVSWYQLIPGSAP VLCL
RTLIYGDTSRASGVPDRFSGSRSGNTATLTISSLQAEDEADYFCAS
AEDSSSNAVFGSGTTLTVLGQPKSPPSVTLFPPSTEELNGNKATLV
CLISDFYPGSVTVVWKADGSTITRNVETTRASKQSNSKYAASSYL
SLTSSDWKSKGSYSCEVTHEGSTVTKTVKPSECS
TABLE-US-00027 TABLE 23 Exemplary cysteine motifs SEQ ID NO:
Sequence 45 CX.sub.10CX.sub.5CX.sub.5CXCX.sub.7C 46
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.15C 47 CX.sub.11CXCX.sub.5C 48
CX.sub.11CX.sub.5CX.sub.5CXCX.sub.7C 49
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.13C 50
CX.sub.10CX.sub.5CXCX.sub.4CX.sub.8C 51
CX.sub.10CX.sub.6CX.sub.6CXCX.sub.7C 52
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.8C 53
CX.sub.10CX.sub.4CX.sub.7CXCX.sub.7C 54 CX.sub.13CX.sub.8CX.sub.8C
55 CX.sub.10CX.sub.6CX.sub.5CXCX.sub.7C 56
CX.sub.10CX.sub.5CX.sub.5C 57 CX.sub.10CX.sub.5CX.sub.6CXCX.sub.7C
58 CX.sub.10CX.sub.6CX.sub.5CX.sub.7CX.sub.9C 59
CX.sub.9CX.sub.7CX.sub.5CXCX.sub.7C 60
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.9C 61
CX.sub.10CXCX.sub.4CX.sub.5CX.sub.11C 62
CX.sub.7CX.sub.3CX.sub.6CX.sub.5CXCX.sub.5CX.sub.10C 63
CX.sub.10CXCX.sub.4CX.sub.5CXCX.sub.2CX.sub.3C 64
CX.sub.16CX.sub.5CXC 65 CX.sub.6CX.sub.4CXCX.sub.4CX.sub.5C 66
CX.sub.11CX.sub.4CX.sub.5CX.sub.6CX.sub.3C 67
CX.sub.8CX.sub.2CX.sub.6CX.sub.5C 68
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.10C 69
CX.sub.10CXCX.sub.6CX.sub.4CXC 70
CX.sub.10CX.sub.5CX.sub.5CXCX.sub.2C 71
CX.sub.14CX.sub.2CX.sub.3CXCXC 72 CX.sub.15CX.sub.5CXC 73
CX.sub.4CX.sub.6CX.sub.9CX.sub.2CX.sub.11C 74
CX.sub.6CX.sub.4CX.sub.5CX.sub.5CX.sub.12C 75
CX.sub.7CX.sub.3CXCXCX.sub.4CX.sub.5CX.sub.9C 76
CX.sub.10CX.sub.6CX.sub.5C 77
CX.sub.7CX.sub.3CX.sub.5CX.sub.5CX.sub.9C 78
CX.sub.7CX.sub.5CXCX.sub.2C 79 CX.sub.10CXCX.sub.6C 80
CX.sub.10CX.sub.3CX.sub.3CX.sub.5CX.sub.7CXCX.sub.6C 81
CX.sub.10CX.sub.4CX.sub.5CX.sub.12CX.sub.2C 82
CX.sub.12CX.sub.4CX.sub.5CXCXCX.sub.9CX.sub.3C 83
CX.sub.12CX.sub.4CX.sub.5CX.sub.12CX.sub.2C 84
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.11C 85
CX.sub.16CX.sub.5CXCXCX.sub.14C 86
CX.sub.10CX.sub.5CXCX.sub.8CX.sub.6C 87
CX.sub.12CX.sub.4CX.sub.5CX.sub.8CX.sub.2C 88
CX.sub.12CX.sub.5CX.sub.5CXCX.sub.8C 89
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.4CXCX.sub.9C 90
CX.sub.11CX.sub.4CX.sub.5CX.sub.8CX.sub.2C 91
CX.sub.10CX.sub.6CX.sub.5CX.sub.8CX.sub.2C 92
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.8C 93
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.3CX.sub.8CX.sub.2C 94
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.6CX.sub.5C 95
CX.sub.10CX.sub.6CX.sub.5CX.sub.3CX.sub.8C 96
CX.sub.7CX.sub.6CX.sub.3CX.sub.3CX.sub.9C 97
CX.sub.9CX.sub.8CX.sub.5CX.sub.6CX.sub.5C 98
CX.sub.10CX.sub.2CX.sub.2CX.sub.7CXCX.sub.11CX.sub.5C 99
CX.sub.10CX.sub.6CX.sub.5CXCX.sub.2CX.sub.8CX.sub.4C 100
CCX.sub.3CXCX.sub.3CX.sub.2CCXCX.sub.5CX.sub.9CX.sub.5CXC 101
CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC 102
CX.sub.6CX.sub.2CX.sub.5CX.sub.4CCXCX.sub.4CX.sub.6CXC 103
CX.sub.9CX.sub.3CXCX.sub.2CXCCCX.sub.6CX.sub.4C 104
CX.sub.5CX.sub.3CXCX.sub.4CX.sub.4CCX.sub.10CX.sub.2CC 105
CX.sub.5CXCX.sub.1CXCX.sub.3CCX.sub.3CX.sub.4CX.sub.10C 106
CX.sub.9CCCX.sub.3CX.sub.4CCCX.sub.5CX.sub.6C 107
CCX.sub.8CX.sub.5CX.sub.4CX.sub.3CX.sub.4CCXCX.sub.1C 108
CCX.sub.6CCX.sub.5CCCX.sub.4CX.sub.4CX.sub.12C 109
CX.sub.6CX.sub.2CX.sub.3CCCX.sub.4CX.sub.5CX.sub.3CX.sub.3C 110
CX.sub.3CX.sub.5CX.sub.6CX.sub.4CCXCX.sub.5CX.sub.4CXC 111
CX.sub.4CX.sub.4CCX.sub.4CX.sub.4CXCX.sub.11CX.sub.2CXC 112
CX.sub.5CX.sub.2CCX.sub.5CX.sub.4CCX.sub.3CCX.sub.7C 113
CX.sub.5CX.sub.5CX.sub.3CX.sub.2CXCCX.sub.4CX.sub.7CXC 114
CX.sub.3CX.sub.7CX.sub.3CX.sub.4CCXCX.sub.2CX.sub.5CX.sub.2C 115
CX.sub.9CX.sub.3CXCX.sub.4CCX.sub.5CCCX.sub.6C 116
CX.sub.9CX.sub.3CXCX.sub.2CXCCX.sub.6CX.sub.3CX.sub.3C 117
CX.sub.8CCXCX.sub.3CCX.sub.3CXCX.sub.3CX.sub.4C 118
CX.sub.9CCX.sub.4CX.sub.2CXCCXCX.sub.4CX.sub.3C 119
CX.sub.10CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC 120
CX.sub.9CXCX.sub.3CX.sub.2CXCCX.sub.4CX.sub.5CXC 121
CX.sub.6CCXCX.sub.5CX.sub.4CCXCX.sub.5CX.sub.2C 122
CX.sub.6CCXCX.sub.3CXCCX.sub.3CX.sub.4CC 123
CX.sub.6CCXCX.sub.3CXCX.sub.2CXCX.sub.4CX.sub.8C 124
CX.sub.4CX.sub.2CCX.sub.3CXCX.sub.4CCX.sub.2CX.sub.3C 125
CX.sub.3CX.sub.5CX.sub.3CCCX.sub.4CX.sub.9C 126
CCX.sub.9CX.sub.3CXCCX.sub.3CX.sub.5C 127
CX.sub.9CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5C 128
CX.sub.9CX.sub.7CX.sub.4CCXCX.sub.7CX.sub.3C 129
CX.sub.9CX.sub.3CCCX.sub.10CX.sub.2CX.sub.3C 130
CX.sub.3CX.sub.5CX.sub.5CX.sub.4CCX.sub.10CX.sub.6C 131
CX.sub.9CX.sub.5CX.sub.4CCXCX.sub.5CX.sub.4C 132
CX.sub.7CXCX.sub.6CX.sub.4CCCX.sub.10C 133
CX.sub.8CX.sub.2CX.sub.4CCX.sub.4CX.sub.3CX.sub.3C 134
CX.sub.7CX.sub.5CXCX.sub.4CCX.sub.7CX.sub.4C 135
CX.sub.11CX.sub.3CX.sub.4CCCX.sub.8CX.sub.2C 136
CX.sub.2CX.sub.3CX.sub.4CCX.sub.4CX.sub.5CX.sub.15C 137
CX.sub.9CX.sub.5CX.sub.4CCX.sub.7C 138
CX.sub.9CX.sub.7CX.sub.3CX.sub.2CX.sub.6C 139
CX.sub.9CX.sub.5CX.sub.4CCX.sub.14C 140
CX.sub.9CX.sub.5CX.sub.4CCX.sub.8C 141 CX.sub.9CX.sub.6CX.sub.4CCXC
142 CX.sub.5CCX.sub.7CX.sub.4CX.sub.12 143
CX.sub.10CX.sub.3CX.sub.4CCX.sub.4C 144
CX.sub.9CX.sub.4CCX.sub.5CX.sub.4C 145
CX.sub.10CX.sub.3CX.sub.4CX.sub.7CXC 146
CX.sub.7CX.sub.7CX.sub.2CX.sub.2CX.sub.3C 147
CX.sub.9CX.sub.4CX.sub.4CCX.sub.6C 148
CX.sub.7CXCX.sub.3CXCX.sub.6C 149 CX.sub.7CXCX.sub.4CXCX.sub.4C 150
CX.sub.9CX.sub.5CX.sub.4C 151 CX.sub.3CX.sub.6CX.sub.8C 152
CX.sub.10CXCX.sub.4C 153 CX.sub.10CCX.sub.4C 154 CX.sub.15C 155
CX.sub.10C 156 CX.sub.9C
TABLE-US-00028 TABLE 24 Exemplary conserved motifs with the stalk
domain SEQ ID NO: Sequence 157 TSVHQETKKYQ 158 VHQETKKYQ 159 TTVHQ
160 TSVHQ 161 SSVTQ 162 STVHQ 163 ATVRQ 164 TTVYQ 165 SPVHQ 166
ATVYQ 167 TAVYQ 168 TNVHQ 169 ATVHQ 170 STVYQ 171 TIVHQ 172 ATVYQ
173 TTVFQ 174 AAVFQ 175 GTVHQ 176 ASVHQ 177 TAVFQ 178 ATVFQ 179
AAAHQ 180 VVVYQ 181 GTVFQ 182 TAVHQ 183 ITVHQ 184 ITAHQ 185 VTVHQ
186 AAVHQ 187 GTVYQ 188 TTVLQ 189 TTTHQ 190 TTDYQ 191 TTDYQ 192
CTSVHQ 193 CSSVTQ 194 CSTVHQ 195 CATVRQ 196 CTTVYQ 197 CSPVHQ 198
CATVYQ 199 CTAVYQ 200 CTNVHQ 201 CATVHQ 202 CSTVYQ 203 CTIVHQ 204
CAIVYQ 205 CTTVFQ 206 CAAVFQ 207 CGTVHQ 208 CASVHQ 209 CTAVFQ 210
CATVFQ 211 CAAAHQ 212 CVVVYQ 213 CGTVFQ 214 CTAVHQ 215 CITVHQ 216
CITAHQ 217 CVTVHQ 218 CAAVHQ 219 CGTVYQ 220 CTTVLQ 221 CTTTHQ 222
CTTDYQ 223 CTTVHQX.sub.n 224 CTSVHQX.sub.n 225 VHQ 226 KKQ 227 VYQ
228 CX.sup.1 X.sup.2X.sup.3 X.sup.4Q 229 X.sup.1 X.sup.2VHQ 230
CX.sup.1 X.sup.2VHQ 231 X.sup.1 X.sup.2VX.sup.3Q 232 CX.sup.1
X.sup.2VX.sup.3Q 233 X.sup.1 X.sup.2KKQ 234 CX.sup.1 X.sup.2KKQ 235
YTYNYEW 236 YTYNYE 237 YLYTYEH 238 YLYTYE 239 CYTYNYEF 240 HYTYTYDF
241 HYTYTYEW 242 KHRYTYEW 243 NYIYKYSF 244 PYIYTYQF 245 SFTYTYEW
246 SYIYIYQW 247 SYNYTYSW 248 SYSYSYEY 249 SYTYNYDF 250 SYTYNYEW
251 SYTYNYQF 252 SYVWTHNF 253 TYKYVYEW 254 TYTYTYEF 255 TYTYTYEW
256 VFTYTYEF 257 AYTYEW 258 DYIYTY 259 IHSYEF 260 SFTYEF 261 SHSYEF
262 THTYEF 263 TWTYEF 264 TYNYEW 265 TYSYEF 266 TYSYEH 267 TYTYDF
268 TYTYEF 269 TYTYEW 270 AYEF 271 AYSF 272 AYSY 273 CYSF 274 DYTY
275 KYEH 276 KYEW 277 MYEF 278 NWIY
279 NYDY 280 NYQW 281 NYSF 282 PYEW 283 RYNW 284 RYTY 285 SYEF 286
SYEH 287 SYEW 288 SYKW 289 SYTY 290 TYDF 291 TYEF 292 TYEW 293 TYQW
294 TYTY 295 VYEW 296 YX.sup.1YX.sup.2 297 YX.sup.1YX.sup.2 Y 298
YX.sup.1YX.sup.2 YX.sup.3 299 YX.sup.1YX.sup.2 YX.sup.3X.sup.4 300
YEX 301 YDX 302 XYE 303 XYD 304 YEX.sup.1X.sub.nW 305
YDX.sup.1X.sub.nW 306 YEX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5W 307
YDX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5W 333 YEXXXW 334 YEXXXXW 335
YDXXXW 336 YDXXXXW
TABLE-US-00029 TABLE 25 Exemplary Linker Sequences SEQ ID NO:
Sequence 337 GGGSGGGGS 338 GGGGSGGGS 339 (GGGS)n 342 (GSG)n
TABLE-US-00030 TABLE 26 Exemplary non-antibody sequences SEQ ID
Description NO: Sequence IL8 317
PRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLS
DGRELCLDPKENWVQRVVEKFLKRAENS ziconotide 318
CKGKGAKCSRLMYDCCTGSCRSGKC somatostatin 319 AGCKNFFWKTFTSCG
chlorotoxin 320 MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCL SDF1(alpha) 321
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLK
NNNRQVCIDPKLKWIQEYLEKALNK IL21 322
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCE
WSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRR
QKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGS EDS Protoxin2 323
YCQKWMWTCDSERKCCEGMVCRLWCKKKLW IFN-beta 324
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPE
EIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN bGCSF 325
TPLGPARSLPQSFLLKCLEQVRKIQADGAELQERLCAAHKLCH
PEELMLLRHSLGIPQAPLSSCSSQSLQLTSCLNQLHGGLFLYQG
LLQALAGISPELAPTLDTLQLDVTDFATNIWLQMEDLGAAPAV
QPTQGAMPTFTSAFQRRAGGVLVASQLHRFLELAYRGLRYLA EP GMCSF 326
APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVIS
EMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQ
HCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE hFGF21 327
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGG
AADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLH
FDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAP
RGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGR SPSYAS Ex-4 328
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS hGLP-1 329
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR hEPO 330
PPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTK
VNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQ
PWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAP
LRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR Moka 331
INVKCSLPQQCIKPCKDAGMRFGKCMNKKCRCYS VM-24 332
AAAISCVGSPECPPKCRAQGCKNGKCMNRKCKCYYC
Sequence CWU 1
1
5111648DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Light Chain fusion polynucleotide 1caggccgtcc tgaaccagcc
aagcagcgtc tccgggtctc tggggcagcg ggtctcaatc 60acctgtagcg ggtcttcctc
caatgtcggc aacggctacg tgtcttggta tcagctgatc 120cctggcagtg
ccccacgaac cctgatctac ggcgacacat ccagagcttc tggggtcccc
180gatcggttct cagggagcag atccggaaac acagctactc tgaccatcag
ctccctgcag 240gctgaggacg aagcagatta tttctgcgca tctgccgagg
actctagttc aaatgccgtg 300tttggaagcg gcaccacact gacagtcctg
gggcagccca agagtccccc ttcagtgact 360ctgttcccac cctctaccga
ggaactgaac ggaaacaagg ccacactggt gtgtctgatc 420agcgactttt
accctggatc cgtcactgtg gtctggaagg cagatggcag cacaattact
480aggaacgtgg aaactacccg cgcctccaag cagtctaata gtaaatacgc
cgccagctcc 540tatctgagcc tgacctctag tgattggaag tccaaagggt
catatagctg cgaagtgacc 600catgaaggct caaccgtgac taagactgtg
aaaccatccg agtgctcc 6482507DNAArtificial SequenceDescription of
Artificial Sequence Synthetic Heavy Chain -no insertion fusion
polynucleotide 2caggtccagc 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 ctctagt
50731905DNAArtificial SequenceDescription of Artificial Sequence
Synthetic IFN-beta fusion polynucleotide 3caggtccagc 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 cgggggtggc
ggaagcatga gctacaactt gcttggattc 360ctacaaagaa gcagcaattt
tcagtgtcag aagctcctgt ggcaattgaa tgggaggctt 420gaatactgcc
tcaaggacag gatgaacttt gacatccctg aggagattaa gcagctgcag
480cagttccaga aggaggacgc cgcattgacc atctatgaga tgctccagaa
catctttgct 540attttcagac aagattcatc tagcactggc tggaatgaga
ctattgttga gaacctcctg 600gctaatgtct atcatcagat aaaccatctg
aagacagtcc tggaagaaaa actggagaaa 660gaagatttca ccaggggaaa
actcatgagc agtctgcacc tgaaaagata ttatgggagg 720attctgcatt
acctgaaggc caaggagtac agtcactgtg cctggaccat agtcagagtg
780gaaatcctaa ggaactttta cttcattaac agacttacag gttacctccg
aaacggcgga 840ggtgggagtt cttataccta caattatgaa tggcatgtgg
atgtctgggg acagggcctg 900ctggtgacag tctctagtgc ttccacaact
gcaccaaagg tgtaccccct gtcaagctgc 960tgtggggaca aatcctctag
taccgtgaca ctgggatgcc tggtctcaag ctatatgccc 1020gagcctgtga
ctgtcacctg gaactcagga gccctgaaaa gcggagtgca caccttccca
1080gctgtgctgc agtcctctgg cctgtatagc ctgagttcaa tggtgacagt
ccccggcagt 1140acttcagggc agaccttcac ctgtaatgtg gcccatcctg
ccagctccac caaagtggac 1200aaagcagtgg aacccaaatc ttgcgacaaa
actcacacat gcccaccgtg cccagcacct 1260gaactcctgg ggggaccgtc
agtcttcctc ttccccccaa aacccaagga caccctcatg 1320atctcccgga
cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag
1380gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac
aaagccgcgg 1440gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc
tcaccgtcct gcaccaggac 1500tggctgaatg gcaaggagta caagtgcaag
gtctccaaca aagccctccc agcccccatc 1560gagaaaacca tctccaaagc
caaagggcag ccccgagaac cacaggtgta caccctgccc 1620ccatcccggg
atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc
1680tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa
caactacaag 1740accacgcctc ccgtgctgga ctccgacggc tccttcttcc
tctacagcaa gctcaccgtg 1800gacaagagca ggtggcagca ggggaacgtc
ttctcatgct ccgtgatgca tgaggctctg 1860cacaaccact acacgcagaa
gagcctctcc ctgtctccgg gtaaa 190541899DNAArtificial
SequenceDescription of Artificial Sequence Synthetic bGCSF-L0
fusion polynucleotide 4caggtccagc 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 cacccccctt ggccctgccc gatccctgcc
ccagagcttc 360ctgctcaagt gcttagagca agtgaggaaa atccaggctg
atggcgccga gctgcaggag 420aggctgtgtg ccgcccacaa gctgtgccac
ccggaggagc tgatgctgct caggcactct 480ctgggcatcc cccaggctcc
cctaagcagc tgctccagcc agtccctgca gctgacgagc 540tgcctgaacc
aactacacgg cggcctcttt ctctaccagg gcctcctgca ggccctggcg
600ggcatctccc cagagctggc ccccaccttg gacacactgc agctggacgt
cactgacttt 660gccacgaaca tctggctgca gatggaggac ctgggggcgg
cccccgctgt gcagcccacc 720cagggcgcca tgccgacctt cacttcagcc
ttccaacgca gagcaggagg ggtcctggtt 780gcttcccagc tgcatcgttt
cctggagctg gcataccgtg gcctgcgcta ccttgctgag 840ccctcttata
cctacaatta tgaatggcat gtggatgtct ggggacaggg cctgctggtg
900acagtctcta gtgcttccac aactgcacca aaggtgtacc ccctgtcaag
ctgctgtggg 960gacaaatcct ctagtaccgt gacactggga tgcctggtct
caagctatat gcccgagcct 1020gtgactgtca cctggaactc aggagccctg
aaaagcggag tgcacacctt cccagctgtg 1080ctgcagtcct ctggcctgta
tagcctgagt tcaatggtga cagtccccgg cagtacttca 1140gggcagacct
tcacctgtaa tgtggcccat cctgccagct ccaccaaagt ggacaaagca
1200gtggaaccca aatcttgcga caaaactcac acatgcccac cgtgcccagc
acctgaactc 1260ctggggggac cgtcagtctt cctcttcccc ccaaaaccca
aggacaccct catgatctcc 1320cggacccctg aggtcacatg cgtggtggtg
gacgtgagcc acgaagaccc tgaggtcaag 1380ttcaactggt acgtggacgg
cgtggaggtg cataatgcca agacaaagcc gcgggaggag 1440cagtacaaca
gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg
1500aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc
catcgagaaa 1560accatctcca aagccaaagg gcagccccga gaaccacagg
tgtacaccct gcccccatcc 1620cgggatgagc tgaccaagaa ccaggtcagc
ctgacctgcc tggtcaaagg cttctatccc 1680agcgacatcg ccgtggagtg
ggagagcaat gggcagccgg agaacaacta caagaccacg 1740cctcccgtgc
tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag
1800agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc
tctgcacaac 1860cactacacgc agaagagcct ctccctgtct ccgggtaaa
189951929DNAArtificial SequenceDescription of Artificial Sequence
Synthetic bGCSF-L1 fusion polynucleotide 5caggtccagc 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 cggtggcgga
ggatctaccc cccttggccc tgcccgatcc 360ctgccccaga gcttcctgct
caagtgctta gagcaagtga ggaaaatcca ggctgatggc 420gccgagctgc
aggagaggct gtgtgccgcc cacaagctgt gccacccgga ggagctgatg
480ctgctcaggc actctctggg catcccccag gctcccctaa gcagctgctc
cagccagtcc 540ctgcagctga cgagctgcct gaaccaacta cacggcggcc
tctttctcta ccagggcctc 600ctgcaggccc tggcgggcat ctccccagag
ctggccccca ccttggacac actgcagctg 660gacgtcactg actttgccac
gaacatctgg ctgcagatgg aggacctggg ggcggccccc 720gctgtgcagc
ccacccaggg cgccatgccg accttcactt cagccttcca acgcagagca
780ggaggggtcc tggttgcttc ccagctgcat cgtttcctgg agctggcata
ccgtggcctg 840cgctaccttg ctgagcccgg tggcggagga tcttcttata
cctacaatta tgaatggcat 900gtggatgtct ggggacaggg cctgctggtg
acagtctcta gtgcttccac aactgcacca 960aaggtgtacc ccctgtcaag
ctgctgtggg gacaaatcct ctagtaccgt gacactggga 1020tgcctggtct
caagctatat gcccgagcct gtgactgtca cctggaactc aggagccctg
1080aaaagcggag tgcacacctt cccagctgtg ctgcagtcct ctggcctgta
tagcctgagt 1140tcaatggtga cagtccccgg cagtacttca gggcagacct
tcacctgtaa tgtggcccat 1200cctgccagct ccaccaaagt ggacaaagca
gtggaaccca aatcttgcga caaaactcac 1260acatgcccac cgtgcccagc
acctgaactc ctggggggac cgtcagtctt cctcttcccc 1320ccaaaaccca
aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg
1380gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg
cgtggaggtg 1440cataatgcca agacaaagcc gcgggaggag cagtacaaca
gcacgtaccg tgtggtcagc 1500gtcctcaccg tcctgcacca ggactggctg
aatggcaagg agtacaagtg caaggtctcc 1560aacaaagccc tcccagcccc
catcgagaaa accatctcca aagccaaagg gcagccccga 1620gaaccacagg
tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc
1680ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg
ggagagcaat 1740gggcagccgg agaacaacta caagaccacg cctcccgtgc
tggactccga cggctccttc 1800ttcctctaca gcaagctcac cgtggacaag
agcaggtggc agcaggggaa cgtcttctca 1860tgctccgtga tgcatgaggc
tctgcacaac cactacacgc agaagagcct ctccctgtct 1920ccgggtaaa
192961788DNAArtificial SequenceDescription of Artificial Sequence
Synthetic GMCSF fusion polynucleotide 6caggtccagc 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 cgggggtggc
ggaagcgcac ccgcccgctc gcccagcccc 360agcacgcagc cctgggagca
tgtgaatgcc atccaggagg cccggcgtct cctgaacctg 420agtagagaca
ctgctgctga gatgaatgaa acagtagaag tcatctcaga aatgtttgac
480ctccaggagc cgacctgcct acagacccgc ctggagctgt acaagcaggg
cctgcggggc 540agcctcacca agctcaaggg ccccttgacc atgatggcca
gccactacaa gcagcactgc 600cctccaaccc cggaaacttc ctgtgcaacc
cagattatca cctttgaaag tttcaaagag 660aacctgaagg actttctgct
tgtcatcccc tttgactgct gggagccagt ccaggagggc 720ggaggtggga
gttcttatac ctacaattat gaatggcatg tggatgtctg gggacagggc
780ctgctggtga cagtctctag tgcttccaca actgcaccaa aggtgtaccc
cctgtcaagc 840tgctgtgggg acaaatcctc tagtaccgtg acactgggat
gcctggtctc aagctatatg 900cccgagcctg tgactgtcac ctggaactca
ggagccctga aaagcggagt gcacaccttc 960ccagctgtgc tgcagtcctc
tggcctgtat agcctgagtt caatggtgac agtccccggc 1020agtacttcag
ggcagacctt cacctgtaat gtggcccatc ctgccagctc caccaaagtg
1080gacaaagcag tggaacccaa atcttgcgac aaaactcaca catgcccacc
gtgcccagca 1140cctgaactcc tggggggacc gtcagtcttc ctcttccccc
caaaacccaa ggacaccctc 1200atgatctccc ggacccctga ggtcacatgc
gtggtggtgg acgtgagcca cgaagaccct 1260gaggtcaagt tcaactggta
cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 1320cgggaggagc
agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag
1380gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct
cccagccccc 1440atcgagaaaa ccatctccaa agccaaaggg cagccccgag
aaccacaggt gtacaccctg 1500cccccatccc gggatgagct gaccaagaac
caggtcagcc tgacctgcct ggtcaaaggc 1560ttctatccca gcgacatcgc
cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1620aagaccacgc
ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc
1680gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat
gcatgaggct 1740ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa
178871950DNAArtificial SequenceDescription of Artificial Sequence
Synthetic hFGF21 fusion polynucleotide 7caggtccagc 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 cgggggtggc
ggaagccacc ccatccctga ctccagtcct 360ctcctgcaat tcgggggcca
agtccggcag cggtacctct acacagatga tgcccagcag 420acagaagccc
acctggagat cagggaggat gggacggtgg ggggcgctgc tgaccagagc
480cccgaaagtc tcctgcagct gaaagccttg aagccgggag ttattcaaat
cttgggagtc 540aagacatcca ggttcctgtg ccagcggcca gatggggccc
tgtatggatc gctccacttt 600gaccctgagg cctgcagctt ccgggagctg
cttcttgagg acggatacaa tgtttaccag 660tccgaagccc acggcctccc
gctgcacctg ccagggaaca agtccccaca ccgggaccct 720gcaccccgag
gaccagctcg cttcctgcca ctaccaggcc tgccccccgc acccccggag
780ccacccggaa tcctggcccc ccagcccccc gatgtgggct cctcggaccc
tctgagcatg 840gtgggacctt cccagggccg aagccccagc tacgcttccg
gcggaggtgg gagttcttat 900acctacaatt atgaatggca tgtggatgtc
tggggacagg gcctgctggt gacagtctct 960agtgcttcca caactgcacc
aaaggtgtac cccctgtcaa gctgctgtgg ggacaaatcc 1020tctagtaccg
tgacactggg atgcctggtc tcaagctata tgcccgagcc tgtgactgtc
1080acctggaact caggagccct gaaaagcgga gtgcacacct tcccagctgt
gctgcagtcc 1140tctggcctgt atagcctgag ttcaatggtg acagtccccg
gcagtacttc agggcagacc 1200ttcacctgta atgtggccca tcctgccagc
tccaccaaag tggacaaagc agtggaaccc 1260aaatcttgcg acaaaactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 1320ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
1380gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 1440tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtacaac 1500agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 1560gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1620aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag
1680ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 1740gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 1800ctggactccg acggctcctt cttcctctac
agcaagctca ccgtggacaa gagcaggtgg 1860cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1920cagaagagcc
tctccctgtc tccgggtaaa 195081515DNAArtificial SequenceDescription of
Artificial Sequence Synthetic Ex-4 fusion polynucleotide
8caggtccagc 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
ctgcgggggt ggcggaagca tcgaaggtcg tcacgctgag 360ggaacattca
cttccgatgt gtcctcctac ctggagggcc aggctgccaa agagttcatc
420gcttggctcg tcaagggcag gggcggaggt gggagttgct cttataccta
caattatgaa 480tggcatgtgg atgtctgggg acagggcctg ctggtgacag
tctctagtgc ttccacaact 540gcaccaaagg tgtaccccct gtcaagctgc
tgtggggaca aatcctctag taccgtgaca 600ctgggatgcc tggtctcaag
ctatatgccc gagcctgtga ctgtcacctg gaactcagga 660gccctgaaaa
gcggagtgca caccttccca gctgtgctgc agtcctctgg cctgtatagc
720ctgagttcaa tggtgacagt ccccggcagt acttcagggc agaccttcac
ctgtaatgtg 780gcccatcctg ccagctccac caaagtggac aaagcagtgg
aacccaaatc ttgcgacaaa 840actcacacat gcccaccgtg cccagcacct
gaactcctgg ggggaccgtc agtcttcctc 900ttccccccaa aacccaagga
caccctcatg atctcccgga cccctgaggt cacatgcgtg 960gtggtggacg
tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg
1020gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac
gtaccgtgtg 1080gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg
gcaaggagta caagtgcaag 1140gtctccaaca aagccctccc agcccccatc
gagaaaacca tctccaaagc caaagggcag 1200ccccgagaac cacaggtgta
caccctgccc ccatcccggg atgagctgac caagaaccag 1260gtcagcctga
cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag
1320agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 1380tccttcttcc tctacagcaa gctcaccgtg gacaagagca
ggtggcagca ggggaacgtc 1440ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacgcagaa gagcctctcc 1500ctgtctccgg gtaaa
151591515DNAArtificial SequenceDescription of Artificial Sequence
Synthetic hGLP-1 fusion polynucleotide 9caggtccagc 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 ctgcgggggt
ggcggaagca tcgaaggtcg tcacgctgag 360ggaacattca cttccgatgt
gtcctcctac ctggagggcc aggctgccaa agagttcatc 420gcttggctcg
tcaagggcag gggcggaggt gggagttgct cttataccta caattatgaa
480tggcatgtgg atgtctgggg acagggcctg ctggtgacag tctctagtgc
ttccacaact 540gcaccaaagg tgtaccccct gtcaagctgc tgtggggaca
aatcctctag taccgtgaca 600ctgggatgcc tggtctcaag ctatatgccc
gagcctgtga ctgtcacctg gaactcagga 660gccctgaaaa gcggagtgca
caccttccca gctgtgctgc agtcctctgg cctgtatagc 720ctgagttcaa
tggtgacagt ccccggcagt acttcagggc agaccttcac ctgtaatgtg
780gcccatcctg ccagctccac caaagtggac aaagcagtgg aacccaaatc
ttgcgacaaa 840actcacacat gcccaccgtg cccagcacct gaactcctgg
ggggaccgtc agtcttcctc 900ttccccccaa aacccaagga caccctcatg
atctcccgga cccctgaggt cacatgcgtg 960gtggtggacg tgagccacga
agaccctgag gtcaagttca actggtacgt ggacggcgtg 1020gaggtgcata
atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg
1080gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta
caagtgcaag 1140gtctccaaca aagccctccc agcccccatc gagaaaacca
tctccaaagc caaagggcag 1200ccccgagaac cacaggtgta caccctgccc
ccatcccggg atgagctgac caagaaccag 1260gtcagcctga cctgcctggt
caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1320agcaatgggc
agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc
1380tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca
ggggaacgtc 1440ttctcatgct ccgtgatgca tgaggctctg cacaaccact
acacgcagaa gagcctctcc 1500ctgtctccgg gtaaa 1515101905DNAArtificial
SequenceDescription of Artificial Sequence Synthetic hEPO fusion
polynucleotide 10caggtccagc 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 cgggggtggc
ggaagcgccc caccacgcct catctgtgac 360agccgagtcc tggagaggta
cctcttggag gccaaggagg ccgagaatat cacgacgggc 420tgtgctgaac
actgcagctt gaatgagaat atcactgtcc cagacaccaa agttaatttc
480tatgcctgga agaggatgga ggtcgggcag caggccgtag aagtctggca
gggcctggcc 540ctgctgtcgg aagctgtcct gcggggccag gccctgttgg
tcaactcttc ccagccgtgg 600gagcccctgc agctgcatgt ggataaagcc
gtcagtggcc ttcgcagcct caccactctg 660cttcgggctc tgggagccca
gaaggaagcc atctcccctc cagatgcggc ctcagctgct 720ccactccgaa
caatcactgc tgacactttc cgcaaactct tccgagtcta ctccaatttc
780ctccggggaa agctgaagct gtacacaggg gaggcctgca ggacagggga
cagaggcgga 840ggtgggagtt cttataccta caattatgaa tggcatgtgg
atgtctgggg acagggcctg 900ctggtgacag tctctagtgc ttccacaact
gcaccaaagg tgtaccccct gtcaagctgc 960tgtggggaca aatcctctag
taccgtgaca ctgggatgcc tggtctcaag ctatatgccc 1020gagcctgtga
ctgtcacctg gaactcagga gccctgaaaa gcggagtgca caccttccca
1080gctgtgctgc agtcctctgg cctgtatagc ctgagttcaa tggtgacagt
ccccggcagt 1140acttcagggc agaccttcac ctgtaatgtg gcccatcctg
ccagctccac caaagtggac 1200aaagcagtgg aacccaaatc ttgcgacaaa
actcacacat gcccaccgtg cccagcacct 1260gaactcctgg ggggaccgtc
agtcttcctc ttccccccaa aacccaagga caccctcatg 1320atctcccgga
cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag
1380gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac
aaagccgcgg 1440gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc
tcaccgtcct gcaccaggac 1500tggctgaatg gcaaggagta caagtgcaag
gtctccaaca aagccctccc agcccccatc 1560gagaaaacca tctccaaagc
caaagggcag ccccgagaac cacaggtgta caccctgccc 1620ccatcccggg
atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc
1680tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa
caactacaag 1740accacgcctc ccgtgctgga ctccgacggc tccttcttcc
tctacagcaa gctcaccgtg 1800gacaagagca ggtggcagca ggggaacgtc
ttctcatgct ccgtgatgca tgaggctctg 1860cacaaccact acacgcagaa
gagcctctcc ctgtctccgg gtaaa 1905111479DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Moka-L0 fusion
polynucleotide 11caggtccagc 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 catcaacgtg aagtgcagcc tgccccagca
gtgcatcaag 360ccctgcaagg acgccggcat gcggttcggc aagtgcatga
acaagaagtg caggtgctac 420agctcttata cctacaatta tgaatggcat
gtggatgtct ggggacaggg cctgctggtg 480acagtctcta gtgcttccac
aactgcacca aaggtgtacc ccctgtcaag ctgctgtggg 540gacaaatcct
ctagtaccgt gacactggga tgcctggtct caagctatat gcccgagcct
600gtgactgtca cctggaactc aggagccctg aaaagcggag tgcacacctt
cccagctgtg 660ctgcagtcct ctggcctgta tagcctgagt tcaatggtga
cagtccccgg cagtacttca 720gggcagacct tcacctgtaa tgtggcccat
cctgccagct ccaccaaagt ggacaaagca 780gtggaaccca aatcttgcga
caaaactcac acatgcccac cgtgcccagc acctgaactc 840ctggggggac
cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc
900cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc
tgaggtcaag 960ttcaactggt acgtggacgg cgtggaggtg cataatgcca
agacaaagcc gcgggaggag 1020cagtacaaca gcacgtaccg tgtggtcagc
gtcctcaccg tcctgcacca ggactggctg 1080aatggcaagg agtacaagtg
caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1140accatctcca
aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc
1200cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg
cttctatccc 1260agcgacatcg ccgtggagtg ggagagcaat gggcagccgg
agaacaacta caagaccacg 1320cctcccgtgc tggactccga cggctccttc
ttcctctaca gcaagctcac cgtggacaag 1380agcaggtggc agcaggggaa
cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1440cactacacgc
agaagagcct ctccctgtct ccgggtaaa 1479121509DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Moka-L1 fusion
polynucleotide 12caggtccagc 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 cggtggcgga ggatctatca acgtgaagtg
cagcctgccc 360cagcagtgca tcaagccctg caaggacgcc ggcatgcggt
tcggcaagtg catgaacaag 420aagtgcaggt gctacagcgg aggtggtggt
tcatcttata cctacaatta tgaatggcat 480gtggatgtct ggggacaggg
cctgctggtg acagtctcta gtgcttccac aactgcacca 540aaggtgtacc
ccctgtcaag ctgctgtggg gacaaatcct ctagtaccgt gacactggga
600tgcctggtct caagctatat gcccgagcct gtgactgtca cctggaactc
aggagccctg 660aaaagcggag tgcacacctt cccagctgtg ctgcagtcct
ctggcctgta tagcctgagt 720tcaatggtga cagtccccgg cagtacttca
gggcagacct tcacctgtaa tgtggcccat 780cctgccagct ccaccaaagt
ggacaaagca gtggaaccca aatcttgcga caaaactcac 840acatgcccac
cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc
900ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg
cgtggtggtg 960gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt
acgtggacgg cgtggaggtg 1020cataatgcca agacaaagcc gcgggaggag
cagtacaaca gcacgtaccg tgtggtcagc 1080gtcctcaccg tcctgcacca
ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140aacaaagccc
tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga
1200gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa
ccaggtcagc 1260ctgacctgcc tggtcaaagg cttctatccc agcgacatcg
ccgtggagtg ggagagcaat 1320gggcagccgg agaacaacta caagaccacg
cctcccgtgc tggactccga cggctccttc 1380ttcctctaca gcaagctcac
cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440tgctccgtga
tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct
1500ccgggtaaa 1509131515DNAArtificial SequenceDescription of
Artificial Sequence Synthetic VM-24-L1 fusion polynucleotide
13caggtccagc 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
cgggggtggc ggaagcgccg ctgcaatctc ctgcgtcggc 360agccccgaat
gtcctcccaa gtgccgggct cagggatgca agaacggcaa gtgtatgaac
420cggaagtgca agtgctacta ttgcggcgga ggtgggagtt cttataccta
caattatgaa 480tggcatgtgg atgtctgggg acagggcctg ctggtgacag
tctctagtgc ttccacaact 540gcaccaaagg tgtaccccct gtcaagctgc
tgtggggaca aatcctctag taccgtgaca 600ctgggatgcc tggtctcaag
ctatatgccc gagcctgtga ctgtcacctg gaactcagga 660gccctgaaaa
gcggagtgca caccttccca gctgtgctgc agtcctctgg cctgtatagc
720ctgagttcaa tggtgacagt ccccggcagt acttcagggc agaccttcac
ctgtaatgtg 780gcccatcctg ccagctccac caaagtggac aaagcagtgg
aacccaaatc ttgcgacaaa 840actcacacat gcccaccgtg cccagcacct
gaactcctgg ggggaccgtc agtcttcctc 900ttccccccaa aacccaagga
caccctcatg atctcccgga cccctgaggt cacatgcgtg 960gtggtggacg
tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg
1020gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac
gtaccgtgtg 1080gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg
gcaaggagta caagtgcaag 1140gtctccaaca aagccctccc agcccccatc
gagaaaacca tctccaaagc caaagggcag 1200ccccgagaac cacaggtgta
caccctgccc ccatcccggg atgagctgac caagaaccag 1260gtcagcctga
cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag
1320agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 1380tccttcttcc tctacagcaa gctcaccgtg gacaagagca
ggtggcagca ggggaacgtc 1440ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacgcagaa gagcctctcc 1500ctgtctccgg gtaaa
1515141539DNAArtificial SequenceDescription of Artificial Sequence
Synthetic VM-24-L2 fusion polynucleotide 14caggtccagc 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 cggcggtgga
tctgggggtg gcggaagcgc cgctgcaatc 360tcctgcgtcg gcagccccga
atgtcctccc aagtgccggg ctcagggatg caagaacggc 420aagtgtatga
accggaagtg caagtgctac tattgcggcg gaggtgggag tggaggcggt
480agctcttata cctacaatta tgaatggcat gtggatgtct ggggacaggg
cctgctggtg 540acagtctcta gtgcttccac aactgcacca aaggtgtacc
ccctgtcaag ctgctgtggg 600gacaaatcct ctagtaccgt gacactggga
tgcctggtct caagctatat gcccgagcct 660gtgactgtca cctggaactc
aggagccctg aaaagcggag tgcacacctt cccagctgtg 720ctgcagtcct
ctggcctgta tagcctgagt tcaatggtga cagtccccgg cagtacttca
780gggcagacct tcacctgtaa tgtggcccat cctgccagct ccaccaaagt
ggacaaagca 840gtggaaccca aatcttgcga caaaactcac acatgcccac
cgtgcccagc acctgaactc 900ctggggggac cgtcagtctt cctcttcccc
ccaaaaccca aggacaccct catgatctcc 960cggacccctg aggtcacatg
cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 1020ttcaactggt
acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag
1080cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca
ggactggctg 1140aatggcaagg agtacaagtg caaggtctcc aacaaagccc
tcccagcccc catcgagaaa 1200accatctcca aagccaaagg gcagccccga
gaaccacagg tgtacaccct gcccccatcc 1260cgggatgagc tgaccaagaa
ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1320agcgacatcg
ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg
1380cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac
cgtggacaag 1440agcaggtggc agcaggggaa cgtcttctca tgctccgtga
tgcatgaggc tctgcacaac 1500cactacacgc agaagagcct ctccctgtct
ccgggtaaa 1539151497DNAArtificial SequenceDescription of Artificial
Sequence Synthetic Protoxin2-L1 fusion polynucleotide 15caggtccagc
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
cgggggtggc ggaagctact gccagaaatg gatgtggacc 360tgcgactctg
aacgtaaatg ctgcgaaggt atggtttgcc gtctgtggtg caaaaaaaaa
420ctgtggggcg gaggtgggag ttcttatacc tacaattatg aatggcatgt
ggatgtctgg 480ggacagggcc tgctggtgac agtctctagt gcttccacaa
ctgcaccaaa ggtgtacccc 540ctgtcaagct gctgtgggga caaatcctct
agtaccgtga cactgggatg cctggtctca 600agctatatgc ccgagcctgt
gactgtcacc tggaactcag gagccctgaa aagcggagtg 660cacaccttcc
cagctgtgct gcagtcctct ggcctgtata gcctgagttc aatggtgaca
720gtccccggca gtacttcagg gcagaccttc acctgtaatg tggcccatcc
tgccagctcc 780accaaagtgg acaaagcagt ggaacccaaa tcttgcgaca
aaactcacac atgcccaccg 840tgcccagcac ctgaactcct ggggggaccg
tcagtcttcc tcttcccccc aaaacccaag 900gacaccctca tgatctcccg
gacccctgag gtcacatgcg tggtggtgga cgtgagccac 960gaagaccctg
aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag
1020acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt
cctcaccgtc 1080ctgcaccagg actggctgaa tggcaaggag tacaagtgca
aggtctccaa caaagccctc 1140ccagccccca tcgagaaaac catctccaaa
gccaaagggc agccccgaga accacaggtg 1200tacaccctgc ccccatcccg
ggatgagctg accaagaacc aggtcagcct gacctgcctg 1260gtcaaaggct
tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag
1320aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt
cctctacagc 1380aagctcaccg tggacaagag caggtggcag caggggaacg
tcttctcatg ctccgtgatg 1440catgaggctc tgcacaacca ctacacgcag
aagagcctct ccctgtctcc gggtaaa 149716615DNAArtificial
SequenceDescription of Artificial Sequence Synthetic BLV1H12-IL8
fusion polynucleotide 16caggtccagc 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 cccaaggagt gctaaagaac ttagatgtca
gtgcataaag 360acatactcca aacctttcca ccccaagttc atcaaggagc
tgagagtgat tgagagtgga 420ccacactgcg ccaacacaga gattattgta
aagctttctg atgggagaga gctctgcctg 480gaccccaagg aaaactgggt
gcagagggtc gtggagaagt tcttgaagag ggctgagaac 540tcaggcagcg
gttcttatac ctacaattat gaatggcatg tggatgtctg gggacagggc
600ctgctggtga cagtc 61517459DNAArtificial SequenceDescription of
Artificial Sequence Synthetic BLV1H12-Ziconotide fusion
polynucleotide 17caggtccagc 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 ctgcaagggc aaaggtgcga aatgcagccg
cctgatgtat 360gattgctgta ccgggtcctg ccgcagtggc aagtgctctt
atacctacaa ttatgaatgg 420catgtggatg tctggggaca gggcctgctg gtgacagtc
45918429DNAArtificial SequenceDescription of Artificial Sequence
Synthetic BLV1H12-Somatostatin fusion polynucleotide 18caggtccagc
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
cgctggctgc aagaatttct tctggaagac tttcacatcc 360tgtggttctt
atacctacaa ttatgaatgg catgtggatg tctggggaca gggcctgctg 420gtgacagtc
42919486DNAArtificial SequenceDescription of Artificial Sequence
Synthetic BLV1H12-Chlorotoxin fusion polynucleotide 19caggtccagc
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
catgtgtatg ccctgcttca cgaccgatca ccagatggcg 360cgcaaatgcg
atgactgttg cggcggtaaa ggtcgcggaa agtgctatgg cccgcagtgt
420ctgtcttata cctacaatta tgaatggcat gtggatgtct ggggacaggg
cctgctggtg 480acagtc 48620597DNAArtificial SequenceDescription of
Artificial Sequence Synthetic BLV1H12-SDF1(alpha) fusion
polynucleotide 20caggtccagc 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 caagcccgtc agcctgagct acagatgccc
atgccgattc 360ttcgaaagcc atgttgccag agccaacgtc aagcatctca
aaattctcaa cactccaaac 420tgtgcccttc agattgtagc ccggctgaag
aacaacaaca gacaagtgtg cattgacccg 480aagctaaagt ggattcagga
gtacctggag aaagctttaa acaagggcag cggttcttat 540acctacaatt
atgaatggca tgtggatgtc tggggacagg gcctgctggt gacagtc
59721783DNAArtificial SequenceDescription of Artificial Sequence
Synthetic BLV1H12-IL21 fusion polynucleotide 21caggtccagc
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
ccaaggtcaa gatcgccaca tgatcagaat gcgtcagctc 360atagatattg
ttgatcagct gaagaactac gtgaacgact tggtccctga atttctgcca
420gctcccgaag atgtagagac aaactgtgag tggtcagcct tctcctgctt
tcagaaggcc 480caactaaagt cagcaaatac cggcaacaac gagaggataa
tcaatgtatc aatcaaaaag 540ctgaagagga agccaccttc cacaaatgca
gggagacggc agaaacaccg cctgacatgc 600ccttcatgtg attcttacga
gaagaagcca cccaaagagt tcctagagcg gttcaagtca 660cttctccaaa
agatgattca tcagcatctg tcctctcgca cacacggaag tgaagattcc
720tcttatacct acaattatga atggcatgtg gatgtctggg gacagggcct
gctggtgaca 780gtc 78322474DNAArtificial SequenceDescription of
Artificial Sequence Synthetic BLV1H12-ProTxII fusion polynucleotide
22caggtccagc 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
ctattgccag aagtggatgt ggacctgcga tagcgaacgg 360aaatgttgcg
aaggcatggt gtgccgcctg tggtgcaaga agaaactctg gtcttatacc
420tacaattatg aatggcatgt ggatgtctgg ggacagggcc tgctggtgac agtc
47423216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Light Chain fusion polypeptide 23Gln Ala Val Leu Asn Gln
Pro Ser Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15 Arg Val Ser Ile
Thr Cys
Ser Gly Ser Ser Ser Asn Val Gly Asn Gly 20 25 30 Tyr Val Ser Trp
Tyr Gln Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu 35 40 45 Ile Tyr
Gly Asp Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60
Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln 65
70 75 80 Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu Asp
Ser Ser 85 90 95 Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr
Val Leu Gly Gln 100 105 110 Pro Lys Ser Pro Pro Ser Val Thr Leu Phe
Pro Pro Ser Thr Glu Glu 115 120 125 Leu Asn Gly Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ser Val Thr Val
Val Trp Lys Ala Asp Gly Ser Thr Ile Thr 145 150 155 160 Arg Asn Val
Glu Thr Thr Arg Ala Ser Lys Gln Ser Asn Ser Lys Tyr 165 170 175 Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Ser Ser Asp Trp Lys Ser Lys 180 185
190 Gly Ser Tyr Ser Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys
195 200 205 Thr Val Lys Pro Ser Glu Cys Ser 210 215
24169PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Heavy Chain fusion polypeptide 24Gln Val Gln Leu Arg Glu
Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu
Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val
Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45
Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50
55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser
Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr
Tyr Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser
Cys Pro Asp Gly Tyr 100 105 110 Arg Glu Arg Ser Asp Cys Ser Asn Arg
Pro Ala Cys Gly Thr Ser Asp 115 120 125 Cys Cys Arg Val Ser Val Phe
Gly Asn Cys Leu Thr Thr Leu Pro Val 130 135 140 Ser Tyr Ser Tyr Thr
Tyr Asn Tyr Glu Trp His Val Asp Val Trp Gly 145 150 155 160 Gln Gly
Leu Leu Val Thr Val Ser Ser 165 25635PRTArtificial
SequenceDescription of Artificial Sequence Synthetic IFN-beta
fusion polypeptide 25Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu
Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser
Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Val Arg Gln
Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Ser Ile Asp Thr
Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55 60 Ser Arg Leu
Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Ser
Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr 85 90
95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Gly Gly Gly Gly Ser
100 105 110 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn
Phe Gln 115 120 125 Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu
Glu Tyr Cys Leu 130 135 140 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu
Glu Ile Lys Gln Leu Gln 145 150 155 160 Gln Phe Gln Lys Glu Asp Ala
Ala Leu Thr Ile Tyr Glu Met Leu Gln 165 170 175 Asn Ile Phe Ala Ile
Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 180 185 190 Glu Thr Ile
Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 195 200 205 His
Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 210 215
220 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg
225 230 235 240 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys
Ala Trp Thr 245 250 255 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr
Phe Ile Asn Arg Leu 260 265 270 Thr Gly Tyr Leu Arg Asn Gly Gly Gly
Gly Ser Ser Tyr Thr Tyr Asn 275 280 285 Tyr Glu Trp His Val Asp Val
Trp Gly Gln Gly Leu Leu Val Thr Val 290 295 300 Ser Ser Ala Ser Thr
Thr Ala Pro Lys Val Tyr Pro Leu Ser Ser Cys 305 310 315 320 Cys Gly
Asp Lys Ser Ser Ser Thr Val Thr Leu Gly Cys Leu Val Ser 325 330 335
Ser Tyr Met Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu 340
345 350 Lys Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu 355 360 365 Tyr Ser Leu Ser Ser Met Val Thr Val Pro Gly Ser Thr
Ser Gly Gln 370 375 380 Thr Phe Thr Cys Asn Val Ala His Pro Ala Ser
Ser Thr Lys Val Asp 385 390 395 400 Lys Ala Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro 405 410 415 Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 420 425 430 Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 435 440 445 Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 450 455 460
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 465
470 475 480 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val 485 490 495 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 500 505 510 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys 515 520 525 Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp 530 535 540 Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe 545 550 555 560 Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 565 570 575 Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 580 585
590 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
595 600 605 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr 610 615 620 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 625
630 635 26633PRTArtificial SequenceDescription of Artificial
Sequence Synthetic bGCSF-L0 fusion polypeptide 26Gln Val Gln Leu
Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30
Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35
40 45 Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu
Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln
Val Ser Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala
Thr Tyr Tyr Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr
Gln Ser Thr Pro Leu Gly Pro 100 105 110 Ala Arg Ser Leu Pro Gln Ser
Phe Leu Leu Lys Cys Leu Glu Gln Val 115 120 125 Arg Lys Ile Gln Ala
Asp Gly Ala Glu Leu Gln Glu Arg Leu Cys Ala 130 135 140 Ala His Lys
Leu Cys His Pro Glu Glu Leu Met Leu Leu Arg His Ser 145 150 155 160
Leu Gly Ile Pro Gln Ala Pro Leu Ser Ser Cys Ser Ser Gln Ser Leu 165
170 175 Gln Leu Thr Ser Cys Leu Asn Gln Leu His Gly Gly Leu Phe Leu
Tyr 180 185 190 Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile Ser Pro Glu
Leu Ala Pro 195 200 205 Thr Leu Asp Thr Leu Gln Leu Asp Val Thr Asp
Phe Ala Thr Asn Ile 210 215 220 Trp Leu Gln Met Glu Asp Leu Gly Ala
Ala Pro Ala Val Gln Pro Thr 225 230 235 240 Gln Gly Ala Met Pro Thr
Phe Thr Ser Ala Phe Gln Arg Arg Ala Gly 245 250 255 Gly Val Leu Val
Ala Ser Gln Leu His Arg Phe Leu Glu Leu Ala Tyr 260 265 270 Arg Gly
Leu Arg Tyr Leu Ala Glu Pro Ser Tyr Thr Tyr Asn Tyr Glu 275 280 285
Trp His Val Asp Val Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser 290
295 300 Ala Ser Thr Thr Ala Pro Lys Val Tyr Pro Leu Ser Ser Cys Cys
Gly 305 310 315 320 Asp Lys Ser Ser Ser Thr Val Thr Leu Gly Cys Leu
Val Ser Ser Tyr 325 330 335 Met Pro Glu Pro Val Thr Val Thr Trp Asn
Ser Gly Ala Leu Lys Ser 340 345 350 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 355 360 365 Leu Ser Ser Met Val Thr
Val Pro Gly Ser Thr Ser Gly Gln Thr Phe 370 375 380 Thr Cys Asn Val
Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Ala 385 390 395 400 Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 405 410
415 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
420 425 430 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val 435 440 445 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr 450 455 460 Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu 465 470 475 480 Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His 485 490 495 Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 500 505 510 Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 515 520 525 Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 530 535
540 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
545 550 555 560 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn 565 570 575 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu 580 585 590 Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val 595 600 605 Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln 610 615 620 Lys Ser Leu Ser Leu
Ser Pro Gly Lys 625 630 27643PRTArtificial SequenceDescription of
Artificial Sequence Synthetic bGCSF-L1 fusion polypeptide 27Gln Val
Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20
25 30 Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp
Leu 35 40 45 Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro
Gly Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys
Ser Gln Val Ser Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp
Ser Ala Thr Tyr Tyr Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys
Lys Tyr Gln Ser Gly Gly Gly Gly Ser 100 105 110 Thr Pro Leu Gly Pro
Ala Arg Ser Leu Pro Gln Ser Phe Leu Leu Lys 115 120 125 Cys Leu Glu
Gln Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu Gln 130 135 140 Glu
Arg Leu Cys Ala Ala His Lys Leu Cys His Pro Glu Glu Leu Met 145 150
155 160 Leu Leu Arg His Ser Leu Gly Ile Pro Gln Ala Pro Leu Ser Ser
Cys 165 170 175 Ser Ser Gln Ser Leu Gln Leu Thr Ser Cys Leu Asn Gln
Leu His Gly 180 185 190 Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
Leu Ala Gly Ile Ser 195 200 205 Pro Glu Leu Ala Pro Thr Leu Asp Thr
Leu Gln Leu Asp Val Thr Asp 210 215 220 Phe Ala Thr Asn Ile Trp Leu
Gln Met Glu Asp Leu Gly Ala Ala Pro 225 230 235 240 Ala Val Gln Pro
Thr Gln Gly Ala Met Pro Thr Phe Thr Ser Ala Phe 245 250 255 Gln Arg
Arg Ala Gly Gly Val Leu Val Ala Ser Gln Leu His Arg Phe 260 265 270
Leu Glu Leu Ala Tyr Arg Gly Leu Arg Tyr Leu Ala Glu Pro Gly Gly 275
280 285 Gly Gly Ser Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val
Trp 290 295 300 Gly Gln Gly Leu Leu Val Thr Val Ser Ser Ala Ser Thr
Thr Ala Pro 305 310 315 320 Lys Val Tyr Pro Leu Ser Ser Cys Cys Gly
Asp Lys Ser Ser Ser Thr 325 330 335 Val Thr Leu Gly Cys Leu Val Ser
Ser Tyr Met Pro Glu Pro Val Thr 340 345 350 Val Thr Trp Asn Ser Gly
Ala Leu Lys Ser Gly Val His Thr Phe Pro 355 360 365 Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr 370 375 380 Val Pro
Gly Ser Thr Ser Gly Gln Thr Phe Thr Cys Asn Val Ala His 385 390 395
400 Pro Ala Ser Ser Thr Lys Val Asp Lys Ala Val Glu Pro Lys Ser Cys
405 410 415 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly 420 425 430 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 435 440 445 Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 450 455 460 Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 465 470 475 480 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 485 490 495 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 500 505 510 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 515 520
525 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
530 535 540 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 545 550 555 560 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 565 570 575 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 580 585 590 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 595 600 605 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 610 615 620 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 625 630 635 640 Pro Gly Lys
28596PRTArtificial SequenceDescription of Artificial Sequence
Synthetic GMCSF fusion polypeptide 28Gln Val Gln Leu Arg Glu Ser
Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly
Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly
Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55
60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr
Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Gly
Gly Gly Gly Ser 100 105 110 Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr
Gln Pro Trp Glu His Val 115 120 125 Asn Ala Ile Gln Glu Ala Arg Arg
Leu Leu Asn Leu Ser Arg Asp Thr 130 135 140 Ala Ala Glu Met Asn Glu
Thr Val Glu Val Ile Ser Glu Met Phe Asp 145 150 155 160 Leu Gln Glu
Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln 165 170 175 Gly
Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met 180 185
190 Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser Cys
195 200 205 Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu
Lys Asp 210 215 220 Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro
Val Gln Glu Gly 225 230 235 240 Gly Gly Gly Ser Ser Tyr Thr Tyr Asn
Tyr Glu Trp His Val Asp Val 245 250 255 Trp Gly Gln Gly Leu Leu Val
Thr Val Ser Ser Ala Ser Thr Thr Ala 260 265 270 Pro Lys Val Tyr Pro
Leu Ser Ser Cys Cys Gly Asp Lys Ser Ser Ser 275 280 285 Thr Val Thr
Leu Gly Cys Leu Val Ser Ser Tyr Met Pro Glu Pro Val 290 295 300 Thr
Val Thr Trp Asn Ser Gly Ala Leu Lys Ser Gly Val His Thr Phe 305 310
315 320 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met
Val 325 330 335 Thr Val Pro Gly Ser Thr Ser Gly Gln Thr Phe Thr Cys
Asn Val Ala 340 345 350 His Pro Ala Ser Ser Thr Lys Val Asp Lys Ala
Val Glu Pro Lys Ser 355 360 365 Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu 370 375 380 Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 385 390 395 400 Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 405 410 415 His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 420 425 430
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 435
440 445 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn 450 455 460 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro 465 470 475 480 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 485 490 495 Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val 500 505 510 Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 515 520 525 Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 530 535 540 Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 545 550 555
560 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
565 570 575 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 580 585 590 Ser Pro Gly Lys 595 29650PRTArtificial
SequenceDescription of Artificial Sequence Synthetic hFGF21 fusion
polypeptide 29Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys
Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe
Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Val Arg Gln Ala Pro
Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Ser Ile Asp Thr Gly Gly
Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55 60 Ser Arg Leu Ser Ile
Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Ser Val Ser
Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr 85 90 95 Ser
Val His Gln Glu Thr Lys Lys Tyr Gln Ser Gly Gly Gly Gly Ser 100 105
110 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
115 120 125 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu
Ala His 130 135 140 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala
Ala Asp Gln Ser 145 150 155 160 Pro Glu Ser Leu Leu Gln Leu Lys Ala
Leu Lys Pro Gly Val Ile Gln 165 170 175 Ile Leu Gly Val Lys Thr Ser
Arg Phe Leu Cys Gln Arg Pro Asp Gly 180 185 190 Ala Leu Tyr Gly Ser
Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 195 200 205 Glu Leu Leu
Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 210 215 220 Gly
Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro 225 230
235 240 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro
Pro 245 250 255 Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro
Pro Asp Val 260 265 270 Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro
Ser Gln Gly Arg Ser 275 280 285 Pro Ser Tyr Ala Ser Gly Gly Gly Gly
Ser Ser Tyr Thr Tyr Asn Tyr 290 295 300 Glu Trp His Val Asp Val Trp
Gly Gln Gly Leu Leu Val Thr Val Ser 305 310 315 320 Ser Ala Ser Thr
Thr Ala Pro Lys Val Tyr Pro Leu Ser Ser Cys Cys 325 330 335 Gly Asp
Lys Ser Ser Ser Thr Val Thr Leu Gly Cys Leu Val Ser Ser 340 345 350
Tyr Met Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu Lys 355
360 365 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr 370 375 380 Ser Leu Ser Ser Met Val Thr Val Pro Gly Ser Thr Ser
Gly Gln Thr 385 390 395 400 Phe Thr Cys Asn Val Ala His Pro Ala Ser
Ser Thr Lys Val Asp Lys 405 410 415 Ala Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys 420 425 430 Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 435 440 445 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 450 455 460 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 465 470 475
480 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
485 490 495 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 500 505 510 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 515 520 525 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 530 535 540 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu 545 550 555 560 Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 565 570 575 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 580 585 590 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 595 600
605 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
610 615 620 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 625 630 635 640 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 645
650 30514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ex-4 fusion polypeptide 30Gln Val Gln Leu Arg Glu Ser Gly
Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp
Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Ser
Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55 60
Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65
70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr
Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Cys
Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser 115 120 125 Lys Gln Met Glu Glu Glu Ala Val
Arg Leu Phe Ile Glu Trp Leu Lys 130 135 140 Asn Gly Gly Pro Ser Ser
Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly 145 150 155 160 Ser Cys Ser
Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val Trp Gly 165 170 175 Gln
Gly Leu Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Lys 180 185
190 Val Tyr Pro Leu Ser Ser Cys Cys Gly Asp Lys Ser Ser Ser Thr Val
195 200 205 Thr Leu Gly Cys Leu Val Ser Ser Tyr Met Pro Glu Pro Val
Thr Val 210 215 220 Thr Trp Asn Ser Gly Ala Leu Lys Ser Gly Val His
Thr Phe Pro Ala 225 230 235 240 Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Met Val Thr Val 245 250 255 Pro Gly Ser Thr Ser Gly Gln
Thr Phe Thr Cys Asn Val Ala His Pro 260 265 270 Ala Ser Ser Thr Lys
Val Asp Lys Ala Val Glu Pro Lys Ser Cys Asp 275 280 285 Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 290 295 300 Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 305 310
315 320 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu 325 330 335 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His 340 345 350 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg 355 360 365 Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys 370 375 380 Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu 385 390 395 400 Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 405 410 415 Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 420 425 430
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 435
440 445 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val 450 455 460 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp 465 470 475 480 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His 485 490 495 Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 500 505 510 Gly Lys
31505PRTArtificial SequenceDescription of Artificial Sequence
Synthetic hGLP-1 fusion polypeptide 31Gln Val Gln Leu Arg Glu Ser
Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly
Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly
Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55
60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr
Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Cys
Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser 115 120 125 Ser Tyr Leu Glu Gly Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val 130 135 140 Lys Gly Arg Gly Gly Gly
Gly Ser Cys Ser Tyr Thr Tyr Asn Tyr Glu 145 150 155 160 Trp His Val
Asp Val Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser 165 170 175 Ala
Ser Thr Thr Ala Pro Lys Val Tyr Pro Leu Ser Ser Cys Cys Gly 180 185
190 Asp Lys Ser Ser Ser Thr Val Thr Leu Gly Cys Leu Val Ser Ser Tyr
195 200 205 Met Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu
Lys Ser 210 215 220 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 225 230 235 240 Leu Ser Ser Met Val Thr Val Pro Gly
Ser Thr Ser Gly Gln Thr Phe 245 250 255 Thr Cys Asn Val Ala His Pro
Ala Ser Ser Thr Lys Val Asp Lys Ala 260 265 270 Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 275 280 285 Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 290 295 300 Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 305 310
315 320 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr 325 330 335 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu 340 345 350 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His 355 360 365 Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys 370 375 380 Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln 385 390 395 400 Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 405 410 415 Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 420 425 430
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 435
440 445 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu 450 455 460 Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 465 470 475 480 Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 485 490 495
Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 32635PRTArtificial
SequenceDescription of Artificial Sequence Synthetic hEPO fusion
polypeptide 32Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys
Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe
Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Val Arg Gln Ala Pro
Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Ser Ile Asp Thr Gly Gly
Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55 60 Ser Arg Leu Ser Ile
Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Ser Val Ser
Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr 85 90 95 Ser
Val His Gln Glu Thr Lys Lys Tyr Gln Ser Gly Gly Gly Gly Ser 100 105
110 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu
115 120 125 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala
Glu His 130 135 140 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr
Lys Val Asn Phe 145 150 155 160 Tyr Ala Trp Lys Arg Met Glu Val Gly
Gln Gln Ala Val Glu Val Trp 165 170 175 Gln Gly Leu Ala Leu Leu Ser
Glu Ala Val Leu Arg Gly Gln Ala Leu 180 185 190 Leu Val Asn Ser Ser
Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 195 200 205 Lys Ala Val
Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 210 215 220 Gly
Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 225 230
235 240 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg
Val 245 250 255 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr
Gly Glu Ala 260 265 270 Cys Arg Thr Gly Asp Arg Gly Gly Gly Gly Ser
Ser Tyr Thr Tyr Asn 275 280 285 Tyr Glu Trp His Val Asp Val Trp Gly
Gln Gly Leu Leu Val Thr Val 290 295 300 Ser Ser Ala Ser Thr Thr Ala
Pro Lys Val Tyr Pro Leu Ser Ser Cys 305 310 315 320 Cys Gly Asp Lys
Ser Ser Ser Thr Val Thr Leu Gly Cys Leu Val Ser 325 330 335 Ser Tyr
Met Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu 340 345 350
Lys Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 355
360 365 Tyr Ser Leu Ser Ser Met Val Thr Val Pro Gly Ser Thr Ser Gly
Gln 370 375 380 Thr Phe Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr
Lys Val Asp 385 390 395 400 Lys Ala Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro 405 410 415 Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro 420 425 430 Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr 435 440 445 Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 450 455 460 Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 465 470 475
480 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
485 490 495 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser 500 505 510 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys 515 520 525 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp 530 535 540 Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe 545 550 555 560 Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 565 570 575 Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 580 585 590 Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 595 600
605 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
610 615 620 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 625 630 635
33493PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Moka-L0 fusion polypeptide 33Gln Val Gln Leu Arg Glu Ser
Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly
Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly
Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55
60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr
Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Ile
Asn Val Lys Cys 100 105 110 Ser Leu Pro Gln Gln Cys Ile Lys Pro Cys
Lys Asp Ala Gly Met Arg 115 120 125 Phe Gly Lys Cys Met Asn Lys Lys
Cys Arg Cys Tyr Ser Ser Tyr Thr 130 135 140 Tyr Asn Tyr Glu Trp His
Val Asp Val Trp Gly Gln Gly Leu Leu Val 145 150 155 160 Thr Val Ser
Ser Ala Ser Thr Thr Ala Pro Lys Val Tyr Pro Leu Ser 165 170 175 Ser
Cys Cys Gly Asp Lys Ser Ser Ser Thr Val Thr Leu Gly Cys Leu 180 185
190 Val Ser Ser Tyr Met Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly
195 200 205 Ala Leu Lys Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser 210 215 220 Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro
Gly Ser Thr Ser 225 230 235 240 Gly Gln Thr Phe Thr Cys Asn Val Ala
His Pro Ala Ser Ser Thr Lys 245 250 255 Val Asp Lys Ala Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys 260 265 270 Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 275 280 285 Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 290 295 300 Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 305 310
315 320 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys 325 330 335 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu 340 345 350 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys 355 360 365 Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys 370 375 380 Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser 385 390 395 400 Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 405 410 415 Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 420 425 430
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 435
440 445 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln 450 455 460 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn 465 470 475 480 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 485 490 34503PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Moka-L1 fusion polypeptide 34Gln Val
Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20
25 30 Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp
Leu 35 40 45 Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro
Gly Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys
Ser Gln Val Ser Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp
Ser Ala Thr Tyr Tyr Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys
Lys Tyr Gln Ser Gly Gly Gly Gly Ser 100 105 110 Ile Asn Val Lys Cys
Ser Leu Pro Gln Gln Cys Ile Lys Pro Cys Lys 115 120 125 Asp Ala Gly
Met Arg Phe Gly Lys Cys Met Asn Lys Lys Cys Arg Cys 130 135 140 Tyr
Ser Gly Gly Gly Gly Ser Ser Tyr Thr Tyr Asn Tyr Glu Trp His 145 150
155 160 Val Asp Val Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser Ala
Ser 165 170 175 Thr Thr Ala Pro Lys Val Tyr Pro Leu Ser Ser Cys Cys
Gly Asp Lys 180 185 190 Ser Ser Ser Thr Val Thr Leu Gly Cys Leu Val
Ser Ser Tyr Met Pro 195 200 205 Glu Pro Val Thr Val Thr Trp Asn Ser
Gly Ala Leu Lys Ser Gly Val 210 215 220 His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser 225 230 235 240 Ser Met Val Thr
Val Pro Gly Ser Thr Ser Gly Gln Thr Phe Thr Cys 245 250 255 Asn Val
Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Ala Val Glu 260 265 270
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 275
280 285 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys 290 295 300 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val 305 310 315 320 Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp 325 330 335 Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr 340 345 350 Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp 355 360 365 Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 370 375 380 Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 385 390 395
400 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
405 410 415 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp 420 425 430 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys 435 440 445 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser 450 455 460 Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser 465 470 475 480 Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 485 490 495 Leu Ser Leu
Ser Pro Gly Lys 500 35505PRTArtificial SequenceDescription of
Artificial Sequence Synthetic VM-24-L1 fusion polypeptide 35Gln Val
Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20
25 30 Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp
Leu 35 40 45 Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro
Gly Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys
Ser Gln Val Ser Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp
Ser Ala Thr Tyr Tyr Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys
Lys Tyr Gln Ser Gly Gly Gly Gly Ser 100 105 110 Ala Ala Ala Ile Ser
Cys Val Gly Ser Pro Glu Cys Pro Pro Lys Cys 115 120 125 Arg Ala Gln
Gly Cys Lys Asn Gly Lys Cys Met Asn Arg Lys Cys Lys 130 135 140 Cys
Tyr Tyr Cys Gly Gly Gly Gly Ser Ser Tyr Thr Tyr Asn Tyr Glu 145 150
155 160 Trp His Val Asp Val Trp Gly Gln Gly Leu Leu Val Thr Val Ser
Ser 165 170 175 Ala Ser Thr Thr Ala Pro Lys Val Tyr Pro Leu Ser Ser
Cys Cys Gly 180 185 190 Asp Lys Ser Ser Ser Thr Val Thr Leu Gly Cys
Leu Val Ser Ser Tyr 195 200 205 Met Pro Glu Pro Val Thr Val Thr Trp
Asn Ser Gly Ala Leu Lys Ser 210 215 220 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 225 230 235 240 Leu Ser Ser Met
Val Thr Val Pro Gly Ser Thr Ser Gly Gln Thr Phe 245 250 255 Thr Cys
Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Ala 260 265 270
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 275
280 285 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys 290 295 300 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val 305 310 315 320 Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr 325 330 335 Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu 340 345 350 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 355 360 365 Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 370 375 380 Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 385 390 395
400 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
405 410 415 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 420 425 430 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn 435 440 445 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 450 455 460 Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val 465 470 475 480 Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 485 490 495 Lys Ser Leu
Ser Leu Ser Pro Gly Lys 500 505 36513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic VM-24-L2
fusion polypeptide 36Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu
Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser
Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Val Arg Gln
Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Ser Ile Asp Thr
Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55
60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr
Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Gly
Gly Gly Ser Gly 100 105 110 Gly Gly Gly Ser Ala Ala Ala Ile Ser Cys
Val Gly Ser Pro Glu Cys 115 120 125 Pro Pro Lys Cys Arg Ala Gln Gly
Cys Lys Asn Gly Lys Cys Met Asn 130 135 140 Arg Lys Cys Lys Cys Tyr
Tyr Cys Gly Gly Gly Gly Ser Gly Gly Gly 145 150 155 160 Ser Ser Tyr
Thr Tyr Asn Tyr Glu Trp His Val Asp Val Trp Gly Gln 165 170 175 Gly
Leu Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Lys Val 180 185
190 Tyr Pro Leu Ser Ser Cys Cys Gly Asp Lys Ser Ser Ser Thr Val Thr
195 200 205 Leu Gly Cys Leu Val Ser Ser Tyr Met Pro Glu Pro Val Thr
Val Thr 210 215 220 Trp Asn Ser Gly Ala Leu Lys Ser Gly Val His Thr
Phe Pro Ala Val 225 230 235 240 Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Met Val Thr Val Pro 245 250 255 Gly Ser Thr Ser Gly Gln Thr
Phe Thr Cys Asn Val Ala His Pro Ala 260 265 270 Ser Ser Thr Lys Val
Asp Lys Ala Val Glu Pro Lys Ser Cys Asp Lys 275 280 285 Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 290 295 300 Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 305 310
315 320 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp 325 330 335 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 340 345 350 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val 355 360 365 Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu 370 375 380 Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys 385 390 395 400 Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 405 410 415 Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 420 425 430
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 435
440 445 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu 450 455 460 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys 465 470 475 480 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 485 490 495 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 500 505 510 Lys 37499PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Protoxin2-L1
fusion polypeptide 37Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu
Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser
Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Val Arg Gln
Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Ser Ile Asp Thr
Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55 60 Ser Arg Leu
Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Ser
Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr 85 90
95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Gly Gly Gly Gly Ser
100 105 110 Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Glu Arg Lys
Cys Cys 115 120 125 Glu Gly Met Val Cys Arg Leu Trp Cys Lys Lys Lys
Leu Trp Gly Gly 130 135 140 Gly Gly Ser Ser Tyr Thr Tyr Asn Tyr Glu
Trp His Val Asp Val Trp 145 150 155 160 Gly Gln Gly Leu Leu Val Thr
Val Ser Ser Ala Ser Thr Thr Ala Pro 165 170 175 Lys Val Tyr Pro Leu
Ser Ser Cys Cys Gly Asp Lys Ser Ser Ser Thr 180 185 190 Val Thr Leu
Gly Cys Leu Val Ser Ser Tyr Met Pro Glu Pro Val Thr 195 200 205 Val
Thr Trp Asn Ser Gly Ala Leu Lys Ser Gly Val His Thr Phe Pro 210 215
220 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr
225 230 235 240 Val Pro Gly Ser Thr Ser Gly Gln Thr Phe Thr Cys Asn
Val Ala His 245 250 255 Pro Ala Ser Ser Thr Lys Val Asp Lys Ala Val
Glu Pro Lys Ser Cys 260 265 270 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 275 280 285 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 290 295 300 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 305 310 315 320 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 325 330 335
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 340
345 350 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 355 360 365 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 370 375 380 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 385 390 395 400 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 405 410 415 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 420 425 430 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 435 440 445 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 450 455 460
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 465
470 475 480 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 485 490 495 Pro Gly Lys 38205PRTArtificial
SequenceDescription of Artificial Sequence Synthetic BLV1H12-IL8
fusion polypeptide 38Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu
Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser
Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Val Arg Gln
Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Ser Ile Asp Thr
Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55 60 Ser Arg Leu
Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Ser
Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr 85 90
95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Pro Arg Ser Ala Lys
100 105 110 Glu Leu Arg Cys Gln Cys Ile Lys Thr Tyr Ser Lys Pro Phe
His Pro 115 120 125 Lys Phe Ile Lys Glu Leu Arg Val Ile Glu Ser Gly
Pro His Cys Ala 130 135 140 Asn Thr Glu Ile Ile Val Lys Leu Ser Asp
Gly Arg Glu Leu Cys Leu 145 150 155 160 Asp Pro Lys Glu Asn Trp Val
Gln Arg Val Val Glu Lys Phe Leu Lys 165 170 175 Arg Ala Glu Asn Ser
Gly Ser Gly Ser Tyr Thr Tyr Asn Tyr Glu Trp 180 185 190 His Val Asp
Val Trp Gly Gln Gly Leu Leu Val Thr Val 195 200 205
39153PRTArtificial SequenceDescription of Artificial Sequence
Synthetic BLV1H12-Ziconotide fusion polypeptide 39Gln Val Gln Leu
Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30
Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35
40 45 Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu
Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln
Val Ser Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala
Thr Tyr Tyr Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr
Gln Ser Cys Lys Gly Lys Gly 100 105 110 Ala Lys Cys Ser Arg Leu Met
Tyr Asp Cys Cys Thr Gly Ser Cys Arg 115 120 125 Ser Gly Lys Cys Ser
Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val 130 135 140 Trp Gly Gln
Gly Leu Leu Val Thr Val 145 150 40143PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
BLV1H12-Somatostatin fusion polypeptide 40Gln Val Gln Leu Arg Glu
Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu
Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val
Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45
Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50
55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser
Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr
Tyr Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser
Ala Gly Cys Lys Asn 100 105 110 Phe Phe Trp Lys Thr Phe Thr Ser Cys
Gly Ser Tyr Thr Tyr Asn Tyr 115 120 125 Glu Trp His Val Asp Val Trp
Gly Gln Gly Leu Leu Val Thr Val 130 135 140 41162PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
BLV1H12-Chlorotoxin fusion polypeptide 41Gln Val Gln Leu Arg Glu
Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu
Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val
Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45
Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50
55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser
Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr
Tyr Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser
Met Cys Met Pro Cys 100 105 110 Phe Thr Thr Asp His Gln Met Ala Arg
Lys Cys Asp Asp Cys Cys Gly 115 120 125 Gly Lys Gly Arg Gly Lys Cys
Tyr Gly Pro Gln Cys Leu Ser Tyr Thr 130 135 140 Tyr Asn Tyr Glu Trp
His Val Asp Val Trp Gly Gln Gly Leu Leu Val 145 150 155 160 Thr Val
42199PRTArtificial SequenceDescription of Artificial Sequence
Synthetic BLV1H12-SDF1(alpha) fusion polypeptide 42Gln Val Gln Leu
Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30
Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35
40 45 Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu
Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln
Val Ser Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala
Thr Tyr Tyr Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr
Gln Ser Lys Pro Val Ser Leu 100 105 110 Ser Tyr Arg Cys Pro Cys Arg
Phe Phe Glu Ser His Val Ala Arg Ala 115 120 125 Asn Val Lys His Leu
Lys Ile Leu Asn Thr Pro Asn Cys Ala Leu Gln 130 135 140 Ile Val Ala
Arg Leu Lys Asn Asn Asn Arg Gln Val Cys Ile Asp Pro 145 150 155 160
Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys Ala Leu Asn Lys Gly 165
170 175 Ser Gly Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val Trp
Gly 180 185 190 Gln Gly Leu Leu Val Thr Val 195 43261PRTArtificial
SequenceDescription of Artificial Sequence Synthetic BLV1H12-IL21
fusion polypeptide 43Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu
Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser
Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Val Arg Gln
Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Ser Ile Asp Thr
Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55 60 Ser Arg Leu
Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Ser
Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr 85 90
95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Gln Gly Gln Asp Arg
100 105 110 His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln
Leu Lys 115 120 125 Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro
Ala Pro Glu Asp 130 135 140 Val Glu Thr Asn Cys Glu Trp Ser Ala Phe
Ser Cys Phe Gln Lys Ala 145 150 155 160 Gln Leu Lys Ser Ala Asn Thr
Gly Asn Asn Glu Arg Ile Ile Asn Val 165 170 175 Ser Ile Lys Lys Leu
Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg 180 185 190 Arg Gln Lys
His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys 195 200 205 Lys
Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys 210 215
220 Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser
225 230 235 240 Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val Trp
Gly Gln Gly 245 250 255 Leu Leu Val Thr Val 260 44158PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
BLV1H12-ProTxII fusion polypeptide 44Gln Val Gln Leu Arg Glu Ser
Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly
Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly
Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55
60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr
Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Tyr
Cys Gln Lys Trp 100 105
110 Met Trp Thr Cys Asp Ser Glu Arg Lys Cys Cys Glu Gly Met Val Cys
115 120 125 Arg Leu Trp Cys Lys Lys Lys Leu Trp Ser Tyr Thr Tyr Asn
Tyr Glu 130 135 140 Trp His Val Asp Val Trp Gly Gln Gly Leu Leu Val
Thr Val 145 150 155 4534PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif polypeptide 45Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20
25 30 Xaa Cys 4643PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 46Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 4721PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
peptide 47Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Cys Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys 20 4835PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 48Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Cys 35 4941PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 49Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35
40 5034PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 50Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Cys Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Cys
5136PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 51Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Cys 35 5236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 52Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Cys 35 5335PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 53Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Cys 35
5433PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 54Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Cys
5535PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 55Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Cys 35
5624PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif peptide 56Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Xaa Xaa
Xaa Cys 20 5735PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 57Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa
Xaa Cys 35 5843PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 58Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 5935PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 59Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Cys 35 6037PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 60Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Cys 35
6137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 61Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Cys 35 6245PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 62Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 20 25 30 Xaa
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 45
6334PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 63Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Cys Xaa Xaa 20 25 30 Xaa Cys
6426PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif peptide 64Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Xaa Xaa
Xaa Cys Xaa Cys 20 25 6526PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif peptide 65Cys Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 15 Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys 20 25 6635PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 66Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa 20 25 30 Xaa Xaa Cys 35 6726PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
peptide 67Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa
Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys 20 25
6837PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 68Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Cys 35 6928PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif peptide 69Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Cys Xaa Cys 20 25 7029PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
peptide 70Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa
Cys 20 25 7127PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif peptide 71Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 15 Xaa Xaa Cys Xaa
Xaa Xaa Cys Xaa Cys Xaa Cys 20 25 7225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
peptide 72Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 20 25
7338PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 73Cys Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Xaa Cys 35 7438PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 74Cys Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa
Xaa Xaa Xaa Xaa Cys 35 7538PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif polypeptide 75Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 15
Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 20
25 30 Xaa Xaa Xaa Xaa Xaa Cys 35 7625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
peptide 76Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys 20 25
7735PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 77Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Cys 35
7820PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif peptide 78Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 15 Cys Xaa Xaa Cys 20
7921PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif peptide 79Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys 20
8043PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 80Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 1 5 10 15 Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Cys Xaa
Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 8139PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 81Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Cys Xaa Xaa Cys 35
8243PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 82Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Cys Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 35 40 8341PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 83Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 35
40 8439PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 84Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Xaa Xaa Cys 35 8543PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif polypeptide 85Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 20
25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40
8636PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 86Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Cys 35 8737PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 87Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Cys Xaa
Xaa Cys 35 8837PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 88Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 20 25 30 Xaa
Xaa Xaa Xaa Cys 35 8944PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif polypeptide 89Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys 20
25 30 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40
9036PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 90Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Cys Xaa Xaa
Cys 35 9137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 91Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Cys Xaa Xaa Cys 35
9236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 92Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Cys 35 9343PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 93Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa 20 25 30 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 35 40 9443PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 94Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys
Xaa Xaa Cys Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Cys 35 40 9538PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 95Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 20 25 30 Xaa
Xaa Xaa Xaa Xaa Cys 35 9634PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif polypeptide 96Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 15
Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20
25 30 Xaa Cys 9739PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 97Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Cys
Xaa Xaa Xaa Xaa Xaa Cys 35 9846PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif polypeptide 98Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa 1 5 10 15
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 20
25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys 35 40
45 9944PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 99Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Cys Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys 35 40 10042PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 100Cys Cys Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa
Xaa Cys Cys 1 5 10 15 Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa
Cys 35 40 10139PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 101Cys Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Cys Xaa
Xaa Xaa Xaa Cys Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa 20 25 30
Xaa Xaa Xaa Xaa Cys Xaa Cys 35 10238PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 102Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa Cys Cys Cys Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Cys Xaa Cys 35
10336PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 103Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa 1 5 10 15 Cys Xaa Xaa Cys Xaa
Cys Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 20 25 30 Xaa Xaa Xaa
Cys 35 10439PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 104Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 15 Xaa Cys
Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30
Xaa Xaa Cys Xaa Xaa Cys Cys 35 10538PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 105Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Cys Xaa Cys
Xaa Xaa Xaa 1 5 10 15 Cys Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Cys 35
10637PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 106Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Cys Cys Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa
Cys Cys Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Cys 35 10736PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 107Cys Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Cys Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Cys 20 25 30
Xaa Cys Xaa Cys 35 10841PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif polypeptide 108Cys Cys
Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 15
Cys Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 20
25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 10936PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 109Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa
Xaa Cys Cys 1 5 10 15 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Cys 20 25 30 Xaa Xaa Xaa Cys 35 11039PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 110Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Cys Xaa
Xaa Xaa Xaa Xaa Cys 20 25 30 Xaa Xaa Xaa Xaa Cys Xaa Cys 35
11141PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 111Cys Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa
Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Cys Xaa Xaa Cys Xaa Cys 35 40 11236PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 112Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Cys Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Cys
Cys Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Cys 35 11338PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 113Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Cys Xaa Cys Cys Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Cys Xaa Cys 35
11437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 114Cys Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa
Cys Cys Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa 20 25 30 Xaa Cys Xaa
Xaa Cys 35 11538PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 115Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa 1 5 10 15 Cys Xaa
Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Cys Cys Cys Xaa 20 25 30
Xaa Xaa Xaa Xaa Xaa Cys 35 11638PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Cysteine motif polypeptide 116Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa 1 5 10
15 Cys Xaa Xaa Cys Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
20 25 30 Xaa Cys Xaa Xaa Xaa Cys 35 11733PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 117Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Cys
Xaa Xaa Xaa 1 5 10 15 Cys Cys Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa 20 25 30 Cys 11834PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 118Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa
Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Cys Xaa Cys Cys Xaa Cys Xaa Xaa
Xaa Xaa Cys Xaa Xaa 20 25 30 Xaa Cys 11937PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 119Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Cys Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Cys Xaa Cys Cys Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa 20 25 30 Xaa Xaa Cys Xaa Cys 35
12036PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 120Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Cys Xaa
Cys Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 20 25 30 Xaa Cys Xaa
Cys 35 12134PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 121Cys Xaa Xaa Xaa
Xaa Xaa Xaa Cys Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Cys Xaa
Xaa Xaa Xaa Cys Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa 20 25 30
Xaa Cys 12228PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif peptide 122Cys Xaa Xaa Xaa Xaa
Xaa Xaa Cys Cys Xaa Cys Xaa Xaa Xaa Cys Xaa 1 5 10 15 Cys Cys Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Cys 20 25 12336PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 123Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Cys Xaa Xaa
Xaa Cys Xaa 1 5 10 15 Cys Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Cys 35 12429PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
peptide 124Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Cys Xaa Xaa Xaa Cys
Xaa Cys 1 5 10 15 Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Cys Xaa Xaa Xaa
Cys 20 25 12532PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 125Cys Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Cys 1 5 10 15 Cys Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 20 25 30
12629PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif peptide 126Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 1 5 10 15 Xaa Cys Cys Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Cys 20 25 12735PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 127Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Cys Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa 20 25 30 Xaa Xaa Cys 35 12839PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 128Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Cys
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Cys Xaa Xaa Xaa Cys 35
12935PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 129Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Cys 1 5 10 15 Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa 20 25 30 Xaa Xaa Cys 35
13041PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif polypeptide 130Cys Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa
Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Cys 35 40 13136PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 131Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Cys Xaa Xaa
Xaa Xaa Xaa Cys Xaa 20 25 30 Xaa Xaa Xaa Cys 35 13236PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 132Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Xaa Xaa Cys Cys Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Cys 35 13332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 133Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys
Xaa Xaa Xaa 1 5 10 15 Xaa Cys Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Cys Xaa Xaa Xaa Cys 20 25 30 13436PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Cysteine motif polypeptide 134Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10
15 Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
20 25 30 Xaa Xaa Xaa Cys 35 13536PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Cysteine motif polypeptide 135Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10
15 Cys Xaa Xaa Xaa Xaa Cys Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30 Cys Xaa Xaa Cys 35 13641PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Cysteine motif polypeptide 136Cys
Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa 1 5 10
15 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25
30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 13731PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 137Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys 20 25 30 13833PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif polypeptide 138Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20
25 30 Cys 13938PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif polypeptide 139Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Cys Xaa
Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30
Xaa Xaa Xaa Xaa Xaa Cys 35 14032PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Cysteine motif polypeptide 140Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Cys Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
20 25 30 14126PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif peptide 141Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa
Xaa Xaa Xaa Cys Cys Xaa Cys 20 25 14233PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 142Cys Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys 1 5 10 15 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa 14327PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
peptide 143Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Cys 1 5 10 15 Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Cys 20 25
14428PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif peptide 144Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys 1 5 10 15 Cys Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Cys 20 25 14531PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
polypeptide 145Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Cys 1 5 10 15 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Cys 20 25 30 14627PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif peptide 146Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Cys
Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Cys 20 25 14729PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
peptide 147Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Cys 1 5 10 15 Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa
Cys 20 25 14824PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif peptide 148Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa 1 5 10 15 Cys Xaa Xaa
Xaa Xaa Xaa Xaa Cys 20 14923PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif peptide 149Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys 1 5 10 15 Xaa
Cys Xaa Xaa Xaa Xaa Cys 20 15022PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Cysteine motif peptide 150Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 15
Cys Xaa Xaa Xaa Xaa Cys 20 15121PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Cysteine motif peptide 151Cys Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Cys 20 15219PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Cysteine motif peptide 152Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 15 Xaa
Xaa Cys 15318PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Cysteine motif peptide 153Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa 1 5 10 15 Xaa Cys
15417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif peptide 154Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Cys 15512PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Cysteine motif
peptide 155Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10
15611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Cysteine motif peptide 156Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys 1 5 10 15711PRTBos taurusStalk motif 1 157Thr Ser Val
His Gln Glu Thr Lys Lys Tyr Gln 1 5 10 1589PRTBos taurusStalk motif
1 158Val His Gln Glu Thr Lys Lys Tyr Gln 1 5 1595PRTBos taurusStalk
motif 1 159Thr Thr Val His Gln 1 5 1605PRTBos taurusStalk motif 1
160Thr Ser Val His Gln 1 5 1615PRTBos taurusStalk motif 1 161Ser
Ser Val Thr Gln 1 5 1625PRTBos taurusStalk motif 1 162Ser Thr Val
His Gln 1 5 1635PRTBos taurusStalk motif 1 163Ala Thr Val Arg Gln 1
5 1645PRTBos taurusStalk motif 1 164Thr Thr Val Tyr Gln 1 5
1655PRTBos taurusStalk motif 1 165Ser Pro Val His Gln 1 5
1665PRTBos taurusStalk motif 1 166Ala Thr Val Tyr Gln 1 5
1675PRTBos taurusStalk motif 1 167Thr Ala Val Tyr Gln 1 5
1685PRTBos taurusStalk motif 1 168Thr Asn Val His Gln 1 5
1695PRTBos taurusStalk motif 1 169Ala Thr Val His Gln 1 5
1705PRTBos taurusStalk motif 1 170Ser Thr Val Tyr Gln 1 5
1715PRTBos taurusStalk motif 1 171Thr Ile Val His Gln 1 5
1725PRTBos taurusStalk motif 1 172Ala Ile Val Tyr Gln 1 5
1735PRTBos taurusStalk motif 1 173Thr Thr Val Phe Gln 1 5
1745PRTBos taurusStalk motif 1 174Ala Ala Val Phe Gln 1 5
1755PRTBos taurusStalk motif 1 175Gly Thr Val His Gln 1 5
1765PRTBos taurusStalk motif 1 176Ala Ser Val His Gln 1 5
1775PRTBos taurusStalk motif 1 177Thr Ala Val Phe Gln 1 5
1785PRTBos taurusStalk motif 1 178Ala Thr Val Phe Gln 1 5
1795PRTBos taurusStalk motif 1 179Ala Ala Ala His Gln 1 5
1805PRTBos taurusStalk motif 1 180Val Val Val Tyr Gln 1 5
1815PRTBos taurusStalk motif 1 181Gly Thr Val Phe Gln 1 5
1825PRTBos taurusStalk motif 1 182Thr Ala Val His Gln 1 5
1835PRTBos taurusStalk motif 1 183Ile Thr Val His Gln 1 5
1845PRTBos taurusStalk motif 1 184Ile Thr Ala His Gln 1 5
1855PRTBos taurusStalk motif 1 185Val Thr Val His Gln 1 5
1865PRTBos taurusStalk motif 1 186Ala Ala Val His Gln 1 5
1875PRTBos taurusStalk motif 1 187Gly Thr Val Tyr Gln 1 5
1885PRTBos taurusStalk motif 1 188Thr Thr Val Leu Gln 1 5
1895PRTBos taurusStalk motif 1 189Thr Thr Thr His Gln 1 5
1905PRTBos taurusStalk motif 1 190Thr Thr Asp Tyr Gln 1 5
1915PRTBos taurusStalk motif 1 191Thr Thr Asp Tyr Gln 1 5
1926PRTBos taurusStalk motif 1 192Cys Thr Ser Val His Gln 1 5
1936PRTBos taurusStalk motif 1 193Cys Ser Ser Val Thr Gln 1 5
1946PRTBos taurusStalk motif 1 194Cys Ser Thr Val His Gln 1 5
1956PRTBos taurusStalk motif 1 195Cys Ala Thr Val Arg Gln 1 5
1966PRTBos taurusStalk motif 1 196Cys Thr Thr Val Tyr Gln 1 5
1976PRTBos taurusStalk motif 1 197Cys Ser Pro Val His Gln 1 5
1986PRTBos taurusStalk motif 1 198Cys Ala Thr Val Tyr Gln 1 5
1996PRTBos taurusStalk motif 1 199Cys Thr Ala Val Tyr Gln 1 5
2006PRTBos taurusStalk motif 1 200Cys Thr Asn Val His Gln 1 5
2016PRTBos taurusStalk motif 1 201Cys Ala Thr Val His Gln 1 5
2026PRTBos taurusStalk motif 1 202Cys Ser Thr Val Tyr Gln 1 5
2036PRTBos taurusStalk motif 1 203Cys Thr Ile Val His Gln 1 5
2046PRTBos taurusStalk motif 1 204Cys Ala Ile Val Tyr Gln 1 5
2056PRTBos taurusStalk motif 1 205Cys Thr Thr Val Phe Gln 1 5
2066PRTBos taurusStalk motif 1 206Cys Ala Ala Val Phe Gln 1 5
2076PRTBos taurusStalk motif 1 207Cys Gly Thr Val His Gln 1 5
2086PRTBos taurusStalk motif 1 208Cys Ala Ser Val His Gln 1 5
2096PRTBos taurusStalk motif 1 209Cys Thr Ala Val Phe Gln 1 5
2106PRTBos taurusStalk motif 1 210Cys Ala Thr Val Phe Gln 1 5
2116PRTBos taurusStalk motif 1 211Cys Ala Ala Ala His Gln 1 5
2126PRTBos taurusStalk motif 1 212Cys Val Val Val Tyr Gln 1 5
2136PRTBos taurusStalk motif 1 213Cys Gly Thr Val Phe Gln 1 5
2146PRTBos taurusStalk motif 1 214Cys Thr Ala Val His Gln 1 5
2156PRTBos taurusStalk motif 1 215Cys Ile Thr Val His Gln 1 5
2166PRTBos taurusStalk motif 1 216Cys Ile Thr Ala His Gln 1 5
2176PRTBos taurusStalk motif 1 217Cys Val Thr Val His Gln 1 5
2186PRTBos taurusStalk motif 1 218Cys Ala Ala Val His Gln 1 5
2196PRTBos taurusStalk motif 1 219Cys Gly Thr Val Tyr Gln 1 5
2206PRTBos taurusStalk motif 1 220Cys Thr Thr Val Leu Gln 1 5
2216PRTBos taurusStalk motif 1 221Cys Thr Thr Thr His Gln 1 5
2226PRTBos taurusStalk motif 1 222Cys Thr Thr Asp Tyr Gln 1 5
2237PRTBos taurusStalk motif 1 223Cys Thr Thr Val His Gln Xaa 1 5
22414PRTBos taurusStalk motif 1 224Cys Thr Ser Val His Gln Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 2253PRTBos taurusStalk motif 1
225Val His Gln 1 2263PRTBos taurusStalk motif 1 226Lys Lys Gln 1
2273PRTBos taurusStalk motif 1 227Val Tyr Gln 1 2286PRTBos
taurusStalk motif 1 228Cys Xaa Xaa Xaa Xaa Gln 1 5 2295PRTBos
taurusStalk motif 1 229Xaa Xaa Val His Gln 1 5 2306PRTBos
taurusStalk motif 1 230Cys Xaa Xaa Val His Gln 1 5 2315PRTBos
taurusStalk motif 1 231Xaa Xaa Val Xaa Gln 1 5 2326PRTBos
taurusStalk motif 1 232Cys Xaa Xaa Val Xaa Gln 1 5 2335PRTBos
taurusStalk motif 1 233Xaa Xaa Lys Lys Gln 1 5 2346PRTBos
taurusStalk motif 1 234Cys Xaa Xaa Lys Lys Gln 1 5 2357PRTBos
taurusStalk motif 2 235Tyr Thr Tyr Asn Tyr Glu Trp 1 5 2366PRTBos
taurusStalk motif 2 236Tyr Thr Tyr Asn Tyr Glu 1 5 2377PRTBos
taurusStalk motif 2 237Tyr Leu Tyr Thr Tyr Glu His 1 5 2386PRTBos
taurusStalk motif 2 238Tyr Leu Tyr Thr Tyr Glu 1 5 2398PRTBos
taurusStalk motif 2 239Cys Tyr Thr Tyr Asn Tyr Glu Phe 1 5
2408PRTBos taurusStalk motif 2 240His Tyr Thr Tyr Thr Tyr Asp Phe 1
5 2418PRTBos taurusStalk motif 2 241His Tyr Thr Tyr Thr Tyr Glu Trp
1 5 2428PRTBos taurusStalk motif 2 242Lys His Arg Tyr Thr Tyr Glu
Trp 1 5 2438PRTBos taurusStalk motif 2 243Asn Tyr Ile Tyr Lys Tyr
Ser Phe 1 5 2448PRTBos taurusStalk motif 2 244Pro Tyr Ile Tyr Thr
Tyr Gln Phe 1 5 2458PRTBos taurusStalk motif 2 245Ser Phe Thr Tyr
Thr Tyr Glu Trp 1 5 2468PRTBos taurusStalk motif 2 246Ser Tyr Ile
Tyr Ile Tyr Gln Trp 1 5 2478PRTBos taurusStalk motif 2 247Ser Tyr
Asn Tyr Thr Tyr Ser Trp 1 5 2488PRTBos taurusStalk motif 2 248Ser
Tyr Ser Tyr Ser Tyr Glu Tyr 1 5 2498PRTBos taurusStalk motif 2
249Ser Tyr Thr Tyr Asn Tyr Asp Phe 1 5 2508PRTBos taurusStalk motif
2 250Ser Tyr Thr Tyr Asn Tyr Glu Trp 1 5 2518PRTBos taurusStalk
motif 2 251Ser Tyr Thr Tyr Asn Tyr Gln Phe 1 5 2528PRTBos
taurusStalk motif 2 252Ser Tyr Val Trp Thr His Asn Phe 1 5
2538PRTBos taurusStalk motif 2 253Thr Tyr Lys Tyr Val Tyr Glu Trp 1
5 2548PRTBos taurusStalk motif 2 254Thr Tyr Thr Tyr Thr Tyr Glu Phe
1 5 2558PRTBos taurusStalk motif 2 255Thr Tyr Thr Tyr Thr Tyr Glu
Trp 1 5 2568PRTBos taurusStalk motif 2 256Val Phe Thr Tyr Thr Tyr
Glu Phe 1 5 2576PRTBos taurusStalk motif 2 257Ala Tyr Thr Tyr Glu
Trp 1 5 2586PRTBos taurusStalk motif 2 258Asp Tyr Ile Tyr Thr Tyr 1
5 2596PRTBos taurusStalk motif 2 259Ile His Ser Tyr Glu Phe 1 5
2606PRTBos taurusStalk motif 2 260Ser Phe Thr Tyr Glu Phe 1 5
2616PRTBos taurusStalk motif 2 261Ser His Ser Tyr Glu Phe 1 5
2626PRTBos taurusStalk motif 2 262Thr His Thr Tyr Glu Phe 1 5
2636PRTBos taurusStalk motif 2 263Thr Trp Thr Tyr Glu Phe 1 5
2646PRTBos taurusStalk motif 2 264Thr Tyr Asn Tyr Glu Trp 1 5
2656PRTBos taurusStalk motif 2 265Thr Tyr Ser Tyr Glu Phe 1 5
2666PRTBos taurusStalk motif 2 266Thr Tyr Ser Tyr Glu His 1 5
2676PRTBos taurusStalk motif 2 267Thr Tyr Thr Tyr Asp Phe 1 5
2686PRTBos taurusStalk motif 2 268Thr Tyr Thr Tyr Glu Phe 1 5
2696PRTBos taurusStalk motif 2 269Thr Tyr Thr Tyr Glu Trp 1 5
2704PRTBos taurusStalk motif 2 270Ala Tyr Glu Phe 1 2714PRTBos
taurusStalk motif 2 271Ala Tyr Ser Phe 1 2724PRTBos taurusStalk
motif 2 272Ala Tyr Ser Tyr 1 2734PRTBos taurusStalk motif 2 273Cys
Tyr Ser Phe 1 2744PRTBos taurusStalk motif 2 274Asp Tyr Thr Tyr 1
2754PRTBos taurusStalk motif 2 275Lys Tyr Glu His 1 2764PRTBos
taurusStalk motif 2 276Lys Tyr Glu Trp 1 2774PRTBos taurusStalk
motif 2 277Met Tyr Glu Phe 1 2784PRTBos taurusStalk motif 2 278Asn
Trp Ile Tyr 1 2794PRTBos taurusStalk motif 2 279Asn Tyr Asp Tyr 1
2804PRTBos taurusStalk motif 2 280Asn Tyr Gln Trp 1 2814PRTBos
taurusStalk motif 2 281Asn Tyr Ser Phe 1 2824PRTBos taurusStalk
motif 2 282Pro Tyr Glu Trp 1 2834PRTBos taurusStalk motif 2 283Arg
Tyr Asn Trp 1 2844PRTBos taurusStalk motif 2 284Arg Tyr Thr Tyr 1
2854PRTBos taurusStalk motif 2 285Ser Tyr Glu Phe 1 2864PRTBos
taurusStalk motif 2 286Ser Tyr Glu His 1 2874PRTBos taurusStalk
motif 2 287Ser Tyr Glu Trp 1 2884PRTBos taurusStalk motif 2 288Ser
Tyr Lys Trp 1 2894PRTBos taurusStalk motif 2 289Ser Tyr Thr Tyr 1
2904PRTBos taurusStalk motif 2 290Thr Tyr Asp Phe 1 2914PRTBos
taurusStalk motif 2 291Thr Tyr Glu Phe 1 2924PRTBos taurusStalk
motif 2 292Thr Tyr Glu Trp 1 2934PRTBos taurusStalk motif 2 293Thr
Tyr Gln Trp 1 2944PRTBos taurusStalk motif 2 294Thr Tyr Thr Tyr 1
2954PRTBos taurusStalk motif 2 295Val Tyr Glu Trp 1 2964PRTBos
taurusStalk motif 2 296Tyr Xaa Tyr Xaa 1 2975PRTBos taurusStalk
motif 2 297Tyr Xaa Tyr Xaa Tyr 1 5 2986PRTBos taurusStalk motif 2
298Tyr Xaa Tyr Xaa Tyr Xaa 1 5 2997PRTBos taurusStalk motif 2
299Tyr Xaa Tyr Xaa Tyr Xaa Xaa 1 5 3003PRTBos taurusStalk motif 2
300Tyr Glu Xaa 1 3013PRTBos taurusStalk motif 2 301Tyr Asp Xaa 1
3023PRTBos taurusStalk motif 2 302Xaa Tyr Glu 1 3033PRTBos
taurusStalk motif 2 303Xaa Tyr Asp 1 3045PRTBos taurusStalk motif 2
304Tyr Glu Xaa Xaa Trp 1 5 3055PRTBos taurusStalk motif 2 305Tyr
Asp Xaa Xaa Trp 1 5 3068PRTBos taurusStalk motif 2 306Tyr Glu Xaa
Xaa Xaa Xaa Xaa Trp 1 5
3078PRTBos taurusStalk motif 2 307Tyr Asp Xaa Xaa Xaa Xaa Xaa Trp 1
5 30863DNAArtificial SequenceDescription of Artificial Sequence
Synthetic MID1 FW primer 308cctatcccct gtgtgccttg gcagtctcag
acgagtgcgt ttgagcgaca aggctgtagg 60ctg 6330962DNAArtificial
SequenceDescription of Artificial Sequence Synthetic MID1 RV primer
309ccatctcatc cctgcgtgtc tccgactcag acgagtgcgt ctttcggggc
tgtggtggag 60gc 6231063DNAArtificial SequenceDescription of
Artificial Sequence Synthetic MID10 FW primer 310cctatcccct
gtgtgccttg gcagtctcag tctctatgcg ttgagcgaca aggctgtagg 60ctg
6331166DNAArtificial SequenceDescription of Artificial Sequence
Synthetic MID10 RV primer 311ccatctcatc cctgcgtgtc tccgactcag
tctctatgcg agtgaagact ctcgggtgtg 60attcac 6631263DNAArtificial
SequenceDescription of Artificial Sequence Synthetic MID11 FW
primer 312cctatcccct gtgtgccttg gcagtctcag tgatacgtct ttgagcgaca
aggctgtagg 60ctg 6331366DNAArtificial SequenceDescription of
Artificial Sequence Synthetic MID11 RV primer 313ccatctcatc
cctgcgtgtc tccgactcag tgatacgtct agtgaagact ctcgggtgtg 60attcac
6631423DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer A 314ttgagcgaca aggctgtagg ctg
2331522DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer B 315ctttcggggc tgtggtggag gc 2231620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer C
316agatccaagc tgtgaccggc 2031774PRTHomo sapiensIL8 317Pro Arg Ser
Ala Lys Glu Leu Arg Cys Gln Cys Ile Lys Thr Tyr Ser 1 5 10 15 Lys
Pro Phe His Pro Lys Phe Ile Lys Glu Leu Arg Val Ile Glu Ser 20 25
30 Gly Pro His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly
35 40 45 Arg Glu Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln Arg
Val Val 50 55 60 Glu Lys Phe Leu Lys Arg Ala Glu Asn Ser 65 70
31825PRTUnknownDescription of Unknown Ziconotide peptide 318Cys Lys
Gly Lys Gly Ala Lys Cys Ser Arg Leu Met Tyr Asp Cys Cys 1 5 10 15
Thr Gly Ser Cys Arg Ser Gly Lys Cys 20 25
31915PRTUnknownDescription of Unknown Somatostatin peptide 319Ala
Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Gly 1 5 10 15
32034PRTUnknownDescription of Unknown Chlorotoxin polypeptide
320Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys
1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly
Pro Gln 20 25 30 Cys Leu 32168PRTUnknownDescription of Unknown
SDF1(alpha) polypeptide 321Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro
Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys
His Leu Lys Ile Leu Asn Thr Pro 20 25 30 Asn Cys Ala Leu Gln Ile
Val Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45 Val Cys Ile Asp
Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50 55 60 Ala Leu
Asn Lys 65 322133PRTHomo sapiensIL21 322Gln Gly Gln Asp Arg His Met
Ile Arg Met Arg Gln Leu Ile Asp Ile 1 5 10 15 Val Asp Gln Leu Lys
Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu 20 25 30 Pro Ala Pro
Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser 35 40 45 Cys
Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu 50 55
60 Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser
65 70 75 80 Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro
Ser Cys 85 90 95 Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu
Glu Arg Phe Lys 100 105 110 Ser Leu Leu Gln Lys Met Ile His Gln His
Leu Ser Ser Arg Thr His 115 120 125 Gly Ser Glu Asp Ser 130
32330PRTUnknownDescription of Unknown Protoxin2 polypeptide 323Tyr
Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Glu Arg Lys Cys Cys 1 5 10
15 Glu Gly Met Val Cys Arg Leu Trp Cys Lys Lys Lys Leu Trp 20 25 30
324166PRTHomo sapiensIFN-beta 324Met Ser Tyr Asn Leu Leu Gly Phe
Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu Trp
Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met
Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe
Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60
Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65
70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln
Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys
Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu His Leu
Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys
Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile
Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr
Leu Arg Asn 165 325174PRTBos taurusbGCSF 325Thr Pro Leu Gly Pro Ala
Arg Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gln
Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu Gln 20 25 30 Glu Arg
Leu Cys Ala Ala His Lys Leu Cys His Pro Glu Glu Leu Met 35 40 45
Leu Leu Arg His Ser Leu Gly Ile Pro Gln Ala Pro Leu Ser Ser Cys 50
55 60 Ser Ser Gln Ser Leu Gln Leu Thr Ser Cys Leu Asn Gln Leu His
Gly 65 70 75 80 Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Ala
Gly Ile Ser 85 90 95 Pro Glu Leu Ala Pro Thr Leu Asp Thr Leu Gln
Leu Asp Val Thr Asp 100 105 110 Phe Ala Thr Asn Ile Trp Leu Gln Met
Glu Asp Leu Gly Ala Ala Pro 115 120 125 Ala Val Gln Pro Thr Gln Gly
Ala Met Pro Thr Phe Thr Ser Ala Phe 130 135 140 Gln Arg Arg Ala Gly
Gly Val Leu Val Ala Ser Gln Leu His Arg Phe 145 150 155 160 Leu Glu
Leu Ala Tyr Arg Gly Leu Arg Tyr Leu Ala Glu Pro 165 170
326127PRTHomo sapiensGMCSF 326Ala Pro Ala Arg Ser Pro Ser Pro Ser
Thr Gln Pro Trp Glu His Val 1 5 10 15 Asn Ala Ile Gln Glu Ala Arg
Arg Leu Leu Asn Leu Ser Arg Asp Thr 20 25 30 Ala Ala Glu Met Asn
Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp 35 40 45 Leu Gln Glu
Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln 50 55 60 Gly
Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met 65 70
75 80 Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser
Cys 85 90 95 Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn
Leu Lys Asp 100 105 110 Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu
Pro Val Gln Glu 115 120 125 327181PRTHomo sapienshFGF21 327His Pro
Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20
25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln
Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly
Val Ile Gln 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys
Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp
Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly
Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His
Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg
Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala
Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150
155 160 Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg
Ser 165 170 175 Pro Ser Tyr Ala Ser 180 32839PRTHomo sapiensEx-4
328His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 32930PRTHomo
sapienshGLP-1 329His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Lys Gly Arg 20 25 30 330165PRTHomo sapienshEPO 330Pro Pro Arg
Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu 1 5 10 15 Glu
Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys 20 25
30 Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr
35 40 45 Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val
Trp Gln 50 55 60 Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly
Gln Ala Leu Leu 65 70 75 80 Val Asn Ser Ser Gln Pro Trp Glu Pro Leu
Gln Leu His Val Asp Lys 85 90 95 Ala Val Ser Gly Leu Arg Ser Leu
Thr Thr Leu Leu Arg Ala Leu Gly 100 105 110 Ala Gln Lys Glu Ala Ile
Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro 115 120 125 Leu Arg Thr Ile
Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr 130 135 140 Ser Asn
Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys 145 150 155
160 Arg Thr Gly Asp Arg 165 33134PRTUnknownDescription of Unknown
Moka toxin polypeptide 331Ile Asn Val Lys Cys Ser Leu Pro Gln Gln
Cys Ile Lys Pro Cys Lys 1 5 10 15 Asp Ala Gly Met Arg Phe Gly Lys
Cys Met Asn Lys Lys Cys Arg Cys 20 25 30 Tyr Ser
33236PRTUnknownDescription of Unknown VM-24 polypeptide 332Ala Ala
Ala Ile Ser Cys Val Gly Ser Pro Glu Cys Pro Pro Lys Cys 1 5 10 15
Arg Ala Gln Gly Cys Lys Asn Gly Lys Cys Met Asn Arg Lys Cys Lys 20
25 30 Cys Tyr Tyr Cys 35 3336PRTBos taurusStalk motif 2 333Tyr Glu
Xaa Xaa Xaa Trp 1 5 3347PRTBos taurusStalk motif 2 334Tyr Glu Xaa
Xaa Xaa Xaa Trp 1 5 3356PRTBos taurusStalk motif 2 335Tyr Asp Xaa
Xaa Xaa Trp 1 5 3367PRTBos taurusStalk motif 2 336Tyr Asp Xaa Xaa
Xaa Xaa Trp 1 5 3379PRTArtificial SequenceDescription of Artificial
Sequence Synthetic linker peptide 337Gly Gly Gly Ser Gly Gly Gly
Gly Ser 1 5 3389PRTArtificial SequenceDescription of Artificial
Sequence Synthetic linker peptide 338Gly Gly Gly Gly Ser Gly Gly
Gly Ser 1 5 33925PRTArtificial SequenceDescription of Artificial
Sequence Synthetic linker peptide 339Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly
Gly Gly Gly Ser 20 25 340269PRTBos taurusBLV5B8 VHCH1 340Gln Val
Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Gln Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20
25 30 Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp
Leu 35 40 45 Gly Ser Ile Asp Thr Gly Gly Ser Thr Gly Tyr Asn Pro
Gly Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys
Ser Gln Val Ser Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp
Ser Ala Thr Tyr Tyr Cys Thr 85 90 95 Thr Val His Gln Glu Thr Arg
Lys Thr Cys Ser Asp Gly Tyr Ile Ala 100 105 110 Val Asp Ser Cys Gly
Arg Gly Gln Ser Asp Gly Cys Val Asn Asp Cys 115 120 125 Asn Ser Cys
Tyr Tyr Gly Trp Arg Asn Cys Arg Arg Gln Pro Ala Ile 130 135 140 His
Ser Tyr Glu Phe His Val Asp Ala Trp Gly Arg Gly Leu Leu Val 145 150
155 160 Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Lys Val Tyr Pro Leu
Ser 165 170 175 Ser Cys Cys Gly Asp Lys Ser Ser Ser Thr Val Thr Leu
Gly Cys Leu 180 185 190 Val Ser Ser Tyr Met Pro Glu Pro Val Thr Val
Thr Trp Asn Ser Gly 195 200 205 Ala Leu Lys Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser 210 215 220 Gly Leu Tyr Ser Leu Ser Ser
Met Val Thr Val Pro Gly Ser Thr Ser 225 230 235 240 Gly Gln Thr Phe
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys 245 250 255 Val Asp
Lys Ala Val Glu Pro Lys Ser Cys Asp Gly Ser 260 265 341216PRTBos
taurusBLV5B8 VLCL 341Gln Ala Val Leu Asn Gln Pro Ser Ser Val Ser
Gly Ser Leu Gly Gln 1 5 10 15 Arg Val Ser Ile Thr Cys Ser Gly Ser
Ser Ser Asn Val Gly Asn Gly 20 25 30 Tyr Val Ser Trp Tyr Gln Leu
Ile Pro Gly Ser Ala Pro Arg Thr Leu 35 40 45 Ile Tyr Gly Asp Thr
Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg
Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ala
Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu Asp Ser Ser 85 90
95 Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu Gly Gln
100 105 110 Pro Lys Ser Pro Pro Ser Val Thr Leu Phe Pro Pro Ser Thr
Glu Glu 115 120 125 Leu Asn Gly Asn Lys Ala Thr Leu Val Cys Leu Ile
Ser Asp Phe Tyr 130 135 140 Pro Gly Ser Val Thr Val Val Trp Lys Ala
Asp Gly Ser Thr Ile Thr 145 150 155 160 Arg Asn Val Glu Thr Thr Arg
Ala Ser Lys Gln Ser Asn Ser Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr
Leu Ser Leu Thr Ser Ser Asp Trp Lys Ser Lys 180 185 190 Gly Ser Tyr
Ser Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys 195 200 205
Thr Val Lys Pro Ser Glu Cys Ser 210 215 3423PRTArtificial
SequenceDescription of Artificial Sequence Synthetic linker peptide
342Gly Ser Gly 1 34311PRTMus sp.D44.1 343Cys Ala Arg Gly Asp Gly
Asn Tyr Gly Tyr Trp 1 5 10 34413PRTMus sp.93F3 344Cys Ala Lys His
Thr Tyr Gly Gly Pro Gly Asp Ser Trp 1 5 10 34514PRTMus sp.OKT3
345Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp 1 5 10
34614PRTHomo sapiensYvo 346Cys Ala Arg Thr Ser Gly Trp Asp Ile Glu
Phe Glu Tyr Trp 1 5 10 34716PRTHomo sapiensCR6261 347Cys Ala Lys
His Met Gly Tyr Gln Val Arg Glu Thr Met Asp Val Trp 1 5 10 15
34832PRTHomo sapiensPG9 348Cys Val Arg Glu Ala Gly Gly Pro Asp Tyr
Arg Asn Gly Tyr Asn Tyr 1 5 10 15 Tyr Asp Phe Tyr Asp Gly Tyr Tyr
Asn Tyr His Tyr Met Asp Val Trp 20 25 30 34920PRTBos taurusB-S1
349Cys Ala Lys Ser Ser Gly Thr Asn Phe Ala Val Ala Thr Trp Asp Val
1 5 10 15 Ile Asp Ala Trp 20 35026PRTBos taurusB-S2 350Cys Ala Lys
Ser Ser Gly Asn Val Gly Phe Tyr Gln Ser Tyr Asn Ser 1 5 10 15 Arg
Ser Trp Lys Gln Tyr Val Asp Ala Trp 20 25 35127PRTBos taurusB-S3
351Cys Ala Lys His Phe Ala Gly Ala Asn Ile Ile Cys Asp Leu Asn His
1 5 10 15 Asp Ala Trp Gly Ser Gly Ser Leu Asp Ala Trp 20 25
35230PRTBos taurusB-S4 352Cys Thr Lys Glu Thr Trp Thr Gly Pro Gly
Tyr Asn Ala Asn Gly Cys 1 5 10 15 Tyr Cys Val Gly Gly Arg Gly Glu
Cys Tyr Val Asp Ala Trp 20 25 30 35360PRTBos taurusBF4E9 353Cys Thr
Thr Val His Gln Ile Phe Cys Pro Asp Gly Tyr Ser Tyr Gly 1 5 10 15
Tyr Gly Cys Gly Tyr Gly Tyr Gly Cys Ser Gly Tyr Asp Cys Tyr Gly 20
25 30 Tyr Gly Gly Tyr Gly Tyr Gly Gly Tyr Gly Gly Tyr Ser Ser Tyr
Ser 35 40 45 Tyr Ser Tyr Ser Tyr Glu Tyr Tyr Gly Asp Ala Trp 50 55
60 35460PRTBos taurusBLV5B8 354Cys Thr Thr Val His Gln Glu Thr Arg
Lys Thr Cys Ser Asp Gly Tyr 1 5 10 15 Ile Ala Val Asp Ser Cys Gly
Arg Gly Gln Ser Asp Gly Cys Val Asn 20 25 30 Asp Cys Asn Ser Cys
Tyr Tyr Gly Trp Arg Asn Cys Arg Arg Gln Pro 35 40 45 Ala Ile His
Ser Tyr Glu Phe His Val Asp Ala Trp 50 55 60 35561PRTBos
taurusBLV5D3 355Cys Ser Ser Val Thr Gln Arg Thr His Val Ser Arg Ser
Cys Pro Asp 1 5 10 15 Gly Cys Ser Asp Gly Asp Gly Cys Val Asp Gly
Cys Cys Cys Ser Ala 20 25 30 Tyr Arg Cys Tyr Thr Pro Gly Val Arg
Asp Leu Ser Cys Thr Ser Tyr 35 40 45 Ser Ile Thr Tyr Thr Tyr Glu
Trp Asn Val Asp Ala Trp 50 55 60 35662PRTBos taurusBLV8C11 356Cys
Thr Thr Val His Gln Lys Thr Thr Arg Lys Thr Cys Cys Ser Asp 1 5 10
15 Ala Tyr Arg Tyr Asp Ser Gly Cys Gly Ser Gly Cys Asp Cys Cys Gly
20 25 30 Ala Asp Cys Tyr Val Phe Gly Ala Cys Thr Phe Gly Leu Asp
Ser Ser 35 40 45 Tyr Ser Tyr Ile Tyr Ile Tyr Gln Trp Tyr Val Asp
Ala Trp 50 55 60 35763PRTBos taurusB-L2 357Cys Ala Thr Val Arg Gln
Thr Thr Leu Arg Asp Cys Pro Gly Gly Tyr 1 5 10 15 Thr Glu Asp Arg
Ser Cys Val Asn Thr Tyr Ser Cys Gly Ala Asp Asp 20 25 30 Cys Cys
Gly Arg Gly Asp Val Gly Tyr Pro Ala Leu Tyr Gly Tyr Arg 35 40 45
Cys Ala Ala His Ile Gln Arg Tyr Asn Trp His Ala Asp Ala Trp 50 55
60 35865PRTBos taurusBLV1H12 358Cys Thr Ser Val His Gln Glu Thr Lys
Lys Tyr Gln Ser Cys Pro Asp 1 5 10 15 Gly Tyr Arg Glu Arg Ser Asp
Cys Ser Asn Arg Pro Ala Cys Gly Thr 20 25 30 Ser Asp Cys Cys Arg
Val Ser Val Phe Gly Asn Cys Leu Thr Thr Leu 35 40 45 Pro Val Ser
Tyr Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val 50 55 60 Trp 65
35970PRTBos taurusB-L1 359Cys Ser Thr Val His Gln Lys Thr Arg Thr
Thr Gln Gly Asn Thr Cys 1 5 10 15 Pro Asp Gly Tyr Thr Leu Lys Asp
Asp Cys Pro Arg Cys Arg Gly Gly 20 25 30 Cys Asp Gly Tyr Asp Cys
Cys Trp Gly Asp Ala Cys Arg Ser Ser Gly 35 40 45 Leu Cys Trp Gly
His Asn Pro Leu Val Thr Glu Thr Tyr Thr Tyr Glu 50 55 60 Phe Tyr
Ile Asp Ala Trp 65 70 36048PRTBos taurusBLV1H12 (2) 360Ser Cys Pro
Asp Gly Tyr Arg Glu Arg Ser Asp Cys Ser Asn Arg Pro 1 5 10 15 Ala
Cys Gly Thr Ser Asp Cys Cys Arg Val Ser Val Phe Gly Asn Cys 20 25
30 Leu Thr Thr Leu Pro Val Ser Tyr Ser Tyr Thr Tyr Asn Tyr Glu Trp
35 40 45 36144PRTBos taurusBLV5B8 (2) 361Cys Ser Asp Gly Tyr Ile
Ala Val Asp Ser Cys Gly Arg Gly Gln Ser 1 5 10 15 Asp Gly Cys Val
Asn Asp Cys Asn Ser Cys Tyr Tyr Gly Trp Arg Asn 20 25 30 Cys Arg
Arg Gln Pro Ala Ile His Ser Tyr Glu Phe 35 40 362133PRTBos
taurusVHBUL 362Leu Lys Arg Leu Val Gly Val Val Thr Leu Ile Cys Ser
Lys Met Asn 1 5 10 15 Pro Leu Trp Thr Leu Leu Phe Val Leu Ser Ala
Pro Arg Gly Val Leu 20 25 30 Ser Gln Val Gln Leu Arg Glu Ser Gly
Pro Ser Leu Val Lys Pro Ser 35 40 45 Gln Thr Leu Ser Leu Thr Cys
Thr Ala Ser Gly Phe Ser Leu Ser Asp 50 55 60 Lys Ala Val Gly Trp
Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp 65 70 75 80 Leu Gly Gly
Ile Asp Thr Gly Gly Ser Thr Gly Tyr Asn Pro Gly Leu 85 90 95 Lys
Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser 100 105
110 Leu Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys
115 120 125 Thr Thr Val His Gln 130 363521DNABos taurusVHBUL 363ttg
aag aga ctt gtg gga gtg gtg act ctc atc tgc tcc aag atg aac 48Leu
Lys Arg Leu Val Gly Val Val Thr Leu Ile Cys Ser Lys Met Asn 1 5 10
15 cca ctg tgg acc ctc ctc ttt gtg ctc tca gcc ccc aga ggt 90Pro
Leu Trp Thr Leu Leu Phe Val Leu Ser Ala Pro Arg Gly 20 25 30
gagtgtctct gggtcagaca taggcacgtg gggaagctgc ctctgagccc acgggtcacc
150gtgcttctct ctctccacag gg gtc ctg tcc cag gtg cag ctg cgg gag tcg
202 Val Leu Ser Gln Val Gln Leu Arg Glu Ser 35 40 ggc ccc agc ctg
gtg aag ccc tca cag acc ctc tcg ctc acc tgc acg 250Gly Pro Ser Leu
Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr 45 50 55 gcc tct
gga ttc tca ttg agc gac aag gct gta ggc tgg gtc cgc cag 298Ala Ser
Gly Phe Ser Leu Ser Asp Lys Ala Val Gly Trp Val Arg Gln 60 65 70
gct cca ggg aag gcg ctg gag tgg ctc ggt ggt ata gac act ggt gga
346Ala Pro Gly Lys Ala Leu Glu Trp Leu Gly Gly Ile Asp Thr Gly Gly
75 80 85 agc aca ggc tat aac cca ggc ctg aaa tcc cgg ctc agc atc
acc aag 394Ser Thr Gly Tyr Asn Pro Gly Leu Lys Ser Arg Leu Ser Ile
Thr Lys 90 95 100 gac aac tcc aag agc caa gtc tct ctg tca gtg agc
agc gtg aca act 442Asp Asn Ser Lys Ser Gln Val Ser Leu Ser Val Ser
Ser Val Thr Thr 105 110 115 120 gag gac tcg gcc aca tac tac tgt act
act gtg cac cag acacagtgag 491Glu Asp Ser Ala Thr Tyr Tyr Cys Thr
Thr Val His Gln 125 130 gggaaatcag tgtgagccca gacaaaaacc
5213646PRTBos taurusVH germline 364Cys Thr Thr Val His Gln 1 5
36548PRTBos taurusDH2 germline 365Ser Cys Pro Asp Gly Tyr Ser Tyr
Gly Tyr Gly Cys Gly Tyr Gly Tyr 1 5 10 15 Gly Cys Ser Gly Tyr Asp
Cys Tyr Gly Tyr Gly Gly Tyr Gly Gly Tyr 20 25 30 Gly Gly Tyr Gly
Tyr Ser Ser Tyr Ser Tyr Ser Tyr Thr Tyr Glu Tyr 35 40 45 3665PRTBos
taurusJH1 germline 366Tyr Val Asp Ala Trp 1 5 36764PRTBos
taurusBLV1H12 367Cys Thr Ser Val His Gln Glu Thr Lys Lys Tyr Ser
Cys Pro Asp Gly 1 5 10 15 Tyr Arg Glu Arg Ser Asp Cys Ser Asn Arg
Pro Ala Cys Gly Thr Ser 20 25 30 Asp Cys Cys Arg Val Ser Val Phe
Gly Asn Cys Leu Thr Thr Leu Pro 35 40 45 Val Ser Tyr Ser Tyr Thr
Tyr Asn Tyr Glu Trp His Val Asp Val Trp 50 55 60 36860PRTBos
taurusBLV5B8 368Cys Thr Thr Val His Gln Glu Thr Arg Lys Thr Cys Ser
Asp Gly Tyr 1 5 10 15 Ile Ala Val Asp Ser Cys Gly Arg Gly Gln Ser
Asp Gly Cys Val Asn 20 25 30 Asp Cys Asn Ser Cys Tyr Tyr Gly Trp
Arg Asn Cys Arg Arg Gln Pro 35 40 45 Ala Ile His Ser Tyr Glu Phe
His Val Asp Ala Trp 50 55 60 36963PRTBos taurusBovine VH CDRH3 (1)
369Cys Ser Pro Val His Gln Glu Ile Arg Lys Cys Cys Pro Ala Gly Cys
1 5 10 15 Gln Cys Gly Arg Ser Cys Gly Ala Cys Cys Gly Cys Ala Gly
Asp Glu 20 25 30 Phe Cys Gly Ile Asn Val Tyr Gly Tyr Val Thr Cys
Gly Gly Tyr Arg 35 40 45 Thr Cys Ser Cys Ile Asp Thr Tyr Asp Phe
Tyr Val Asp Ala Trp 50 55 60 37063PRTBos taurusBovine VH CDRH3 (2)
370Cys Thr Thr Val His Gln Lys Thr Lys Lys Leu Cys Pro Asn Gly Arg
1 5 10 15 Thr Cys Gly Cys Gly Cys Asp Cys Gly Ser Gly Cys Cys Thr
Ser Tyr 20 25 30 Cys Asp Ser Phe Gly Cys Trp Gly Gly Arg Asp Thr
Phe Gly Ser Ser 35 40 45 Cys Thr Ser Ala Thr Tyr Thr Tyr Glu Trp
Gly Val Asp Ala Trp 50 55 60 37164PRTBos taurusBovine VH CDRH3 (3)
371Cys Ala Thr Val His Gln His Thr Asn Lys Lys Arg Cys Pro Asp Gly
1 5 10 15 Tyr Glu Phe Ser Ala Gly Cys Cys Cys Gly Glu Gly Cys Ser
Gly Ser 20 25 30 Asp Cys Cys Cys Asn Ser Arg Leu Arg Cys Ser Trp
Tyr Glu Ile Tyr 35 40 45 Cys Ser Val Ser Pro Ser Asp Thr Tyr Glu
Phe His Val Asp Ala Trp 50 55 60 37270PRTBos taurusBovine VH CDRH3
(4) 372Cys Ser Thr Val His Gln Lys Thr Arg Thr Thr Gln Gly Asn Thr
Cys 1 5 10 15 Pro Asp Gly Tyr Thr Leu Lys Asp Asp Cys Pro Arg Cys
Arg Gly Gly 20 25 30 Cys Asp Gly Tyr Asp Cys Cys Trp Gly Asp Ala
Cys Arg Ser Ser Gly 35 40 45 Leu Cys Trp Gly His Asn Pro Leu Val
Thr Glu Thr Tyr Thr Tyr Glu 50 55 60 Phe Tyr Ile Asp Ala Trp 65 70
37363PRTBos taurusBovine VH CDRH3 (5) 373Cys Thr Thr Val His Gln
Glu Thr His Lys Arg Cys Pro Asp Gly Tyr 1 5 10 15 Thr Tyr Gly Tyr
Tyr Cys Gly Tyr Ala Cys Thr Cys Ser Gly Asp Glu 20 25 30 Cys Tyr
Arg Tyr Asp Tyr Cys Ala Ala Tyr Gly Ser Leu Gly Cys Cys 35 40 45
Thr Asn Asp His Thr Tyr Thr Tyr Glu Phe His Val Asp Ser Trp 50 55
60 37463PRTBos taurusBovine VH CDRH3 (6) 374Cys Thr Ala Val Tyr Gln
Gln Thr Arg Lys Ser Cys Pro Asp Gly Tyr 1 5 10 15 Arg Ser Gly Asn
Asp Cys Ser Ser Ala Cys Ser Cys Ser Asn Tyr Glu 20 25 30 Cys Tyr
Arg Tyr Gly Ser Tyr Gly Ser Asn Gly Lys Cys Gly Tyr Asp 35 40 45
Ala His Ala Tyr Thr Tyr Thr Tyr Glu Ile His Ile Asp Ala Trp 50 55
60 37564PRTBos taurusBovine VH CDRH3 (7) 375Cys Gly Ala Val His Gln
Lys Thr Ala Arg Ser Cys Pro Asn Ile Tyr 1 5 10 15 Ser Thr Tyr Tyr
Gly Gly Arg Ser Gly Ser Val Gly Cys Ser Ala Tyr 20 25 30 Asp Cys
Glu Asn Cys Cys Thr Tyr Asp Gly Met Gly Arg Tyr Ser Val 35 40 45
Ser Thr Cys Ser Gly Ser Val Ile Tyr Glu Phe Tyr Val Asp Thr Trp 50
55 60 37660PRTBos taurusBovine VH CDRH3 (8) 376Cys Ala Thr Lys Lys
Gln Ile Cys Cys Pro Asp Asp Ser Ser Leu Glu 1 5 10 15 Val Ala Cys
Ser His Gly Ala Gly Cys Ser Gly Cys Val Gly Tyr Thr 20 25 30 Gly
Gly Thr Trp Gly Thr Leu Ser Asp Tyr Phe His Gly Lys Tyr Thr 35 40
45 Cys Thr Tyr Thr Tyr Glu His Asn Val Asp Ala Trp 50 55 60
37763PRTBos taurusBovine VH CDRH3 (9) 377Cys Thr Ile Val His Gln
Gln Thr Thr Lys Arg Cys Pro Asp Asp Asp 1 5 10 15 Asn Tyr Pro Tyr
Trp Cys Ser Val Ala Asn Gly Gly Gly Ser Asp Ala 20 25 30 Cys Tyr
Gly Cys Ser Gly Arg Ser Ser Asp Thr Phe Trp Arg Cys Ser 35 40 45
Thr Val Arg Tyr Arg Tyr Thr Tyr Glu Trp His Val Asp Ala Trp 50 55
60 37863PRTBos taurusBovine VH CDRH3 (10) 378Cys Ala Thr Val His
Gln Leu Thr Arg Ala His Cys Pro Asp Asp Tyr 1 5 10 15 Ser Tyr Leu
Tyr Thr Ser Arg Trp Asp Cys Ala Ser Cys Asp Asp Gly 20 25 30 Cys
Tyr Ala Ala Arg Asp Trp Arg Gly Cys Phe Asp Cys Glu Ser Ser 35 40
45 Lys Thr Ser Val Ser Tyr Ile Tyr Glu His His Val Asn Ala Trp 50
55 60 37965PRTBos taurusBovine VH CDRH3 (11) 379Cys Ala Thr Val His
Gln Arg Thr Glu Lys Ser Cys Ser Ala Gly His 1 5 10 15 Ile Asp Gly
Val Gln Cys Cys Cys Ser Gly Val Ala Cys Asp Gly Ala 20 25 30 Gly
Cys Val Arg Gly Cys Ser Tyr Gly Thr Asp Gly Trp Tyr Gly Trp 35 40
45 Cys Asn Arg Tyr Ser Tyr Thr Ile Thr Tyr Glu Phe Tyr Val Thr Ala
50 55 60 Trp 65 38067PRTBos taurusBovine VH CDRH3 (12) 380Cys Thr
Thr Val His Gln Arg Thr Lys Arg Ser Cys Pro Asp Asp Tyr 1 5 10 15
Thr Tyr Thr Tyr Thr Cys Val Ser Glu Ser Asp His Gln Ala Glu Arg 20
25 30 Gly Cys Tyr Gly Pro Gly Gly Tyr Gly Trp Cys Asp Trp Thr Gly
Ser 35 40 45 Thr Thr Val Ser Arg Glu Gly Glu Arg Asn Asn Tyr Glu
Phe His Ile 50 55 60 Asp Ala Trp 65 38163PRTBos taurusBovine VH
CDRH3 (13) 381Cys Thr Thr Val His Gln Ile Thr His Lys Glu Cys Pro
Asp Gly Tyr 1 5 10
15 Ser Asp Gly Cys Thr Cys Thr Arg Ser Trp Tyr Tyr Ser Gly Trp Asn
20 25 30 Cys Tyr Pro Gly Glu Val Cys Trp Ser Arg Gly Gly Cys Gly
Ile Ser 35 40 45 Gly Val Thr Tyr Ser Asp Thr Tyr Glu Phe Tyr Ile
Asp Ala Trp 50 55 60 38265PRTBos taurusBovine VH CDRH3 (14) 382Cys
Gly Thr Val His Gln His Thr Thr Thr Lys Asn Thr Cys Pro Asp 1 5 10
15 Gly Tyr Thr Phe Arg Ala Gly Cys Cys Cys Ser Ser Gly Cys Ile Ser
20 25 30 Cys Asp Ser Ser Ile Cys Asp Asn Thr Ser Pro Ser Trp Phe
Cys Ser 35 40 45 Arg Thr Ser Pro Thr Tyr Thr Tyr Thr Tyr Glu Phe
Tyr Ile Thr Ala 50 55 60 Trp 65 38362PRTBos taurusBovine VH CDRH3
(15) 383Cys Ala Thr Val His Gln Lys Thr Leu Glu Lys Thr Cys Pro Asp
Gly 1 5 10 15 Tyr Ala Tyr Gly Asp Thr Asp Asn Gly His Cys Ser Ala
Tyr Asp Cys 20 25 30 Trp Arg Met Gly Thr Tyr Cys Thr Glu Asp Met
Tyr Gly Cys Ser Cys 35 40 45 Tyr Ser Gly Thr Thr Thr Tyr Glu Trp
Tyr Val Glu Ala Trp 50 55 60 38464PRTBos taurusBovine VH CDRH3 (16)
384Cys Ala Thr Val His Gln Glu Val Gln Lys Lys Thr Cys Pro Asp Gly
1 5 10 15 Tyr Ala His Leu Gly Phe Cys Asn Asp Asp Asp Gly Arg Leu
Gly Ser 20 25 30 Ala Cys Cys Ser Gly Gly Ala Phe Gly Ser Asp Gly
Asp Thr Asp Cys 35 40 45 His Cys Tyr Ser Asp Ser Tyr Asn Tyr Glu
Asn His Val Asp Glu Trp 50 55 60 38559PRTBos taurusBovine VH CDRH3
(17) 385Cys Ser Thr Val His Gln Lys Thr Gln Arg Ser Cys Pro Asp Gly
Tyr 1 5 10 15 Arg Thr Gly Tyr Gly Cys Asp Asp Gly Ser Cys Cys Ser
Gly Ser Asn 20 25 30 Cys Tyr Ser Tyr Leu Ser Arg Ile Asn Arg Gly
Thr Cys Arg Thr Lys 35 40 45 Ile Thr Thr Tyr Glu His His Ile Asp
Ala Trp 50 55 38667PRTBos taurusBovine VH CDRH3 (18) 386Cys Thr Thr
Val His Gln Glu Thr Lys Thr Arg Ser Thr Cys Pro Asp 1 5 10 15 Gly
Tyr Gly Cys Thr Val Gly Cys Tyr Tyr Gly Thr Tyr Ser Cys Ser 20 25
30 Gly Ser Asp Cys Thr Cys Ser Arg Ile Arg Arg Val Tyr Gly Ala Thr
35 40 45 Gly Gly Leu Ser Ile Cys Thr Ser Thr His Thr Tyr Glu Trp
His Val 50 55 60 Asp Thr Trp 65 38764PRTBos taurusBovine VH CDRH3
(19) 387Cys Thr Thr Val His Gln Arg Thr Thr Thr Glu Arg Ser Cys Pro
Glu 1 5 10 15 Gly Tyr Asn Trp Arg Tyr Gly Cys Asp Gly Trp Val Arg
Gly Cys Ser 20 25 30 Asp Ala Cys Trp Thr Gly Asp Thr Asp Gly Ala
Arg Gly Glu Tyr Gly 35 40 45 Gly Asp Gly Ser Val Arg Thr Ser Tyr
Glu Trp Tyr Ala Asp Ala Trp 50 55 60 38862PRTBos taurusBovine VH
CDRH3 (20) 388Cys Thr Thr Val His Gln Lys Thr Gln Arg Thr Cys Pro
Asp Gly Trp 1 5 10 15 Thr Asp Ile Trp Asp Cys Cys Arg Lys Ser Thr
Cys Ser Gly Ser Asp 20 25 30 Cys Pro Thr Asn Asp Asp Cys Arg Leu
Ile Phe Pro Tyr Ala Trp Ser 35 40 45 Thr Thr Tyr Leu Tyr Thr Tyr
Glu His His Val Asp Thr Trp 50 55 60 389144DNABos taurusDH2
germline 389agttgtcctg atggttatag ttatggttat ggttgtggtt atggttatgg
ttgtagtggt 60tatgattgtt atggttatgg tggttatggt ggttatggtg gttatggtta
tagtagttat 120agttatagtt atacttacga atat 144390155DNABos
taurusMID10 390tgtactactg tgcaccagaa aacacaaaaa gttgtcctga
tggttatatg atgtgtatgt 60gttgtgcgtg ttgtgtggtg gtgttgtgtt gtggtgtatg
gtttgtggta ctgtatgtag 120tatacttata cttacgaatt cacgtcgatg cctgg
155391155DNABos taurusMID1 391tgtactactg tgcaccagaa aacaacaaac
agttgtcctg atggttatag tatggtatct 60gtgtgtactt atggttgtgt gtgatgattg
tgtgtagtta tggtcgtgtg gtatgtgtga 120gttatatata cttacgaatt
cacgtcgatg cctgg 155392157DNABos taurusMID11 392tgtactactg
tgcaccagaa aacaaaaaag ttgtcctgat ggttatagta tgatgtgtgt 60tgtggttgtg
ttgtagtgat tgattgttgt gtgtggtgtg tggtgtgtag ttgtagttgt
120attatactta tacttacgaa ttcacgtcga tgcctgg 157393160DNAArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
sequence 393tgtactactg tgcaccagaa aacacaaaaa gttgtcctga tggttatagt
atgrtgtgtg 60tgtgtttrtg gttgtggtgt rgtgrttgtg tgtgttrtgg tgtgtggttt
gtggtttggt 120tgtattatac ttatacttac gaattcacgt cgatgcctgg
160394148DNABos taurusBovine DH2 394gt agt tgt cct gat ggt tat agt
tat ggt tat ggt tgt ggt tat ggt 47 Ser Cys Pro Asp Gly Tyr Ser Tyr
Gly Tyr Gly Cys Gly Tyr Gly 1 5 10 15 tat ggt tgt agt ggt tat gat
tgt tat ggt tat ggt ggt tat ggt ggt 95Tyr Gly Cys Ser Gly Tyr Asp
Cys Tyr Gly Tyr Gly Gly Tyr Gly Gly 20 25 30 tat ggt ggt tat ggt
tat agt agt tat agt tat agt tat act tac gaa 143Tyr Gly Gly Tyr Gly
Tyr Ser Ser Tyr Ser Tyr Ser Tyr Thr Tyr Glu 35 40 45 tat ac 148Tyr
39548PRTBos taurusBovine DH2 395Ser Cys Pro Asp Gly Tyr Ser Tyr Gly
Tyr Gly Cys Gly Tyr Gly Tyr 1 5 10 15 Gly Cys Ser Gly Tyr Asp Cys
Tyr Gly Tyr Gly Gly Tyr Gly Gly Tyr 20 25 30 Gly Gly Tyr Gly Tyr
Ser Ser Tyr Ser Tyr Ser Tyr Thr Tyr Glu Tyr 35 40 45 39661PRTBos
taurusUL CDRH3 (1) 396Cys Thr Thr Val Tyr Gln Glu Thr His Lys Asn
Cys Pro Glu Gly Trp 1 5 10 15 Met Ser Arg Asp Thr Cys Arg Ile Asp
Ala Cys Ser Gly Asp Cys Cys 20 25 30 Arg Val Tyr Asp Ala Ser Thr
Gln Glu Arg Trp Arg Arg Gln Val Gln 35 40 45 Ser Tyr Ile Tyr Thr
Tyr Glu Leu His Val Asp Thr Trp 50 55 60 39761PRTBos taurusUL CDRH3
(2) 397Cys Thr Ala Val Tyr Gln Glu Thr His Lys Asn Cys Pro Glu Gly
Tyr 1 5 10 15 Met Asp Arg Gly Ser Cys Arg Ile Asp Ala Cys Ser Gly
Ala Cys Cys 20 25 30 Arg Val Tyr Asp Ala Arg Thr Gln Glu Cys Trp
Arg Arg Asp Val Gln 35 40 45 Ser Tyr Ile Tyr Thr Tyr Glu Leu His
Val Asp Thr Trp 50 55 60 39858PRTBos taurusUL CDRH3 (3) 398Cys Ile
Thr Ala His Gln Glu Thr Gln Lys Ser Cys Ser Asp Asp Tyr 1 5 10 15
Thr Tyr Tyr Gly Asp Ala Thr Cys Ala Tyr Val Cys Ser Thr Asp Glu 20
25 30 Cys Cys Cys Gly Arg Thr Trp Leu Ser Ala Gly Tyr Arg Pro Cys
Arg 35 40 45 Tyr Thr Tyr Asn Leu His Val Asp Ala Trp 50 55
39958PRTBos taurusUL CDRH3 (4) 399Cys Ile Thr Val His Gln Glu Thr
Gln Lys Ser Cys Pro Asp Asp Tyr 1 5 10 15 Thr Tyr Tyr Gly Asp Gly
Thr Cys Val Tyr Ile Cys Ser Ile Asp Lys 20 25 30 Cys Cys Cys Gly
Arg Thr Trp Leu Ser Ser Gly Cys His Pro Cys Arg 35 40 45 Tyr Thr
Tyr Asn Leu His Val Asp Ala Trp 50 55 40058PRTBos taurusUL CDRH3
(5) 400Cys Ile Thr Val His Gln Glu Thr Gln Lys Ser Cys Pro Asp Asp
Tyr 1 5 10 15 Thr Tyr Tyr Gly Asp Gly Thr Cys Ala Tyr Val Cys Ser
Ile Asp Asn 20 25 30 Cys Cys Cys Gly Arg Thr Trp Leu Ser Ser Gly
Cys Leu Pro Cys Arg 35 40 45 Tyr Thr Tyr Asn Leu His Val Asp Ala
Trp 50 55 40158PRTBos taurusUL CDRH3 (6) 401Cys Ile Thr Val His Gln
Glu Thr Gln Lys Ser Cys Pro Asp Asp Tyr 1 5 10 15 Thr Ser Tyr Gly
Asp Ala Thr Cys Ala Tyr Val Cys Ser Thr Asp Glu 20 25 30 Cys Cys
Cys Gly Arg Thr Trp Leu Ser Ala Gly Cys Arg Pro Cys Arg 35 40 45
Tyr Thr Tyr Asn Leu His Val Asp Ala Trp 50 55 40258PRTBos taurusUL
CDRH3 (7) 402Cys Ile Thr Val His Gln Glu Thr Gln Lys Ser Cys Phe
Asp Asp Tyr 1 5 10 15 Thr Tyr Tyr Gly Asp Ala Ser Cys Ala Tyr Val
Cys Ser Thr Asp Glu 20 25 30 Cys Cys Cys Gly Arg Thr Trp Leu Ser
Ala Gly Cys Arg Pro Cys Arg 35 40 45 Tyr Thr Tyr Asn Leu His Val
Asp Ala Trp 50 55 40358PRTBos taurusUL CDRH3 (8) 403Cys Ile Thr Val
His Gln Glu Thr Gln Lys Ser Cys Pro Asp Asp Tyr 1 5 10 15 Thr Tyr
Tyr Gly Asp Gly Thr Cys Ala Tyr Val Cys Ser Ile Asp Lys 20 25 30
Cys Cys Cys Gly Arg Thr Trp Leu Ser Ser Gly Cys Leu Pro Cys Arg 35
40 45 Tyr Thr Tyr Asn Leu His Val Asp Ala Trp 50 55 40458PRTBos
taurusUL CDRH3 (9) 404Cys Ile Thr Val His Gln Glu Thr Gln Lys Ser
Cys Pro Asp Asp Tyr 1 5 10 15 Thr Tyr Tyr Gly Asp Ala Thr Cys Ala
Tyr Val Cys Ser Thr Asp Glu 20 25 30 Cys Cys Cys Gly Arg Thr Trp
Leu Ser Ala Gly Cys Arg Pro Cys Arg 35 40 45 Tyr Thr Tyr Asn Leu
His Val Asp Ala Trp 50 55 40558PRTBos taurusUL CDRH3 (10) 405Cys
Ile Thr Val His Gln Glu Thr Gln Lys Ser Cys Pro Asp Asp Tyr 1 5 10
15 Thr Tyr Tyr Gly Asp Ala Ser Cys Ala Tyr Val Cys Ser Thr Asp Glu
20 25 30 Cys Cys Cys Gly Arg Thr Trp Leu Ser Ala Gly Cys Arg Pro
Cys Arg 35 40 45 Tyr Thr Tyr Asn Leu His Val Asp Ala Trp 50 55
40658PRTBos taurusUL CDRH3 (11) 406Cys Ile Thr Ala His Gln Glu Thr
Gln Lys Ser Cys Ser Asp Asp Tyr 1 5 10 15 Thr Tyr Tyr Gly Asp Ala
Thr Cys Ala Tyr Val Cys Ser Thr Asp Glu 20 25 30 Cys Cys Cys Gly
Arg Thr Trp Leu Ser Ala Gly Cys Arg Pro Cys Arg 35 40 45 Tyr Thr
Tyr Asn Leu His Val Asp Ala Trp 50 55 40758PRTBos taurusUL CDRH3
(12) 407Cys Gly Thr Val Tyr Gln His Thr Lys Glu Ile Lys Thr Cys Pro
Asp 1 5 10 15 Gly Tyr Ser Asp Cys Phe Thr Tyr Cys Pro Val Thr Cys
Pro Gly Trp 20 25 30 Asp Cys Cys Arg Arg Asn Asp Cys Gly Arg Thr
Arg Tyr Thr Val Ala 35 40 45 Tyr Ser Tyr Ala Leu His Val Asp Val
Trp 50 55 40858PRTBos taurusUL CDRH3 (13) 408Cys Gly Thr Val Tyr
Gln His Thr Lys Glu Ile Lys Thr Cys Pro Asp 1 5 10 15 Gly Tyr Ser
Asp Val Phe Thr Tyr Cys Pro Val Ser Cys Pro Gly Trp 20 25 30 Asp
Cys Cys Arg Arg Ala Asp Cys Arg Arg Thr Arg Tyr Thr Val Ala 35 40
45 Tyr Ser Tyr Ala Leu His Val Asp Val Trp 50 55 40958PRTBos
taurusUL CDRH3 (14) 409Cys Gly Thr Val Tyr Gln His Thr Lys Glu Ile
Lys Thr Cys Pro Asp 1 5 10 15 Gly Tyr Ser Asp Val Phe Thr Tyr Cys
Pro Val Thr Cys Pro Gly Trp 20 25 30 Asp Cys Cys Arg Arg Asn Asp
Cys Gly Arg Thr Arg Tyr Thr Val Ala 35 40 45 Tyr Ser Tyr Ala Leu
His Val Asp Val Trp 50 55 41058PRTBos taurusUL CDRH3 (15) 410Cys
Gly Thr Val Tyr Gln His Thr Lys Glu Ile Lys Thr Cys Pro Asp 1 5 10
15 Gly Tyr Ser Asp Val Phe Thr Tyr Cys Pro Val Ser Cys Pro Gly Trp
20 25 30 Asp Cys Cys Arg Arg Asn Asp Cys Gly Arg Thr Arg Tyr Thr
Val Ala 35 40 45 Tyr Ser Tyr Ala Leu His Val Asp Val Trp 50 55
41148PRTBos taurusDH2 germline 411Ser Cys Pro Asp Gly Tyr Ser Tyr
Gly Tyr Gly Cys Gly Tyr Gly Tyr 1 5 10 15 Gly Cys Ser Gly Tyr Asp
Cys Tyr Gly Tyr Gly Gly Tyr Gly Gly Tyr 20 25 30 Gly Gly Tyr Gly
Tyr Ser Ser Tyr Ser Tyr Ser Tyr Thr Tyr Glu Tyr 35 40 45
41265PRTBos taurusBLV1H12 412Cys Thr Ser Val His Gln Glu Thr Lys
Lys Tyr Gln Ser Cys Pro Asp 1 5 10 15 Gly Tyr Arg Glu Arg Ser Asp
Cys Ser Asn Arg Pro Ala Cys Gly Thr 20 25 30 Ser Asp Cys Cys Arg
Val Ser Val Phe Gly Asn Cys Leu Thr Thr Leu 35 40 45 Pro Val Ser
Tyr Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val 50 55 60 Trp 65
41363PRTBos taurusB08 413Cys Ala Ala Val His Gln Gln Thr Thr Asn
Arg Cys Pro Ala Gly Ser 1 5 10 15 Ser Val Arg Asn Gly Cys Cys Val
Asn Pro Val Trp His Pro Asn Ser 20 25 30 Cys Ala Arg Asn Val Val
Tyr Thr Lys Asp Gln His Gly Val Cys Cys 35 40 45 Ser Glu Arg Leu
Ile Tyr Thr Tyr Glu His His Val Asp Thr Trp 50 55 60 41463PRTBos
taurusB09 414Cys Thr Ile Val Asn Gln Leu Thr Lys Lys Thr Cys Pro
Asp Thr Tyr 1 5 10 15 Thr Asp Ala Glu Ser Cys Cys Gly Gly Ser Gly
Cys Tyr Leu Asp Ser 20 25 30 Cys Tyr Thr Ile Lys Lys Tyr Gly Cys
Gly Arg Ile Gly Arg Trp Pro 35 40 45 Thr Thr Thr Tyr Ser Tyr Thr
Tyr Asp Arg Tyr Val Asp Ala Trp 50 55 60 41567PRTBos taurusH12
415Cys Val Ile Val His Gln Lys Thr Thr Gln Gln Ser Ser Cys Pro Ala
1 5 10 15 Gly Phe Arg Asp Cys Val Ala Cys Thr Pro Gly Pro Glu Ser
Cys Cys 20 25 30 Arg Ser Gly Cys Asp Gly Ala Arg Arg Arg Val Gly
Leu Arg Tyr Phe 35 40 45 Phe Asp Ser Thr Ser Pro Ile Thr Thr Tyr
Thr Tyr Glu His His Ile 50 55 60 Asp Ala Trp 65 41615PRTBos
taurusJH1 germline 416Tyr Val Asp Ala Trp Gly Gln Gly Leu Leu Val
Thr Val Ser Ser 1 5 10 15 41771PRTBos taurusUL1 417Cys Ser Thr Val
His Gln Lys Thr Arg Thr Thr Gln Gly Glu Tyr Leu 1 5 10 15 Ser Leu
Met Val Thr Leu Leu Lys Asp Asp Cys Pro Arg Cys Arg Gly 20 25 30
Gly Cys Asp Gly Tyr Asp Cys Cys Trp Gly Asp Ala Cys Arg Ser Ser 35
40 45 Gly Leu Cys Trp Gly His Asn Pro Leu Val Thr Glu Thr Tyr Thr
Tyr 50 55 60 Glu Phe Tyr Ile Asp Ala Trp 65 70 41871PRTBos
taurusUL2 418Cys Ser Thr Val His Gln Lys Thr Arg Thr Thr Gln Gly
Asn Asn Leu 1 5 10 15 Ser Leu Met Val Thr Leu Leu Lys Asp Asp Cys
Pro Arg Cys Arg Gly 20 25 30 Gly Cys Asp Gly Tyr Asp Cys Cys Trp
Gly Asp Ala Cys Arg Ser Ser 35 40 45 Gly Leu Cys Trp Gly His Asn
Pro Leu Val Thr Glu Thr Tyr Thr Tyr 50 55 60 Glu Phe Tyr Ile Asp
Ala Trp 65
70 41970PRTBos taurusUL3 419Cys Ser Thr Val His Gln Lys Thr Arg Thr
Thr Gln Gly Asn Thr Cys 1 5 10 15 Pro Asp Gly Tyr Thr Leu Lys Asp
Asp Cys Pro Arg Cys Arg Gly Gly 20 25 30 Cys Asp Gly Tyr Asp Cys
Cys Trp Gly Asp Ala Cys Arg Ser Ser Gly 35 40 45 Leu Cys Trp Gly
His Asn Pro Leu Val Thr Glu Thr Tyr Thr Tyr Glu 50 55 60 Phe Tyr
Ile Asp Ala Trp 65 70 42070PRTBos taurusUL4 420Cys Ser Thr Val His
Gln Lys Thr Arg Thr Thr Gln Gly Asn Thr Cys 1 5 10 15 Pro Asp Gly
Tyr Thr Phe Lys Asp Asp Cys Pro Arg Cys Arg Gly Gly 20 25 30 Cys
Asp Gly Tyr Asp Cys Cys Trp Gly Asp Ala Cys Arg Ser Ser Gly 35 40
45 Leu Cys Trp Gly His Asn Pro Leu Val Thr Glu Thr Tyr Thr Tyr Glu
50 55 60 Phe Tyr Ile Asp Ala Trp 65 70 42170PRTBos taurusUL5 421Cys
Thr Thr Val His Gln Lys Thr Arg Thr Thr Gln Gly Asn Thr Cys 1 5 10
15 Pro Asp Gly Tyr Thr Leu Lys Asp Asp Cys Pro Arg Cys Arg Gly Gly
20 25 30 Cys Asp Gly Tyr Asp Cys Cys Trp Gly Asp Ala Cys Arg Ser
Ser Gly 35 40 45 Leu Cys Trp Gly His Asn Pro Leu Val Thr Glu Thr
Tyr Thr Tyr Glu 50 55 60 Phe Tyr Ile Asp Ala Trp 65 70 42270PRTBos
taurusUL6 422Cys Ser Thr Val His Gln Lys Thr Arg Thr Thr Gln Gly
Asn Thr Cys 1 5 10 15 Pro Asp Gly Tyr Thr Leu Lys Asn Asp Cys Pro
Arg Cys Arg Gly Gly 20 25 30 Cys Asp Gly Tyr Asp Cys Cys Trp Gly
Asp Ala Cys Arg Ser Ser Gly 35 40 45 Leu Cys Trp Gly His Asn Pro
Leu Val Thr Glu Thr Tyr Thr Tyr Glu 50 55 60 Phe Tyr Ile Asp Ala
Trp 65 70 42370PRTBos taurusUL7 423Cys Thr Thr Val Tyr Gln Lys Thr
Arg Thr Thr Gln Gly Asn Thr Cys 1 5 10 15 Pro Asp Gly Tyr Thr Leu
Lys Asp Asp Cys Pro Arg Cys Arg Gly Gly 20 25 30 Cys Asp Gly Tyr
Asp Cys Cys Trp Gly Asp Ala Cys Arg Ser Ser Gly 35 40 45 Leu Cys
Trp Gly His Asn Pro Leu Val Thr Glu Thr Tyr Thr Tyr Glu 50 55 60
Phe Tyr Ile Asp Ala Trp 65 70 42470PRTBos taurusUL8 424Cys Ser Thr
Val His Gln Lys Pro Gly Gln His Lys Gly Ile Leu Val 1 5 10 15 Leu
Met Val Thr Leu Leu Lys Asp Asp Cys Pro Arg Cys Arg Gly Gly 20 25
30 Cys Asp Gly Tyr Asp Cys Cys Trp Gly Asp Ala Cys Arg Ser Ser Gly
35 40 45 Leu Cys Trp Gly His Asn Pro Leu Val Thr Glu Thr Tyr Thr
Tyr Glu 50 55 60 Phe Tyr Ile Asp Ala Trp 65 70 42570PRTBos
taurusUL9 425Cys Ser Thr Val His Gln Lys Thr Arg Thr Thr Gln Gly
Ile Leu Val 1 5 10 15 Leu Met Val Thr Leu Leu Lys Asp Asp Cys Pro
Arg Cys Arg Gly Gly 20 25 30 Cys Asp Gly Tyr Asp Cys Cys Trp Gly
Asp Ala Cys Arg Ser Ser Gly 35 40 45 Leu Cys Trp Gly His Asn Pro
Leu Val Thr Glu Thr Tyr Thr Tyr Glu 50 55 60 Phe Tyr Ile Asp Ala
Trp 65 70 42663PRTBos taurusUL10 426Cys Ser Pro Val His Gln Glu Ile
Arg Lys Cys Cys Pro Ala Gly Cys 1 5 10 15 Gln Cys Gly Arg Ser Cys
Gly Ala Cys Cys Gly Cys Ala Gly Asp Glu 20 25 30 Phe Cys Gly Ile
Asn Val Tyr Gly Tyr Val Thr Cys Gly Gly Tyr Arg 35 40 45 Thr Cys
Ser Cys Ile Asp Thr Tyr Asp Phe Tyr Val Asp Ala Trp 50 55 60
42763PRTBos taurusUL11 427Cys Ser Pro Val His Gln Gln Thr Arg Lys
Cys Cys Pro Ala Gly Cys 1 5 10 15 Gln Cys Gly Arg Ser Cys Gly Ala
Cys Cys Gly Cys Ala Gly Asp Glu 20 25 30 Phe Cys Gly Ile Asn Val
Tyr Gly Tyr Ile Thr Cys Gly Gly Tyr Arg 35 40 45 Thr Cys Ser Cys
Ile Asp Thr Tyr Asp Phe Tyr Val Glu Ala Trp 50 55 60 42868PRTBos
taurusUL12 428Cys Ala Thr Val Tyr Gln Lys Thr Asn Gln Ser Lys Asn
Cys Pro Glu 1 5 10 15 Gly Ser Ala Trp Cys Arg Ser Cys Asp Gly Gly
Ala Gly Cys Ala Asp 20 25 30 Tyr Glu Cys Cys Arg Cys Gly Trp Ser
Gly Cys Ser Trp Arg Asn Gly 35 40 45 Ala Cys Glu Cys Ser Ser Leu
Ser Ser Ser Tyr Thr Tyr Glu Leu His 50 55 60 Val Asp Ala Trp 65
42965PRTBos taurusUL13 429Cys Ser Thr Val His Gln Thr Thr His Gln
Ile His Thr Cys Pro Asn 1 5 10 15 Gly Trp Thr Gly Gly Cys Val Cys
Ser Ser Arg Phe Asn Cys Arg Gly 20 25 30 Asn Asn Cys Cys Cys Arg
Thr Ala Tyr Cys Ser Val Asp Arg Tyr Val 35 40 45 Cys Ala Cys Pro
Thr Val Thr Tyr Thr Tyr Glu Phe Asn Val Asp Ser 50 55 60 Trp 65
43065PRTBos taurusUL14 430Cys Thr Ala Val Tyr Gln Lys Thr Thr Ser
Ile Arg Ser Cys Pro Gly 1 5 10 15 Gly Thr Thr Leu Arg Asn Gly Cys
Arg Ser Ala Cys Gly Cys Asn Asp 20 25 30 Cys Asp Cys Cys Cys Gly
Ser Ser Trp Asp Ile Cys Tyr Met Ser Lys 35 40 45 Cys Thr Ser Ala
Pro Glu Thr Tyr Thr Tyr Glu Leu His Ile Asp Ala 50 55 60 Trp 65
43165PRTBos taurusUL15 431Cys Thr Asn Val His Gln Lys Thr Lys Lys
Thr Cys Pro Asp Asp Tyr 1 5 10 15 Thr Cys Gly Val Ser Cys Ser Cys
Ser Ser Ser Gly Cys Ala Asp Tyr 20 25 30 Gly Cys Cys Ser Tyr Ile
Thr Tyr Gly Val Pro Gly Asp Cys Gly Gly 35 40 45 Cys Cys Ser Tyr
Lys His Arg Tyr Thr Tyr Glu Trp Asn Val Asp Ala 50 55 60 Trp 65
43263PRTBos taurusUL16 432Cys Thr Thr Val His Gln Lys Thr Lys Lys
Leu Cys Pro Asn Gly Arg 1 5 10 15 Thr Cys Gly Cys Gly Cys Asp Cys
Gly Ser Gly Cys Cys Thr Ser Tyr 20 25 30 Cys Asp Ser Phe Gly Cys
Trp Gly Gly Arg Asp Thr Phe Gly Ser Ser 35 40 45 Cys Thr Ser Ala
Thr Tyr Thr Tyr Glu Trp Gly Val Asp Ala Trp 50 55 60 43364PRTBos
taurusUL17 433Cys Ala Thr Val His Gln His Thr Asn Lys Lys Arg Cys
Pro Asp Gly 1 5 10 15 Tyr Glu Phe Ser Ala Gly Cys Cys Cys Gly Glu
Gly Cys Ser Gly Ser 20 25 30 Asp Cys Cys Cys Asn Ser Arg Leu Arg
Cys Ser Trp Tyr Glu Ile Tyr 35 40 45 Cys Ser Val Ser Pro Ser Asp
Thr Tyr Glu Phe His Val Asp Ala Trp 50 55 60 43464PRTBos taurusUL18
434Cys Thr Thr Val His Gln His Thr Asn Lys Lys Arg Cys Pro Asp Gly
1 5 10 15 Tyr Arg Phe Ser Ala Ala Cys Cys Cys Gly Glu Gly Cys Ser
Gly Asn 20 25 30 Glu Cys Cys Cys Asn Thr Arg Leu Arg Cys Ser Trp
Tyr Glu Ile Tyr 35 40 45 Cys Ser Val Ser Pro Ser Asp Thr Tyr Glu
Phe His Val Asp Ala Trp 50 55 60 43564PRTBos taurusUL19 435Cys Thr
Thr Val His Gln His Thr Asn Gln Asn Arg Cys Pro Thr Gly 1 5 10 15
Tyr Lys His Ser Ala Gly Cys Cys Cys Gly Val Gly Cys Ser Gly Asn 20
25 30 Asp Cys Cys Cys Asn Ser Arg Leu Arg Cys Ser Trp Tyr Glu Thr
Tyr 35 40 45 Cys Ser Leu Ser Pro Thr Asp Met Tyr Glu Phe Tyr Val
Asp Ala Trp 50 55 60 43664PRTBos taurusUL20 436Cys Ser Thr Val His
Gln His Thr Asn Gln Asn Arg Cys Pro Ala Gly 1 5 10 15 Tyr Lys His
Ser Ala Gly Cys Cys Cys Gly Val Gly Cys Ser Gly Asn 20 25 30 Asp
Cys Cys Cys Asn Ser Arg Leu Arg Cys Ser Trp Tyr Glu Thr Tyr 35 40
45 Cys Ser Leu Ser Pro Thr Asp Met Tyr Glu Phe Tyr Val Asp Ala Trp
50 55 60 43763PRTBos taurusUL21 437Cys Thr Thr Val His Gln Lys Thr
Asn Glu Arg Cys Cys Arg Val Val 1 5 10 15 Ser Asp Asp Gly Glu Cys
Gly Asp Gly Asn Ser Cys His Arg Trp Leu 20 25 30 Cys Ser Asp Tyr
Cys Tyr Ser Gly Asp Cys Cys Ala Cys Gly Cys Arg 35 40 45 Ala Tyr
His Tyr Thr Tyr Thr Tyr Glu Trp Asn Ile Asp Ala Trp 50 55 60
43863PRTBos taurusUL22 438Cys Thr Thr Val His Gln Lys Thr Asn Glu
Arg Cys Cys Arg Val Val 1 5 10 15 Ser Asp Asp Gly Glu Cys Gly Asp
Gly Asn Ser Cys His Arg Trp Leu 20 25 30 Cys Ser Asp Tyr Cys Tyr
Ser Gly Asp Cys Cys Ala Cys Gly Cys Arg 35 40 45 Ala Tyr His Tyr
Thr Tyr Thr Tyr Asp Phe Arg Ile Asp Val Trp 50 55 60 43964PRTBos
taurusUL23 439Cys Thr Thr Val His Gln Lys Thr Asn Arg Glu Arg Cys
Cys Pro Asp 1 5 10 15 Gly Tyr Tyr Tyr Cys Cys Arg Ser Val Ser Asp
Cys Cys Cys Ser Thr 20 25 30 Arg Ala Cys Val Gly Asp Ser Cys Gly
Trp Thr Asp Phe Gly Ser Thr 35 40 45 His Asn Val Asp Cys Ser Phe
Thr Tyr Glu Phe His Val Asp Ala Trp 50 55 60 44063PRTBos taurusUL24
440Cys Thr Thr Val His Gln Gln Thr Arg Lys Ser Cys Pro Asp Gly Tyr
1 5 10 15 Thr Tyr Cys His Asp Cys Gly Tyr Gly Cys Cys Cys Gly Ala
Ser Phe 20 25 30 Cys Arg Asp Tyr Gly Gly Cys Gly Ser Leu Cys Gly
Arg Tyr Cys Thr 35 40 45 Ser Phe Asp Tyr Ile Tyr Thr Tyr Glu Asn
Tyr Val Glu Thr Trp 50 55 60 44164PRTBos taurusUL25 441Cys Thr Thr
Val His Gln Glu Thr Lys Lys Asn Cys Pro Asp Asn Cys 1 5 10 15 Tyr
Tyr Glu Asn Ser Cys Gly Asp Tyr Gly Ser Gly Cys Asn Gly Gly 20 25
30 Asp Cys Cys Arg Cys Gly Thr Trp Leu Thr Cys Ser Val Ser Gly Cys
35 40 45 Thr Cys Ile Arg Ala Thr Asn Thr Tyr Gln Trp Tyr Val Asn
Ala Trp 50 55 60 44264PRTBos taurusUL26 442Cys Thr Thr Val His Gln
Ser Thr Asn Lys Lys Ser Cys Pro Asp Arg 1 5 10 15 Val Cys Trp Ala
Val Gly Cys Cys Phe Gly Glu Asp Cys Thr Ser Ser 20 25 30 Asp Cys
Thr Cys Tyr Ala Ser Pro Gly Asn Pro Tyr Arg His Asp Cys 35 40 45
Gly Asn Cys Asp Cys Arg Ser Ser Tyr Glu His His Val Asp Ala Trp 50
55 60 44363PRTBos taurusUL27 443Cys Thr Thr Val Arg Gln Glu Thr Leu
Ile Arg Cys Arg Asp Gly Pro 1 5 10 15 Ser Cys Ala Ala Cys Cys Arg
Ser Gly Arg Arg Cys Ser Gly Tyr Gly 20 25 30 Cys Cys Thr Asp Gly
Cys Cys Ser Asp Asn Asp Tyr Ala Asp Cys Ile 35 40 45 Arg Gly Glu
Phe Val Asp Val Tyr Glu Trp Asn Val Asp Ala Trp 50 55 60
44463PRTBos taurusUL28 444Cys Ser Thr Val Tyr Gln Lys Thr Arg Thr
Thr Cys Pro Asp Gly Tyr 1 5 10 15 Thr Cys Gly Asp Gly Ala Arg Cys
Glu Lys Ala Cys Arg Gly Cys Asp 20 25 30 Cys Cys Arg Thr Thr Val
Cys Asp Thr Val Trp Ser Ser Tyr Cys Ser 35 40 45 Cys Tyr Ser Phe
Thr Asp Ser Tyr Glu Phe Tyr Val Asp Ala Trp 50 55 60 44566PRTBos
taurusUL29 445Cys Ala Thr Val Tyr Gln Lys Thr Asn Arg Glu Met Ser
Cys Pro Asp 1 5 10 15 Gly Cys Arg Ile His Asn Ala Arg Leu Cys Leu
Ser Gly Cys Ser Gly 20 25 30 Ser Asp Cys Cys Ser Cys Gly Asp Cys
Val Ser Asp Ala Arg Cys Tyr 35 40 45 Asn Cys Arg Ser Ala Val Phe
Thr Tyr Thr Tyr Glu Phe His Val Asp 50 55 60 Ala Trp 65 44663PRTBos
taurusUL30 446Cys Thr Ile Val His Gln Glu Thr Lys Arg Ser Cys Pro
Asp Gly Tyr 1 5 10 15 Asn Thr Gly Thr Arg Cys Phe Gly Ser Cys Gly
Cys Ile Gly Ser Asn 20 25 30 Cys Cys Arg Ser Thr Thr Ser Cys Cys
Cys Ala Gly Ile Tyr Ser Gln 35 40 45 Cys Thr Thr Ser Thr Leu Thr
Tyr Glu Trp His Ala Asp Val Trp 50 55 60 44763PRTBos taurusUL31
447Cys Ala Ile Val Tyr Gln Arg Thr Arg Gln Arg Cys Pro Asp Gly Tyr
1 5 10 15 Asn Thr Gly Thr Arg Cys Phe Gly Thr Cys Gly Cys Asn Gly
Ser Asn 20 25 30 Cys Cys Arg Phe Thr Thr Ser Cys Cys Cys Ala Gly
Val Tyr Ser Gln 35 40 45 Cys Thr Thr Ser Thr Leu Thr Tyr Glu Trp
His Ala Asp Val Trp 50 55 60 44863PRTBos taurusUL32 448Cys Thr Thr
Val His Gln Lys Thr Glu Thr Arg Cys Pro Asp Gly Tyr 1 5 10 15 Ser
Ser Thr Asn Gly Cys Asp Ala Arg Cys Gly Cys Ser Asp Cys Asp 20 25
30 Cys Cys Asn Val Gly Arg Trp Gly Cys Pro Leu Ile Cys Ser Arg Asn
35 40 45 Cys Arg Ser Phe Thr Tyr Thr Tyr Glu Trp Tyr Ala Asp Ala
Trp 50 55 60 44963PRTBos taurusUL33 449Cys Thr Thr Val His Gln Lys
Thr Asn Lys Lys Glu Ser Cys Pro Asp 1 5 10 15 Gly Tyr Thr Met Asn
Glu Cys Cys Gly Cys Gly Tyr Gly Cys Cys Arg 20 25 30 Gly Gly Cys
Val Cys Ser Ala Tyr Cys Ser Arg Pro Asn Cys Trp Arg 35 40 45 Glu
Leu Thr Tyr Thr Tyr Thr Tyr Glu Phe Tyr Val Asp Thr Trp 50 55 60
45063PRTBos taurusUL34 450Cys Thr Thr Val Tyr Gln Lys Ser Arg Lys
Glu Ser Ser Cys Pro Asn 1 5 10 15 Gly Trp Ile Tyr Gly Lys Asp Cys
Cys Ser Trp Ser Tyr Cys Thr Asp 20 25 30 Cys Asp Cys Cys Leu Cys
Gly Asp Leu His Cys Tyr Asp Gly Cys Ser 35 40 45 Ser Phe Gly Val
Thr Trp Thr Tyr Glu Phe His Val Asp Ala Trp 50 55 60 45162PRTBos
taurusUL35 451Cys Thr Thr Val Phe Gln Glu Thr Arg Lys Ser Cys Pro
Thr Gly Phe 1 5 10 15 Tyr Val Asp Gly Ser Thr Cys Gly Cys Ala Thr
Tyr Cys Arg Thr Cys 20 25 30 Asp Cys Cys Gly Gly Tyr Arg Cys Ser
Gly Gly Gly Ser Cys Ala Cys 35 40 45 Ser Ser Tyr Thr Tyr Asn Tyr
Asp Phe His Val Asp Ala Trp 50 55 60 45261PRTBos taurusUL36 452Cys
Ala Ala Val Phe Gln Glu Thr Arg Thr Asn Cys Pro Ser Gly Tyr 1 5 10
15 Gly Asn Ala Phe Ser Cys Gly Cys Pro Ile Ala Cys Arg Asp Cys Asp
20 25 30 Cys
Cys Gly Gly Tyr Trp Cys Ser Gly Gly Ala Asp Cys His Cys Val 35 40
45 Ser Tyr Asn Tyr Thr Tyr Ser Trp His Val Asp Ala Trp 50 55 60
45362PRTBos taurusUL37 453Cys Ala Thr Val Tyr Gln Lys Thr Glu Lys
His Cys Pro Leu Phe His 1 5 10 15 Ser Ile Cys Cys His Cys Gly Glu
Gly Val Gly Cys Ser Gly Gly Asp 20 25 30 Cys Cys Gly Cys Glu Arg
Arg Ser Gly Cys Val Val Cys Thr Met Arg 35 40 45 Asn Ser Tyr Thr
Tyr Asn Tyr Gln Phe His Val Asp Ala Trp 50 55 60 45463PRTBos
taurusUL38 454Cys Gly Thr Val His Gln Lys Thr Lys Glu Leu Cys Pro
Asp Asp Ser 1 5 10 15 Thr Tyr Cys Cys Gly Cys Val Ser Gly Cys Ala
Cys Cys Thr Tyr Gly 20 25 30 Cys Asp Gly Val Gly Cys Cys Arg Val
Ser Leu Trp Thr Thr Tyr Ile 35 40 45 Lys Asp Ile Val Gly Val Ser
Tyr Glu Trp His Val Asp Ala Trp 50 55 60 45561PRTBos taurusUL39
455Cys Ala Ser Val His Gln His Thr Glu Pro Thr Cys Pro Ala Gly Tyr
1 5 10 15 Thr Tyr Cys Cys Gly Cys Leu Tyr Lys Cys Asn Cys Gly Asp
Cys Gly 20 25 30 Cys Tyr Asn Val Gly Cys Gly Ser Gly Trp Leu Gly
Lys Ala Cys Gly 35 40 45 Asp Tyr Arg Glu Thr Tyr Glu Trp Tyr Val
Asp Ala Trp 50 55 60 45661PRTBos taurusUL40 456Cys Ala Ser Val His
Gln His Thr Glu Pro Thr Cys Pro Ala Gly Tyr 1 5 10 15 Thr Tyr Cys
Cys Gly Cys Leu Tyr Lys Cys Asn Cys Gly Asp Cys Gly 20 25 30 Cys
Tyr Asn Ala Gly Cys Gly Ser Gly Trp Leu Gly Lys Ala Cys Gly 35 40
45 Asp Tyr Arg Glu Thr Tyr Glu Trp Tyr Val Asp Ala Trp 50 55 60
45761PRTBos taurusUL41 457Cys Thr Thr Val Phe Gln Glu Thr Arg Lys
Ser Cys Pro Ser Gly Phe 1 5 10 15 Arg Asp Arg Asp Ala Cys Gly Cys
Ala Val Thr Cys Arg Asn Cys Asp 20 25 30 Cys Cys Gly Gly Gly Pro
Cys Asn Gly Gly Gly Ser Cys Arg Cys Asn 35 40 45 Asn Tyr Ile Tyr
Lys Tyr Ser Phe His Val Asp Ala Trp 50 55 60 45861PRTBos taurusUL42
458Cys Thr Ala Val Phe Gln Glu Thr Arg Lys Asp Cys Pro Ser Gly Tyr
1 5 10 15 Gly Ser Ala Phe Thr Cys Gly Cys Leu Ala Ala Cys His Gly
Cys Asp 20 25 30 Cys Cys Gly Gly Gly Trp Cys Ser Gly Gly Gly Asp
Cys Arg Cys Arg 35 40 45 Ser Tyr Ser Thr Ala Tyr Ser Phe His Ile
Asp Ala Trp 50 55 60 45961PRTBos taurusUL43 459Cys Ala Thr Val Phe
Gln Glu Thr Arg Lys Ser Cys Pro Ser Gly Tyr 1 5 10 15 Ala Asp Arg
Phe Thr Cys Asp Cys Val Tyr Tyr Cys Gln Thr Cys Asp 20 25 30 Cys
Cys Gly Gly Asn Arg Cys Ser Gly Gly Gly Pro Cys Arg Cys Ser 35 40
45 Ser Tyr Ser Ile Asn Tyr Ser Phe His Val Asp Thr Trp 50 55 60
46058PRTBos taurusUL44 460Cys Ala Ala Ala His Gln Glu Thr Lys Lys
Ser Cys Pro Asp Gly Thr 1 5 10 15 Cys Arg Gln Cys Cys Gly Gly Val
Cys Arg Cys His Ala Ser Gly Cys 20 25 30 Cys Tyr Trp Cys Thr Thr
Gly Cys Val Gly Arg Ala Leu Ser Glu Ser 35 40 45 His Ser Tyr Glu
Phe His Val Asp Thr Trp 50 55 46170PRTBos taurusUL45 461Cys Ser Thr
Val His Gln Lys Thr Arg Thr Thr Gln Gly Asn Thr Cys 1 5 10 15 Pro
Asp Gly Tyr Thr Leu Lys Asp Asp Cys Pro Arg Cys Arg Gly Gly 20 25
30 Cys Asp Gly Tyr Asp Cys Cys Trp Gly Asp Ala Cys Arg Ser Ser Gly
35 40 45 Leu Cys Trp Gly His Asn Pro Leu Val Thr Glu Thr Tyr Thr
Tyr Glu 50 55 60 Phe Tyr Ile Asp Ala Trp 65 70 46268PRTBos
taurusUL46 462Cys Val Val Val Tyr Gln Lys Thr Asn Ser Gln Lys Ser
Cys Pro Arg 1 5 10 15 Gly Tyr Thr Glu Arg Glu Thr Cys Asn Arg Arg
Tyr Gly Trp Gly Cys 20 25 30 Gly Arg Tyr Asp Cys Cys Asp Cys Asp
Arg Trp Val Ser Gly Asn Cys 35 40 45 Ala Asn Ile Cys Thr Asp Tyr
Thr Asp Thr His Thr Tyr Glu Phe His 50 55 60 Ala Asp Ala Trp 65
46364PRTBos taurusUL47 463Cys Gly Thr Val Phe Gln Gln Thr His Lys
Val Arg Asp Cys Pro Asp 1 5 10 15 Gly Phe Thr Ala Ala Pro Arg Cys
Gly Gly Glu Cys Cys Cys Ser Asn 20 25 30 Val Asn Ser Arg Ser Gly
Gly Trp Cys Arg Tyr Cys Gly Arg Asp Cys 35 40 45 Thr Ala Pro Thr
Glu Thr Ser Thr Tyr Glu Phe His Val Asp Ala Trp 50 55 60
46463PRTBos taurusUL48 464Cys Thr Ala Val Tyr Gln Arg Thr Gly Gln
Lys Cys Pro Glu Gly Cys 1 5 10 15 Glu Ser Arg Asn Thr Cys Leu Tyr
Ser Arg Asn Cys Gly Asp Tyr Thr 20 25 30 Cys Cys Gly Gly Ser Arg
Ala Ser Gly Ser Gly Ala Cys Gly Trp Asn 35 40 45 Ser Val Asp Cys
Lys Asn Lys Tyr Glu His His Val Asp Ala Trp 50 55 60 46563PRTBos
taurusUL49 465Cys Thr Thr Val Tyr Gln Lys Thr Lys Gln Asn Cys Pro
Asp Gly Tyr 1 5 10 15 Asp Phe Arg Asp Thr Cys Gly Ser Gln Ser Tyr
Cys Ser Gly Tyr Asp 20 25 30 Cys Cys Arg Cys Ser Arg Phe Gly Gly
Cys Ser Ile Gly Thr Cys Ile 35 40 45 Ser Tyr Ser Asp Ala Tyr Thr
Tyr Glu Trp Tyr Val Asp Ala Trp 50 55 60 46663PRTBos taurusUL50
466Cys Thr Thr Val His Gln Gln Thr His Glu Lys Arg Ser Cys Pro Glu
1 5 10 15 Ser Tyr Ser Tyr Ser Cys Ser Cys Ala Ser Gly Val Val Gly
Cys Gly 20 25 30 Pro Asp Asp Cys Cys Cys Thr Tyr Arg Ile Ser Ile
Arg Gly Tyr Thr 35 40 45 Cys Ser Ser Leu Ser Asn Ser Tyr Glu Trp
Tyr Val Asp Ala Trp 50 55 60 46761PRTBos taurusUL51 467Cys Thr Ala
Val His Gln Gln Thr Lys Arg Lys Ser Gly Cys Pro Asp 1 5 10 15 Gly
Tyr Ser Asp Glu Ser Cys Ser Tyr Cys Gly Ser Ser Trp Cys Cys 20 25
30 Pro Val Tyr Trp Cys Gly Ser Pro Cys Ser Tyr Arg Cys Leu Arg His
35 40 45 Thr Asp Thr Tyr Ser Tyr Glu His His Val Asp Ala Trp 50 55
60 46860PRTBos taurusUL52 468Cys Ala Thr Val Tyr Gln Glu Thr Lys
Arg Thr Cys Ala Gly Gly His 1 5 10 15 Ser Val Glu Cys Asp Ser Pro
Tyr Asp Cys Asn Cys Arg Gly Gly Asp 20 25 30 Cys Cys Arg Ser Pro
Ile Phe Asn Asp Cys Trp Ala Ala Ser Cys Ser 35 40 45 Ala Thr Lys
Thr Tyr Glu Trp His Val Glu Ser Trp 50 55 60 46958PRTBos taurusUL53
469Cys Ile Thr Val His Gln Glu Thr Gln Lys Ser Cys Pro Asp Asp Tyr
1 5 10 15 Thr Tyr Tyr Gly Asp Gly Thr Cys Ala Tyr Val Cys Ser Ile
Asp Lys 20 25 30 Cys Cys Cys Gly Arg Thr Trp Leu Ser Ser Gly Cys
Leu Pro Cys Arg 35 40 45 Tyr Thr Tyr Asn Leu His Val Asp Ala Trp 50
55 47058PRTBos taurusUL54 470Cys Ile Thr Val His Gln Glu Thr Gln
Lys Ser Cys Pro Asp Asp Tyr 1 5 10 15 Thr Ser Tyr Gly Asp Ala Thr
Cys Ala Tyr Val Cys Ser Thr Asp Glu 20 25 30 Cys Cys Cys Gly Arg
Thr Trp Leu Ser Ala Gly Cys Arg Pro Cys Arg 35 40 45 Tyr Thr Tyr
Asn Leu His Val Asp Ala Trp 50 55 47158PRTBos taurusUL55 471Cys Ile
Thr Val His Gln Glu Thr Gln Lys Ser Cys Phe Asp Asp Tyr 1 5 10 15
Thr Tyr Tyr Gly Asp Ala Ser Cys Ala Tyr Val Cys Ser Thr Asp Glu 20
25 30 Cys Cys Cys Gly Arg Thr Trp Leu Ser Ala Gly Cys Arg Pro Cys
Arg 35 40 45 Tyr Thr Tyr Asn Leu His Val Asp Ala Trp 50 55
47258PRTBos taurusUL56 472Cys Ile Thr Ala His Gln Glu Thr Gln Lys
Ser Cys Ser Asp Asp Tyr 1 5 10 15 Thr Tyr Tyr Gly Asp Ala Thr Cys
Ala Tyr Val Cys Ser Thr Asp Glu 20 25 30 Cys Cys Cys Gly Arg Thr
Trp Leu Ser Ala Gly Cys Arg Pro Cys Arg 35 40 45 Tyr Thr Tyr Asn
Leu His Val Asp Ala Trp 50 55 47358PRTBos taurusUL57 473Cys Ile Thr
Val His Gln Glu Thr Gln Lys Ser Cys Pro Asp Asp Tyr 1 5 10 15 Thr
Tyr Tyr Gly Asp Gly Thr Cys Ala Tyr Val Cys Ser Ile Asp Asn 20 25
30 Cys Cys Cys Gly Arg Thr Trp Leu Ser Ser Gly Cys Leu Pro Cys Arg
35 40 45 Tyr Thr Tyr Asn Leu His Val Asp Ala Trp 50 55 47461PRTBos
taurusUL58 474Cys Val Thr Val His Gln Gln Thr His Ala Thr Arg Arg
Cys Pro Asp 1 5 10 15 Gly Tyr Gly Asp Ser Tyr Ala Cys Lys Ser Asn
Tyr Gly Cys Ser Ala 20 25 30 Glu Gly Cys Cys Arg Trp Gly Pro Gly
Ser Gly Ala Cys Thr Gly Ala 35 40 45 Ile Tyr Thr Ser Pro Tyr Glu
Trp Tyr Val Asp Ala Trp 50 55 60 47560PRTBos taurusUL59 475Cys Ala
Ala Val His Gln Arg Thr Glu Gly Gln Gln Ser Cys Pro Asp 1 5 10 15
Gly Tyr Leu Glu Thr Arg Val Cys Pro Tyr Arg Met Tyr Arg Cys Ile 20
25 30 Gly Trp Asp Cys Cys Arg Cys Ser Asp Gly Ser Arg Asp Asn Tyr
Ile 35 40 45 Met Thr Tyr Ser Tyr Glu Phe His Val Asp Val Trp 50 55
60 47659PRTBos taurusUL60 476Cys Thr Thr Val Tyr Gln Glu Thr Lys
Thr Lys Ser Gly Cys Pro Asp 1 5 10 15 Gly Tyr Ser Cys Cys Tyr Asn
Gly Arg Ser Arg Ser Cys Arg Pro Asn 20 25 30 Asp Cys Ser Thr Tyr
Gly Glu Val Arg Ser Leu Ser Arg Ser Cys Tyr 35 40 45 Thr Tyr Asn
Tyr Glu Phe Tyr Val Asp Ala Trp 50 55 47758PRTBos taurusUL61 477Cys
Gly Thr Val Tyr Gln His Thr Lys Glu Ile Lys Thr Cys Pro Asp 1 5 10
15 Gly Tyr Ser Asp Val Phe Thr Tyr Cys Pro Val Thr Cys Pro Gly Trp
20 25 30 Asp Cys Cys Arg Arg Asn Asp Cys Gly Arg Thr Arg Tyr Thr
Val Ala 35 40 45 Tyr Ser Tyr Ala Leu His Val Asp Val Trp 50 55
47857PRTBos taurusUL62 478Cys Thr Thr Val Leu Gln Glu Thr His Gln
Gln Arg Gly Cys Pro Ala 1 5 10 15 Gly Tyr Gln Val Val Asp Gly Cys
Pro Tyr Gly Asp Cys Cys Arg Thr 20 25 30 Ser Tyr Val Cys Gly Pro
Leu Thr Cys Thr Ser Asn Thr Ala Thr Arg 35 40 45 Asn Tyr Gln Trp
Tyr Val Asp Ala Trp 50 55 47956PRTBos taurusUL63 479Cys Ser Thr Val
Tyr Gln Lys Thr Glu Lys Lys Cys Pro Asp Gly Tyr 1 5 10 15 Thr Asp
Arg Arg Asp Glu Cys Pro Asn Thr Cys Lys Asn Phe Asp Cys 20 25 30
Glu Asn Glu Gly Gly Leu Arg Cys Leu Cys Ser Ala Tyr Ile Ser Ala 35
40 45 Tyr Glu Phe His Val Asp Ala Trp 50 55 48055PRTBos taurusUL64
480Cys Thr Thr Thr His Gln Arg Thr Gln Lys Ser Cys Pro Asp Tyr Ala
1 5 10 15 Ser Tyr Asp Cys Gly Ser Pro Asp Asp Glu Glu Cys Ser Ser
Cys Arg 20 25 30 Ser Cys Thr Arg Trp Cys Ala Pro Thr Ala Pro Tyr
Ile Tyr Thr Tyr 35 40 45 Gln Phe Tyr Ile Asp Ala Trp 50 55
48154PRTBos taurusUL65 481Cys Thr Thr Val His Gln Gln Thr Asn Lys
Arg Cys Pro Thr Gly Tyr 1 5 10 15 Asn Ser Gly Thr Leu Cys Asn Met
Ile Gly Cys Ser Gly Asp Glu Cys 20 25 30 Cys Asn Tyr Gly Arg Val
Glu Cys Thr Ser Tyr Val Trp Thr His Asn 35 40 45 Phe Tyr Val Asp
Ala Trp 50 48253PRTBos taurusUL66 482Cys Thr Thr Val His Gln Glu
Thr Gln Arg Thr Ser Cys Pro Ser Gly 1 5 10 15 Trp Thr Tyr Thr Cys
Asn Cys Arg Asn Gly Cys Gly Cys Tyr Arg Pro 20 25 30 Ser Gln Leu
Cys Gly Ala Tyr Val Ala Val Thr His Thr Tyr Glu Phe 35 40 45 His
Val Asp Ala Trp 50 48353PRTBos taurusUL67 483Cys Ala Thr Val His
Gln Lys Asp Lys His Cys Pro Ala Gly Tyr Arg 1 5 10 15 Ser Gly Thr
Leu Cys Arg Met Ile Gly Cys Thr Gly Asp Asp Cys Cys 20 25 30 Asn
Tyr Asp Arg Val Glu Cys Thr Asn Tyr Asp Tyr Thr Asn Asn Phe 35 40
45 Tyr Val Asp Ala Trp 50 48452PRTBos taurusUL68 484Cys Thr Ala Val
His Gln Gln Thr Thr Glu Lys Gly Lys Thr Cys Pro 1 5 10 15 Pro Arg
Ser Arg Asp Met Gly Thr Arg Cys Arg Asp Asp Arg Tyr Tyr 20 25 30
Pro Trp Arg Tyr Ser Asp Tyr Thr Tyr Thr Tyr Thr Tyr Glu Trp His 35
40 45 Val Asp Ala Trp 50 48551PRTBos taurusUL69 485Cys Thr Ser Val
His Gln Lys Thr Asp Val Thr Cys Pro Ser Gly Ala 1 5 10 15 Thr Tyr
Arg Cys Asp Cys Gly Gly Arg Gly Cys Gly Cys Tyr Asp Pro 20 25 30
Trp Cys Ser Thr Thr Tyr Arg Gly Thr Tyr Thr Tyr Asp Phe His Val 35
40 45 Glu Thr Trp 50 48650PRTBos taurusUL70 486Cys Gly Thr Val His
Gln Glu Thr His Thr Gln Arg Thr Cys Pro Asp 1 5 10 15 Ala Cys Asp
Val Thr Gly Asp Asn Cys Lys Val Arg Arg Asn Gly Asp 20 25 30 Trp
Cys Gly Arg Ala Ser Lys Thr Asp Thr Tyr Asp Phe Tyr Val Asp 35 40
45 Ala Trp 50 48749PRTBos taurusUL71 487Cys Thr Thr Asp Tyr Gln Lys
Thr Glu Lys Ser Cys Pro Glu Asn Tyr 1 5 10 15 Tyr Ala Glu Thr Gly
Tyr Cys Met Cys Gly Ser Trp Arg Cys Gly Tyr 20 25 30 Gly Ser Thr
Thr Ser Leu Ile Val Ser Tyr Lys Trp Tyr Val Asp Ala 35 40 45 Trp
48849PRTBos taurusUL72 488Cys Thr Thr Val His Gln Lys Thr Asn Gln
Lys Trp Gly Cys Pro Asp 1 5 10 15 Gly Tyr Val His Met Ser Gly Ser
Cys Cys Arg Gly Ser Ile Cys Thr 20 25 30 Asn Gly Leu Phe Arg Asn
Thr Tyr Thr Tyr Glu Phe Asn Val Glu Ala 35 40 45 Trp 48948PRTBos
taurusUL73 489Cys Thr Thr Val Tyr Gln Glu Thr Arg Thr Asn Cys Pro
Asp Gly Tyr 1 5 10 15 Asn Tyr Arg Ser Gly Asp Cys Arg Arg Trp Asn
His Trp Leu Gly Glu 20 25 30 Gln Arg Val Ser Pro Thr Tyr Asn Tyr
Glu Trp Tyr
Val Asp Ser Trp 35 40 45 49047PRTBos taurusUL74 490Cys Thr Thr Val
Tyr Gln Lys Thr Thr Thr Thr Lys Ser Cys Pro Gly 1 5 10 15 Gly Phe
Asp Asn Gly Arg Arg Cys Ile Met Gly Leu Gly Asp Leu Arg 20 25 30
Asp Tyr Thr Tyr Phe Asn Lys Tyr Glu Trp Tyr Val Glu Thr Trp 35 40
45 49146PRTBos taurusUL75 491Cys Ser Thr Val His Gln Lys Thr Glu
Gln Arg Cys Leu Asp Gly Tyr 1 5 10 15 Asp Asp Arg Gly Ala Tyr Cys
Tyr Asp Ser Val Arg Gly Leu Met Ser 20 25 30 Trp Thr Tyr Lys Tyr
Val Tyr Glu Trp Arg Val Asp Thr Trp 35 40 45 49245PRTBos taurusUL76
492Cys Thr Asn Val His Gln Met Thr Ile Lys Thr Cys Pro Asp Gly Gly
1 5 10 15 Ser Tyr Gly Trp Tyr Trp Pro Tyr Gly Tyr Gly Cys Asn Gly
Gly Val 20 25 30 Ser Ala Thr Tyr Thr Tyr Glu Phe Tyr Val Asp Ala
Trp 35 40 45 49344PRTBos taurusUL77 493Cys Thr Thr Val Tyr Gln Lys
Thr Glu Ser Val Arg Ser Cys Pro Asp 1 5 10 15 Gly Ser Met Asp Gly
Trp Arg Cys Arg Leu Gly Thr Met Asn Trp Ile 20 25 30 Tyr Ser Asn
Thr Tyr Glu Phe Tyr Val Asp Ala Trp 35 40 49459PRTArtificial
SequenceDescription of Artificial Sequence Synthetic ultralong CDR3
consensus motif polypeptide 494Xaa Xaa Xaa Xaa Gln Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 4955PRTArtificial
SequenceDescription of Artificial Sequence Synthetic ultralong CDR3
consensus motif peptide 495Xaa Xaa Xaa Xaa Gln 1 5
4968PRTArtificial SequenceDescription of Artificial Sequence
Synthetic ultralong CDR3 consensus motif peptide 496Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 49767PRTArtificial SequenceDescription of
Artificial Sequence Synthetic ultralong CDR3 consensus motif
polypeptide 497Xaa Xaa Xaa Xaa Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa 65
4988PRTArtificial SequenceDescription of Artificial Sequence
Synthetic ultralong CDR3 consensus motif peptide 498Tyr Xaa Xaa Xaa
Xaa Xaa Xaa Trp 1 5 49925PRTArtificial SequenceDescription of
Artificial Sequence Synthetic linker peptide 499Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly
Ser Gly Gly Gly Gly Ser 20 25 5004PRTBos taurusStalk motif 500Cys
Pro Asp Gly 1 5015PRTArtificial SequenceDescription of Artificial
Sequence Synthetic linker peptide 501Gly Gly Gly Gly Ser 1 5
5026PRTBos taurusStalk motif 502Cys Thr Xaa Val His Gln 1 5
50314PRTBos taurusStalk motif 503Cys Thr Xaa Val His Gln Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 5043PRTBos taurusStalk motif 504Tyr
Xaa Xaa 1 5053PRTBos taurusStalk motif 505Xaa Tyr Xaa 1 5068PRTBos
taurusStalk motif 506Tyr Xaa Xaa Xaa Xaa Xaa Xaa Trp 1 5 5074PRTBos
taurusknob domain 507Cys Xaa Asp Gly 1 5085PRTBos taurusStalk motif
508Thr Xaa Val His Gln 1 5 5095PRTArtificial SequenceDescription of
Artificial Sequence Synthetic linker peptide 509Gly Gly Gly Gly Ser
1 5 5104PRTUnknownDescription of Unknown Cleavage site peptide
510Ile Glu Gly Arg 1 5116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic 6xHis tag 511His His His His His His1
5
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References