U.S. patent application number 16/388646 was filed with the patent office on 2019-11-21 for lag-3 targeted heterodimeric fusion proteins containing il-15/il-15ra fc-fusion proteins and lag-3 antigen binding domains.
The applicant listed for this patent is Xencor, Inc.. Invention is credited to Matthew Bernett, Christine Bonzon, John Desjarlais, Rumana Rashid, Rajat Varma.
Application Number | 20190352362 16/388646 |
Document ID | / |
Family ID | 66429614 |
Filed Date | 2019-11-21 |
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United States Patent
Application |
20190352362 |
Kind Code |
A1 |
Bernett; Matthew ; et
al. |
November 21, 2019 |
LAG-3 TARGETED HETERODIMERIC FUSION PROTEINS CONTAINING
IL-15/IL-15RA Fc-FUSION PROTEINS AND LAG-3 ANTIGEN BINDING
DOMAINS
Abstract
The present invention is directed to novel targeted
heterodimeric fusion proteins comprising an IL-15/IL-15R.alpha.
Fc-fusion protein and a LAG-3 antibody fragment-Fc fusion
protein.
Inventors: |
Bernett; Matthew; (Monrovia,
CA) ; Desjarlais; John; (Pasadena, CA) ;
Rashid; Rumana; (Temple City, CA) ; Varma; Rajat;
(Monrovia, CA) ; Bonzon; Christine; (Los Angeles,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Xencor, Inc. |
Monrovia |
CA |
US |
|
|
Family ID: |
66429614 |
Appl. No.: |
16/388646 |
Filed: |
April 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62659624 |
Apr 18, 2018 |
|
|
|
62783107 |
Dec 20, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/53 20130101;
C07K 2317/56 20130101; C07K 14/7155 20130101; A61K 2039/505
20130101; C07K 2317/622 20130101; C07K 16/2803 20130101; C07K
14/5443 20130101; C07K 2319/00 20130101; C07K 2317/526 20130101;
C07K 2317/524 20130101; A61K 38/00 20130101; C07K 2317/66 20130101;
A61K 2039/507 20130101; A61P 35/00 20180101; C07K 2319/30
20130101 |
International
Class: |
C07K 14/54 20060101
C07K014/54 |
Claims
1. A heterodimeric fusion protein comprising: a) a first monomer
comprising, from N-to C-terminal: i) an IL-15R.alpha.(sushi)
domain; ii) a first domain linker; iii) an IL-15 variant; iv) a
hinge; and v) a first variant Fc domain comprising CH2-CH3; and b)
a second monomer comprising, from N-to C-terminal,
VH-CH1-hinge-CH2-CH3, wherein the CH2-CH3 is a second variant Fc
domain; and c) a third monomer comprising a VL-CL, wherein the VH
and VL are a variable heavy domain and a variable light domain,
respectively, that form a human LAG-3 antigen binding domain,
wherein the first variant Fc domain comprises skew variants
L368D/K370S and the second variant Fc domain comprises skew
variants S364K/E357Q, wherein the first and second variant Fc
domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, wherein the first variant Fc
domain comprises pI variants Q295E/N384D/Q418E/N421D, and wherein
numbering is according to EU numbering.
2. A heterodimeric fusion protein according to claim 1, wherein the
hinge of the first monomer comprises amino acid substitution C220S,
and wherein numbering is according to EU numbering.
3. A heterodimeric fusion protein according to claim 1 or 2,
wherein the first and second variant Fc domains each further
comprise half-life extension variants
4. A heterodimeric fusion protein according to any one of claims 1
to 3, wherein the IL-15 variant comprises an amino acid
substitution(s) selected from the group consisting of N1D, N4D,
D8N, D30N, D61N, E64Q, N65D, Q108E, N4D/N65D, D30N/N65D, and
D30N/E64Q/N65D.
5. A heterodimeric fusion protein according to claim 4, wherein the
IL-15 variant comprises amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D
6. A heterodimeric fusion protein according to any one of claims 1
to 5, wherein the VH and VL are the variable heavy domain and
variable domain of any of the LAG-3 antigen binding domains in
FIGS. 12 and 13.
7. A heterodimeric fusion protein according to claim 6, wherein the
LAG-3 antigen binding domain is selected from 2A11_H1.144_L2.142
and 7G8_H3.30_L1.34.
8. A heterodimeric fusion protein according to claim 1, wherein the
heterodimeric fusion protein is selected from the group consisting
of: XENP27972, XENP27973, XENP27977, XENP27978, XENP029486,
XENP029487, XENC1000, XENC1001, XENC1002, XENC1003, XENC1004 and
XENC1005.
9. A nucleic acid composition comprising: a) a first nucleic acid
encoding said first monomer of any of claims 1 to 8; b) a second
nucleic acid encoding said second monomer of any of claims 1 to 8;
a) a third nucleic acid encoding said third monomer of any of
claims 1 to 8; respectively.
10. An expression vector composition comprising: a) a first
expression vector comprising said first nucleic acid of claim 9; b)
a second expression vector comprising said second nucleic acid of
claim 9; and c) a third expression vector comprising said third
nucleic acid of claim 9.
11. A host cell comprising the expression vector composition
according to claim 10.
12. A method of making a heterodimeric fusion protein comprising
culturing the host cell of claim 11 and recovering the
heterodimeric fusion protein from the cell culture.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Nos. 62/659,624 filed Apr. 18, 2018 and 62/783,107,
filed Dec. 20, 2018, which are expressly incorporated herein by
reference in their entirety, with particular reference to the
figures, legends, and claims therein.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been filed electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 31, 2019, is named 067461-5222-US_SL.txt and is 421,438
bytes in size.
BACKGROUND OF THE INVENTION
[0003] Two very promising approaches in cancer immunotherapy
include cytokine-based treatments and blockade of immune checkpoint
proteins such as PD-1.
[0004] Cytokines such as IL-2 and IL-15 function in aiding the
proliferation and differentiation of B cells, T cells, and NK
cells. Both cytokines exert their cell signaling function through
binding to a trimeric complex consisting of two shared receptors,
the common gamma chain (.gamma.c; CD132) and IL-2 receptor
beta-chain (IL-2R1; CD122), as well as an alpha chain receptor
unique to each cytokine: IL-2 receptor alpha (IL-2R.alpha.; CD25)
or IL-15 receptor alpha (IL-15R.alpha.; CD215). Both cytokines are
considered as potentially valuable therapeutics in oncology, and
IL-2 has been approved for use in patients with metastatic
renal-cell carcinoma and malignant melanoma. Currently, there are
no approved uses of recombinant IL-15, although several clinical
trials are ongoing. However, as potential drugs, both cytokines
suffer from a very fast clearance, with half-lives measured in
minutes. IL-2 immunotherapy has been associated with systemic
toxicity when administered in high doses to overcome fast
clearance. Such systemic toxicity has also been reported with IL-15
immunotherapy in recent clinical trials (Guo et al., J Immunol,
2015, 195(5):2353-64).
[0005] Immune checkpoint proteins such as PD-1 are up-regulated
following T cell activation to preclude autoimmunity by exhausting
activated T cells upon binding to immune checkpoint ligands such as
PD-L1. However, immune checkpoint proteins are also up-regulated in
tumor-infiltrating lymphocytes (TILs), and immune checkpoint
ligands are overexpressed on tumor cells, contributing to immune
escape by tumor cells. De-repression of TILs by blockade of immune
checkpoint interactions by drugs such as Opdivo.RTM. (nivolumab)
and Keytruda.RTM. (pembrolizumab) have proven highly effective in
treatment of cancer. Despite the promise of checkpoint blockade
therapies such as nivolumab and pembrolizumab, many patients still
fail to achieve sufficient response to checkpoint blockade
alone.
[0006] Therefore, there remains an unmet need in oncology treatment
for therapeutic strategies with cytokines that do not require high
doses and are targeted to tumors to avoid systemic toxicity.
Further, there is a need to identify additional therapeutic
modalities to stack with checkpoint blockade that could increase
patient response rate.
[0007] To address these needs and caveats, provided herein are
novel LAG-3-targeted IL-15 heterodimeric fusion proteins with
enhanced half-life and more selective targeted of TILs to improve
safety profile, and which synergistically combine with checkpoint
blockade antibodies (FIG. 1).
BRIEF SUMMARY OF THE INVENTION
[0008] In one aspect, the present invention provides a targeted
IL-15/IL-15R.alpha. heterodimeric protein comprising: (a) a first
monomer comprising, from N-to C-terminal: i) an IL-15 sushi domain;
ii) a first domain linker; iii) a variant IL-15 domain; iv) a
second domain linker; v) a first variant Fc domain comprising
CH2-CH3; and (b) a second monomer comprising, from N-to C-terminal:
i) a scFv domain; ii) a third domain linker; iii) a second variant
Fc domain comprising CH2-CH3; wherein the scFv domain comprises a
first variable heavy domain, an scFv linker and a first variable
light domain, wherein the scFv domain binds human LAG-3.
[0009] In other aspects of the present invention, provided herein
is a targeted IL-15/IL-15R.alpha. heterodimeric protein comprising:
(a) a first monomer comprising, from N-to C-terminal: i) an IL-15
sushi domain; ii) a first domain linker; iii) a first variant Fc
domain comprising CH2-CH3; (b) a second monomer comprising, from
N-to C-terminal: i) a scFv domain; ii) a third domain linker; iii)
a second variant Fc domain comprising CH2-CH3; wherein the scFv
domain comprises a first variable heavy domain, an scFv linker and
a first variable light domain; and (c) a third monomer comprising a
variant IL-15 domain; wherein the scFv domain binds human
LAG-3.
[0010] In one aspect, provided are "scIL-15/R.alpha. X Fab" format
heterodimeric proteins. Such "scIL-15/R.alpha. X Fab" format
heterodimeric proteins include: a) a first monomer comprising, from
N-to C-terminal: i) an IL-15R.alpha.(sushi) domain; ii) a first
domain linker; iii) an IL-15 variant; iv) a second domain linker;
v) a first variant Fc domain comprising CH2-CH3; b) a second
monomer comprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3,
wherein CH2-CH3 is a second variant Fc domain; and c) a third
monomer comprising a VL-CL. The VH and VL are a variable heavy
domain and a variable light domain, respectively, that form a human
LAG-3 antigen binding domain. In some embodiments, the second
domain linker is an antibody hinge.
[0011] In certain embodiments of the "scIL-15/R.alpha. X Fab"
format heterodimeric protein, the first variant Fc domain the
second variant Fc domain comprises one of the following skew
variant sets: S267K/L368D/K370S:S267K/S364K/E357Q;
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411E/K360E/Q362E:D401K; L368D/K370S:S364K/E357L and
K370S:S364K/E357Q according to EU numbering. In an exemplary
embodiment, the skew variant set is S364K/E357Q:L368D/K370S.
[0012] In an exemplary embodiment of the "scIL-15/R.alpha. X Fab"
format heterodimeric protein, the "scIL-15/R.alpha. X Fab" format
heterodimeric protein includes: a) a first monomer comprising, from
N-to C-terminal: i) an IL-15R.alpha.(sushi) domain; ii) a first
domain linker; iii) an IL-15 variant; iv) a hinge; v) a first
variant Fc domain comprising CH2-CH3; b) a second monomer
comprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3, wherein the
CH2-CH3 is a second variant Fc domain; and c) a third monomer
comprising a VL-CL. The VH and VL are a variable heavy domain and a
variable light domain, respectively, that form a human LAG-3
antigen binding domain. In such embodiments, the first variant Fc
domain comprises skew variants L368D/K370S and the second variant
Fc domain comprises skew variants S364K/E357Q the first and second
variant Fc domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, the first variant Fc domain
comprises pI variants Q295E/N384D/Q418E/N421D, wherein numbering is
according to EU numbering. In some embodiments, the hinge of the
first monomer comprises amino acid substitution C220S, wherein
numbering is according to EU numbering. In an exemplary embodiment,
the first and second variant Fc domains each further comprise
half-life extension variants M428:/N434S.
[0013] In some embodiments of the "scIL-15/R.alpha. X Fab" format
heterodimeric protein, the IL-15 variant of the heterodimeric
protein provided herein comprises an amino acid substitution(s)
selected from the group consisting of N1D, N4D, D8N, D30N, D61N,
E64Q, N65D, Q108E, N4D/N65D, D30N/N65D, and D30N/E64Q/N65D. In an
exemplary embodiment, the IL-15 variant comprises amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D
[0014] In an exemplary embodiment, the "scIL-15/R.alpha. X Fab"
format heterodimeric protein is XENP27972, XENP27973, XENP27977,
XENP27978, XENP029486, XENP029487, XENC1000, XENC1001, XENC1002,
XENC1003, XENC1004 or XENC1005.
[0015] In certain embodiment, the VH and VL of the scIL-15/R.alpha.
X Fab" format heterodimeric proteins provided herein are the
variable heavy domain and variable domain of any of the LAG-3
antigen binding domains in FIGS. 12 and 13. In an exemplary
embodiment, the LAG-3 antigen binding domain is 2A11_H1.144_L2.142
and 7G8_H3.30_L1.34.
[0016] In one aspect, provided herein is a heterodimeric protein
having the "scIL-15/R.alpha. X scFv" format. In one embodiment, the
heterodimeric protein includes: a) a first monomer comprising, from
N-to C-terminal: i) an IL-15R.alpha.(sushi) domain; ii) a first
domain linker; iii) an IL-15 variant; iv) a second domain linker;
and v) a first variant Fc domain comprising CH2-CH3; and b) a
second monomer comprising, from N-to C-terminal: i) a scFv domain;
ii) a third domain linker; and iii) a second variant Fc domain
comprising CH2-CH3. In some embodiments, the scFv domain comprises
a variable heavy domain (VH), an scFv linker and a variable light
domain (VL), and the scFv domain binds human LAG-3. In some
embodiments of the "scIL-15/R.alpha. X scFv" format heterodimeric
protein, the second domain linker and the third domain linker are
each an antibody hinge.
[0017] In certain embodiments, the first variant Fc domain the
second variant Fc domain comprises one of the following skew
variant sets: S267K/L368D/K370S:S267K/S364K/E357Q;
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411E/K360E/Q362E:D401K; L368D/K370S:S364K/E357L and
K370S:S364K/E357Q, according to EU numbering. In an exemplary
embodiment, the skew variant set is S364K/E357Q:L368D/K370S.
[0018] In some embodiments of the "scIL-15/R.alpha. X scFv" format,
the heterodimeric protein includes: a) a first monomer comprising,
from N-to C-terminal: i) an IL-15R.alpha.(sushi) domain; ii) a
first domain linker; iii) an IL-15 variant; iv) a hinge; and v) a
first variant Fc domain comprising CH2-CH3; and b) a second monomer
comprising, from N-to C-terminal: i) a scFv domain; ii) a hinge;
and iii) a second variant Fc domain comprising CH2-CH3. In some
embodiments, the scFv domain comprises a variable heavy domain
(VH), an scFv linker and a variable light domain (VL), and the scFv
domain binds human LAG-3. In such embodiments, the first variant Fc
domain comprises skew variants L368D/K370S and the second variant
Fc domain comprises skew variants S364K/E357Q, the first and second
variant Fc domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, the first variant Fc domain
comprises pI variants Q295E/N384D/Q418E/N421D, and the numbering is
according to EU numbering. In certain embodiments, the first and
second hinges each comprise amino acid substitution C220S, wherein
numbering is according to EU numbering. In one embodiment, the
first and second variant Fc domains each further comprise half-life
extension variants M428:/N434S.
[0019] In another aspect, provided herein are "scFv X
ncIL-15/R.alpha." format heterodimeric proteins. Such heterodimeric
proteins include: a) a first monomer comprising, from N-to
C-terminal: i) a scFv domain; ii) a first domain linker; and iii) a
first variant Fc domain comprising CH2-CH3; b) a second monomer
comprising, from N-to C-terminal: i) an IL-15R.alpha.(sushi)
domain; ii) a second domain linker; and iii) a second variant Fc
domain comprising CH2-CH3; and c) a third monomer comprising an
IL-15 variant. The scFv domain comprises a variable heavy domain
(VH), an scFv linker and a variable light domain (VL), and the scFv
domain binds human LAG-3. In one embodiment, the first domain
linker and the second domain linker are each an antibody hinge.
[0020] In some embodiments of the "scFv X ncIL-15/R.alpha." format
heterodimeric protein, the first variant Fc domain the second
variant Fc domain comprises one of the following skew variant sets:
S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;
L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;
L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EU
numbering. In an exemplary embodiment, the skew variant set is
S364K/E357Q:L368D/K370S.
[0021] In an exemplary embodiment, the "scFv X ncIL-15/R.alpha."
format heterodimeric protein includes: a) a first monomer
comprising, from N-to C-terminal: i) a scFv domain; ii) a hinge;
and iii) a first variant Fc domain comprising CH2-CH3; b) a second
monomer comprising, from N-to C-terminal: i) an
IL-15R.alpha.(sushi) domain; ii) a hinge; and iii) a second variant
Fc domain comprising CH2-CH3; and c) a third monomer comprising an
IL-15 variant. Further, the scFv domain comprises a variable heavy
domain (VH), an scFv linker and a variable light domain (VL), and
the scFv domain binds human LAG-3. In such embodiments, the first
variant Fc domain comprises skew variants L368D/K370S and the
second variant Fc domain comprises skew variants S364K/E357Q, the
first and second variant Fc domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, the first variant Fc domain
comprises pI variants Q295E/N384D/Q418E/N421D, and wherein
numbering is according to EU numbering. In certain embodiments, the
first and second hinges each comprise amino acid substitution
C220S, wherein numbering is according to EU numbering. In one
embodiment, the first and second variant Fc domains each further
comprise half-life extension variants M428:/N434S.
[0022] In another aspect, provided herein are "scFv x
dsIL-15/R.alpha." format heterodimeric proteins. The "scFv x
dsIL-15/R.alpha." format heterodimeric protein includes: a) a first
monomer comprising, from N-to C-terminal: i) a variant
IL-15R.alpha.(sushi) domain comprising an amino acid substituted
for a cysteine residue; ii) a first domain linker; and iii) a first
variant Fc domain comprising CH2-CH3; b) a second monomer
comprising, from N-to C-terminal: i) a scFv domain; ii) a second
domain linker; iii) a second variant Fc domain comprising CH2-CH3;
an c) a third monomer comprising an IL-15 variant comprising an
amino acid substituted for a cysteine residue. The scFv domain
comprises a variable heavy domain (VH), an scFv linker and a
variable light domain (VL), wherein the cysteine residue of the
variant IL-15R.alpha.(sushi) domain and the cysteine residue of the
IL-15 variant form a disulfide bond and the scFv domain binds human
LAG-3. In certain embodiments, the first domain linker and the
second domain linker are each an antibody hinge.
[0023] In some embodiments of the "scFv x dsIL-15/R.alpha." format
heterodimeric protein, the first variant Fc domain the second
variant Fc domain comprises one of the following skew variant sets:
S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;
L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;
L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EU
numbering. In an exemplary embodiment, the skew variant set is
S364K/E357Q:L368D/K370S.
[0024] In an exemplary embodiment, the "scFv x dsIL-15/R.alpha."
format heterodimeric protein includes: a) a first monomer
comprising, from N-to C-terminal: i) a variant IL-15R.alpha.(sushi)
domain comprising an amino acid substituted for a cysteine residue;
ii) a hinge; and iii) a first variant Fc domain comprising CH2-CH3;
b) a second monomer comprising, from N-to C-terminal: i) a scFv
domain; ii) a hinge; iii) a second variant Fc domain comprising
CH2-CH3; an c) a third monomer comprising an IL-15 variant
comprising an amino acid substituted for a cysteine residue. The
scFv domain comprises a variable heavy domain (VH), an scFv linker
and a variable light domain (VL), wherein the cysteine residue of
the variant IL-15R.alpha.(sushi) domain and the cysteine residue of
the IL-15 variant form a disulfide bond and the scFv domain binds
human LAG-3. In such embodiments, the first variant Fc domain
comprises skew variants L368D/K370S and the second variant Fc
domain comprises skew variants S364K/E357Q, the first and second
variant Fc domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, the first variant Fc domain
comprises pI variants Q295E/N384D/Q418E/N421D, wherein numbering is
according to EU numbering. In certain embodiments, the hinges of
the first and second monomers each comprise amino acid substitution
C220S, wherein numbering is according to EU numbering. In some
embodiments, the first and second variant Fc domains each comprise
half-life extension variants M428:/N434S.
[0025] In one aspect, provided herein are "Fab X ncIL-15/R.alpha."
format heterodimeric proteins. Such heterodimeric proteins include:
a) a first monomer comprising, from N-to C-terminal,
VH-CH1-hinge-CH2-CH3, wherein the CH2-CH3 is a first variant Fc
domain; b) a second monomer comprising, from N-to C-terminal: i) an
IL-15R.alpha.(sushi) domain; ii) a first domain linker; iii) a
first variant Fc domain comprising CH2-CH3; c) a third monomer
comprising a light chain comprising VL-CL; and d) a fourth monomer
comprising an IL-15 variant. The VH and VL are a variable heavy
domain and a variable light domain, respectively, that form a human
LAG-3 antigen binding domain. In some embodiments, the first domain
linker is an antibody hinge.
[0026] In some embodiments of the "Fab X ncIL-15/R.alpha." format
heterodimeric protein, the first variant Fc domain the second
variant Fc domain comprises one of the following skew variant sets:
S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;
L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;
L368D/K370S:S364K/E357L and K370S:S364K/E357Q according to EU
numbering. In an exemplary embodiment, the skew variant set is
S364K/E357Q:L368D/K370S.
[0027] In exemplary embodiments, the "Fab X ncIL-15/R.alpha."
format heterodimeric protein includes: a) a first monomer
comprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3, wherein the
CH2-CH3 is a first variant Fc domain; b) a second monomer
comprising, from N-to C-terminal: i) an IL-15R.alpha.(sushi)
domain; ii) a hinge; iii) a first variant Fc domain comprising
CH2-CH3; c) a third monomer comprising a light chain comprising
VL-CL; and d) a fourth monomer comprising an IL-15 variant. The VH
and VL are a variable heavy domain and a variable light domain,
respectively, that form a human LAG-3 antigen binding domain. In
such embodiments, the first variant Fc domain comprises skew
variants L368D/K370S and the second variant Fc domain comprises
skew variants S364K/E357Q, the first and second variant Fc domains
each comprise FcKO variants E233P/L234V/L235A/G236del/S267K, and
the hinge-first variant Fc domain of the first monomer comprises pI
variants N208D/Q295E/N384D/Q418E/N421D, wherein numbering is
according to EU numbering. In some embodiments, the hinge of the
second monomer comprises amino acid substitution C220S, wherein
numbering is according to EU numbering. In certain embodiments, the
first and second variant Fc domains each further comprise half-life
extension variants M428:/N434S.
[0028] In another aspect, provided herein are "Fab X
dsIL-15/R.alpha." format heterodimeric proteins. Such "Fab X
dsIL-15/R.alpha." format heterodimeric proteins include: a) a first
monomer comprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3,
wherein CH2-CH3 is a first variant Fc domain; b) a second monomer
comprising, from N-to C-terminal: i) a variant IL-15R.alpha.(sushi)
domain comprising an amino acid substituted for a cysteine residue;
ii) a first domain linker; and a first variant Fc domain comprising
CH2-CH3; c) a third monomer comprising, from N- to C-terminal,
VL-CL; and d) a fourth monomer comprising an IL-15 variant
comprising an amino acid substituted for a cysteine residue.
Further, the cysteine residue of the variant IL-15R.alpha.(sushi)
domain and the cysteine residue of the IL-15 variant form a
disulfide bond, and the VH and VL are a variable heavy domain and a
variable light domain, respectively, that form a human LAG-3
antigen binding domain. In some embodiments, the first domain
linker is an antibody hinge.
[0029] In some embodiments of the "Fab X dsIL-15/R.alpha." format
heterodimeric protein, the first variant Fc domain the second
variant Fc domain comprises one of the following skew variant sets:
S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;
L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;
L368D/K370S:S364K/E357L and K370S:S364K/E357Q according to EU
numbering. In an exemplary embodiment, the skew variant set is
S364K/E357Q:L368D/K370S.
[0030] In an exemplary embodiment, the "Fab X dsIL-15/R.alpha."
format heterodimeric protein includes: a) a first monomer
comprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3, wherein
CH2-CH3 is a first variant Fc domain; b) a second monomer
comprising, from N-to C-terminal: i) a variant IL-15R.alpha.(sushi)
domain comprising an amino acid substituted for a cysteine residue;
a hinge; and a first variant Fc domain comprising CH2-CH3; c) a
third monomer comprising, from N- to C-terminal, VL-CL; and d) a
fourth monomer comprising an IL-15 variant comprising an amino acid
substituted for a cysteine residue. Further, the cysteine residue
of the variant IL-15R.alpha.(sushi) domain and the cysteine residue
of the IL-15 variant form a disulfide bond, and the VH and VL are a
variable heavy domain and a variable light domain, respectively,
that form a human LAG-3 antigen binding domain. In such
embodiments, the first variant Fc domain comprises skew variants
L368D/K370S and the second variant Fc domain comprises skew
variants S364K/E357Q the first and second variant Fc domains each
comprise FcKO variants E233P/L234V/L235A/G236del/S267K, and the
hinge-first variant Fc domain of the first monomer comprises pI
variants N208D/Q295E/N384D/Q418E/N421D, wherein numbering is
according to EU numbering. In certain embodiments, the hinge of the
second monomer comprises amino acid substitution C220S, wherein
numbering is according to EU numbering. In some embodiments, the
first and second variant Fc domains each further comprise half-life
extension variants M428:/N434S.
[0031] In one aspect, provided herein are "mAb-scIL-15/R.alpha."
format heterodimeric proteins. The "mAb-scIL-15/R.alpha." format
heterodimeric proteins include: a) a first monomer comprising, from
N-to C-terminal, VH-CH1-hinge-CH2-CH3, wherein the CH2-CH3 is a
first variant Fc domain; b) a second monomer comprising, from N-to
C-terminal, VH-CH1-hinge-CH2-CH3-domain linker-IL-15R.alpha.(sushi)
domain-domain linker-IL-15 variant, wherein the CH2-CH3 is a second
variant Fc domain; and c) a third monomer and fourth monomer that
each comprises, from N- to C-terminal, VL-CL. Further, the VH of
the first monomer and the VL of the third monomer form a first
human LAG-3 binding domain, and the VH of the second monomer and
the VL of the fourth monomer form a second human LAG-3 binding
domain.
[0032] In some embodiments of the "mAb-scIL-15/R.alpha." format
heterodimeric protein, the first variant Fc domain the second
variant Fc domain comprises one of the following skew variant sets:
S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;
L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;
L368D/K370S:S364K/E357L and K370S:S364K/E357Q according to EU
numbering. In an exemplary embodiment, the skew variant set is
S364K/E357Q:L368D/K370S.
[0033] In some embodiments of the "mAb-scIL-15/R.alpha." format
heterodimeric protein, the first variant Fc domain comprises skew
variants L368D/K370S and the second variant Fc domain comprises
skew variants S364K/E357Q and the first and second variant Fc
domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, wherein numbering is according to
EU numbering. In certain embodiments, a) the hinge-first variant Fc
domain of the first monomer further comprises pI substitutions
N208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc
domain of the second monomer further comprises pI variants
Q196K/I199T/P271R/P228R/N276K; b) the hinge-first variant Fc domain
of the first monomer further comprises pI substitutions
N208D/Q295E/N384D/Q418D/N421D; or c) the hinge-second variant Fc
domain of the second monomer further comprises pI variants
Q196K/I199T/P271R/P228R/N276K, wherein numbering is according to EU
numbering.
[0034] In some embodiments of the "mAb-scIL-15/R.alpha." format
heterodimeric protein, the first variant Fc domain comprises skew
variants S364K/E357Q and the second variant Fc domain comprises
skew variants L368D/K370S, and the first and second variant Fc
domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, wherein numbering is according to
EU numbering. In such embodiments, a) the hinge-first variant Fc
domain of the first monomer further comprises pI substitutions
Q196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc
domain of the second monomer further comprises pI variants
N208D/Q295E/N384D/Q418D/N421D; b) the hinge-first variant Fc domain
of the first monomer further comprises pI substitutions
Q196K/I199T/P271R/P228R/N276K; or c) the hinge-second variant Fc
domain of the second monomer further comprises pI variants
N208D/Q295E/N384D/Q418D/N421D, wherein numbering is according to EU
numbering.
[0035] In some embodiments of the "mAb-scIL-15/R.alpha." format
heterodimeric protein, the first and second variant Fc domains each
further comprise half-life extension variants M428:/N434S.
[0036] In another aspect, provided herein are
"mAb-ncIL-15/R.alpha." format heterodimeric proteins. Such
heterodimeric protein include: a) a first monomer comprising, from
N-to C-terminal, VH-CH1-hinge-CH2-CH3, wherein the CH2-CH3 is a
first variant Fc domain; b) a second monomer comprising, from N-to
C-terminal, VH-CH1-hinge-CH2-CH3-domain linker-IL-15R.alpha.(sushi)
domain, wherein the CH2-CH3 is a second variant Fc domain; c) a
third monomer comprising an IL-15 variant; and d) a fourth and
fifth monomer that each comprises, from N- to C-terminal, VL-CL.
The VH of the first monomer and the VL of the fourth monomer form a
first human LAG-3 binding domain, and the VH of the second monomer
and the VL of the fifth monomer form a second human LAG-3 binding
domain.
[0037] In some embodiments of the "mAb-ncIL-15/R.alpha." format
heterodimeric protein, the first variant Fc domain the second
variant Fc domain comprises one of the following skew variant sets:
S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;
L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;
L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EU
numbering. In an exemplary embodiment, the skew variant set is
S364K/E357Q:L368D/K370S.
[0038] In an exemplary embodiment of the "mAb-ncIL-15/R.alpha."
format heterodimeric protein, the first variant Fc domain comprises
skew variants L368D/K370S and the second variant Fc domain
comprises skew variants S364K/E357Q, and the first and second
variant Fc domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, wherein numbering is according to
EU numbering. In some embodiments, a) the hinge-first variant Fc
domain of the first monomer further comprises pI substitutions
N208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc
domain of the second monomer further comprises pI variants
Q196K/I199T/P271R/P228R/N276K; b) the hinge-first variant Fc domain
of the first monomer further comprises pI substitutions
N208D/Q295E/N384D/Q418D/N421D; or c) the hinge-second variant Fc
domain of the second monomer further comprises pI variants
Q196K/I199T/P271R/P228R/N276K, wherein numbering is according to EU
numbering.
[0039] In another exemplary embodiment of the
"mAb-ncIL-15/R.alpha." format heterodimeric protein, the first
variant Fc domain comprises skew variants S364K/E357Q and the
second variant Fc domain comprises skew variants L368D/K370S, and
the first and second variant Fc domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, wherein numbering is according to
EU numbering. In certain embodiments, a) the hinge-first variant Fc
domain of the first monomer further comprises pI substitutions
Q196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc
domain of the second monomer further comprises pI variants
N208D/Q295E/N384D/Q418D/N421D; b) the hinge-first variant Fc domain
of the first monomer further comprises pI substitutions
Q196K/I199T/P271R/P228R/N276K; or c) the hinge-second variant Fc
domain of the second monomer comprises pI variants
N208D/Q295E/N384D/Q418D/N421D, wherein numbering is according to EU
numbering.
[0040] In certain embodiments, the first and second variant Fc
domains each further comprise half-life extension variants
M428:/N434S.
[0041] In another aspect, provided herein are
"mAb-dsIL-15/R.alpha." heterodimeric proteins. Such
"mAb-dsIL-15/R.alpha." heterodimeric proteins include: a) a first
monomer comprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3,
wherein the CH2-CH3 is a first variant Fc domain; b) a second
monomer comprising, from N-to C-terminal,
VH-CH1-hinge-CH2-CH3-domain linker-variant IL-15R.alpha.(sushi)
domain, wherein the variant IL-15R.alpha.(sushi) domain an amino
acid substituted for a cysteine residue and wherein the CH2-CH3 is
a second variant Fc domain; c) a third monomer comprising an IL-15
variant comprising an amino acid substituted for a cysteine
residue; and d) a fourth and fifth monomer that each comprises,
from N- to C-terminal, VL-CL. The cysteine residue of the variant
IL-15R.alpha.(sushi) domain and the cysteine residue of the IL-15
variant form a disulfide bond, the VH of the first monomer and the
VL of the fourth monomer form a first human LAG-3 binding domain,
and the VH of the second monomer and the VL of the fifth monomer
form a second human LAG-3 binding domain.
[0042] In some embodiments, the first variant Fc domain the second
variant Fc domain comprises one of the following skew variant sets:
S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;
L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;
L368D/K370S:S364K/E357L and K370S:S364K/E357Q according to EU
numbering. In an exemplary embodiment, the skew variant set is
S364K/E357Q:L368D/K370S.
[0043] In an exemplary embodiment of the "mAb-dsIL-15/R.alpha."
heterodimeric proteins, the first variant Fc domain comprises skew
variants L368D/K370S and the second variant Fc domain comprises
skew variants S364K/E357Q and the first and second variant Fc
domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, wherein numbering is according to
EU numbering. In some embodiments, a) the hinge-first variant Fc
domain of the first monomer further comprises pI substitutions
N208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc
domain of the second monomer further comprises pI variants
Q196K/I199T/P271R/P228R/N276K; b) the hinge-first variant Fc domain
of the first monomer further comprises pI substitutions
N208D/Q295E/N384D/Q418D/N421D; or c) the hinge-second variant Fc
domain of the second monomer further comprises pI variants
Q196K/I199T/P271R/P228R/N276K, wherein numbering is according to EU
numbering.
[0044] In another exemplary embodiment of the
"mAb-dsIL-15/R.alpha." heterodimeric proteins, the first variant Fc
domain comprises skew variants S364K/E357Q and the second variant
Fc domain comprises skew variants L368D/K370S, and the first and
second variant Fc domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, wherein numbering is according to
EU numbering. In certain embodiments, a) the hinge-first variant Fc
domain of the first monomer further comprises pI substitutions
Q196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc
domain of the second monomer further comprises pI variants
N208D/Q295E/N384D/Q418D/N421D; b) the hinge-first variant Fc domain
of the first monomer further comprises pI substitutions
Q196K/I199T/P271R/P228R/N276K; or c) the hinge-second variant Fc
domain of the second monomer further comprises pI variants
N208D/Q295E/N384D/Q418D/N421D, wherein numbering is according to EU
numbering. In certain embodiments, the first and second variant Fc
domains each further comprise half-life extension variants
M428:/N434S.
[0045] In one aspect, provided herein are "central-IL-15/R.alpha."
format heterodimeric proteins. Such "central-IL-15/R.alpha." format
heterodimeric proteins include: a) a first monomer comprising, from
N- to C-terminal, a VH-CH1-domain linker-IL-15
variant-hinge-CH2-CH3, wherein the CH2-CH3 is a first variant Fc
domain; b) a second monomer comprising, from N- to C-terminal, a
VH-CH1-domain linker-IL-15R.alpha.(sushi) domain-hinge-CH2-CH3,
wherein the CH2-CH3 is a second variant Fc domain; and c) a third
and fourth monomer that each comprises, from N- to C-terminal,
VL-CL. The VH of the first monomer and the VL of the third monomer
form a first human LAG-3 binding domain, and the VH of the second
monomer and the VL of the fourth monomer form a second human LAG-3
binding domain.
[0046] In some embodiments of the "central-IL-15/R.alpha." format
heterodimeric protein, the first variant Fc domain the second
variant Fc domain comprises one of the following skew variant sets:
S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;
L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;
L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EU
numbering. In an exemplary embodiment, the skew variant set is
S364K/E357Q:L368D/K370S.
[0047] In an exemplary embodiment, the first variant Fc domain
comprises skew variants L368D/K370S and the second variant Fc
domain comprise the skew variant pair S364K/E357Q, the first and
second variant Fc domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, and the first variant Fc domain
comprises pI substitutions Q295E/N384D/Q418D/N421D, wherein
numbering is according to EU numbering.
[0048] In an exemplary embodiment of the "central-IL-15/R.alpha."
format heterodimeric protein, the first variant Fc domain comprises
skew variants S364K/E357Q and the second variant Fc domain comprise
the skew variant pair L368D/K370S, the first and second variant Fc
domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, and the second variant Fc domain
of the second monomer comprises pI substitutions
Q295E/N384D/Q418D/N421D, wherein numbering is according to EU
numbering. In some embodiments of the "central-IL-15/R.alpha."
format heterodimeric protein, the hinge of the first and second
monomers each comprise amino acid substitution C220S, wherein
numbering is according to EU numbering. In certain embodiments, the
first and second variant Fc domains each further comprise half-life
extension variants M428:/N434S.
[0049] In another aspect, provided herein are
"central-scIL-15/R.alpha." format heterodimeric proteins. Such
"central-scIL-15/R.alpha." format heterodimeric proteins include:
a) a first monomer comprising, from N-to C-terminal, VH-CH1-domain
linker-IL-15R.alpha.(sushi) domain-domain linker-IL-15
variant-hinge-CH2-CH3, wherein the CH2-CH3 is a first variant Fc
domain; b) a second monomer comprising, from N-to C-terminal, a
VH-CH1-hinge-CH2-CH3, wherein the CH2-CH3 is a second variant Fc
domain; and c) a third and fourth monomer that each comprises, from
N-to C-terminal, VL-CL. The VH of the first monomer and the VL of
the third monomer form a first human LAG-3 binding domain, and the
VH of the second monomer and the VL of the fourth monomer form a
second human LAG-3 binding domain.
[0050] In some embodiments of the "central-scIL-15/R.alpha." format
heterodimeric protein, the first variant Fc domain the second
variant Fc domain comprises one of the following skew variant sets:
S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;
L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;
L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EU
numbering. In an exemplary embodiment, the skew variant set is
S364K/E357Q:L368D/K370S.
[0051] In an exemplary embodiment, the first variant Fc domain
comprises skew variants L368D/K370S and the second variant Fc
domain comprises skew variants S364K/E357Q, the first and second
variant Fc domains each comprise FcKO variants
E233P/L234V/L235A/G236del/S267K, and the first variant Fc domain
comprises pI variants Q295E/N384D/Q418E/N421D, wherein numbering is
according to EU numbering. In some embodiments, the hinge of the
first monomer comprises amino acid substitution C220S, wherein
numbering is according to EU numbering. In certain embodiments, the
first and second variant Fc domains each further comprise half-life
extension variants M428:/N434S.
[0052] In certain embodiment, the VH and VL of any of the
heterodimeric proteins provided herein are the variable heavy
domain and variable domain of any of the LAG-3 antigen binding
domains in FIGS. 12 and 13. In an exemplary embodiment, the LAG-3
antigen binding domain is 2A11_H1.144_L2.142 and
7G8_H3.30_L1.34.
[0053] In some embodiments, the IL-15 variant of the heterodimeric
protein provided herein comprises an amino acid substitution(s)
selected from the group consisting of N1D, N4D, D8N, D30N, D61N,
E64Q, N65D, Q108E, N4D/N65D, D30N/N65D, and D30N/E64Q/N65D. In an
exemplary embodiment, the IL-15 variant comprises amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D
[0054] In one aspect, provided herein is a pharmaceutical
composition that includes any of the heterodimeric proteins
disclosed herein and a pharmaceutically acceptable carrier.
[0055] In another aspect, provided herein is a method of treating a
patient in need thereof comprising administering to the patient any
one of the heterodimeric proteins or pharmaceutical compositions
disclosed herein. In some embodiments, the method further
comprising administering an antibody, where the antibody is an
anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4
antibody, an anti-TIM-3 antibody or an-anti-TIGIT antibody.
[0056] In another aspect, provided herein are nucleic acid
compositions that include one or more nucleic acids encoding any of
the heterodimeric proteins disclosed herein, expression vectors
that include the nucleic acids, host cells that include the nucleic
acids or expression vectors. Also provided herein are methods of
making subject heterodimeric proteins by culturing host cells under
suitable conditions and recovering the heterodimeric proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 depicts selectivity of LAG-3-targeted
IL-15/R.alpha.-Fc fusion proteins for tumor-reactive
tumor-infiltrating lymphocytes expressing PD-1, and its combination
with PD-1 blockade antibody.
[0058] FIGS. 2A-2B depict the sequences for IL-15 and its
receptors.
[0059] FIG. 3 depicts the sequences for LAG-3, including both human
and cyno (predicted), to facilitate the development of antigen
binding domains that bind to both for ease of clinical
development.
[0060] FIGS. 4A-4E depict useful pairs of Fc heterodimerization
variant sets (including skew and pI variants). There are variants
for which there are no corresponding "monomer 2" variants; these
are pI variants which can be used alone on either monomer.
[0061] FIG. 5 depicts a list of isosteric variant antibody constant
regions and their respective substitutions. pI_(-) indicates lower
pI variants, while pI_(+) indicates higher pI variants. These can
be optionally and independently combined with other
heterodimerization variants of the inventions (and other variant
types as well, as outlined herein.)
[0062] FIG. 6 depicts useful ablation variants that ablate
Fc.gamma.R binding (sometimes referred to as "knock outs" or "KO"
variants). Generally, ablation variants are found on both monomers,
although in some cases they may be on only one monomer.
[0063] FIGS. 7A-7F show particularly useful embodiments of
"non-cytokine"/"non-Fv" components of the LAG-3-targeting
IL-15/R.alpha.-Fc fusion proteins of the invention.
[0064] FIG. 8 depicts a number of exemplary variable length linkers
for use in IL-15/R.alpha.-Fc fusion proteins. In some embodiments,
these linkers find use linking the C-terminus of IL-15 and/or
IL-15R.alpha.(sushi) to the N-terminus of the Fc region. In some
embodiments, these linkers find use fusing IL-15 to the
IL-15R.alpha.(sushi).
[0065] FIGS. 9A-9C depict a number of charged scFv linkers that
find use in increasing or decreasing the pI of heterodimeric
antibodies that utilize one or more scFv as a component. The (+H)
positive linker finds particular use herein. A single prior art
scFv linker with single charge is referenced as "Whitlow", from
Whitlow et al., Protein Engineering 6(8):989-995 (1993). It should
be noted that this linker was used for reducing aggregation and
enhancing proteolytic stability in scFvs.
[0066] FIG. 10 show the sequences of several useful LAG-3-targeting
IL-15/R.alpha.-Fc fusion format backbones based on human IgG1,
without the cytokine sequences (e.g., the 11-15 and/or
IL-15R.alpha.(sushi)) or VH, and further excluding light chain
backbones which are depicted in FIG. 11. Backbone 1 is based on
human IgG1 (356E/358M allotype), and includes the
S364K/E357Q:L368D/K370S skew variants, C220S and the
Q295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370S
skew variants and the E233P/L234V/L235A/G236del/S267K ablation
variants on both chains. Backbone 2 is based on human IgG1
(356E/358M allotype), and includes the S364K/E357Q:L368D/K370S skew
variants, the N208D/Q295E/N384D/Q418E/N421D pI variants on the
chain with L368D/K370S skew variants, C220S in the chain with
S364K/E357Q variants, and the E233P/L234V/L235A/G236del/S267K
ablation variants on both chains. Backbone 3 is based on human IgG1
(356E/358M allotype), and includes the S364K/E357Q:L368D/K370S skew
variants, the N208D/Q295E/N384D/Q418E/N421D pI variants on the
chains with L368D/K370S skew variants, the
Q196K/I199T/P217R/P228R/N276K pI variants on the chains with
S364K/E357Q variants, and the E233P/L234V/L235A/G236del/S267K
ablation variants on both chains. Such backbone sequences can be
included, for example, in the "scIL-15/R.alpha. X Fab" format
heterodimeric proteins described herein.). In some embodiments, the
"scIL-15/R.alpha. X Fab" format heterodimeric protein includes: a)
a first monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(hinge)-CH2-CH3, where hinge-CH2-CH3 has the amino acid
sequence of "Chain 2" of any of the backbone sequences in FIG. 10
(SEQ ID NO: 58, 60 and 62); b) a second monomer that includes, from
N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable
heavy domain and CH1-hinge-CH2-CH3 has the amino acid sequence of
Chain 1 of any one of the backbone sequences in FIG. 10 (SEQ ID NO:
57, 59 and 61), and c) a light chain that includes from, N- to
C-terminus, VL-VC, where VL is a variable light domain and VC has
the sequence of "Constant Light Chain--Kappa" or "Constant Light
Chain--Lambda "in FIG. 11 (SEQ ID NO: 63-64). In an exemplary
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In exemplary embodiments,
the VH and VL are the variable heavy domain and variable light
domain, respectively, of any of the LAG-3 ABDs provided in FIGS. 12
and 13A-C.
[0067] In certain embodiments, these sequences can be of the
356D/358L allotype. In other embodiments, these sequences can
include either the N297A or N297S substitutions. In some other
embodiments, these sequences can include the M428L/N434S Xtend
mutations. In yet other embodiments, these sequences can instead be
based on human IgG4, and include a S228P (EU numbering, this is
S241P in Kabat) variant on both chains that ablates Fab arm
exchange as is known in the art. In yet further embodiments, these
sequences can instead be based on human IgG2. Further, these
sequences may instead utilize the other skew variants, pI variants,
and ablation variants depicted in the Figures.
[0068] As will be appreciated by those in the art and outlined
below, these sequences can be used with any IL-15 and
IL-15R.alpha.(sushi) pairs outlined herein, including but not
limited to scIL-15/R.alpha., ncIL-15/R.alpha., and dsIL-15R.alpha.,
as schematically depicted in FIG. 21. Further as will be
appreciated by those in the art and outlined below, any IL-15
and/or IL-15R.alpha.(sushi) variants can be incorporated in these
backbones. Furthermore as will be appreciated by those in the art
and outlined below, these sequences can be used with any VH and VL
pairs outlined herein, including either a scFv or a Fab.
[0069] Included within each of these backbones are sequences that
are 90, 95, 98 and 99% identical (as defined herein) to the recited
sequences, and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
additional amino acid substitutions (as compared to the "parent" of
the Figure, which, as will be appreciated by those in the art,
already contain a number of amino acid modifications as compared to
the parental human IgG1 (or IgG2 or IgG4, depending on the
backbone). That is, the recited backbones may contain additional
amino acid modifications (generally amino acid substitutions) in
addition to the skew, pI and ablation variants contained within the
backbones of this figure.
[0070] FIG. 11 depicts the "non-Fv" backbone of light chains (i.e.
constant light chain) which find use in LAG-3-targeting
IL-15/R.alpha.-Fc fusion proteins of the invention.
[0071] FIG. 12 depicts the variable region sequences for a select
number of anti-LAG-3 antibody binding domains. The CDRs are
underlined. As noted herein and is true for every sequence herein
containing CDRs, the exact identification of the CDR locations may
be slightly different depending on the numbering used as is shown
in Table 2, and thus included herein are not only the CDRs that are
underlined but also CDRs included within the VH and VL domains
using other numbering systems. Furthermore, as for all the
sequences in the Figures, these VH and VL sequences can be used
either in a scFv format or in a Fab format.
[0072] FIGS. 13A-13C depict the variable regions of additional
LAG-3 ABDs which may find use in the LAG-3-targeting
IL-15/R.alpha.-Fc fusion proteins of the invention. The CDRs are
underlined. As noted herein and is true for every sequence herein
containing CDRs, the exact identification of the CDR locations may
be slightly different depending on the numbering used as is shown
in Table 2, and thus included herein are not only the CDRs that are
underlined but also CDRs included within the VH and VL domains
using other numbering systems. Furthermore, as for all the
sequences in the Figures, these VH and VL sequences can be used
either in a scFv format or in a Fab format.
[0073] FIG. 14 depicts a structural model of the IL-15/R.alpha.
heterodimer showing locations of engineered disulfide bond
pairs.
[0074] FIG. 15 depicts sequences for illustrative
IL-15R.alpha.(sushi) variants engineered with additional residues
at the C-terminus to serve as a scaffold for engineering cysteine
residues.
[0075] FIG. 16 depicts sequences for illustrative IL-15 variants
engineered with cysteines in order to form covalent disulfide bonds
with IL-15R.alpha.(sushi) variants engineered with cysteines.
[0076] FIG. 17 depicts sequences for illustrative
IL-15R.alpha.(sushi) variants engineered with cysteines in order to
form covalent disulfide bonds with IL-15 variants engineered with
cysteines.
[0077] FIG. 18 depicts the structure of IL-15 complexed with
IL-15R.alpha., IL-2R.beta., and common gamma chain. Locations of
substitutions designed to reduce potency are shown.
[0078] FIGS. 19A-19C depict sequences for illustrative IL-15
variants engineered for reduced potency. Included within each of
these variant IL-15 sequences are sequences that are 90, 95, 98 and
99% identical (as defined herein) to the recited sequences, and/or
contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid
substitutions. In a non-limiting example, the recited sequences may
contain additional amino acid modifications such as those
contributing to formation of covalent disulfide bonds as shown in
FIG. 14, FIG. 16, and FIG. 17.
[0079] FIG. 20 depicts EC50 for induction of NK and CD8.sup.+ T
cells proliferation by variant IL-15/R.alpha.-Fc fusion proteins,
and fold reduction in EC50 relative to XENP20818, the wild type.
These fusion proteins do not contain a LAG-3 ABD.
[0080] FIG. 21A-FIG. 21K depict several formats for the
LAG-3-targeting IL-15/R.alpha.-Fc fusion proteins of the present
invention. The "scIL-15/R.alpha. x scFv" format (FIG. 21A)
comprises IL-15R.alpha.(sushi) fused to IL-15 by a variable length
linker (termed "scIL-15/R.alpha.") which is then fused to the
N-terminus of a heterodimeric Fc-region, with an scFv fused to the
other side of the heterodimeric Fc. The "scFv x ncIL-15/R.alpha."
format (FIG. 21B) comprises an scFv fused to the N-terminus of a
heterodimeric Fc-region, with IL-15R.alpha.(sushi) fused to the
other side of the heterodimeric Fc, while IL-15 is transfected
separately so that a non-covalent IL-15/R.alpha. complex is formed.
The "scFv x dsIL-15/R.alpha." format (FIG. 21C) is the same as the
"scFv x ncIL-15/R.alpha." format, but wherein IL-15R.alpha.(sushi)
and IL-15 are covalently linked as a result of engineered
cysteines. The "scIL-15/R.alpha. x Fab" format (FIG. 21D) comprises
IL-15R.alpha.(sushi) fused to IL-15 by a variable length linker
(termed "scIL-15/R.alpha.") which is then fused to the N-terminus
of a heterodimeric Fc-region, with a variable heavy chain (VH)
fused to the other side of the heterodimeric Fc, while a
corresponding light chain is transfected separately so as to form a
Fab with the VH. The "ncIL-15/R.alpha. x Fab" format (FIG. 21E)
comprises a VH fused to the N-terminus of a heterodimeric
Fc-region, with IL-15R.alpha.(sushi) fused to the other side of the
heterodimeric Fc, while a corresponding light chain is transfected
separately so as to form a Fab with the VH, and while IL-15 is
transfected separately so that a non-covalent IL-15/R.alpha.
complex is formed. The "dsIL-15/R.alpha. x Fab" format (FIG. 21F)
is the same as the "ncIL-15/R.alpha. x Fab" format, but wherein
IL-15R.alpha.(sushi) and IL-15 are covalently linked as a result of
engineered cysteines. The "mAb-scIL-15/R.alpha." format (FIG. 21G)
comprises VH fused to the N-terminus of a first and a second
heterodimeric Fc, with IL-15 is fused to IL-15R.alpha.(sushi) which
is then further fused to the C-terminus of one of the heterodimeric
Fc-region, while corresponding light chains are transfected
separately so as to form a Fabs with the VHs. The
"mAb-ncIL-15/R.alpha." format (FIG. 21H) comprises VH fused to the
N-terminus of a first and a second heterodimeric Fc, with
IL-15R.alpha.(sushi) fused to the C-terminus of one of the
heterodimeric Fc-region, while corresponding light chains are
transfected separately so as to form a Fabs with the VHs, and while
and while IL-15 is transfected separately so that a non-covalent
IL-15/R.alpha. complex is formed. The "mAb-dsIL-15/R.alpha." format
(FIG. 21I) is the same as the "mAb-ncIL-15/R.alpha." format, but
wherein IL-15R.alpha.(sushi) and IL-15 are covalently linked as a
result of engineered cysteines. The "central-IL-15/R.alpha." format
(FIG. 21J) comprises a VH recombinantly fused to the N-terminus of
IL-15 which is then further fused to one side of a heterodimeric Fc
and a VH recombinantly fused to the N-terminus of
IL-15R.alpha.(sushi) which is then further fused to the other side
of the heterodimeric Fc, while corresponding light chains are
transfected separately so as to form a Fabs with the VHs. The
"central-scIL-15/R.alpha." format (FIG. 21K) comprises a VH fused
to the N-terminus of IL-15R.alpha.(sushi) which is fused to IL-15
which is then further fused to one side of a heterodimeric Fc and a
VH fused to the other side of the heterodimeric Fc, while
corresponding light chains are transfected separately so as to form
a Fabs with the VHs.
[0081] FIGS. 22A-22B depict sequences of XENP27972 and XENP27973,
illustrative LAG-3-targeting IL-15/R.alpha.-Fc fusion protein of
the "scIL-15/R.alpha. x Fab" format. The CDRs are in bold. As noted
herein and is true for every sequence herein containing CDRs, the
exact identification of the CDR locations may be slightly different
depending on the numbering used as is shown in Table 2, and thus
included herein are not only the CDRs that are underlined but also
CDRs included within the V.sub.H and V.sub.L domains using other
numbering systems. IL-15 and IL-15R.alpha.(sushi) are underlined,
linkers are double underlined (although as will be appreciated by
those in the art, the linkers can be replaced by other linkers,
some of which are depicted in the Figures, and slashes (/) indicate
the border(s) between IL-15, IL-15R.alpha., linkers, variable
regions, and constant/Fc regions.
[0082] FIG. 23 depict the sequences for XENP16432, a bivalent
anti-PD-1 mAb with an ablation variant
(E233P/L234V/L235A/G236del/S267K, "IgG1_PVA_/S267k"). The CDRs are
underlined. As noted herein and is true for every sequence herein
containing CDRs, the exact identification of the CDR locations may
be slightly different depending on the numbering used as is shown
in Table 2, and thus included herein are not only the CDRs that are
underlined but also CDRs included within the VH and VL domains
using other numbering systems.
[0083] FIGS. 24A-24B depict CD8.sup.+ T cell counts in whole blood
of PBMC-engrafted NSG mice on Days A) 6 and B) 10 after first dose
of the indicated test articles.
[0084] FIGS. 25A-25B depict CD4.sup.+ T cell counts in whole blood
of PBMC-engrafted NSG mice on Days A) 6 and B) 10 after first dose
of the indicated test articles.
[0085] FIGS. 26A-26B depict CD45.sup.+ T cell counts in whole blood
of PBMC-engrafted NSG mice on Days A) 6 and B) 10 after first dose
of the indicated test articles.
[0086] FIGS. 27A-27B depict CD16.sup.+CD56.sup.+ NK cell counts in
whole blood of PBMC-engrafted NSG mice on Days A) 6 and B) 10 after
first dose of the indicated test articles.
[0087] FIG. 28 depicts the change in body weight (as percentage of
initial body weight) of PBMC-engrafted NSG mice after dosing with
the indicated test articles.
[0088] FIGS. 29A-29B depict the sequences of XENP27977 and 27978
that include M428L/N434S variants in both Fc domains.
[0089] FIG. 30 depicts induction of A) CD8.sup.+ T cells and B)
CD4.sup.+ T cells proliferation by LAG-3-targeted IL-15/R.alpha.-Fc
fusions (and controls) as indicated by percentage proliferating
cells (determined based on CFSE dilution). The data show that
LAG-3-targeted IL-15/R.alpha.-Fc fusions are more potent in
inducing proliferation of CD8.sup.+ T cells in comparison to
untargeted IL-15(D30N/E64Q/N65D)/R.alpha.-Fc fusion (as well as
control RSV-targeted IL-15/R.alpha.-Fc fusion).
[0090] FIG. 31 depicts induction of A) CD8 memory T cell and B) CD8
naive T cell proliferation by LAG-3-targeted IL-15/R.alpha.-Fc
fusions (and controls) as indicated by percentage proliferating
cells (determined based on CFSE dilution). The data show that
LAG-3-targeted IL-15/R.alpha.-Fc fusions are much more potent in
inducing proliferation of CD8 memory T cells in comparison to
untargeted IL-15(D30N/E64Q/N65D)/R.alpha.-Fc fusion (as well as
control RSV-targeted IL-15/R.alpha.-Fc fusion).
[0091] FIG. 32 depicts induction of A) CD8 memory T cell and B) CD8
naive T cell proliferation by LAG-3-targeted IL-15/R.alpha.-Fc
fusions (and controls) as indicated by cell counts. The data show
that LAG-3-targeted IL-15/R.alpha.-Fc fusions are much more potent
in expanding CD8 memory T cells in comparison to untargeted
IL-15(D30N/E64Q/N65D)/R.alpha.-Fc fusion (as well as control
RSV-targeted IL-15/R.alpha.-Fc fusion).
[0092] FIG. 33 depicts induction of A) CD4 memory T cell and B) CD4
naive T cell proliferation by LAG-3-targeted IL-15/R.alpha.-Fc
fusions (and controls) as indicated by percentage proliferating
cells (determined based on CFSE dilution). The data show that
LAG-3-targeted IL-15/R.alpha.-Fc fusions are more potent in
expanding CD4 memory T cells in comparison to untargeted
IL-15(D30N/E64Q/N65D)/R.alpha.-Fc fusion (as well as control
RSV-targeted IL-15/R.alpha.-Fc fusion).
[0093] FIG. 34 depicts induction of A) CD4 memory T cell and B) CD4
naive T cell proliferation by LAG-3-targeted IL-15/R.alpha.-Fc
fusions (and controls) as indicated by cell counts.
[0094] FIG. 35 depicts induction of NK cells proliferation by
LAG-3-targeted IL-15/R.alpha.-Fc fusions (and controls) as
indicated A) percentage proliferating cells (determined based on
CFSE dilution) and B) by cell counts.
[0095] FIG. 36 depicts activation of CD8.sup.+ T cells as indicated
by A) percentage CD8 memory T cells expressing CD25, B) percentage
CD8 naive T cells expressing CD25, C) percentage CD4 memory T cells
expressing CD25, and D) percentage CD4 naive T cells expressing
CD25 following incubation with LAG-3-targeted IL-15/R.alpha.-Fc
fusions (and controls). The data show that LAG-3-targeted
IL-15/R.alpha.-Fc fusions, in particular XENP27972, appear to
upregulate CD25 in both CD8 and CD4 memory T cells in comparison to
untargeted IL-15(D30N/E64Q/N65D)/R.alpha.-Fc fusion (as well as
control RSV-targeted IL-15/R.alpha.-Fc fusion).
[0096] FIG. 37 depicts activation of CD8.sup.+ T cells as indicated
by A) HLA-DR MFI on CD8 memory T cells, B) percentage CD8 memory T
cells expressing HLA-DR, C) HLA-DR MFI on CD8 naive T cells, and D)
percentage CD8 naive T cells expressing HLA-DR following incubation
with LAG-3-targeted IL-15/R.alpha.-Fc fusions (and controls). The
data show that LAG-3-targeted IL-15/R.alpha.-Fc fusions appear to
upregulate HLA-DR in CD8 memory T cells more potently in comparison
to untargeted IL-15(D30N/E64Q/N65D)/R.alpha.-Fc fusion (as well as
control RSV-targeted IL-15/R.alpha.-Fc fusion).
[0097] FIG. 38 depicts activation of CD4.sup.+ T cells as indicated
by A) HLA-DR MFI on CD4 memory T cells, B) percentage CD4 memory T
cells expressing HLA-DR, C) HLA-DR MFI on CD4 naive T cells, and D)
percentage CD4 naive T cells expressing HLA-DR following incubation
with LAG-3-targeted IL-15/R.alpha.-Fc fusions (and controls).
[0098] FIG. 39 depicts the sequences of XENP22853, an
IL-15/R.alpha.-heteroFc fusion comprising a wild-type IL-15 and
Xtend Fc (M428L/N434S) variant. IL-15 and IL-15R.alpha.(sushi) are
underlined, linkers are double underlined (although as will be
appreciated by those in the art, the linkers can be replaced by
other linkers, some of which are depicted in the Figures, and
slashes (/) indicate the border(s) between IL-15, IL-15R.alpha.,
linkers, and constant/Fc regions.
[0099] FIG. 40 depicts the sequences of XENP24113, an
IL-15/R.alpha.-heteroFc fusion comprising an IL-15(N4D/N65D)
variant and Xtend Fc (M428L/N434S) variant. IL-15 and
IL-15R.alpha.(sushi) are underlined, linkers are double underlined
(although as will be appreciated by those in the art, the linkers
can be replaced by other linkers, some of which are depicted in the
Figures, and slashes (/) indicate the border(s) between IL-15,
IL-15R.alpha., linkers, and constant/Fc regions.
[0100] FIG. 41 depicts the sequences of XENP24294, an
scIL-15/R.alpha.-Fc fusion comprising an IL-15(N4D/N65D) variant
and Xtend Fc (M428L/N434S) substitution. IL-15 and
IL-15R.alpha.(sushi) are underlined, linkers are double underlined
(although as will be appreciated by those in the art, the linkers
can be replaced by other linkers, some of which are depicted in the
Figures, and slashes (/) indicate the border(s) between IL-15,
IL-15R.alpha., linkers, and constant/Fc regions.
[0101] FIG. 42 depicts the sequences of XENP24306, an
IL-15/R.alpha.-heteroFc fusion comprising an IL-15(D30N/E64Q/N65D)
variant and Xtend Fc (M428L/N434S) substitution. IL-15 and
IL-15R.alpha.(sushi) are underlined, linkers are double underlined
(although as will be appreciated by those in the art, the linkers
can be replaced by other linkers, some of which are depicted in the
Figures, and slashes (/) indicate the border(s) between IL-15,
IL-15R.alpha., linkers, and constant/Fc regions.
[0102] FIG. 43 depicts the serum concentration of the indicated
test articles over time in cynomolgus monkeys following a first
dose at the indicated relative concentrations.
[0103] FIGS. 44A-44C depict sequences of illustrative
scIL-15/R.alpha.-Fc fusions comprising additional IL-15 potency
variants. IL-15 and IL-15R.alpha.(sushi) are underlined, linkers
are double underlined (although as will be appreciated by those in
the art, the linkers can be replaced by other linkers, some of
which are depicted in Figures some of which are depicted in FIGS. 9
and 10), and slashes (/) indicate the border(s) between IL-15,
IL-15R.alpha., linkers, variable regions, and constant/Fc
regions.
[0104] FIGS. 45A-45G depicts percentage of A)
CD4.sup.+CD45RA.sup.-, B) CD4.sup.+CD45RA.sup.+, C)
CD8.sup.+CD45RA.sup.-, D) CD8.sup.+CD45RA.sup.+, E) CD16.sup.+ NK
cells, F) CD56.sup.+ NK cells, and G) .gamma..delta. cells
expression Ki67 following incubation of PBMCs with the indicated
test articles for 3 days.
[0105] FIGS. 46A and 46B depict sequences of illustrative
LAG-3-targeted IL-15/R.alpha.-Fc fusions comprising
IL-15(D30N/N65D) variant. The CDRs are in bold. As noted herein and
is true for every sequence herein containing CDRs, the exact
identification of the CDR locations may be slightly different
depending on the numbering used as is shown in Table 2, and thus
included herein are not only the CDRs that are underlined but also
CDRs included within the V.sub.H and V.sub.L domains using other
numbering systems. IL-15 and IL-15R.alpha.(sushi) are underlined,
linkers are double underlined (although as will be appreciated by
those in the art, the linkers can be replaced by other linkers,
some of which are depicted in FIGS. 9 and 10), and slashes (/)
indicate the border(s) between IL-15, IL-15R.alpha., linkers,
variable regions, and constant/Fc regions.
[0106] FIGS. 47A and 47B depicts sequences of illustrative
LAG-3-targeted IL-15/R.alpha.-Fc fusions comprising
IL-15(D30N/E64Q/N65D) variant. The CDRs are in bold. As noted
herein and is true for every sequence herein containing CDRs, the
exact identification of the CDR locations may be slightly different
depending on the numbering used as is shown in Table 2, and thus
included herein are not only the CDRs that are underlined but also
CDRs included within the VH and VL domains using other numbering
systems. IL-15 and IL-15R.alpha.(sushi) are underlined, linkers are
double underlined (although as will be appreciated by those in the
art, the linkers can be replaced by other linkers, some of which
are depicted in FIGS. 9 and 10), and slashes (/) indicate the
border(s) between IL-15, IL-15R.alpha., linkers, variable regions,
and constant/Fc regions.
[0107] FIGS. 48A-48D depict sequences of illustrative
LAG-3-targeted IL-15/R.alpha.-Fc fusions comprising Xtend
(M428L/N434S) substitutions for enhancing serum half-life. The CDRs
are in bold. As noted herein and is true for every sequence herein
containing CDRs, the exact identification of the CDR locations may
be slightly different depending on the numbering used as is shown
in Table 2, and thus included herein are not only the CDRs that are
underlined but also CDRs included within the V.sub.H and V.sub.L
domains using other numbering systems. IL-15 and
IL-15R.alpha.(sushi) are underlined, linkers are double underlined
(although as will be appreciated by those in the art, the linkers
can be replaced by other linkers, some of which are depicted in
FIGS. 9 and 10), and slashes (/) indicate the border(s) between
IL-15, IL-15R.alpha., linkers, variable regions, and constant/Fc
regions. It should be noted that any of the sequences depicted
herein may include or exclude the M428L/N434S substitutions.
[0108] FIGS. 49A and 49B depict the sequences of XENP26007,
XENP29481, and XENP30432, control RSV-targeted IL-15/R.alpha.-Fc
fusions. The CDRs are underlined. As noted herein and is true for
every sequence herein containing CDRs, the exact identification of
the CDR locations may be slightly different depending on the
numbering used as is shown in Table 2, and thus included herein are
not only the CDRs that are underlined but also CDRs included within
the V.sub.H and V.sub.L domains using other numbering systems.
IL-15 and IL-15R.alpha.(sushi) are italicized, linkers are double
underlined (although as will be appreciated by those in the art,
the linkers can be replaced by other linkers, some of which are
depicted in Figures some of which are depicted in FIGS. 9 and 10),
and slashes (/) indicate the border(s) between IL-15,
IL-15R.alpha., linkers, variable regions, and constant/Fc
regions.
DETAILED DESCRIPTION
I. DEFINITIONS
[0109] In order that the application may be more completely
understood, several definitions are set forth below. Such
definitions are meant to encompass grammatical equivalents.
[0110] By "ablation" herein is meant a decrease or removal of
activity. Thus for example, "ablating Fc.gamma.R binding" means the
Fc region amino acid variant has less than 50% starting binding as
compared to an Fc region not containing the specific variant, with
less than 70-80-90-95-98% loss of activity being preferred, and in
general, with the activity being below the level of detectable
binding in a Biacore assay. Of particular use in the ablation of
Fc.gamma.R binding are those shown in FIG. 6. However, unless
otherwise noted, the Fc monomers of the invention retain binding to
the FcRn receptor.
[0111] By "ADCC" or "antibody dependent cell-mediated cytotoxicity"
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell. ADCC is correlated with binding to Fc.gamma.RIIIa;
increased binding to Fc.gamma.RIIIa leads to an increase in ADCC
activity. As is discussed herein, many embodiments of the invention
ablate ADCC activity entirely.
[0112] By "ADCP" or antibody dependent cell-mediated phagocytosis
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause phagocytosis
of the target cell.
[0113] By "antigen binding domain" or "ABD" herein is meant a set
of six Complementary Determining Regions (CDRs) that, when present
as part of a polypeptide sequence, specifically binds a target
antigen as discussed herein. Thus, a "LAG-3 antigen binding domain"
binds a human LAG-3 antigen as outlined herein. As is known in the
art, these CDRs are generally present as a first set of variable
heavy CDRs (vhCDRs or V.sub.HCDRs) and a second set of variable
light CDRs (vlCDRs or V.sub.LCDRs), each comprising three CDRs:
vhCDR1, vhCDR2, vhCDR3 for the heavy chain and vlCDR1, vlCDR2 and
vlCDR3 for the light. The CDRs are present in the variable heavy
and variable light domains, respectively, and together form an Fv
region. Thus, in some cases, the six CDRs of the antigen binding
domain are contributed by a variable heavy and variable light
chain. In a "Fab" format, the set of 6 CDRs are contributed by two
different polypeptide sequences, the variable heavy domain (VH or
vh or V.sub.H; containing the vhCDR1, vhCDR2 and vhCDR3) and the
variable light domain (VL or vl or V.sub.L; containing the vlCDR1,
vlCDR2 and vlCDR3), with the C-terminus of the VH domain being
attached to the N-terminus of the CH1 domain of the heavy chain and
the C-terminus of the vl domain being attached to the N-terminus of
the constant light domain (and thus forming the light chain). In a
scFv format, the VH and VL domains are covalently attached,
generally through the use of a linker as outlined herein, into a
single polypeptide sequence, which can be either (starting from the
N-terminus) VH-linker-VL or VL-linker-VH, with the former being
generally preferred (including optional domain linkers on each
side, depending on the format used (e.g., from FIG. 1 of U.S.
62/353,511).
[0114] By "modification" herein is meant an amino acid
substitution, insertion, and/or deletion in a polypeptide sequence
or an alteration to a moiety chemically linked to a protein. For
example, a modification may be an altered carbohydrate or PEG
structure attached to a protein. By "amino acid modification"
herein is meant an amino acid substitution, insertion, and/or
deletion in a polypeptide sequence. For clarity, unless otherwise
noted, the amino acid modification is always to an amino acid coded
for by DNA, e.g., the 20 amino acids that have codons in DNA and
RNA.
[0115] By "amino acid substitution" or "substitution" herein is
meant the replacement of an amino acid at a particular position in
a parent polypeptide sequence with a different amino acid. In
particular, in some embodiments, the substitution is to an amino
acid that is not naturally occurring at the particular position,
either not naturally occurring within the organism or in any
organism. For example, the substitution E272Y refers to a variant
polypeptide, in this case an Fc variant, in which the glutamic acid
at position 272 is replaced with tyrosine. For clarity, a protein
which has been engineered to change the nucleic acid coding
sequence but not change the starting amino acid (for example
exchanging CGG (encoding arginine) to CGA (still encoding arginine)
to increase host organism expression levels) is not an "amino acid
substitution"; that is, despite the creation of a new gene encoding
the same protein, if the protein has the same amino acid at the
particular position that it started with, it is not an amino acid
substitution.
[0116] By "amino acid insertion" or "insertion" as used herein is
meant the addition of an amino acid sequence at a particular
position in a parent polypeptide sequence. For example, -233E or
233E designates an insertion of glutamic acid after position 233
and before position 234. Additionally, -233ADE or A233ADE
designates an insertion of AlaAspGlu after position 233 and before
position 234.
[0117] By "amino acid deletion" or "deletion" as used herein is
meant the removal of an amino acid sequence at a particular
position in a parent polypeptide sequence. For example, E233- or
E233#, E233( ) or E233del designates a deletion of glutamic acid at
position 233. Additionally, EDA233- or EDA233# designates a
deletion of the sequence GluAspAla that begins at position 233.
[0118] By "variant protein" or "protein variant", or "variant" as
used herein is meant a protein that differs from that of a parent
protein by virtue of at least one amino acid modification. Protein
variant may refer to the protein itself, a composition comprising
the protein, or the amino sequence that encodes it. Preferably, the
protein variant has at least one amino acid modification compared
to the parent protein, e.g., from about one to about seventy amino
acid modifications, and preferably from about one to about five
amino acid modifications compared to the parent. As described
below, in some embodiments the parent polypeptide, for example an
Fc parent polypeptide, is a human wild type sequence, such as the
Fc region from IgG1, IgG2, IgG3 or IgG4. The protein variant
sequence herein will preferably possess at least about 80% identity
with a parent protein sequence, and most preferably at least about
90% identity, more preferably at least about 95-98-99% identity.
Variant protein can refer to the variant protein itself,
compositions comprising the protein variant, or the DNA sequence
that encodes it.
[0119] "Variant," as used herein can also refer to particular amino
acid modifications (e.g., substitutions, deletions, insertions) in
a variant protein (e.g., a variant Fc domain), for example,
heterodimerization variants, ablation variants, FcKO variants,
etc., as disclosed in Section II.C. below.
[0120] Accordingly, by "Fc variant" or "variant Fc" as used herein
is meant a protein comprising an amino acid modification in an Fc
domain. The Fc variants of the present invention are defined
according to the amino acid modifications that compose them. Thus,
for example, N434S or 434S is an Fc variant with the substitution
serine at position 434 relative to the parent Fc polypeptide,
wherein the numbering is according to the EU index. Likewise,
M428L/N434S defines an Fc variant with the substitutions M428L and
N434S relative to the parent Fc polypeptide. The identity of the WT
amino acid may be unspecified, in which case the aforementioned
variant is referred to as 428L/434S. It is noted that the order in
which substitutions are provided is arbitrary, that is to say that,
for example, 428L/434S is the same Fc variant as M428L/N434S, and
so on. For all positions discussed in the present invention that
relate to antibodies, unless otherwise noted, amino acid position
numbering is according to the EU index. The EU index or EU index as
in Kabat or EU numbering scheme refers to the numbering of the EU
antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85,
hereby entirely incorporated by reference). The modification can be
an addition, deletion, or substitution. Substitutions can include
naturally occurring amino acids and, in some cases, synthetic amino
acids. Examples include U.S. Pat. No. 6,586,207; WO 98/48032; WO
03/073238; US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W.
Chin et al., (2002), Journal of the American Chemical Society
124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem
11:1135-1137; J. W. Chin, et al., (2002), PICAS United States of
America 99:11020-11024; and, L. Wang, & P. G. Schultz, (2002),
Chem. 1-10, all entirely incorporated by reference.
[0121] As used herein, "protein" herein is meant at least two
covalently attached amino acids, which includes proteins,
polypeptides, oligopeptides and peptides.
[0122] By "residue" as used herein is meant a position in a protein
and its associated amino acid identity. For example, Asparagine 297
(also referred to as Asn297 or N297) is a residue at position 297
in the human antibody IgG1.
[0123] By "Fab" or "Fab region" as used herein is meant the
polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin
domains. Fab may refer to this region in isolation, or this region
in the context of a full-length antibody, antibody fragment or Fab
fusion protein.
[0124] By "Fv" or "Fv fragment" or "Fv region" as used herein is
meant a polypeptide that comprises the VL and VH domains of a
single antibody. As will be appreciated by those in the art, these
generally are made up of two chains, or can be combined (generally
with a linker as discussed herein) to form an scFv.
[0125] By "single chain Fv" or "scFv" herein is meant a variable
heavy domain covalently attached to a variable light domain,
generally using a scFv linker as discussed herein, to form a scFv
or scFv domain. A scFv domain can be in either orientation from N-
to C-terminus (VH-linker-VL or VL-linker-VH).
[0126] By "IgG subclass modification" or "isotype modification" as
used herein is meant an amino acid modification that converts one
amino acid of one IgG isotype to the corresponding amino acid in a
different, aligned IgG isotype. For example, because IgG1 comprises
a tyrosine and IgG2 a phenylalanine at EU position 296, a F296Y
substitution in IgG2 is considered an IgG subclass
modification.
[0127] By "non-naturally occurring modification" as used herein is
meant an amino acid modification that is not isotypic. For example,
because none of the IgGs comprise a serine at position 434, the
substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof)
is considered a non-naturally occurring modification.
[0128] By "amino acid" and "amino acid identity" as used herein is
meant one of the 20 naturally occurring amino acids that are coded
for by DNA and RNA.
[0129] By "effector function" as used herein is meant a biochemical
event that results from the interaction of an antibody Fc region
with an Fc receptor or ligand. Effector functions include but are
not limited to ADCC, ADCP, and CDC.
[0130] By "Fc gamma receptor", "Fc.gamma.R" or "FcgammaR" as used
herein is meant any member of the family of proteins that bind the
IgG antibody Fc region and is encoded by an Fc.gamma.R gene. In
humans this family includes but is not limited to Fc.gamma.RI
(CD64), including isoforms Fc.gamma.RIa, Fc.gamma.RIb, and
Fc.gamma.RIc; Fc.gamma.RII (CD32), including isoforms Fc.gamma.RIIa
(including allotypes H131 and R131), Fc.gamma.RIIb (including
Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2), and Fc.gamma.RIIc; and
Fc.gamma.RIII (CD16), including isoforms Fc.gamma.RIIIa (including
allotypes V158 and F158) and Fc.gamma.RIIIb (including allotypes
Fc.gamma.RIIb-NA1 and Fc.gamma.RIIb-NA2) (Jefferis et al., 2002,
Immunol Lett 82:57-65, entirely incorporated by reference), as well
as any undiscovered human Fc.gamma.Rs or Fc.gamma.R isoforms or
allotypes.
[0131] By "FcRn" or "neonatal Fc Receptor" as used herein is meant
a protein that binds the IgG antibody Fc region and is encoded at
least in part by an FcRn gene. As is known in the art, the
functional FcRn protein comprises two polypeptides, often referred
to as the heavy chain and light chain. The light chain is
beta-2-microglobulin and the heavy chain is encoded by the FcRn
gene. Unless otherwise noted herein, FcRn or an FcRn protein refers
to the complex of FcRn heavy chain with beta-2-microglobulin. A
variety of FcRn variants can be used to increase binding to the
FcRn receptor, and in some cases, to increase serum half-life. In
general, unless otherwise noted, the Fc monomers of the invention
retain binding to the FcRn receptor (and, as noted below, can
include amino acid variants to increase binding to the FcRn
receptor).
[0132] By "parent polypeptide" as used herein is meant a starting
polypeptide that is subsequently modified to generate a variant.
The parent polypeptide may be a naturally occurring polypeptide, or
a variant or engineered version of a naturally occurring
polypeptide. Parent polypeptide may refer to the polypeptide
itself, compositions that comprise the parent polypeptide, or the
amino acid sequence that encodes it.
[0133] By "Fc" or "Fc region" or "Fc domain" as used herein is
meant the polypeptide comprising the constant region of an antibody
excluding the first constant region immunoglobulin domain (e.g.,
CH1) and in some cases, part of the hinge. For IgG, the Fc domain
comprises immunoglobulin domains CH2 and CH3 (C.gamma.2 and
C.gamma.3) and the lower hinge region between CH1 (C.gamma.1) and
CH2 (C.gamma.2). Thus, in some cases, the Fc domain includes, from
N- to C-terminal, CH2-CH3 and hinge-CH2-CH3. In some embodiments,
the Fc domain is that from IgG1, IgG2, IgG3 or IgG4, with IgG1
hinge-CH2-CH3 and IgG4 hinge-CH2-CH3 finding particular use in many
embodiments. Additionally, in certain embodiments, wherein the Fc
domain is a human IgG1 Fc domain, the hinge includes a C220S amino
acid substitution. Furthermore, in some emboidments where the Fc
domain is a human IgG4 Fc domain, the hinge includes a S228P amino
acid substitution. Although the boundaries of the Fc region may
vary, the human IgG heavy chain Fc region is usually defined to
include residues C226 or P230 to its carboxyl-terminus, wherein the
numbering is according to the EU index as in Kabat. Accordingly,
"CH" domains in the context of IgG are as follows: "CH1" refers to
positions 118-215 according to the EU index as in Kabat. "Hinge"
refers to positions 216-230 according to the EU index as in Kabat.
"CH2" refers to positions 231-340 according to the EU index as in
Kabat, and "CH3" refers to positions 341-447 according to the EU
index as in Kabat. Thus, the "Fc domain" includes the -CH2-CH3
domain, and optionally a hinge domain (hinge-CH2-CH3).
[0134] As will be appreciated by those in the art, the exact
numbering and placement of the heavy constant region domains can be
different among different numbering systems. A useful comparison of
heavy constant region numbering according to EU and Kabat is as
below, see Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85
and Kabat et al., 1991, Sequences of Proteins of Immunological
Interest, 5th Ed., United States Public Health Service, National
Institutes of Health, Bethesda, entirely incorporated by
reference.
TABLE-US-00001 TABLE 1 EU Numbering Kabat Numbering CH1 118-215
114-223 Hinge 216-230 226-243 CH2 231-340 244-360 CH3 341-447
361-478
[0135] In the embodiments herein, when a scFv or IL-15 complex is
attached to an Fc domain, it is the C-terminus of the scFv, IL-15
or IL-15R.alpha. construct that is attached to the Fc domain via a
domain linker; for example, a hinge domain as depicted in FIG. 8.
In some embodiments, as is more fully described below, amino acid
modifications are made to the Fc region, for example to alter
binding to one or more Fc.gamma.R receptors or to the FcRn
receptor, and to enable heterodimer formation and purification, as
outlined herein.
[0136] By "heavy constant region" herein is meant the
CH1-hinge-CH2-CH3 portion of an antibody.
[0137] By "Fc fusion protein" or "immunoadhesin" herein is meant a
protein comprising an Fc region, generally linked (optionally
through a linker moiety, as described herein) to a different
protein, such as to IL-15 and/or IL-15R, as described herein. In
some instances, two Fc fusion proteins can form a homodimeric Fc
fusion protein or a heterodimeric fusion protein with the latter
being preferred. In some cases, one monomer of the heterodimeric
fusion protein comprises an Fc domain alone (e.g., an empty Fc
domain) and the other monomer is a Fc fusion, comprising a variant
Fc domain and a protein domain, such as a receptor, ligand or other
binding partner.
[0138] By "position" as used herein is meant a location in the
sequence of a protein. Positions may be numbered sequentially, or
according to an established format, for example the EU index for
antibody numbering.
[0139] By "strandedness" in the context of the monomers of the
heterodimeric antibodies of the invention herein is meant that,
similar to the two strands of DNA that "match", heterodimerization
variants are incorporated into each monomer so as to preserve the
ability to "match" to form heterodimers. For example, if some pI
variants are engineered into monomer A (e.g., making the pI higher)
then steric variants that are "charge pairs" that can be utilized
as well do not interfere with the pI variants, e.g., the charge
variants that make a pI higher are put on the same "strand" or
"monomer" to preserve both functionalities. Similarly, for "skew"
variants that come in pairs of a set as more fully outlined below,
the skilled artisan will consider pI in deciding into which strand
or monomer that incorporates one set of the pair will go, such that
pI separation is maximized using the pI of the skews as well.
[0140] By "target cell" as used herein is meant a cell that
expresses the target antigen, in this case, LAG-3.
[0141] By "variable region" as used herein is meant the region of
an immunoglobulin that comprises one or more Ig domains
substantially encoded by any of the V.kappa., V.lamda., and/or VH
genes that make up the kappa, lambda, and heavy chain
immunoglobulin genetic loci respectively.
[0142] By "wild type or WT" herein is meant an amino acid sequence
or a nucleotide sequence that is found in nature, including allelic
variations. A WT protein has an amino acid sequence or a nucleotide
sequence that has not been intentionally modified.
[0143] The subject LAG-3 targeted heterodimeric proteins are
generally isolated or recombinant. "Isolated," when used to
describe the various polypeptides disclosed herein, means a
polypeptide that has been identified and separated and/or recovered
from a cell or cell culture from which it was expressed.
Ordinarily, an isolated polypeptide will be prepared by at least
one purification step. An "isolated protein," refers to a protein
which is substantially free of other proteins having different
binding specificities. "Recombinant" means the proteins are
generated using recombinant nucleic acid techniques in exogeneous
host cells.
[0144] "Percent (%) amino acid sequence identity" with respect to a
protein sequence is defined as the percentage of amino acid
residues in a candidate sequence that are identical with the amino
acid residues in the specific (parental) 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. One particular program is the ALIGN-2 program
outlined at paragraphs [0279] to [0280] of US Pub. No. 20160244525,
hereby incorporated by reference.
[0145] The degree of identity between an amino acid sequence of the
present invention ("invention sequence") and the parental amino
acid sequence is calculated as the number of exact matches in an
alignment of the two sequences, divided by the length of the
"invention sequence," or the length of the parental sequence,
whichever is the shortest. The result is expressed in percent
identity.
[0146] In some embodiments, two or more amino acid sequences are at
least 50%, 60%, 70%, 80%, or 90% identical. In some embodiments,
two or more amino acid sequences are at least 95%, 97%, 98%, 99%,
or even 100% identical.
[0147] "Specific binding" or "specifically binds to" or is
"specific for" a particular antigen or an epitope (in this case,
human LAG-3) means binding that is measurably different from a
non-specific interaction. Specific binding can be measured, for
example, by determining binding of a molecule compared to binding
of a control molecule, which generally is a molecule of similar
structure that does not have binding activity. For example,
specific binding can be determined by competition with a control
molecule that is similar to the target.
[0148] Specific binding for a particular antigen or an epitope can
be exhibited, for example, by an antibody having a KD for an
antigen or epitope of at least about 10.sup.-4 M, at least about
10.sup.-5 M, at least about 10.sup.-6 M, at least about 10.sup.-7
M, at least about 10.sup.-8M, at least about 10.sup.-9 M,
alternatively at least about 10.sup.-10 M, at least about
10.sup.-13M, at least about 10.sup.-12M, or greater, where KD
refers to a dissociation rate of a particular antibody-antigen
interaction. Typically, an antibody that specifically binds an
antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-,
10,000- or more times greater for a control molecule relative to
the antigen or epitope.
[0149] Also, specific binding for a particular antigen or an
epitope can be exhibited, for example, by an antibody having a KA
or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-,
1000-, 5,000-, 10,000- or more times greater for the epitope
relative to a control, where KA or Ka refers to an association rate
of a particular antibody-antigen interaction. Binding affinity is
generally measured using a Biacore assay.
II. INTRODUCTION
[0150] The invention provides heterodimeric fusion proteins that
contain an IL-15 complex on one side and an anti-human LAG-3
antigen binding domain on the other. Thus, the heterodimeric fusion
proteins of the invention can bind to the checkpoint LAG-3 antigen
and can complex with the common gamma chain (.gamma.c; CD132)
and/or the IL-2 receptor .beta.-chain (IL-2R.beta.; CD122). In
general, the heterodimeric fusion proteins of the invention have
three functional components: an IL-15/IL-15R.alpha.(sushi)
component, generally referred to herein as an "IL-15 complex", an
anti-LAG-3 ABD component which serves as a "targeting" moiety by
bringing the fusion protein to a cell expressing LAG-3, and an Fc
component, each of which can take different forms and each of which
can be combined with the other components in any configuration.
[0151] In general, as is more fully described herein, the fusion
proteins of the invention are heterodimeric proteins that are based
on the association of antibody Fc domains. That is, by using two
different variant Fc domains that have been engineered to favor the
formation of heterodimers over homodimers, the heterodimeric
proteins are formed. In this case, one of the variant Fc domains is
fused to an IL-15/RA complex and the other has a LAG-3 ABD as more
fully outlined herein. By including optional pI variants, the
heterodimers can be more easily purified away from the homodimers.
Additionally, the inclusion of ablation variants eliminates the
effector functions of the Fc domains.
A. IL-15/IL-15R.alpha.(Sushi) Domains
[0152] As shown in the figures, the IL-15 complex can take several
forms. As stated above, the IL-15 protein on its own is less stable
than when complexed with the IL-15R.alpha. protein. As is known in
the art, the IL-15R.alpha. protein contains a "sushi domain", which
is the shortest region of the receptor that retains IL-15 binding
activity. Thus, while heterodimeric fusion proteins comprising the
entire IL-15R.alpha. protein can be made, preferred embodiments
herein include complexes that just use the sushi domain, the
sequence of which is shown in the figures.
[0153] Accordingly, the IL-15 complex generally comprises the IL-15
protein and the sushi domain of IL IL-15R.alpha. (unless otherwise
noted that the full length sequence is used, "IL-15R.alpha.",
"IL-15R.alpha.(sushi)", "IL-15RA" and "sushi" are used
interchangeably throughout).
[0154] Importantly, the IL-15 component is generally engineered to
reduce its potency. In many embodiments, the wild-type IL-15 is too
potent and can cause undesirable toxicity. Accordingly, the IL-15
component of the IL-15 complex can have one or more amino acid
substitutions that result in decreased activity. Various amino acid
substitutions were made (see FIG. 19) and tested (see FIG. 20). Of
particular interest in some embodiments are a double variant,
N4D/N65D or D30N/N65D, or a triple variant, D30N/E64Q/N65D.
[0155] The targeted IL-15/IL-15R.alpha. heterodimeric fusion
proteins of the present invention include an IL-15/IL-15 receptor
alpha (IL-15R.alpha.)-Fc fusion monomer; reference is made to
US2018/0118828, filed 16, October 2017, U.S. Ser. No. 62/408,655,
filed on Oct. 14, 2016, U.S. Ser. No. 62/416,087, filed on October
Nov. 1, 2016, U.S. Ser. No. 62/443,465, filed on Jan. 6, 2017, U.S.
Ser. No. 62/477,926, filed on Mar. 28, 2017, and U.S. Ser. No.
62/659,571, filed on Apr. 18, 2018, hereby incorporated by
reference in their entirety and in particular for the sequences
outlined therein. In some cases, the IL-15 and IL-15 receptor alpha
(IL-15R.alpha.) protein domains are in different orientations.
Exemplary embodiments of IL-15/IL-15R.alpha.-Fc fusion monomers are
provided in XENP21480 (chain 1; FIG. 64A), XENP22022 (chain 1, FIG.
64D), XENP22112, (chains 1 and 3; FIG. 64E), XENP22641 (chains 2
and 4; FIG. 64F), XENP22642, (chains 1 and 4; FIG. 64H) and
XENP22644 (chains 1 and 4; FIG. 64I) as described, for example, in
US 2018/0118828.
[0156] 1. IL-15 Variants
[0157] In some embodiments, the human IL-15 protein has the amino
acid sequence set forth in NCBI Ref. Seq. No. NP_000576.1 as shown
in FIG. 2. In some cases, the coding sequence of human IL-15 is set
forth in NCBI Ref. Seq. No. NM_000585. An exemplary IL-15 protein
of the Fc fusion heterodimeric protein outlined herein can have the
amino acid sequence of SEQ ID NO:2 or amino acids 49-162 of SEQ ID
NO:1. In some embodiments, the IL-15 protein has at least 90%,
e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more
sequence identity to SEQ ID NO:2. In some embodiments, the IL-15
protein has the amino acid sequence set forth in SEQ ID NO:2 except
with the amino acid substitution N72D. In other embodiments, the
IL-15 protein has the amino acid sequence of SEQ ID NO:2 except
with one or more amino acid substitutions selected from the group
consisting of C42S, L45C, Q48C, V49C, L52C, E53C, E87C, and E89C.
In some aspects, the IL-15 protein has one or more amino acid
substitutions selected from the group consisting of N1D, N4D, D8N,
D30N, D61N, E64Q, N65D, and Q108E. In other embodiments, the amino
acid substitutions are N4D/N65D. In certain embodiments, the amino
acid substitutions are D30N/N65D. In some embodiments, the amino
acid substitution is Q108E. In certain embodiments, the amino acid
substitution is N65D. In other embodiments, the amino acid
substitutions are D30N/E64Q/N65D. In certain embodiments, the amino
acid substitution is N65D. In some instances, the amino acid
substitutions are N1D/N65D. In some instances, the amino acid
substitutions are D30N/N65D. Optionally, the IL-15 protein also has
an N72D substitution. The IL-15 protein of the Fc fusion protein
can have 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions. In
some embodiments, the IL-15 protein of the Fc fusion protein
comprises a D30N substitution. In some embodiments, the IL-15
protein of the Fc fusion protein comprises a N65D substitution. In
some embodiments, the IL-15 protein of the Fc fusion contains one
or more amino acid substitutions at the IL-15:CD132 interface. In
certain embodiments, the Fc fusion protein described herein induces
proliferation of NK cells and CD8+ T cells. Additionally, IL-15
variants that can be used with the subject targeted
IL-15/IL-15R.alpha. heterodimer proteins are included in FIGS.
19A-C.
[0158] In some embodiments, the human IL-15 receptor alpha
(IL-15R.alpha.) protein has the amino acid sequence set forth in
NCBI Ref. Seq. No. NP_002180.1 or SEQ ID NO:3. In some cases, the
coding sequence of human IL-15R.alpha. is set forth in NCBI Ref.
Seq. No. NM_002189.3. An exemplary the IL-15R.alpha. protein of the
Fc fusion heterodimeric protein outlined herein can comprise or
consist of the sushi domain of SEQ ID NO:3 (e.g., amino acids 31-95
of SEQ ID NO:3), or in other words, the amino acid sequence of SEQ
ID NO:4. In some embodiments, the IL-15R.alpha. protein has the
amino acid sequence of SEQ ID NO:4 and an amino acid insertion
selected from the group consisting of D96, P97, A98, D96/P97,
D96/C97, D96/P97/A98, D96/P97/C98, and D96/C97/A98, wherein the
amino acid position is relative to full-length human IL-15R.alpha.
protein or SEQ ID NO:3. For instance, amino acid(s) such as D
(e.g., Asp), P (e.g., Pro), A (e.g., Ala), DP (e.g., Asp-Pro), DC
(e.g., Asp-Cys), DPA (e.g., Asp-Pro-Ala), DPC (e.g., Asp-Pro-Cys),
or DCA (e.g., Asp-Cys-Ala) can be added to the C-terminus of the
IL-15R.alpha. protein of SEQ ID NO:4. In some embodiments, the
IL-15R.alpha. protein has the amino acid sequence of SEQ ID NO:4
and one or more amino acid substitutions selected from the group
consisting of K34C, A37C, G38C, S40C, and L42C, wherein the amino
acid position is relative to SEQ ID NO:4. The IL-15R.alpha. protein
can have 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid mutations (e.g.,
substitutions, insertions and/or deletions).
[0159] 2. IL-15/RA Complexes
[0160] As outlined herein, the IL-15 variants and the sushi domain
can be complexed in at least three different ways to form an IL-15
complex.
[0161] In some embodiments, as shown in FIG. 21B, for example, the
IL-15 protein and the IL-15R.alpha.(sushi) are not covalently
attached, but rather are self-assembled through regular
ligand-ligand interactions. As is more fully described herein, it
can be either the IL-15 domain or the sushi domain that is
covalently linked to the Fc domain (generally using an optional
domain linker). Again, of particular use in this embodiment are a
double variant, N4D/N65D or D30N/N65D, or a triple variant,
D30N/E64Q/N65D, used with a wild type sushi domain.
[0162] In alternative embodiments, the variant IL-15 can be
complexed to the sushi domain using a domain linker, such that they
are covalently attached as generally shown in FIG. 21D; this figure
depicts the sushi domain as the N-terminal domain, although this
can be reversed. Again, of particular use in this embodiment are a
double variant, N4D/N65D or D30N/N65D, or a triple variant,
D30N/E64Q/N65D, used with a wild type sushi domain. Exemplary
domain linkers that can be used to attach the IL-15 variant to the
sushi domain are depicted in FIG. 8.
[0163] Alternatively, each of the IL-15 and sushi domains can be
engineered to contain a cysteine amino acid, that forms a disulfide
bond to form the complex as is generally shown in FIG. 21C, again,
with either the IL-15 domain or the sushi domain being covalently
attached (using an optional domain linker) to the Fc domain. Again,
of particular use in this embodiment are a double variant, N4D/N65D
or D30N/N65D, (additionally including an amino acid substitution to
cysteine), or a triple variant, D30N/E64Q/N65D (additionally
including an amino acid substitution to cysteine), used with a
sushi domain also comprising an amino acid substitution to provide
a cysteine.
[0164] Additional particular embodiments are outlined below.
B. Anti-LAG-3 Components
[0165] In some embodiments, the heterodimeric fusion proteins
provided herein include some antibody components.
[0166] Traditional antibody structural units typically comprise a
tetramer. Each tetramer is typically composed of two identical
pairs of polypeptide chains, each pair having one "light"
(typically having a molecular weight of about 25 kDa) and one
"heavy" chain (typically having a molecular weight of about 50-70
kDa). Human light chains are classified as kappa and lambda light
chains. The present invention is directed to antibodies or antibody
fragments (antibody monomers) that generally are based on the IgG
class, which has several subclasses, including, but not limited to
IgG1, IgG2, IgG3, and IgG4. In general, IgG1, IgG2 and IgG4 are
used more frequently than IgG3. It should be noted that IgG1 has
different allotypes with polymorphisms at 356 (D or E) and 358 (L
or M). The sequences depicted herein use the 356D/358M allotype,
however the other allotype is included herein. That is, any
sequence inclusive of an IgG1 Fc domain included herein can have
356E/358L replacing the 356D/358M allotype.
[0167] In addition, many of the monomer sequences herein have at
least one the cysteines at position 220 replaced by a serine, to
reduce disulfide formation. Specifically included within the
sequences herein are one or both of these cysteines replaced
(C220S).
[0168] Thus, "isotype" as used herein is meant any of the
subclasses of immunoglobulins defined by the chemical and antigenic
characteristics of their constant regions.
[0169] The amino-terminal portion of each chain includes a variable
region of about 100 to 110 or more amino acids primarily
responsible for antigen recognition, generally referred to in the
art and herein as the "Fv domain" or "Fv region". In the variable
region, three loops are gathered for each of the V domains of the
heavy chain and light chain to form an antigen-binding site. Each
of the loops is referred to as a complementarity-determining region
(hereinafter referred to as a "CDR"), in which the variation in the
amino acid sequence is most significant. "Variable" refers to the
fact that certain segments of the variable region differ
extensively in sequence among antibodies. Variability within the
variable region is not evenly distributed. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-15 amino
acids long or longer.
[0170] Each VH and VL is composed of three hypervariable regions
("complementary determining regions," "CDRs") and four FRs,
arranged from amino-terminus to carboxy-terminus in the following
order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
[0171] The hypervariable region generally encompasses amino acid
residues from about amino acid residues 24-34 (LCDR1; "L" denotes
light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain
variable region and around about 31-35B (HCDR1; "H" denotes heavy
chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain
variable region; Kabat et al., SEQUENCES OF PROTEINS OF
IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991) and/or those residues
forming a hypervariable loop (e.g., residues 26-32 (LCDR1), 50-52
(LCDR2) and 91-96 (LCDR3) in the light chain variable region and
26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain
variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.
Specific CDRs of the invention are described below.
[0172] As will be appreciated by those in the art, the exact
numbering and placement of the CDRs can be different among
different numbering systems. However, it should be understood that
the disclosure of a variable heavy and/or variable light sequence
includes the disclosure of the associated (inherent) CDRs.
Accordingly, the disclosure of each variable heavy region is a
disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2 and vhCDR3) and the
disclosure of each variable light region is a disclosure of the
vlCDRs (e.g., vlCDR1, vlCDR2 and vlCDR3).
[0173] A useful comparison of CDR numbering is as below, see
Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003):
TABLE-US-00002 TABLE 2 Kabat + Chothia IMGT Kabat AbM Chothia
Contact Xencor vhCDR1 26-35 27-38 31-35 26-35 26-32 30-35 27-35
vhCDR2 50-65 56-65 50-65 50-58 52-56 47-58 54-61 vhCDR3 95-102
105-117 95-102 95-102 95-102 93-101 103-116 vlCDR1 24-34 27-38
24-34 24-34 24-34 30-36 27-38 vlCDR2 50-56 56-65 50-56 50-56 50-56
46-55 56-62 vlCDR3 89-97 105-117 89-97 89-97 89-97 89-96 97-105
[0174] Throughout the present specification, the Kabat numbering
system is generally used when referring to a residue in the
variable domain (approximately, residues 1-107 of the light chain
variable region and residues 1-113 of the heavy chain variable
region) and the EU numbering system for Fc regions (e.g., Kabat et
al., supra (1991)).
[0175] The present invention provides a large number of different
CDR sets. In this case, a "full CDR set" comprises the three
variable light and three variable heavy CDRs, e.g., a vlCDR1,
vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. These can be part of a
larger variable light or variable heavy domain, respectfully. In
addition, as more fully outlined herein, the variable heavy and
variable light domains can be on separate polypeptide chains, when
a heavy and light chain is used (for example when Fabs are used),
or on a single polypeptide chain in the case of scFv sequences.
[0176] The CDRs contribute to the formation of the antigen-binding,
or more specifically, epitope binding site of antibodies. "Epitope"
refers to a determinant that interacts with a specific antigen
binding site in the variable region of an antibody molecule known
as a paratope. Epitopes are groupings of molecules such as amino
acids or sugar side chains and usually have specific structural
characteristics, as well as specific charge characteristics. A
single antigen may have more than one epitope.
[0177] The epitope may comprise amino acid residues directly
involved in the binding (also called immunodominant component of
the epitope) and other amino acid residues, which are not directly
involved in the binding, such as amino acid residues which are
effectively blocked by the specifically antigen binding peptide; in
other words, the amino acid residue is within the footprint of the
specifically antigen binding peptide.
[0178] Epitopes may be either conformational or linear. A
conformational epitope is produced by spatially juxtaposed amino
acids from different segments of the linear polypeptide chain. A
linear epitope is one produced by adjacent amino acid residues in a
polypeptide chain. Conformational and nonconformational epitopes
may be distinguished in that the binding to the former but not the
latter is lost in the presence of denaturing solvents.
[0179] An epitope typically includes at least 3, and more usually,
at least 5 or 8-10 amino acids in a unique spatial conformation.
Antibodies that recognize the same epitope can be verified in a
simple immunoassay showing the ability of one antibody to block the
binding of another antibody to a target antigen, for example
"binning." As outlined below, the invention not only includes the
enumerated antigen binding domains and antibodies herein, but those
that compete for binding with the epitopes bound by the enumerated
antigen binding domains.
[0180] The carboxy-terminal portion of each chain defines a
constant region primarily responsible for effector function. Kabat
et al. collected numerous primary sequences of the variable regions
of heavy chains and light chains. Based on the degree of
conservation of the sequences, they classified individual primary
sequences into the CDR and the framework and made a list thereof
(see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH
publication, No. 91-3242, E. A. Kabat et al., entirely incorporated
by reference).
[0181] In the IgG subclass of immunoglobulins, there are several
immunoglobulin domains in the heavy chain. By "immunoglobulin (Ig)
domain" herein is meant a region of an immunoglobulin having a
distinct tertiary structure. Of interest in the present invention
are the heavy chain domains, including, the constant heavy (CH)
domains and the hinge domains. In the context of IgG antibodies,
the IgG isotypes each have three CH regions. Accordingly, "CH"
domains in the context of IgG are as follows: "CH1" refers to
positions 118-220 according to the EU index as in Kabat. "CH2"
refers to positions 237-340 according to the EU index as in Kabat,
and "CH3" refers to positions 341-447 according to the EU index as
in Kabat. As shown herein and described below, the pI variants can
be in one or more of the CH regions, as well as the hinge region,
discussed below.
[0182] Another type of Ig domain of the heavy chain is the hinge
region. By "hinge" or "hinge region" or "antibody hinge region" or
"immunoglobulin hinge region" herein is meant the flexible
polypeptide comprising the amino acids between the first and second
constant domains of an antibody. Structurally, the IgG CH1 domain
ends at EU position 220, and the IgG CH2 domain begins at residue
EU position 237. Thus for IgG the antibody hinge is herein defined
to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1),
wherein the numbering is according to the EU index as in Kabat. In
some embodiments, for example in the context of an Fc region, the
lower hinge is included, with the "lower hinge" generally referring
to positions 226 or 230. As noted herein, pI variants can be made
in the hinge region as well.
[0183] The light chain generally comprises two domains, the
variable light domain (containing the light chain CDRs and together
with the variable heavy domains forming the Fv region), and a
constant light chain region (often referred to as CL or
C.kappa.).
[0184] Another region of interest for additional substitutions,
outlined herein, is the Fc region.
[0185] Thus, the present heterodimeric fusion proteins provided
herein include one or more antibody domains. As described herein
and known in the art, the heterodimeric antibodies provided herein
comprise different domains within the heavy and light chains, which
can be overlapping as well. These domains include, but are not
limited to, the Fc domain, the CH1 domain, the CH2 domain, the CH3
domain, the hinge domain, the heavy constant domain (CH1-hinge-Fc
domain or CH1-hinge-CH2-CH3), the variable heavy domain, the
variable light domain, the light constant domain, Fab domains and
scFv domains.
[0186] As generally outlined herein, the heterodimeric proteins of
the invention include one or more LAG-3 antigen binding domains
(e.g., Fvs) that binds human LAG-3. "Lymphocyte-activation gene 3,"
"LAG-3," "LAG3," "CD223," and "duster of differentiation 3," (e.g.,
Genebank Accession Numbers NM_002286 and NP_002277 (human)) as used
herein is meant a member of an immunoglobulin (Ig) superfamily,
that is a type I transmembrane protein with four extracellular
Ig-like domains. LAG-3 is a negative regulator of T cells and
appears to work in concert with other checkpoint molecules,
including CTLA-4 and PD-1. In 1990, Triebel et al. discovered LAG3
as a transmembrane protein expressed by activated human natural
killer (NK) and T-cell lines, although it can also be expressed on
B cells. The human LAG3 gene has 20% identity with the human CD4
gene, resulting in surface-expressed LAG-3 protein being able to
bind major histocompatibility complex (MHC) class II molecules with
high affinity. LAG-3 either exists as a cell surface dimer or as a
soluble form released by interferon-gamma-producing CD4-positive T
cells via proteolytic cleavage of a membrane-proximal connecting
peptide. Exemplary LAG-3 sequences are depicted in FIG. 3.
[0187] This Fv, or anti-LAG-3 component (the anti-LAG-3 antigen
binding domain or LAG-3 ABD) of the subject heterodimer fusion
proteins is generally a set of 6 CDRs and/or a variable heavy
domain and a variable light domain that form an Fv domain that can
bind human LAG-3. As described herein, there are a number of
different formats that can be used, generally either by using a
scFv or a Fab as outlined herein.
[0188] In certain embodiments, the ABDs of the invention comprise a
heavy chain variable region with frameworks from a particular
germline heavy chain immunoglobulin gene and/or a light chain
variable region from a particular germline light chain
immunoglobulin gene. For example, such ABDs may comprise or consist
of a human ABD comprising heavy or light chain variable regions
that are "the product of" or "derived from" a particular germline
sequence. An ABD that is "the product of" or "derived from" a human
germline immunoglobulin sequence can be identified as such by
comparing the amino acid sequence of the ABD to the amino acid
sequences of human germline immunoglobulins and selecting the human
germline immunoglobulin sequence that is closest in sequence (i.e.,
greatest % identity) to the sequence of the ABD. An ABD that is
"the product of" or "derived from" a particular human germline
immunoglobulin sequence may contain amino acid differences as
compared to the germline sequence, due to, for example, CDRs,
naturally-occurring somatic mutations or intentional introduction
of site-directed mutation. However, a humanized ABD typically is at
least 90% identical in amino acids sequence to an amino acid
sequence encoded by a human germline immunoglobulin gene and
contains amino acid residues that identify the ABD as being derived
from human sequences when compared to the germline immunoglobulin
amino acid sequences of other species (e.g., murine germline
sequences). In certain cases, a humanized ABD may be at least 95,
96, 97, 98 or 99%, or even at least 96%, 97%, 98%, or 99% identical
in amino acid sequence to the amino acid sequence encoded by the
germline immunoglobulin gene. Typically, a humanized ABD derived
from a particular human germline sequence will display no more than
10-20 amino acid differences from the amino acid sequence encoded
by the human germline immunoglobulin gene (prior to the
introduction of any skew, pI and ablation variants herein; that is,
the number of variants is generally low, prior to the introduction
of the variants of the invention). In certain cases, the humanized
ABD may display no more than 5, or even no more than 4, 3, 2, or 1
amino acid difference from the amino acid sequence encoded by the
germline immunoglobulin gene (again, prior to the introduction of
any skew, pI and ablation variants herein; that is, the number of
variants is generally low, prior to the introduction of the
variants of the invention). In one embodiment, the parent ABD has
been affinity matured, as is known in the art. Structure-based
methods may be employed for humanization and affinity maturation,
for example as described in U.S. Ser. No. 11/004,590. Selection
based methods may be employed to humanize and/or affinity mature
antibody variable regions, including but not limited to methods
described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et
al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996,
J. Biol. Chem. 271(37): 22611-22618; Rader et al., 1998, Proc.
Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein
Engineering 16(10):753-759, all entirely incorporated by reference.
Other humanization methods may involve the grafting of only parts
of the CDRs, including but not limited to methods described in U.S.
Ser. No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125;
De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirely
incorporated by reference.
[0189] As shown herein, the anti-LAG-3 ABD can be in the form of
either a Fab or an scFv.
[0190] In some embodiments, for example as depicted in FIGS. 21B
and C, the anti-LAG-3 ABD is a scFv, wherein the VH and VL domains
are joined using an scFv linker, which can be optionally a charged
scFv linker. As will be appreciated by those in the art, the scFv
can be assembled from N- to C-terminus, as N-VH-scFv linker-VL-C or
as N-VL-scFv linker-VH-C, with the C terminus of the scFv domain
generally being linked to the hinge-CH2-CH3 Fc domain, wherein the
hinge in this case serving as a domain linker. Suitable Fvs
(including CDR sets and variable heavy/variable light domains) can
be used in scFv formats or Fab formats are shown in the Figures as
well as disclosed in WO2017/218707, the contents are hereby
incorporated in its entirety for all purposes, and in particular
for the LAG-3 ABDs in FIG. 11, the data in FIG. 18, FIG. 55, FIG.
56, FIG. 63 and SEQ ID NO:s 36819-36962, SEQ ID NO:s 35417-35606,
SEQ ID NO:s 25914-32793 and SEQ ID NO:s 32794-33002 sequences in
the sequence listing.
[0191] As will further be appreciated by those in the art, all or
part of the hinge (which can also be a wild type hinge from IgG1,
IgG2 or IgG4 or a variant thereof, such as the IgG4 S241P or S228P
hinge variant with the substitution proline at position 228
relative to the parent IgG4 hinge polypeptide (wherein the
numbering S228P is according to the EU index and the S241P is the
Kabat numbering)) can be used as the domain linker between the scFv
and the CH2-CH3 domain, or a different domain linker such as
depicted in the Figures can be used.
[0192] Alternatively, the LAG-3 ABD can be in the form of a Fab
fragment. In this embodiment, the ABD is made up of a variable
heavy domain, contributed by a heavy chain, and a variable light
domain, contributed by a light chain. Suitable Fvs (including CDR
sets and variable heavy/variable light domains) can be used in scFv
formats or Fab formats are shown in the Figures as well as
disclosed in in WO2017/218707, the contents are hereby incorporated
in its entirety for all purposes, and in particular for the LAG-3
ABDs in FIG. 11, the data in FIG. 18, FIG. 55, FIG. 56, FIG. 63 and
SEQ ID NO:s 36819-36962, SEQ ID NO:s 35417-35606, SEQ ID NO:s
25914-32793 and SEQ ID NO:s 32794-33002 sequences in the sequence
listing.
[0193] As will be appreciated by those in the art, suitable LAG-3
binding domains can comprise a set of 6 CDRs as depicted in the
sequence listing and figures (e.g., FIGS. 12 and 13), either as
they are underlined/bolded or, in the case where a different
numbering scheme is used as described herein and as shown in Table
2, as the CDRs that are identified using other alignments within
the variable heavy (VH) domain and variable light domain (VL)
sequences of those depicted in the figures (e.g., FIGS. 12 and
13A-C)and the sequence listing. Suitable LAG-3 ABDs that find use
in the subject targeted IL-15/IL-15R.alpha. heterodimeric fusion
proteins can also include the entire VH and VL sequences as
depicted in these sequences and figures, used as scFvs or as
Fabs.
[0194] In one embodiment, the LAG-3 antigen binding domain includes
the 6 CDRs (i.e., vhCDR1-3 and vlCDR1-3) of any of the LAG-3
binding domains described in FIGS. 12 and 13A-C or the sequence
listing.
[0195] In addition to the parental CDR sets disclosed in the
figures and sequence listing that form an ABD to LAG-3, provided
herein are variant LAG-3 ABDS having CDRs that include at least one
modification of the LAG-3 ABD CDRs disclosed herein (e.g., FIGS. 12
and 13A-C). In one embodiment, the heterodimeric fusion protein
includes a LAG-3 ABD that includes a set of 6 CDRs with 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 amino acid modifications as compared to the 6
CDRs of a LAG-3 ABD as depicted in FIGS. 112 and 3A-C or the
sequence listing. In certain embodiments, the LAG-3 ABD is capable
of binding LAG-3 antigen, as measured by at least one of a Biacore,
surface plasmon resonance (SPR) and/or BLI (biolayer
interferometry, e.g., Octet assay) assay, with the latter finding
particular use in many embodiments.
[0196] In one embodiment, the LAG-3 ABD of the subject targeted
IL-15/IL-15R.alpha. heterodimeric fusion protein includes 6 CDRs
that are at least 90, 95, 97, 98 or 99% identical to the 6 CDRs of
a LAG-3 ABD as depicted in FIGS. 12 and 13A-C or the sequence
listing. In certain embodiments, the LAG-3 ABD is capable of
binding to the LAG-3, as measured by at least one of a Biacore,
surface plasmon resonance (SPR) and/or BLI (biolayer
interferometry, e.g., Octet assay) assay, with the latter finding
particular use in many embodiments.
[0197] In one embodiment of the subject targeted
IL-15/IL-15R.alpha. heterodimeric fusion protein, the LAG-3 antigen
binding domain includes the 6 CDRs (i.e., vhCDR1-3 and vlCDR1-3) of
one of the following LAG-3 ABDs: 7G8[LAG-3]_H.sub.0_L0,
7G8[LAG-3]_H3_L1, 7G8[LAG-3]_H3.30_L1.34. 2A11[LAG-3]_H.sub.0_L0,
2A11[LAG-3]_H1_L2, 2A11[LAG-3]_H1.44_L2.142, BMS-986016[LAG-3],
IMP731[LAG-3], 13E2[LAG-3], 34F4[LAG-3], IMP761[LAG-3],
H5L7[LAG-3], hu22D2[LAG-3], H4sH15482P[LAG-3], L35D4[LAG-3],
L35G6[LAG-3], L33H11[LAG-3], L32A9[LAG-3], L32D10[LAG-3],
L32A4[LAG-3], L3A1[LAG-3], L3A10[LAG-3], L3C5[LAG-3], and
L3E3[LAG-3] (see, e.g., FIGS. 12 and 13A-C). In an exemplary
embodiment, the LAG-3 ABD is 7G8[LAG-3]_H3.30_L1.34 or
2A11[LAG-3]_H1.144_L2.142 LAG-3 ABD (see, e.g., FIG. 12).
[0198] In one embodiment, the LAG-3 antigen binding domain is a
variant LAG-3 antigen binding domain that includes 6 CDRs (i.e.,
vhCDR1-3 and vlCDR1-3), where the 6 CDRs include 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 modifications as compared to the 6 CDRs of one of
the following LAG-3 ABDs: 7G8[LAG-3]_H.sub.0_L0, 7G8[LAG-3]_H3_L1,
7G8[LAG-3]_H3.30_L1.34. 2A11[LAG-3]_H.sub.0_L0, 2A11[LAG-3]_H1_L2,
2A11[LAG-3]_H1.44_L2.142, BMS-986016[LAG-3], IMP731[LAG-3],
13E2[LAG-3], 34F4[LAG-3], IMP761[LAG-3], H5L7[LAG-3],
hu22D2[LAG-3], H4sH15482P[LAG-3], L35D4[LAG-3], L35G6[LAG-3],
L33H11[LAG-3], L32A9[LAG-3], L32D10[LAG-3], L32A4[LAG-3],
L3A1[LAG-3], L3A10[LAG-3], L3C5[LAG-3], and L3E3[LAG-3] (see, e.g.,
FIGS. 12 and 13A-C). In an exemplary embodiment, the LAG-3 ABD is
7G8[LAG-3]_H3.30_L1.34 or 2A11[LAG-3]_H1.144_L2.142 LAG-3 ABD (see,
e.g., FIG. 12).
[0199] In one embodiment, the LAG-3 antigen binding domain of the
IL-15/IL-15R.alpha. heterodimeric fusion protein is a variant LAG-3
antigen binding domain that includes 6 CDRs (i.e., vhCDR1-3 and
vlCDR1-3), where the 6 CDRs are at least 90, 95, 97, 98 or 99%
identical as compared to the 6 CDRs) of one of the following LAG-3
ABDs: 7G8[LAG-3]_H0_L0, 7G8[LAG-3]_H3_L1, 7G8[LAG-3]_H3.30_L1.34.
2A11[LAG-3]_H.sub.0_L0, 2A11[LAG-3]_H1_L2,
2A11[LAG-3]_H1.44_L2.142, BMS-986016[LAG-3], IMP731[LAG-3],
13E2[LAG-3], 34F4[LAG-3], IMP761[LAG-3], H5L7[LAG-3],
hu22D2[LAG-3], H4sH15482P[LAG-3], L35D4[LAG-3], L35G6[LAG-3],
L33H11[LAG-3], L32A9[LAG-3], L32D10[LAG-3], L32A4[LAG-3],
L3A1[LAG-3], L3A10[LAG-3], L3C5[LAG-3], and L3E3[LAG-3] (see, e.g.,
FIGS. 12 and 13A-C). In an exemplary embodiment, the LAG-3 ABD is
7G8[LAG-3]_H3.30_L1.34 or 2A11[LAG-3]_H1.144_L2.142 LAG-3 ABD (see,
e.g., FIG. 12).
[0200] In some embodiments, the LAG-3 ABD of the
IL-15/IL-15R.alpha. heterodimeric fusion protein includes the
variable heavy domain (VH) and variable light domain (VL) of any of
the LAG-ABDs disclosed herein, including, but not limited to those
disclosed in FIGS. 12 and 13A-C. In addition to the parental LAG-3
variable heavy and variable light domains disclosed herein,
provided herein are subject targeted IL-15/IL-15R.alpha.
heterodimeric fusion proteins having one or more LAG-3 ABDs that
include a variable heavy domain and/or a variable light domain that
are variants of a LAG-3 ABD VH and VL domain disclosed herein. In
one embodiment, the variant VH domain and/or VL domain has from 1,
2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL
domain of a LAG-3 ABD depicted in FIGS. 12, 13A-C, 29A and B, or
the sequence listing. In certain embodiments, the LAG-3 ABD is
capable of binding to LAG-3, as measured at least one of a Biacore,
surface plasmon resonance (SPR) and/or BLI (biolayer
interferometry, e.g., Octet assay) assay, with the latter finding
particular use in many embodiments.
[0201] In one embodiment, the variant VH and/or VL domain of the
IL-15/IL-15R.alpha. heterodimeric fusion protein is at least 90,
95, 97, 98 or 99% identical to the VH and/or VL of a LAG-3 ABD as
depicted in FIGS. 12 and 13A-C or the sequence listing. In certain
embodiments, the LAG-3 ABD is capable of binding to LAG-3, as
measured by at least one of a Biacore, surface plasmon resonance
(SPR) and/or BLI (biolayer interferometry, e.g., Octet assay)
assay, with the latter finding particular use in many
embodiments.
[0202] In some embodiments, the LAG-3 ABD includes the VH and VL of
one of the following LAG-3 ABDs: 7G8[LAG-3]_H.sub.0_L0,
7G8[LAG-3]_H3_L1, 7G8[LAG-3]_H3.30_L1.34. 2A11[LAG-3]_H.sub.0_L0,
2A11[LAG-3]_H1_L2, 2A11[LAG-3]_H1.44_L2.142, BMS-986016[LAG-3],
IMP731[LAG-3], 13E2[LAG-3], 34F4[LAG-3], IMP761[LAG-3],
H5L7[LAG-3], hu22D2[LAG-3], H4sH15482P[LAG-3], L35D4[LAG-3],
L35G6[LAG-3], L33H11[LAG-3], L32A9[LAG-3], L32D10[LAG-3],
L32A4[LAG-3], L3A1[LAG-3], L3A10[LAG-3], L3C5[LAG-3], and
L3E3[LAG-3] (see, e.g., FIGS. 12 and 13A-C). In an exemplary
embodiment, the LAG-3 ABD is 7G8[LAG-3]_H3.30_L1.34 or
2A11[LAG-3]_H1.144_L2.142 LAG-3 ABD (see, e.g., FIG. 12).
[0203] In some embodiments, the LAG-3 ABD includes a VH and VL,
where the VH and/or VL includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
amino acid modifications as compared to a VH and/or VL of a
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142 LAG-3 ABD (see, e.g., FIG.
12).
[0204] In certain embodiments, the LAG-3 ABD includes a VH and VL,
where the VH and VL are at least 90, 95, 97, 98 or 99% identical as
compared to a VH and VL of one of the following LAG-3 ABDs:
7G8[LAG-3]_H.sub.0_L0, 7G8[LAG-3]_H3_L1, 7G8[LAG-3]_H3.30_L1.34.
2A11[LAG-3]_H.sub.0_L0, 2A11[LAG-3]_H1_L2,
2A11[LAG-3]_H1.44_L2.142, BMS-986016[LAG-3], IMP731[LAG-3],
13E2[LAG-3], 34F4[LAG-3], IMP761[LAG-3], H5L7[LAG-3],
hu22D2[LAG-3], H4sH15482P[LAG-3], L35D4[LAG-3], L35G6[LAG-3],
L33H11[LAG-3], L32A9[LAG-3], L32D10[LAG-3], L32A4[LAG-3],
L3A1[LAG-3], L3A10[LAG-3], L3C5[LAG-3], and L3E3[LAG-3] (see, e.g.,
FIGS. 12 and 13A-C). In an exemplary embodiment, the LAG-3 ABD is
7G8[LAG-3]_H3.30_L1.34 or 2A11[LAG-3]_H1.144_L2.142 LAG-3 ABD (see,
e.g., FIG. 12).
C. Fc Domains
[0205] The Fc domain component of the invention is as described
herein, which generally contains skew variants and/or optional pI
variants and/or ablation variants are outlined herein. See for
example the disclosure of WO2017/218707 under the heading "IV
Heterodimeric Antibodies", including sections IV.A, IV.B, IV.C,
IV.D, IV.E, IV.F, IV.G, IV.H and IV.I, all of which are expressly
incorporated by reference in their entirety. Of particular use in
the heterodimeric proteins of the present invention are Fc domains
containing "skew variants", "pI variants", "ablation variants" and
FcRn variants as outlined therein. Particularly useful combinations
of such variants are depicted, for example, FIGS. 7A-F.
[0206] The Fc domains can be derived from IgG Fc domains, e.g.,
IgG1, IgG2, IgG3 or IgG4 Fc domains. In an exemplary embodiment,
the subject heterodimeric fusion protein provided herein includes
an IgG1 Fc domain. The following describes Fc domains that are
useful for IL-15/IL-15R.alpha. Fc fusion monomers and anti-LAG-3
antibody fragments of the targeted IL-15/IL-15R.alpha.
heterodimeric fusion proteins.
[0207] Thus, the "Fc domain" includes the -CH2-CH3 domain, and
optionally a hinge domain, and can be from human IgG1, IgG2, IgG3
or IgG4, with Fc domains derived from IgG1. In some of the
embodiments herein, when a protein fragment, e.g., IL-15 or
IL-15R.alpha. is attached to an Fc domain, it is the C-terminus of
the IL-15 or IL-15R.alpha. construct that is attached to all or
part of the hinge of the Fc domain. In other embodiments, when a
protein fragment, e.g., IL-15 or IL-15R.alpha., is attached to an
Fc domain, it is the C-terminus of the IL-15 or IL-15R.alpha.
construct that is attached to the CH1 domain of the Fc domain.
[0208] In some of the constructs and sequences outlined herein of
an Fc domain protein, the C-terminus of the IL-15 or IL-15R.alpha.
protein fragment is attached to the N-terminus of a domain linker,
the C-terminus of which is attached to the N-terminus of a constant
Fc domain (N-IL-15 or IL-15R.alpha. protein fragment-linker-Fc
domain-C) although that can be switched (N-Fc domain-linker-IL-15
or IL-15R.alpha. protein fragment -C). In other constructs and
sequence outlined herein, C-terminus of a first protein fragment is
attached to the N-terminus of a second protein fragment, optionally
via a domain linker, the C-terminus of the second protein fragment
is attached to the N-terminus of a constant Fc domain, optionally
via a domain linker. In yet other constructs and sequences outlined
herein, a constant Fc domain that is not attached to a first
protein fragment or a second protein fragment is provided. A
heterodimeric fusion protein can contain two or more of the
exemplary monomeric Fc domain proteins described herein. Any domain
linker can be used to attach a IL-15 or IL-15R.alpha. protein
fragment to an Fc domain of the heterodimeric fusion protein
provided herein. In some embodiments, the linker is any one of the
linkers in FIG. 8.
[0209] In some embodiments, the linker is a "domain linker", used
to link any two domains (e.g., IL-15 or IL-15R.alpha. protein
fragment to Fc domain or scFv to Fc domain) as outlined herein
together, some of which are depicted in FIG. 8. While any suitable
linker can be used, many embodiments utilize a glycine-serine
polymer, including for example (GS)n, (GSGGS)n (SEQ ID NO: 382),
(GGGGS)n (SEQ ID NO: 14), and (GGGS)n (SEQ ID NO: 383), where n is
an integer of at least one (and generally from 1 to 2 to 3 to 4 to
5) as well as any peptide sequence that allows for recombinant
attachment of the two domains with sufficient length and
flexibility to allow each domain to retain its biological function.
In some cases, and with attention being paid to "strandedness", as
outlined below, charged domain linkers.
[0210] In one embodiment, the heterodimeric fusion proteins contain
at least two constant domains which can be engineered to produce
heterodimers, such as pI engineering. Other Fc domains that can be
used include fragments that contain one or more of the CH1, CH2,
CH3, and hinge domains of the invention that have been pI
engineered. In particular, the formats depicted in FIG. 21 are
heterodimeric fusion proteins, meaning that the protein has two
associated Fc sequences self-assembled into a heterodimeric Fc
domain and at least one fusion protein (e.g., 1, 2 or more fusion
proteins) as more fully described below. In some cases, a first
fusion protein is linked to a first Fc and a second fusion protein
is linked to a second Fc. In other cases, a first fusion protein is
linked to a first Fc, and the first fusion protein is
non-covalently attached to a second fusion protein that is not
linked to an Fc. In some cases, the heterodimeric fusion protein
contains a first fusion protein linked to a second fusion protein
which is linked a first Fc sequence, and a second Fc sequence that
is not linked to either the first or second fusion proteins.
[0211] Accordingly, in some embodiments the present invention
provides heterodimeric fusion proteins that rely on the use of two
different heavy chain variant Fc sequences, that will self-assemble
to form a heterodimeric Fc domain fusion polypeptide.
[0212] The present invention is directed to novel constructs to
provide heterodimeric fusion proteins that allow binding to one or
more binding partners, ligands or receptors. The heterodimeric
fusion constructs are based on the self-assembling nature of the
two Fc domains of the heavy chains of antibodies, e.g., two
"monomers" that assemble into a "dimer". Heterodimeric Fc fusions
are made by altering the amino acid sequence of each monomer as
more fully discussed below. Thus, the present invention is
generally directed to the creation of heterodimeric fusion proteins
which can co-engage binding partner(s) or ligand(s) or receptor(s)
in several ways, relying on amino acid variants in the constant
regions that are different on each chain to promote heterodimeric
formation and/or allow for ease of purification of heterodimers
over the homodimers. Specific variants that are included in the Fc
domains of specific embodiments of the subject heterodimeric fusion
protein are described in greater detail below.
[0213] 1. Heterodimerization Variants
[0214] The present invention provides heterodimeric proteins,
including heterodimeric fusion proteins in a variety of formats.
Such heterodimeric proteins include two different Fc domains (one
on each of the first and second monomers) that include
modifications that facilitate the heterodimerization of the first
and second monomers and/or allow for ease of purification of
heterodimers over homodimers, collectively referred to herein as
"heterodimerization variants." As discussed below,
heterodimerization variants can include skew variants (e.g., the
"knobs and holes" and "charge pairs" variants described below) as
well as "pI variants" that facilitates the separation of homodimers
away from heterodimers. As is generally described in U.S. Pat. No.
9,605,084, hereby incorporated by reference in its entirety and
specifically as below for the discussion of heterodimerization
variants, useful mechanisms for heterodimerization include "knobs
and holes" ("KIH") as described in U.S. Pat. No. 9,605,084,
"electrostatic steering" or "charge pairs" as described in U.S.
Pat. No. 9,605,084, pI variants as described in U.S. Pat. No.
9,605,084, and general additional Fc variants as outlined in U.S.
Pat. No. 9,605,084 and below.
[0215] a. Skew Variants
[0216] In some embodiments, the subject heterodimeric protein
includes skew variants, which are one or more amino acid
modifications in a first Fc domain (A) and/or a second Fc domain
(B) that favor the formation of Fc heterodimers (Fc dimers that
include the first and the second Fc domain; A-B) over Fc homodimers
(Fc dimers that include two of the first Fc domain or two of the
second Fc domain; A-A or B-B). Suitable skew variants are included
in the FIG. 29 of US Publ. App. No. 2016/0355608, hereby
incorporated by reference in its entirety and specifically for its
disclosure of skew variants, as well as in FIG. 4.
[0217] One mechanism for skew variants is generally referred to in
the art as "knobs and holes," referring to amino acid engineering
that creates steric influences to favor heterodimeric formation and
disfavor homodimeric formation, as described in USSN 61/596,846,
Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al.,
J. Mol. Biol. 1997 270:26; U.S. Pat. No. 8,216,805, all of which
are hereby incorporated by reference in their entirety and
specifically for the disclosure of "knobs and holes" mutations.
This is sometime referred to herein as "steric variants." The
figures identify a number of "monomer A--monomer B" pairs that rely
on "knobs and holes". In addition, as described in Merchant et al.,
Nature Biotech. 16:677 (1998), these "knobs and holes" mutations
can be combined with disulfide bonds to further favor formation of
Fc heterodimers.
[0218] An additional mechanism for skew variants that finds use in
the generation of heterodimers is sometimes referred to as
"electrostatic steering" as described in Gunasekaran et al., J.
Biol. Chem. 285(25):19637 (2010), hereby incorporated by reference
in its entirety. This is sometimes referred to herein as "charge
pairs." In this embodiment, electrostatics are used to skew the
formation towards heterodimerization. As those in the art will
appreciate, these may also have an effect on pI, and thus on
purification, and thus could in some cases also be considered pI
variants. However, as these were generated to force
heterodimerization and were not used as purification tools, they
are classified as "skew variants." These include, but are not
limited to, D221E/P228E/L368E paired with D221R/P228R/K409R (e.g.,
these are "monomer" corresponding sets) and C220E/P228E/368E paired
with C220R/E224R/P228R/K409R.
[0219] In some embodiments, the skew variants advantageously and
simultaneously favor heterodimerization based on both the "knobs
and holes" mechanism as well as the "electrostatic steering"
mechanisms described above. In some embodiments, the heterodimeric
protein includes one or more sets of such heterodimerization skew
variants. These variants come in "pairs" of "sets." That is, one
set of the pair is incorporated into the first monomer and the
other set of the pair is incorporated into the second monomer.
Exemplary "skew variants` in this category include
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L;
K370S:S364K/E357Q; or a T366S/L368A/Y407V:T366W (optionally
including a bridging disulfide,
T366S/L368A/Y407V/Y349C:T366W/S354C) "skew" variant amino acid
substitution sets. In terms of nomenclature, the pair
"S364K/E357Q:L368D/K370S" means that one of the monomers includes
an Fc domain that includes the amino acid substitutions S364K and
E357Q and the other monomer includes an Fc domain that includes the
amino acid substitutions L368D and K370S; as above, the
"strandedness" of these pairs depends on the starting pI. It should
be noted that these sets do not necessarily behave as "knobs in
holes" variants, with a one-to-one correspondence between a residue
on one monomer and a residue on the other. That is, these pairs of
sets may instead form an interface between the two monomers that
encourages heterodimer formation and discourages homodimer
formation, allowing the percentage of heterodimers that
spontaneously form under biological conditions to be over 90%,
rather than the expected 50% (25% homodimer A/A:50% heterodimer
A/B:25% homodimer B/B).
[0220] In exemplary embodiments, the heterodimeric fusion protein
includes a S364K/E357Q:L368D/K370S; L368D/K370S:S364K;
L368E/K370S:S364K; T411T/E360E/Q362E:D401K;
L368D/K370S:S364K/E357L; K370S:S364K/E357Q; or a
T366S/L368A/Y407V:T366W (optionally including a bridging disulfide,
T366S/L368A/Y407V/Y349C:T366W/S354C) "skew" variant amino acid
substitution set. In an exemplary embodiment, the heterodimeric
fusion protein includes a "S364K/E357Q:L368D/K370S" amino acid
substitution set.
[0221] In some embodiments, the skew variants provided herein are
independently incorporated with other modifications, including, but
not limited to, other skew variants (see, e.g., in FIG. 37 of US
Publ. App. No. 2012/0149876, herein incorporated by reference,
particularly for its disclosure of skew variants), pI variants,
isotypic variants, FcRn variants, ablation variants, etc. into one
or both of the first and second Fc domains of the heterodimeric
fusion protein. Further, individual modifications can also
independently and optionally be included or excluded from the
subject heterodimeric fusion proteins.
[0222] b. pI (Isoelectric Point) Variants for Heterodimers
[0223] In some embodiments, the heterodimeric fusion protein
includes purification variants that advantageously allow for the
separation of heterodimeric fusion proteins from homodimeric
proteins ("pI variants").
[0224] In general, as will be appreciated by those in the art,
there are two general categories of pI variants: those that
increase the pI of the protein (basic changes) and those that
decrease the pI of the protein (acidic changes). As described
herein, all combinations of these variants can be done: one monomer
may be wild type, or a variant that does not display a
significantly different pI from wild-type, and the other can be
either more basic or more acidic. Alternatively, each monomer is
changed, one to more basic and one to more acidic.
[0225] There are several basic mechanisms that can lead to ease of
purifying heterodimeric proteins. One such mechanism relies on the
use of pI variants which include one or more modifications that
affect the isoelectric point of one or both of the monomers of the
fusion protein, such that each monomer, and subsequently each
dimeric species, has a different pI, thus allowing the isoelectric
purification of A-A, A-B and B-B dimeric proteins. Alternatively,
some formats also allow separation on the basis of size. As is
further outlined above, it is also possible to "skew" the formation
of heterodimers over homodimers using skew variants. Thus, a
combination of heterodimerization skew variants and pI variants
find particular use in the subject heterodimeric fusion proteins
provided herein.
[0226] Additionally, as more fully outlined below, depending on the
format of the heterodimeric fusion protein, pI variants can be
either contained within the constant region and/or Fc domains of a
monomer, and/or domain linkers can be used. In some embodiments,
the heterodimeric fusion protein includes additional modifications
for alternative functionalities can also create pI changes, such as
Fc, FcRn and KO variants.
[0227] In the embodiments that utilizes pI as a separation
mechanism to allow the purification of heterodimeric fusion
proteins, amino acid modifications can be introduced into one or
both of the monomers of the heterodimeric fusion protein. That is,
the pI of one of the monomers (referred to herein for simplicity as
"monomer A") can be engineered away from monomer B, or both monomer
A and B can be changed, with the pI of monomer A increasing and the
pI of monomer B decreasing. As discussed, the pI changes of either
or both monomers can be done by removing or adding a charged
residue (e.g., a neutral amino acid is replaced by a positively or
negatively charged amino acid residue, e.g., glutamine to glutamic
acid), changing a charged residue from positive or negative to the
opposite charge (e.g., aspartic acid to lysine) or changing a
charged residue to a neutral residue (e.g., loss of a charge;
lysine to serine.). A number of these variants are shown in the
figures, including, FIGS. 4 and 5.
[0228] Creating a sufficient change in pI in at least one of the
monomers such that heterodimers can be separated from homodimers
can be done by using a "wild type" heavy chain constant region and
a variant region that has been engineered to either increase or
decrease its pI (wt A:B+ or wt A:B-), or by increasing one region
and decreasing the other region (A+:B- or A-:B+).
[0229] Thus, in general, a component of some embodiments of the
present subject fusion proteins are amino acid variants in the Fc
domains or constant domain regions that are directed to altering
the isoelectric point (pI) of at least one, if not both, of the
monomers of a dimeric protein by incorporating amino acid
substitutions ("pI variants" or "pI substitutions") into one or
both of the monomers. The separation of the heterodimers from the
two homodimers can be accomplished if the pIs of the two monomers
differ by as little as 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 or
greater all finding use in the present invention.
[0230] As will be appreciated by those in the art, the number of pI
variants to be included on each or both monomer(s) of a
heterodimeric fusion protein to achieve good separation will depend
in part on the starting pI of the components. That is, to determine
which monomer to engineer or in which "direction" (e.g., more
positive or more negative), the sequences of the Fc domains and any
IL-15, IL-15R.alpha. or linker included in each monomer are
calculated and a decision is made from there based on the pIs of
the monomers. As is known in the art, different Fc domains, linkers
IL-15, and IL-15R.alpha. will have different starting pIs. In
general, as outlined herein, the pIs are engineered to result in a
total pI difference of each monomer of at least about 0.1 logs,
with 0.2 to 0.5 being preferred as outlined herein.
[0231] In general, as will be appreciated by those in the art,
there are two general categories of amino acid modifications that
affect pI: those that increase the pI of the protein (basic
changes) and those that decrease the pI of the protein (acidic
changes). As described herein, all combinations of these variants
can be used: one monomer may include a wild type Fc domain, or a
variant Fc domain that does not display a significantly different
pI from wild-type, and the other monomer includes a Fc domain that
is either more basic or more acidic. Alternatively, each monomer
may be changed, one to more basic and one to more acidic.
[0232] In the case where pI variants are used to achieve
heterodimerization, a more modular approach to designing and
purifying heterodimeric fusion proteins is provided. Thus, in some
embodiments, heterodimerization variants (including skew and pI
variants) must be engineered. In addition, in some embodiments, the
possibility of immunogenicity resulting from the pI variants is
significantly reduced by importing pI variants from different IgG
isotypes such that pI is changed without introducing significant
immunogenicity (see isotypic variants below). Thus, an additional
problem to be solved is the elucidation of low pI constant domains
with high human sequence content, e.g., the minimization or
avoidance of non-human residues at any particular position.
Alternatively or in addition to isotypic substitutions, the
possibility of immunogenicity resulting from the pI variants is
significantly reduced by utilizing isosteric substitutions (e.g.,
Asn to Asp; and Gln to Glu).
[0233] A side benefit that can occur with this pI engineering is
also the extension of serum half-life and increased FcRn binding.
That is, as described in US Publ. App. No. US 2012/0028304
(incorporated by reference in its entirety and specifically for the
disclosure of pI variants that provide additional function),
lowering the pI of antibody constant domains (including those found
in Fc fusions) can lead to longer serum retention in vivo. These pI
variants for increased serum half-life also facilitate pI changes
for purification.
[0234] In addition, it should be noted that the pI variants of the
heterodimerization variants give an additional benefit for the
analytics and quality control process of Fc fusion proteins, as the
ability to either eliminate, minimize and distinguish when
homodimers are present is significant. Similarly, the ability to
reliably test the reproducibility of the heterodimeric fusion
protein production is important.
[0235] Exemplary combinations of pI variants are shown in FIGS. 4
and 5, and FIG. 30 of US Publ. App. No. 2016/0355608, all of which
are herein incorporated by reference in its entirety and
specifically for the disclosure of pI variants. As outlined herein
and shown in the figures, these changes are shown relative to IgG1,
but all isotypes can be altered this way, as well as isotype
hybrids. In the case where the heavy chain constant domain is from
IgG2-4, R133E and R133Q can also be used.
[0236] In some embodiments, modifications are made in the hinge of
the Fc domain, including positions 208, 216, 217, 218, 219, 220,
221, 222, 223, 224, 225, 226, 227, 228, 229, and 230 based on EU
numbering. Thus, pI mutations and particularly substitutions can be
made in one or more of positions 216-230, with 1, 2, 3, 4 or 5
mutations finding use. Again, all possible combinations are
contemplated, alone or with other pI variants in other domains.
[0237] Specific substitutions that find use in lowering the pI of
hinge domains include, but are not limited to, a deletion at
position 221, a non-native valine or threonine at position 222, a
deletion at position 223, a non-native glutamic acid at position
224, a deletion at position 225, a deletion at position 235 and a
deletion or a non-native alanine at position 236. In some cases,
only pI substitutions are done in the hinge domain, and in others,
these substitution(s) are added to other pI variants in other
domains in any combination.
[0238] In some embodiments, mutations can be made in the CH2
region, including positions 233, 234, 235, 236, 274, 296, 300, 309,
320, 322, 326, 327, 334 and 339, based on EU numbering. It should
be noted that changes in 233-236 can be made to increase effector
function (along with 327A) in the IgG2 backbone. Again, all
possible combinations of these 14 positions can be made; e.g., a
heterodimeric fusion protein may include a variant Fc domain with
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 CH2 pI substitutions.
[0239] Specific substitutions that find use in lowering the pI of
CH2 domains include, but are not limited to, a non-native glutamine
or glutamic acid at position 274, a non-native phenylalanine at
position 296, a non-native phenylalanine at position 300, a
non-native valine at position 309, a non-native glutamic acid at
position 320, a non-native glutamic acid at position 322, a
non-native glutamic acid at position 326, a non-native glycine at
position 327, a non-native glutamic acid at position 334, a
non-native threonine at position 339, and all possible combinations
within CH2 and with other domains.
[0240] In this embodiment, the modifications can be independently
and optionally selected from position 355, 359, 362, 384, 389,392,
397, 418, 419, 444 and 447 (EU numbering) of the CH3 region.
Specific substitutions that find use in lowering the pI of CH3
domains include, but are not limited to, a non-native glutamine or
glutamic acid at position 355, a non-native serine at position 384,
a non-native asparagine or glutamic acid at position 392, a
non-native methionine at position 397, a non-native glutamic acid
at position 419, a non-native glutamic acid at position 359, a
non-native glutamic acid at position 362, a non-native glutamic
acid at position 389, a non-native glutamic acid at position 418, a
non-native glutamic acid at position 444, and a deletion or
non-native aspartic acid at position 447. Exemplary embodiments of
pI variants are provided in FIG. 5.
[0241] In one embodiment, the heterodimeric fusion protein includes
a monomer with a variant Fc domain having pI variant modifications
295E/384D/418E/421D (Q295E/N384D/Q418E/N421D when relative to human
IgG1). In one embodiment, the heterodimeric fusion protein includes
a monomer with a variant Fc domain having pI variant modifications
208D/295E/384D/418E/421D (N208D/Q295E/N384D/Q418E/N421D when
relative to human IgG1). In some embodiments, the heterodimeric
fusion protein includes a monomer with a variant Fc domain having
pI variant modifications 295E/384D/418E/421D
(Q295E/N384D/Q418E/N421D when relative to human IgG1). In one
embodiment, the heterodimeric fusion protein includes a monomer
with a variant Fc domain having pI variant modifications
196K/199T/217R/228R/276K (Q196K/I199T/P217R/P228R/N276K) when
relative to human IgG1).
[0242] In one embodiment, the heterodimeric fusion protein includes
a monomer with a variant Fc domain having pI variant modifications
217R/228R/276K (P217R/P228R/N276K when relative to human IgG1).
Additional exemplary pI variant modification that can be
incorporated into the Fc domain of a subject are depicted in FIG.
5.
[0243] c. Isotypic Variants
[0244] In addition, many embodiments of the invention rely on the
"importation" of pI amino acids at particular positions from one
IgG isotype into another, thus reducing or eliminating the
possibility of unwanted immunogenicity being introduced into the
variants. A number of these are shown in FIG. 21 of US Publ. App.
No. 2014/0370013, hereby incorporated by reference. That is, IgG1
is a common isotype for therapeutic antibodies for a variety of
reasons, including high effector function. However, the heavy
constant region of IgG1 has a higher pI than that of IgG2 (8.10
versus 7.31). By introducing IgG2 residues at particular positions
into the IgG1 backbone, the pI of the resulting monomer is lowered
(or increased) and additionally exhibits longer serum half-life.
For example, IgG1 has a glycine (pI 5.97) at position 137, and IgG2
has a glutamic acid (pI 3.22); importing the glutamic acid will
affect the pI of the resulting protein. As is described below, a
number of amino acid substitutions are generally required to
significant affect the pI of the variant Fc fusion protein.
However, it should be noted as discussed below that even changes in
IgG2 molecules allow for increased serum half-life.
[0245] In other embodiments, non-isotypic amino acid changes are
made, either to reduce the overall charge state of the resulting
protein (e.g., by changing a higher pI amino acid to a lower pI
amino acid), or to allow accommodations in structure for stability,
etc. as is more further described below.
[0246] In addition, by pI engineering both the heavy and light
constant domains, significant changes in each monomer of the
heterodimer can be seen. As discussed herein, having the pIs of the
two monomers differ by at least 0.5 can allow separation by ion
exchange chromatography or isoelectric focusing, or other methods
sensitive to isoelectric point.
[0247] d. Calculating pI
[0248] The pI of each monomer can depend on the pI of the variant
heavy chain constant domain and the pI of the total monomer,
including the variant heavy chain constant domain and the fusion
partner. Thus, in some embodiments, the change in pI is calculated
on the basis of the variant heavy chain constant domain, using the
chart in the FIG. 19 of US2014/0370013. As discussed herein, which
monomer to engineer is generally decided by the inherent pI of each
monomer.
[0249] 2. Additional Fc Variants for Additional Functionality
[0250] In addition to pI amino acid variants, there are a number of
useful Fc amino acid modification that can be made for a variety of
reasons, including, but not limited to, altering binding to one or
more Fc.gamma.R receptors, altered binding to FcRn receptors,
etc.
[0251] Accordingly, the proteins of the invention can include amino
acid modifications, including the heterodimerization variants
outlined herein, which includes the pI variants and steric
variants. Each set of variants can be independently and optionally
included or excluded from any particular heterodimeric protein.
[0252] a. Fc.gamma.R Variants
[0253] Accordingly, there are a number of useful Fc substitutions
that can be made to alter binding to one or more of the Fc.gamma.R
receptors. Substitutions that result in increased binding as well
as decreased binding can be useful. For example, it is known that
increased binding to Fc.gamma.RIIIa results in increased ADCC
(antibody dependent cell-mediated cytotoxicity; the cell-mediated
reaction wherein nonspecific cytotoxic cells that express
Fc.gamma.Rs recognize bound antibody on a target cell and
subsequently cause lysis of the target cell). Similarly, decreased
binding to Fc.gamma.RIIb (an inhibitory receptor) can be beneficial
as well in some circumstances. Amino acid substitutions that find
use in the present invention include those listed in U.S. Ser. No.
11/124,620 (particularly FIG. 41), U.S. Ser. Nos. 11/174,287,
11/396,495, 11/538,406, all of which are expressly incorporated
herein by reference in their entirety and specifically for the
variants disclosed therein. Particular variants that find use
include, but are not limited to, 236A, 239D, 239E, 332E, 332D,
239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y,
239D, 332E/330L, 243A, 243L, 264A, 264V and 299T.
[0254] In addition, amino acid substitutions that increase affinity
for Fc.gamma.RIIc can also be included in the Fc domain variants
outlined herein. The substitutions described in, for example, U.S.
Ser. Nos. 11/124,620 and 14/578,305 are useful.
[0255] In addition, there are additional Fc substitutions that find
use in increased binding to the FcRn receptor and increased serum
half-life, as specifically disclosed in U.S. Ser. No. 12/341,769,
hereby incorporated by reference in its entirety, including, but
not limited to, 434S, 434A, 428L, 308F, 259I, 428L/434S, 259I/308F,
436I/428L, 436I or V/434S, 436V/428L and 259I/308F/428L.
[0256] b. Ablation Variants
[0257] Similarly, another category of functional variants are
"Fc.gamma.R ablation variants" or "Fc knock out (FcKO or KO)"
variants. In these embodiments, for some therapeutic applications,
it is desirable to reduce or remove the normal binding of the Fc
domain to one or more or all of the Fc.gamma. receptors (e.g.,
Fc.gamma.R1, Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIIa, etc.) to
avoid additional mechanisms of action. That is, for example, in
many embodiments, particularly in the use of bispecific
immunomodulatory antibodies desirable to ablate Fc.gamma.RIIIa
binding to eliminate or significantly reduce ADCC activity such
that one of the Fc domains comprises one or more Fc.gamma. receptor
ablation variants. These ablation variants are depicted in FIG. 31
of U.S. Ser. No. 15/141,350, all of which are herein incorporated
by reference in its entirety, and each can be independently and
optionally included or excluded, with preferred aspects utilizing
ablation variants selected from the group consisting of
G236R/L328R, E233P/L234V/L235A/G236del/S239K,
E233P/L234V/L235A/G236del/S267K,
E233P/L234V/L235A/G236del/S239K/A327G,
E233P/L234V/L235A/G236del/S267K/A327G and
E233P/L234V/L235A/G236del, according to the EU index. It should be
noted that the ablation variants referenced herein ablate
Fc.gamma.R binding but generally not FcRn binding.
[0258] Exemplary ablation variants are provided in FIG. 5.
[0259] c. Combination of Heterodimeric and Fc Variants
[0260] As will be appreciated by those in the art, all of the
recited heterodimerization variants (including skew and/or pI
variants) can be optionally and independently combined in any way,
as long as they retain their "strandedness" or "monomer partition".
In addition, all of these variants can be combined into any of the
heterodimerization formats.
[0261] In the case of pI variants, while embodiments finding
particular use are shown in the Figures, other combinations can be
generated, following the basic rule of altering the pI difference
between two monomers to facilitate purification.
[0262] In addition, any of the heterodimerization variants, skew
and pI, are also independently and optionally combined with Fc
ablation variants, Fc variants, FcRn variants, as generally
outlined herein.
[0263] In addition, a monomeric Fc domain can comprise a set of
amino acid substitutions that includes C220S/S267K/L368D/K370S or
C220S/S267K/S364K/E357Q.
[0264] In addition, the heterodimeric fusion proteins can comprise
skew variants (e.g., a set of amino acid substitutions as shown in
FIGS. 1A-1C of U.S. Ser. No. 15/141,350, all of which are herein
incorporated by reference in its entirety), with particularly
useful skew variants being selected from the group consisting of
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411E/K360E/Q362E:D401K; L368D/K370S:S364K/E357L,
K370S:S364K/E357Q, T366S/L368A/Y407V:T366W and
T366S/L368A/Y407V/Y349C:T366W/S354C, optionally ablation variants,
optionally charged domain linkers and the heavy chain comprises pI
variants.
[0265] In some embodiments, the Fc domain comprising an amino acid
substitution selected from the group consisting of: 236R, 239D,
239E, 243L, M252Y, V259I, 267D, 267E, 298A, V308F, 328F, 328R,
330L, 332D, 332E, M428L, N434A, N434S, 236R/328R, 239D/332E, M428L,
236R/328F, V259I/V308F, 267E/328F, M428L/N434S, Y436I/M428L,
Y436V/M428L, Y436I/N434S, Y436V/N434S, 239D/332E/330L,
M252Y/S254T/T256E, V259I/V308F/M428L,
E233P/L234V/L235A/G236del/S267K, G236R/L328R and PVA/S267K. In some
cases, the Fc domain comprises the amino acid substitution
239D/332E. In other cases, the Fc domain comprises the amino acid
substitution G236R/L328R or PVA/S267K.
[0266] In one embodiment, a particular combination of skew and pI
variants that finds use in the present invention is
T366S/L368A/Y407V:T366W (optionally including a bridging disulfide,
T366S/L368A/Y407V/Y349C:T366W/S354C) with one monomer comprises
Q295E/N384D/Q418E/N481D and the other a positively charged domain
linker. As will be appreciated in the art, the "knobs in holes"
variants do not change pI, and thus can be used on either monomer.
Useful combination of variants that can be used in particular
formats of the invention are included in FIGS. 7A-7F.
III. IL-15/IL-15R.alpha. FC FUSION X LAG-3 ABD HETERODIMERIC
PROTEINS
[0267] Provided herein are heterodimeric fusion proteins that can
bind to the checkpoint inhibitor LAG-3 antigen and can complex with
the common gamma chain (.gamma.c; CD132) and/or the Il-2 receptor
.beta.-chain (IL-2R.beta.; CD122). The heterodimeric fusion
proteins can contain an IL-15/IL-15R.alpha.-Fc fusion protein and
an antibody fusion protein. The IL-15/IL-15R.alpha.-Fc fusion
protein can include as IL-15 protein (generally including amino
acid substitutions) covalently attached to an IL-15R.alpha., and an
Fc domain. Optionally, the IL-15 protein and IL-15R.alpha. protein
are noncovalently attached.
IV. USEFUL FORMATS OF THE INVENTION
[0268] As shown in FIG. 21, there are a number of useful formats of
the subject targeted IL-15/IL-15R.alpha. heterodimeric fusion
proteins. In general, the heterodimeric fusion proteins provided
herein have three functional components: an
IL-15/IL-15R.alpha.(sushi) component, an anti-LAG-3 component (also
referred to as a "LAG-3 binding domain" or "LAG-3 antigen binding
domain"), and an Fc component that includes a first Fc domain and
second Fc domain, each of which can take different forms as
outlined herein and each of which can be combined with the other
components in any configuration.
[0269] The first and the second Fc domains can have a set of amino
acid skew substitutions selected from the following skew variants:
a) S267K/L368D/K370S:S267K/S364K/E357Q; b) S364K/E357Q:L368D/K370S;
c) L368D/K370S:S364K; d) L368E/K370S:S364K; e)
T411E/K360E/Q362E:D401K; f) L368D/K370S:S364K/E357L and g)
K370S:S364K/E357Q according to EU numbering. In an exemplary
embodiment, the skew variants are S364K/E357Q:L368D/K370S.
[0270] In some embodiments, the first and/or the second Fc domains
have an additional set of pI amino acid substitutions selected from
the following pI variants: Q295E/N384D/Q418E/N421D,
N208/Q295E/N384D/Q418E/N421D or Q196K/I199T/P217R/P228R/N276K,
according to EU numbering.
[0271] Optionally, the first and/or the second Fc domains have an
additional set of ablation ("FcKO") variants selected from the
following FcKO variants: G236R/L328R,
E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,
E233P/L234V/L235A/G236del/S239K/A327G,
E233P/L234V/L235A/G236del/S267K/A327G and
E233P/L234V/L235A/G236del, according to EU numbering.
[0272] Optionally, the first and/or second Fc domains have
428L/434S variants for half-life extension.
[0273] In embodiments wherein a hinge or partial hinge is used to
link an Fc domain to a scFv, IL-15 or IL-15R.alpha. domain, the
hinge may optional include a C220S substitution to prevent the
hinge from forming undesirable disulfide bonds with any light
chains.
[0274] Exemplary formats of the subject heterodimeric fusion
proteins are provided below.
A. scIL-15/R.alpha. X scFv
[0275] One embodiment is shown in FIG. 21A, and comprises two
monomers. The first monomer comprises, from N- to C-terminus, the
IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant-(domain
linker)-CH2-CH3 (with the second domain linker frequently being a
hinge domain), and the second monomer comprises VH-scFv
linker-VL-hinge-CH2-CH3 or VL-scFv linker-VH-hinge-CH2-CH3,
although in either orientation a domain linker can be substituted
for the hinge. This is generally referred to as "scIL-15/R.alpha. X
scFv", with the "sc" standing for "single chain" referring to the
attachment of the IL-15 variant and IL-15R.alpha.(sushi) domain
using a covalent linker. Preferred combinations of variants for
this embodiment are found in FIGS. 21A and B.
[0276] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scIL-15/R.alpha. X scFv" format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first Fc domain; and b) a second monomer that
includes, from N- to C-terminus, an anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a second Fc domain. Any useful
domain linker can be used to attach the various components of the
heterodimeric protein including, but not limited to those in FIGS.
8 and 9A-C. In an exemplary embodiment, the domain linkers that
attach the IL-15 variant to the first Fc domain and the anti-LAG-3
scFv to the second Fc domain are each antibody hinge domains.
[0277] In some embodiments, the anti-LAG-3 scFv includes a variable
heavy domain (VH) covalently attached to a variable light domain
(VL) by an scFv linker (e.g., FIGS. 9A-C). In one embodiment, the
anti-LAG-3 scFv is from N- to C-terminus VH-scFv linker-VL. In
another embodiment, the anti-LAG-3 scFv is from N- to C-terminus
VL-scFv linker-VH. The C-terminus of the anti-LAG-3 scFv is
attached to the N terminus of the first Fc domain by a domain
linker (e.g., an antibody hinge domain).
[0278] In the scIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having any of the variable
heavy and light domain pairs as shown in FIGS. 12 and 13A-C.
[0279] In the scIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12. In one
embodiment, the "scIL-15/R.alpha. X scFv" format heterodimeric
protein includes: a) a first monomer that includes, from N- to
C-terminus, an IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(hinge)-CH2-CH3, where CH2-CH3 is a first Fc domain; and b)
a second monomer that includes, from N- to C-terminus, an anti
-LAG-3 scFv-(hinge)-CH2-CH3, where CH2-CH3 is a second Fc domain,
and where the anti-LAG-3 scFv includes the variable heavy domain
and variable light domain of 7G8_H3.30_L1.34. In one embodiment,
the "scIL-15/R.alpha. X scFv" format heterodimeric protein
includes: a) a first monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(hinge)-CH2-CH3, where CH2-CH3 is a first Fc domain; and b)
a second monomer that includes, from N- to C-terminus, an
anti-LAG-3 scFv-(hinge)-CH2-CH3, where CH2-CH3 is a second Fc
domain, and where the anti-LAG-3 scFv includes the variable heavy
domain and variable light domain of 2A11_H1.144_L2.142.
[0280] In the scIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an IL-15 variant that includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In one
embodiment, the "scIL-15/R.alpha. X scFv" format heterodimeric
protein includes: a) a first monomer that includes, from N- to
C-terminus, an IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(hinge)-CH2-CH3, where CH2-CH3 is a first Fc domain; and b)
a second monomer that includes, from N- to C-terminus, anti-LAG-3
scFv-(hinge)-CH2-CH3, where CH2-CH3 is a second Fc domain, and
where the IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment, the LAG-3
scFv includes the VH and VL of any of the LAG-3 ABDs in FIGS. 12
and 13A-C and the IL-15 variant includes amino acid substitutions
N4D/N65D. In another exemplary embodiment, the LAG-3 scFv includes
the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and
the IL-15 variant includes amino acid substitutions D30N/N65D. In
yet another exemplary embodiment, the LAG-3 scFv includes the VH
and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/E64Q/N65D.
[0281] In the scIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12, with
either the IL-15 N4D/N65D variant or the IL-15 D30N/N65D variant or
the IL-15 D30N/E64Q/N65D variant. In one embodiment, the
"scIL-15/R.alpha. X scFv" format heterodimeric protein includes: a)
a first monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(hinge)-CH2-CH3, where CH2-CH3 is a first Fc domain; and b)
a second monomer that includes, from N- to C-terminus, anti-LAG-3
scFv-(hinge)-CH2-CH3, where CH2-CH3 is a second Fc domain, where
the anti-LAG-3 scFv includes the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and where
the IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In a particular embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and the scFv
includes the variable heavy and light domain pair of
7G8_H3.30_L1.34. In another embodiment of the scIL-15/R.alpha. X
scFv format heterodimeric protein, the IL-15 variant includes amino
acid substitutions N4D/N65D and the scFv includes the variable
heavy and light domain pair of 2A11_H1.144_L2.142. In one
embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and the scFv includes the variable heavy and light domain
pair of 7G8_H3.30_L1.34. In another embodiment of the
scIL-15/R.alpha. X scFv format heterodimeric protein, the IL-15
variant includes amino acid substitutions D30N/N65D and the scFv
includes the variable heavy and light domain pair of
2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variant
is the IL-15 D30N/E64Q/N65D variant and the scFv includes the
variable heavy and light domain pair of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D and the scFv includes the variable heavy and light
domain pair of 2A11_H1.144_L2.142.
[0282] In the scIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes the skew variant pair S364K/E357Q:L368D/K370S.
In one embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric
protein is an "scIL-15/R.alpha. X scFv" format heterodimeric
protein that includes: a) a first monomer that includes, from N- to
C-terminus, an IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant
Fc domain; and b) a second monomer that includes, from N- to
C-terminus, anti-LAG-3 scFv-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain, and where the first and second
variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants L368D/K370S.
[0283] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scIL-15/R.alpha. X scFv" format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; and b) a second monomer that
includes, from N- to C-terminus, anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a second variant Fc domain, where
the IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D, and where the first and second
variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants L368D/K370S. In an
exemplary embodiment, the LAG-3 scFv includes the VH and VL of any
of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant
includes amino acid substitutions N4D/N65D. In another exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/N65D. In yet another exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0284] In the scIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
"scIL-15/R.alpha. X scFv" format heterodimeric protein includes: a)
a first monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc
domain; and b) a second monomer that includes, from N- to
C-terminus, anti-LAG-3 scFv-(hinge)-CH2-CH3, where CH2-CH3 is a
second variant Fc domain, where the anti-LAG-3 scFv includes the
variable heavy domain and variable light domain of 7G8_H3.30_L1.34,
and where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
"scIL-15/R.alpha. X scFv" format heterodimeric protein includes: a)
a first monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(hinge)-CH2-CH3, where CH2-CH3 is a first Fc domain; and b)
a second monomer that includes, from N- to C-terminus, anti-LAG-3
scFv-(hinge)-CH2-CH3, where CH2-CH3 is a second Fc domain, and
where the anti-LAG-3 scFv includes the variable heavy domain and
variable light domain of 2A11_H1.144_L2.142, and where the first
and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants L368D/K370S.
[0285] In the scIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S with either the IL-15
N4D/N65D variant or the IL-15 D30N/N65D variant or the IL-15
D30N/E64Q/N65D variant. In one embodiment, the "scIL-15/R.alpha. X
scFv" format heterodimeric protein includes: a) a first monomer
that includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(hinge)-CH2-CH3, where CH2-CH3
is a first variant Fc domain; and b) a second monomer that
includes, from N- to C-terminus, anti-LAG-3 scFv-(hinge)-CH2-CH3,
where CH2-CH3 is a second variant Fc domain, where the anti-LAG-3
scFv includes the variable heavy domain and variable light domain
of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the IL-15 variant
includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S. In an
exemplary embodiment, the first variant Fc domain includes skew
variants L368D/K370S, and the second variant Fc domain includes
skew variants L368D/K370S. In a particular embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and the scFv
includes the variable heavy and light domain pair of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and the scFv includes the
variable heavy and light domain pair of 2A11_H1.144_L2.142. In one
embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and the scFv includes the variable heavy and light domain
pair of 7G8_H3.30_L1.34. In another embodiment of the
scIL-15/R.alpha. X scFv format heterodimeric protein, the IL-15
variant includes amino acid substitutions D30N/N65D and the scFv
includes the variable heavy and light domain pair of
2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variant
is the IL-15 D30N/E64Q/N65D variant and the scFv includes the
variable heavy and light domain pair of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D and the scFv includes the variable heavy and light
domain pair of 2A11_H1.144_L2.142.
[0286] In the scIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0287] In one embodiment, the "scIL-15/R.alpha. X scFv" format
heterodimeric protein includes: a) a first monomer that includes,
from N- to C-terminus, an IL-15R.alpha.(sushi) domain-(domain
linker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a
first variant Fc domain; and b) a second monomer that includes,
from N- to C-terminus, anti-LAG-3 scFv-(hinge)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain; where the first variant Fc
domain includes skew variants L368D/K370S and the second variant Fc
domain includes skew variants S364K/E357Q, where the first and
second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the first variant Fc domain
includes pI variants Q295E/N384D/Q418E/N421D, and where numbering
is according to EU numbering. In some embodiments, the hinge of the
first and second monomers also each include amino acid substitution
C220S. In certain embodiments, the first and second variant Fc
domains each further include half-life extension variants
M428L/N434S. In an exemplary embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In
an exemplary embodiment, the LAG-3 scFv includes the VH and VL of
any of the LAG-3 ABDs in FIGS. 12 and 13A-C. In an exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the LAG-3 scFv includes the VH and VL of any of the LAG-3 ABDs in
FIGS. 12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
LAG-3 scFv includes the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/E64Q/N65D.
[0288] In the scIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 with
the FIG. 21A format, the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0289] In one embodiment, the "scIL-15/R.alpha. X scFv" format
heterodimeric protein includes: a) a first monomer that includes,
from N- to C-terminus, an IL-15R.alpha.(sushi) domain-(domain
linker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a
first variant Fc domain; and b) a second monomer that includes,
from N- to C-terminus, anti-LAG-3 scFv-(hinge)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain; where the anti-LAG-3 scFv
includes the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants L368D/K370S and the second variant Fc
domain includes skew variants S364K/E357Q, where the first and
second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the first variant Fc domain
includes pI variants Q295E/N384D/Q418E/N421D, and where numbering
is according to EU numbering. In some embodiments, the hinge of the
first and second monomers also each include amino acid substitution
C220S. In certain embodiments, the first and second variant Fc
domains each further include half-life extension variants
M428L/N434S. In a particular embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and the scFv includes the
variable heavy and light domain pair of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D and the scFv includes the variable heavy and light domain
pair of 2A11_H1.144_L2.142. In one embodiment, the IL-15 variant
includes amino acid substitutions D30N/N65D and the scFv includes
the variable heavy and light domain pair of 7G8_H3.30_L1.34. In
another embodiment of the scIL-15/R.alpha. X scFv format
heterodimeric protein, the IL-15 variant includes amino acid
substitutions D30N/N65D and the scFv includes the variable heavy
and light domain pair of 2A11_H1.144_L2.142. In yet another of
embodiment, the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D and the scFv includes the variable heavy and light
domain pair of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions D30N/E64Q/N65D and the
scFv includes the variable heavy and light domain pair of
2A11_H1.144_L2.142.
B. scFv X ncIL-15/R.alpha.
[0290] This embodiment is shown in FIG. 21B, and comprises three
monomers. The first monomer comprises, from N- to C-terminus, the
IL-15R.alpha.(sushi) domain-domain linker-CH2-CH3, and the second
monomer comprises VH-scFv linker-VL-hinge-CH2-CH3 or VL-scFv
linker-VH-hinge-CH2-CH3, although in either orientation a domain
linker can be substituted for the hinge. The third monomer is the
variant IL-15 domain. This is generally referred to as
"ncIL-15/R.alpha. X scFv" or "scFv X ncIL-15/R.alpha." with the
"nc" standing for "non-covalent" referring to the self-assembling
non-covalent attachment of the IL-15 variant and
IL-15R.alpha.(sushi) domain.
[0291] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second Fc domain; and c) an IL-15 variant, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex.
Any useful domain linker can be used to attach the various
components of the heterodimeric protein including, but not limited
to those in FIGS. 8 and 9A-C. In an exemplary embodiment, the
domain linkers that attach the anti-LAG-3 scFv to the first Fc
domain and the IL-15R.alpha.(sushi) domain to the second Fc domain
are each antibody hinge domains.
[0292] In some embodiments, the anti-LAG-3 scFv includes a variable
heavy domain (VH) covalently attached to a variable light domain
(VL) by an scFv linker (e.g., FIGS. 9A-C). In one embodiment, the
anti-LAG-3 scFv is, from N- to C-terminus, VH-scFv linker-VL. In
another embodiment, the anti-LAG-3 scFv is from, N- to C-terminus,
VL-scFv linker-VH. The C-terminus of the anti-LAG-3 scFv is
attached to the N terminus of the first Fc domain by a domain
linker (e.g., an antibody hinge domain).
[0293] In the ncIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having any of the variable
heavy and light domain pairs as shown in FIGS. 12 and 13A-C.
[0294] In the ncIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12.
[0295] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second Fc domain; and c) an IL-15 variant, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
and where the anti-LAG-3 scFv includes the variable heavy domain
and variable light domain of 7G8_H3.30_L1.34. In another
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "scFv X ncIL-15/R.alpha." format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
an anti-LAG-3 scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a
first Fc domain; b) a second monomer that includes, from N- to
C-terminus, an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3,
where CH2-CH3 is a second Fc domain; and c) an IL-15 variant, where
the IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, and where the anti-LAG-3 scFv includes the variable heavy
domain and variable light domain of 2A11_H1.144_L2.142.
[0296] In the ncIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an IL-15 variant that includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In one
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "scFv X ncIL-15/R.alpha." format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
an anti-LAG-3 scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a
first Fc domain; b) a second monomer that includes, from N- to
C-terminus, an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3,
where CH2-CH3 is a second Fc domain; and c) an IL-15 variant, where
the IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, and where the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an
exemplary embodiment, the LAG-3 scFv includes the VH and VL of any
of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant
includes amino acid substitutions N4D/N65D. In another exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/N65D. In yet another exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0297] In the ncIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12, with
either the IL-15 N4D/N65D variant or the IL-15 D30N/N65D variant or
the IL-15 D30N/E64Q/N65D variant. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an "scFv X
ncIL-15/R.alpha." format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, an anti-LAG-3
scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc domain;
b) a second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second Fc domain; and c) an IL-15 variant, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
where the anti-LAG-3 scFv includes the variable heavy domain and
variable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and
where the IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In a particular embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and the scFv
includes the variable heavy and light domain pair of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and the scFv includes the
variable heavy and light domain pair of 2A11_H1.144_L2.142. In one
embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and the scFv includes the variable heavy and light domain
pair of 7G8_H3.30_L1.34. In another embodiment, the IL-15 variant
includes amino acid substitutions D30N/N65D and the scFv includes
the variable heavy and light domain pair of 2A11_H1.144_L2.142. In
yet another of embodiment, the IL-15 variant includes amino acid
substitutions D30N/E64Q/N65D and the scFv includes the variable
heavy and light domain pair of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D and the scFv includes the variable heavy and light
domain pair of 2A11_H1.144_L2.142.
[0298] In the ncIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes the skew variant pair S364K/E357Q:L368D/K370S.
In one embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric
protein is an "scFv X ncIL-15/R.alpha." format heterodimeric
protein that includes: a) a first monomer that includes, from N- to
C-terminus, an anti-LAG-3 scFv-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant
Fc domain; and c) an IL-15 variant, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, and where the
first and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants S364K/E357Q.
[0299] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain; and c) an IL-15 variant, where the
IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, where the IL-15 variant includes amino acid substitutions
N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, and where the first and
second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants S364K/E357Q. In an
exemplary embodiment, the LAG-3 scFv includes the VH and VL of any
of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant
includes amino acid substitutions N4D/N65D. In another exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/N65D. In yet another exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0300] In the ncIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an "scFv X
ncIL-15/R.alpha." format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, an anti-LAG-3
scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant Fc
domain; b) a second monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain; and c) an IL-15 variant,
where the IL-15 variant and the IL-15R.alpha.(sushi) domain form an
IL-15 complex, where the anti-LAG-3 scFv includes the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34, and
where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S. In another embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an "scFv X
ncIL-15/R.alpha." format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, an anti-LAG-3
scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant Fc
domain; b) a second monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain; and c) an IL-15 variant,
where the IL-15 variant and the IL-15R.alpha.(sushi) domain form an
IL-15 complex, where the anti-LAG-3 scFv includes the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142, and
where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S.
[0301] In the ncIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S with either the IL-15
N4D/N65D variant or the IL-15 D30N/N65D variant or the IL-15
D30N/E64Q/N65D variant. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants L368D/K370S. In one
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "scFv X ncIL-15/R.alpha." format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
an anti-LAG-3 scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a
first variant Fc domain; b) a second monomer that includes, from N-
to C-terminus, an IL-15R.alpha.(sushi) domain-(domain
linker)-CH2-CH3, where CH2-CH3 is a second variant Fc domain; and
c) an IL-15 variant, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the
anti-LAG-3 scFv includes the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the
IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D, and where the first and second
variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In a particular embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and the scFv
includes the variable heavy and light domain pair of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and the scFv includes the
variable heavy and light domain pair of 2A11_H1.144_L2.142. In one
embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and the scFv includes the variable heavy and light domain
pair of 7G8_H3.30_L1.34. In another embodiment, the IL-15 variant
includes amino acid substitutions D30N/N65D and the scFv includes
the variable heavy and light domain pair of 2A11_H1.144_L2.142. In
yet another of embodiment, the IL-15 variant includes amino acid
substitutions D30N/E64Q/N65D and the scFv includes the variable
heavy and light domain pair of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D and the scFv includes the variable heavy and light
domain pair of 2A11_H1.144_L2.142.
[0302] In the ncIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0303] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain; and c) an IL-15 variant, where the
IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, where the first variant Fc domain includes skew variants
L368D/K370S and the second variant Fc domain includes skew variants
S364K/E357Q, where the first and second variant Fc domains each
include FcKO variants E233P/L234V/L235A/G236del/S267K, where the
first variant Fc domain includes pI variants
Q295E/N384D/Q418E/N421D, and where numbering is according to EU
numbering. In some embodiments, the hinge of the first and second
monomers also each include amino acid substitution C220S. In
certain embodiments, the first and second variant Fc domains each
further include half-life extension variants M428L/N434S. In an
exemplary embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an
exemplary embodiment, the LAG-3 scFv includes the VH and VL of any
of the LAG-3 ABDs in FIGS. 12 and 13A-C. In an exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the LAG-3 scFv includes the VH and VL of any of the LAG-3 ABDs in
FIGS. 12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
LAG-3 scFv includes the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/E64Q/N65D.
[0304] In the ncIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of7G8_H3.30_L1. or the variable heavy and light
domain pair 2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG.
21B format, the skew variant set S364K/E357Q:L368D/K370S, the pI
variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0305] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain; and c) an IL-15 variant, where the
IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, where the anti-LAG-3 scFv includes the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, where the first variant Fc domain includes skew
variants L368D/K370S and the second variant Fc domain includes skew
variants S364K/E357Q, where the first and second variant Fc domains
each include FcKO variants E233P/L234V/L235A/G236del/S267K, where
the first variant Fc domain includes pI variants
Q295E/N384D/Q418E/N421D, and where numbering is according to EU
numbering. In some embodiments, the hinge of the first and second
monomers also each include amino acid substitution C220S. In
certain embodiments, the first and second variant Fc domains each
further include half-life extension variants M428L/N434S. In a
particular embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and the scFv includes the variable heavy and
light domain pair of 7G8_H3.30_L1.34. In another embodiment, the
IL-15 variant includes amino acid substitutions N4D/N65D and the
scFv includes the variable heavy and light domain pair of
2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes
amino acid substitutions D30N/N65D and the scFv includes the
variable heavy and light domain pair of 7G8_H3.30_L1.34. In another
embodiment of the scIL-15/R.alpha. X scFv format heterodimeric
protein, the IL-15 variant includes amino acid substitutions
D30N/N65D and the scFv includes the variable heavy and light domain
pair of 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15
variant includes amino acid substitutions D30N/E64Q/N65D and the
scFv includes the variable heavy and light domain pair of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and the scFv includes the
variable heavy and light domain pair of 2A11_H1.144_L2.142.
C. scFv X dsIL-15/R.alpha.
[0306] This embodiment is shown in FIG. 21C, and comprises three
monomers. The first monomer comprises, from N- to C-terminus, the
IL-15R.alpha.(sushi) domain-domain linker-CH2-CH3, wherein the
IL-15R.alpha.(sushi) domain has an engineered cysteine residue and
the second monomer comprises VH-scFv linker-VL-hinge-CH2-CH3 or
VL-scFv linker-VH-hinge-CH2-CH3, although in either orientation a
domain linker can be substituted for the hinge. The third monomer
is the variant IL-15 domain, also engineered to have a cysteine
variant amino acid, thus allowing a disulfide bridge to form
between the IL-15R.alpha.(sushi) domain and the variant IL-15
domain. This is generally referred to as "scFv X dsIL-15/R.alpha."
or "dsIL-15/R.alpha. X scFv", with the "ds" standing for
"disulfide".
[0307] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second Fc domain and the IL-15R.alpha.(sushi) domain includes
an amino acid substitution for a cysteine residue; and c) an IL-15
variant that includes an amino acid substitution for a cysteine
residue, and where the cysteine residue on the IL-15 variant and
the cysteine residue on the IL-15R.alpha.(sushi) domain form a
disulfide bond. Any useful domain linker can be used to attach the
various components of the heterodimeric protein including, but not
limited to those in FIGS. 8 and 9A-C. In an exemplary embodiment,
the domain linkers that attach the anti-LAG-3 scFv to the first Fc
domain and the IL-15R.alpha.(sushi) domain to the second Fc domain
and are each antibody hinge domains.
[0308] Any useful domain linker can be used to attach the various
components of the heterodimeric protein including, but not limited
to those in FIGS. 8 and 9A-C. In an exemplary embodiment, the
domain linkers that attach the anti-LAG-3 scFv to the first Fc
domain and the IL-15R.alpha.(sushi) domain to the second Fc domain
and are each antibody hinge domains (e.g., an antibody hinge
domain).
[0309] In some embodiments, the anti-LAG-3 scFv includes a variable
heavy domain (VH) covalently attached to a variable light domain
(VL) by an scFv linker (e.g., FIGS. 9A-C). In one embodiment, the
anti-LAG-3 scFv is from N- to C-terminus VH-scFv linker-VL. In
another embodiment, the anti-LAG-3 scFv is from N- to C-terminus
VL-scFv linker-VH. The C-terminus of the anti-LAG-3 scFv is
attached to the N terminus of the first Fc domain by a domain
linker (e.g., an antibody hinge domain).
[0310] In the dsIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having any of the variable
heavy and light domain pairs as shown in FIGS. 12 and 13A-C.
[0311] In the dsIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12.
[0312] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second Fc domain and the IL-15R.alpha.(sushi) domain includes
an amino acid substitution for a cysteine residue; and c) an IL-15
variant that includes an amino acid substitution for a cysteine
residue, where the cysteine residue on the IL-15 variant and the
cysteine residue on the IL-15R.alpha.(sushi) domain form a
disulfide bond, and where the anti-LAG-3 scFv includes the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34. In
another embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric
protein is an "scFv X dsIL-15/R.alpha." format heterodimeric
protein that includes: a) a first monomer that includes, from N- to
C-terminus, an anti-LAG-3 scFv-(domain linker)-CH2-CH3, where
CH2-CH3 is a first Fc domain; b) a second monomer that includes,
from N- to C-terminus, an IL-15R.alpha.(sushi) domain-(domain
linker)-CH2-CH3, where CH2-CH3 is a second Fc domain and the
IL-15R.alpha.(sushi) domain includes an amino acid substitution for
a cysteine residue; and c) an IL-15 variant that includes an amino
acid substitution for a cysteine residue, where the cysteine
residue on the IL-15 variant and the cysteine residue on the
IL-15R.alpha.(sushi) domain form a disulfide bond, and where the
anti-LAG-3 scFv includes the variable heavy domain and variable
light domain of 2A11_H1.144_L2.142.
[0313] In the dsIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes the IL-15 N4D/N65D variant or the IL-15
D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant, as well as
appropriate cysteine substitutions. In on embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an "scFv X
dsIL-15/Re.alpha." format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, an anti-LAG-3
scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc domain;
b) a second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second Fc domain and the IL-15R.alpha.(sushi) domain includes
an amino acid substitution for a cysteine residue; and c) an IL-15
variant that includes an amino acid substitution for a cysteine
residue, where the cysteine residue on the IL-15 variant and the
cysteine residue on the IL-15R.alpha.(sushi) domain form a
disulfide bond, and where the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an
exemplary embodiment, the LAG-3 scFv includes the VH and VL of any
of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant
includes amino acid substitutions N4D/N65D. In another exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/N65D. In yet another exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0314] In the dsIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12, with
either the IL-15 N4D/N65D variant or the IL-15 D30N/N65D variant or
the IL-15 D30N/E64Q/N65D variant, as well as appropriate cysteine
substitutions. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second Fc domain and the IL-15R.alpha.(sushi) domain includes
an amino acid substitution for a cysteine residue; and c) an IL-15
variant that includes an amino acid substitution for a cysteine
residue, where the cysteine residue on the IL-15 variant and the
cysteine residue on the IL-15R.alpha.(sushi) domain form a
disulfide bond, where the anti-LAG-3 scFv includes the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, and where the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In a
particular embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and the scFv includes the variable heavy and
light domain pair of 7G8_H3.30_L1.34. In another embodiment, the
IL-15 variant includes amino acid substitutions N4D/N65D and the
scFv includes the variable heavy and light domain pair of
2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes
amino acid substitutions D30N/N65D and the scFv includes the
variable heavy and light domain pair of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and the scFv includes the variable heavy and light domain
pair of 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15
variant includes amino acid substitutions D30N/E64Q/N65D and the
scFv includes the variable heavy and light domain pair of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and the scFv includes the
variable heavy and light domain pair of 2A11_H1.144_L2.142.
[0315] In the dsIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes the skew variant pair S364K/E357Q:L368D/K370S.
In one embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric
protein is an "scFv X dsIL-15/R.alpha." format heterodimeric
protein that includes: a) a first monomer that includes, from N- to
C-terminus, an anti-LAG-3 scFv-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant
Fc domain and the IL-15R.alpha.(sushi) domain includes an amino
acid substitution for a cysteine residue; and c) an IL-15 variant
that includes an amino acid substitution for a cysteine residue,
where the cysteine residue on the IL-15 variant and the cysteine
residue on the IL-15R.alpha.(sushi) domain form a disulfide bond,
and where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S. In an exemplary embodiment,
the first variant Fc domain includes skew variants L368D/K370S, and
the second variant Fc domain includes skew variants
S364K/E357Q.
[0316] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain and the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue; and c)
an IL-15 variant that includes an amino acid substitution for a
cysteine residue, where the cysteine residue on the IL-15 variant
and the cysteine residue on the IL-15R.alpha.(sushi) domain form a
disulfide bond, where the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, and where the
first and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants S364K/E357Q. In an
exemplary embodiment, the LAG-3 scFv includes the VH and VL of any
of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant
includes amino acid substitutions N4D/N65D. In another exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/N65D. In yet another exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0317] In the dsIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an "scFv X
dsIL-15/R.alpha." format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, an anti-LAG-3
scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant Fc
domain; b) a second monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain and the IL-15R.alpha.(sushi)
domain includes an amino acid substitution for a cysteine residue;
and c) an IL-15 variant that includes an amino acid substitution
for a cysteine residue, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where the anti-LAG-3 scFv includes the
variable heavy domain and variable light domain of 7G8_H3.30_L1.34,
and where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S. In another embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an "scFv X
dsIL-15/R.alpha." format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, an anti-LAG-3
scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant Fc
domain; b) a second monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain and the IL-15R.alpha.(sushi)
domain includes an amino acid substitution for a cysteine residue;
and c) an IL-15 variant that includes an amino acid substitution
for a cysteine residue, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where the anti-LAG-3 scFv includes the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142, and where the first and second variant Fc
domains include the skew variant pair S364K/E357Q:L368D/K370S.
[0318] In the dsIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S with either the IL-15
N4D/N65D variant or the IL-15 D30N/N65D variant or the IL-15
D30N/E64Q/N65D variant with the appropriate cysteine
substitutions.
[0319] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3 scFv-(domain
linker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain and the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue; and c)
an IL-15 variant that includes an amino acid substitution for a
cysteine residue, where the cysteine residue on the IL-15 variant
and the cysteine residue on the IL-15R.alpha.(sushi) domain form a
disulfide bond, where the anti-LAG-3 scFv includes the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, where the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, and where the
first and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants S364K/E357Q. In a
particular embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and the scFv includes the variable heavy and
light domain pair of 7G8_H3.30_L1.34. In another embodiment, the
IL-15 variant includes amino acid substitutions N4D/N65D and the
scFv includes the variable heavy and light domain pair of
2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes
amino acid substitutions D30N/N65D and the scFv includes the
variable heavy and light domain pair of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and the scFv includes the variable heavy and light domain
pair of 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15
variant includes amino acid substitutions D30N/E64Q/N65D and the
scFv includes the variable heavy and light domain pair of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and the scFv includes the
variable heavy and light domain pair of 2A11_H1.144_L2.142.
[0320] In the dsIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0321] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3
scFv-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc domain;
b) a second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain and the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue; and c)
an IL-15 variant that includes an amino acid substitution for a
cysteine residue, where the cysteine residue on the IL-15 variant
and the cysteine residue on the IL-15R.alpha.(sushi) domain form a
disulfide bond, where the first variant Fc domain includes skew
variants L368D/K370S and the second variant Fc domain includes skew
variants S364K/E357Q, where the first and second variant Fc domains
each include FcKO variants E233P/L234V/L235A/G236del/S267K, where
the first variant Fc domain includes pI variants
Q295E/N384D/Q418E/N421D, and where numbering is according to EU
numbering. In some embodiments, the hinge of the first monomer and
second monomer also each include amino acid substitution C220S. In
certain embodiments, the first and second variant Fc domains each
further include half-life extension variants M428L/N434S. In an
exemplary embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an
exemplary embodiment, the LAG-3 scFv includes the VH and VL of any
of the LAG-3 ABDs in FIGS. 12 and 13A-C. In an exemplary
embodiment, the LAG-3 scFv includes the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the LAG-3 scFv includes the VH and VL of any of the LAG-3 ABDs in
FIGS. 12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
LAG-3 scFv includes the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/E64Q/N65D.
[0322] In the dsIL-15/R.alpha. X scFv format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 with
the FIG. 21C format, the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0323] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scFv X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an anti-LAG-3
scFv-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc domain;
b) a second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain and the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue; and c)
an IL-15 variant that includes an amino acid substitution for a
cysteine residue, where the cysteine residue on the IL-15 variant
and the cysteine residue on the IL-15R.alpha.(sushi) domain form a
disulfide bond, where the anti-LAG-3 scFv includes the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, where the first variant Fc domain includes skew
variants L368D/K370S and the second variant Fc domain includes skew
variants S364K/E357Q, where the first and second variant Fc domains
each include FcKO variants E233P/L234V/L235A/G236del/S267K, where
the first variant Fc domain includes pI variants
Q295E/N384D/Q418E/N421D, and where numbering is according to EU
numbering. In some embodiments, the hinge of the first monomer and
second monomer also each include amino acid substitution C220S. In
certain embodiments, the first and second variant Fc domains each
further include half-life extension variants M428L/N434S. In a
particular embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and the scFv includes the variable heavy and
light domain pair of 7G8_H3.30_L1.34. In another embodiment, the
IL-15 variant includes amino acid substitutions N4D/N65D and the
scFv includes the variable heavy and light domain pair of
2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes
amino acid substitutions D30N/N65D and the scFv includes the
variable heavy and light domain pair of 7G8_H3.30_L1.34. In another
embodiment of the scIL-15/R.alpha. X scFv format heterodimeric
protein, the IL-15 variant includes amino acid substitutions
D30N/N65D and the scFv includes the variable heavy and light domain
pair of 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15
variant includes amino acid substitutions D30N/E64Q/N65D and the
scFv includes the variable heavy and light domain pair of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and the scFv includes the
variable heavy and light domain pair of 2A11_H1.144_L2.142.
D. scIL-15/R.alpha. X Fab
[0324] This embodiment is shown in FIG. 21D, and comprises three
monomers. The first monomer comprises, from N- to C-terminus, the
IL-15R.alpha.(sushi) domain-(domain linker)-variant IL-15-domain
linker-CH2-CH3 and the second monomer comprises a heavy chain,
VH-CH1-hinge-CH2-CH3. The third monomer is a light chain, VL-CL.
This is generally referred to as "scIL-15/R.alpha. X Fab", with the
"sc" standing for "single chain". The scIL-15/R.alpha. x Fab format
(see FIG. 21 FIG. 21D) comprises IL-15R.alpha.(sushi) fused to a
variant IL-15 by a variable length linker (termed
"scIL-15/R.alpha.") which is then fused to the N-terminus of a
heterodimeric Fc-region (inclusive of the hinge). The second
monomer is a heavy chain, VH-CH1-hinge-CH2-CH3, while a
corresponding light chain (the third monomer) is transfected
separately so as to form a Fab with the VH.
[0325] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scIL-15/R.alpha. X Fab" format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a
variable heavy domain and CH2-CH3 is a second Fc domain, and c) a
light chain that includes from, N- to C-terminus, VL-VC, where VL
is a variable light domain, where VH and VL form a LAG-3 binding
domain. Any useful domain linker can be used to attach the various
components of the heterodimeric protein including, but not limited
to those in FIGS. 8 and 9A-C. In an exemplary embodiment, the
domain linkers that attach the IL-15 variant to the first Fc domain
is an antibody hinge domain (e.g., an antibody hinge domain).
[0326] In the scIL-15/R.alpha. X Fab format, one preferred
embodiment utilizes an anti-LAG-3 ABD having any of the variable
heavy and light domain pairs as shown in FIGS. 12 and 13A-C.
[0327] In the scIL-15/R.alpha. X Fab format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12.
[0328] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric fusion protein is an "scIL-15/R.alpha. X Fab" format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a
variable heavy domain and CH2-CH3 is a second Fc domain, and c) a
light chain that includes from, N- to C-terminus, VL-VC, where VL
is a variable light domain, and where VH and VL are the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34,
respectively. In another embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an "scIL-15/R.alpha. X
Fab" format heterodimeric protein that includes: a) a first monomer
that includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a
variable heavy domain and CH2-CH3 is a second Fc domain, and c) a
light chain that includes from, N- to C-terminus, VL-VC, where VL
is a variable light domain, and where VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142,
respectively.
[0329] In the scIL-15/R.alpha. X Fab format, one preferred
embodiment utilizes an IL-15 variant that includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In one
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "scIL-15/R.alpha. X Fab" format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc
domain; b) a second monomer that includes, from N- to C-terminus, a
VH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and
CH2-CH3 is a second Fc domain, and c) a light chain that includes
from, N- to C-terminus, VL-VC, where VL is a variable light domain,
wherein the VH and VL form a LAG-3 binding domain, and where the
IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment, the VH
and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and
13A-C and the IL-15 variant includes amino acid substitutions
N4D/N65D. In another exemplary embodiment, the VH and VL are the VH
and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0330] In the scIL-15/R.alpha. X Fab format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12, with
either the IL-15 N4D/N65D variant or the IL-15 D30N/N65D variant or
the IL-15 D30N/E64Q/N65D variant. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an "scIL-15/R.alpha. X
Fab" format heterodimeric protein that includes: a) a first monomer
that includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a
variable heavy domain and CH2-CH3 is a second Fc domain, and c) a
light chain that includes from, N- to C-terminus, VL-VC, where VL
is a variable light domain, where VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, and where the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In a
particular embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and VH and VL are the variable heavy domain
and variable light domain of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D and VH and VL are the variable heavy domain and variable
light domain of 2A11_H1.144_L2.142. In one embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
yet another of embodiment, the IL-15 variant includes amino acid
substitutions D30N/E64Q/N65D and VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D and VH and VL are the variable heavy domain and
variable light domain of 2A11_H1.144_L2.142.
[0331] In the scIL-15/R.alpha. X Fab format, one preferred
embodiment utilizes the skew variant pair S364K/E357Q:L368D/K370S.
In one embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric
protein is an "scIL-15/R.alpha. X Fab" format heterodimeric protein
that includes: a) a first monomer that includes, from N- to
C-terminus, an IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant
Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable heavy
domain and CH2-CH3 is a second variant Fc domain, and c) a light
chain that includes from, N- to C-terminus, VL-VC, where VL is a
variable light domain, where VH and VL form a LAG-3 binding domain,
and where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S. In an exemplary embodiment,
the first variant Fc domain includes skew variants L368D/K370S, and
the second variant Fc domain includes skew variants
S364K/E357Q.
[0332] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scIL-15/R.alpha. X Fab" format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH
is a variable heavy domain and CH2-CH3 is a second variant Fc
domain, and c) a light chain that includes from, N- to C-terminus,
VL-VC, where VL is a variable light domain, where VH and VL form a
LAG-3 binding domain, where the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, and where the
first and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants S364K/E357Q. In an
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12
and 13A-C and the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D.
[0333] In the scIL-15/R.alpha. X Fab format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S. In one embodiment,
2A11_H1.144_L2.142 the targeted IL-15/IL-15R.alpha. heterodimeric
protein is an "scIL-15/R.alpha. X Fab" format heterodimeric protein
that includes: a) a first monomer that includes, from N- to
C-terminus, an IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant
Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable heavy
domain and CH2-CH3 is a second variant Fc domain, and c) a light
chain that includes from, N- to C-terminus, VL-VC, where VL is a
variable light domain, where VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34, respectively,
and where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S. In another embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"scIL-15/R.alpha. X Fab" format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant
Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable heavy
domain and CH2-CH3 is a second variant Fc domain, and c) a light
chain that includes from, N- to C-terminus, VL-VC, where VL is a
variable light domain, where VH and VL are the variable heavy
domain and variable light domain of 2A11_H1.144_L2.142,
respectively, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S.
[0334] In the scIL-15/R.alpha. X Fab format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S with either the IL-15
N4D/N65D variant or the IL-15 D30N/N65D variant or the IL-15
D30N/E64Q/N65D variant. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an "scIL-15/R.alpha. X
Fab" format heterodimeric protein that includes: a) a first monomer
that includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, variant
here VH is a variable heavy domain and CH2-CH3 is a second variant
Fc domain, and c) a light chain that includes from, N- to
C-terminus, VL-VC, where VL is a variable light domain, where VH
and VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the IL-15 variant
includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S. In an
exemplary embodiment, the first variant Fc domain includes skew
variants L368D/K370S, and the second variant Fc domain includes
skew variants S364K/E357Q. In a particular embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
one embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
[0335] In the scIL-15/R.alpha. X Fab format, one preferred
embodiment utilizes the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0336] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scIL-15/R.alpha. X Fab" format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, variant
here VH is a variable heavy domain and CH2-CH3 is a second variant
Fc domain, and c) a light chain that includes from, N- to
C-terminus, VL-VC, where VL is a variable light domain, where VH
and VL form a LAG-3 binding domain, where the first variant Fc
domain includes skew variants L368D/K370S and the second variant Fc
domain includes skew variants S364K/E357Q, where the first and
second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the first variant Fc domain
includes pI variants Q295E/N384D/Q418E/N421D, and where numbering
is according to EU numbering. In some embodiments, the hinge of the
first monomer also includes amino acid substitution C220S. In
certain embodiments, the first and second variant Fc domains each
further include half-life extension variants M428L/N434S. In an
exemplary embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C. In an exemplary embodiment, the
VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12
and 13A-C and the IL-15 variant includes amino acid substitutions
N4D/N65D. In another exemplary embodiment, the VH and VL are the VH
and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0337] In the scIL-15/R.alpha. X Fab format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 with
the FIG. 21D format, the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0338] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scIL-15/R.alpha. X Fab" format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, variant
here VH is a variable heavy domain and CH2-CH3 is a second variant
Fc domain, and c) a light chain that includes from, N- to
C-terminus, VL-VC, where VL is a variable light domain, where VH
and VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants L368D/K370S and the second variant Fc
domain includes skew variants S364K/E357Q, where the first and
second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the first variant Fc domain
includes pI variants Q295E/N384D/Q418E/N421D, and where numbering
is according to EU numbering. In some embodiments, the hinge of the
first monomer also includes amino acid substitution C220S. In
certain embodiments, the first and second variant Fc domains each
further include half-life extension variants M428L/N434S. In a
particular embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and the scFv includes the variable heavy and
light domain pair of 7G8_H3.30_L1.34. In another embodiment, the
IL-15 variant includes amino acid substitutions N4D/N65D and the
scFv includes the variable heavy and light domain pair of
2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes
amino acid substitutions D30N/N65D and the scFv includes the
variable heavy and light domain pair of 7G8_H3.30_L1.34. In another
embodiment of the scIL-15/R.alpha. X scFv format heterodimeric
protein, the IL-15 variant includes amino acid substitutions
D30N/N65D and the scFv includes the variable heavy and light domain
pair of 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15
variant includes amino acid substitutions D30N/E64Q/N65D and the
scFv includes the variable heavy and light domain pair of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and the scFv includes the
variable heavy and light domain pair of 2A11_H1.144_L2.142.
E. Fab X ncIL-15/R.alpha.
[0339] This embodiment is shown in FIG. 21E, and comprises four
monomers. The first monomer comprises, from N- to C-terminus, the
IL-15R.alpha.(sushi)domain-(domain linker)-CH2-CH3, and the second
monomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3. The third
monomer is the light chain that includes, from N-to C-terminus, a
variable light domain (VL) and a light constant domain(CL). The
fourth monomer is a variant IL-15 domain. This is generally
referred to as "Fab X ncIL-15/R.alpha.", with the "nc" standing for
"non-covalent" referring to the self-assembling non-covalent
attachment of the IL-15 variant and IL-15R.alpha.(sushi)domain.
[0340] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "Fab X ncIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH
is a variable heavy domain and CH2-CH3 is a first Fc domain; b) a
second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second Fc domain; c) a third monomer that includes from, N- to
C-terminus, VL-VC, where VL is a variable light domain, and d) a
fourth monomer comprising an IL-15 variant, where the VH and the VL
form a LAG-3 binding domain, and where the IL-15 and
IL-15R.alpha.(sushi) domain form an IL-15 complex. Any useful
domain linker can be used to attach the various components of the
heterodimeric protein including, but not limited to those in FIGS.
8 and 9A-C. In an exemplary embodiment, the domain linkers that
attach the IL-15R.alpha.(sushi) domain to the second Fc domain is
an antibody hinge domain (e.g., an antibody hinge domain).
[0341] In the Fab X ncIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having any of the variable
heavy and light domain pairs as shown in FIG. 12.
[0342] In the Fab X ncIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12.
[0343] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is a "Fab X ncIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH
is a variable heavy domain and CH2-CH3 is a first Fc domain; b) a
second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second Fc domain; c) a third monomer that includes from, N- to
C-terminus, VL-VC, where VL is a variable light domain, and d) a
fourth monomer comprising an IL-15 variant, where the VH and the VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34, respectively, and where the IL-15 and
IL-15R.alpha.(sushi) domain form an IL-15 complex. In another
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "Fab X ncIL-15/R.alpha."format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and
CH2-CH3 is a first Fc domain; b) a second monomer that includes,
from N- to C-terminus, an IL-15R.alpha.(sushi) domain-(domain
linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; c) a third
monomer that includes from, N- to C-terminus, VL-VC, where VL is a
variable light domain, and d) a fourth monomer comprising an IL-15
variant, where the VH and the VL are the variable heavy domain and
variable light domain of 2A11_H1.144_L2.142, respectively, and
where the IL-15 and IL-15R.alpha.(sushi) domain form an IL-15
complex.
[0344] In the Fab X ncIL-15/R.alpha. format, one preferred
embodiment utilizes an IL-15 variant that includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In one
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is a "Fab X ncIL-15/R.alpha."format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and
CH2-CH3 is a first Fc domain; b) a second monomer that includes,
from N- to C-terminus, an IL-15R.alpha.(sushi) domain-(domain
linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; c) a third
monomer that includes from, N- to C-terminus, VL-VC, where VL is a
variable light domain, and d) a fourth monomer comprising an IL-15
variant, where the VH and the VL form a LAG-3 binding domain, where
the IL-15 and IL-15R.alpha.(sushi) domain form an IL-15 complex,
and where the IL-15 variant includes amino acid substitutions
N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions N4D/N65D. In another exemplary embodiment, the VH and
VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C
and the IL-15 variant includes amino acid substitutions D30N/N65D.
In yet another exemplary embodiment, the VH and VL are the VH and
VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/E64Q/N65D.
[0345] In the Fab X ncIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12, with
either the IL-15 N4D/N65D variant or the IL-15 D30N/N65D variant or
the IL-15 D30N/E64Q/N65D variant. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is a "Fab X
ncIL-15/R.alpha."format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, a
VH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and
CH2-CH3 is a first Fc domain; b) a second monomer that includes,
from N- to C-terminus, an IL-15R.alpha.(sushi) domain-(domain
linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; c) a third
monomer that includes from, N- to C-terminus, VL-VC, where VL is a
variable light domain, and d) a fourth monomer comprising an IL-15
variant, where the VH and the VL form a LAG-3 binding domain, where
the IL-15 and IL-15R.alpha.(sushi) domain form an IL-15 complex,
where VH and VL are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and where the
IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In a particular embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
one embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
[0346] In the Fab X ncIL-15/R.alpha. format, one preferred
embodiment utilizes the skew variant pair S364K/E357Q:L368D/K370S.
In one embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric
protein is an "Fab X ncIL-15/R.alpha."format heterodimeric protein
that includes: a) a first monomer that includes, from N- to
C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable heavy
domain and CH2-CH3 is a first variant Fc domain; b) a second
monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes
from, N- to C-terminus, VL-VC, where VL is a variable light domain,
and d) a fourth monomer comprising an IL-15 variant, where the VH
and the VL form a LAG-3 binding domain, where the IL-15 and
IL-15R.alpha.(sushi) domain form an IL-15 complex, and where the
first and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants S364K/E357Q.
[0347] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "Fab X ncIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH
is a variable heavy domain and CH2-CH3 is a first variant Fc
domain; b) a second monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain; c) a third monomer that
includes from, N- to C-terminus, VL-VC, where VL is a variable
light domain, and d) a fourth monomer comprising an IL-15 variant,
where the VH and the VL form a LAG-3 binding domain, where the
IL-15 and IL-15R.alpha.(sushi) domain form an IL-15 complex, where
the IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D, and where the first and second
variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants S364K/E357Q. In an
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12
and 13A-C and the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D.
[0348] In the Fab X ncIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is a "Fab X
ncIL-15/R.alpha."format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, a
VH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant
Fc domain; c) a third monomer that includes from, N- to C-terminus,
VL-VC, where VL is a variable light domain, and d) a fourth monomer
comprising an IL-15 variant, where the VH and the VL are the
variable heavy domain and variable light domain of 7G8_H3.30_L1.34,
respectively, where the IL-15 and IL-15R.alpha.(sushi) domain form
an IL-15 complex and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S. In another
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "Fab X ncIL-15/R.alpha."format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant
Fc domain; c) a third monomer that includes from, N- to C-terminus,
VL-VC, where VL is a variable light domain, and d) a fourth monomer
comprising an IL-15 variant, where the VH and the VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142, respectively, where the IL-15 and
IL-15R.alpha.(sushi) domain form an IL-15 complex, and where the
first and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S.
[0349] In the Fab X ncIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S with either the IL-15
N4D/N65D variant or the IL-15 D30N/N65D variant or the IL-15
D30N/E64Q/N65D variant.
[0350] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is a "Fab X ncIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH
is a variable heavy domain and CH2-CH3 is a first variant Fc
domain; b) a second monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain; c) a third monomer that
includes from, N- to C-terminus, VL-VC, where VL is a variable
light domain, and d) a fourth monomer comprising an IL-15 variant,
where the VH and the VL form a LAG-3 binding domain, where the
IL-15 and IL-15R.alpha.(sushi) domain form an IL-15 complex, where
VH and VL are the variable heavy domain and variable light domain
of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the IL-15 variant
includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S. In an
exemplary embodiment, the first variant Fc domain includes skew
variants L368D/K370S, and the second variant Fc domain includes
skew variants S364K/E357Q. In a particular embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
one embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
[0351] In the Fab X ncIL-15/R.alpha. format, one preferred
embodiment utilizes the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0352] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is a "Fab X ncIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH
is a variable heavy domain and CH2-CH3 is a first variant Fc
domain; b) a second monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain; c) a third monomer that
includes from, N- to C-terminus, VL-VC, where VL is a variable
light domain, and d) a fourth monomer comprising an IL-15 variant,
where the VH and the VL form a LAG-3 binding domain, where the
IL-15 and IL-15R.alpha.(sushi) domain form an IL-15 complex, where
the first variant Fc domain includes skew variants L368D/K370S and
the second variant Fc domain includes skew variants S364K/E357Q,
where the first and second variant Fc domains each include FcKO
variants E233P/L234V/L235A/G236del/S267K, where the hinge-first
variant Fc domain of the first monomer includes pI variants
N208D/Q295E/N384D/Q418E/N421D, and where numbering is according to
EU numbering. In some embodiments, the hinge of the second monomer
also includes amino acid substitution C220S. In certain
embodiments, the first and second variant Fc domains each further
include half-life extension variants M428L/N434S. In an exemplary
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions N4D/N65D. In another exemplary embodiment, the VH and
VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C
and the IL-15 variant includes amino acid substitutions D30N/N65D.
In yet another exemplary embodiment, the VH and VL are the VH and
VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/E64Q/N65D.
[0353] In the Fab X ncIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 with
the FIG. 21E format, the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0354] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is a "Fab X ncIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH
is a variable heavy domain and CH2-CH3 is a first variant Fc
domain; b) a second monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain; c) a third monomer that
includes from, N- to C-terminus, VL-VC, where VL is a variable
light domain, and d) a fourth monomer comprising an IL-15 variant,
where the VH and the VL form a LAG-3 binding domain, where the
IL-15 and IL-15R.alpha.(sushi) domain form an IL-15 complex, where
VH and VL are the variable heavy domain and variable light domain
of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant
Fc domain includes skew variants L368D/K370S and the second variant
Fc domain includes skew variants S364K/E357Q, where the first and
second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI variants
N208D/Q295E/N384D/Q418E/N421D, and where numbering is according to
EU numbering. In some embodiments, the hinge of the second monomer
also includes amino acid substitution C220S. In certain
embodiments, the first and second variant Fc domains each further
include half-life extension variants M428L/N434S. In a particular
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D and the scFv includes the variable heavy and light domain
pair of 7G8_H3.30_L1.34. In a particular embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
one embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
F. Fab X dsIL-15/R.alpha.
[0355] This embodiment is shown in FIG. 21F, and comprises four
monomers. The first monomer comprises, from N- to C-terminus, the
IL-15R.alpha.(sushi)domain-domain linker-CH2-CH3, wherein the
IL-15R.alpha.(sushi)domain has been engineered to contain a
cysteine residue, and the second monomer comprises a heavy chain,
VH-CH1-hinge-CH2-CH3. The third monomer is a light chain that
includes, from N-to C-terminus, a variable light domain (VL) and a
constant light domain (CL). The fourth monomer is the variant IL-15
domain, also engineered to have a cysteine residue, such that a
disulfide bridge is formed under native cellular conditions. This
is generally referred to as "Fab X dsIL-15/R.alpha.", with the "ds"
standing for "disulfide".
[0356] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "Fab X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where VH is a
variable heavy domain and CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second Fc domain and the IL-15R.alpha.(sushi) domain includes
an amino acid substitution for a cysteine residue; c) a third
monomer that includes, from N- to C-terminus, a VL-CL, where VL is
a variable light domain; and d) an IL-15 variant that includes an
amino acid substitution for a cysteine residue, where the VH and VL
form a LAG-3 binding domain, and where the cysteine residue on the
IL-15 variant and the cysteine residue on the IL-15R.alpha.(sushi)
domain form a disulfide bond. Any useful domain linker can be used
to attach the various components of the heterodimeric protein
including, but not limited to those in FIGS. 8 and 9A-C. In an
exemplary embodiment, the domain linkers that attach the
IL-15R.alpha.(sushi) domain to the second Fc domain is an antibody
hinge domain (e.g., an antibody hinge domain).
[0357] In the Fab X dsIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having any of the variable
heavy and light domain pairs as shown in FIG. 12.
[0358] In the Fab X dsIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12.
[0359] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "Fab X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where VH is a
variable heavy domain and CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second Fc domain and the IL-15R.alpha.(sushi) domain includes
an amino acid substitution for a cysteine residue; c) a third
monomer that includes, from N- to C-terminus, a VL-CL, where VL is
a variable light domain; and d) an IL-15 variant that includes an
amino acid substitution for a cysteine residue, where the VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34, respectively, and where the cysteine residue on
the IL-15 variant and the cysteine residue on the
IL-15R.alpha.(sushi) domain form a disulfide bond. In another
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "Fab X dsIL-15/R.alpha." format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-hinge-CH2-CH3, where VH is a variable heavy domain and CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3,
where CH2-CH3 is a second Fc domain and the IL-15R.alpha.(sushi)
domain includes an amino acid substitution for a cysteine residue;
c) a third monomer that includes, from N- to C-terminus, a VL-CL,
where VL is a variable light domain; and d) an IL-15 variant that
includes an amino acid substitution for a cysteine residue, where
the VH and VL are the variable heavy domain and variable light
domain of 2A11_H1.144_L2.142, respectively, and where the cysteine
residue on the IL-15 variant and the cysteine residue on the
IL-15R.alpha.(sushi) domain form a disulfide bond.
[0360] In the Fab X dsIL-15/R.alpha. format, one preferred
embodiment utilizes an IL-15 variant that includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, with the
appropriate cysteine amino acid substitutions. In one embodiment,
the targeted IL-15/IL-15R.alpha. heterodimeric protein is an "Fab X
dsIL-15/R.alpha." format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where VH is a variable heavy domain and CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3,
where CH2-CH3 is a second Fc domain and the IL-15R.alpha.(sushi)
domain includes an amino acid substitution for a cysteine residue;
c) a third monomer that includes, from N- to C-terminus, a VL-CL,
where VL is a variable light domain; and d) an IL-15 variant that
includes an amino acid substitution for a cysteine residue, where
the VH and VL form a LAG-3 binding domain, where the cysteine
residue on the IL-15 variant and the cysteine residue on the
IL-15R.alpha.(sushi) domain form a disulfide bond, and where the
IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment, the VH
and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and
13A-C and the IL-15 variant includes amino acid substitutions
N4D/N65D. In another exemplary embodiment, the VH and VL are the VH
and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0361] In the Fab X dsIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12, with
either the IL-15 N4D/N65D variant or the IL-15 D30N/N65D variant or
the IL-15 D30N/E64Q/N65D variant, with the appropriate cysteine
amino acid substitutions. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an "Fab X
dsIL-15/R.alpha." format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where VH is a variable heavy domain and CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, an IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3,
where CH2-CH3 is a second Fc domain and the IL-15R.alpha.(sushi)
domain includes an amino acid substitution for a cysteine residue;
c) a third monomer that includes, from N- to C-terminus, a VL-CL,
where VL is a variable light domain; and d) an IL-15 variant that
includes an amino acid substitution for a cysteine residue, where
the VH and VL form a LAG-3 binding domain, where the cysteine
residue on the IL-15 variant and the cysteine residue on the
IL-15R.alpha.(sushi) domain form a disulfide bond, where VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and where the IL-15 variant
includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D. In a particular embodiment, the IL-15 variant
includes amino acid substitutions N4D/N65D and VH and VL are the
variable heavy domain and variable light domain of 7G8_H3.30_L1.34.
In another embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and VH and VL are the variable heavy domain
and variable light domain of 2A11_H1.144_L2.142. In one embodiment,
the IL-15 variant includes amino acid substitutions D30N/N65D and
VH and VL are the variable heavy domain and variable light domain
of 7G8_H3.30_L1.34. In another embodiment, the IL-15 variant
includes amino acid substitutions D30N/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
[0362] In the Fab X dsIL-15/R.alpha. format, one preferred
embodiment utilizes the skew variant pair S364K/E357Q:L368D/K370S.
In one embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric
protein is an "Fab X dsIL-15/R.alpha." format heterodimeric protein
that includes: a) a first monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3, where VH is a variable heavy domain
and CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant
Fc domain and the IL-15R.alpha.(sushi) domain includes an amino
acid substitution for a cysteine residue; c) a third monomer that
includes, from N- to C-terminus, a VL-CL, where VL is a variable
light domain; and d) an IL-15 variant that includes an amino acid
substitution for a cysteine residue, where the VH and VL form a
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, and where the first and second variant Fc
domains include the skew variant pair S364K/E357Q:L368D/K370S. In
an exemplary embodiment, the first variant Fc domain includes skew
variants L368D/K370S, and the second variant Fc domain includes
skew variants S364K/E357Q.
[0363] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "Fab X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where VH is a
variable heavy domain and CH2-CH3 is a first variant Fc domain; b)
a second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain and the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue; c) a
third monomer that includes, from N- to C-terminus, a VL-CL, where
VL is a variable light domain; and d) an IL-15 variant that
includes an amino acid substitution for a cysteine residue, where
the VH and VL form a LAG-3 binding domain, where the cysteine
residue on the IL-15 variant and the cysteine residue on the
IL-15R.alpha.(sushi) domain form a disulfide bond, where the IL-15
variant includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S. In an
exemplary embodiment, the first variant Fc domain includes skew
variants L368D/K370S, and the second variant Fc domain includes
skew variants S364K/E357Q. In an exemplary embodiment, the VH and
VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C
and the IL-15 variant includes amino acid substitutions N4D/N65D.
In another exemplary embodiment, the VH and VL are the VH and VL of
any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant
includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0364] In the Fab X dsIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an "Fab X
dsIL-15/R.alpha." format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where VH is a variable heavy domain and CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, an IL-15R.alpha.(sushi) domain-(domain
linker)-CH2-CH3, where CH2-CH3 is a variant second Fc domain and
the IL-15R.alpha.(sushi) domain includes an amino acid substitution
for a cysteine residue; c) a third monomer that includes, from N-
to C-terminus, a VL-CL, where VL is a variable light domain; and d)
an IL-15 variant that includes an amino acid substitution for a
cysteine residue, where the VH and VL are the variable heavy domain
and variable light domain of 7G8_H3.30_L1.34, respectively, where
the cysteine residue on the IL-15 variant and the cysteine residue
on the IL-15R.alpha.(sushi) domain form a disulfide bond, and where
the first and second variant Fc domains include the skew variant
pair S364K/E357Q:L368D/K370S. In another embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an "Fab X
dsIL-15/R.alpha." format heterodimeric protein that includes: a) a
first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where VH is a variable heavy domain and CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, an IL-15R.alpha.(sushi) domain-(domain
linker)-CH2-CH3, where CH2-CH3 is a second variant Fc domain and
the IL-15R.alpha.(sushi) domain includes an amino acid substitution
for a cysteine residue; c) a third monomer that includes, from N-
to C-terminus, a VL-CL, where VL is a variable light domain; and d)
an IL-15 variant that includes an amino acid substitution for a
cysteine residue, where the VH and VL are the variable heavy domain
and variable light domain of 2A11_H1.144_L2.142, respectively,
where the cysteine residue on the IL-15 variant and the cysteine
residue on the IL-15R.alpha.(sushi) domain form a disulfide bond,
and where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S.
[0365] In the Fab X dsIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S with either the IL-15
N4D/N65D variant or the IL-15 D30N/N65D variant or the IL-15
D30N/E64Q/N65D variant with appropriate cysteine substitutions. In
one embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric
protein is an "Fab X dsIL-15/R.alpha." format heterodimeric protein
that includes: a) a first monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3, where VH is a variable heavy domain
and CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant
Fc domain and the IL-15R.alpha.(sushi) domain includes an amino
acid substitution for a cysteine residue; c) a third monomer that
includes, from N- to C-terminus, a VL-CL, where VL is a variable
light domain; and d) an IL-15 variant that includes an amino acid
substitution for a cysteine residue, where the VH and VL form a
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, where the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, and where the
first and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants S364K/E357Q. In a
particular embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and VH and VL are the variable heavy domain
and variable light domain of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D and VH and VL are the variable heavy domain and variable
light domain of 2A11_H1.144_L2.142. In one embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
yet another of embodiment, the IL-15 variant includes amino acid
substitutions D30N/E64Q/N65D and VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D and VH and VL are the variable heavy domain and
variable light domain of 2A11_H1.144_L2.142.
[0366] In the Fab X dsIL-15/R.alpha. format, one preferred
embodiment utilizes the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0367] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "Fab X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where VH is a
variable heavy domain and CH2-CH3 is a first variant Fc domain; b)
a second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain and the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue; c) a
third monomer that includes, from N- to C-terminus, a VL-CL, where
VL is a variable light domain; and d) an IL-15 variant that
includes an amino acid substitution for a cysteine residue, where
the VH and VL form a LAG-3 binding domain, where the cysteine
residue on the IL-15 variant and the cysteine residue on the
IL-15R.alpha.(sushi) domain form a disulfide bond, where the first
variant Fc domain includes skew variants L368D/K370S and the second
variant Fc domain includes skew variants S364K/E357Q, where the
first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI variants
N208D/Q295E/N384D/Q418E/N421D, and where numbering is according to
EU numbering. In some embodiments, the hinge of the second monomer
also includes amino acid substitution C220S. In certain
embodiments, the first and second variant Fc domains each further
include half-life extension variants M428L/N434S. In an exemplary
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions N4D/N65D. In another exemplary embodiment, the VH and
VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C
and the IL-15 variant includes amino acid substitutions D30N/N65D.
In yet another exemplary embodiment, the VH and VL are the VH and
VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/E64Q/N65D.
[0368] In the Fab X dsIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 with
the FIG. 21F format, the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0369] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "Fab X dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where VH is a
variable heavy domain and CH2-CH3 is a first variant Fc domain; b)
a second monomer that includes, from N- to C-terminus, an
IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3
is a second variant Fc domain and the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue; c) a
third monomer that includes, from N- to C-terminus, a VL-CL, where
VL is a variable light domain; and d) an IL-15 variant that
includes an amino acid substitution for a cysteine residue, where
the VH and VL form a LAG-3 binding domain, where the cysteine
residue on the IL-15 variant and the cysteine residue on the
IL-15R.alpha.(sushi) domain form a disulfide bond, where VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants L368D/K370S and the second variant Fc
domain includes skew variants S364K/E357Q where the first and
second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI variants
N208D/Q295E/N384D/Q418E/N421D, and where numbering is according to
EU numbering. In some embodiments, the hinge of the second monomer
also includes amino acid substitution C220S. In certain
embodiments, the first and second variant Fc domains each further
include half-life extension variants M428L/N434S. In a particular
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes
amino acid substitutions D30N/N65D and VH and VL are the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34. In
another embodiment, the IL-15 variant includes amino acid
substitutions D30N/N65D and VH and VL are the variable heavy domain
and variable light domain of 2A11_H1.144_L2.142. In yet another of
embodiment, the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D and VH and VL are the variable heavy domain and
variable light domain of 7G8_H3.30_L1.34. In another embodiment,
the IL-15 variant includes amino acid substitutions D30N/E64Q/N65D
and VH and VL are the variable heavy domain and variable light
domain of 2A11_H1.144_L2.142.
G. mAb-scIL-15/R.alpha.
[0370] This embodiment is shown in FIG. 21G, and comprises three
monomers (although the fusion protein is a tetramer). The first
monomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3. The second
monomer comprises a heavy chain with a scIL-15 complex,
VH-CH1-hinge-CH2-CH3-domain
linker-IL-15R.alpha.(sushi)domain-domain linker-IL-15 variant. The
third (and fourth) monomer are light chains, VL-CL. This is
generally referred to as "mAb-scIL-15/R.alpha.", with the "sc"
standing for "single chain". This binds the LAG-3 molecule
bivalently.
[0371] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second Fc
domain; and c) a third and fourth monomer that each include, from
N- to C-terminus, a VL-CL, where VL is a variable light domain,
where the VH of the first monomer and the VL of the third monomer
form a first LAG-3 binding domain, where the VH of the second
monomer and the VL of the fourth monomer form a second LAG-3
binding domain, and where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex. Any useful
domain linker can be used to attach the various components of the
heterodimeric protein including, but not limited to those in FIGS.
8 and 9A-C.
[0372] In the mAb-scIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having any of the variable heavy and
light domain pairs as shown in FIG. 12.
[0373] In the mAb-scIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12.
[0374] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second Fc
domain; and c) a third and fourth monomer that each include, from
N- to C-terminus, a VL-CL, where VL is a variable light domain,
where the VH of the first monomer and the VL of the third monomer
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34, respectively, where the VH of the second monomer
and the VL of the fourth monomer are the variable heavy domain and
variable light domain of 7G8_H3.30_L1.34, respectively, and where
the IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex. In another embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second Fc
domain; and c) a third and fourth monomer that each include, from
N- to C-terminus, a VL-CL, where VL is a variable light domain,
where the VH of the first monomer and the VL of the third monomer
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142, respectively, where the VH of the second
monomer and the VL of the fourth monomer are the variable heavy
domain and variable light domain of 2A11_H1.144_L2.142,
respectively, and where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex.
[0375] In the mAb-scIL-15/R.alpha. format, one preferred embodiment
utilizes an IL-15 variant that includes amino acid substitutions
N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-scIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second Fc
domain; and c) a third and fourth monomer that each include, from
N- to C-terminus, a VL-CL, where VL is a variable light domain,
where the VH of the first monomer and the VL of the third monomer
form a first LAG-3 binding domain, where the VH of the second
monomer and the VL of the fourth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, and where the
IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment, the VH
and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and
13A-C and the IL-15 variant includes amino acid substitutions
N4D/N65D. In another exemplary embodiment, the VH and VL are the VH
and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0376] In the mAb-scIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12, with either
the IL-15 N4D/N65D variant or the IL-15 D30N/N65D variant or the
IL-15 D30N/E64Q/N65D variant. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-scIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second Fc
domain; and c) a third and fourth monomer that each include, from
N- to C-terminus, a VL-CL, where VL is a variable light domain,
where the VH of the first monomer and the VL of the third monomer
form a first LAG-3 binding domain, where the VH of the second
monomer and the VL of the fourth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and where the IL-15 variant
includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D. In a particular embodiment, the IL-15 variant
includes amino acid substitutions N4D/N65D and VH and VL are the
variable heavy domain and variable light domain of 7G8_H3.30_L1.34.
In another embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and VH and VL are the variable heavy domain
and variable light domain of 2A11_H1.144_L2.142. In one embodiment,
the IL-15 variant includes amino acid substitutions D30N/N65D and
VH and VL are the variable heavy domain and variable light domain
of 7G8_H3.30_L1.34. In another embodiment, the IL-15 variant
includes amino acid substitutions D30N/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
[0377] In the mAb-scIL-15/R.alpha. format, one preferred embodiment
utilizes the skew variant pair S364K/E357Q:L368D/K370S. In one
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "mAb-scIL-15/R.alpha." format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b)
a second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second
variant Fc domain; and c) a third and fourth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
third monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fourth monomer form a second
LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, and where the
first and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D and K370S, and the
second variant Fc domain includes skew variants S364K and E357Q. In
an exemplary embodiment, the first variant Fc domain includes skew
variants S364K and E357Q, and the second variant Fc domain includes
skew variants L368D and K370S.
[0378] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where
the IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D, and where the first and second
variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D and K370S, and the
second variant Fc domain includes skew variants S364K and E357Q. In
an exemplary embodiment, the first variant Fc domain includes skew
variants S364K and E357Q, and the second variant Fc domain includes
skew variants L368D and K370S. In an exemplary embodiment, the VH
and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and
13A-C and the IL-15 variant includes amino acid substitutions
N4D/N65D. In another exemplary embodiment, the VH and VL are the VH
and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0379] In the mAb-scIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the skew
variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-scIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second
variant Fc domain; and c) a third and fourth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
third monomer are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34, respectively, where the VH of the second
monomer and the VL of the fourth monomer are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34, respectively,
where the IL-15 variant and the IL-15R.alpha.(sushi) domain form an
IL-15 complex, where the IL-15 variant and the IL-15R.alpha.(sushi)
domain form an IL-15 complex, and where the first and second
variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In another embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-scIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second
variant Fc domain; and c) a third and fourth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
third monomer are the variable heavy domain and variable light
domain of 2A11_H1.144_L2.142, respectively, where the VH of the
second monomer and the VL of the fourth monomer are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142,
respectively, where the IL-15 variant and the IL-15R.alpha.(sushi)
domain form an IL-15 complex, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, and where the
first and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D and K370S, and the
second variant Fc domain includes skew variants S364K and E357Q. In
an exemplary embodiment, the first variant Fc domain includes skew
variants S364K and E357Q, and the second variant Fc domain includes
skew variants L368D and K370S.
[0380] In the mAb-scIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the skew
variant pair S364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D
variant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D
variant. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH
and VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the IL-15 variant
includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S. In an
exemplary embodiment, the first variant Fc domain includes skew
variants L368D and K370S, and the second variant Fc domain includes
skew variants S364K and E357Q. In a particular embodiment, the
IL-15 variant includes amino acid substitutions N4D/N65D and VH and
VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
one embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In an exemplary embodiment, the first variant
Fc domain includes skew variants L368D and K370S, and the second
variant Fc domain includes skew variants S364K and E357Q. In an
exemplary embodiment, the first variant Fc domain includes skew
variants S364K and E357Q, and the second variant Fc domain includes
skew variants L368D and K370S.
[0381] In the mAb-scIL-15/R.alpha. format, one preferred embodiment
utilizes the skew variant set S364K/E357Q:L368D/K370S, the pI
variants N208D/Q295E/N384D/Q418D/N421D and/or
Q196K/I199T/P271R/P228R/N276K, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0382] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where
the first variant Fc domain includes skew variants L368D/K370S and
the second variant Fc domain include the skew variant pair
S364K/E357Q, where the first and second variant Fc domains each
include FcKO variants E233P/L234V/L235A/G236del/S267K, where the
hinge-first variant Fc domain of the first monomer includes pI
substitutions N208D/Q295E/N384D/Q418D/N421D and the hinge-second
variant Fc domain of the second monomer includes pI variants
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where
the first variant Fc domain includes skew variants L368D/K370S and
the second variant Fc domain include the skew variant pair
S364K/E357Q, where the first and second variant Fc domains each
include FcKO variants E233P/L234V/L235A/G236del/S267K, where the
hinge-first variant Fc domain of the first monomer includes pI
substitutions N208D/Q295E/N384D/Q418D/N421D, and where numbering is
according to EU numbering. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-scIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second
variant Fc domain; and c) a third and fourth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
third monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fourth monomer form a second
LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the first
variant Fc domain includes skew variants L368D/K370S and the second
variant Fc domain include the skew variant pair S364K/E357Q, where
the first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fc
domain of the second monomer includes pI variants
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where
the first variant Fc domain includes skew variants S364K/E357Q and
the second variant Fc domain include the skew variant pair
L368D/K370S, where the first and second variant Fc domains each
include FcKO variants E233P/L234V/L235A/G236del/S267K, where the
hinge-first variant Fc domain of the first monomer includes pI
substitutions Q196K/I199T/P271R/P228R/N276K and the hinge-second
variant Fc domain of the second monomer includes pI variants
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where
the first variant Fc domain includes skew variants S364K/E357Q and
the second variant Fc domain include the skew variant pair
L368D/K370S, where the first and second variant Fc domains each
include FcKO variants E233P/L234V/L235A/G236del/S267K, where the
hinge-first variant Fc domain of the first monomer includes pI
substitutions Q196K/I199T/P271R/P228R/N276K, and where numbering is
according to EU numbering. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-scIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second
variant Fc domain; and c) a third and fourth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
third monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fourth monomer form a second
LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the first
variant Fc domain includes skew variants S364K/E357Q and the second
variant Fc domain include the skew variant pair L368D/K370S, where
the first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the hinge-second variant Fc domain of the second monomer
includes pI variants N208D/Q295E/N384D/Q418D/N421D, and where
numbering is according to EU numbering. In certain embodiments, the
first and second variant Fc domains each further include half-life
extension variants M428L/N434S. In an exemplary embodiment, the
IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment, the VH
and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and
13A-C and the IL-15 variant includes amino acid substitutions
N4D/N65D. In another exemplary embodiment, the VH and VL are the VH
and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0383] In the mAb-scIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG.
21G format, the skew variant set S364K/E357Q:L368D/K370S, the pI
variants N208D/Q295E/N384D/Q418D/N421D and/or
Q196K/I199T/P271R/P228R/N276K, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0384] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH
and VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants L368D/K370S and the second variant Fc
domain include the skew variant pair S364K/E357Q where the first
and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
N208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc
domain of the second monomer includes pI variants
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH
and VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants L368D/K370S and the second variant Fc
domain include the skew variant pair S364K/E357Q where the first
and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH
and VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants L368D/K370S and the second variant Fc
domain include the skew variant pair S364K/E357Q, where the first
and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fc
domain of the second monomer includes pI variants
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH
and VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants S364K/E357Q and the second variant Fc
domain include the skew variant pair L368D/K370S, where the first
and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
Q196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc
domain of the second monomer includes pI variants
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH
and VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants S364K/E357Q and the second variant Fc
domain include the skew variant pair L368D/K370S, where the first
and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-scIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant,
where CH2-CH3 is a second variant Fc domain; and c) a third and
fourth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH
and VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants S364K/E357Q and the second variant Fc
domain include the skew variant pair L368D/K370S, where the first
and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the hinge-second variant Fc domain of the second monomer
includes pI variants N208D/Q295E/N384D/Q418D/N421D, and where
numbering is according to EU numbering. In certain embodiments, the
first and second variant Fc domains each further include half-life
extension variants M428L/N434S. In a particular embodiment, the
IL-15 variant includes amino acid substitutions N4D/N65D and VH and
VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
one embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
H. mAb-ncIL-15/R.alpha.
[0385] This embodiment is shown in FIG. 21H, and comprises four
monomers (although the heterodimeric fusion protein is a pentamer).
The first monomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3.
The second monomer comprises a heavy chain with an
IL-15R.alpha.(sushi) domain: e.g., VH-CH1-hinge-CH2-CH3-domain
linker-IL-15R.alpha.(sushi) domain. The third monomer is a variant
IL-15 domain. The fourth (and fifth) monomer are light chains,
VL-CL. This is generally referred to as "mAb-ncIL-15/R.alpha.",
with the "nc" standing for "non-covalent". This also binds the
LAG-3 bivalently.
[0386] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where CH2-CH3 is a second Fc domain; c) a
third monomer that includes an IL-15 variant; and d) a fourth and
fifth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the fourth monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fifth
monomer form a second LAG-3 binding domain, and where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex.
Any useful domain linker can be used to attach the various
components of the heterodimeric protein including, but not limited
to those in FIGS. 8 and 9A-C.
[0387] In the mAb-ncIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having any of the variable heavy and
light domain pairs as shown in FIG. 12.
[0388] In the mAb-ncIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12.
[0389] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where CH2-CH3 is a second Fc domain; c) a
third monomer that includes an IL-15 variant; and d) a fourth and
fifth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the fourth monomer are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34, respectively,
where the VH of the second monomer and the VL of the fifth monomer
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34, respectively, and where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex. In another
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "mAb-ncIL-15/R.alpha." format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where CH2-CH3 is a second Fc domain; c) a
third monomer that includes an IL-15 variant; and d) a fourth and
fifth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the fourth monomer are the variable heavy
domain and variable light domain of 2A11_H1.144_L2.142,
respectively, where the VH of the second monomer and the VL of the
fifth monomer are the variable heavy domain and variable light
domain of 2A11_H1.144_L2.142, respectively, and where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex.
[0390] In the mAb-ncIL-15/R.alpha. format, one preferred embodiment
utilizes an IL-15 variant that includes amino acid substitutions
N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-ncIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where CH2-CH3 is a second Fc domain; c) a
third monomer that includes an IL-15 variant; and d) a fourth and
fifth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the fourth monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fifth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where
the IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, and where the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12
and 13A-C and the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D.
[0391] In the mAb-ncIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12, with either
the IL-15 N4D/N65D variant or the IL-15 D30N/N65D variant or the
IL-15 D30N/E64Q/N65D variant. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-ncIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where CH2-CH3 is a second Fc domain; c) a
third monomer that includes an IL-15 variant; and d) a fourth and
fifth monomer that each include, from N- to C-terminus, a VL-CL,
where VL is a variable light domain, where the VH of the first
monomer and the VL of the fourth monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fifth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where
the IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, where VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and where
the IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In a particular embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
one embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
[0392] In the mAb-ncIL-15/R.alpha. format, one preferred embodiment
utilizes the skew variant pair S364K/E357Q:L368D/K370S. In one
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "mAb-ncIL-15/R.alpha." format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b)
a second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where CH2-CH3 is a second variant Fc
domain; c) a third monomer that includes an IL-15 variant; and d) a
fourth and fifth monomer that each include, from N- to C-terminus,
a VL-CL, where VL is a variable light domain, where the VH of the
first monomer and the VL of the fourth monomer form a first LAG-3
binding domain, where the VH of the second monomer and the VL of
the fifth monomer form a second LAG-3 binding domain, where the
IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, and where the first and second variant Fc domains include
the skew variant pair S364K/E357Q:L368D/K370S. In an exemplary
embodiment, the first variant Fc domain includes skew variants
L368D/K370S, and the second variant Fc domain includes skew
variants S364K/E357Q. In another exemplary embodiment, the first
variant Fc domain includes skew variants S364K/E357Q, and the
second variant Fc domain includes skew variants L368D/K370S.
[0393] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes an
IL-15 variant; and d) a fourth and fifth monomer that each include,
from N- to C-terminus, a VL-CL, where VL is a variable light
domain, where the VH of the first monomer and the VL of the fourth
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fifth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S. In an
exemplary embodiment, the first variant Fc domain includes skew
variants L368D/K370S, and the second variant Fc domain includes
skew variants S364K/E357Q. In another exemplary embodiment, the
first variant Fc domain includes skew variants S364K/E357Q, and the
second variant Fc domain includes skew variants L368D/K370S. In an
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12
and 13A-C and the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D.
[0394] In the mAb-ncIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the skew
variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-ncIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where CH2-CH3 is a second variant Fc
domain; c) a third monomer that includes an IL-15 variant; and d) a
fourth and fifth monomer that each include, from N- to C-terminus,
a VL-CL, where VL is a variable light domain, where the VH of the
first monomer and the VL of the fourth monomer are the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34,
respectively, where the VH of the second monomer and the VL of the
fifth monomer are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34, respectively, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, and
where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S. In another embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-ncIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where CH2-CH3 is a second variant Fc
domain; c) a third monomer that includes an IL-15 variant; and d) a
fourth and fifth monomer that each include, from N- to C-terminus,
a VL-CL, where VL is a variable light domain, where the VH of the
first monomer and the VL of the fourth monomer are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142,
respectively, where the VH of the second monomer and the VL of the
fifth monomer are the variable heavy domain and variable light
domain of 2A11_H1.144_L2.142, respectively, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, and
where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S.
[0395] In the mAb-ncIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the skew
variant pair S364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D
variant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D
variant. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes an
IL-15 variant; and d) a fourth and fifth monomer that each include,
from N- to C-terminus, a VL-CL, where VL is a variable light
domain, where the VH of the first monomer and the VL of the fourth
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fifth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
where VH and VL are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the IL-15
variant includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S. In an
exemplary embodiment, the first variant Fc domain includes skew
variants L368D/K370S, and the second variant Fc domain includes
skew variants S364K/E357Q. In an exemplary embodiment, the first
variant Fc domain includes skew variants S364K/E357Q, and the
second variant Fc domain includes skew variants L368D/K370S. In a
particular embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and VH and VL are the variable heavy domain
and variable light domain of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D and VH and VL are the variable heavy domain and variable
light domain of 2A11_H1.144_L2.142. In one embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
yet another embodiment, the IL-15 variant includes amino acid
substitutions D30N/E64Q/N65D and VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D and VH and VL are the variable heavy domain and
variable light domain of 2A11_H1.144_L2.142.
[0396] In the mAb-ncIL-15/R.alpha. format, one preferred embodiment
utilizes the skew variant set S364K/E357Q:L368D/K370S, the pI
variants N208D/Q295E/N384D/Q418D/N421D and/or
Q196K/I199T/P271R/P228R/N276K, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0397] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes an
IL-15 variant; and d) a fourth and fifth monomer that each include,
from N- to C-terminus, a VL-CL, where VL is a variable light
domain, where the VH of the first monomer and the VL of the fourth
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fifth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
where the first variant Fc domain includes skew variants
L368D/K370S and the second variant Fc domain include the skew
variant pair S364K/E357Q, where the first and second variant Fc
domains each include FcKO variants E233P/L234V/L235A/G236del/S267K,
where the hinge-first variant Fc domain of the first monomer
includes pI substitutions N208D/Q295E/N384D/Q418D/N421D and the
hinge-second variant Fc domain of the second monomer includes pI
variants Q196K/I199T/P271R/P228R/N276K, and where numbering is
according to EU numbering. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-ncIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where CH2-CH3 is a second variant Fc
domain; c) a third monomer that includes an IL-15 variant; and d) a
fourth and fifth monomer that each include, from N- to C-terminus,
a VL-CL, where VL is a variable light domain, where the VH of the
first monomer and the VL of the fourth monomer form a first LAG-3
binding domain, where the VH of the second monomer and the VL of
the fifth monomer form a second LAG-3 binding domain, where the
IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, where the IL-15 variant and the IL-15R.alpha.(sushi)
domain form an IL-15 complex, where the first variant Fc domain
includes skew variants L368D/K370S and the second variant Fc domain
include the skew variant pair S364K/E357Q, where the first and
second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes an
IL-15 variant; and d) a fourth and fifth monomer that each include,
from N- to C-terminus, a VL-CL, where VL is a variable light
domain, where the VH of the first monomer and the VL of the fourth
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fifth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
where the first variant Fc domain includes skew variants
L368D/K370S and the second variant Fc domain include the skew
variant pair S364K/E357Q, where the first and second variant Fc
domains each include FcKO variants E233P/L234V/L235A/G236del/S267K,
where the hinge-second variant Fc domain of the second monomer
includes pI variants Q196K/I199T/P271R/P228R/N276K, and where
numbering is according to EU numbering. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-ncIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where CH2-CH3 is a second variant Fc
domain; c) a third monomer that includes an IL-15 variant; and d) a
fourth and fifth monomer that each include, from N- to C-terminus,
a VL-CL, where VL is a variable light domain, where the VH of the
first monomer and the VL of the fourth monomer form a first LAG-3
binding domain, where the VH of the second monomer and the VL of
the filth monomer form a second LAG-3 binding domain, where the
IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, where the IL-15 variant and the IL-15R.alpha.(sushi)
domain form an IL-15 complex, where the first variant Fc domain
includes skew variants S364K/E357Q and the second variant Fc domain
include the skew variant pair L368D/K370S, where the first and
second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
Q196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc
domain of the second monomer includes pI variants
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes an
IL-15 variant; and d) a fourth and fifth monomer that each include,
from N- to C-terminus, a VL-CL, where VL is a variable light
domain, where the VH of the first monomer and the VL of the fourth
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fifth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
where the first variant Fc domain includes skew variants
S364K/E357Q and the second variant Fc domain include the skew
variant pair L368D/K370S, where the first and second variant Fc
domains each include FcKO variants E233P/L234V/L235A/G236del/S267K,
where the hinge-first variant Fc domain of the first monomer
includes pI substitutions Q196K/I199T/P271R/P228R/N276K, and where
numbering is according to EU numbering. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-ncIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where CH2-CH3 is a second variant Fc
domain; c) a third monomer that includes an IL-15 variant; and d) a
fourth and fifth monomer that each include, from N- to C-terminus,
a VL-CL, where VL is a variable light domain, where the VH of the
first monomer and the VL of the fourth monomer form a first LAG-3
binding domain, where the VH of the second monomer and the VL of
the fifth monomer form a second LAG-3 binding domain, where the
IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, where the IL-15 variant and the IL-15R.alpha.(sushi)
domain form an IL-15 complex, where the first variant Fc domain
includes skew variants S364K/E357Q and the second variant Fc domain
include the skew variant pair L368D/K370S, where the first and
second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fc
domain of the second monomer includes pI variants
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In certain embodiments, the first and second variant
Fc domains each further include half-life extension variants
M428L/N434S. In an exemplary embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In
an exemplary embodiment, the VH and VL are the VH and VL of any of
the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12
and 13A-C and the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D.
[0398] In the mAb-ncIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142 as shown in
FIG. 12 with the FIG. 21H format, the skew variant set
S364K/E357Q:L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418D/N421D and/or Q196K/I199T/P271R/P228R/N276K,
the ablation variants E233P/L234V/L235A/G236_/S267K on both first
and second monomers, and optionally the 428L/434S variants on both
first and second monomers. In an exemplary embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D.
[0399] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes an
IL-15 variant; and d) a fourth and fifth monomer that each include,
from N- to C-terminus, a VL-CL, where VL is a variable light
domain, where the VH of the first monomer and the VL of the fourth
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fifth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
where VH and VL are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first
variant Fc domain includes skew variants L368D/K370S and the second
variant Fc domain include the skew variant pair S364K/E357Q, where
the first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
N208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc
domain of the second monomer includes pI variants
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes an
IL-15 variant; and d) a fourth and fifth monomer that each include,
from N- to C-terminus, a VL-CL, where VL is a variable light
domain, where the VH of the first monomer and the VL of the fourth
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fifth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
where VH and VL are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first
variant Fc domain includes skew variants L368D/K370S and the second
variant Fc domain include the skew variant pair S364K/E357Q, where
the first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes an
IL-15 variant; and d) a fourth and fifth monomer that each include,
from N- to C-terminus, a VL-CL, where VL is a variable light
domain, where the VH of the first monomer and the VL of the fourth
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fifth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
where VH and VL are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first
variant Fc domain includes skew variants L368D/K370S and the second
variant Fc domain include the skew variant pair S364K/E357Q, where
the first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fc
domain of the second monomer includes pI variants
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes an
IL-15 variant; and d) a fourth and fifth monomer that each include,
from N- to C-terminus, a VL-CL, where VL is a variable light
domain, where the VH of the first monomer and the VL of the fourth
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fifth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
where VH and VL are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first
variant Fc domain includes skew variants S364K/E357Q and the second
variant Fc domain include the skew variant pair L368D/K370S, where
the first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
Q196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc
domain of the second monomer includes pI variants
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes an
IL-15 variant; and d) a fourth and fifth monomer that each include,
from N- to C-terminus, a VL-CL, where VL is a variable light
domain, where the VH of the first monomer and the VL of the fourth
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fifth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
where VH and VL are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first
variant Fc domain includes skew variants S364K/E357Q and the second
variant Fc domain include the skew variant pair L368D/K370S, where
the first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-ncIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where CH2-CH3
is a second variant Fc domain; c) a third monomer that includes an
IL-15 variant; and d) a fourth and fifth monomer that each include,
from N- to C-terminus, a VL-CL, where VL is a variable light
domain, where the VH of the first monomer and the VL of the fourth
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fifth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant and the IL-15R.alpha.(sushi) domain form an IL-15 complex,
where VH and VL are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first
variant Fc domain includes skew variants S364K/E357Q and the second
variant Fc domain include the skew variant pair L368D/K370S, where
the first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fc
domain of the second monomer includes pI variants
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In certain embodiments, the first and second variant
Fc domains each further include half-life extension variants
M428L/N434S. In a particular embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34. In
another embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and VH and VL are the variable heavy domain
and variable light domain of 2A11_H1.144_L2.142. In one embodiment,
the IL-15 variant includes amino acid substitutions D30N/N65D and
VH and VL are the variable heavy domain and variable light domain
of 7G8_H3.30_L1.34. In another embodiment, the IL-15 variant
includes amino acid substitutions D30N/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
I. mAb-dsIL-15/R.alpha.
[0400] This embodiment is shown in FIG. 21I, and comprises four
monomers (although the heterodimeric fusion protein is a pentamer).
The first monomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3.
The second monomer comprises a heavy chain with an
IL-15R.alpha.(sushi) domain: e.g., VH-CH1-hinge-CH2-CH3-domain
linker-IL-15R.alpha.(sushi) domain, where the IL-15R.alpha.(sushi)
domain has been engineered to contain a cysteine residue. The third
monomer is a variant IL-15 domain, which has been engineered to
contain a cysteine residue, such that the IL-15 complex is formed
under physiological conditions. The fourth (and fifth) monomer are
light chains, VL-CL. This is generally referred to as
"mAb-dsIL-15/R.alpha.", with the "ds" standing for "disulfide", and
it binds LAG-3 bivalently.
[0401] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, and where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond. Any useful domain linker can be used to
attach the various components of the heterodimeric protein
including, but not limited to those in FIGS. 8 and 9A-C.
[0402] In the mAb-dsIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having any of the variable heavy and
light domain pairs as shown in FIG. 12.
[0403] In the mAb-dsIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12.
[0404] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34, respectively, where the VH of the second
monomer and the VL of the fifth monomer are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34, respectively,
and where the cysteine residue on the IL-15 variant and the
cysteine residue on the IL-15R.alpha.(sushi) domain form a
disulfide bond. In another embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-dsIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer are the variable heavy domain and variable light
domain of 2A11_H1.144_L2.142, respectively, where the VH of the
second monomer and the VL of the fifth monomer are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142,
respectively, and where the cysteine residue on the IL-15 variant
and the cysteine residue on the IL-15R.alpha.(sushi) domain form a
disulfide bond.
[0405] In the mAb-dsIL-15/R.alpha. format, one preferred embodiment
utilizes an IL-15 variant that includes amino acid substitutions
N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, with the appropriate
cysteine amino acid substitutions. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-dsIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, and where the IL-15 variant includes amino
acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12
and 13A-C and the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D.
[0406] In the mAb-dsIL-15/R.alpha. format, one preferred embodiment
7G8_H3.30_L1.34 or the variable heavy and light domain pair of
2A11_H1.144_L2.142 as shown in FIG. 12, with either the IL-15
N4D/N65D variant or the IL-15 D30N/N65D variant or the IL-15
D30N/E64Q/N65D variant, with the appropriate cysteine amino acid
substitutions. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, and where the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In a
particular embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and VH and VL are the variable heavy domain
and variable light domain of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D and VH and VL are the variable heavy domain and variable
light domain of 2A11_H1.144_L2.142. In one embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
yet another embodiment, the IL-15 variant includes amino acid
substitutions D30N/E64Q/N65D and VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34. In another
embodiment, the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D and VH and VL are the variable heavy domain and
variable light domain of 2A11_H1.144_L2.142.
[0407] In the mAb-dsIL-15/R.alpha. format, one preferred embodiment
utilizes the skew variant pair S364K/E357Q:L368D/K370S. In one
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "mAb-dsIL-15/R.alpha." format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b)
a second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second variant Fc domain; c) a third monomer that
includes an IL-15 variant that includes an amino acid substitution
for a cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, and where the first and second variant Fc
domains include the skew variant pair S364K/E357Q:L368D/K370S. In
an exemplary embodiment, the first variant Fc domain includes skew
variants L368D/K370S, and the second variant Fc domain includes
skew variants S364K/E357Q. In another exemplary embodiment, the
first variant Fc domain includes skew variants S364K/E357Q, and the
second variant Fc domain includes skew variants L368D/K370S.
[0408] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first variant Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-hinge-CH2-CH3-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker), where the
IL-15R.alpha.(sushi) domain includes an amino acid substitution for
a cysteine residue and CH2-CH3 is a second variant Fc domain; c) a
third monomer that includes an IL-15 variant that includes an amino
acid substitution for a cysteine residue; and d) a fourth and fifth
monomer that each include, from N- to C-terminus, a VL-CL, where VL
is a variable light domain, where the VH of the first monomer and
the VL of the fourth monomer form a first LAG-3 binding domain,
where the VH of the second monomer and the VL of the fifth monomer
form a second LAG-3 binding domain, where the cysteine residue on
the IL-15 variant and the cysteine residue on the
IL-15R.alpha.(sushi) domain form a disulfide bond, where the IL-15
variant includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S. In an
exemplary embodiment, the first variant Fc domain includes skew
variants L368D/K370S, and the second variant Fc domain includes
skew variants S364K/E357Q. In another exemplary embodiment, the
first variant Fc domain includes skew variants S364K/E357Q, and the
second variant Fc domain includes skew variants L368D/K370S. In an
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12
and 13A-C and the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D.
[0409] In the mAb-dsIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the skew
variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-dsIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second variant Fc domain; c) a third monomer that
includes an IL-15 variant that includes an amino acid substitution
for a cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34, respectively, where the VH of the second
monomer and the VL of the fifth monomer are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34, respectively,
where the cysteine residue on the IL-15 variant and the cysteine
residue on the IL-15R.alpha.(sushi) domain form a disulfide bond,
and where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S. In another embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-dsIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second variant Fc domain; c) a third monomer that
includes an IL-15 variant that includes an amino acid substitution
for a cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer are the variable heavy domain and variable light
domain of 2A11_H1.144_L2.142, respectively, where the VH of the
second monomer and the VL of the fifth monomer are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142,
respectively, where the cysteine residue on the IL-15 variant and
the cysteine residue on the IL-15R.alpha.(sushi) domain form a
disulfide bond, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S.
[0410] In the mAb-dsIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the skew
variant pair S364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D
variant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D
variant with appropriate cysteine substitutions. In one embodiment,
the targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"mAb-dsIL-15/R.alpha." format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, where the IL-15 variant includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, and where the
first and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants L368D/K370S, and the
second variant Fc domain includes skew variants S364K/E357Q. In an
exemplary embodiment, the first variant Fc domain includes skew
variants S364K/E357Q, and the second variant Fc domain includes
skew variants L368D/K370S. In a particular embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
one embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
[0411] In the mAb-dsIL-15/R.alpha. format, one preferred embodiment
utilizes the skew variant set S364K/E357Q:L368D/K370S, the pI
variants N208D/Q295E/N384D/Q418D/N421D and/or
Q196K/I199T/P271R/P228R/N276K, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0412] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where the first variant Fc domain includes
skew variants L368D/K370S and the second variant Fc domain include
the skew variant pair S364K/E357Q, where the first and second
variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
N208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc
domain of the second monomer includes pI variants
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where the first variant Fc domain includes
skew variants L368D/K370S and the second variant Fc domain include
the skew variant pair S364K/E357Q, where the first and second
variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where the first variant Fc domain includes
skew variants L368D/K370S and the second variant Fc domain include
the skew variant pair S364K/E357Q, where the first and second
variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fc
domain of the second monomer includes pI variants
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where the first variant Fc domain includes
skew variants S364K/E357Q and the second variant Fc domain include
the skew variant pair L368D/K370S, where the first and second
variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
Q196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc
domain of the second monomer includes pI variants
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where the first variant Fc domain includes
skew variants S364K/E357Q and the second variant Fc domain include
the skew variant pair L368D/K370S, where the first and second
variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where the first variant Fc domain includes
skew variants S364K/E357Q and the second variant Fc domain include
the skew variant pair L368D/K370S, where the first and second
variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fc
domain of the second monomer includes pI variants
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In certain embodiments, the first and second variant
Fc domains each further include half-life extension variants
M428L/N434S. In an exemplary embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In
an exemplary embodiment, the VH and VL are the VH and VL of any of
the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12
and 13A-C and the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D.
[0413] In the mAb-dsIL-15/R.alpha. format, one preferred embodiment
utilizes an anti-LAG-3 ABD having the variable heavy and light
domain pair of 7G8_H3.30_L1.34 or the variable heavy and light
domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG.
21I format, the skew variant set S364K/E357Q:L368D/K370S, the pI
variants N208D/Q295E/N384D/Q418D/N421D and/or
Q196K/I199T/P271R/P228R/N276K, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers. In an exemplary embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D.
[0414] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, where the first variant Fc domain includes skew
variants L368D/K370S and the second variant Fc domain include the
skew variant pair S364K/E357Q, where the first and second variant
Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
N208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc
domain of the second monomer includes pI variants
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, where the first variant Fc domain includes skew
variants L368D/K370S and the second variant Fc domain include the
skew variant pair S364K/E357Q, where the first and second variant
Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, where the first variant Fc domain includes skew
variants L368D/K370S and the second variant Fc domain include the
skew variant pair S364K/E357Q, where the first and second variant
Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fc
domain of the second monomer includes pI variants
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, where the first variant Fc domain includes skew
variants S364K/E357Q and the second variant Fc domain include the
skew variant pair L368D/K370S, where the first and second variant
Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
Q196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc
domain of the second monomer includes pI variants
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, where the first variant Fc domain includes skew
variants S364K/E357Q and the second variant Fc domain include the
skew variant pair L368D/K370S, where the first and second variant
Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc
domain of the first monomer includes pI substitutions
Q196K/I199T/P271R/P228R/N276K, and where numbering is according to
EU numbering. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "mAb-dsIL-15/R.alpha." format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker), where the IL-15R.alpha.(sushi) domain
includes an amino acid substitution for a cysteine residue and
CH2-CH3 is a second Fc domain; c) a third monomer that includes an
IL-15 variant that includes an amino acid substitution for a
cysteine residue; and d) a fourth and fifth monomer that each
include, from N- to C-terminus, a VL-CL, where VL is a variable
light domain, where the VH of the first monomer and the VL of the
fourth monomer form a first LAG-3 binding domain, where the VH of
the second monomer and the VL of the fifth monomer form a second
LAG-3 binding domain, where the cysteine residue on the IL-15
variant and the cysteine residue on the IL-15R.alpha.(sushi) domain
form a disulfide bond, where VH and VL are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34 or
2A11_H1.144_L2.142, where the first variant Fc domain includes skew
variants S364K/E357Q and the second variant Fc domain include the
skew variant pair L368D/K370S, where the first and second variant
Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fc
domain of the second monomer includes pI variants
N208D/Q295E/N384D/Q418D/N421D, and where numbering is according to
EU numbering. In certain embodiments, the first and second variant
Fc domains each further include half-life extension variants
M428L/N434S. In a particular embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34. In
another embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and VH and VL are the variable heavy domain
and variable light domain of 2A11_H1.144_L2.142. In one embodiment,
the IL-15 variant includes amino acid substitutions D30N/N65D and
VH and VL are the variable heavy domain and variable light domain
of 7G8_H3.30_L1.34. In another embodiment, the IL-15 variant
includes amino acid substitutions D30N/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
J. Central-IL-15/R.alpha.
[0415] This embodiment is shown in FIG. 21J, and comprises four
monomers forming a tetramer. The first monomer comprises a
VH-CH1-[optional domain linker]-IL-15 variant-[optional domain
linker]-CH2-CH3, with the second optional domain linker sometimes
being the hinge domain. The second monomer comprises a
VH-CH1-[optional domain linker]-IL-15R.alpha.(sushi)
domain-[optional domain linker]-CH2-CH3, with the second optional
domain linker sometimes being the hinge domain. The third (and
fourth) monomers are light chains, VL-CL. This is generally
referred to as "central-IL-15/R.alpha.".
[0416] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "central-IL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc
domain; b) a second monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15R.alpha.(sushi) domain-(domain
linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; and d) a
third and fourth monomer that each include from N-to C-terminus, a
VL-CL, where the VH of the first monomer and the VL of the third
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fourth monomer form a second LAG-3
binding domain, and where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex. Any useful
domain linker can be used to attach the various components of the
heterodimeric protein including, but not limited to those in FIGS.
8 and 9A-C. In an exemplary embodiment, the domain linkers that
attach the IL-15 variant to the first Fc domain and the
IL-15R.alpha.(sushi) domain to the second Fc domain are each
antibody hinge domains.
[0417] In the central-IL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having any of the variable
heavy and light domain pairs as shown in FIG. 12.
[0418] In the central-IL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12.
[0419] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "central-IL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc
domain; b) a second monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15R.alpha.(sushi) domain-(domain
linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; and d) a
third and fourth monomer that each include from N-to C-terminus, a
VL-CL, where the VH of the first monomer and the VL of the third
monomer are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34, respectively, where the VH of the second monomer
and the VL of the fourth monomer are the variable heavy domain and
variable light domain of 7G8_H3.30_L1.34, respectively, and where
the IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex. In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "central-IL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc
domain; b) a second monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15R.alpha.(sushi) domain-(domain
linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; and d) a
third and fourth monomer that each include from N-to C-terminus, a
VL-CL, where the VH of the first monomer and the VL of the third
monomer are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142, respectively, where the VH of the second
monomer and the VL of the fourth monomer are the variable heavy
domain and variable light domain of 2A11_H1.144_L2.142,
respectively, and where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex.
[0420] In the "central-IL-15/R.alpha."format, one preferred
embodiment utilizes an IL-15 variant that includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In one
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "central-IL-15/R.alpha."format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc
domain; and d) a third and fourth monomer that each include from
N-to C-terminus, a VL-CL, where the VH of the first monomer and the
VL of the third monomer form a first LAG-3 binding domain, where
the VH of the second monomer and the VL of the fourth monomer form
a second LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, and where the
IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment, the VH
and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and
13A-C and the IL-15 variant includes amino acid substitutions
N4D/N65D. In another exemplary embodiment, the VH and VL are the VH
and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0421] In the central-IL-15/R.alpha.format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair 7G8_H3.30_L1.34 or the variable heavy and light
domain pair 2A11_H1.144_L2.142 as shown in FIG. 12, with either the
IL-15 N4D/N65D variant or the IL-15 D30N/N65D variant or the IL-15
D30N/E64Q/N65D variant. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"central-IL-15/R.alpha."format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first Fc domain; b) a second monomer that includes,
from N- to C-terminus, a VH-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc
domain; and d) a third and fourth monomer that each include from
N-to C-terminus, a VL-CL, where the VH of the first monomer and the
VL of the third monomer form a first LAG-3 binding domain, where
the VH of the second monomer and the VL of the fourth monomer form
a second LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and where the IL-15 variant
includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D. In a particular embodiment, the IL-15 variant
includes amino acid substitutions N4D/N65D and VH and VL are the
variable heavy domain and variable light domain of 7G8_H3.30_L1.34.
In another embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and VH and VL are the variable heavy domain
and variable light domain of 2A11_H1.144_L2.142. In one embodiment,
the IL-15 variant includes amino acid substitutions D30N/N65D and
VH and VL are the variable heavy domain and variable light domain
of 7G8_H3.30_L1.34. In another embodiment, the IL-15 variant
includes amino acid substitutions D30N/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
[0422] In the central-IL-15/R.alpha. format, one preferred
embodiment utilizes the skew variant pair S364K/E357Q:L368D/K370S.
In one embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric
protein is an "central-IL-15/R.alpha."format heterodimeric protein
that includes: a) a first monomer that includes, from N- to
C-terminus, a VH-(domain linker)-IL-15 variant-(domain
linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second
monomer that includes, from N- to C-terminus, a VH-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second Fc domain; and d) a third and fourth monomer
that each include from N-to C-terminus, a VL-CL, where the VH of
the first monomer and the VL of the third monomer form a first
LAG-3 binding domain, where the VH of the second monomer and the VL
of the fourth monomer form a second LAG-3 binding domain, where the
IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, and where the first and second variant Fc domains include
the skew variant pair S364K/E357Q:L368D/K370S. In an exemplary
embodiment, the first variant Fc domain includes skew variants
S364K and E357Q, and the second variant Fc domain includes skew
variants L368D and K370S. In another exemplary embodiment, the
first variant Fc domain includes skew variants L368D and K370S, and
the second variant Fc domain includes skew variants S364K and
E357Q.
[0423] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "central-IL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc
domain; b) a second monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15R.alpha.(sushi) domain-(domain
linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; and d) a
third and fourth monomer that each include from N-to C-terminus, a
VL-CL, where the VH of the first monomer and the VL of the third
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fourth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S. In an
exemplary embodiment, the first variant Fc domain includes skew
variants S364K and E357Q, and the second variant Fc domain includes
skew variants L368D and K370S. In another exemplary embodiment, the
first variant Fc domain includes skew variants L368D and K370S, and
the second variant Fc domain includes skew variants S364K and
E357Q. In an exemplary embodiment, the VH and VL are the VH and VL
of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions N4D/N65D. In another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/N65D. In yet another exemplary
embodiment, the VH and VL are the VH and VL of any of the LAG-3
ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes amino
acid substitutions D30N/E64Q/N65D.
[0424] In the central-IL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"central-IL-15/R.alpha."format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, a VH-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain; and d) a third and fourth
monomer that each include from N-to C-terminus, a VL-CL, where the
VH of the first monomer and the VL of the third monomer are the
variable heavy domain and variable light domain of 7G8_H3.30_L1.34,
respectively, where the VH of the second monomer and the VL of the
fourth monomer are the variable heavy domain and variable light
domain of 7G8_H3.30_L1.34, respectively, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, and
where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"central-IL-15/R.alpha."format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, a VH-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain; and d) a third and fourth
monomer that each include from N-to C-terminus, a VL-CL, where the
VH of the first monomer and the VL of the third monomer are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142, respectively, where the VH of the second
monomer and the VL of the fourth monomer are the variable heavy
domain and variable light domain of 2A11_H1.144_L2.142,
respectively, where the IL-15 variant and the IL-15R.alpha.(sushi)
domain form an IL-15 complex, and where the first and second
variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S.
[0425] In the central-IL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S with either the IL-15
N4D/N65D variant or the IL-15 D30N/E64Q/N65D variant with
appropriate cysteine substitutions. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"central-IL-15/R.alpha."format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, a VH-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-CH2-CH3, where
CH2-CH3 is a second variant Fc domain; and d) a third and fourth
monomer that each include from N- to C-terminus, a VL-CL, where the
VH of the first monomer and the VL of the third monomer form a
first LAG-3 binding domain, where the VH of the second monomer and
the VL of the fourth monomer form a second LAG-3 binding domain,
where the IL-15 variant and the IL-15R.alpha.(sushi) domain form an
IL-15 complex, where VH and VL are the variable heavy domain and
variable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142,
where the IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D, and where the first and second
variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In an exemplary embodiment, the first
variant Fc domain includes skew variants S364K and E357Q, and the
second variant Fc domain includes skew variants L368D and K370S. In
another exemplary embodiment, the first variant Fc domain includes
skew variants L368D and K370S, and the second variant Fc domain
includes skew variants S364K and E357Q. In a particular embodiment,
the IL-15 variant includes amino acid substitutions N4D/N65D and VH
and VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
one embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
[0426] In the central-IL-15/R.alpha. format, one preferred
embodiment utilizes the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0427] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "central-IL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-(domain linker)-IL-15
variant-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc
domain; b) a second monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15R.alpha.(sushi) domain-(hinge)-CH2-CH3,
where CH2-CH3 is a second variant Fc domain; and d) a third and
fourth monomer that each include from N-to C-terminus, a VL-CL,
where the VH of the first monomer and the VL of the third monomer
form a first LAG-3 binding domain, where the VH of the second
monomer and the VL of the fourth monomer form a second LAG-3
binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the first
variant Fc domain includes skew variants L368D/K370S and the second
variant Fc domain include the skew variant pair S364K/E357Q, where
the first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the first variant Fc domain
includes pI substitutions Q295E/N384D/Q418D/N421D, and where
numbering is according to EU numbering. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"central-IL-15/R.alpha."format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15 variant-(hinge)-CH2-CH3, where CH2-CH3 is
a first variant Fc domain; b) a second monomer that includes, from
N- to C-terminus, a VH-(domain linker)-IL-15R.alpha.(sushi)
domain-(hinge)-CH2-CH3, where CH2-CH3 is a second variant Fc
domain; and d) a third and fourth monomer that each include from
N-to C-terminus, a VL-CL, where the VH of the first monomer and the
VL of the third monomer form a first LAG-3 binding domain, where
the VH of the second monomer and the VL of the fourth monomer form
a second LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the first
variant Fc domain includes skew variants S364K/E357Q and the second
variant Fc domain include the skew variant pair L368D/K370S, where
the first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the second variant Fc domain
of the second monomer includes pI substitutions
Q295E/N384D/Q418D/N421D, and where numbering is according to EU
numbering. In certain embodiments, the first and second variant Fc
domains each further include half-life extension variants
M428L/N434S. In an exemplary embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In
an exemplary embodiment, the VH and VL are the VH and VL of any of
the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions N4D/N65D. In another exemplary embodiment,
the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS.
12 and 13A-C and the IL-15 variant includes amino acid
substitutions D30N/N65D. In yet another exemplary embodiment, the
VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12
and 13A-C and the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D.
[0428] In the central-IL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142 as shown
in FIG. 12 with the FIG. 21K, the skew variant set
S364K/E357Q:L368D/K370S, the pI variants Q295E/N384D/Q418E/N421D,
the ablation variants E233P/L234V/L235A/G236_/S267K on both first
and second monomers, and optionally the 428L/434S variants on both
first and second monomers. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"central-IL-15/R.alpha."format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15 variant-(hinge)-CH2-CH3, where CH2-CH3 is
a first variant Fc domain; b) a second monomer that includes, from
N- to C-terminus, a VH-(domain linker)-IL-15R.alpha.(sushi)
domain-(hinge)-CH2-CH3, where CH2-CH3 is a second variant Fc
domain; and d) a third and fourth monomer that each include from
N-to C-terminus, a VL-CL, where the VH of the first monomer and the
VL of the third monomer form a first LAG-3 binding domain, where
the VH of the second monomer and the VL of the fourth monomer form
a second LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants L368D/K370S and the second variant Fc
domain include the skew variant pair S364K/E357Q, where the first
and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the first variant Fc domain
includes pI substitutions Q295E/N384D/Q418D/N421D, and where
numbering is according to EU numbering. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"central-IL-15/R.alpha."format heterodimeric protein that includes:
a) a first monomer that includes, from N- to C-terminus, a
VH-(domain linker)-IL-15 variant-(hinge)-CH2-CH3, where CH2-CH3 is
a first variant Fc domain; b) a second monomer that includes, from
N- to C-terminus, a VH-(domain linker)-IL-15R.alpha.(sushi)
domain-(hinge)-CH2-CH3, where CH2-CH3 is a second variant Fc
domain; and d) a third and fourth monomer that each include from
N-to C-terminus, a VL-CL, where the VH of the first monomer and the
VL of the third monomer form a first LAG-3 binding domain, where
the VH of the second monomer and the VL of the fourth monomer form
a second LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants S364K/E357Q and the second variant Fc
domain include the skew variant pair L368D/K370S, where the first
and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the second variant Fc domain
of the second monomer includes pI substitutions
Q295E/N384D/Q418D/N421D, and where numbering is according to EU
numbering. In certain embodiments, the first and second variant Fc
domains each further include half-life extension variants
M428L/N434S. In a particular embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34. In
another embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and VH and VL are the variable heavy domain
and variable light domain of 2A11_H1.144_L2.142. In one embodiment,
the IL-15 variant includes amino acid substitutions D30N/N65D and
VH and VL are the variable heavy domain and variable light domain
of 7G8_H3.30_L1.34. In another embodiment, the IL-15 variant
includes amino acid substitutions D30N/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
K. Central scIL-15/R.alpha.
[0429] This embodiment is shown in FIG. 21K, and comprises four
monomers forming a tetramer. The first monomer comprises a
VH-CH1-[optional domain linker]-IL-15R.alpha.(sushi) domain-domain
linker-IL-15 variant-[optional domain linker]-CH2-CH3, with the
second optional domain linker sometimes being the hinge domain. The
second monomer comprises a VH-CH1-hinge-CH2-CH3. The third (and
fourth) monomers are light chains, VL-CL. This is generally
referred to as "central-scIL-15/R.alpha.", with the "sc" standing
for "single chain".
[0430] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "central-scIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc
domain; b) a second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a second Fc domain; and d) a
third and fourth monomer that each include from N-to C-terminus, a
VL-CL, where the VH of the first monomer and the VL of the third
monomer form a first LAG-3 binding domain, where the VH of the
second monomer and the VL of the fourth monomer form a second LAG-3
binding domain, and where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex. Any useful
domain linker can be used to attach the various components of the
heterodimeric protein including, but not limited to those in FIGS.
8 and 9A-C. In an exemplary embodiment, the domain linker that
attaches the IL-15 variant to the first Fc domain is an antibody
hinge domain.
[0431] In the central-scIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having any of the variable
heavy and light domain pairs as shown in FIG. 12.
[0432] In the central-scIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair 7G8_H3.30_L1.34 34 or the variable heavy and
light domain pair 2A11_H1.144_L2.142 as shown in as shown in FIG.
12.
[0433] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "central-scIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc
domain; b) a second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a second Fc domain; and d) a
third and fourth monomer that each include from N-to C-terminus, a
VL-CL, where the VH of the first monomer and the VL of the third
monomer are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34, respectively, where the VH of the second monomer
and the VL of the fourth monomer are the variable heavy domain and
variable light domain of 7G8_H3.30_L1.34, respectively, and where
the IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex. In another embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "central-scIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-(domain
linker)-IL-15 variant-CH2-CH3, where CH2-CH3 is a first Fc domain;
b) a second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a second Fc domain; and d) a
third and fourth monomer that each include from N-to C-terminus, a
VL-CL, where the VH of the first monomer and the VL of the third
monomer are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142, respectively, where the VH of the second
monomer and the VL of the fourth monomer are the variable heavy
domain and variable light domain of 2A11_H1.144_L2.142,
respectively, and where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex.
[0434] In the central-scIL-15/R.alpha. format, one preferred
embodiment utilizes an IL-15 variant that includes amino acid
substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In one
embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric protein
is an "central-scIL-15/R.alpha."format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-(domain linker)-IL-15R.alpha.(sushi) domain-(domain
linker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a
first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a second Fc
domain; and d) a third and fourth monomer that each include from
N-to C-terminus, a VL-CL, where the VH of the first monomer and the
VL of the third monomer form a first LAG-3 binding domain, where
the VH of the second monomer and the VL of the fourth monomer form
a second LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, and where the
IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment, the VH
and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and
13A-C and the IL-15 variant includes amino acid substitutions
N4D/N65D. In another exemplary embodiment, the VH and VL are the VH
and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0435] In the central-scIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12, with
either the IL-15 N4D/N65D variant or the IL-15 D30N/N65D variant or
the IL-15 D30N/E64Q/N65D variant. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"central-scIL-15/R.alpha."format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-(domain linker)-IL-15R.alpha.(sushi) domain-(domain
linker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a
first Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a second Fc
domain; and d) a third and fourth monomer that each include from
N-to C-terminus, a VL-CL, where the VH of the first monomer and the
VL of the third monomer form a first LAG-3 binding domain, where
the VH of the second monomer and the VL of the fourth monomer form
a second LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and where the IL-15 variant
includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D. In a particular embodiment, the IL-15 variant
includes amino acid substitutions N4D/N65D and VH and VL are the
variable heavy domain and variable light domain of 7G8_H3.30_L1.34.
In another embodiment, the IL-15 variant includes amino acid
substitutions N4D/N65D and VH and VL are the variable heavy domain
and variable light domain of 2A11_H1.144_L2.142. In one embodiment,
the IL-15 variant includes amino acid substitutions D30N/N65D and
VH and VL are the variable heavy domain and variable light domain
of 7G8_H3.30_L1.34. In another embodiment, the IL-15 variant
includes amino acid substitutions D30N/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
[0436] In the central-scIL-15/R.alpha. format, one preferred
embodiment utilizes the skew variant pair S364K/E357Q:L368D/K370S.
In one embodiment, the targeted IL-15/IL-15R.alpha. heterodimeric
protein is an "central-scIL-15/R.alpha."format heterodimeric
protein that includes: a) a first monomer that includes, from N- to
C-terminus, a VH-(domain linker)-IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3
is a second variant Fc domain; and d) a third and fourth monomer
that each include from N-to C-terminus, a VL-CL, where the VH of
the first monomer and the VL of the third monomer form a first
LAG-3 binding domain, where the VH of the second monomer and the VL
of the fourth monomer form a second LAG-3 binding domain, where the
IL-15 variant and the IL-15R.alpha.(sushi) domain form an IL-15
complex, and where the first and second variant Fc domains include
the skew variant pair S364K/E357Q:L368D/K370S. In an exemplary
embodiment, the first variant Fc domain includes skew variants
L368D and K370S, and the second variant Fc domain includes skew
variants S364K and E357Q.
[0437] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "central-scIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant
Fc domain; b) a second monomer that includes, from N- to
C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a second variant
Fc domain; and d) a third and fourth monomer that each include from
N-to C-terminus, a VL-CL, where the VH of the first monomer and the
VL of the third monomer form a first LAG-3 binding domain, where
the VH of the second monomer and the VL of the fourth monomer form
a second LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the IL-15
variant includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D, and where the first and second variant Fc domains
include the skew variant pair S364K/E357Q:L368D/K370S. In an
exemplary embodiment, the first variant Fc domain includes skew
variants L368D and K370S, and the second variant Fc domain includes
skew variants S364K and E357Q. In an exemplary embodiment, the VH
and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and
13A-C and the IL-15 variant includes amino acid substitutions
N4D/N65D. In another exemplary embodiment, the VH and VL are the VH
and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0438] In the central-scIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S. In one embodiment, the
targeted IL-15/IL-15R.alpha. heterodimeric protein is an
"central-scIL-15/R.alpha."format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-(domain linker)-IL-15R.alpha.(sushi) domain-(domain
linker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a
first variant Fc domain; b) a second monomer that includes, from N-
to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a second
variant Fc domain; and d) a third and fourth monomer that each
include from N-to C-terminus, a VL-CL, where the VH of the first
monomer and the VL of the third monomer are the variable heavy
domain and variable light domain of 7G8_H3.30_L1.34, respectively,
where the VH of the second monomer and the VL of the fourth monomer
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34, respectively, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, and where the
first and second variant Fc domains include the skew variant pair
S364K/E357Q:L368D/K370S. In another embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"central-scIL-15/R.alpha."format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-(domain linker)-IL-15R.alpha.(sushi) domain-(domain
linker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a
first variant Fc domain; b) a second monomer that includes, from N-
to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a second
variant Fc domain; and d) a third and fourth monomer that each
include from N-to C-terminus, a VL-CL, where the VH of the first
monomer and the VL of the third monomer are the variable heavy
domain and variable light domain of 2A11_H1.144_L2.142,
respectively, where the VH of the second monomer and the VL of the
fourth monomer are the variable heavy domain and variable light
domain of 2A11_H1.144_L2.142, respectively, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, and
where the first and second variant Fc domains include the skew
variant pair S364K/E357Q:L368D/K370S. In an exemplary embodiment,
the first variant Fc domain includes skew variants L368D and K370S,
and the second variant Fc domain includes skew variants S364K and
E357Q.
[0439] In the central-scIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or the variable heavy and
light domain pair of 2A11_H1.144_L2.142 as shown in FIG. 12 and the
skew variant pair S364K/E357Q:L368D/K370S with either the IL-15
N4D/N65D variant or the IL-15 D30N/N65D variant or the IL-15
D30N/E64Q/N65D variant. In one embodiment, the targeted
IL-15/IL-15R.alpha. heterodimeric protein is an
"central-scIL-15/R.alpha."format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
a VH-(domain linker)-IL-15R.alpha.(sushi) domain-(domain
linker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a
first variant Fc domain; b) a second monomer that includes, from N-
to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a second
variant Fc domain; and d) a third and fourth monomer that each
include from N-to C-terminus, a VL-CL, where the VH of the first
monomer and the VL of the third monomer form a first LAG-3 binding
domain, where the VH of the second monomer and the VL of the fourth
monomer form a second LAG-3 binding domain, where the IL-15 variant
and the IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH
and VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the IL-15 variant
includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D, and where the first and second variant Fc domains
include the skew variant pair L368D/K370S:S364K/E357Q. In an
exemplary embodiment, the first variant Fc domain includes skew
variants L368D/K370S, and the second variant Fc domain includes
skew variants S364K/E357Q. In a particular embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions N4D/N65D and VH and VL are the variable
heavy domain and variable light domain of 2A11_H1.144_L2.142. In
one embodiment, the IL-15 variant includes amino acid substitutions
D30N/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions D30N/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variant
includes amino acid substitutions D30N/E64Q/N65D and VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D and VH and VL are the
variable heavy domain and variable light domain of
2A11_H1.144_L2.142.
[0440] In the central-scIL-15/R.alpha. format, one preferred
embodiment utilizes the skew variant set S364K/E357Q:L368D/K370S,
the pI variants Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236_/S267K on both first and second monomers,
and optionally the 428L/434S variants on both first and second
monomers.
[0441] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "central-scIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant
-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a second variant Fc domain; and
d) a third and fourth monomer that each include from N-to
C-terminus, a VL-CL, where the VH of the first monomer and the VL
of the third monomer form a first LAG-3 binding domain, where the
VH of the second monomer and the VL of the fourth monomer form a
second LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where the first
variant Fc domain includes skew variants L368D/K370S and the second
variant Fc domain includes skew variants S364K/E357Q where the
first and second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the first variant Fc domain
includes pI variants Q295E/N384D/Q418E/N421D, and where numbering
is according to EU numbering. In an exemplary embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D, D30N/N65D, or
D30N/E64Q/N65D. In an exemplary embodiment, the VH and VL are the
VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the
IL-15 variant includes amino acid substitutions N4D/N65D. In
another exemplary embodiment, the VH and VL are the VH and VL of
any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant
includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0442] In the central-scIL-15/R.alpha. format, one preferred
embodiment utilizes an anti-LAG-3 ABD having the variable heavy and
light domain pair of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142 as shown
in FIG. 12 with the FIG. 21K format, the skew variant set
S364K/E357Q:L368D/K370S, the pI variants Q295E/N384D/Q418E/N421D,
the ablation variants E233P/L234V/L235A/G236_/S267K on both first
and second monomers, and optionally the 428L/434S variants on both
first and second monomers.
[0443] In one embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "central-scIL-15/R.alpha."format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, a VH-(domain
linker)-IL-15R.alpha.(sushi) domain-(domain linker)-IL-15 variant
-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a
second monomer that includes, from N- to C-terminus, a
VH-hinge-CH2-CH3, where CH2-CH3 is a second variant Fc domain; and
d) a third and fourth monomer that each include from N-to
C-terminus, a VL-CL, where the VH of the first monomer and the VL
of the third monomer form a first LAG-3 binding domain, where the
VH of the second monomer and the VL of the fourth monomer form a
second LAG-3 binding domain, where the IL-15 variant and the
IL-15R.alpha.(sushi) domain form an IL-15 complex, where VH and VL
are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc
domain includes skew variants L368D/K370S and the second variant Fc
domain includes skew variants S364K/E357Q where the first and
second variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the first variant Fc domain
includes pI variants Q295E/N384D/Q418E/N421D, and where numbering
is according to EU numbering. In certain embodiments, the hinge of
the first monomer further includes variant C220S. In certain
embodiments, the first and second variant Fc domains each further
include half-life extension variants M428L/N434S. In a particular
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D and VH and VL are the variable heavy domain and variable
light domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15
variant includes amino acid substitutions N4D/N65D and VH and VL
are the variable heavy domain and variable light domain of
2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes
amino acid substitutions D30N/N65D and VH and VL are the variable
heavy domain and variable light domain of 7G8_H3.30_L1.34. In
another embodiment, the IL-15 variant includes amino acid
substitutions D30N/N65D and VH and VL are the variable heavy domain
and variable light domain of 2A11_H1.144_L2.142. In yet another
embodiment, the IL-15 variant includes amino acid substitutions
D30N/E64Q/N65D and VH and VL are the variable heavy domain and
variable light domain of 7G8_H3.30_L1.34. In another embodiment,
the IL-15 variant includes amino acid substitutions D30N/E64Q/N65D
and VH and VL are the variable heavy domain and variable light
domain of 2A11_H1.144_L2.142.
V. PARTICULARLY USEFUL EMBODIMENTS OF THE INVENTION
[0444] The present invention provides a targeted
IL-15/IL-15R.alpha. heterodimeric protein comprising at least two
monomers, one of which contains an anti-LAG-3 ABD and the other
that contains an IL-15/RA complex, joined using heterodimeric Fc
domains.
[0445] In some embodiments, the first and the second Fc domains
have a set of amino acid substitutions selected from the group
consisting of S267K/L368D/K370S:S267K/S364K/E357Q;
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411E/K360E/Q362E:D401K; L368D/K370S:S364K/E357L and
K370S:S364K/E357Q, according to EU numbering.
[0446] In some instances, the first and/or the second Fc domains
have an additional set of amino acid substitutions comprising
Q295E/N384D/Q418E/N421D, according to EU numbering. In some cases,
the first and/or the second Fc domains have an additional set of
amino acid substitutions consisting of G236R/L328R,
E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,
E233P/L234V/L235A/G236del/S239K/A327G,
E233P/L234V/L235A/G236del/S267K/A327G and
E233P/L234V/L235A/G236del, according to EU numbering.
[0447] In some embodiments, the IL-15 protein has a polypeptide
sequence selected from the group consisting of SEQ ID NO:1
(full-length human IL-15) and SEQ ID NO:2 (truncated human IL-15),
and the IL-15R.alpha. protein has a polypeptide sequence selected
from the group consisting of SEQ ID NO:3 (full-length human
IL-15R.alpha.) and SEQ ID NO:4 (sushi domain of human
IL-15R.alpha.).
[0448] In embodiments the IL-15 protein and the IL-15R.alpha.
protein can have a set of amino acid substitutions selected from
the group consisting of E87C:D96/P97/C98; E87C:D96/C97/A98;
V49C:S40C; L52C:S40C; E89C:K34C; Q48C:G38C; E53C:L42C; C42S:A37C;
and L45C:A37C, respectively.
[0449] In some embodiments, the IL-15 protein is a variant protein
that has a sequence selected from FIGS. 19 and FIG. 20 to reduce
potency. In some embodiments, the IL-15 protein is a variant
protein having one or more amino acid substitutions at the
IL-15:CD132 interface.
[0450] In some embodiments, the LAG-3 antigen binding domain
comprises an anti-LAG-3 scFv or an anti-LAG-3 Fab. In an exemplary
embodiment, the LAG-3 ABD includes the VH and VL of any of the
LAG-3 ABDs depicted in FIGS. 12 and 13A-C.
[0451] In an exemplary embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scIL-15/R.alpha. X Fab" format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, variant
here VH is a variable heavy domain and CH2-CH3 is a second variant
Fc domain, and c) a light chain that includes from, N- to
C-terminus, VL-VC, where VL is a variable light domain, where VH
and VL form a LAG-3 binding domain, where the IL-15 variant is an
IL-15 N4D/N65D variant, where the first variant Fc domain includes
skew variants L368D/K370S and the second variant Fc domain includes
skew variants S364K/E357Q, where the first and second variant Fc
domains each include FcKO variants E233P/L234V/L235A/G236del/S267K,
where the first variant Fc domain includes pI variants
Q295E/N384D/Q418E/N421D, and where numbering is according to EU
numbering. In certain embodiments, the first and second variant Fc
domains each further include half-life extension variants
M428L/N434S. In certain embodiments, the hinge of the first monomer
includes also includes amino acid substitution C220S and the first
and second variant Fc domains each further include half-life
extension variants M428L/N434S. In some embodiments, the VH and VL
are the variable heavy domain and variable light domain of any of
the LAG-3 ABDs in FIG. 12 or 13A-C. In some embodiments, the VH and
VL are the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142 (FIG. 12).
[0452] In an exemplary embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scIL-15/R.alpha. X Fab" format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, variant
here VH is a variable heavy domain and CH2-CH3 is a second variant
Fc domain, and c) a light chain that includes from, N- to
C-terminus, VL-VC, where VL is a variable light domain, where VH
and VL form a LAG-3 binding domain, where the IL-15 variant is an
IL-15 D30N/N65D variant, where the first variant Fc domain includes
skew variants L368D/K370S and the second variant Fc domain includes
skew variants S364K/E357Q, where the first and second variant Fc
domains each include FcKO variants E233P/L234V/L235A/G236del/S267K,
where the first variant Fc domain includes pI variants
Q295E/N384D/Q418E/N421D, and where numbering is according to EU
numbering. In some embodiments, the hinge of the first monomer also
includes amino acid substitution C220S. In certain embodiments, the
first and second variant Fc domains each further include half-life
extension variants M428L/N434S. In certain embodiments, the hinge
of the first monomer includes also includes amino acid substitution
C220S and the first and second variant Fc domains each further
include half-life extension variants M428L/N434S. In some
embodiments, the VH and VL are the variable heavy domain and
variable light domain of any of the LAG-3 ABDs in FIG. 12 or 13A-C.
In some embodiments, the VH and VL are the variable heavy domain
and variable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142
(FIG. 12).
[0453] In an exemplary embodiment, the targeted IL-15/IL-15R.alpha.
heterodimeric protein is an "scIL-15/R.alpha. X Fab" format
heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where
CH2-CH3 is a first variant Fc domain; b) a second monomer that
includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, variant
here VH is a variable heavy domain and CH2-CH3 is a second variant
Fc domain, and c) a light chain that includes from, N- to
C-terminus, VL-VC, where VL is a variable light domain, where VH
and VL form a LAG-3 binding domain, where the IL-15 variant is an
IL-15 D30N/E64Q/N65D variant, where the first variant Fc domain
includes skew variants L368D/K370S and the second variant Fc domain
includes skew variants S364K/E357Q, where the first and second
variant Fc domains each include FcKO variants
E233P/L234V/L235A/G236del/S267K, where the first variant Fc domain
includes pI variants Q295E/N384D/Q418E/N421D, and where numbering
is according to EU numbering. In some embodiments, the hinge of the
first monomer also includes amino acid substitution C220S. In
certain embodiments, the first and second variant Fc domains each
further include half-life extension variants M428L/N434S. In
certain embodiments, the hinge of the first monomer includes also
includes amino acid substitution C220S and the first and second
variant Fc domains each further include half-life extension
variants M428L/N434S. In some embodiments, the VH and VL are the
variable heavy domain and variable light domain of any of the LAG-3
ABDs in FIG. 12 or 13A-C. In some embodiments, the VH and VL are
the variable heavy domain and variable light domain of
7G8_H3.30_L1.34 or 2A11_H1.144_L2.142 (FIG. 12).
[0454] Useful "backbone" sequences that can be included in the
"scIL-15/R.alpha. X Fab" format heterodimeric protein are depicted
in FIG. 10. In some embodiments, the "scIL-15/R.alpha. X Fab"
format heterodimeric protein that includes: a) a first monomer that
includes, from N- to C-terminus, an IL-15R.alpha.(sushi)
domain-(domain linker)-IL-15 variant-(hinge)-CH2-CH3, where
hinge-CH2-CH3 has the amino acid sequence of Chain 2 of "Backbone
1" in FIG. 10 (SEQ ID NO: 58); b) a second monomer that includes,
from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a
variable heavy domain and CH1-hinge-CH2-CH3 has the amino acid
sequence of Chain 1 of "Backbone 1" in FIG. 10 (SEQ ID NO: 57), and
c) a light chain that includes from, N- to C-terminus, VL-VC, where
VL is a variable light domain and VC has the sequence of "Constant
Light Chain--Kappa" in FIG. 11 (SEQ ID NO: 63). In certain
embodiments, the "scIL-15/R.alpha. X Fab" format heterodimeric
protein that includes: a) a first monomer that includes, from N- to
C-terminus, an IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(hinge)-CH2-CH3, where hinge-CH2-CH3 has the amino acid
sequence of Chain 2 of "Backbone 2" in FIG. 10 (SEQ ID NO: 60); b)
a second monomer that includes, from N- to C-terminus, a
VH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and
CH1-hinge-CH2-CH3 has the amino acid sequence of Chain 1 of
"Backbone 2" in FIG. 10 (SEQ ID NO: 59), and c) a light chain that
includes from, N- to C-terminus, VL-VC, where VL is a variable
light domain and VC has the sequence of "Constant Light
Chain--Kappa" in FIG. 11 (SEQ ID NO: 63). In some embodiments, the
"scIL-15/R.alpha. X Fab" format heterodimeric protein that
includes: a) a first monomer that includes, from N- to C-terminus,
an IL-15R.alpha.(sushi) domain-(domain linker)-IL-15
variant-(hinge)-CH2-CH3, where hinge-CH2-CH3 has the amino acid
sequence of Chain 2 of "Backbone 3" in FIG. 10 (SEQ ID NO: 62); b)
a second monomer that includes, from N- to C-terminus, a
VH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and
CH1-hinge-CH2-CH3 has the amino acid sequence of Chain 1 of
"Backbone 3" in FIG. 10 (SEQ ID NO: 61), and c) a light chain that
includes from, N- to C-terminus, VL-VC, where VL is a variable
light domain and VC has the sequence of "Constant Light
Chain--Kappa" in FIG. 11 (SEQ ID NO: 63). In an exemplary
embodiment, the IL-15 variant includes amino acid substitutions
N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment,
the IL-15 variant includes amino acid substitutions N4D/N65D,
D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment, the VH
and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and
13A-C and the IL-15 variant includes amino acid substitutions
N4D/N65D. In another exemplary embodiment, the VH and VL are the VH
and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15
variant includes amino acid substitutions D30N/N65D. In yet another
exemplary embodiment, the VH and VL are the VH and VL of any of the
LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes
amino acid substitutions D30N/E64Q/N65D.
[0455] Particular preferred LAG-3 targeted IL-15/IL-15R.alpha.-Fc
heterodimeric fusion proteins include XENP27972, XENP27973,
XENP27977, XENP27978, XENP029486, XENP029487, XENC1000, XENC1001,
XENC1002, XENC1003, XENC1004 and XENC1005 "scIL-15/R.alpha. X Fab"
format heterodimeric protein. Exemplary embodiments of the LAG-3
targeted IL-15/IL-15R.alpha.-Fc heterodimeric fusion proteins are
shown in FIGS. 22A and B, FIGS. 29A and B, FIGS. 46A and B, FIGS.
47A and B, and FIGS. 48A-D.
VI. NUCLEIC ACIDS OF THE INVENTION
[0456] In another aspect, provided herein are nucleic acid
compositions encoding the targeted heterodimeric fusion proteins
(or, in the case of a monomer Fc domain protein, nucleic acids
encoding those as well).
[0457] As will be appreciated by those in the art, the nucleic acid
compositions will depend on the format of the targeted
heterodimeric fusion protein. Thus, for example, when the format
requires three amino acid sequences, three nucleic acid sequences
can be incorporated into one or more expression vectors for
expression. Similarly, some formats only two nucleic acids are
needed; again, they can be put into one or two expression vectors,
or four or 5. As noted herein, some constructs have two copies of a
light chain, for example.
[0458] As is known in the art, the nucleic acids encoding the
components of the invention can be incorporated into expression
vectors as is known in the art, and depending on the host cells
used to produce the targeted heterodimeric fusion proteins of the
invention. Generally the nucleic acids are operably linked to any
number of regulatory elements (promoters, origin of replication,
selectable markers, ribosomal binding sites, inducers, etc.). The
expression vectors can be extra-chromosomal or integrating
vectors.
[0459] The nucleic acids and/or expression vectors provided herein
are then transformed into any number of different types of host
cells as is well known in the art, including mammalian, bacterial,
yeast, insect and/or fungal cells, with mammalian cells (e.g., CHO
cells), finding use in many embodiments.
[0460] In some embodiments, nucleic acids encoding each monomer, as
applicable depending on the format, are each contained within a
single expression vector, generally under different or the same
promoter controls. In embodiments of particular use in the present
invention, each of these two or three nucleic acids are contained
on a different expression vector.
[0461] The targeted heterodimeric fusion protein of the invention
are made by culturing host cells comprising the expression
vector(s) as is well known in the art. Once produced, traditional
fusion protein or antibody purification steps are done, including
an ion exchange chromatography step. As discussed herein, having
the pIs of the two monomers differ by at least 0.5 can allow
separation by ion exchange chromatography or isoelectric focusing,
or other methods sensitive to isoelectric point. That is, the
inclusion of pI substitutions that alter the isoelectric point (pI)
of each monomer so that such that each monomer has a different pI
and the heterodimer also has a distinct pI, thus facilitating
isoelectric purification of the heterodimer (e.g., anionic exchange
columns, cationic exchange columns). These substitutions also aid
in the determination and monitoring of any contaminating homodimers
post-purification (e.g., IEF gels, cIEF, and analytical IEX
columns).
VII. BIOLOGICAL AND BIOCHEMICAL FUNCTIONALITY OF TARGETED LAG-3
ANTIBODY X IL-15/IL-15R.alpha. HETERODIMERIC IMMUNOMODULATORY
FUSION PROTEINS
[0462] Generally the targeted heterodimeric fusion proteins of the
invention are administered to patients with cancer, and efficacy is
assessed, in a number of ways as described herein. Thus, while
standard assays of efficacy can be run, such as cancer load, size
of tumor, evaluation of presence or extent of metastasis, etc.,
immuno-oncology treatments can be assessed on the basis of immune
status evaluations as well. This can be done in a number of ways,
including both in vitro and in vivo assays. For example, evaluation
of changes in immune status along with "old fashioned" measurements
such as tumor burden, size, invasiveness, LN involvement,
metastasis, etc. can be done. Thus, any or all of the following can
be evaluated: the inhibitory effects of the heterodimeric proteins
on CD4.sup.+ T cell activation or proliferation, CD8.sup.+ T (CTL)
cell activation or proliferation, CD8.sup.+ T cell-mediated
cytotoxic activity and/or CTL mediated cell depletion, NK cell
activity and NK mediated cell depletion, the potentiating effects
of the heterodimeric protein on Treg cell differentiation and
proliferation and Treg- or myeloid derived suppressor cell
(MDSC)-mediated immunosuppression or immune tolerance, and/or the
effects of heterodimeric protein on proinflammatory cytokine
production by immune cells, e.g., IL-2, IFN-.gamma. or TNF-.alpha.
production by T or other immune cells.
[0463] In some embodiments, assessment of treatment is done by
evaluating immune cell proliferation, using for example, CFSE
dilution method, Ki67 intracellular staining of immune effector
cells, and .sup.3H-thymidine incorporation method.
[0464] In some embodiments, assessment of treatment is done by
evaluating the increase in gene expression or increased protein
levels of activation-associated markers, including one or more of:
CD25, CD69, CD137, ICOS, PD1, GITR, OX40, and cell degranulation
measured by surface expression of CD107A.
[0465] In general, gene expression assays are done as is known in
the art.
[0466] In general, protein expression measurements are also
similarly done as is known in the art.
[0467] In some embodiments, assessment of treatment is done by
assessing cytotoxic activity measured by target cell viability
detection via estimating numerous cell parameters such as enzyme
activity (including protease activity), cell membrane permeability,
cell adherence, ATP production, co-enzyme production, and
nucleotide uptake activity. Specific examples of these assays
include, but are not limited to, Trypan Blue or PI staining,
.sup.51Cr or .sup.35S release method, LDH activity, MTT and/or WST
assays, Calcein-AM assay, Luminescent based assay, and others.
[0468] In some embodiments, assessment of treatment is done by
assessing T cell activity measured by cytokine production, measure
either intracellularly in culture supernatant using cytokines
including, but not limited to, IFN.gamma., TNF.alpha., GM-CSF, IL2,
IL6, IL4, IL5, IL10, IL13 using well known techniques.
[0469] Accordingly, assessment of treatment can be done using
assays that evaluate one or more of the following: (i) increases in
immune response, (ii) increases in activation of .alpha..beta.
and/or .gamma..delta. T cells, (iii) increases in cytotoxic T cell
activity, (iv) increases in NK and/or NKT cell activity, (v)
alleviation of .alpha..beta. and/or .gamma..delta. T-cell
suppression, (vi) increases in pro-inflammatory cytokine secretion,
(vii) increases in IL-2 secretion; (viii) increases in
interferon-.gamma. production, (ix) increases in Th1 response, (x)
decreases in Th2 response, (xi) decreases or eliminates cell number
and/or activity of at least one of regulatory T cells (Tregs).
A. Assays to Measure Efficacy
[0470] In some embodiments, T cell activation is assessed using a
Mixed Lymphocyte Reaction (MLR) assay as is known in the art. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0471] In one embodiment, the signaling pathway assay measures
increases or decreases in immune response as measured for an
example by phosphorylation or de-phosphorylation of different
factors, or by measuring other post translational modifications. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0472] In one embodiment, the signaling pathway assay measures
increases or decreases in activation of .alpha..beta. and/or
.gamma..delta. T cells as measured for an example by cytokine
secretion or by proliferation or by changes in expression of
activation markers like for an example CD137, CD107a, PD1, etc. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0473] In one embodiment, the signaling pathway assay measures
increases or decreases in cytotoxic T cell activity as measured for
an example by direct killing of target cells like for an example
cancer cells or by cytokine secretion or by proliferation or by
changes in expression of activation markers like for an example
CD137, CD107a, PD1, etc. An increase in activity indicates
immunostimulatory activity. Appropriate increases in activity are
outlined below.
[0474] In one embodiment, the signaling pathway assay measures
increases or decreases in NK and/or NKT cell activity as measured
for an example by direct killing of target cells like for an
example cancer cells or by cytokine secretion or by changes in
expression of activation markers like for an example CD107a, etc.
An increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0475] In one embodiment, the signaling pathway assay measures
increases or decreases in .alpha..beta. and/or .gamma..delta.
T-cell suppression, as measured for an example by cytokine
secretion or by proliferation or by changes in expression of
activation markers like for an example CD137, CD107a, PD1, etc. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0476] In one embodiment, the signaling pathway assay measures
increases or decreases in pro-inflammatory cytokine secretion as
measured for example by ELISA or by Luminex or by Multiplex bead
based methods or by intracellular staining and FACS analysis or by
Alispot etc. An increase in activity indicates immunostimulatory
activity. Appropriate increases in activity are outlined below.
[0477] In one embodiment, the signaling pathway assay measures
increases or decreases in IL-2 secretion as measured for example by
ELISA or by Luminex or by Multiplex bead based methods or by
intracellular staining and FACS analysis or by Alispot etc. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0478] In one embodiment, the signaling pathway assay measures
increases or decreases in interferon-y production as measured for
example by ELISA or by Luminex or by Multiplex bead based methods
or by intracellular staining and FACS analysis or by Alispot etc.
An increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0479] In one embodiment, the signaling pathway assay measures
increases or decreases in Th1 response as measured for an example
by cytokine secretion or by changes in expression of activation
markers. An increase in activity indicates immunostimulatory
activity. Appropriate increases in activity are outlined below.
[0480] In one embodiment, the signaling pathway assay measures
increases or decreases in Th2 response as measured for an example
by cytokine secretion or by changes in expression of activation
markers. An increase in activity indicates immunostimulatory
activity. Appropriate increases in activity are outlined below.
[0481] In one embodiment, the signaling pathway assay measures
increases or decreases cell number and/or activity of at least one
of regulatory T cells (Tregs), as measured for example by flow
cytometry or by IHC. A decrease in response indicates
immunostimulatory activity. Appropriate decreases are the same as
for increases, outlined below.
[0482] In one embodiment, the signaling pathway assay measures
increases or decreases in M2 macrophages cell numbers, as measured
for example by flow cytometry or by IHC. A decrease in response
indicates immunostimulatory activity. Appropriate decreases are the
same as for increases, outlined below.
[0483] In one embodiment, the signaling pathway assay measures
increases or decreases in M2 macrophage pro-tumorigenic activity,
as measured for an example by cytokine secretion or by changes in
expression of activation markers. A decrease in response indicates
immunostimulatory activity. Appropriate decreases are the same as
for increases, outlined below.
[0484] In one embodiment, the signaling pathway assay measures
increases or decreases in N2 neutrophils increase, as measured for
example by flow cytometry or by IHC. A decrease in response
indicates immunostimulatory activity. Appropriate decreases are the
same as for increases, outlined below.
[0485] In one embodiment, the signaling pathway assay measures
increases or decreases in N2 neutrophils pro-tumorigenic activity,
as measured for an example by cytokine secretion or by changes in
expression of activation markers. A decrease in response indicates
immunostimulatory activity. Appropriate decreases are the same as
for increases, outlined below.
[0486] In one embodiment, the signaling pathway assay measures
increases or decreases in inhibition of T cell activation, as
measured for an example by cytokine secretion or by proliferation
or by changes in expression of activation markers like for an
example CD137, CD107a, PD1, etc. An increase in activity indicates
immunostimulatory activity. Appropriate increases in activity are
outlined below.
[0487] In one embodiment, the signaling pathway assay measures
increases or decreases in inhibition of CTL activation as measured
for an example by direct killing of target cells like for an
example cancer cells or by cytokine secretion or by proliferation
or by changes in expression of activation markers like for an
example CD137, CD107a, PD1, etc. An increase in activity indicates
immunostimulatory activity. Appropriate increases in activity are
outlined below.
[0488] In one embodiment, the signaling pathway assay measures
increases or decreases in .alpha..beta. and/or .gamma..delta. T
cell exhaustion as measured for an example by changes in expression
of activation markers. A decrease in response indicates
immunostimulatory activity. Appropriate decreases are the same as
for increases, outlined below.
[0489] In one embodiment, the signaling pathway assay measures
increases or decreases .alpha..beta. and/or .gamma..delta. T cell
response as measured for an example by cytokine secretion or by
proliferation or by changes in expression of activation markers
like for an example CD137, CD107a, PD1, etc. An increase in
activity indicates immunostimulatory activity. Appropriate
increases in activity are outlined below.
[0490] In one embodiment, the signaling pathway assay measures
increases or decreases in stimulation of antigen-specific memory
responses as measured for an example by cytokine secretion or by
proliferation or by changes in expression of activation markers
like for an example CD45RA, CCR7 etc. An increase in activity
indicates immunostimulatory activity. Appropriate increases in
activity are outlined below.
[0491] In one embodiment, the signaling pathway assay measures
increases or decreases in apoptosis or lysis of cancer cells as
measured for an example by cytotoxicity assays such as for an
example MTT, Cr release, Calcine AM, or by flow cytometry based
assays like for an example CFSE dilution or propidium iodide
staining etc. An increase in activity indicates immunostimulatory
activity. Appropriate increases in activity are outlined below.
[0492] In one embodiment, the signaling pathway assay measures
increases or decreases in stimulation of cytotoxic or cytostatic
effect on cancer cells, as measured for an example by cytotoxicity
assays such as for an example MTT, Cr release, Calcine AM, or by
flow cytometry based assays like for an example CFSE dilution or
propidium iodide staining etc. An increase in activity indicates
immunostimulatory activity. Appropriate increases in activity are
outlined below.
[0493] In one embodiment, the signaling pathway assay measures
increases or decreases direct killing of cancer cells as measured
for an example by cytotoxicity assays such as for an example MTT,
Cr release, Calcine AM, or by flow cytometry based assays like for
an example CFSE dilution or propidium iodide staining etc. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0494] In one embodiment, the signaling pathway assay measures
increases or decreases Th17 activity as measured for an example by
cytokine secretion or by proliferation or by changes in expression
of activation markers. An increase in activity indicates
immunostimulatory activity. Appropriate increases in activity are
outlined below.
[0495] In one embodiment, the signaling pathway assay measures
increases or decreases in induction of complement dependent
cytotoxicity and/or antibody dependent cell-mediated cytotoxicity,
as measured for an example by cytotoxicity assays such as for an
example MTT, Cr release, Calcine AM, or by flow cytometry based
assays like for an example CFSE dilution or propidium iodide
staining etc. An increase in activity indicates immunostimulatory
activity. Appropriate increases in activity are outlined below.
[0496] In one embodiment, T cell activation is measured for an
example by direct killing of target cells like for an example
cancer cells or by cytokine secretion or by proliferation or by
changes in expression of activation markers like for an example
CD137, CD107a, PD1, etc. For T-cells, increases in proliferation,
cell surface markers of activation (e.g., CD25, CD69, CD137, PD1),
cytotoxicity (ability to kill target cells), and cytokine
production (e.g., IL-2, IL-4, IL-6, IFN-.gamma., TNF-.alpha.,
IL-10, IL-17A) would be indicative of immune modulation that would
be consistent with enhanced killing of cancer cells.
[0497] In one embodiment, NK cell activation is measured for
example by direct killing of target cells like for an example
cancer cells or by cytokine secretion or by changes in expression
of activation markers like for an example CD107a, etc. For NK
cells, increases in proliferation, cytotoxicity (ability to kill
target cells and increases CD107a, granzyme, and perforin
expression), cytokine production (e.g., IFN.gamma. and TNF), and
cell surface receptor expression (e.g., CD25) would be indicative
of immune modulation that would be consistent with enhanced killing
of cancer cells.
[0498] In one embodiment, .gamma..delta. T cell activation is
measured for example by cytokine secretion or by proliferation or
by changes in expression of activation markers.
[0499] In one embodiment, Th1 cell activation is measured for
example by cytokine secretion or by changes in expression of
activation markers.
[0500] Appropriate increases in activity or response (or decreases,
as appropriate as outlined above), are increases of 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the
signal in either a reference sample or in control samples, for
example test samples that do not contain a heterodimeric protein of
the invention. Similarly, increases of at least one-, two-, three-,
four- or five-fold as compared to reference or control samples show
efficacy.
VIII. TREATMENTS
[0501] Once made, the compositions of the invention find use in a
number of oncology applications, by treating cancer, generally by
promoting T cell activation (e.g., T cells are no longer
suppressed) with the binding of the heterodimeric fusion proteins
of the invention.
[0502] Accordingly, the targeted heterodimeric compositions of the
invention find use in the treatment of these cancers.
A. Targeted Heterodimeric Protein Compositions for In Vivo
Administration
[0503] Formulations of the antibodies used in accordance with the
present invention are prepared for storage by mixing an antibody
having the desired degree of purity with optional pharmaceutically
acceptable carriers, excipients or stabilizers (as generally
outlined in Remington's Pharmaceutical Sciences 16th edition, Osol,
A. Ed. [1980]), in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, buffers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
B. Combination Therapies
[0504] In some embodiments, the heterodimeric proteins of the
invention can be used in combination therapies with antibodies that
bind to different checkpoint proteins, e.g., not LAG-3 antibodies.
In this way, the antigen binding domains of the additional antibody
do not compete for binding with the targeted heterodimeric protein.
In this way, a sort of "triple combination" therapy is achieved, as
three receptors are engaged (two from the targeted heterodimeric
protein and one from the additional antibody). As discussed herein,
the heterodimeric protein can have different valencies and
specifities as outlined herein.
[0505] Surprisingly, as shown herein, these combinations can result
in synergistic effects when co-administered. In this context,
"co-administration" means that the two moieties can be administered
simultaneously or sequentially. That is, in some cases, the drugs
may be administered simultaneously, although generally this is
through the use of two separate IV infusions; that is, the drugs
are generally not combined into a single dosage unit.
Alternatively, co-administration includes the sequential
administration of the two separate drugs, either in a single day or
separate days (including separate days over time).
[0506] 1. Anti-PD-1 Antibodies for Use in Co-Administration
Therapies
[0507] As is known in the art, there are two currently approved
anti-PD-1 antibodies and many more in clinical testing. Thus,
suitable anti-PD-1 antibodies for use in combination therapies as
outlined herein include, but are not limited to, the two currently
FDA approved antibodies, pembrolizumab and nivolizumab, as well as
those in clinical testing currently, including, but not limited to,
tislelizumab, Sym021, REGN2810 (developed by Rengeneron),
JNJ-63723283 (developed by J and J), SHR-1210, pidilizumab,
AMP-224, MEDIo680, PDR001 and CT-001, as well as others outlined in
Liu et al., J. Hemat. & Oncol. (2017)10:136, the antibodies
therein expressly incorporated by reference. As above, anti-PD-1
antibodies are used in combination when the targeted heterodimeric
proteins of the invention do not have an antigen binding domain
that binds PD-1.
[0508] 2. Anti-PD-L1 Antibodies for Use in Co-Administration
Therapies
[0509] In some embodiments, anti-PD-L1 antibodies are used in
combination. As is known in the art, there are three currently
approved anti-PD-L1 antibodies and many more in clinical testing.
Thus, suitable anti-PD-L1 antibodies for use in combination
therapies as outlined herein include, but are not limited to, the
three currently FDA approved antibodies, atezolizumab, avelumab,
durvalumab, as well as those in clinical testing currently,
including, but not limited to, LY33000054 and CS1001, as well as
others outlined in Liu et al., J. Hemat. & Oncol. (2017)10:136,
the antibodies therein expressly incorporated by reference. As
above, anti-PD-L1 antibodies are used in combination when the
targeted heterodimeric proteins of the invention do not have an
antigen binding domain that binds PD-L1.
[0510] 3. Anti-TIM-3 Antibodies for Use in Co-Administration
Therapies
[0511] In some embodiments, anti-TIM-3 antibodies can be used in
combination with the targeted heterodimeric proteins of the
invention. There are several TIM-3 antibodies in clinical
development, including MBG453 and TSR-022. As above, anti-TIM-3
antibodies are used in combination when the targeted heterodimeric
proteins of the invention do not have an antigen binding domain
that binds TIM-3.
[0512] 4. Anti-TIGIT Antibodies for Use in Co-Administration
Therapies
[0513] In some embodiments, anti-TIGIT antibodies can be used in
combination with the targeted heterodimeric proteins of the
invention. There are several TIGIT antibodies in clinical
development, BMS-986207, OMP-313M32 and MTIG7192A. As above,
anti-TIGIT antibodies are used in combination when the targeted
heterodimeric proteins of the invention do not have an antigen
binding domain that binds TIGIT.
[0514] 5. Anti-CTLA-4 Antibodies for Use in Co-Administration
Therapies
[0515] In some embodiments, anti-CTLA-4 antibodies can be used in
combination with the targeted heterodimeric proteins of the
invention. Ipilimumab has been approved, and there are several more
in development, including CP-675,206 and AGEN-1884. As above,
anti-CTLA-4 antibodies are used in combination when the targeted
heterodimeric proteins of the invention do not have an antigen
binding domain that binds CTLA-4.
C. Administrative Modalities
[0516] The targeted heterodimeric proteins and chemotherapeutic
agents of the invention are administered to a subject, in accord
with known methods, such as intravenous administration as a bolus
or by continuous infusion over a period of time.
D. Treatment Modalities
[0517] In the methods of the invention, therapy is used to provide
a positive therapeutic response with respect to a disease or
condition. By "positive therapeutic response" is intended an
improvement in the disease or condition, and/or an improvement in
the symptoms associated with the disease or condition. For example,
a positive therapeutic response would refer to one or more of the
following improvements in the disease: (1) a reduction in the
number of neoplastic cells; (2) an increase in neoplastic cell
death; (3) inhibition of neoplastic cell survival; (5) inhibition
(i.e., slowing to some extent, preferably halting) of tumor growth;
(6) an increased patient survival rate; and (7) some relief from
one or more symptoms associated with the disease or condition.
[0518] Positive therapeutic responses in any given disease or
condition can be determined by standardized response criteria
specific to that disease or condition. Tumor response can be
assessed for changes in tumor morphology (i.e., overall tumor
burden, tumor size, and the like) using screening techniques such
as magnetic resonance imaging (MRI) scan, x-radiographic imaging,
computed tomographic (CT) scan, bone scan imaging, endoscopy, and
tumor biopsy sampling including bone marrow aspiration (BMA) and
counting of tumor cells in the circulation.
[0519] In addition to these positive therapeutic responses, the
subject undergoing therapy may experience the beneficial effect of
an improvement in the symptoms associated with the disease.
[0520] Treatment according to the present invention includes a
"therapeutically effective amount" of the medicaments used. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve a desired
therapeutic result.
[0521] A therapeutically effective amount may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the medicaments to elicit a desired
response in the individual. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the antibody
or antibody portion are outweighed by the therapeutically
beneficial effects.
[0522] A "therapeutically effective amount" for tumor therapy may
also be measured by its ability to stabilize the progression of
disease. The ability of a compound to inhibit cancer may be
evaluated in an animal model system predictive of efficacy in human
tumors.
[0523] Alternatively, this property of a composition may be
evaluated by examining the ability of the compound to inhibit cell
growth or to induce apoptosis by in vitro assays known to the
skilled practitioner. A therapeutically effective amount of a
therapeutic compound may decrease tumor size, or otherwise
ameliorate symptoms in a subject. One of ordinary skill in the art
would be able to determine such amounts based on such factors as
the subject's size, the severity of the subject's symptoms, and the
particular composition or route of administration selected.
[0524] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. Parenteral compositions may be formulated in dosage unit
form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited
as unitary dosages for the subjects to be treated; each unit
contains a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the
required pharmaceutical carrier.
[0525] The specification for the dosage unit forms of the present
invention are dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0526] The efficient dosages and the dosage regimens for the
targeted heterodimeric protein used in the present invention depend
on the disease or condition to be treated and may be determined by
the persons skilled in the art.
[0527] An exemplary, non-limiting range for a therapeutically
effective amount of a targeted heterodimeric protein used in the
present invention is about 0.1-100 mg/kg.
[0528] All cited references are herein expressly incorporated by
reference in their entirety.
[0529] Whereas particular embodiments of the invention have been
described above for purposes of illustration, it will be
appreciated by those skilled in the art that numerous variations of
the details may be made without departing from the invention as
described in the appended claims.
IX. EXAMPLES
[0530] Examples are provided below to illustrate the present
invention. These examples are not meant to constrain the present
invention to any particular application or theory of operation. For
all constant region positions discussed in the present invention,
numbering is according to the EU index as in Kabat (Kabat et al.,
1991, Sequences of Proteins of Immunological Interest, 5th Ed.,
United States Public Health Service, National Institutes of Health,
Bethesda, entirely incorporated by reference). Those skilled in the
art of antibodies will appreciate that this convention consists of
nonsequential numbering in specific regions of an immunoglobulin
sequence, enabling a normalized reference to conserved positions in
immunoglobulin families. Accordingly, the positions of any given
immunoglobulin as defined by the EU index will not necessarily
correspond to its sequential sequence.
[0531] General and specific scientific techniques are outlined in
US Patent Publications 2015/0307629, 2014/0288275 and
WO2014/145806, all of which are expressly incorporated by reference
in their entirety and particularly for the techniques outlined
therein. Examples 1 and 2 from U.S. Ser. No. 62,416, 087, filed on
Nov. 1, 2016 are expressly incorporated by reference in their
entirety, including the corresponding figures. Additionally, U.S.
Ser. Nos. 62/408,655, 62/443,465, 62/477,926, 15/785,401,
62/416,087 and 15/785,393 are expressly incorporated by reference
in their entirety, and specifically for all the sequences, Figures
and Legends therein.
A. Example 1
Anti-LAG-3 ABDs
[0532] Examples of antigen-binding domains which bind LAG-3 were
described in in WO2017/218707, the contents are hereby incorporated
in its entirety for all purposes, and in particular for the LAG-3
ABDs in FIG. 11, the data in FIG. 18, FIG. 55, FIG. 56, FIG. 63 and
SEQ ID NO:s 36819-36962, SEQ ID NO:s 35417-35606, SEQ ID NO:s
25914-32793 and SEQ ID NO:s 32794-33002 sequences in the sequence
listing.
B. Example 2
LAG-3-Targeted IL-15/R.alpha.-Fc Fusions
[0533] Reference is made to WO2018/071919 which describes
IL-15/RA-Fc fusions that do not contain ABDs as are generally
depicted in FIGS. 9A-9G and FIGS. 39A-39D. WO2018/071919 is
expressly incorporated by reference herein, and specifically for
all of the sequences, formats, Figures and Legends therein.
[0534] 2A: Generation of LAG-3-Targeted IL-15/R.alpha.-Fc
Fusions
[0535] Plasmids coding for IL-15, IL-15R.alpha. sushi domain, or
the anti-LAG-3 variable regions were constructed by standard gene
synthesis, followed by subcloning into a pTT5 expression vector
containing Fc fusion partners (e.g., constant regions as depicted
in Figure). Cartoon schematics of illustrative LAG-3-targeted
IL-15/R.alpha.-Fc fusions are depicted in FIG. 21.
[0536] The "scIL-15/R.alpha. x scFv" format (FIG. 21A) comprises
IL-15R.alpha.(sushi) fused to IL-15 by a variable length linker
(termed "scIL-15/R.alpha.") which is then fused to the N-terminus
of a heterodimeric Fc-region, with an scFv fused to the other side
of the heterodimeric Fc.
[0537] The "scFv x ncIL-15/R.alpha." format (FIG. 21B) comprises an
scFv fused to the N-terminus of a heterodimeric Fc-region, with
IL-15R.alpha.(sushi) fused to the other side of the heterodimeric
Fc, while IL-15 is transfected separately so that a non-covalent
IL-15/R.alpha. complex is formed.
[0538] The "scFv x dsIL-15/R.alpha." format (FIG. 21C) is the same
as the "scFv x ncIL-15/R.alpha." format, but wherein
IL-15R.alpha.(sushi) and IL-15 are covalently linked as a result of
engineered cysteines.
[0539] The "scIL-15/R.alpha. x Fab" format (FIG. 21D) comprises
IL-15R.alpha.(sushi) fused to IL-15 by a variable length linker
(termed "scIL-15/R.alpha.") which is then fused to the N-terminus
of a heterodimeric Fc-region, with a variable heavy chain (VH)
fused to the other side of the heterodimeric Fc, while a
corresponding light chain is transfected separately so as to form a
Fab with the VH. Sequences for illustrative LAG-3-targeted
IL-15/R.alpha.-Fc fusion proteins of this format are depicted in
Figure.
[0540] The "ncIL-15/R.alpha. x Fab" format (FIG. 21E) comprises a
VH fused to the N-terminus of a heterodimeric Fc-region, with
IL-15R.alpha.(sushi) fused to the other side of the heterodimeric
Fc, while a corresponding light chain is transfected separately so
as to form a Fab with the VH, and while IL-15 is transfected
separately so that a non-covalent IL-15/R.alpha. complex is
formed.
[0541] The "dsIL-15/R.alpha. x Fab" format (FIG. 21F) is the same
as the "ncIL-15/R.alpha. x Fab" format, but wherein
IL-15R.alpha.(sushi) and IL-15 are covalently linked as a result of
engineered cysteines.
[0542] The "mAb-scIL-15/R.alpha." format (FIG. 21G) comprises VH
fused to the N-terminus of a first and a second heterodimeric Fc,
with IL-15 is fused to IL-15R.alpha.(sushi) which is then further
fused to the C-terminus of one of the heterodimeric Fc-region,
while corresponding light chains are transfected separately so as
to form a Fabs with the VHs.
[0543] The "mAb-ncIL-15/R.alpha." format (FIG. 21H) comprises VH
fused to the N-terminus of a first and a second heterodimeric Fc,
with IL-15R.alpha.(sushi) fused to the C-terminus of one of the
heterodimeric Fc-region, while corresponding light chains are
transfected separately so as to form a Fabs with the VHs, and while
and while IL-15 is transfected separately so that a non-covalent
IL-15/R.alpha. complex is formed.
[0544] The "mAb-dsIL-15/R.alpha." format (FIG. 21I) is the same as
the "mAb-ncIL-15/R.alpha." format, but wherein IL-15R.alpha.(sushi)
and IL-15 are covalently linked as a result of engineered
cysteines.
[0545] The "central-IL-15/R.alpha." format (FIG. 21J) comprises a
VH recombinantly fused to the N-terminus of IL-15 which is then
further fused to one side of a heterodimeric Fc and a VH
recombinantly fused to the N-terminus of IL-15R.alpha.(sushi) which
is then further fused to the other side of the heterodimeric Fc,
while corresponding light chains are transfected separately so as
to form a Fabs with the VHs.
[0546] The "central-scIL-15/R.alpha." format (FIG. 21K) comprises a
VH fused to the N-terminus of IL-15R.alpha.(sushi) which is fused
to IL-15 which is then further fused to one side of a heterodimeric
Fc and a VH fused to the other side of the heterodimeric Fc, while
corresponding light chains are transfected separately so as to form
a Fabs with the VHs.
[0547] 2B: LAG-3-Targeted IL-15/R.alpha.-Fc Fusions Enhance GVHD,
and Combines Synergistically with Anti-PD-1 Antibody
[0548] Illustrative LAG-3-targeted IL-15/R.alpha.-Fc fusion
proteins, XENP27972 and XENP27973 alone or in combination with (a
bivalent anti-PD-1 mAb based on nivolumab with ablated effector
function; sequences for which is depicted in FIG. 23), were
evaluated in a Graft-versus-Host Disease (GVHD) model conducted in
NSG (NOD-SCID-gamma) immunodeficient mice. When the NSG mice are
injected with human PBMCs, the human PBMCs develop an autoimmune
response against mouse cells. Dosing of NSG mice injected with
human PBMCs followed with LAG-3-targeted IL-15/R.alpha.-Fc fusion
proteins proliferate the engrafted T cells and enhances
engraftment.
[0549] 10 million human PBMCs were engrafted into NSG mice via
IV-OSP on Day -1 followed by dosing with the indicated test
articles at the indicated concentrations on Days 0, 7, 14, and 21.
Counts of various lymphocyte populations were performed on Days 6
and 10, data for which are depicted in Figures--. Body weights of
mice were measured over time and depicted in Figure as percentage
of initial body weight. The data show that dosing with either
XENP27972 or XENP27973 following engraftment with human PBMCs
enhanced GVHD as indicated by increased T cell (CD8+ and CD4+), NK
cell, and CD45+ cell counts as well as decreased body weight in
comparison to engraftment with PBMC alone. Notably, both XENP27972
and XENP27973 enhanced GVHD to a greater extent than dosing with
XENP16432 alone. Additionally, the data show that XENP27972 and
XENP27973 combine synergistically with XENP16432 in enhancing GVHD
as indicated by the death of all mice by Day 19 following dosing
with a combination of XENP27972 and XENP16432, and death of all but
one mice by Day 19 following dosing with a combination of XENP27973
and XENP16432. This suggests that, in an immuno-oncology setting,
treatment with LAG-3-targeted IL-15/R.alpha.-Fc fusion proteins
alone or in combination with checkpoint blockade antibodies will
proliferate tumor-infiltrating lymphocytes and enhance anti-tumor
activity.
[0550] 2C: In Vitro Characterization of LAG-3-Targeted
IL-15/R.alpha.-Fc Fusions
[0551] The LAG-3-targeted IL-15/R.alpha.-Fc fusions were further
characterized in a cell proliferation assay. Human PBMCs were
stimulated for 48 hours with 500 ng/ml plate-bound anti-CD3 (OKT3)
and then labeled with CFSE and incubated with the following test
articles for 4 days at 37.degree. C.: XENP27972 (LAG-3-targeted
IL-15/R.alpha.-Fc fusion based on anti-LAG-3 clone 7G8); XENP27973
(LAG-3-targeted IL-15/R.alpha.-Fc fusion based on anti-LAG-3 done
2A11); XENP24306 (control untargeted
IL-15(D30N/E64Q/N65D)/R.alpha.-Fc fusion having D30N/E64Q/N65D
IL-15 variant); and XENP26007 (control RSV-targeted
IL-15/R.alpha.-Fc fusion having N4D/N65D IL-15 variant). Cells were
stained with the following antibodies:, anti-CD8-PerCP-Cy5.5 (SK1),
anti-CD3-PE-Cy7 (OKT3), anti-CD45RO-APC-Fire750 (UCHL1),
anti-HLA-DR-Alexa700 (L243), anti-CD16-BV605 (3G6), anti-CD56-BV605
(HCD56), anti-CD25-BV711 (M-A251), anti-CD45RA-BV785 (HI100),
anti-CD4-BUV395 (SK3), and Zombie Aqua-BV510 and analyzed by flow
for various cell populations.
[0552] The proliferation of various T cell and NK cell populations
based on CFSE dilution (Zombie Aqua to exclude dead cells) was
investigated, data for which are depicted in FIGS. 30-35. The data
show that the LAG-3-targeted IL-15/R.alpha.-Fc fusions, in
particular XENP27972, are more potent in inducing proliferation of
both CD8.sup.+ and CD4.sup.+ T cells in comparison to untargeted
IL-15(D30N/E64Q/N65D)/R.alpha.-Fc fusion (as well as control
RSV-targeted IL-15/R.alpha.-Fc fusion), with a preference for CD8+
T cells. Notably, the LAG-3-targeted IL-15/R.alpha.-Fc fusions
preferentially targets memory T cells, suggesting that in a
clinical setting, the LAG-3-targeted IL-15/R.alpha.-Fc fusions will
be selective for activated tumor-infiltrating lymphocytes in the
tumor environment.
[0553] The activation of various T cell populations based on
expression of CD25 (a late stage T cell activation marker) and
HLA-DR (another activation marker) were also investigated, data for
which are depicted in FIGS. 36-38. The data show that
LAG-3-targeted IL-15/R.alpha.-Fc fusions generally appear more
potent in inducing activation of CD8 memory T cell populations in
comparison to untargeted IL-15(D30N/E64Q/N65D)/R.alpha.-Fc fusion
(as well as control RSV-targeted IL-15/R.alpha.-Fc fusion).
C. Example 3
LAG-3-Targeted IL-15/R.alpha.-Fc Fusions with Tuned IL-15 Potency
3A: IL-15(D30N/N65D) Variant
[0554] In a study investigating the pharmacokinetics of IL-15-Fc
potency variants with Xtend, cynomolgus monkeys were administered a
first single intravenous (i.v.) dose of XENP22853 (WT
IL-15/R.alpha.-heteroFc with Xtend; sequences depicted in FIG. 39),
XENP24306 (IL-15(D30N/E64Q/N65D)/R.alpha.-heteroFc with Xtend;
sequences depicted in FIG. 42), XENP24113
(IL-15(N4D/N65D)/R.alpha.-heteroFc with Xtend; sequences depicted
in FIG. 40), and XENP24294 (scIL-15(N4D/N65D)/R.alpha.-Fc with
Xtend; sequences depicted in FIG. 41) at varying
concentrations.
[0555] FIG. 43 depicts the serum concentration of the test articles
over time following the first dose. As expected, incorporating
potency variants in addition to Xtend substitution (as in XENP24306
and XENP24113) greatly improves the pharmacokinetics of IL-15-Fc
fusions (in comparison to XENP22583). Unexpectedly, however,
IL-15/R.alpha.-heteroFc fusion XENP24113 and scIL-15/R.alpha.-Fc
fusion XENP24294 (which have the same IL-15(N4D/N65D) potency
variant) demonstrated reduced pharmacokinetics in comparison to
XENP24306. This suggests that the reduced pharmacokinetics was due
to the particular IL-15 potency variant rather than the format of
the IL-15-Fc fusion. While a decrease in pharmacokinetics for
XENP24113 and XENP24294 was expected on the basis of previous
findings which demonstrated that the IL-15-Fc fusions having
IL-15(N4D/N65D) variant had greater in vitro potency than IL-15-Fc
fusions having the IL-15(D30N/E64Q/N65D) variant, the decrease in
pharmacokinetics was unexpectedly disproportionate to the increase
in potency. Accordingly, identification of alternative IL-15
potency variants for use in the LAG-3-targeted IL-15-Fc fusions of
the invention was carried out.
[0556] It is noted that IL-15(N4D/N65D) has both its substitutions
at the IL-15 interface responsible for binding to CD122, while
IL-15(D30N/E64Q/N65D) has two substitutions (E64Q and N65D) at
IL-15:CD122 interface; and one substitution (D30N) at the IL-15
interface responsible for binding to CD132. Accordingly, it is
believed that the modification at the IL-15:CD132 interface may
contribute to the superior pharmacokinetics observed for XENP24306.
Notably, it was determined that scIL-15/R.alpha.-Fc fusions
comprising IL-15(N4D/N65D) variant and IL-15(D30N/N65D) variant
demonstrated very similar potency in vitro, as depicted in FIG. 45.
In view of the above, illustrative LAG-3-targeted IL-15-Fc fusion
comprising the IL-15(D30N/N65D) variants were conceived, sequences
for which are depicted in FIG. 46. A control RSV-targeted
IL-15/R.alpha.-Fc fusion protein XENP29481 with IL-15(D30N/N65D)
variant was also generated, sequences for which are depicted in
FIG. 49.
[0557] 3B: IL-15(D30N/E64Q/N65D) Variant
[0558] Although the LAG-3-targeted IL-15/R.alpha.-Fc fusions were
designed with the aim to be targeted to the tumor environment via
the LAG-3-targeting arm, the cytokine moiety is still capable of
signaling before reaching the tumor site and may contribute to
systemic toxicity. Accordingly, LAG-3-targeted IL-15/R.alpha.-Fc
fusions with IL-15(D30N/E64Q/N65D) variant were constructed to
further reduce the IL-15 potency, which as illustrated in Example
2C has drastically reduced activity and in FIG. 45. Sequences for
illustrative LAG-3-targeted IL-15/R.alpha.-Fc fusions comprising
IL-15(D30N/E64Q/N65D) variant are depicted in FIG. 47.
Additionally, XENP30432, a RSV-targeted IL-15/R.alpha.-Fc fusion
comprising IL-15(D30N/E64Q/N65D) variant (sequences for which are
depicted in FIG. 49) was constructed, to act as a surrogate for
investigating the behavior of LAG-3-targeted IL-15/R.alpha.-Fc
fusions comprising IL-15(D30N/E64Q/N65D) variant outside of the
tumor environment.
Sequence CWU 1
1
3831162PRTHomo sapiens 1Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile
Ser Ile Gln Cys Tyr1 5 10 15Leu Cys Leu Leu Leu Asn Ser His Phe Leu
Thr Glu Ala Gly Ile His 20 25 30Val Phe Ile Leu Gly Cys Phe Ser Ala
Gly Leu Pro Lys Thr Glu Ala 35 40 45Asn Trp Val Asn Val Ile Ser Asp
Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60Gln Ser Met His Ile Asp Ala
Thr Leu Tyr Thr Glu Ser Asp Val His65 70 75 80Pro Ser Cys Lys Val
Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95Val Ile Ser Leu
Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110Asn Leu
Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 115 120
125Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
130 135 140Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe
Ile Asn145 150 155 160Thr Ser2114PRTHomo sapiens 2Asn Trp Val Asn
Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met
His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser
Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val
Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55
60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65
70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn
Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe
Ile Asn 100 105 110Thr Ser3267PRTHomo sapiens 3Met Ala Pro Arg Arg
Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala1 5 10 15Leu Leu Leu Leu
Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr 20 25 30Cys Pro Pro
Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 35 40 45Tyr Ser
Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 50 55 60Arg
Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala65 70 75
80Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp
85 90 95Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr
Thr 100 105 110Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser
Gly Lys Glu 115 120 125Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr
Ala Ala Thr Thr Ala 130 135 140Ala Ile Val Pro Gly Ser Gln Leu Met
Pro Ser Lys Ser Pro Ser Thr145 150 155 160Gly Thr Thr Glu Ile Ser
Ser His Glu Ser Ser His Gly Thr Pro Ser 165 170 175Gln Thr Thr Ala
Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln 180 185 190Pro Pro
Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile 195 200
205Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu
210 215 220Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser
Val Glu225 230 235 240Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp
Gly Thr Ser Ser Arg 245 250 255Asp Glu Asp Leu Glu Asn Cys Ser His
His Leu 260 265465PRTHomo sapiens 4Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55
60Arg655175PRTHomo sapiens 5Ile Thr Cys Pro Pro Pro Met Ser Val Glu
His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu
Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser
Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His
Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Asp Pro Ala Leu Val
His Gln Arg Pro Ala Pro Pro Ser Thr Val65 70 75 80Thr Thr Ala Gly
Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly 85 90 95Lys Glu Pro
Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr 100 105 110Thr
Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro 115 120
125Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr
130 135 140Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser
Ala Ser145 150 155 160His Gln Pro Pro Gly Val Tyr Pro Gln Gly His
Ser Asp Thr Thr 165 170 1756551PRTHomo sapiens 6Met Ala Ala Pro Ala
Leu Ser Trp Arg Leu Pro Leu Leu Ile Leu Leu1 5 10 15Leu Pro Leu Ala
Thr Ser Trp Ala Ser Ala Ala Val Asn Gly Thr Ser 20 25 30Gln Phe Thr
Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp 35 40 45Ser Gln
Asp Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp 50 55 60Pro
Asp Arg Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser65 70 75
80Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln
85 90 95Lys Leu Thr Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg
Glu 100 105 110Gly Val Arg Trp Arg Val Met Ala Ile Gln Asp Phe Lys
Pro Phe Glu 115 120 125Asn Leu Arg Leu Met Ala Pro Ile Ser Leu Gln
Val Val His Val Glu 130 135 140Thr His Arg Cys Asn Ile Ser Trp Glu
Ile Ser Gln Ala Ser His Tyr145 150 155 160Phe Glu Arg His Leu Glu
Phe Glu Ala Arg Thr Leu Ser Pro Gly His 165 170 175Thr Trp Glu Glu
Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp 180 185 190Ile Cys
Leu Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val 195 200
205Arg Val Lys Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser
210 215 220Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys
Asp Thr225 230 235 240Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu
Ser Gly Ala Phe Gly 245 250 255Phe Ile Ile Leu Val Tyr Leu Leu Ile
Asn Cys Arg Asn Thr Gly Pro 260 265 270Trp Leu Lys Lys Val Leu Lys
Cys Asn Thr Pro Asp Pro Ser Lys Phe 275 280 285Phe Ser Gln Leu Ser
Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu 290 295 300Ser Ser Pro
Phe Pro Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro305 310 315
320Glu Ile Ser Pro Leu Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu
325 330 335Leu Leu Gln Gln Asp Lys Val Pro Glu Pro Ala Ser Leu Ser
Ser Asn 340 345 350His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr
Phe Phe Phe His 355 360 365Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys
Gln Val Tyr Phe Thr Tyr 370 375 380Asp Pro Tyr Ser Glu Glu Asp Pro
Asp Glu Gly Val Ala Gly Ala Pro385 390 395 400Thr Gly Ser Ser Pro
Gln Pro Leu Gln Pro Leu Ser Gly Glu Asp Asp 405 410 415Ala Tyr Cys
Thr Phe Pro Ser Arg Asp Asp Leu Leu Leu Phe Ser Pro 420 425 430Ser
Leu Leu Gly Gly Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser 435 440
445Gly Ala Gly Glu Glu Arg Met Pro Pro Ser Leu Gln Glu Arg Val Pro
450 455 460Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro Pro Thr Pro Gly
Val Pro465 470 475 480Asp Leu Val Asp Phe Gln Pro Pro Pro Glu Leu
Val Leu Arg Glu Ala 485 490 495Gly Glu Glu Val Pro Asp Ala Gly Pro
Arg Glu Gly Val Ser Phe Pro 500 505 510Trp Ser Arg Pro Pro Gly Gln
Gly Glu Phe Arg Ala Leu Asn Ala Arg 515 520 525Leu Pro Leu Asn Thr
Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly 530 535 540Gln Asp Pro
Thr His Leu Val545 5507214PRTHomo sapiens 7Ala Val Asn Gly Thr Ser
Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala1 5 10 15Asn Ile Ser Cys Val
Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser 20 25 30Cys Gln Val His
Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys 35 40 45Glu Leu Leu
Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60Gly Ala
Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu65 70 75
80Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln
85 90 95Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser
Leu 100 105 110Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser
Trp Glu Ile 115 120 125Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu
Glu Phe Glu Ala Arg 130 135 140Thr Leu Ser Pro Gly His Thr Trp Glu
Glu Ala Pro Leu Leu Thr Leu145 150 155 160Lys Gln Lys Gln Glu Trp
Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175Gln Tyr Glu Phe
Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr 180 185 190Thr Trp
Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200
205Ala Leu Gly Lys Asp Thr 2108369PRTHomo sapiens 8Met Leu Lys Pro
Ser Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln Leu1 5 10 15Pro Leu Leu
Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly 20 25 30Asn Glu
Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp 35 40 45Ser
Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val 50 55
60Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro65
70 75 80Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp
Asn 85 90 95Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu
Ile Thr 100 105 110Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu
Tyr Gln Thr Phe 115 120 125Val Val Gln Leu Gln Asp Pro Arg Glu Pro
Arg Arg Gln Ala Thr Gln 130 135 140Met Leu Lys Leu Gln Asn Leu Val
Ile Pro Trp Ala Pro Glu Asn Leu145 150 155 160Thr Leu His Lys Leu
Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn 165 170 175Arg Phe Leu
Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp 180 185 190Trp
Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe 195 200
205Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg
210 215 220Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser
Glu Trp225 230 235 240Ser His Pro Ile His Trp Gly Ser Asn Thr Ser
Lys Glu Asn Pro Phe 245 250 255Leu Phe Ala Leu Glu Ala Val Val Ile
Ser Val Gly Ser Met Gly Leu 260 265 270Ile Ile Ser Leu Leu Cys Val
Tyr Phe Trp Leu Glu Arg Thr Met Pro 275 280 285Arg Ile Pro Thr Leu
Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His 290 295 300Gly Asn Phe
Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser305 310 315
320Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile Pro
325 330 335Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Ala Ser Pro
Cys Asn 340 345 350Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr Thr
Leu Lys Pro Glu 355 360 365Thr9240PRTHomo sapiens 9Leu Asn Thr Thr
Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala1 5 10 15Asp Phe Phe
Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser Thr 20 25 30Leu Pro
Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu Tyr Met 35 40 45Asn
Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn Leu Thr 50 55
60Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys65
70 75 80Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu
Gln 85 90 95Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu
Gln Asp 100 105 110Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu
Lys Leu Gln Asn 115 120 125Leu Val Ile Pro Trp Ala Pro Glu Asn Leu
Thr Leu His Lys Leu Ser 130 135 140Glu Ser Gln Leu Glu Leu Asn Trp
Asn Asn Arg Phe Leu Asn His Cys145 150 155 160Leu Glu His Leu Val
Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr 165 170 175Glu Gln Ser
Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp 180 185 190Gly
Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu 195 200
205Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp
210 215 220Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu
Glu Ala225 230 235 24010525PRTHomo sapiens 10Met Trp Glu Ala Gln
Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp1 5 10 15Val Ala Pro Val
Lys Pro Leu Gln Pro Gly Ala Glu Val Pro Val Val 20 25 30Trp Ala Gln
Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile 35 40 45Pro Leu
Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln 50 55 60His
Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu65 70 75
80Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro
85 90 95Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser
Gly 100 105 110Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg
Gly Arg Gln 115 120 125Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala
Arg Arg Ala Asp Ala 130 135 140Gly Glu Tyr Arg Ala Ala Val His Leu
Arg Asp Arg Ala Leu Ser Cys145 150 155 160Arg Leu Arg Leu Arg Leu
Gly Gln Ala Ser Met Thr Ala Ser Pro Pro 165 170 175Gly Ser Leu Arg
Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser 180 185 190Arg Pro
Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln 195 200
205Gly Arg Val Pro Val Arg Glu Ser Pro His His His Leu Ala Glu Ser
210 215 220Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro
Trp Gly225 230 235 240Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val
Ser Ile Met Tyr Asn 245 250 255Leu Thr Val Leu Gly Leu Glu Pro Pro
Thr Pro Leu Thr Val Tyr Ala 260 265 270Gly Ala Gly Ser Arg Val Gly
Leu Pro Cys Arg Leu Pro Ala Gly Val 275 280 285Gly Thr Arg Ser Phe
Leu Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly 290 295 300Pro Asp
Leu
Leu Val Thr Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu305 310 315
320Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His
325 330 335Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile
Ile Thr 340 345 350Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu
Gly Lys Leu Leu 355 360 365Cys Glu Val Thr Pro Val Ser Gly Gln Glu
Arg Phe Val Trp Ser Ser 370 375 380Leu Asp Thr Pro Ser Gln Arg Ser
Phe Ser Gly Pro Trp Leu Glu Ala385 390 395 400Gln Glu Ala Gln Leu
Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln 405 410 415Gly Glu Arg
Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser 420 425 430Pro
Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly 435 440
445His Leu Leu Leu Phe Leu Ile Leu Gly Val Leu Ser Leu Leu Leu Leu
450 455 460Val Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp
Arg Pro465 470 475 480Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His
Pro Pro Gln Ala Gln 485 490 495Ser Lys Ile Glu Glu Leu Glu Gln Glu
Pro Glu Pro Glu Pro Glu Pro 500 505 510Glu Pro Glu Pro Glu Pro Glu
Pro Glu Pro Glu Gln Leu 515 520 52511422PRTHomo sapiens 11Val Pro
Val Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys1 5 10 15Ser
Pro Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly 20 25
30Val Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro
35 40 45Gly His Pro Leu Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser
Trp 50 55 60Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly Pro
Gly Gly65 70 75 80Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg Val
Gln Leu Asp Glu 85 90 95Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu Trp
Leu Arg Pro Ala Arg 100 105 110Arg Ala Asp Ala Gly Glu Tyr Arg Ala
Ala Val His Leu Arg Asp Arg 115 120 125Ala Leu Ser Cys Arg Leu Arg
Leu Arg Leu Gly Gln Ala Ser Met Thr 130 135 140Ala Ser Pro Pro Gly
Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn145 150 155 160Cys Ser
Phe Ser Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg 165 170
175Asn Arg Gly Gln Gly Arg Val Pro Val Arg Glu Ser Pro His His His
180 185 190Leu Ala Glu Ser Phe Leu Phe Leu Pro Gln Val Ser Pro Met
Asp Ser 195 200 205Gly Pro Trp Gly Cys Ile Leu Thr Tyr Arg Asp Gly
Phe Asn Val Ser 210 215 220Ile Met Tyr Asn Leu Thr Val Leu Gly Leu
Glu Pro Pro Thr Pro Leu225 230 235 240Thr Val Tyr Ala Gly Ala Gly
Ser Arg Val Gly Leu Pro Cys Arg Leu 245 250 255Pro Ala Gly Val Gly
Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro 260 265 270Pro Gly Gly
Gly Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe 275 280 285Thr
Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr 290 295
300Cys His Ile His Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr
Leu305 310 315 320Ala Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser
Pro Gly Ser Leu 325 330 335Gly Lys Leu Leu Cys Glu Val Thr Pro Val
Ser Gly Gln Glu Arg Phe 340 345 350Val Trp Ser Ser Leu Asp Thr Pro
Ser Gln Arg Ser Phe Ser Gly Pro 355 360 365Trp Leu Glu Ala Gln Glu
Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys 370 375 380Gln Leu Tyr Gln
Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr385 390 395 400Glu
Leu Ser Ser Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala 405 410
415Leu Pro Ala Gly His Leu 42012533PRTMacaca
fascicularisMOD_RES(74)..(74)Any amino acid 12Met Trp Glu Ala Gln
Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp1 5 10 15Val Ala Pro Val
Lys Pro Pro Gln Pro Gly Ala Glu Ile Ser Val Val 20 25 30Trp Ala Gln
Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile 35 40 45Pro Leu
Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln 50 55 60His
Gln Pro Asp Ser Gly Pro Pro Ala Xaa Ala Pro Gly His Pro Pro65 70 75
80Val Pro Gly His Arg Pro Ala Ala Pro Tyr Ser Trp Gly Pro Arg Pro
85 90 95Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser
Gly 100 105 110Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg
Gly Arg Gln 115 120 125Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala
Arg Arg Ala Asp Ala 130 135 140Gly Glu Tyr Arg Ala Thr Val His Leu
Arg Asp Arg Ala Leu Ser Cys145 150 155 160Arg Leu Arg Leu Arg Val
Gly Gln Ala Ser Met Thr Ala Ser Pro Pro 165 170 175Gly Ser Leu Arg
Thr Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser 180 185 190Arg Pro
Asp Arg Pro Ala Ser Val His Trp Phe Arg Ser Arg Gly Gln 195 200
205Gly Arg Val Pro Val Gln Gly Ser Pro His His His Leu Ala Glu Ser
210 215 220Phe Leu Phe Leu Pro His Val Gly Pro Met Asp Ser Gly Leu
Trp Gly225 230 235 240Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val
Ser Ile Met Tyr Asn 245 250 255Leu Thr Val Leu Gly Leu Glu Pro Ala
Thr Pro Leu Thr Val Tyr Ala 260 265 270Gly Ala Gly Ser Arg Val Glu
Leu Pro Cys Arg Leu Pro Pro Ala Val 275 280 285Gly Thr Gln Ser Phe
Leu Thr Ala Lys Trp Ala Pro Pro Gly Gly Gly 290 295 300Pro Asp Leu
Leu Val Ala Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu305 310 315
320Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Ile Cys His Ile Arg
325 330 335Leu Gln Gly Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile
Ile Thr 340 345 350Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu
Gly Lys Leu Leu 355 360 365Cys Glu Val Thr Pro Ala Ser Gly Gln Glu
His Phe Val Trp Ser Pro 370 375 380Leu Asn Thr Pro Ser Gln Arg Ser
Phe Ser Gly Pro Trp Leu Glu Ala385 390 395 400Gln Glu Ala Gln Leu
Leu Ser Gln Pro Trp Gln Cys Gln Leu His Gln 405 410 415Gly Glu Arg
Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser 420 425 430Pro
Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Arg Ala Gly 435 440
445His Leu Pro Leu Phe Leu Ile Leu Gly Val Leu Phe Leu Leu Leu Leu
450 455 460Val Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp
Arg Pro465 470 475 480Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His
Pro Pro Gln Ala Gln 485 490 495Ser Lys Ile Glu Glu Leu Glu Gln Glu
Pro Glu Leu Glu Pro Glu Pro 500 505 510Glu Leu Glu Arg Glu Leu Gly
Pro Glu Pro Glu Pro Gly Pro Glu Pro 515 520 525Glu Pro Glu Gln Leu
53013422PRTMacaca fascicularisMOD_RES(46)..(46)Any amino acid 13Ile
Ser Val Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys1 5 10
15Ser Pro Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly
20 25 30Val Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Xaa Ala
Pro 35 40 45Gly His Pro Pro Val Pro Gly His Arg Pro Ala Ala Pro Tyr
Ser Trp 50 55 60Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly
Pro Gly Gly65 70 75 80Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg
Val Gln Leu Asp Glu 85 90 95Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu
Trp Leu Arg Pro Ala Arg 100 105 110Arg Ala Asp Ala Gly Glu Tyr Arg
Ala Thr Val His Leu Arg Asp Arg 115 120 125Ala Leu Ser Cys Arg Leu
Arg Leu Arg Val Gly Gln Ala Ser Met Thr 130 135 140Ala Ser Pro Pro
Gly Ser Leu Arg Thr Ser Asp Trp Val Ile Leu Asn145 150 155 160Cys
Ser Phe Ser Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg 165 170
175Ser Arg Gly Gln Gly Arg Val Pro Val Gln Gly Ser Pro His His His
180 185 190Leu Ala Glu Ser Phe Leu Phe Leu Pro His Val Gly Pro Met
Asp Ser 195 200 205Gly Leu Trp Gly Cys Ile Leu Thr Tyr Arg Asp Gly
Phe Asn Val Ser 210 215 220Ile Met Tyr Asn Leu Thr Val Leu Gly Leu
Glu Pro Ala Thr Pro Leu225 230 235 240Thr Val Tyr Ala Gly Ala Gly
Ser Arg Val Glu Leu Pro Cys Arg Leu 245 250 255Pro Pro Ala Val Gly
Thr Gln Ser Phe Leu Thr Ala Lys Trp Ala Pro 260 265 270Pro Gly Gly
Gly Pro Asp Leu Leu Val Ala Gly Asp Asn Gly Asp Phe 275 280 285Thr
Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Ile 290 295
300Cys His Ile Arg Leu Gln Gly Gln Gln Leu Asn Ala Thr Val Thr
Leu305 310 315 320Ala Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser
Pro Gly Ser Leu 325 330 335Gly Lys Leu Leu Cys Glu Val Thr Pro Ala
Ser Gly Gln Glu His Phe 340 345 350Val Trp Ser Pro Leu Asn Thr Pro
Ser Gln Arg Ser Phe Ser Gly Pro 355 360 365Trp Leu Glu Ala Gln Glu
Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys 370 375 380Gln Leu His Gln
Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr385 390 395 400Glu
Leu Ser Ser Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala 405 410
415Leu Arg Ala Gly His Leu 420145PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 14Gly Gly Gly Gly Ser1
51510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5
101615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser1 5 10 151720PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 17Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser
201825PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser 20
251930PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 19Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 20 25 302035PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 20Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30Gly Gly Ser
352130PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro
Pro Gly Gly Gly Gly1 5 10 15Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly 20 25 30225PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 22Gly Lys Pro Gly Ser1
52325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly Lys
Pro Gly Ser Gly1 5 10 15Lys Pro Gly Ser Gly Lys Pro Gly Ser 20
252430PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 24Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly
Lys Pro Gly Ser Gly1 5 10 15Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly
Lys Pro Gly Ser 20 25 30255PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 25Gly Gly Gly Glu Ser1
5269PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Lys Thr His Thr Cys Pro Pro Cys Pro1
52715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
Pro Cys Pro1 5 10 152819PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 28Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Lys Thr His Thr Cys Pro1 5 10 15Pro Cys
Pro2919PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Lys Thr
His Thr Cys Pro1 5 10 15Pro Cys Pro3014PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 30Gly
Lys Pro Gly Ser Lys Thr His Thr Cys Pro Pro Cys Pro1 5
103115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser1 5 10 153218PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 32Gly Ser Thr Ser Gly Ser Gly
Lys Pro Gly Ser Gly Glu Gly Ser Thr1 5 10 15Lys
Gly3314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Ile Arg Pro Arg Ala Ile Gly Gly Ser Lys Pro Arg
Val Ala1 5 103415PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 34Gly Lys Gly Gly Ser Gly Lys Gly Gly
Ser Gly Lys Gly Gly Ser1 5 10 153515PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Gly
Gly Lys Gly Ser Gly Gly Lys Gly Ser Gly Gly Lys Gly Ser1 5 10
153615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Gly Gly Gly Lys Ser Gly Gly Gly Lys Ser Gly Gly
Gly Lys Ser1 5 10 153715PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 37Gly Lys Gly Lys Ser Gly Lys
Gly Lys Ser Gly Lys Gly Lys Ser1 5 10 153815PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Gly
Gly Gly Lys Ser Gly Gly Lys Gly Ser Gly Lys Gly Gly Ser1 5 10
153915PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly Lys
Pro Gly Ser1 5 10 154020PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 40Gly Lys Pro Gly Ser Gly Lys
Pro Gly Ser Gly Lys Pro Gly Ser Gly1 5 10 15Lys Pro Gly Ser
204120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Gly Lys Gly Lys Ser Gly Lys Gly Lys Ser Gly Lys
Gly Lys Ser Gly1 5 10 15Lys Gly Lys Ser 204220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 42Gly
Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser
204314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Ser Thr Ala Gly Asp Thr His Leu Gly Gly Glu Asp
Phe Asp1 5 104415PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 44Gly Glu Gly Gly Ser Gly Glu Gly Gly
Ser Gly Glu Gly Gly Ser1 5 10 154515PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Gly
Gly Glu Gly Ser Gly Gly Glu Gly Ser Gly Gly Glu Gly Ser1 5 10
154615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Gly Gly Gly Glu Ser Gly Gly Gly Glu Ser Gly Gly
Gly Glu Ser1 5 10 154715PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 47Gly Glu Gly Glu Ser Gly Glu
Gly Glu Ser Gly Glu Gly Glu Ser1 5 10 154815PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 48Gly
Gly Gly Glu Ser Gly Gly Glu Gly Ser Gly Glu Gly Gly Ser1 5 10
154920PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Gly Glu Gly Glu Ser Gly Glu Gly Glu Ser Gly Glu
Gly Glu Ser Gly1 5 10 15Glu Gly Glu Ser 205015PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 50Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10
155120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser 205218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Gly
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr1 5 10
15Lys Gly5319PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 53Pro Arg Gly Ala Ser Lys Ser Gly Ser
Ala Ser Gln Thr Gly Ser Ala1 5 10 15Pro Gly Ser5419PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Gly
Thr Ala Ala Ala Gly Ala Gly Ala Ala Gly Gly Ala Ala Ala Gly1 5 10
15Ala Ala Gly5519PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 55Gly Thr Ser Gly Ser Ser Gly Ser Gly
Ser Gly Gly Ser Gly Ser Gly1 5 10 15Gly Gly Gly5620PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 56Gly
Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly1 5 10
15Lys Pro Gly Ser 2057329PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 57Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 115 120 125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 130 135 140Val Val Asp Val Lys His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr145 150 155 160Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 180 185 190Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200
205Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
210 215 220Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Gln Met225 230 235 240Thr Lys Asn Gln Val Lys Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280 285Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 290 295 300Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln305 310 315
320Lys Ser Leu Ser Leu Ser Pro Gly Lys 32558231PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
58Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala1
5 10 15Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys 20 25 30Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 35 40 45Asp Val Lys His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp 50 55 60Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Glu Tyr65 70 75 80Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp 85 90 95Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu 100 105 110Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 130 135 140Asn Gln Val
Ser Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp145 150 155
160Ile Ala Val Glu Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys
165 170 175Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 180 185 190Lys Leu Thr Val Asp Lys Ser Arg Trp Glu Gln Gly
Asp Val Phe Ser 195 200 205Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser 210 215 220Leu Ser Leu Ser Pro Gly Lys225
23059329PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 59Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asp Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120
125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
130 135 140Val Val Asp Val Lys His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr145 150 155 160Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 165 170 175Glu Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met225 230 235
240Thr Lys Asn Gln Val Ser Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro
245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser Asp Gly Gln Pro Glu
Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 275 280 285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Glu Gln Gly Asp Val 290 295 300Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln305 310 315 320Lys Ser Leu Ser Leu
Ser Pro Gly Lys 32560231PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 60Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala1 5 10 15Pro Pro Val Ala Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45Asp Val Lys
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr65 70 75
80Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
85 90 95Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu 100 105 110Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 115 120 125Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Gln Met Thr Lys 130 135 140Asn Gln Val Lys Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp145 150 155 160Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200
205Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
210 215 220Leu Ser Leu Ser Pro Gly Lys225 23061329PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
61Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asp Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120 125Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135 140Val Val Asp
Val Lys His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr145 150 155
160Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
165 170 175Glu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met225 230 235 240Thr Lys Asn Gln Val
Ser Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro 245 250 255Ser Asp Ile
Ala Val Glu Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn 260 265 270Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280
285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Glu Gln Gly Asp Val
290 295 300Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln305 310 315 320Lys Ser Leu Ser Leu Ser Pro Gly Lys
32562329PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 62Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Arg
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Arg Cys 100 105 110Pro Ala
Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120
125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
130 135 140Val Val Asp Val Lys His Glu Asp Pro Glu Val Lys Phe Lys
Trp Tyr145 150 155 160Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Gln Met225 230 235
240Thr Lys Asn Gln Val Lys Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 275 280 285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val 290 295 300Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln305 310 315 320Lys Ser Leu Ser Leu
Ser Pro Gly Lys 32563107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 63Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75
80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10564106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 64Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser1 5 10 15Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp 20 25 30Phe Tyr Pro Gly Ala Val Thr Val Ala
Trp Lys Ala Asp Ser Ser Pro 35 40 45Val Lys Ala Gly Val Glu Thr Thr
Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60Lys Tyr Ala Ala Ser Ser Tyr
Leu Ser Leu Thr Pro
Glu Gln Trp Lys65 70 75 80Ser His Arg Ser Tyr Ser Cys Gln Val Thr
His Glu Gly Ser Thr Val 85 90 95Glu Lys Thr Val Ala Pro Thr Glu Cys
Ser 100 10565121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 65Glu Val Lys Leu Glu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Met Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30Trp Met Asp Trp Val Arg
Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile Arg Thr
Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75 80Val Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95Tyr
Cys Thr Arg Leu Ala Asn Trp Asp Trp Tyr Phe Asp Val Trp Gly 100 105
110Ala Gly Thr Thr Val Thr Val Ser Ser 115 120665PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 66Asp
Ala Trp Met Asp1 56719PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 67Glu Ile Arg Thr Lys Ala Asn
Asn His Ala Thr Tyr Tyr Ala Glu Ser1 5 10 15Val Lys
Gly6810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 68Leu Ala Asn Trp Asp Trp Tyr Phe Asp Val1 5
1069111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 69Asp Thr Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser
Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser
Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Leu Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu Glu Glu
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro Phe
Thr Phe Gly Ser Gly Thr Lys Leu Glu Val Lys 100 105
1107015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 70Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser
Tyr Met Asn1 5 10 15717PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 71Ala Ala Ser Asn Leu Glu
Ser1 5729PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 72Gln Gln Ser Asn Glu Asp Pro Phe Thr1
573121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 73Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asp Ala 20 25 30Trp Met Asp Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile Arg Thr Lys Ala Asn
Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75 80Val Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Arg
Leu Ala Asn Trp Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120745PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 74Asp
Ala Trp Met Asp1 57519PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 75Glu Ile Arg Thr Lys Ala Asn
Asn His Ala Thr Tyr Tyr Ala Glu Ser1 5 10 15Val Lys
Gly7610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 76Leu Ala Asn Trp Asp Trp Tyr Phe Asp Val1 5
1077111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 77Asp Thr Val Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser
Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Leu Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro Phe
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105
1107815PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 78Arg Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser
Tyr Met Asn1 5 10 15797PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 79Ala Ala Ser Asn Leu Glu
Ser1 5809PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 80Gln Gln Ser Asn Glu Asp Pro Phe Thr1
581121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 81Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asp Asp Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile Ser Thr Lys Ala Asn
Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75 80Val Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Arg
Leu Ala Thr Trp Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120825PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 82Asp
Ala Trp Met Ser1 58319PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 83Glu Ile Ser Thr Lys Ala Asn
Asn His Ala Thr Tyr Tyr Ala Glu Ser1 5 10 15Val Lys
Gly8410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 84Leu Ala Thr Trp Asp Trp Tyr Phe Asp Val1 5
1085111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 85Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser
Glu Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro Phe
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105
1108615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 86Arg Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser
Tyr Met Asn1 5 10 15877PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 87Ala Ala Ser Glu Leu Glu
Ser1 5889PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 88Gln Gln Ser Asn Glu Asp Pro Phe Thr1
589118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 89Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Ser Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly
Phe Asn Ile Lys Asp Tyr 20 25 30Tyr Met His Trp Val Lys Gln Arg Pro
Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Asp Pro Glu Asn Gly
Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Met Thr
Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Ser Ser
Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Tyr Ala Arg Gly
Val Arg Gln Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110Ser Val
Thr Val Ser Ser 115905PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 90Asp Tyr Tyr Met His1
59117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 91Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala
Pro Lys Phe Gln1 5 10 15Gly929PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 92Arg Gly Val Arg Gln Ala Leu
Asp Tyr1 593107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 93Asp Ile Gln Met Thr Gln Thr Thr
Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys
Arg Ala Ser Gln Asp Ile Gly Asn Tyr 20 25 30Leu Asn Trp Phe Gln Gln
Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp
Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 1059411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Arg
Ala Ser Gln Asp Ile Gly Asn Tyr Leu Asn1 5 10957PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 95Tyr
Thr Ser Arg Leu His Ser1 5969PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 96Gln Gln Gly Asn Thr Leu Pro
Tyr Thr1 597118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 97Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys
Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30Tyr Met His Trp Val Gln
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro
Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Tyr
Ala Arg Gly Val Arg Gln Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 115985PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 98Asp Tyr Tyr Met His1
59917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 99Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala
Pro Lys Phe Gln1 5 10 15Gly1009PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 100Arg Gly Val Arg Gln Ala
Leu Asp Tyr1 5101107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 101Asp Ile Gln Met Thr Gln Ser Pro
Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys
Gln Ala Ser Gln Asp Ile Gly Asn Tyr 20 25 30Leu Asn Trp Phe Gln Gln
Lys Pro Asp Gln Thr Val Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala65 70 75 80Glu Asp
Ala Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10510211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 102Gln
Ala Ser Gln Asp Ile Gly Asn Tyr Leu Asn1 5 101037PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 103Tyr
Thr Ser Arg Leu His Ser1 51049PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 104Gln Gln Gly Asn Thr Leu
Pro Tyr Thr1 5105118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 105Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys
Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30Phe Met His Trp Val Gln
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro
Glu Leu Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Tyr
Ala Arg Gly Val Tyr Gln Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 1151065PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 106Asp Tyr Phe Met His1
510717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 107Trp Ile Asp Pro Glu Leu Gly Asp Thr Glu Tyr
Ala Pro Lys Phe Gln1 5 10 15Gly1089PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 108Arg
Gly Val Tyr Gln Ala Leu Asp Tyr1 5109107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
109Asp Ile Gln Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1
5 10 15Glu Lys Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn
Tyr 20 25 30Leu Asn Trp Phe Gln Gln Lys Pro Gly Gln Thr Val Lys Leu
Leu Ile 35 40 45Tyr Phe Thr Ser Tyr Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser
Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln
Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 10511011PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 110Gln Ala Ser Gln Asp Ile Gly Asn Tyr
Leu Asn1 5 101117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 111Phe Thr Ser Tyr Leu His Ser1
51129PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 112Gln Gln Gly Asn Thr Leu Pro Tyr Thr1
5113120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 113Gln Val Gln Leu Gln Gln Trp Gly Ala Gly
Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr
Gly Gly Ser Phe Ser Asp Tyr 20 25 30Tyr Trp Asn Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn His Arg Gly
Ser Thr Asn Ser Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Leu Ser
Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Arg Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Phe Gly Tyr
Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln 100 105 110Gly
Thr Leu Val Thr Val Ser Ser 115 1201145PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 114Asp
Tyr Tyr Trp Asn1 511516PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 115Glu Ile Asn His Arg Gly
Ser Thr Asn Ser Asn Pro Ser Leu Lys Ser1 5 10 1511612PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 116Gly
Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro1 5
10117107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 117Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala
Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95Thr Phe Gly
Gln Gly Thr Asn Leu Glu Ile Lys 100 10511811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 118Arg
Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala1 5 101197PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 119Asp
Ala Ser Asn Arg Ala Thr1 51209PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 120Gln Gln Arg Ser Asn Trp
Pro Leu Thr1 5121116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 121Gln Val Gln Leu Lys Glu Ser Gly
Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr
Val Ser Gly Phe Ser Leu Thr Ala Tyr 20 25 30Gly Val Asn Trp Val Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Met Ile Trp Asp
Asp Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys 50 55 60Ser Arg Leu Ser
Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65 70 75 80Lys Met
Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala 85 90 95Arg
Glu Gly Asp Val Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105
110Thr Val Ser Ser 1151225PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 122Ala Tyr Gly Val Asn1
512316PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 123Met Ile Trp Asp Asp Gly Ser Thr Asp Tyr Asn
Ser Ala Leu Lys Ser1 5 10 151248PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 124Glu Gly Asp Val Ala Phe
Asp Tyr1 5125114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 125Asp Ile Val Met Thr Gln Ser Pro
Ser Ser Leu Ala Val Ser Val Gly1 5 10 15Gln Lys Val Thr Met Ser Cys
Lys Ser Ser Gln Ser Leu Leu Asn Gly 20 25 30Ser Asn Gln Lys Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu
Val Tyr Phe Ala Ser Thr Arg Asp Ser Gly Val 50 55 60Pro Asp Arg Phe
Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser
Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln 85 90 95His
Phe Gly Thr Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105
110Lys Arg12617PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 126Lys Ser Ser Gln Ser Leu Leu Asn Gly
Ser Asn Gln Lys Asn Tyr Leu1 5 10 15Ala1277PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 127Phe
Ala Ser Thr Arg Asp Ser1 51289PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 128Leu Gln His Phe Gly Thr
Pro Pro Thr1 5129123PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 129Gln Val Thr Leu Lys Glu Ser Gly
Pro Gly Ile Leu Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Gly Leu Gly Trp
Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu Thr His Ile
Trp Trp Asp Asp Ile Lys Arg Tyr Asn Pro Asp 50 55 60Leu Arg Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Ser Ser Gln Ile65 70 75 80Phe Leu
Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys
Ala Arg Ile Val Glu Gly Ser Tyr Ser Ser Ser Tyr Phe Asp Val 100 105
110Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115
1201307PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 130Thr Ser Gly Met Gly Leu Gly1
513116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 131His Ile Trp Trp Asp Asp Ile Lys Arg Tyr Asn
Pro Asp Leu Arg Ser1 5 10 1513213PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 132Ile Val Glu Gly Ser Tyr
Ser Ser Ser Tyr Phe Asp Val1 5 10133107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
133Asp Ile Val Met Thr Gln Pro His Lys Phe Met Ser Thr Ser Val Glu1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ile Phe
Asp 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Ser Arg Val Ser Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser Val Gln Ala65 70 75 80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
His Tyr Ser Thr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Thr Leu Glu
Ile Lys 100 10513411PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 134Lys Ala Ser Gln Asp Val Ile Phe Asp
Val Ala1 5 101357PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 135Ser Ala Ser Ser Arg Val Ser1
51369PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 136Gln Gln His Tyr Ser Thr Pro Tyr Thr1
5137123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 137Gln Val Thr Leu Lys Glu Ser Gly Pro Gly
Ile Leu Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser Phe Ser
Gly Phe Ser Leu Asn Thr Ser 20 25 30Gly Met Gly Val Gly Trp Ile Arg
Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu Thr His Ile Trp Trp
Asp Asp Val Lys Arg Tyr Asn Pro Ala 50 55 60Leu Lys Ser Arg Leu Thr
Ile Ser Lys Asp Thr Ser Ser Ser Gln Val65 70 75 80Phe Leu Lys Ile
Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg
Ile Glu Gly Asp Thr Tyr Tyr Asp Tyr Tyr Phe Asp Tyr 100 105 110Trp
Gly Gln Gly Val Thr Leu Thr Val Ser Ser 115 1201387PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 138Thr
Ser Gly Met Gly Val Gly1 513916PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 139His Ile Trp Trp Asp Asp
Val Lys Arg Tyr Asn Pro Ala Leu Lys Ser1 5 10 1514013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 140Ile
Glu Gly Asp Thr Tyr Tyr Asp Tyr Tyr Phe Asp Tyr1 5
10141107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 141Asp Ile Val Met Thr Gln Ser His Lys Leu
Met Ser Thr Ser Val Gly1 5 10 15Asp Gly Leu Ser Ile Thr Cys Lys Ala
Ser Gln Asp Val Ser Ile Ala 20 25 30Val Val Trp Tyr Gln Gln Lys Pro
Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Arg Tyr
Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp Leu Ala
Val Tyr Tyr Cys Gln Gln His Tyr Ser Ile Pro Trp 85 90 95Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 10514211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 142Lys
Ala Ser Gln Asp Val Ser Ile Ala Val Val1 5 101437PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 143Ser
Ala Ser Phe Arg Tyr Thr1 51449PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 144Gln Gln His Tyr Ser Ile
Pro Trp Thr1 5145123PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 145Gln Ile Thr Leu Lys Glu Ser Gly
Pro Thr Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Gly Leu Gly Trp
Ile Arg Gln Pro Pro Gly Lys Thr Leu Glu 35 40 45Trp Leu Thr His Ile
Trp Trp Asp Asp Ile Lys Arg Tyr Asn Pro Asp 50 55 60Leu Arg Ser Arg
Leu Ser Ile Thr Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu
Thr Met Thr Asn Met Asp Pro Leu Asp Thr Gly Thr Tyr Tyr 85 90 95Cys
Ala Arg Ile Val Glu Gly Ser Tyr Ser Ser Ser Tyr Phe Asp Val 100 105
110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
1201467PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 146Thr Ser Gly Met Gly Leu Gly1
514716PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 147His Ile Trp Trp Asp Asp Ile Lys Arg Tyr Asn
Pro Asp Leu Arg Ser1 5 10 1514813PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 148Ile Val Glu Gly Ser Tyr
Ser Ser Ser Tyr Phe Asp Val1 5 10149107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
149Asp Ile Val Met Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ile Phe
Asp 20 25 30Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Ser Arg Val Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
His Tyr Ser Thr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Asp
Ile Lys 100 10515011PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 150Lys Ala Ser Gln Asp Val Ile Phe Asp
Val Ala1 5 101517PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 151Ser Ala Ser Ser Arg Val Ser1
51529PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 152Gln Gln His Tyr Ser Thr Pro Tyr Thr1
5153116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 153Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Phe Ser Leu Thr Ala Tyr 20 25 30Gly Val Asn Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Met Ile Trp Asp Asp Gly
Ser Thr Asp Tyr Asp Ser Ala Leu Lys 50 55 60Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Glu Gly
Asp Val Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr
Val Ser Ser 1151545PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 154Ala Tyr Gly Val Asn1
515516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 155Met Ile Trp Asp Asp Gly Ser Thr Asp Tyr Asp
Ser Ala Leu Lys Ser1 5 10 151568PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 156Glu Gly Asp Val Ala Phe
Asp Tyr1 5157114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 157Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ser Ser Gln Ser Leu Leu Asn Pro 20 25 30Ser Asn Gln Lys Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45Ala Pro Lys Leu Leu
Val Tyr Phe Ala Ser Thr Arg Asp Ser Gly Val 50 55 60Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 85 90 95His
Phe Gly Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105
110Lys Arg15817PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 158Lys Ser Ser Gln Ser Leu Leu Asn Pro
Ser Asn Gln Lys Asn Tyr Leu1 5 10 15Ala1597PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 159Phe
Ala Ser Thr Arg Asp Ser1 51609PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 160Leu Gln His Phe Gly Thr
Pro Pro Thr1 5161119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 161Gln Met Gln Leu Val Gln Ser Gly
Pro Glu Val Lys Lys Pro Gly Thr1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Asn Val Asp Trp Val Arg
Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile 35 40 45Gly Asp Ile Asn Pro
Asn Asn Gly Gly Thr Ile Tyr Ala Gln Lys Phe 50 55 60Gln Glu Arg Val
Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asn Tyr Arg Trp Phe Gly Ala Met Asp His Trp Gly Gln Gly 100 105
110Thr Thr Val Thr Val Ser Ser 1151625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 162Asp
Tyr Asn Val Asp1 516317PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 163Asp Ile Asn Pro Asn Asn
Gly Gly Thr Ile Tyr Ala Gln Lys Phe Gln1 5 10
15Glu16410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 164Asn Tyr Arg Trp Phe Gly Ala Met Asp His1 5
10165111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 165Asp Ile Val Met Thr Gln Thr Pro Leu Ser
Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ala
Ser Gln Ser Leu Asp Tyr Glu 20 25 30Gly Asp Ser Asp Met Asn Trp Tyr
Leu Gln Lys Pro Gly Gln Pro Pro 35 40 45Gln Leu Leu Ile Tyr Gly Ala
Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser65 70 75 80Arg Val Glu Ala
Glu
Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Thr 85 90 95Glu Asp Pro Arg
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11016615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 166Lys Ala Ser Gln Ser Leu Asp Tyr Glu Gly Asp
Ser Asp Met Asn1 5 10 151677PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 167Gly Ala Ser Asn Leu Glu
Ser1 51689PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 168Gln Gln Ser Thr Glu Asp Pro Arg Thr1
5169123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 169Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ile Ile Trp Tyr Asp Gly
Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Gln Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Val
Ala Thr Ser Gly Asp Phe Asp Tyr Tyr Gly Met Asp Val 100 105 110Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 1201705PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 170Ser
Tyr Gly Met His1 517117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 171Ile Ile Trp Tyr Asp Gly
Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly17214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 172Val Ala Thr Ser Gly Asp Phe Asp Tyr Tyr Gly
Met Asp Val1 5 10173107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 173Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Thr Thr
Leu Ser Cys Arg Ala Ser Gln Arg Ile Ser Thr Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Lys Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10517411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 174Arg Ala Ser Gln Arg Ile Ser Thr Tyr Leu Ala1 5
101757PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 175Asp Ala Ser Lys Arg Ala Thr1
51769PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 176Gln Gln Arg Ser Asn Trp Pro Leu Thr1
5177119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 177Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Ala
Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 1151785PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 178Ser Tyr Tyr Met His1
517917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 179Ile Ile Asn Pro Ser Ala Gly Ser Thr Ser Tyr
Ala Gln Lys Phe Gln1 5 10 15Gly18010PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 180Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr1 5 10181112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
181Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln1
5 10 15Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln Tyr Pro Gly Lys Ala Pro
Arg Leu 35 40 45Met Ile Phe Glu Val Thr Glu Arg Ala Ser Gly Val Pro
Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Val Ser Gly Leu65 70 75 80Gln Thr Glu Asp Glu Ala Val Tyr Phe Cys
Ser Ser Tyr Ser Gly Ser 85 90 95Asn Asn Pro Gly Ala Met Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 11018214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 182Thr
Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser1 5
101837PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 183Glu Val Thr Glu Arg Ala Ser1
518412PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 184Ser Ser Tyr Ser Gly Ser Asn Asn Pro Gly Ala
Met1 5 10185119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 185Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro
Ser Ala Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Glu Leu Met Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser 1151865PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 186Ser
Tyr Tyr Met His1 518717PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 187Ile Ile Asn Pro Ser Ala
Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln1 5 10
15Gly18810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 188Glu Leu Met Ala Thr Gly Gly Phe Asp Tyr1 5
10189110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 189Gln Ala Gly Leu Thr Gln Pro Ala Ser Val
Ser Gly Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Ser
Ser Ser Asp Val Gly Gly Tyr 20 25 30Ser Tyr Val Ser Trp Tyr Gln Lys
His Pro Gly Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr Asp Val Thr Asn
Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly
Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Ser Thr Tyr Thr Arg Ser 85 90 95Asn Thr Leu
Val Phe Gly Pro Gly Thr Lys Val Thr Val Leu 100 105
11019014PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 190Thr Gly Ser Ser Ser Asp Val Gly Gly Tyr Ser
Tyr Val Ser1 5 101917PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 191Asp Val Thr Asn Arg Pro
Ser1 519210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 192Ser Thr Tyr Thr Arg Ser Asn Thr Leu Val1 5
10193119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 193Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Ala
Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 1151945PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 194Ser Tyr Tyr Met His1
519517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 195Ile Ile Asn Pro Ser Ala Gly Ser Thr Ser Tyr
Ala Gln Lys Phe Gln1 5 10 15Gly19610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 196Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr1 5 10197110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
197Leu Pro Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1
5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45Met Ile Tyr Asp Val Thr Asn Arg Pro Ser Gly Val Ser
Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Ser Ser Tyr Thr Ser Ser 85 90 95Asn Thr Leu Leu Phe Gly Gly Gly Thr
Gln Leu Thr Val Leu 100 105 11019814PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 198Thr
Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser1 5
101997PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 199Asp Val Thr Asn Arg Pro Ser1
520010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 200Ser Ser Tyr Thr Ser Ser Asn Thr Leu Leu1 5
10201119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 201Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Ala
Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 1152025PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 202Ser Tyr Tyr Met His1
520317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 203Ile Ile Asn Pro Ser Ala Gly Ser Thr Ser Tyr
Ala Gln Lys Phe Gln1 5 10 15Gly20410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 204Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr1 5 10205110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
205Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1
5 10 15Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn
Asn 20 25 30Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Asp Asn Asn Lys Arg His Ser Gly Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Thr Ser Asp Thr Ser Ala Thr Leu Gly Ile
Thr Arg Leu Gln65 70 75 80Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly
Thr Trp Asp Ser Ser Leu 85 90 95Ser Ala Tyr Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu 100 105 11020613PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 206Ser
Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser1 5
102077PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 207Asp Asn Asn Lys Arg His Ser1
520811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 208Gly Thr Trp Asp Ser Ser Leu Ser Ala Tyr Val1 5
10209119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 209Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Ala
Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 1152105PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 210Ser Tyr Tyr Met His1
521117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 211Ile Ile Asn Pro Ser Ala Gly Ser Thr Ser Tyr
Ala Gln Lys Phe Gln1 5 10 15Gly21210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 212Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr1 5 10213110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
213Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln1
5 10 15Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr 20 25 30Asp Tyr Val Ser Trp Tyr Gln Gln His Gln Gly Lys Ala Pro
Lys Leu 35 40 45Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser
Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Ser Ser Tyr Thr Ser Ser 85 90 95Thr Thr Leu Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 11021414PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 214Thr
Gly Thr Ser Ser Asp Val Gly Gly Tyr Asp Tyr Val Ser1 5
102157PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 215Asp Val Ser Asn Arg Pro Ser1
521610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 216Ser Ser Tyr Thr Ser Ser Thr Thr Leu Val1 5
10217119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 217Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Ala Gly
Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Leu
Met Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 1152185PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 218Ser Tyr Tyr Met His1
521917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 219Ile Ile Asn Pro Ser Ala Gly Ser Thr Ser Tyr
Ala Gln Lys Phe Gln1 5 10 15Gly22010PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 220Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr1 5 10221110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
221Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1
5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly Ala
Tyr 20 25 30Asn Phe Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45Met Ile Tyr Gly Val Ser Asn Arg Pro Ser Gly Val Ser
Ser Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Ser Thr Ala Ser Leu Thr
Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Ser Ser Tyr Thr Thr Ser 85 90 95Gly Ser Ala Val Phe Gly Thr Gly Thr
Lys Leu Thr Val Leu 100 105 11022214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 222Thr
Gly Thr Ser Ser Asp Ile Gly Ala Tyr Asn Phe Val Ser1 5
102237PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 223Gly Val Ser Asn Arg Pro Ser1
522410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 224Ser Ser Tyr Thr Thr Ser Gly Ser Ala Val1 5
10225119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 225Glu Val Gln Leu Leu Glu Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Ala
Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 1152265PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 226Ser Tyr Tyr Met His1
522717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 227Ile Ile Asn Pro Ser Ala Gly Ser Thr Ser Tyr
Ala Gln Lys Phe Gln1 5 10 15Gly22810PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 228Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr1 5 10229110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
229Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1
5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly Ala
Tyr 20 25 30Asn Phe Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45Met Ile Tyr Gly Val Ser Asn Arg Pro Ser Gly Val Ser
Ser Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Ser Thr Ala Ser Leu Thr
Ile Thr Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Ser Ser Tyr Thr Thr Ser 85 90 95Gly Ser Ala Val Phe Gly Thr Gly Thr
Lys Leu Thr Val Leu 100 105 11023014PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 230Thr
Gly Thr Ser Ser Asp Ile Gly Ala Tyr Asn Phe Val Ser1 5
102317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 231Gly Val Ser Asn Arg Pro Ser1
523210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 232Ser Ser Tyr Thr Thr Ser Gly Ser Ala Val1 5
10233121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 233Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Val Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn His 20 25 30Ala Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly
Ser Lys Lys Phe Tyr Ser Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly
Ala His Gly Tyr Thr Ser Gly Trp His Asp Tyr Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser 115 1202345PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 234Asn
His Ala Met His1 523517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 235Val Ile Ser Tyr Asp Gly
Ser Lys Lys Phe Tyr Ser Asp Ser Val Arg1 5 10
15Gly23612PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 236Gly Ala His Gly Tyr Thr Ser Gly Trp His Asp
Tyr1 5 10237108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 237Asp Val Val Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys
Arg Ala Ser Gln Asn Ile Gly Arg Tyr 20 25 30Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45Ser Ala Ala Ser Ser
Leu Gln Gly Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Phe Cys Gln Gln Thr Tyr Ser Ser Pro Gln 85 90 95Cys
Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys 100
10523811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 238Arg Ala Ser Gln Asn Ile Gly Arg Tyr Leu Asn1 5
102397PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 239Ala Ala Ser Ser Leu Gln Gly1
524010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 240Gln Gln Thr Tyr Ser Ser Pro Gln Cys Thr1 5
10241121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 241Gln Val Gln Leu Val Gln Ser Gly Ser Glu
Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Gly
Gly Ala Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Ser Gly Tyr Asp Leu Gly Tyr Gly Met Asp Val Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser 115 1202425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 242Asn
Tyr Tyr Met His1 524317PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 243Ile Ile Asn Pro Ser Gly
Gly Ala Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10
15Gly24412PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 244Asp Ser Gly Tyr Asp Leu Gly Tyr Gly Met Asp
Val1 5 10245110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 245Gln Ser Val Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys Thr
Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr Asp Val
Ser Asn Arg Pro Ser Gly Ala Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys
Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala
Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Asn Arg 85 90 95Asn
Thr Leu Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
11024614PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 246Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn
Tyr Val Ser1 5 102477PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 247Asp Val Ser Asn Arg Pro
Ser1 524810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 248Ser Ser Tyr Thr Asn Arg Asn Thr Leu Leu1 5
10249119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 249Glu Val Gln Leu Leu Glu Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Ala
Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 1152505PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 250Ser Tyr Tyr Met His1
525117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 251Ile Ile Asn Pro Ser Ala Gly Ser Thr Ser Tyr
Ala Gln Lys Phe Gln1 5 10 15Gly25210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 252Glu
Leu Met Ala Thr Gly Gly Phe Asp Tyr1 5 10253110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
253Gln Ser Val Leu Thr Gln Pro Ala Ser Ala Ser Gly Ser Pro Gly Gln1
5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser
Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asn Tyr Tyr Cys
Ser Ser Tyr Thr Ser Ser 85 90 95Ser Thr Asn Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu 100 105 11025414PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 254Thr
Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser1 5
102557PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 255Asp Val Ser Asn Arg Pro Ser1
525610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 256Ser Ser Tyr Thr Ser Ser Ser Thr Asn Val1 5
1025766PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 257Ile Thr Cys Pro Pro Pro Met Ser Val Glu
His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu
Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser
Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His
Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg
Asp6525867PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 258Ile Thr Cys Pro Pro Pro Met Ser Val Glu
His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu
Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser
Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His
Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Asp
Pro6525968PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 259Ile Thr Cys Pro Pro Pro Met Ser Val Glu
His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu
Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser
Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His
Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Asp Pro
Ala65260114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 260Asn Trp Val Asn Val Ile Ser Asp Leu Lys
Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala Thr Leu
Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr Ala Met
Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly
Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala
Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly
Cys Lys Cys Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe
Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110Thr
Ser261114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 261Asn Trp Val Asn Val Ile Ser Asp Leu Lys
Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala Thr Leu
Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr Ala Met
Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Cys Ile Ser Leu Glu Ser Gly
Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala
Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly
Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe
Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110Thr
Ser262114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 262Asn Trp Val Asn Val Ile Ser Asp Leu Lys
Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala Thr Leu
Tyr Thr Glu Ser Asp Val His
20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu
Gln 35 40 45Val Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr
Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn
Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu
Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His Ile
Val Gln Met Phe Ile Asn 100 105 110Thr Ser263114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
263Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Cys Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser264114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
264Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Cys 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser265114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
265Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Cys Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser266114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
266Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Ser Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser267114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
267Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Cys Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser26867PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
268Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1
5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser
Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val
Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu
Lys Cys Ile 50 55 60Arg Asp Cys6526968PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
269Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1
5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser
Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val
Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu
Lys Cys Ile 50 55 60Arg Asp Pro Cys6527068PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
270Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1
5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser
Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val
Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu
Lys Cys Ile 50 55 60Arg Asp Cys Ala6527165PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
271Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1
5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser
Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu Thr Glu Cys Val
Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu
Lys Cys Ile 50 55 60Arg6527265PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 272Ile Thr Cys Pro Pro
Pro Met Ser Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser
Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Cys Arg
Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala
Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55
60Arg6527365PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 273Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Cys
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55
60Arg6527465PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 274Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Cys Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55
60Arg6527565PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 275Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Cys Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55
60Arg65276114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 276Asp Trp Val Asn Val Ile Ser Asp
Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala
Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr
Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu
Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile
Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu
Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys
Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105
110Thr Ser277114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 277Asn Trp Val Asp Val Ile Ser Asp
Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala
Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr
Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu
Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile
Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu
Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys
Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105
110Thr Ser278114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 278Asn Trp Val Asn Val Ile Ser Asn
Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala
Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr
Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu
Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile
Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu
Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys
Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105
110Thr Ser279114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 279Asn Trp Val Asn Val Ile Ser Asp
Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala
Thr Leu Tyr Thr Glu Ser Asn Val His 20 25 30Pro Ser Cys Lys Val Thr
Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu
Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile
Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu
Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys
Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105
110Thr Ser280114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 280Asn Trp Val Asn Val Ile Ser Asp
Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala
Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr
Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu
Ser Gly Asp Ala Ser Ile His Asn Thr Val Glu 50 55 60Asn Leu Ile Ile
Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu
Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys
Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105
110Thr Ser281114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 281Asn Trp Val Asn Val Ile Ser Asp
Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala
Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr
Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu
Ser Gly Asp Ala Ser Ile His Asp Thr Val Gln 50 55 60Asn Leu Ile Ile
Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu
Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys
Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105
110Thr Ser282114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 282Asn Trp Val Asn Val Ile Ser Asp
Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala
Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr
Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu
Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asp Leu Ile Ile
Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu
Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys
Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105
110Thr Ser283114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 283Asn Trp Val Asn Val Ile Ser Asp
Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala
Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr
Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu
Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile
Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu
Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys
Glu Phe Leu Gln Ser Phe Val His Ile Val Glu Met Phe Ile Asn 100 105
110Thr Ser284114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 284Asp Trp Val Asn Val Ile Ser Asp
Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala
Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr
Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu
Ser Gly Asp Ala Ser Ile His Asn Thr Val Glu 50 55 60Asn Leu Ile Ile
Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu
Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys
Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105
110Thr Ser285114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 285Asp Trp Val Asn Val Ile Ser Asp
Leu Lys Lys Ile Glu Asp Leu Ile1 5
10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Gln 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser286114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
286Asn Trp Val Asp Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asn
Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser287114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
287Asn Trp Val Asp Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Gln 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser288114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
288Asn Trp Val Asn Val Ile Ser Asn Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asn
Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser289114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
289Asn Trp Val Asn Val Ile Ser Asn Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Gln 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser290114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
290Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asn
Thr Val Gln 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser291114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
291Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Gln 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Glu Met Phe Ile Asn 100 105 110Thr Ser292114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
292Asp Trp Val Asp Val Ile Ser Asn Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser293114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
293Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asn
Thr Val Gln 50 55 60Asp Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser294114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
294Asp Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asn
Thr Val Gln 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Glu Met Phe Ile Asn 100 105 110Thr Ser295114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
295Asn Trp Val Asp Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asn
Thr Val Gln 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Glu Met Phe Ile Asn 100 105 110Thr Ser296114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
296Asp Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asp Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser297114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
297Asp Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Glu Met Phe Ile Asn 100 105 110Thr Ser298114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
298Asn Trp Val Asp Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asp Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser299114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
299Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asp Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser300114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
300Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Glu Met Phe Ile Asn 100 105 110Thr Ser301114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
301Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asp Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Glu Met Phe Ile Asn 100 105 110Thr Ser302114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
302Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Gln 50 55 60Asp Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser303114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
303Asp Trp Val Asp Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asp Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser304114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
304Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Gln 50 55 60Asp Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr
Ser305114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
305Asn Trp Val Asp Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1
5 10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asn
Thr Val Glu 50 55 60Asp Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser306435PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
306Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1
5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser
Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val
Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu
Lys Cys Ile 50 55 60Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser65 70 75 80Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asn Trp Val Asp Val Ile 85 90 95Ser Asp Leu Lys Lys Ile Glu Asp Leu
Ile Gln Ser Met His Ile Asp 100 105 110Ala Thr Leu Tyr Thr Glu Ser
Asp Val His Pro Ser Cys Lys Val Thr 115 120 125Ala Met Lys Cys Phe
Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser 130 135 140Gly Asp Ala
Ser Ile His Asp Thr Val Glu Asp Leu Ile Ile Leu Ala145 150 155
160Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys
165 170 175Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu
Gln Ser 180 185 190Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser
Glu Pro Lys Ser 195 200 205Ser Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Pro Val Ala 210 215 220Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Lys His 245 250 255Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 260 265 270His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Tyr Asn Ser Thr Tyr 275 280
285Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile305 310 315 320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 325 330 335Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 340 345 350Leu Thr Cys Asp Val Ser Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 355 360 365Trp Glu Ser Asp Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 370 375 380Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val385 390 395
400Asp Lys Ser Arg Trp Glu Gln Gly Asp Val Phe Ser Cys Ser Val Met
405 410 415His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 420 425 430Pro Gly Lys 43530765PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
307Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1
5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser
Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val
Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu
Lys Cys Ile 50 55 60Arg65308114PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 308Asn Trp Val Asp Val
Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His
Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys
Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile
Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asp
Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75
80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile
Asn 100 105 110Thr Ser309231PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 309Glu Pro Lys Ser Ser
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala1 5 10 15Pro Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45Asp Val
Lys His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Tyr65 70 75
80Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
85 90 95Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu 100 105 110Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 115 120 125Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys 130 135 140Asn Gln Val Ser Leu Thr Cys Asp Val
Ser Gly Phe Tyr Pro Ser Asp145 150 155 160Ile Ala Val Glu Trp Glu
Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190Lys Leu
Thr Val Asp Lys Ser Arg Trp Glu Gln Gly Asp Val Phe Ser 195 200
205Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
210 215 220Leu Ser Leu Ser Pro Gly Lys225 230310450PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
310Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Glu Ile Ser Thr Lys Ala Asn Asn His Ala Thr Tyr
Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Ser65 70 75 80Val Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Arg Leu Ala Thr Trp Asp
Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala Gly225 230 235 240Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Lys His Glu 260 265 270Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu
Gln Met Thr Lys Asn Gln Val Lys Leu 355 360 365Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395
400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 445Gly Lys 450311121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
311Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Glu Ile Ser Thr Lys Ala Asn Asn His Ala Thr Tyr
Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Ser65 70 75 80Val Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Arg Leu Ala Thr Trp Asp
Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val
Ser Ser 115 1203125PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 312Asp Ala Trp Met Ser1
531319PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 313Glu Ile Ser Thr Lys Ala Asn Asn His Ala Thr
Tyr Tyr Ala Glu Ser1 5 10 15Val Lys Gly31410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 314Leu
Ala Thr Trp Asp Trp Tyr Phe Asp Val1 5 10315218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
315Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr
Asp 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser Glu Leu Glu Ser Gly
Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro Phe Thr Phe Gly Ser Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150 155
160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215316111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 316Asp Ile Val Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Met Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro 35 40 45Lys Leu Leu Ile Tyr
Ala Ala Ser Glu Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu
Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105
11031715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 317Arg Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp
Ser Tyr Met Asn1 5 10 153187PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 318Ala Ala Ser Glu Leu Glu
Ser1 53199PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 319Gln Gln Ser Asn Glu Asp Pro Phe Thr1
5320435PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 320Ile Thr Cys Pro Pro Pro Met Ser Val Glu
His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu
Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser
Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His
Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75 80Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asn Trp Val Asp Val Ile 85 90 95Ser Asp Leu
Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 100 105 110Ala
Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr 115 120
125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser
130 135 140Gly Asp Ala Ser Ile His Asp Thr Val Glu Asp Leu Ile Ile
Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr
Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu Leu Glu Glu Lys Asn
Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val His Ile Val Gln Met
Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200 205Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 210 215 220Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235
240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys His
245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Tyr
Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 340 345 350Leu
Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 355
360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Glu Gln Gly Asp Val
Phe Ser Cys Ser Val Met 405 410 415His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro Gly Lys
435321447PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 321Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser
Gly Phe Asn Ile Lys Asp Tyr 20 25 30Phe Met His Trp Val Gln Gln Ala
Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Leu
Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Tyr Ala Arg
Gly Val Tyr Gln Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val225 230 235
240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Lys His Glu Asp
Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro
Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu Thr Cys Leu 355 360
365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445322214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
322Asp Ile Gln Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1
5 10 15Glu Lys Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn
Tyr 20 25 30Leu Asn Trp Phe Gln Gln Lys Pro Gly Gln Thr Val Lys Leu
Leu Ile 35 40 45Tyr Phe Thr Ser Tyr Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser
Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln
Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
210323442PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 323Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Asp Cys Lys Ala Ser
Gly Ile Thr Phe Ser Asn Ser 20 25 30Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly
Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Asn
Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120
125Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr145 150 155 160Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln 180 185 190Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220Cys Pro Ala
Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro225 230 235
240Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255Val Val Val Asp Val Lys His Glu Asp Pro Glu Val Lys Phe
Asn Trp 260 265 270Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 275 280 285Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 290 295 300His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn305 310 315 320Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360
365Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe385 390 395 400Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn 405 410 415Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr 420 425 430Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 440324214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 324Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210325435PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
325Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1
5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser
Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val
Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu
Lys Cys Ile 50 55 60Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser65 70 75 80Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asn Trp Val Asp Val Ile 85 90 95Ser Asp Leu Lys Lys Ile Glu Asp Leu
Ile Gln Ser Met His Ile Asp 100 105 110Ala Thr Leu Tyr Thr Glu Ser
Asp Val His Pro Ser Cys Lys Val Thr 115 120 125Ala Met Lys Cys Phe
Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser 130 135 140Gly Asp Ala
Ser Ile His Asp Thr Val Glu Asp Leu Ile Ile Leu Ala145 150 155
160Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys
165 170 175Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu
Gln Ser 180 185 190Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser
Glu Pro Lys Ser 195 200 205Ser Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Pro Val Ala 210 215 220Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Lys His 245 250 255Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 260 265 270His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Tyr Asn Ser Thr Tyr 275 280
285Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile305 310 315 320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 325 330 335Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 340 345 350Leu Thr Cys Asp Val Ser Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 355 360 365Trp Glu Ser Asp Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 370 375 380Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val385 390 395
400Asp Lys Ser Arg Trp Glu Gln Gly Asp Val Phe Ser Cys Ser Val Leu
405 410 415His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 420 425 430Pro Gly Lys 435326450PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
326Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Glu Ile Ser Thr Lys Ala Asn Asn His Ala Thr Tyr
Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Ser65 70 75 80Val Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Arg Leu Ala Thr Trp Asp
Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala Gly225 230 235 240Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Lys His Glu 260 265 270Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu
Gln Met Thr Lys Asn Gln Val Lys Leu 355 360 365Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395
400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Leu His 420 425 430Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 445Gly Lys 450327218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
327Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr
Asp 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser Glu Leu Glu Ser Gly
Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro Phe Thr Phe Gly Ser Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro
Ser Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215328435PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 328Ile Thr Cys Pro Pro Pro Met Ser Val Glu
His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu
Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser
Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His
Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75 80Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asn Trp Val Asp Val Ile 85 90 95Ser Asp Leu
Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 100 105 110Ala
Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr 115 120
125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser
130 135 140Gly Asp Ala Ser Ile His Asp Thr Val Glu Asp Leu Ile Ile
Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr
Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu Leu Glu Glu Lys Asn
Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val His Ile Val Gln Met
Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200 205Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 210 215 220Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235
240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys His
245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Tyr
Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 340 345 350Leu
Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 355 360
365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val385 390 395 400Asp Lys Ser Arg Trp Glu Gln Gly Asp Val Phe
Ser Cys Ser Val Leu 405 410 415His Glu Ala Leu His Ser His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro Gly Lys
435329447PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 329Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser
Gly Phe Asn Ile Lys Asp Tyr 20 25 30Phe Met His Trp Val Gln Gln Ala
Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Leu
Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Tyr Ala Arg
Gly Val Tyr Gln Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val225 230 235
240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Lys His Glu Asp
Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro
Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu Thr Cys Leu 355 360
365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Leu His Glu Ala Leu 420 425 430His Ser His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445330214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
330Asp Ile Gln Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1
5 10 15Glu Lys Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn
Tyr 20 25 30Leu Asn Trp Phe Gln Gln Lys Pro Gly Gln Thr Val Lys Leu
Leu Ile 35 40 45Tyr Phe Thr Ser Tyr Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser
Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln
Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
210331350PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 331Asn Trp Val Asn Val Ile Ser Asp Leu Lys
Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala Thr Leu
Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr Ala Met
Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly
Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala
Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly
Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe
Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110Thr
Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His 115 120
125Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
130 135 140Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro145 150 155 160Glu Val Thr Cys Val Val Val Asp Val Lys His
Glu Asp Pro Glu Val 165 170 175Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr 180 185 190Lys Pro Arg Glu Glu Glu Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val 195 200 205Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 210 215 220Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser225 230 235
240Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
245 250 255Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Asp Val 260 265 270Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asp Gly 275 280 285Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp 290 295 300Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp305 310 315 320Glu Gln Gly Asp Val
Phe Ser Cys Ser Val Leu His Glu Ala Leu His 325 330 335Ser His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345
350332301PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 332Ile Thr Cys Pro Pro Pro Met Ser Val Glu
His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu
Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser
Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His
Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly Gly Ser
Glu Pro Lys Ser Ser Asp Lys Thr His Thr65 70 75 80Cys Pro Pro Cys
Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 85 90 95Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 100 105 110Val
Thr Cys Val Val Val Asp Val Lys His Glu Asp Pro Glu Val Lys 115 120
125Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
130 135 140Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu145 150 155 160Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys 165 170 175Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys 180 185 190Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser 195 200 205Arg Glu Gln Met Thr
Lys Asn Gln Val Lys Leu Thr Cys Leu Val Lys 210 215 220Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln225 230 235
240Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
245 250 255Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln 260 265 270Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu
Ala Leu His Ser 275 280 285His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 290 295 300333350PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 333Asn Trp Val Asp Val
Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His
Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys
Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile
Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asp
Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75
80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile
Asn 100 105 110Thr Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp
Lys Thr His 115 120 125Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
Gly Pro Ser Val Phe 130 135 140Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro145 150 155 160Glu Val Thr Cys Val Val
Val Asp Val Lys His Glu Asp Pro Glu Val 165 170 175Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 180 185 190Lys Pro
Arg Glu Glu Glu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 195 200
205Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
210 215 220Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser225 230 235 240Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro 245 250 255Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Asp Val 260 265 270Ser Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asp Gly 275 280 285Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 290 295 300Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp305 310 315
320Glu Gln Gly Asp Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His
325 330 335Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350334301PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 334Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly
Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr65 70 75 80Cys Pro
Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 85 90 95Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 100 105
110Val Thr Cys Val Val Val Asp Val Lys His Glu Asp Pro Glu Val Lys
115 120 125Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 130 135 140Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu145 150 155 160Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys 165 170 175Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys
180 185 190Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser 195 200 205Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu Thr
Cys Leu Val Lys 210 215 220Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln225 230 235 240Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 245 250 255Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 260 265 270Gln Gly Asn
Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser 275 280 285His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 290 295
300335435PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 335Ile Thr Cys Pro Pro Pro Met Ser Val Glu
His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu
Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser
Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His
Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75 80Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asn Trp Val Asp Val Ile 85 90 95Ser Asp Leu
Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 100 105 110Ala
Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr 115 120
125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser
130 135 140Gly Asp Ala Ser Ile His Asp Thr Val Glu Asp Leu Ile Ile
Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr
Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu Leu Glu Glu Lys Asn
Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val His Ile Val Gln Met
Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200 205Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 210 215 220Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235
240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys His
245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Tyr
Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 340 345 350Leu
Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 355 360
365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val385 390 395 400Asp Lys Ser Arg Trp Glu Gln Gly Asp Val Phe
Ser Cys Ser Val Leu 405 410 415His Glu Ala Leu His Ser His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro Gly Lys
435336231PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 336Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala1 5 10 15Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 20 25 30Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 35 40 45Asp Val Lys His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr65 70 75 80Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105 110Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120
125Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Gln Met Thr Lys
130 135 140Asn Gln Val Lys Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp145 150 155 160Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 165 170 175Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 180 185 190Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205Cys Ser Val Leu His
Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser 210 215 220Leu Ser Leu
Ser Pro Gly Lys225 230337350PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 337Asn Trp Val Asn Val
Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His
Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val His 20 25 30Pro Ser Cys
Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile
Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Gln 50 55 60Asp
Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75
80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile
Asn 100 105 110Thr Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp
Lys Thr His 115 120 125Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
Gly Pro Ser Val Phe 130 135 140Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro145 150 155 160Glu Val Thr Cys Val Val
Val Asp Val Lys His Glu Asp Pro Glu Val 165 170 175Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 180 185 190Lys Pro
Arg Glu Glu Glu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 195 200
205Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
210 215 220Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser225 230 235 240Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro 245 250 255Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Asp Val 260 265 270Ser Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asp Gly 275 280 285Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 290 295 300Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp305 310 315
320Glu Gln Gly Asp Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His
325 330 335Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350338301PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 338Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly
Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr65 70 75 80Cys Pro
Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 85 90 95Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 100 105
110Val Thr Cys Val Val Val Asp Val Lys His Glu Asp Pro Glu Val Lys
115 120 125Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 130 135 140Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu145 150 155 160Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys 165 170 175Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 180 185 190Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 195 200 205Arg Glu Gln
Met Thr Lys Asn Gln Val Lys Leu Thr Cys Leu Val Lys 210 215 220Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln225 230
235 240Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly 245 250 255Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 260 265 270Gln Gly Asn Val Phe Ser Cys Ser Val Leu His
Glu Ala Leu His Ser 275 280 285His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 290 295 300339435PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 339Ile Thr Cys Pro Pro
Pro Met Ser Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser
Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg
Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala
Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75
80Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile
85 90 95Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile
Asp 100 105 110Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys
Lys Val Thr 115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val
Ile Ser Leu Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val
Glu Asn Leu Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val
His Ile Val Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200
205Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Lys His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Glu Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315
320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
325 330 335Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 340 345 350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp
Glu Gln Gly Asp Val Phe Ser Cys Ser Val Met 405 410 415His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro
Gly Lys 435340231PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 340Glu Pro Lys Ser Ser Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala1 5 10 15Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45Asp Val Lys His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr65 70 75 80Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105
110Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
115 120 125Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Gln Met
Thr Lys 130 135 140Asn Gln Val Lys Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp145 150 155 160Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 165 170 175Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220Leu
Ser Leu Ser Pro Gly Lys225 230341435PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
341Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1
5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser
Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val
Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu
Lys Cys Ile 50 55 60Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser65 70 75 80Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asn Trp Val Asp Val Ile 85 90 95Ser Asp Leu Lys Lys Ile Glu Asp Leu
Ile Gln Ser Met His Ile Asp 100 105 110Ala Thr Leu Tyr Thr Glu Ser
Asp Val His Pro Ser Cys Lys Val Thr 115 120 125Ala Met Lys Cys Phe
Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser 130 135 140Gly Asp Ala
Ser Ile His Asp Thr Val Glu Asp Leu Ile Ile Leu Ala145 150 155
160Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys
165 170 175Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu
Gln Ser 180 185 190Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser
Glu Pro Lys Ser 195 200 205Ser Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Pro Val Ala 210 215 220Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Lys His 245 250 255Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 260 265 270His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Tyr Asn Ser Thr Tyr 275
280 285Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser 340 345 350Leu Thr Cys Asp Val Ser
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 355 360 365Trp Glu Ser Asp
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 370 375 380Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val385 390 395
400Asp Lys Ser Arg Trp Glu Gln Gly Asp Val Phe Ser Cys Ser Val Met
405 410 415His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 420 425 430Pro Gly Lys 435342231PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
342Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala1
5 10 15Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys 20 25 30Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 35 40 45Asp Val Lys His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp 50 55 60Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr65 70 75 80Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp 85 90 95Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu 100 105 110Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Gln Met Thr Lys 130 135 140Asn Gln Val
Lys Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp145 150 155
160Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
165 170 175Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 180 185 190Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser 195 200 205Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser 210 215 220Leu Ser Leu Ser Pro Gly Lys225
230343435PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 343Ile Thr Cys Pro Pro Pro Met Ser Val Glu
His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu
Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser
Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His
Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75 80Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile 85 90 95Ser Asp Leu
Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 100 105 110Ala
Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys Lys Val Thr 115 120
125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser
130 135 140Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile
Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr
Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu Leu Glu Glu Lys Asn
Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val His Ile Val Gln Met
Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200 205Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 210 215 220Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235
240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys His
245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Tyr
Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 340 345 350Leu
Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 355 360
365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val385 390 395 400Asp Lys Ser Arg Trp Glu Gln Gly Asp Val Phe
Ser Cys Ser Val Met 405 410 415His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro Gly Lys
435344231PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 344Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala1 5 10 15Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 20 25 30Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 35 40 45Asp Val Lys His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr65 70 75 80Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105 110Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120
125Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Gln Met Thr Lys
130 135 140Asn Gln Val Lys Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp145 150 155 160Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 165 170 175Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 180 185 190Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220Leu Ser Leu
Ser Pro Gly Lys225 230345435PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 345Ile Thr Cys Pro Pro
Pro Met Ser Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser
Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg
Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala
Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75
80Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile
85 90 95Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile
Asp 100 105 110Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys
Lys Val Thr 115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val
Ile Ser Leu Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val
Glu Asp Leu Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val
His Ile Val Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200
205Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Lys His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Glu Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315
320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
325 330 335Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 340 345 350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp
Glu Gln Gly Asp Val Phe Ser Cys Ser Val Met 405 410 415His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro
Gly Lys 435346231PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 346Glu Pro Lys Ser Ser Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala1 5 10 15Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45Asp Val Lys His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr65 70 75 80Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105
110Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
115 120 125Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Gln Met
Thr Lys 130 135 140Asn Gln Val Lys Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp145 150 155 160Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 165 170 175Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220Leu
Ser Leu Ser Pro Gly Lys225 230347435PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
347Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1
5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser
Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val
Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu
Lys Cys Ile 50 55 60Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser65 70 75 80Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asn Trp Val Asn Val Ile 85 90 95Ser Asp Leu Lys Lys Ile Glu Asp Leu
Ile Gln Ser Met His Ile Asp 100 105 110Ala Thr Leu Tyr Thr Glu Ser
Asn Val His Pro Ser Cys Lys Val Thr 115 120 125Ala Met Lys Cys Phe
Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser 130 135 140Gly Asp Ala
Ser Ile His Asp Thr Val Gln Asp Leu Ile Ile Leu Ala145 150 155
160Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys
165 170 175Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu
Gln Ser 180 185 190Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser
Glu Pro Lys Ser 195 200 205Ser Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Pro Val Ala 210 215 220Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met225 230 235 240Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Lys His 245 250 255Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 260 265 270His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Tyr Asn Ser Thr Tyr 275 280
285Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
290 295 300Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile305 310 315 320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 325 330 335Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 340 345 350Leu Thr Cys Asp Val Ser Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 355 360 365Trp Glu Ser Asp Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 370 375 380Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val385 390 395
400Asp Lys Ser Arg Trp Glu Gln Gly Asp Val Phe Ser Cys Ser Val Met
405 410 415His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 420 425 430Pro Gly Lys 435348231PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
348Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala1
5 10 15Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys 20 25 30Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 35 40 45Asp Val Lys His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp 50 55 60Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr65 70 75 80Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp 85 90 95Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu 100 105 110Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Gln Met Thr Lys 130 135 140Asn Gln Val
Lys Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp145 150 155
160Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
165 170 175Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 180 185 190Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser 195 200 205Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser 210 215 220Leu Ser Leu Ser Pro Gly Lys225
230349435PRTArtificial SequenceDescription of Artificial
Sequence
Synthetic polypeptide 349Ile Thr Cys Pro Pro Pro Met Ser Val Glu
His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu
Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser
Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His
Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75 80Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile 85 90 95Ser Asp Leu
Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 100 105 110Ala
Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys Lys Val Thr 115 120
125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser
130 135 140Gly Asp Ala Ser Ile His Asp Thr Val Glu Asp Leu Ile Ile
Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr
Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu Leu Glu Glu Lys Asn
Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val His Ile Val Gln Met
Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200 205Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 210 215 220Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230 235
240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys His
245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu Tyr
Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 340 345 350Leu
Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 355 360
365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val385 390 395 400Asp Lys Ser Arg Trp Glu Gln Gly Asp Val Phe
Ser Cys Ser Val Met 405 410 415His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro Gly Lys
435350450PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 350Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asp Asp Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile Ser Thr Lys Ala
Asn Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75 80Val Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr
Arg Leu Ala Thr Trp Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys
His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr
Leu Pro Pro Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu 355 360
365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Lys
450351218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 351Asp Ile Val Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala
Ser Glu Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro
Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120
125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215352435PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 352Ile Thr Cys Pro Pro
Pro Met Ser Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser
Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg
Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala
Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75
80Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile
85 90 95Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile
Asp 100 105 110Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys
Lys Val Thr 115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val
Ile Ser Leu Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val
Glu Asp Leu Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val
His Ile Val Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200
205Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Lys His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Glu Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315
320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
325 330 335Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 340 345 350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp
Glu Gln Gly Asp Val Phe Ser Cys Ser Val Met 405 410 415His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro
Gly Lys 435353447PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 353Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys
Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30Phe Met His Trp Val Gln
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro
Glu Leu Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Tyr
Ala Arg Gly Val Tyr Gln Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val225 230
235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Lys His Glu
Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345
350Pro Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu Thr Cys Leu
355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445354214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 354Asp Ile Gln Met Thr Gln Ser Pro Ala Phe
Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Gln Ala
Ser Gln Asp Ile Gly Asn Tyr 20 25 30Leu Asn Trp Phe Gln Gln Lys Pro
Gly Gln Thr Val Lys Leu Leu Ile 35 40 45Tyr Phe Thr Ser Tyr Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala
Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 210355435PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 355Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75 80Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile 85 90 95Ser
Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 100 105
110Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys Lys Val Thr
115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu
Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val Gln Asp Leu
Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser Asn Gly Asn
Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu Leu Glu Glu
Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val His Ile
Val
Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200 205Ser Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 210 215 220Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230
235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys
His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu
Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 340 345
350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Glu Gln Gly Asp
Val Phe Ser Cys Ser Val Met 405 410 415His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro Gly Lys
435356450PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 356Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asp Asp Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile Ser Thr Lys Ala
Asn Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75 80Val Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr
Arg Leu Ala Thr Trp Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys
His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr
Leu Pro Pro Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu 355 360
365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Lys
450357218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 357Asp Ile Val Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala
Ser Glu Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro
Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120
125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215358435PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 358Ile Thr Cys Pro Pro
Pro Met Ser Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser
Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg
Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala
Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75
80Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile
85 90 95Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile
Asp 100 105 110Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys
Lys Val Thr 115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val
Ile Ser Leu Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val
Gln Asp Leu Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val
His Ile Val Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200
205Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Lys His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Glu Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315
320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
325 330 335Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 340 345 350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp
Glu Gln Gly Asp Val Phe Ser Cys Ser Val Met 405 410 415His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro
Gly Lys 435359447PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 359Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys
Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30Phe Met His Trp Val Gln
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro
Glu Leu Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Tyr
Ala Arg Gly Val Tyr Gln Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val225 230
235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Lys His Glu
Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345
350Pro Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu Thr Cys Leu
355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445360214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 360Asp Ile Gln Met Thr Gln Ser Pro Ala Phe
Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Gln Ala
Ser Gln Asp Ile Gly Asn Tyr 20 25 30Leu Asn Trp Phe Gln Gln Lys Pro
Gly Gln Thr Val Lys Leu Leu Ile 35 40 45Tyr Phe Thr Ser Tyr Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala
Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 210361435PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 361Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75 80Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile 85 90 95Ser
Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 100 105
110Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys Lys Val Thr
115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu
Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val Glu Asp Leu
Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser Asn Gly Asn
Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu Leu Glu Glu
Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val His Ile Val
Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200 205Ser Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 210 215 220Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230
235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys
His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu
Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 340 345
350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr
Val385 390 395 400Asp Lys Ser Arg Trp Glu Gln Gly Asp Val Phe Ser
Cys Ser Val Leu 405 410 415His Glu Ala Leu His Ser His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 420 425 430Pro Gly Lys
435362450PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 362Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asp Asp Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile Ser Thr Lys Ala
Asn Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75 80Val Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr
Arg Leu Ala Thr Trp Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys
His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr
Leu Pro Pro Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu 355 360
365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Leu His 420 425 430Glu Ala Leu His Ser His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Lys
450363218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 363Asp Ile Val Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala
Ser Glu Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro
Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120
125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215364435PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 364Ile Thr Cys Pro Pro
Pro Met Ser Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser
Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg
Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala
Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75
80Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile
85 90 95Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile
Asp 100 105 110Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys
Lys Val Thr 115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val
Ile Ser Leu Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val
Glu Asp Leu Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val
His Ile Val Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200
205Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Lys His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Glu Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315
320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
325 330 335Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 340 345 350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp
Glu Gln Gly Asp Val Phe Ser Cys Ser Val Leu 405 410 415His Glu Ala
Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro
Gly Lys 435365447PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 365Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys
Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30Phe Met His Trp Val Gln
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro
Glu Leu Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Tyr
Ala Arg Gly Val Tyr Gln Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val225 230
235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Lys His Glu
Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345
350Pro Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu Thr Cys Leu
355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Leu His Glu Ala Leu 420 425 430His Ser His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445366214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 366Asp Ile Gln Met Thr Gln Ser Pro Ala Phe
Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Gln Ala
Ser Gln Asp Ile Gly Asn Tyr 20 25 30Leu Asn Trp Phe Gln Gln Lys Pro
Gly Gln Thr Val Lys Leu Leu Ile 35 40 45Tyr Phe Thr Ser Tyr Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala
Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 210367435PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 367Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75 80Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile 85 90 95Ser
Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 100 105
110Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys Lys Val Thr
115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu
Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val Gln Asp Leu
Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser Asn Gly Asn
Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu Leu Glu Glu
Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val His Ile Val
Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200 205Ser Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 210 215 220Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230
235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys
His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu
Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 340 345
350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Glu Gln Gly Asp
Val Phe Ser Cys Ser Val Leu 405 410 415His Glu Ala Leu His Ser His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro Gly Lys
435368450PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 368Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asp Asp Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile Ser Thr Lys Ala
Asn Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75 80Val Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr
Arg Leu Ala Thr Trp Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130
135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly225 230 235 240Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250
255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys His Glu
260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro
Pro Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu 355 360 365Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Leu His 420 425 430Glu Ala Leu His Ser His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Lys
450369218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 369Asp Ile Val Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala
Ser Glu Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro
Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120
125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215370435PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 370Ile Thr Cys Pro Pro
Pro Met Ser Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser
Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg
Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala
Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75
80Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile
85 90 95Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile
Asp 100 105 110Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys
Lys Val Thr 115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val
Ile Ser Leu Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val
Gln Asp Leu Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val
His Ile Val Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200
205Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
210 215 220Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met225 230 235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Lys His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Glu Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315
320Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
325 330 335Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 340 345 350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp
Glu Gln Gly Asp Val Phe Ser Cys Ser Val Leu 405 410 415His Glu Ala
Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro
Gly Lys 435371447PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 371Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys
Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30Phe Met His Trp Val Gln
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro
Glu Leu Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Tyr
Ala Arg Gly Val Tyr Gln Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val225 230
235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Lys His Glu
Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345
350Pro Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu Thr Cys Leu
355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Leu His Glu Ala Leu 420 425 430His Ser His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445372214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 372Asp Ile Gln Met Thr Gln Ser Pro Ala Phe
Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Gln Ala
Ser Gln Asp Ile Gly Asn Tyr 20 25 30Leu Asn Trp Phe Gln Gln Lys Pro
Gly Gln Thr Val Lys Leu Leu Ile 35 40 45Tyr Phe Thr Ser Tyr Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala
Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 210373435PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 373Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75 80Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asp Val Ile 85 90 95Ser
Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 100 105
110Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr
115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu
Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val Glu Asp Leu
Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser Asn Gly Asn
Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu Leu Glu Glu
Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val His Ile Val
Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200 205Ser Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 210 215 220Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230
235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys
His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu
Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 340 345
350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Glu Gln Gly Asp
Val Phe Ser Cys Ser Val Met 405 410 415His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro Gly Lys
435374449PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 374Gln Val Thr Leu Arg Glu Ser Gly Pro Ala
Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser
Gly Phe Ser Leu Ser Thr Ala 20 25 30Gly Met Ser Val Gly Trp Ile Arg
Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile Trp Trp
Asp Asp Lys Lys His Tyr Asn Pro Ser 50 55 60Leu Lys Asp Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Lys Val
Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg
Asp Met Ile Phe Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105 110Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120
125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys His
Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330
335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350Leu Pro Pro Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys
Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445Lys375213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 375Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Ser Ala Ser Ser Arg Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe
Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105
110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly
Glu Cys 210376435PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 376Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75 80Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile 85 90 95Ser
Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 100 105
110Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys Lys Val Thr
115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu
Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val Glu Asp Leu
Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser Asn Gly Asn
Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu Leu Glu Glu
Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val His Ile Val
Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200 205Ser Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 210 215 220Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230
235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys
His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu
Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 340 345
350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Glu Gln Gly Asp
Val Phe Ser Cys Ser Val Met 405 410 415His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro Gly Lys
435377449PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 377Gln Val Thr Leu Arg Glu Ser Gly Pro Ala
Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser
Gly Phe Ser Leu Ser Thr Ala 20 25 30Gly Met Ser Val Gly Trp Ile Arg
Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile Trp Trp
Asp Asp Lys Lys His Tyr Asn Pro Ser 50 55 60Leu Lys Asp Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Lys Val
Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg
Asp Met Ile Phe Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105 110Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120
125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys His
Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445Lys378213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 378Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Ser Ala Ser Ser Arg Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe
Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105
110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly
Glu Cys 210379435PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 379Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser65 70 75 80Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile 85 90 95Ser
Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp 100 105
110Ala Thr Leu Tyr Thr Glu Ser Asn Val His Pro Ser Cys Lys Val Thr
115 120 125Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu
Glu Ser 130 135 140Gly Asp Ala Ser Ile His Asp Thr Val Gln Asp Leu
Ile Ile Leu Ala145 150 155 160Asn Asn Ser Leu Ser Ser Asn Gly Asn
Val Thr Glu Ser Gly Cys Lys 165 170 175Glu Cys Glu Glu Leu Glu Glu
Lys Asn Ile Lys Glu Phe Leu Gln Ser 180 185 190Phe Val His Ile Val
Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser 195 200 205Ser Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 210 215 220Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met225 230
235 240Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys
His 245 250 255Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 260 265 270His Asn Ala Lys Thr Lys Pro Arg Glu Glu Glu
Tyr Asn Ser Thr Tyr 275 280 285Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 290 295 300Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile305 310 315 320Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 340 345
350Leu Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
355 360 365Trp Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 370 375 380Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val385 390 395 400Asp Lys Ser Arg Trp Glu Gln Gly Asp
Val Phe Ser Cys Ser Val Met 405 410 415His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430Pro Gly Lys
435380449PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 380Gln Val Thr Leu Arg Glu Ser Gly Pro Ala
Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser
Gly Phe Ser Leu Ser Thr Ala 20 25 30Gly Met Ser Val Gly Trp Ile Arg
Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile Trp Trp
Asp Asp Lys Lys His Tyr Asn Pro Ser 50 55 60Leu Lys Asp Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Lys Val
Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg
Asp Met Ile Phe Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105 110Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120
125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Lys His
Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445Lys381213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 381Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Ser Ala Ser Ser Arg Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe
Ala Thr Tyr
Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120
125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys
2103825PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 382Gly Ser Gly Gly Ser1 53834PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 383Gly
Gly Gly Ser1
* * * * *