U.S. patent application number 16/758142 was filed with the patent office on 2020-09-10 for recombinant immune cells, methods of making, and methods of use.
The applicant listed for this patent is REGENTS OF THE UNIVERSITY OF MINNESOTA. Invention is credited to Robert Harrison Hullsiek, Yungfang Li, Hemant Kumar Mishra, Bruce Kenneth Walcheck, Jianming Wu.
Application Number | 20200283501 16/758142 |
Document ID | / |
Family ID | 1000004913853 |
Filed Date | 2020-09-10 |
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United States Patent
Application |
20200283501 |
Kind Code |
A1 |
Wu; Jianming ; et
al. |
September 10, 2020 |
RECOMBINANT IMMUNE CELLS, METHODS OF MAKING, AND METHODS OF USE
Abstract
A recombinant immune cell expresses a heterologous IgG Fc
receptor. In some embodiments, the heterologous IgG Fc receptor can
be a chimeric IgG Fc receptor. Generally, the chimeric IgG Fc
receptor includes an extracellular domain, a transmembrane domain,
and an intracellular domain. The extracellular domain generally
includes a sufficient portion of CD64 to bind to an IgG Fc region.
The intracellular domain of the chimeric IgG Fc receptor includes a
sufficient portion of an Fc receptor allowing immunoreceptor
tyrosine-based activation motif (ITAM) to initiate cell signaling
when an IgG Fc region binds to the extracellular domain.
Inventors: |
Wu; Jianming; (Plymouth,
MN) ; Walcheck; Bruce Kenneth; (Lino Lakes, MN)
; Hullsiek; Robert Harrison; (Minneapolis, MN) ;
Li; Yungfang; (Minneapolis, MN) ; Mishra; Hemant
Kumar; (Minneapolis, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REGENTS OF THE UNIVERSITY OF MINNESOTA |
Minneapolis |
MI |
US |
|
|
Family ID: |
1000004913853 |
Appl. No.: |
16/758142 |
Filed: |
October 26, 2018 |
PCT Filed: |
October 26, 2018 |
PCT NO: |
PCT/US2018/057689 |
371 Date: |
April 22, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62577425 |
Oct 26, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/24 20130101;
C07K 16/30 20130101; C07K 14/70535 20130101; C12N 5/0646 20130101;
C07K 2319/74 20130101 |
International
Class: |
C07K 14/735 20060101
C07K014/735; C12N 5/0783 20060101 C12N005/0783; C07K 16/30 20060101
C07K016/30 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under
CA203348 awarded by National Institutes of Health. The government
has certain rights in the invention.
Claims
1. A chimeric IgG Fc receptor comprising: an extracellular domain
comprising a sufficient portion of CD64 to bind to an IgG Fc
region; a transmembrane domain; and an intracellular domain
comprising a sufficient portion of an Fc receptor immunoreceptor
tyrosine-based activation motif (ITAM) to initiate cell signaling
when an IgG Fc region binds to the extracellular domain.
2. The chimeric IgG Fc receptor of claim 1, wherein the
intracellular domain comprises at least a portion of the
intracellular region of CD16A.
3. The chimeric IgG Fc receptor of claim 1, wherein the
intracellular domain comprises at least a portion of the
intracellular region of CD27, CD28, CD134 (OX40), CD137 (4-1BB),
Fc.epsilon.R1, NKG2D, CD244 (2B4), FcR.gamma., DAP10, DAP12, or
CD3.zeta..
4. The chimeric IgG Fc receptor of any preceding claim, wherein the
extracellular domain comprises the CD16A cleavage site.
5. The chimeric IgG Fc receptor of any preceding claim, wherein the
intracellular domain comprises a signaling domain.
6. A polynucleotide encoding the chimeric receptor of any preceding
claim.
7. A recombinant cell comprising the polynucleotide of claim 6.
8. A recombinant cell expressing the IgG Fc chimeric receptor of
any one of claims 1-5.
9. The recombinant cell of claim 8, wherein the recombinant cell is
a natural killer (NK) cell.
10. A recombinant natural killer (NK) cell comprising a
polynucleotide that encodes CD64.
11. A recombinant cell comprising a natural killer (NK) cell
genetically modified to express CD64.
12. A method of killing a tumor cell, the method comprising:
contacting the tumor cell with an antibody that specifically binds
to the tumor cell; and contacting the tumor cell with the
recombinant cell of any one of claims 7-11 under conditions
effective for the recombinant cell to kill the tumor cell.
13. A method of treating a subject having a tumor, the method
comprising: administering to the subject an antibody that
specifically binds to cells of the tumor; and administering to the
subject a composition comprising the recombinant cell of any one of
claims 7-11 under conditions effective for the recombinant cell to
kill cells of the tumor.
14. A composition comprising: the recombinant cell of any one of
claims 7-11; and an antibody bound to the chimeric receptor.
15. A method of treating a subject having a tumor, the method
comprising: administering to the subject the composition of claim
14 wherein the antibody specifically binds to cells of the tumor.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application No. 62/577,425, filed Oct. 26, 2017, which is
incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0003] This application contains a Sequence Listing electronically
submitted to the United States Patent and Trademark Office via
EFS-Web as an ASCII text file entitled
"Sequence-Listing-0589_ST25.txt" having a size of 97 kilobytes and
created on Oct. 26, 2018. Due to the electronic filing of the
Sequence Listing, the electronically submitted Sequence Listing
serves as both the paper copy required by 37 CFR .sctn. 1.821(c)
and the CRF required by .sctn. 1.821(e). The information contained
in the Sequence Listing is incorporated by reference herein.
SUMMARY
[0004] This disclosure describes, in one aspect, this disclosure
describes an immune cell that expresses a heterologous IgG Fc
receptor.
[0005] In some embodiments, the heterologous IgG Fc receptor can be
a chimeric IgG Fc receptor. Generally, the chimeric IgG Fc receptor
includes an extracellular domain, a transmembrane domain, and an
intracellular domain. The extracellular domain generally includes a
sufficient portion of CD64 to bind to an IgG Fc region. The
intracellular domain of the chimeric IgG Fc receptor includes a
sufficient portion of an Fc receptor immunoreceptor tyrosine-based
activation motif (ITAM) to initiate cell signaling when an IgG Fc
region binds to the extracellular domain.
[0006] In some of these embodiments, the intracellular domain
includes at least a portion of the intracellular region of CD16A.
In other embodiments, the intracellular domain can include at least
a portion of the intracellular region of CD27, CD28, CD134 (OX40),
CD137 (4-1BB), FcR.gamma., or CD3.
[0007] In some embodiments, the chimeric IgG Fc receptor can
include the CD16A extracellular cleavage site. In other
embodiments, the extracellular domain of the chimeric IgG Fc
receptor can lack the CD16A extracellular cleavage site.
[0008] In some embodiments, the heterologous IgG Fc receptor can
include an IgG Fc receptor not natively expressed by the immune
cell. In some of these embodiments, the immune cell may be a
natural killer (NK) cell genetically modified to express CD64.
[0009] In another aspect, this disclosure describes a
polynucleotide that encodes any embodiment of the heterologous IgG
Fc receptors summarized above.
[0010] In another aspect, this disclosure describes an immune cell
that is genetically modified to include the polynucleotide that
encodes an embodiment of the heterologous IgG Fc receptors
summarized above.
[0011] In another aspect, this disclosure describes a method of
killing a tumor cell. Generally, the method includes contacting the
tumor cell with an antibody that specifically binds to the tumor
cell and contacting the tumor cell with any embodiment of the
recombinant immune cell summarized above under conditions effective
for the recombinant immune cell to kill the tumor cell.
[0012] In another aspect, this disclosure describes a method of
treating a subject having a tumor. Generally, the method includes
administering to the subject an antibody that specifically binds to
cells of the tumor and administering to the subject a composition
that includes any embodiment of the recombinant immune cell
summarized above under conditions effective for the recombinant
immune cell to kill cells of the tumor.
[0013] In another aspect, this disclosure describes a composition
that include a complex formed between a therapeutic antibody and
any embodiment of the recombinant immune cell summarized above in
which the heterologous IgG Fc receptor is bound to the Fc portion
of the therapeutic antibody.
[0014] In another aspect, this disclosure describes a method of
treating a subject having a tumor. Generally, the method includes
administering to the subject any embodiment of the composition
summarized immediately wherein the therapeutic antibody
specifically binds to cells of the tumor.
[0015] The above summary is not intended to describe each disclosed
embodiment or every implementation of the present invention. The
description that follows more particularly exemplifies illustrative
embodiments. In several places throughout the application, guidance
is provided through lists of examples, which examples can be used
in various combinations. In each instance, the recited list serves
only as a representative group and should not be interpreted as an
exclusive list.
BRIEF DESCRIPTION OF THE FIGURES
[0016] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0017] FIG. 1. Antibody-dependent cell-mediated cytotoxicity
(ADCC).
[0018] FIG. 2. Wildtype CD16A, wildtype CD64, and the CD64/16A
chimeric construct. The scissors and dashed line shown for CD16A
represent the extracellular proteolytic site for ectodomain
shedding.
[0019] FIG. 3. NK92 cells expressing either wildtype CD16A or
CD64/16A. (A) NK92-CD64/16A cells were stained with anti-CD16,
anti-CD64, or control antibodies. (B) NK92-CD16A cells were stained
with anti-CD16, anti-CD64, or control antibodies. (C)
NK92-CD64/16A, NK92-CD16A, or NK92 parent cells were incubated with
trastuzumab then anti-human IgG-APC second stage antibody or
anti-human IgG-APC second stage antibody alone (control). All
antibody staining levels were determined by flow cytometry.
[0020] FIG. 4. NK92 cells expressing CD64/CD16A induce higher
levels of ADCC than wildtype CD16A. (A) A standard ADCC assay was
performed in which trastuzumab (herceptin) was included in the
assay. (B) A standard ADCC assay was performed in which the
NK92-CD64/CD16A and NK92-CD16A cells were pre-incubated with
trastuzumab and then the mAb was washed away prior to the effector
cells being incubated with the SKOV-3 target cells.
[0021] FIG. 5. Flow cytometry data comparing phenotypic markers
expressed by iNK-CD64/CD16A and iNK-pKT2 cells.
[0022] FIG. 6. Bar graph with data showing iNK-CD64/CD16A cells
induce higher levels of ADCC than iNK-CD16A cells using SKOV-3
target cells.
[0023] FIG. 7. Bar graph with data showing iNK-CD64/CD16A, but not
iNK-CD16A cells can be pre-loaded with a therapeutic mAb and
mediate ADCC.
[0024] FIG. 8. Bar graph with data showing iNK-CD64/CD16A cells
induce higher levels of ADCC than iNK-CD16A cells using MA148
target cells.
[0025] FIG. 9. Amino acid sequence of an exemplary CD64/CD16A
chimeric IgG Fc receptor (SEQ ID NO:1).
[0026] FIG. 10. Amino acid sequence of CD64 IgG Fc receptor (SEQ ID
NO:2).
[0027] FIG. 11. Amino acid sequence of an exemplary
CD16A-CD28-BB-.zeta. chain chimeric IgG Fc receptor (SEQ ID
NO:3).
[0028] FIG. 12. Amino acid sequence of an exemplary CD16A-BB-.zeta.
chain chimeric IgG Fc receptor (SEQ ID NO:4).
[0029] FIG. 13. Expression of CD64/16A by NK92 cells. (A) Schematic
representation of the cell membrane forms of CD16A, CD64, and
CD64/16A. CD16A undergoes ectodomain shedding by ADAM17 at a
membrane proximal location, as indicated, which is not present in
CD64 and CD64/16A. (B) NK92 parental cells, NK92-CD16A cells, and
NK92-CD64/16A cells were stained with an anti-CD16, anti-CD64, or
an isotype-matched negative control mAb and examined by flow
cytometry. (C) NK92-CD16A and NK92-CD64/16A cells were incubated
with SKOV-3 cells with or without trastuzumab (5 .mu.g/ml) at
37.degree. C. (E:T=1:1) for two hours. The NK92-CD16A and
NK92-CD64/16A cells were then stained with an anti-CD16 mAb or an
anti-CD64 mAb, respectively, and examined by flow cytometry.
Nonspecific antibody labeling was determined using the appropriate
isotype-negative control mAb. Data is representative of at least
three independent experiments.
[0030] FIG. 14. CD64/16A promotes target cell conjugation, ADCC,
and IFN.gamma. production. (A) NK92-CD64/16A cells expressing eGFP
and SKOV-3 cells labeled CellTrace Violet were mixed at an E:T
ratio of 1:2 with or without trastuzumab (5 .mu.g/ml), incubated at
37.degree. C. for 60 minutes, fixed, and then analyzed by flow
cytometry. Representative data from at least three independent
experiments are shown. (B) NK92-CD64/16A cells were incubated with
SKOV-3 cells (E:T=20:1) and trastuzumab (tras.) at the indicated
concentrations (left panel), or with SKOV-3 cells at the indicated
E:T ratios in the presence or absence of trastuzumab (5 .mu.g/ml)
(right panel) for two hours at 37.degree. C. Data are represented
as % specific release and the mean.+-.SD of three independent
experiments is shown. Statistical significance is indicated as
*p<0.05, **p<0.01. (C) NK92-CD64/16A cells were incubated
with SKOV-3 cells (E:T=20:1) in the presence or absence of
trastuzumab (5 .mu.g/ml) and the anti-CD64 mAb 10.1 (10 .mu.g/ml),
as indicated, for two hours at 37.degree. C. Data are represented
as % specific release and the mean.+-.SD of three independent
experiments is shown. Statistical significance is indicated as
**p<0.01. (D) NK92-CD64/16A cells were incubated with SKOV-3
cells (E:T=1:1) with or without trastuzumab (5 .mu.g/ml) for two
hours at 37.degree. C. Secreted IFN.gamma. levels were quantified
by ELISA. Data is shown as mean of two independent experiments.
[0031] FIG. 15. CD64/16A attaches to soluble tumor-targeting mAbs
and IgG fusion proteins. (A) Relative expression levels of CD16A
and CD64/16A on NK92 cells were determined by cell staining with
anti-CD16 and anti-CD64 mAbs (black bars), respectively, or an
isotype-matched negative control antibody (gray bars). The bar
graph shows mean fluorescence intensity (MFI).+-.SD of three
independent experiments. Representative flow cytometric data are
shown in the histogram overlay. The dashed line histogram shows
CD64 staining of NK92-CD64/16A cells, the orange-filled histogram
shows CD16A staining of NK92-CD16A cells, and the green-filled
histogram shows isotype control antibody staining of the NK92-CD16A
cells. (B) NK92-CD16A and NK92-CD64/16A cells were incubated with
or without trastuzumab (5 .mu.g/ml) for two hours at 37.degree. C.,
washed, stained with a fluorophore-conjugated anti-human secondary
antibody, and analyzed by flow cytometry. Data is representative of
at least three independent experiments. (C) NK92-CD64/16A cells
were incubated with cetuximab or rituximab (5 .mu.g/ml for each),
washed, and then stained with a fluorophore-conjugated anti-human
secondary antibody. Control represents cells stained with the
anti-human secondary antibody only. NK92-CD64/16A cells were also
incubated with L-selectin/Fc (5 .mu.g/ml), washed, and then stained
with a fluorophore-conjugated anti-L-selectin mAb. NK92 cells lack
expression of endogenous L-selectin (data not shown). All staining
was analyzed by flow cytometry. Data shown are representative of
three independent experiments. (D) NK92-CD16A and NK92-CD64/16A
cells were incubated in the presence or absence of trastuzumab (5
.mu.g/ml), washed, and exposed to SKOV-3 cells at the indicated E:T
cell ratios for two hours at 37.degree. C. Data is shown as
mean.+-.SD of three independent experiments. Statistical
significance is indicated as **p<0.01, ***p<0.001. bd=below
detection, (i.e., <spontaneous release by negative control
cells). (E) NK92-CD16A and NK92-CD64/16A cells were incubated with
SKOV-3 cells (E:T=10:1) in the presence or absence of trastuzumab
(5 .mu.g/ml), as indicated, for two hours at 37.degree. C. Data is
shown as mean.+-.SD of three independent experiments. Statistical
significance is indicated as **p<0.01.
[0032] FIG. 16. Generation of iNK cells expressing CD64/CD16A.
iPSCs were transduced to stably express CD64/16A, differentiated
into NK cells, and then expanded using K562-mbIL21-41BBL feeder
cells. iNK-CD64/16A cells and freshly isolated peripheral blood
(PB) NK cells enriched from adult peripheral blood were stained for
CD56, CD3 and various inhibitory and activating receptors, as
indicated. CD64/16A expression was determined by staining the cells
with an anti-CD64 mAb. Representative data from at least three
independent experiments are shown.
[0033] FIG. 17. iNK-CD64/16A cells show enhanced ADCC compared to
iNK-pKT2 control cells. (A) NK cells derived from empty vector
(iNK-pKT2) or CD64/16A (iNK-CD64/16A) transduced iPSCs were stained
for CD56, CD64, and CD16A, as indicated. (B) iNK-pKT2 and
iNK-CD64/16A cells were incubated with SKOV-3 cells (E:T=10:1) in
the presence or absence of trastuzumab (5 .mu.g/ml), the function
blocking anti-CD16 mAb 3G8 (5 .mu.g/ml), and the function blocking
anti-CD64 mAb 10.1 (5 .mu.g/ml), as indicated, for two hours at
37.degree. C. Data is shown as mean.+-.SD of three independent
experiments. Statistical significance is indicated as
***p<0.001; ****p<0.0001. (C) iNK-pKT2 and iNK-CD64/16A cells
were incubated in the presence or absence of trastuzumab (5
.mu.g/ml), washed, and exposed to SKOV-3 cells (E:T=10:1) for two
hours at 37.degree. C. Data is shown as mean.+-.SD of three
independent experiments. Statistical significance is indicated as
***p<0.001.
[0034] FIG. 18. Sequence alignment of canine CD16A (SEQ ID NO:5),
canine CD64sp (SEQ ID NO:25), and human CD16A (SEQ ID NO:6).
[0035] FIG. 19. Sequence alignment of canine CD64 (SEQ ID NO:7) and
human CD64 (SEQ ID NO:8).
[0036] FIG. 20. NK92 cells expressing wildtype human CD64 mediate
ADCC. (A) NK92-CD64 cells were stained with an isotype-matched
negative control mAb or the anti-CD64 mAb (clone 10.1) and examined
by flow cytometry. (B) NK92-CD64 cells were incubated with SKOV-3
cells (at the indicated E:T ratios) in the presence or absence of
trastuzumab (tras.) (5 .mu.g/ml) for two hours at 37.degree. C.
Representative data from at least three independent experiments are
shown.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0037] This disclosure describes recombinant immune cells, methods
of making the recombinant immune cells, and methods of using the
recombinant immune cells. Generally, the recombinant immune cells
are genetically modified to include a heterologous IgG Fc receptor.
In some cases, the heterologous IgG Fc receptor can be a chimeric
receptor engineered include domains from two or more receptors. In
other embodiments, the heterologous may be an IgG Fc receptor that
is not natively expressed by the immune cell. Generally, the
recombinant immune cells provide a sustained cytotoxic immune
response against a target--for example, a tumor cell--that is
targeted for killing by the immune cell because the target binds a
therapeutic antibody that is recognized by the IgG Fc receptor.
[0038] A mechanism of cell-mediated immune defense involves the
engagement of antibodies attached to target cells by Fc receptors
expressed by leukocytes, which results in target cell killing. This
process is referred to antibody-dependent cell-mediated
cytotoxicity (ADCC). Therapeutic monoclonal antibodies (mAbs) have
been generated against a variety of tumor antigens and tested in
clinical trials for treating infectious diseases, chronic diseases,
and cancers including, for example, AML, breast cancer, ovarian
cancer, gastric cancer, neuroblastoma, and lymphoma. Many
clinically successful mAbs use ADCC as a mechanism of action. A
limitation of antibody therapy, however, is the development of
resistance in patients and the non-responsiveness of some
malignancies.
[0039] This disclosure describes an approach for augmenting Fc
receptor interactions with therapeutic antibodies. The approach
involves a chimeric receptor that includes a CD16A domain and a
CD64 domain.
[0040] CD16A (Fc.gamma.RIIIA) is an IgG Fc receptor expressed by
human natural killer (NK) cells, a population of cytotoxic
lymphocytes, and is their sole means of recognizing IgG bound to
tumor cells or virus-infected cells. CD16A is a potent activating
receptor that induces ADCC by NK cells (FIG. 1). The CD16A
transmembrane region is responsible for the association with CD3
and/or FcR .gamma.-chain (FcR.gamma.) that contain immunoreceptor
tyrosine-based activation motifs (FIG. 2; FIG. 13A), and the CD16A
cytoplasmic domain interacts with intracellular molecules critical
for receptor functions. CD16A is a low affinity Fc.gamma.R with
limited capacity to engage therapeutic mAb-coated target cells.
CD16A also undergoes a rapid downregulation in expression upon cell
activation that markedly reduces its cell surface density and
avidity for IgG. CD16A downregulation occurs by a proteolytic event
at an extracellular site proximal to the plasma membrane and is
referred to as ectodomain shedding. The location of this cleavage
site has been reported (Jing et al., 2015. PLoS One 10:e0121788)
and is shown schematically in FIG. 2 and FIG. 13A.
[0041] CD64 (Fc.gamma.RI) is another IgG Fc receptor, and is
expressed by monocytes, macrophages, and activated neutrophils.
CD64 is a high affinity IgG receptor. This receptor does not
undergo ectodomain shedding upon cell activation and does not
naturally transduce signals for ADCC in NK cells.
[0042] This disclosure describes a chimeric Fc.gamma.R that
includes a CD16A domain and a CD64 domain. The chimeric receptor
includes the extracellular region of human CD64 and the cytoplasmic
region of human CD16A, an exemplary embodiment of which is shown
schematically in FIG. 2 and FIG. 13A as CD64/16A. In various
embodiments, the chimeric CD64/CD16A receptor can include the CD64
transmembrane region or the CD16A transmembrane region. The
CD64/16A construct has been engineered so that it lacks the CD16A
extracellular cleavage site and thus is not susceptible to
ectodomain shedding (FIG. 2; FIG. 13A), but includes at least a
portion of the CD16A intracellular region that is involved in
intracellular signaling.
[0043] Also, while described herein in the context of an exemplary
embodiment in which the CD64 domain and the CD16A contain amino
acid sequences of human CD64 and human CD16A, respectively, the
chimeric Fc.gamma.R described herein can include an amino acid
sequence that is, or is derived from, any suitable CD64 or CD16A
natively expressed by any species. FIG. 18 and FIG. 19 provide
amino acid sequence alignments of human and canine amino acid
sequences for CD16A (FIG. 18) and CD64 (FIG. 19).
[0044] As used herein, the amino acid sequence of a domain is
"derived from" a the amino acid sequence of a reference polypeptide
if the amino acid sequence of the domain possesses a specified
amount of sequence similarity and/or sequence identity compared to
the amino acid sequence of the reference polypeptide. Sequence
similarity of can be determined by aligning the residues of the two
polypeptides (for example, the domain amino acid sequence and the
amino acid sequence of the reference CD16A or CD64 polypeptide) to
optimize the number of identical amino acids along the lengths of
their sequences. Gaps in either or both sequences are permitted in
making the alignment in order to optimize the number of identical
amino acids, although the amino acids in each sequence must
nonetheless remain in their proper order.
[0045] A pair-wise comparison analysis of amino acid sequences can
be carried out using the BESTFIT algorithm in the GCG package
(version 10.2, Madison Wis.). Alternatively, polypeptides may be
compared using the Blastp program of the BLAST 2 search algorithm,
as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250
(1999)), and available on the National Center for Biotechnology
Information (NCBI) website. The default values for all BLAST 2
search parameters may be used, including matrix=BLOSUM62; open gap
penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10,
wordsize=3, and filter on.
[0046] The amino acid sequence of a domain is "derived from" a the
amino acid sequence of a reference polypeptide if the amino acid
sequence of the domain possesses a specified degree of amino acid
sequence "identity" or amino acid sequence "similarity." Amino acid
sequence identity refers to the presence of identical amino acids.
Amino acid sequence similarity refers to the presence of not only
identical amino acids but also the presence of conservative
substitutions. A conservative substitution for an amino acid may be
selected from other members of the class to which the substituted
amino acid belongs. For example, it is well-known in the art of
protein biochemistry that an amino acid belonging to a grouping of
amino acids having a particular size or characteristic (such as
charge, hydrophobicity and hydrophilicity) can be substituted for
another amino acid without altering the activity of a protein,
particularly in regions of the protein that are not directly
associated with biological activity. For example, nonpolar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan, and tyrosine. Polar
neutral amino acids include glycine, serine, threonine, cysteine,
tyrosine, asparagine and glutamine. The positively charged (basic)
amino acids include arginine, lysine and histidine. The negatively
charged (acidic) amino acids include aspartic acid and glutamic
acid. Conservative substitutions include, for example, Lys for Arg
and vice versa to maintain a positive charge; Glu for Asp and vice
versa to maintain a negative charge; Ser for Thr so that a free
--OH is maintained; and Gln for Asn to maintain a free --NH.sub.2.
Likewise, biologically active analogs of a polypeptide containing
deletions or additions of one or more contiguous or noncontiguous
amino acids that do not eliminate a functional activity of the
polypeptide are also contemplated.
[0047] The amino acid sequence of a CD16A domain or a CD64 domain
is "derived from" a reference amino acid sequence if the domain
amino acid sequence has at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%
sequence similarity to the reference amino acid sequence.
[0048] The amino acid sequence of a CD16A domain or a CD64 domain
is "derived from" a reference amino acid sequence if the domain
amino acid sequence has at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%
sequence identity to the reference amino acid sequence.
[0049] For the purpose of determining whether a domain amino acid
sequence is "derived from" a specified reference amino acid
sequence, exemplary suitable reference polypeptides include human
CD16A (SEQ ID NO:6), canine CD16A (SEQ ID NO:5), human CD64 (SEQ ID
NO:8), canine CD64 (SEQ ID NO:7), canine CD64sp (SEQ ID NO:25), or
the corresponding domains of any of the constructs listed in Table
1.
[0050] Also, while described herein in the context of an exemplary
embodiment, illustrated in FIG. 2 and FIG. 13A, the chimeric
receptor may be designed to include a different fusion points
between CD64 and CD16A that that shown in FIG. 2 and FIG. 13A, to
include the CD16A cleavage region, modified functional motifs in
regions of CD64 or CD16A, and/or adding an additional signaling
domain, such as, for example, a signaling domain of CD27, CD28,
CD134 (OX40), CD137 (4-1BB), FcR.gamma., or CD3. The chimeric
receptor may therefore be designed to increase the proliferation of
NK cells or other effector cells, increase survival of NK cells or
other effector cells, increase potency of NK cells or other
effector cells, and/or decrease NK cell exhaustion in vivo.
[0051] In certain embodiments, the chimeric Fc.gamma.R can be
modified to include a cytoplasmic domain or a signaling domain that
confers additional functionality to the chimeric receptor. For
example, a chimeric Fc.gamma.R can include a functional portion of
CD28, which transduces signals involved in T-cell proliferation,
survival, and cytokine production. As another example, a chimeric
Fc.gamma.R can include a functional portion of 4-1BB, which
contributes to the clonal expansion, survival, and development of
hematopoietic cells. As another example, a chimeric Fc.gamma.R can
include a functional portion of the CD3.zeta. cytoplasmic domain,
which contains three immunoreceptor tyrosine-based activation
motifs (ITAMs) that trigger intracellular signal-transduction
pathways for ADCC, cytokine production, and cell proliferation and
survival. As another example, a chimeric Fc.gamma.R can include a
functional portion of the FcR.gamma. cytoplasmic domain, which
contains an ITAM that preferentially recruits Syk kinase to mediate
intracellular signals for ADCC, cytokine production, and cell
proliferation and survival. As another example, a chimeric
Fc.gamma.R can include a functional portion of the DAP10
cytoplasmic domain, which contains a YxxM motif that specifically
activates phosphatidylinositol 3-kinase-dependent signaling
pathways for cytotoxicity, cell survival, and proliferation of NK
and T cells. As another example, a chimeric Fc.gamma.R can include
a functional portion of the DAP12 cytoplasmic domain, which
contains an ITAM that triggers signals for cytotoxicity, survival,
and proliferation of NK cells and T cells. As another example, a
chimeric Fc.gamma.R can include a functional portion of the NKG2D
(or CD314) transmembrane domain, which specifically associates with
DAP10 to mediate signaling pathways for cytotoxicity, cell
survival, and proliferation of NK and T cells. As another example,
a chimeric Fc.gamma.R can include a functional portion of the 2B4
(NKR2B4 or CD244) cytoplasmic domain, which transduces signals of
cytolytic granule polarization involved in enhanced cytotoxicity of
NK cells. As another example, a chimeric Fc.gamma.R can include a
functional portion of the high affinity IgE receptor Fc.epsilon.R
transmembrane and cytoplasmic domains, which are constitutively
associated with its .beta.-subunit and FcR .gamma.-chain
(FcR.gamma.) to mediate the most potent degranulation signals in
myeloid cells that initiates the allergic responses, which could be
exploited for cancer therapy with the recombinant high affinity IgG
Fc receptors.
[0052] Exemplary constructs that include exemplary cytoplasmic
domain and/or signaling domain modifications are listed in Table
1.
TABLE-US-00001 TABLE 1 Code Domains (EC/TM/CY/SDs)* Comments SEQ ID
NO: rCD64#1 64/64/64/-- 9 rCD64#2 64/64/mut64/-- CD64 cytoplasmic
domain 10 mutation rCD64#3 64/16A/16A/-- 11 rCD64#4
64/16A/16A/28-BB-CD3c 12 rCD64#5 64/16A/16A/28-BB-FcR.gamma. 13
rCD64#6 64/16A/16A/28-BB-Dap10 14 rCD64#7 64/16A/16A/28-BB-Dap12 15
rCD64#8 64/28/28/BB-CD3c 16 rCD64#9 64/Fc.epsilon.R/Fc.epsilon.R/--
17 rCD64#10 64/16A/mut16A/-- Mutation on PKC 18 phosphorylation
site rCD64#11 64/G2D/2B4/CD3c 19 rCD64#12 64/16A/16A/2B4-CD3c 20
rCD64#13 64/16A/16A/2B4-FcR.gamma. 21 rCD64#14 64/16A/16A/2B4-Dap10
22 rCD64#15 64/16A/16A/2B4-Dap12 23 rCD64#16 64/64/64/2B4-CD3c 24
*EC: Extracellular domain; TM: Transmembrane domain; CY:
Cytoplasmic domain; SD: Signaling domain. 64: CD64, High affinity
IgG Fc receptor Fc.gamma.RI; mutCD64: High affinity IgG Fc receptor
Fc.gamma.R1 with cytoplasmic mutation that results in higher levels
of cytokine production and degranulation; 16A: C16A, Low affinity
IgG Fc receptor Fc.gamma.RIIIA; --: no signaling domain; 28: CD28,
a co-stimulatory receptor for cell proliferation and activation;
BB: 4.1BB or CD137; CD3c: CD3 c-chain or CD247; FcR.gamma.: FcR
.gamma.-chain; D10: DAP10 signaling adaptor; D12: DAP12 signaling
adaptor; FceR: High affinity IgE Fc receptor; 16-PKC.sup.-: CD16A
cytoplasmic domain with mutations on PKC phosphorylation site to
disrupt cytokine productions mediated by CD16A; G2D: NKG2D or
CD314; 2B4: NKR2B4 or CD244.
[0053] The CD64/16A chimeric receptor can be encoded by a cDNA that
can be transcribed and translated from an expression vector
introduced into a host cell to produce a recombinant cell. The host
cell can include a suitable leukocyte-like cell or a primary
leukocyte. Suitable leukocyte-like cells include, but are not
limited to, a hematopoietic cell line or an induced pluripotent
stem cell. Suitable primary leukocytes include, but are not limited
to, NK cells, monocytes, macrophages, neutrophils, or T
lymphocytes. The expression of CD64/16A by genetically-engineered
leukocytes can increase the recombinant cell's effector function in
killing target cells--e.g., tumor cells and virus-infected
cells--in the presence of natural and therapeutic antibodies
compared to the unmodified host cell. Because the CD64/16A chimeric
receptor binds to IgG with high affinity, it may also be feasible
to attach therapeutic mAbs to effector cells expressing the
construct prior to their administration into patients. Hence,
CD64/16A with an attached therapeutic mAb would provide the
effector cells with a targeting element to direct them to cancer
locations.
[0054] NK92 cells are a human NK cell line that lacks expression of
endogenous CD16A. NK cells were generated that express in a stable
manner wildtype CD16A, wildtype CD64, or the exemplary chimeric
receptor CD64/16A. NK92 cells expressing CD64/16A could be stained
with an anti-CD64 mAb, but not with an anti-CD16 mAb (FIG. 3A).
NK92-CD16A cells could be stained with an anti-CD16 mAb, but not
with an anti-CD64 mAb (FIG. 3B). NK92 cells expressing CD64 could
be stained with an anti-CD64 mAb, but not by and isotype-matched
negative control mAb (FIG. 20A).
[0055] FIG. 3C shows the ability of NK92 cells expressing CD64/16A
or CD16A to bind trastuzumab, a therapeutic mAb specific to
HER2/EGFR2 overexpressed by certain malignancies. Non-transduced
NK92 cells, NK92-CD64/16A cells, and NK92-CD16A cells were
incubated with trastuzumab (5 .mu.g/ml) for two hours at room
temperature, washed to remove unbound antibody, incubated with an
anti-human IgG second stage antibody conjugated to a fluorophore,
and then examined by flow cytometry. NK92-hCD64/16A cells bound
much higher levels of trastuzumab than did NK92-CD16A cells and
NK92 cells.
[0056] FIG. 4 presents data that shows the exemplary chimeric
receptor CD64/16A conferred NK92 cells with an ADCC effector
function. NK92 cells expressing CD64/16A or wildtype CD16A were
incubated with the human ovarian cancer cell line SKOV-3 (20:1
ratio) in the presence or absence of trastuzumab at various
concentrations (0.005 .mu.g/ml, 0.05 .mu.g/ml. 0.5 .mu.g/ml, or 5
.mu.g/ml). NK92 cells expressing either CD64/16A or wildtype CD16A
demonstrated SKOV-3 cytotoxicity in the presence of trastuzumab.
NK92 cells expressing CD64/16A had higher levels of target cell
killing than did NK92-CD16A cells at all trastuzumab concentrations
examined (FIG. 4A). NK92 cells expressing either CD64 also
demonstrated SKOV-3 cytotoxicity in the presence of trastuzumab
(FIG. 20B). In addition, NK92-CD64/16A and NK92-CD16A cells were
pretreated with trastuzumab at 5 .mu.g/ml or 10 .mu.g/ml for two
hours, washed to remove unbound antibody, and then incubated with
SKOV-3 cells. In this assay, NK92-CD64/16A cells demonstrated a
marked enhancement in target cell killing when compared to
NK92-CD16A cells (FIG. 4B).
[0057] CD64/16A also was expressed in iPSCs and these cells were
then differentiated into NK cells (referred to here as iNK cells).
As shown in FIG. 5, iNK cells transduced with either CD64/16A or
the empty vector (pKT2) as a control were compared for their
expression of several NK cell markers. iNK-CD64/16A and iNK-pKT2
cells are CD56.sup.+ and CD3.sup.-, indicating that they are indeed
NK cells. They also expressed similar levels of various NK cell
markers. iNK-CD64/16A and iNK-pKT2 cells were found to express
similar levels of CD16A, whereas only iNK-CD64/16A were stained by
an anti-CD64 mAb (FIG. 5).
[0058] iNK-CD64/16A and iNK-pKT2 cells were evaluated for ADCC, as
described above for the NK92 cells. iNK-CD64/16A and iNK-pKT2 cells
were incubated with SKOV-3 cells at 10:1 ratio in the presence or
absence of trastuzumab (5 .mu.g/ml). iNK-CD64/16A cells
demonstrated increased SKOV-3 cytotoxicity in the presence of
trastuzumab and higher levels of ADCC when compared to iNK-pKT2
cells (FIG. 6).
[0059] Although the iNK-CD64/16A and iNK-pKT2 cells expressed
similar levels of CD16A (FIG. 5), the function blocking anti-CD16A
mAb 3G8 only blocked ADCC by the iNK-pKT2 cells (FIG. 6).
Conversely, the function blocking anti-CD64 mAb 10.1 only blocked
ADCC by the iNK-CD64/16A cells (FIG. 6). iNK-CD64/16A and iNK-pKT2
cells were also pretreated with trastuzumab at 10 .mu.g/ml for two
hours, washed to remove unbound antibody, and then incubated with
SKOV-3 cells. In this assay, iNK-CD64/16A cells demonstrated a
distinct enhancement in target cell killing when compared to
iNK-pKT2 cells (FIG. 7). MA148 is a human ovarian cancer cell line
that expresses considerably lower levels of HER2 when compared to
SKOV-3 cells. ADCC was evaluated in iNK-CD64/16A and iNK-pKT2 cells
when exposed to MA148 cells at various ratios in the presence or
absence of trastuzumab (5 .mu.g/ml). Again, iNK-CD64/16A cells
demonstrated significantly higher levels of tumor cell killing than
did iNK-pKT2 cells at all effector to target cell ratios when in
the presence of trastuzumab (FIG. 8).
[0060] The chimeric receptor CD64/16A (FIG. 2; FIG. 13A) was stably
expressed in the human NK cell line NK92. These cells lack
endogenous Fc.gamma.Rs but transduced cells expressing exogenous
CD16A can mediate ADCC. As shown in FIG. 13B, an anti-CD64 mAb
stained NK92 cells expressing CD64/16A cells, but not parent NK92
cells or NK92 cells expressing CD16A. An anti-CD16 mAb stained NK92
cells expressing CD16A, but not NK92 cells expressing CD64/16A or
parent NK92 cells (FIG. 13B). CD16A undergoes ectodomain shedding
by ADAM17 upon NK cell activation resulting in its rapid
downregulation in expression. CD16A and its isoform CD16B on
neutrophils is cleaved by ADAM17, and this occurs at an
extracellular region proximal to the cell membrane. The ADAM17
cleavage region of CD16A is not present in CD64 or CD64/16A (FIG.
13A). CD16A underwent a >50% decrease in expression upon NK92
stimulation by ADCC, whereas CD64/16A demonstrated little to no
downregulation (FIG. 13C).
[0061] To evaluate the function of CD64/16A, the ability of
CD64/16A to initiate E:T conjugation, induce ADCC, and stimulate
cytokine production upon NK cell engagement of antibody-bound tumor
cells was examined. Prior to the release of its granule contents,
an NK cell forms a stable conjugate with a target cell.
NK92-CD64/16A cell and SKOV-3 cell conjugation were examined using
a two-color flow cytometric approach. SKOV-3 cells are an ovarian
cancer cell line that express HER2, and this assay was performed in
the absence and presence of the anti-HER2 therapeutic mAb
trastuzumab. The bicistronic vector containing CD64/16A also
expressed eGFP and its fluorescence was used to identify the NK92
cells. SKOV-3 cells were labeled with the fluorescent dye CellTrace
Violet. E:T conjugation resulted in two-color events that were
evaluated by flow cytometry. Incubating NK92-CD64/16A cells with
SKOV-3 cells resulted in a very low level of conjugation after
initial exposure that increased after 60 minutes of exposure (FIG.
14A). However, in the presence of trastuzumab, NK92-CD64/16A cell
and SKOV-3 conjugation was appreciably enhanced (FIG. 14A). This
increase in conjugation corresponded with higher levels of target
cell killing. As shown in FIG. 14B, SKOV-3 cell cytotoxicity by
NK92-CD64/16A cells varied depending on the trastuzumab
concentration and E:T ratio. To confirm the role of CD64/16A in the
induction of target cell killing, the ADCC assay was performed in
the presence and absence of the anti-CD64 mAb 10.1 (FIG. 14C),
which blocks IgG binding. Cytokine production is also induced
during ADCC and NK cells are major producers of IFN.gamma..
NK92-CD64/16A cells exposed to SKOV-3 cells and trastuzumab
produced considerably higher levels of IFN.gamma. than when exposed
to SKOV-3 cells alone (FIG. 14D). Taken together, the above
findings demonstrate that the CD64 component of the recombinant
receptor engages tumor-bound antibody, and that the CD16A component
promotes intracellular signaling leading to degranulation and
cytokine production.
[0062] CD64 is distinguished from the other Fc.gamma.R members by
its unique third extracellular domain, which contributes to its
high affinity and stable binding to soluble monomeric IgG. NK92
cells expressing CD64/16A or the CD16A-176V higher affinity variant
were compared for their ability to capture soluble therapeutic
mAbs. The NK92 cell transductants examined expressed similar levels
of CD64/16A and CD16A (FIG. 15A). NK92 cell transductants were
incubated with trastuzumab for two hours, excess antibody was
washed away, stained with a fluorophore-conjugated anti-human IgG
antibody, and then evaluated by flow cytometry. As shown in FIG.
15B, NK92-CD64/16A cells captured considerably higher levels of
trastuzumab than did the NK92-CD16A cells (8.1 fold
increase.+-.1.3, mean.+-.SD of three independent experiments).
Moreover, the NK92-CD64/16A cells efficiently captured the
tumor-targeting mAbs cetuximab and rituximab, as well as the fusion
protein L-selectin/Fc (FIG. 15C).
[0063] NK92-CD64/16A cells with a captured tumor-targeting mAb were
tested to determine whether the cells mediated ADCC. For this
assay, equal numbers of NK92-CD64/16A and NK92-CD16A cells were
incubated with the same concentration of soluble trastuzumab,
washed, and exposed to SKOV-3 cells. Target cell killing by
NK92-CD64/16A cells with captured trastuzumab was significantly
higher than NK92-CD64/16A cells alone, and was superior to
NK92-CD16A cells treated with or without trastuzumab at all E:T
ratios examined (FIG. 15D). In contrast, SKOV-3 cytotoxicity by
NK92-CD16A and NK92-CD64/16A cells was not significantly different
when trastuzumab was present and not washed out (FIG. 15E), thus
demonstrating equivalent cytotoxicity by both transductants. Taken
together, these findings show that NK92 cells expressing CD64/16A
can stably bind soluble anti-tumor mAbs and IgG fusion proteins,
and that these can serve as targeting elements to kill cancer
cells.
Expression and Function of CD64/16A in iPSC-Derived NK Cells
[0064] Undifferentiated iPSCs were transduced to express CD64/16A
using a Sleeping Beauty transposon plasmid for nonrandom gene
insertion and stable expression. iPSCs were differentiated into
hematopoietic cells and then iNK cells as described in the EXAMPLE
2. CD34.sup.+CD43.sup.+CD45.sup.+ cells were generated, further
differentiated into iNK cells, and these cells were expanded for
analysis using recombinant IL-2 and aAPCs. CD56.sup.+CD3.sup.- is a
hallmark phenotype of human NK cells, and these cells composed the
majority of our differentiated cell population (FIG. 16).
Expression of activating and inhibitory receptors on the iNK cells
also were assessed and compared to expression by peripheral blood
NK cells. Certain receptors, such as CD16A, were expressed by
similar proportions of the two NK cell populations. The expanded
iNK cells, however, lacked expression of the inhibitory KIR
receptors KIR2DL2/3, KIR2DL1, and KIR3DL1 and also certain
activating receptors (NKp46 and NKG2D) (FIG. 16). Another
difference compared to peripheral blood NK cells was that the iNK
cells were stained with an anti-CD64 mAb (FIG. 16), demonstrating
the expression of CD64/16A.
[0065] To assess the function of CD64/16A in iNK cells, iNK cells
derived from iPSCs transduced with either an pKT2 empty vector or
pKT2-CD64/16A were compared. The NK cell markers mentioned above
were expressed at similar levels and proportions by two iNK cell
populations (data not shown), including CD16A (FIG. 17A), but only
iNK-CD64/16A cells were stained by an anti-CD64 mAb (FIG. 17A).
Both iNK tranductants demonstrated increased SKOV-3 cell killing
when in the presence of trastuzumab, yet iNK-CD64/16A cells
mediated significantly higher levels of ADCC than did the iNK-pKT2
control cells (FIG. 17B). The anti-CD16 function blocking mAb 3G8,
but not the anti-CD64 mAb 10.1, effectively inhibited ADCC by the
iNK-pKT2 cells (FIG. 17B). Conversely, 10.1, but not 3G8, blocked
ADCC by the iNK-CD64/16A cells (FIG. 17B). These findings show that
the iNK cells were cytolytic effectors responsive to CD16A and
CD64/16A engagement of antibody-bound tumor cells.
[0066] Also, iNK-CD64/16A and iNK-pKT2 cells were treated with
soluble trastuzumab, excess antibody was washed away, and the cells
were exposed to SKOV-3 cells. Under these conditions, ADCC by the
iNK-CD64/16A cells was strikingly higher than the iNK-pKT2 cells
(FIG. 17C), further establishing that CD64/16A can capture soluble
anti-tumor mAbs that serve as a targeting element for tumor cell
killing.
[0067] Taken together, the data show that CD64/16A binds
therapeutic mAbs with higher affinity than CD16A. Moreover,
CD64/16A expressed in NK92 cells and iNK cells confers cells with
the ability to mediate higher levels of ADCC than NK92 cells and
iNK cells expressing wildtype or endogenous CD16A, respectively. NK
cells expressing CD64/16A facilitated cell conjugation with
antibody-bound tumor cells, cytotoxicity, and IFN.gamma.
production, demonstrating function by both components of the
recombinant Fc.gamma.R. NK92 cells and iNK cells expressing
CD64/16A can be pre-loaded with a therapeutic mAb prior to their
exposure to target cells. Cells expressing the chimeric receptor
and preloaded with therapeutic antibody may allow one to modify
engineered NK cells with different therapeutic mAbs for targeting
elements of multiple types of cancer. Finally, CD64/16A lacks the
ADAM17 cleavage region found in CD16A and it did not undergo the
same level of downregulation in expression during ADCC.
[0068] CD64/16A was shown to be functional in two NK cell
platforms, the NK92 cell line and primary NK cells derived from
iPSCs. NK92 cells lack inhibitory KIR receptors and show high
levels of natural cytotoxicity compared to other NK cell lines
derived from patients. NK92 cells have been broadly used to express
modified genes to direct their cytolytic effector function, have
been evaluated in preclinical studies, and are undergoing clinical
testing in cancer patients. iPSCs are also very amendable to
genetic engineering and can be differentiated into NK cells
expressing various receptors to direct their effector functions.
The iNK cells generated in this study lacked several inhibitory and
activating receptors that are indicators of an immature state. In
some applications, therapeutic iNK cells lacking inhibitory
receptors and certain activating receptors may allow for more
directed and/or effective tumor cell killing by engineered
receptors.
[0069] The iNK cells expressed endogenous CD16A and mediated ADCC,
thus they were cytotoxic effector cells. An individual NK cell can
kill multiple tumor cells in different manners. This includes by a
process of sequential contacts and degranulations, referred to as
serial killing, and by the localized dispersion of its granule
contents that kills surrounding tumor cells, referred to as
bystander killing. Inhibiting CD16A shedding has been reported to
slow NK cell detachment from target cells and reduce serial killing
by NK cells in vitro. Due to the CD64 component and its lack of
ectodomain shedding, NK cells expressing CD64/16A could be less
efficient at serial killing and more efficient at bystander
killing.
[0070] NK cells expressing CD64/16A have several potential
advantages as a therapeutic in combination with a therapeutic
antibody. Modifying NK cells expressing CD64/16A with an antibody
can reduce the dosage of therapeutic antibodies administered to
patients. Fusion proteins containing a human IgG Fc region, such as
L-selectin/Fc, also can be captured by CD64/16A and this may
provide further options for directing the tissue and tumor antigen
targeting of engineered NK cells. The NK92 and iNK cell platforms
for adoptive cell therapies also can be readily genetically
modified on a clonal level and expanded into clinical-scalable cell
numbers to produce engineered NK cells with improved effector
activities as an off-the-shelf therapeutic for cancer
immunotherapy.
[0071] In some embodiments, iPSC-derived NK cells that express
CD64/16A can exhibit increased in vivo anti-cancer activity in the
presence of therapeutic mAbs. For example,
NOD/SCID/.gamma.c.sup.-/- (NSG) mice and human cancer cell lines
that are stably engineered to express firefly luciferase can be
used for bioluminescent imaging to test iNK cell activity against
cancer cells. The SKOV-3 and MA148 ovarian cancer cell line cans
serve as model in vivo targets. NSG female mice can be subjected to
sublethal irradiation, then injected intraperitoneally with tumor
cells for bioluminescent imaging to quantify tumor growth or
regression. Mice are given IL-2 and/or IL-15 every other day for
four weeks to promote in vivo survival of the NK cells. Therapeutic
antibody (e.g., trastuzumab) can be administered--e.g., once weekly
for four weeks. Tumor growth/regression can be monitored by, for
example, bioluminescent imaging and weighing the mice. Mice also
can be bled (e.g., weekly) to quantify human NK cell survival, and
one can further evaluate the expression/cell surface levels of
various effector function markers by FACS. Evidence of metastasis
can be determined by, for example, harvesting internal organs and
examining the internal organs for metastasis.
[0072] In some embodiments, the number of cells in the therapeutic
composition is at least 0.1.times.10.sup.5 cells, at least
1.times.10.sup.5 cells, at least 5.times.10.sup.5 cells, at least
1.times.10.sup.6 cells, at least 5.times.10.sup.6 cells, at least
1.times.10.sup.7 cells, at least 5.times.10.sup.7 cells, at least
1.times.10.sup.8 cells, at least 5.times.10.sup.8 cells, at least
1.times.10.sup.9 cells, or at least 5.times.10.sup.9 cells, per
dose. In some embodiments, the number of cells in the therapeutic
composition is about 0.1.times.10.sup.5 cells to about
1.times.10.sup.6 cells, per dose; about 0.5.times.10.sup.6 cells to
about 1.times.10.sup.7 cells, per dose; about 0.5.times.10.sup.7
cells to about 1.times.10.sup.8 cells, per dose; about
0.5.times.10.sup.8 cells to about 1.times.10.sup.9 cells, per dose;
about 1.times.10.sup.9 cells to about 5.times.10.sup.9 cells, per
dose; about 0.5.times.10.sup.9 cells to about 8.times.10.sup.9
cells, per dose; about 3.times.10.sup.9 cells to about
3.times.10.sup.10 cells, per dose, or any range in-between.
Generally, 1.times.10.sup.8 cells/dose translates to
1.67.times.10.sup.6 cells/kg for a 60 kg patient.
[0073] In one embodiment, the number of cells in the therapeutic
composition is the number of immune cells in a partial or single
cord of blood, or is at least 0.1.times.10.sup.5 cells/kg of
bodyweight, at least 0.5.times.10.sup.5 cells/kg of bodyweight, at
least 1.times.10.sup.5 cells/kg of bodyweight, at least
5.times.10.sup.5 cells/kg of bodyweight, at least 10.times.10.sup.5
cells/kg of bodyweight, at least 0.75.times.10.sup.6 cells/kg of
bodyweight, at least 1.25.times.10.sup.6 cells/kg of bodyweight, at
least 1.5.times.10.sup.6 cells/kg of bodyweight, at least
1.75.times.10.sup.6 cells/kg of bodyweight, at least
2.times.10.sup.6 cells/kg of bodyweight, at least
2.5.times.10.sup.6 cells/kg of bodyweight, at least
3.times.10.sup.6 cells/kg of bodyweight, at least 4.times.10.sup.6
cells/kg of bodyweight, at least 5.times.10.sup.6 cells/kg of
bodyweight, at least 10.times.10.sup.6 cells/kg of bodyweight, at
least 15.times.10.sup.6 cells/kg of bodyweight, at least
20.times.10.sup.6 cells/kg of bodyweight, at least
25.times.10.sup.6 cells/kg of bodyweight, at least
30.times.10.sup.6 cells/kg of bodyweight, 1.times.10.sup.8 cells/kg
of bodyweight, 5.times.10.sup.8 cells/kg of bodyweight, or
1.times.10.sup.9 cells/kg of bodyweight.
[0074] In one embodiment, a dose of cells is delivered to a
subject. In one illustrative embodiment, the effective amount of
cells provided to a subject is at least 2.times.10.sup.6 cells/kg,
at least 3.times.10.sup.6 cells/kg, at least 4.times.10.sup.6
cells/kg, at least 5.times.10.sup.6 cells/kg, at least
6.times.10.sup.6 cells/kg, at least 7.times.10.sup.6 cells/kg, at
least 8.times.10.sup.6 cells/kg, at least 9.times.10.sup.6
cells/kg, or at least 10.times.10.sup.6 cells/kg, or more cells/kg,
including all intervening doses of cells.
[0075] In another illustrative embodiment, the effective amount of
cells provided to a subject is about 2.times.10.sup.6 cells/kg,
about 3.times.10.sup.6 cells/kg, about 4.times.10.sup.6 cells/kg,
about 5.times.10.sup.6 cells/kg, about 6.times.10.sup.6 cells/kg,
about 7.times.10.sup.6 cells/kg, about 8.times.10.sup.6 cells/kg,
about 9.times.10.sup.6 cells/kg, or about 10.times.10.sup.6
cells/kg, or more cells/kg, including all intervening doses of
cells.
[0076] In another illustrative embodiment, the effective amount of
cells provided to a subject is from about 2.times.10.sup.6 cells/kg
to about 10.times.10.sup.6 cells/kg, about 3.times.10.sup.6
cells/kg to about 10.times.10.sup.6 cells/kg, about
4.times.10.sup.6 cells/kg to about 10.times.10.sup.6 cells/kg,
about 5.times.10.sup.6 cells/kg to about 10.times.10.sup.6
cells/kg, 2.times.10.sup.6 cells/kg to about 6.times.10.sup.6
cells/kg, 2.times.10.sup.6 cells/kg to about 7.times.10.sup.6
cells/kg, 2.times.10.sup.6 cells/kg to about 8.times.10.sup.6
cells/kg, 3.times.10.sup.6 cells/kg to about 6.times.10.sup.6
cells/kg, 3.times.10.sup.6 cells/kg to about 7.times.10.sup.6
cells/kg, 3.times.10.sup.6 cells/kg to about 8.times.10.sup.6
cells/kg, 4.times.10.sup.6 cells/kg to about 6.times.10.sup.6
cells/kg, 4.times.10.sup.6 cells/kg to about 7.times.10.sup.6
cells/kg, 4.times.10.sup.6 cells/kg to about 8.times.10.sup.6
cells/kg, 5.times.10.sup.6 cells/kg to about 6.times.10.sup.6
cells/kg, 5.times.10.sup.6 cells/kg to about 7.times.10.sup.6
cells/kg, 5.times.10.sup.6 cells/kg to about 8.times.10.sup.6
cells/kg, or 6.times.10.sup.6 cells/kg to about 8.times.10.sup.6
cells/kg, including all intervening doses of cells.
[0077] Some variation in dosage will necessarily occur depending on
the condition of the subject being treated. The person responsible
for administration will, in any event, determine the appropriate
dose for the individual subject.
[0078] In some embodiments, the therapeutic use of cells is a
single-dose treatment. In some embodiments, the therapeutic use of
derived hematopoietic lineage cells is a multi-dose treatment. In
some embodiments, the multi-dose treatment is one dose every day,
every 3 days, every 7 days, every 10 days, every 15 days, every 20
days, every 25 days, every 30 days, every 35 days, every 40 days,
every 45 days, or every 50 days, or any number of days
in-between.
[0079] A compositions that includes a population of cells described
herein can be sterile, and can be suitable and ready for
administration (i.e., can be administered without any further
processing) to human patients. A cell-based composition that is
ready for administration means that the composition does not
require any further processing or manipulation prior to transplant
or administration to a subject. In some embodiments, such a
population of cells can include an isolated population of cells
that are expanded and/or modulated prior to administration with one
or more agents.
[0080] In certain embodiments, the primary stimulatory signal and
the co-stimulatory signal for the therapeutic cells can be provided
by different protocols. For example, the agents providing each
signal can be in solution or coupled to a surface. When coupled to
a surface, the agents can be coupled to the same surface (i.e., in
"cis" formation) or to separate surfaces (i.e., in "trans"
formation). Alternatively, one agent can be coupled to a surface
and the other agent in solution. In one embodiment, the agent
providing the co-stimulatory signal can be bound to a cell surface
and the agent providing the primary activation signal is in
solution or coupled to a surface. In certain embodiments, both
agents can be in solution. In another embodiment, the agents can be
in soluble form, and then cross-linked to a surface, such as a cell
expressing Fc receptors or an antibody or other binding agent that
will bind to the agents such as disclosed in U.S. Patent
Application Publication Nos. 20040101519 and 20060034810 for
artificial antigen presenting cells (aAPCs) that are contemplated
for use in activating and expanding T lymphocytes.
[0081] The therapeutic compositions suitable for administration to
a patient can include one or more pharmaceutically acceptable
carriers (additives) and/or diluents (e.g., pharmaceutically
acceptable medium, for example, cell culture medium), or other
pharmaceutically acceptable components. Pharmaceutically acceptable
carriers and/or diluents are determined in part by the particular
composition being administered, as well as by the particular method
used to administer the therapeutic composition. Accordingly, there
is a wide variety of suitable formulations of therapeutic
compositions (see, e.g., Remington's Pharmaceutical Sciences,
17.sup.th ed. 1985, the disclosure of which is hereby incorporated
by reference in its entirety) well known to those of skill in the
art.
[0082] In particular embodiments, therapeutic cell compositions
having an isolated population the cells as described herein also
have a pharmaceutically acceptable cell culture medium, or
pharmaceutically acceptable carriers and/or diluents. A therapeutic
composition comprising a population of the cells as disclosed
herein can be administered separately by intravenous,
intraperitoneal, enteral, or tracheal administration methods or in
combination with other suitable compounds to affect the desired
treatment goals.
[0083] These pharmaceutically acceptable carriers and/or diluents
can be present in amounts sufficient to maintain a pH of the
therapeutic composition of between about 3 and about 10. As such,
the buffering agent can be as much as about 5% on a weight to
weight basis of the total composition. Electrolytes such as, but
not limited to, sodium chloride and potassium chloride can also be
included in the therapeutic composition. In one aspect, the pH of
the therapeutic composition is in the range from about 4 to about
10. Alternatively, the pH of the therapeutic composition is in the
range from about 5 to about 9, from about 6 to about 9, or from
about 6.5 to about 8. In another embodiment, the therapeutic
composition includes a buffer having a pH in one of said pH ranges.
In another embodiment, the therapeutic composition has a pH of
about 7. Alternatively, the therapeutic composition has a pH in a
range from about 6.8 to about 7.4. In still another embodiment, the
therapeutic composition has a pH of about 7.4.
[0084] This disclosure also provides the use of a pharmaceutically
acceptable cell culture medium in particular compositions and/or
cultures of cells as described herein. Such compositions are
suitable for administration to human subjects. Generally speaking,
any medium that supports the maintenance, growth, and/or health of
the iPSC-derived immune cells are suitable for use as a
pharmaceutical cell culture medium. In particular embodiments, the
pharmaceutically acceptable cell culture medium is a serum free,
and/or feeder-free medium. In various embodiments, the serum-free
medium is animal-free, and can optionally be protein-free.
Optionally, the medium can contain biopharmaceutically acceptable
recombinant proteins. Animal-free medium refers to medium wherein
the components are derived from non-animal sources. Recombinant
proteins replace native animal proteins in animal-free medium and
the nutrients are obtained from synthetic, plant or microbial
sources. Protein-free medium, in contrast, is defined as
substantially free of protein. One having ordinary skill in the art
would appreciate that the above examples of media are illustrative
and in no way limit the formulation of suitable media suitable and
that there are many alternative suitable media known and available
to those in the art.
[0085] In the preceding description and following claims, the term
"and/or" means one or all of the listed elements or a combination
of any two or more of the listed elements; the terms "comprises,"
"comprising," and variations thereof are to be construed as open
ended--i.e., additional elements or steps are optional and may or
may not be present; unless otherwise specified, "a," "an," "the,"
and "at least one" are used interchangeably and mean one or more
than one; and the recitations of numerical ranges by endpoints
include all numbers subsumed within that range (e.g., 1 to 5
includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
[0086] In the preceding description, particular embodiments may be
described in isolation for clarity. Unless otherwise expressly
specified that the features of a particular embodiment are
incompatible with the features of another embodiment, certain
embodiments can include a combination of compatible features
described herein in connection with one or more embodiments.
[0087] For any method disclosed herein that includes discrete
steps, the steps may be conducted in any feasible order. And, as
appropriate, any combination of two or more steps may be conducted
simultaneously.
[0088] The present invention is illustrated by the following
examples. It is to be understood that the particular examples,
materials, amounts, and procedures are to be interpreted broadly in
accordance with the scope and spirit of the invention as set forth
herein.
EXAMPLES
Example 1
Generation of Human CD64/16A Expression Constructs
[0089] Total RNA was isolated from peripheral blood leukocytes
(PBL) using TRIzol total RNA isolation reagent (Invitrogen, Thermo
Fisher Scientific, Carlsbad, Calif.). Human PBL cDNA was
synthesized with the SuperScript Preamplification System
(Invitrogen, Thermo Fisher Scientific, Carlsbad, Calif.). The
CD64/16A includes a human CD64 extracellular domain (CD64-EC) and
CD16A transmembrane and cytoplasmic domains (CD16A-TM-CY). The
chimeric construct was generated by overlapping PCR to create EcoR
I-flanked RT-PCR products of chimeric cDNA. CD64-EC cDNA fragment
(885 bps) was amplified from human PBL cDNA using the forward
primer 5'-CGG GAA TTC GGA GAC AAC ATG TGG TTC TTG ACA A-3'(SEQ ID
NO:28) and reverse primer 5'-TTG GTA CCC AGG TGG AAA GAA GCC AAG
CAC TTG AAG CTC CAA-3'; SEQ ID NO:29). CD16A-TM-CY cDNA fragment
(195 bps) was amplified from human PBL cDNA using the forward
primer 5'-TTG GAG CTT CAA GTG CTT GGC TTC TTT CCA CCT GGG TAC
CAA-3' (SEQ ID NO:30) and reverse primer 5'-CCG GAA TTC TCA TTT GTC
TTG AGG GTC CTT TCT-3' (SEQ ID NO:31). The PCR fragments of CD64-EC
cDNA and CD16A-TM-CY cDNA were purified with QIAquick gel
extraction kit (Qiagen, Hilden, Germany) and mixed together with
the forward primer 5'-CGG GAA TTC GGA GAC AAC ATG TGG TTC TTG ACA
A-3' (SEQ ID NO:32) and the reverse primer 5'-CCG GAA TTC TCA TTT
GTC TTG AGG GTC CTT TCT-3' (SEQ ID NO:33). For all primers listed
above, underlined nucleotides are the EcoR I cutting site) to
generate RT-PCR products for the chimeric receptor. RT-PCR was
performed with Pfx50 DNA polymerase (Invitrogen, Thermo Fisher
Scientific, Carlsbad, Calif.). The resultant CD64/CD16a cDNA was
inserted into retrovirus expression vector pBMN-IRES-EGFP (Addgene,
Cambridge, Mass.) and pKT2 Sleeping Beauty transposon vector (Jing
et al. 2015. PLoS One 10:e0121788; Hermanson et al. 2016. Stem
Cells 34:93-101). The sequences of all cloned constructs were
confirmed by direct sequencing from both directions on an ABI 377
sequencer with ABI BigDye terminator cycle sequencing kit (Applied
Biosystems, Thermo Fisher Scientific, Foster City, Calif.).
Stable Expression of CD64/16A in NK Cells
[0090] NK92 cells, a human NK cell line that is deficient for
endogenous CD16A expression, were stably transduced with
pBMN-IRES-EGFP retrovirus expression constructs containing CD64/16A
or wildtype CD16A cDNA using retrovirus infection procedures
described previously (Jing et al. 2015. PLoS One 10:e0121788).
Human iPSCs (UCBiPS7, derived from umbilical cord blood CD34 cells)
were stably transduced with the CD64/16A-pKT2 expression construct
using a Sleeping Beauty transposon system, as previously described
(Jing et al. 2015. PLoS One 10:e0121788). The transduced iPSC cells
were differentiated into hematopoietic cells then mature NK cell as
previously described (Jing et al. 2015. PLoS One 10:e0121788). eGFP
fluorescence and surface expressions of CD64, CD16, and various NK
cell phenotypic markers were determine using flow cytometry
analysis.
Example 2
Antibodies
[0091] All mAbs to human hematopoietic and leukocyte phenotypic
markers are described in Table 2. All isotype-matched negative
control mAbs were purchased from BioLegend (San Diego, Calif.).
APC-conjugated F(ab').sub.2 donkey anti-human or goat anti-mouse
IgG (H+L) were purchased from Jackson ImmunoResearch Laboratories
(West Grove, Pa.). The human IgG1 mAbs trastuzumab/Herceptin and
rituximab/Rituxan, manufactured by Genentech (South San Francisco,
Calif.), and cetuximab/Erbitux, manufactured by Bristol-Myers
Squibb (Lawrence, N.J.), were purchased through the University of
Minnesota Boynton Pharmacy. Recombinant human L-selectin/IgG1 Fc
chimera was purchased from R&D Systems (Minneapolis,
Minn.).
TABLE-US-00002 TABLE 2 Antibodies Antigen Clone Fluorophore Company
CD56 HCD56 PE-CY7 BioLegend, San Diego, CA CD3 HIT3a PE BioLegend
CD16 3G8 APC BioLegend CD16 3G8 none Ancell, Bayport, MN CD7
CD7-6B7 PE/CY5 BioLegend CD336/NKp44 P44-8 APC BioLegend
CD335/NKp46 9E2 APC BioLegend CD159a/NKG2A Z199 APC Beckman
Coulter, Brea, CA CD314/NKG2D 1D11 PerCP/Cy5.5 BioLegend
CD158a/KIR2DL1 HP-MA4 PE BioLegend CD158b1/KIR2DL2/L3 DX27 PE
BioLegend CD158e1/KIR3DL1 DX9 PE BioLegend CD94 DX22 PE BioLegend
CD64 10.1 APC BioLegend CD64 10.1 none Ancell CD34 561 PE BioLegend
CD45 2D1 APC BioLegend CD43 CD43-10G7 APC BioLegend
CD62L/L-selectin LAM1-116 APC Ancell
Generation of Human CD64/16A
[0092] Total RNA was isolated from human peripheral blood
leukocytes using TRIzol total RNA isolation reagent (Invitrogen,
Carlsbad, Calif.) and cDNA was synthesized with the SuperScript
preamplification system (Invitrogen, Carlsbad, Calif.). The
recombinant CD64/16A is comprised of human CD64 extracellular
domain and CD16A transmembrane and cytoplasmic domains. PCR
fragments for CD64 (885 bps) and CD16A (195 bps) were amplified
from the generated cDNA. The PCR fragments were purified and mixed
together with the forward primer 5'-CGG GAA TTC GGA GAC AAC ATG TGG
TTC TTG ACA A-3' (SEQ ID NO:28), the reverse primer 5'-CCG GAA TTC
TCA TTT GTC TTG AGG GTC CTT TCT-3' (SEQ ID NO: 31), and Pfx50 DNA
polymerase (Invitrogen) to generate the recombinant CD64/16A
receptor. For both primers, underlined nucleotides are EcoR I
sites. CD64/CD16A and CD16A cDNA (CD16A-176V variant) was inserted
into the retroviral expression vector pBMN-IRES-EGFP and virus was
generated for NK92 cell transduction, as previously described (Jing
et al., 2015. PLoS One 10(3):e0121788). Additionally, CD64/CD16A
cDNA was inserted into the pKT2 sleeping beauty transposon vector
and used along with SB100X transposase for iPSC transduction, as
previously described (Jing et al., 2015. PLoS One 10(3):e0121788).
The nucleotide sequences of all constructs were confirmed by direct
sequencing from both directions on an ABI 377 sequencer with ABI
BigDye terminator cycle sequencing kit (Applied Biosystems, Foster
City, Calif.).
Cells
[0093] This study was carried out in accordance with and approved
by the Institutional Review Board at the University of Minnesota.
All subjects gave written informed consent in accordance with the
Declaration of Helsinki. Fresh human peripheral blood leukocytes
from plateletpheresis were obtained from Innovative Blood Resources
(St. Paul, Minn.). Peripheral blood mononuclear cells were further
enriched using Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB,
Uppsala, Sweden) and NK cells were purified by negative depletion
using an EasySep human NK cell kit (StemCell Technologies,
Cambridge, Mass.), as per the manufacturer's instructions, with
>95% viability and 90-95% enrichment of CD56.sup.+CD3.sup.-
lymphocytes. Viable cell counting was performed using a Countess II
automated cell counter (Life Technologies Corporation, Bothell,
Wash.). The human NK cell line NK92 and the ovarian cancer cell
line SKOV-3 were obtained from ATCC (Manassas, Va.) and cultured
per the manufacturer's directions. The NK92 cells required IL-2 for
growth (500 IU/ml), which was obtained from R&D Systems
(Minneapolis, Minn.). For all assays described below, cells were
used when in log growth phase.
[0094] The iPSCs UCBiPS7, derived from umbilical cord blood CD34
cells, have been previously characterized and were cultured and
differentiated into hematopoietic progenitor cells as described
with some modifications (Jing et al., 2015. PLoS One 10:e0121788;
Ni et al., 2013. Methods in molecular biology 1029:33-41; Knorr et
al., 2013. Stem Cells Transl Med 2(4):274-283; Ni et al., 2014.
Stem Cells 32(4):1021-1031; Hermanson et al., 2016. Stem Cells
34(1):93-101). iPSCs culture and hematopoietic differentiation was
performed using TeSR-E8 medium and a STEMdiff Hematopoietic Kit
(StemCell Technologies, Cambridge, Mass.), which did not require
the use of mouse embryonic fibroblast feeder cells, TrypLE
adaption, spin embryoid body formation, or CD34.sup.+ cell
enrichment. iPSC cells during passage were dissociated with Gentle
Cell Dissociation Reagent (StemCell Technologies, Cambridge,
Mass.), and iPSC aggregates .gtoreq.50 .mu.m in diameter were
counted with a hemocytometer and diluted to 20-40 aggregates/ml
with TeSR-E8 medium. Each well of a 12-well plate was pre-coated
with Matrigel Matrix (Corning Inc., Tewksbury, Mass.) and seeded
with 40-80 aggregates in 2 ml of TeSR-E8 medium. Cell aggregates
were cultured for 24 hours before differentiation with the STEMdiff
Hematopoietic Kit (StemCell Technologies, Cambridge, Mass.)
according to the manufacturer's instructions. At day 12 of
hematopoietic progenitor cell differentiation, the percentage of
hematopoietic progenitor cells was determined using flow cytometric
analysis with anti-CD34, anti-CD45, and anti-CD43 mAbs. NK cell
differentiation was performed as previously described (Woll et al.,
2009. Blood 113(24):6094-6101). The iPSC-derived NK cells (referred
to here as iNK cells) were expanded for examination using
.gamma.-irradiated K562-mbIL21-41BBL feeder cells (1:2 ratio) in
cell expansion medium [60% DMEM, 30% Ham's F12, 10% human AB serum
(Valley Biomedical, Winchester, Va.), 20 .mu.M 2-mercaptoethanol,
50 .mu.M ethanolamine, 20 .mu.g/ml ascorbic Acid, 5 ng/ml sodium
selenite, 10 mM HEPES, and 100-250 IU/ml IL-2 (R&D Systems)],
as described previously (Jing et al., 2015. PLoS One 10:e0121788;
Knorr et al., 2013. Stem Cells Transl Med 2(4):274-283; Ni et al.,
2014. Stem Cells 32(4):1021-1031; Hermanson et al., 2016. Stem
Cells 34(1):93-101).
Cell Staining, Flow Cytometry, and ELISA
[0095] For cell staining, nonspecific antibody binding sites were
blocked and cells were stained with the indicated antibodies and
examined by flow cytometry, as previously described (Romee et al,
2013. Blood 121(18):3599-3608; Jing et al., 2015. PLoS One
10:e0121788; Mishra et al., 2018. Cancer Immunol Immunother
67(9):1407-1416). For controls, fluorescence minus one was used as
well as appropriate isotype-matched antibodies since the cells of
interest expressed FcRs. An FSC-A/SSC-A plot was used to set an
electronic gate on leukocyte populations and an FSC-A/F SC-H plot
was used to set an electronic gate on single cells. A Zombie
viability kit was used to assess live vs. dead cells, as per the
manufacturer's instructions (BioLegend, San Diego, Calif.).
[0096] To assess the capture of soluble trastuzumab, rituximab,
cetuximab, or L-selectin/Fc chimera, transduced NK cells were
incubated with 5 .mu.g/ml of antibody for two hours at 37.degree.
C. in MEM-.alpha. basal media (Thermo Fisher Scientific, Waltham,
Mass.) supplemented with IL-2 (200 IU/ml), HEPES (10 mM), and
2-mercaptoethanol (0.1 mM), washed with MEM-.alpha. basal media,
and then stained on ice for 30 minutes with a 1:200 dilution of
APC-conjugated F(ab')2 donkey anti-human IgG (H+L). To detect
recombinant human L-selectin/Fc binding, cells were stained with
the anti-L-selectin mAb APC-conjugated Lam1-116.
[0097] To compare CD16A, CD64, and CD64/16A staining levels on NK92
cells, the respective transductants were stained with a saturating
concentration of unconjugated anti-CD16 (3G8) or anti-CD64 (10.1)
mAbs (5 .mu.g/ml), washed extensively with dPBS (USB Corporation,
Cleveland, Ohio) containing 2% goat serum and 2 mM sodium azide,
and then stained with APC-conjugated F(ab')2 goat anti-mouse IgG
(H+L). This approach was used since directly conjugated anti-CD16
and anti-CD64 mAbs can vary in their levels of fluorophore
labeling. ELISA was performed by a cytometric bead-based Flex Set
assay for human IFN.gamma. (BD Biosciences, San Jose, Calif.) per
the manufacturer's instructions. All flow cytometric analyses were
performed on a FACSCelesta instrument (BD Biosciences). Data was
analyzed using FACSDIVA v8 (BD Biosciences) and FlowJo v10
(Ashland, Oreg.).
Cell-Cell Conjugation Assay and ADCC
[0098] The pBMN-IRES-EGFP vector used to express CD64/16A in NK92
cells also expresses eGFP. These cells were incubated for two hours
at 37.degree. C. in MEM-.alpha. basal media (Thermo Fisher
Scientific, Waltham, Mass.) supplemented with IL-2 (200 IU/ml),
HEPES (10 mM), and 2-mercaptoethanol (0.1 mM). SKOV-3 cells were
labeled with CellTrace Violet (Molecular Probes, Eugene, Oreg.) per
the manufacturer's instructions, incubated with 5 .mu.g/ml
trastuzumab for 30 minutes and washed with the MEM-.alpha. basal
media. NK92 cells and SKOV-3 cells were then resuspended in the
supplemented MEM-.alpha. basal media at 1.times.10.sup.6 and
2.times.10.sup.6/ml, respectively. For a 1:2 E:T ratio, 100 .mu.l
of each cell type was mixed together, centrifuged for one minute at
20.times.g and incubated at 37.degree. C. for the indicated time
points. After each time point, the cells were gently vortexed for
three seconds and immediately fixed with 4.degree. C. 1%
paraformaldehyde in dPBS. Samples were immediately analyzed by flow
cytometry. The percentage of conjugated NK cells was calculated by
gating on eGFP and CellTrace Violet double-positive events.
[0099] To evaluate ADCC, a DELFIA EuTDA-based cytotoxicity assay
was used according to the manufacturer's instructions (PerkinElmer,
Waltham, Mass.). Briefly, target cells were labeled with
Bis(acetoxymethyl)-2-2:6,2 terpyridine 6,6 dicarboxylate (BATDA)
for 30 minutes in their culture medium, washed in culture medium,
and pipetted into a 96-well non-tissue culture-treated U-bottom
plates at a density of 8.times.10.sup.4 cells/well. A tumor
targeting mAb was added at the indicated concentrations of 5
.mu.g/mL and NK cells were added at the indicated effector:target
(E:T) ratios. The plates were centrifuged at 400.times.g for one
minute and then incubated for two hours in a humidified 5% CO.sub.2
atmosphere at 37.degree. C. At the end of the incubation, the
plates were centrifuged at 500.times.g for five minutes and
supernatants were transferred to a 96-well DELFIA Yellow Plate
(PerkinElmer, Waltham, Mass.) and combined with europium.
Fluorescence was measured by time-resolved fluorometry using a BMG
Labtech CLARIOstar plate reader (Cary, N.C.). BATDA-labeled target
cells alone with or without therapeutic antibodies were cultured in
parallel to assess spontaneous lysis and in the presence of 2%
Triton-X to measure maximum lysis. The level of ADCC for each
sample was calculated using the formula: Percent Specific
Release=(Experimental release counts-Spontaneous release
counts)/(Maximal release-Spontaneous release counts).times.100%.
For each experiment, measurements were conducted in triplicate
using three replicate wells.
Statistical Analyses
[0100] Statistical analyses were performed by use of GraphPad Prism
(GraphPad Software, La Jolla, Calif., USA). After assessing for
approximate normal distribution, all variables were summarized as
mean.+-.SD. Comparison between two groups was done with Student's
t-test, with p<0.05 taken as statistically significant.
[0101] The complete disclosure of all patents, patent applications,
and publications, and electronically available material (including,
for instance, nucleotide sequence submissions in, e.g., GenBank and
RefSeq, and amino acid sequence submissions in, e.g., SwissProt,
PIR, PRF, PDB, and translations from annotated coding regions in
GenBank and RefSeq) cited herein are incorporated by reference in
their entirety. In the event that any inconsistency exists between
the disclosure of the present application and the disclosure(s) of
any document incorporated herein by reference, the disclosure of
the present application shall govern. The foregoing detailed
description and examples have been given for clarity of
understanding only. No unnecessary limitations are to be understood
therefrom. The invention is not limited to the exact details shown
and described, for variations obvious to one skilled in the art
will be included within the invention defined by the claims.
[0102] Unless otherwise indicated, all numbers expressing
quantities of components, molecular weights, and so forth used in
the specification and claims are to be understood as being modified
in all instances by the term "about." Accordingly, unless otherwise
indicated to the contrary, the numerical parameters set forth in
the specification and claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to
limit the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques.
[0103] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. All numerical values, however,
inherently contain a range necessarily resulting from the standard
deviation found in their respective testing measurements.
[0104] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
TABLE-US-00003 SEQUENCE LISTING FREE TEXT rCD64#1 (pCDH-CD64)
peptide sequence (SEQ ID NO: 9) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW
VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS
GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN
SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL
SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL
KRSPELELQV LGLQLPTPVW FHVLFYLAVG IMFLVNTVLW VTIRKELKRK KKWDLEISLD
SGHEKKVISS LQEDRHLEEE LKCQEQKEEQ LQEGVHRKEP QGAT rCD64#2
(pCDH-mutCD64) peptide sequence (SEQ ID NO: 10) MWFLTTLLLW
VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY
RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK
LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP
APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA
RREDSGLYWC EAATEDGNVL KRSPELELQV LGLQLPTPVW FHVLFYLAVG IMFLVNTVLW
VTIRKELKRK KKWNLEISLD SGHEKKVISS LQEDRHLEEE LKCQEQKEEQ LQEGVHRKEP
QGAT rCD64#3 (pCDH-CD64/16) peptide sequence (SEQ ID NO: 11)
MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG
TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL
ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG
ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN
TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF
AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDK rCD64#4
(pCDH-CD64/16-28-BB-Z) peptide sequence (SEQ ID NO: 12) MWFLTTLLLW
VPVDGQVDTT KAVISLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY
RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK
LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP
APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA
RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV
KTNIRSSTRD WKDHKFKWRK DPQDKRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD
FAAYRSKRGR KKLLYIFKQP FMRPVQTTQE EDGCSCRFPE EEEGGCELRV KFSRSADAPA
YQQGQNQLYN ELNLGRREEY DVLDKRRGRD PEMGGKPRRK NPQEGLYNEL QKDKMAEAYS
EIGMKGERRR GKGHDGLYQG LSTATKDTYD ALHMQALPPR rCD64#5
(pCDH-CD64/16-28-BB-G) peptide sequence (SEQ ID NO: 13) MWFLTTLLLW
VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY
RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK
LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP
APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA
RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV
KTNIRSSTRD WKDHKFKWRK DPQDKRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD
FAAYRSKRGR KKLLYIFKQP FMRPVQTTQE EDGCSCRFPE EEEGGCELRL KIQVRKAAIT
SYEKSDGVYT GLSTRNQETY ETLKHEKPPQ rCD64#6 (pCDH-CD64/16-28-BB-D10)
peptide sequence (SEQ ID NO: 14) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW
VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS
GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN
SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL
SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL
KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK
DPQDKRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKRGR KKLLYIFKQP
FMRPVQTTQE EDGCSCRFPE EEEGGCELAR PRRSPAQEDG KVYINMPGRG rCD64#7
(pCDH-CD64/16-28-BB-D12) peptide sequence (SEQ ID NO: 15)
MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG
TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL
ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG
ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN
TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF
AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDKRSKRS RLLHSDYMNM TPRRPGPTRK
HYQPYAPPRD FAAYRSKRGR KKLLYIFKQP FMRPVQTTQE EDGCSCRFPE EEEGGCELGR
LVPRGRGAAE AATRKQRITE TESPYQELQG QRSDVYSDLN TQRPYYK rCD64#8
(pCDH-CD64-28-28-BB-Z) peptide sequence (SEQ ID NO: 16) MWFLTTLLLW
VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY
RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK
LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP
APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA
RREDSGLYWC EAATEDGNVL KRSPELELQV LGLQLPTPFW VLVVVGGVLA CYSLLVTVAF
IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKRGR KKLLYIFKQP
FMRPVQTTQE EDGCSCRFPE EEEGGCELRV KFSRSADAPA YQQGQNQLYN ELNLGRREEY
DVLDKRRGRD PEMGGKPRRK NPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG
LSTATKDTYD ALHMQALPPR rCD64#9 (pCDH-CD64-FceR) peptide sequence
(SEQ ID NO: 17) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT
LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI
HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI
SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR
PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV
LGAPREKYWL QFFIPLLVVI LFAVDTGLFI STQQQVTFLL KIKRTRKGFR LLNPHPKPNP
KNN rCD64#10 (pCDH-CD64/16-PKC.sup.-) peptide sequence (SEQ ID NO:
18) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG
SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS
SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG
MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY
MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV
SFCLVMVLLF AVDTGLYFSV KTNIRGAGRD WKDHKFKWRK DPQDK rCD64#11
(pCDH-CD64-G2D-2B4-Z) peptide sequence (SEQ ID NO: 19) MWFLTTLLLW
VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY
RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK
LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP
APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA
RREDSGLYWC EAATEDGNVL KRSPELELQV LGSNLFVASW IAVMIIFRIG MAVAIFCCFF
FPSGGSGGGS GWRRKRKEKQ SETSPKEFLT IYEDVKDLKT RRNHEQEQTF PGGGSTIYSM
IQSQSSAPTS QEPAYTLYSL IQPSRKSGSR KRNHSPSFNS TIYEVIGKSQ PKAQNPARLS
RKELENFDVY SGGSGGGSGR VKFSRSADAP AYKQGQNQLY NELNLGRREE YDVLDKRRGR
DPEMGGKPRR KNPQEGLYNE LQKDKMAEAY SEIGMKGERR RGKGHDGLYQ GLSTATKDTY
DALHMQALPP R rCD64#12 (pCDH-CD64/16-2B4-Z) peptide sequence (SEQ ID
NO: 20) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG
SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS
SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG
MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY
MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV
SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDKWRRKR KEKQSETSPK
EFLTIYEDVK DLKTRRNHEQ EQTFPGGGST IYSMIQSQSS APTSQEPAYT LYSLIQPSRK
SGSRKRNHSP SFNSTIYEVI GKSQPKAQNP ARLSRKELEN FDVYSGGSGG GSGRVKFSRS
ADAPAYKQGQ NQLYNELNLG RREEYDVLDK RRGRDPEMGG KPRRKNPQEG LYNELQKDKM
AEAYSEIGMK GERRRGKGHD GLYQGLSTAT KDTYDALHMQ ALPPR rCD64#13
(pCDH-CD64/16-2B4-G) peptide sequence (SEQ ID NO: 21) MWFLTTLLLW
VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY
RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK
LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP
APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA
RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV
KTNIRSSTRD WKDHKFKWRK DPQDKWRRKR KEKQSETSPK EFLTIYEDVK DLKTRRNHEQ
EQTFPGGGST IYSMIQSQSS APTSQEPAYT LYSLIQPSRK SGSRKRNHSP SFNSTIYEVI
GKSQPKAQNP ARLSRKELEN FDVYSGGSGG GSGRLKIQVR KAAITSYEKS DGVYTGLSTR
NQETYETLKH EKPPQ rCD64#14 (pCDH-CD64/16-2B4-D10) peptide sequence
(SEQ ID NO : 22) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT
LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI
HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI
SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR
PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV
LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDKWRRKR
KEKQSETSPK EFLTIYEDVK DLKTRRNHEQ EQTFPGGGST IYSMIQSQSS APTSQEPAYT
LYSLIQPSRK SGSRKRNHSP SFNSTIYEVI GKSQPKAQNP ARLSRKELEN FDVYSGGSGG
GSGARPRRSP AQEDGKVYIN MPGRG rCD64#15 (pCDH-CD64/16-2B4-D12) peptide
sequence (SEQ ID NO: 23) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW
VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS
GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN
SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL
SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL
KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK
DPQDKWRRKR KEKQSETSPK EFLTIYEDVK DLKTRRNHEQ EQTFPGGGST IYSMIQSQSS
APTSQEPAYT LYSLIQPSRK SGSRKRNHSP SFNSTIYEVI GKSQPKAQNP ARLSRKELEN
FDVYSGGSGG GSGGRLVPRG RGAAEAATRK QRITETESPY QELQGQRSDV YSDLNTQRPY
YK rCD64#16 (pCDH-CD64-64-2B4-Z) peptide sequence (SEQ ID NO: 24)
MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG
TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL
ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG
ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN
TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGLQLPTPVW FHVLFYLAVG
IMFLVNTVLW VTIRKELKRK KKWDLEISLD SGHEKKVISS LQEDRHLEEE LKCQEQKEEQ
LQEGVHRKEP QGATGWRRKR KEKQSETSPK EFLTIYEDVK DLKTRRNHEQ EQTFPGGGST
IYSMIQSQSS APTSQEPAYT LYSLIQPSRK SGSRKRNHSP SFNSTIYEVI GKSQPKAQNP
ARLSRKELEN FDVYSGGSGG GSGRVKFSRS ADAPAYKQGQ NQLYNELNLG RREEYDVLDK
RRGRDPEMGG KPRRKNPQEG LYNELQKDKM AEAYSEIGMK GERRRGKGHD GLYQGLSTAT
KDTYDALHMQ ALPPR Canine CD16#17 (pCDH-caCD16) peptide sequence (SEQ
ID NO: 25) MWQLVSSTAL LLLVSAGTQA DVPKAVVVLE PKWNRVLTMD SVTLKCQGDH
LLRDNYTWLH NGRPISNQIS TYIIKNASIK NSGEYRCQTD QSKLSDPVQL EVHTGWLLLQ
VPRLVFQEGE LIQLKCHSWK NTPVRNVQYF QNGRGKKFFY NNSEYHIPAA TSEHNGSYFC
RGIIGKKNES SEAVNIIIQG SSLPSTSLLL SHWPQIPFSL VMALLFAVDT GLYFAVQRDL
RSSMGNLKNS KVIWSQGS Canine CD64#18 (pCDH-caCD64) peptide sequence
(SEQ ID NO: 26) MWLLTVLLLW VPAGAQTDPV KAVITLQPPW VSVFQEESVT
LWCEGPHLPG DSSTQWFLNG TATQTLTPRY RIAAASVNDN GEYRCQTGLS VLSDPIQLGI
HRDWLILQVS GRVFTEGEPL TLRCHGWNNK LVYNVLFYQN GTVLKFSPQN SEFTILKTTL
HHNGIYHCSA MGKHRYESAG VSITIKELFP APVLKASLSS PILEGHVVNL SCETKLLLQR
PGLQLYFSFY MGSKTLLSRN TSSEYQILTA KKEDSGLYWC EATTEDGNVV KRSPELELQV
VGPQTLTPVW FHVLFYVAMG MIFLVDTIFC MIIHKELQRK KKWNLEISLY SGLEKRVDSY
LQKERDLEEP KYQELEQLQE KTPQKPPEGE QQ Canine CD64sp#19
(pCDH-caCD64sp) peptide sequence (SEQ ID NO: 27) MWLLTVLLLW
VPAGAQTDWL ILQVSGRVFT EGEPLTLRCH GWNNKLVYNV LFYQNGTVLK FSPQNSEFTI
LKTTLHHNGI YHCSAMGKHR YESAGVSITI KELFPAPVLK ASLSSPILEG HVVNLSCETK
LLLQRPGLQL YFSFYMGSKT LLSRNTSSEY QILTAKKEDS GLYWCEATTE DGNVVKRSPE
LELQVVGPQT LTPVWFHVLF YVAMGMIFLV DTIFCMIIHK ELQRKKKWNL EISLYSGLEK
RVDSYLQKER DLEEPKYQEL EQLQEKTPQK PPEGEQQ
Sequence CWU 1
1
331335PRTArtificialPolypeptide 1Met Trp Phe Leu Thr Thr Leu Leu Leu
Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val Ile
Ser Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr Val
Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser Thr
Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro Ser
Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu Tyr
Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln Leu
Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys
Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu
Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser
Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val
Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn Ala Ser
Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr Leu Ser
Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230
235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser
Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val
Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Phe
Phe Pro Pro Gly Tyr 275 280 285Gln Val Ser Phe Cys Leu Val Met Val
Leu Leu Phe Ala Val Asp Thr 290 295 300Gly Leu Tyr Phe Ser Val Lys
Thr Asn Ile Arg Ser Ser Thr Arg Asp305 310 315 320Trp Lys Asp His
Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 325 330
3352374PRTArtificialPolypeptide 2Met Trp Phe Leu Thr Thr Leu Leu
Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val
Ile Ser Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr
Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser
Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro
Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu
Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln
Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys
Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu
Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser
Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val
Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn Ala Ser
Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr Leu Ser
Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230
235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser
Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val
Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu
Gln Leu Pro Thr Pro 275 280 285Val Trp Phe His Val Leu Phe Tyr Leu
Ala Val Gly Ile Met Phe Leu 290 295 300Val Asn Thr Val Leu Trp Val
Thr Ile Arg Lys Glu Leu Lys Arg Lys305 310 315 320Lys Lys Trp Asp
Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys 325 330 335Val Thr
Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys 340 345
350Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys
355 360 365Glu Pro Gln Gly Ala Thr 3703449PRTArtificialPolypeptide
3Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5
10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu
Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys
Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe
His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile
Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln
Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His
Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys
Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn
Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly
Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155
160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe
165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile
Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro
Pro Gly Tyr Gln 195 200 205Val Ser Phe Cys Leu Val Met Val Leu Leu
Phe Ala Val Asp Thr Gly 210 215 220Leu Tyr Phe Ser Val Lys Thr Asn
Ile Arg Ser Ser Thr Arg Asp Trp225 230 235 240Lys Asp His Lys Phe
Lys Trp Arg Lys Asp Pro Gln Asp Lys Arg Ser 245 250 255Lys Arg Ser
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg 260 265 270Arg
Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 275 280
285Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr
290 295 300Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
Glu Glu305 310 315 320Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
Glu Gly Gly Cys Glu 325 330 335Leu Arg Val Lys Phe Ser Arg Ser Ala
Asp Ala Pro Ala Tyr Gln Gln 340 345 350Gly Gln Asn Gln Leu Tyr Asn
Glu Leu Asn Leu Gly Arg Arg Glu Glu 355 360 365Tyr Asp Val Leu Asp
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 370 375 380Lys Pro Arg
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln385 390 395
400Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
405 410 415Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
Ser Thr 420 425 430Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
Ala Leu Pro Pro 435 440 445Arg4408PRTArtificialPolypeptide 4Met Trp
Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly
Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25
30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln
35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn
Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala
Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn
Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly
Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu
Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala
Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys
Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala
Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165 170
175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly
Tyr Gln 195 200 205Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala
Val Asp Thr Gly 210 215 220Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg
Ser Ser Thr Arg Asp Trp225 230 235 240Lys Asp His Lys Phe Lys Trp
Arg Lys Asp Pro Gln Asp Lys Lys Arg 245 250 255Gly Arg Lys Lys Leu
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 260 265 270Val Gln Thr
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 275 280 285Glu
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 290 295
300Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
Leu305 310 315 320Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
Lys Arg Arg Gly 325 330 335Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
Arg Lys Asn Pro Gln Glu 340 345 350Gly Leu Tyr Asn Glu Leu Gln Lys
Asp Lys Met Ala Glu Ala Tyr Ser 355 360 365Glu Ile Gly Met Lys Gly
Glu Arg Arg Arg Gly Lys Gly His Asp Gly 370 375 380Leu Tyr Gln Gly
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu385 390 395 400His
Met Gln Ala Leu Pro Pro Arg 4055248PRTCanis lupus familiaris 5Met
Trp Gln Leu Val Ser Ser Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10
15Gly Thr Gln Ala Asp Val Pro Lys Ala Val Val Val Leu Glu Pro Lys
20 25 30Trp Asn Arg Val Leu Thr Met Asp Ser Val Thr Leu Lys Cys Gln
Gly 35 40 45Asp His Leu Leu Arg Asp Asn Tyr Thr Trp Leu His Asn Gly
Arg Pro 50 55 60Ile Ser Asn Gln Ile Ser Thr Tyr Ile Ile Lys Asn Ala
Ser Ile Lys65 70 75 80Asn Ser Gly Glu Tyr Arg Cys Gln Thr Asp Gln
Ser Lys Leu Ser Asp 85 90 95Pro Val Gln Leu Glu Val His Thr Gly Trp
Leu Leu Leu Gln Val Pro 100 105 110Arg Leu Val Phe Gln Glu Gly Glu
Leu Ile Gln Leu Lys Cys His Ser 115 120 125Trp Lys Asn Thr Pro Val
Arg Asn Val Gln Tyr Phe Gln Asn Gly Arg 130 135 140Gly Lys Lys Phe
Phe Tyr Asn Asn Ser Glu Tyr His Ile Pro Ala Ala145 150 155 160Thr
Ser Glu His Asn Gly Ser Tyr Phe Cys Arg Gly Ile Ile Gly Lys 165 170
175Lys Asn Glu Ser Ser Glu Ala Val Asn Ile Ile Ile Gln Gly Ser Ser
180 185 190Leu Pro Ser Thr Ser Leu Leu Leu Ser His Trp Pro Gln Ile
Pro Phe 195 200 205Ser Leu Val Met Ala Leu Leu Phe Ala Val Asp Thr
Gly Leu Tyr Phe 210 215 220Ala Val Gln Arg Asp Leu Arg Ser Ser Met
Gly Asn Leu Lys Asn Ser225 230 235 240Lys Val Ile Trp Ser Gln Gly
Ser 2456254PRTHomo sapiens 6Met Trp Gln Leu Leu Leu Pro Thr Ala Leu
Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys
Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys
Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp
Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln
Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser
Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro
Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala
Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120
125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr
Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe
Cys Arg Gly Leu Phe 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr
Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile
Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205Val Ser Phe Cys Leu
Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220Leu Tyr Phe
Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp225 230 235
240Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245
2507372PRTCanis lupus familiaris 7Met Trp Leu Leu Thr Val Leu Leu
Leu Trp Val Pro Ala Gly Ala Gln1 5 10 15Thr Asp Pro Val Lys Ala Val
Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Ser
Val Thr Leu Trp Cys Glu Gly Pro His Leu 35 40 45Pro Gly Asp Ser Ser
Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Leu Thr Pro
Arg Tyr Arg Ile Ala Ala Ala Ser Val Asn Asp Asn65 70 75 80Gly Glu
Tyr Arg Cys Gln Thr Gly Leu Ser Val Leu Ser Asp Pro Ile 85 90 95Gln
Leu Gly Ile His Arg Asp Trp Leu Ile Leu Gln Val Ser Gly Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Thr Leu Arg Cys His Gly Trp Asn
115 120 125Asn Lys Leu Val Tyr Asn Val Leu Phe Tyr Gln Asn Gly Thr
Val Leu 130 135 140Lys Phe Ser Pro Gln Asn Ser Glu Phe Thr Ile Leu
Lys Thr Thr Leu145 150 155 160His His Asn Gly Ile Tyr His Cys Ser
Ala Met Gly Lys His Arg Tyr 165 170 175Glu Ser Ala Gly Val Ser Ile
Thr Ile Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Lys Ala Ser
Leu Ser Ser Pro Ile Leu Glu Gly His Val Val 195 200 205Asn Leu Ser
Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Leu Ser Arg Asn225 230
235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Lys Lys Glu Asp Ser
Gly 245 250 255Leu Tyr Trp Cys Glu Ala Thr Thr Glu Asp Gly Asn Val
Val Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Val Gly Pro
Gln Thr Leu Thr Pro 275 280 285Val Trp Phe His Val Leu Phe Tyr Val
Ala Met Gly Met Ile Phe Leu 290 295 300Val Asp Thr Ile Phe Cys Met
Ile Ile His Lys Glu Leu Gln Arg Lys305 310 315 320Lys Lys Trp Asn
Leu Glu Ile Ser Leu Tyr Ser Gly Leu Glu Lys Arg 325 330 335Val Asp
Ser Tyr Leu Gln Lys Glu Arg Asp Leu Glu Glu Pro Lys Tyr 340 345
350Gln Glu Leu Glu Gln Leu Gln Glu Lys Thr Pro Gln Lys Pro Pro Glu
355 360 365Gly Glu Gln Gln 3708374PRTHomo sapiens 8Met Trp Phe Leu
Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln1 5 10
15Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser
20 25 30Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His
Leu 35 40 45Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala
Thr Gln 50 55 60Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val
Asn Asp Ser65 70 75 80Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly
Arg Ser Asp Pro Ile 85 90 95Gln Leu Glu Ile His Arg Gly Trp Leu Leu
Leu Gln Val Ser Ser Arg 100 105 110Val Phe Thr Glu Gly Glu Pro Leu
Ala Leu Arg Cys His Ala Trp Lys 115 120 125Asp Lys Leu Val Tyr Asn
Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140Lys Phe Phe His
Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile145 150 155 160Ser
His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170
175Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro
180 185 190Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn
Leu Val 195 200 205Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg
Pro Gly Leu Gln 210 215 220Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys
Thr Leu Arg Gly Arg Asn225 230 235 240Thr Ser Ser Glu Tyr Gln Ile
Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255Leu Tyr Trp Cys Glu
Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270Ser Pro Glu
Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285Val
Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu 290 295
300Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg
Lys305 310 315 320Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly
His Glu Lys Lys 325 330 335Val Ile Ser Ser Leu Gln Glu Asp Arg His
Leu Glu Glu Glu Leu Lys 340 345 350Cys Gln Glu Gln Lys Glu Glu Gln
Leu Gln Glu Gly Val His Arg Lys 355 360 365Glu Pro Gln Gly Ala Thr
3709374PRTArtificialPolypeptide 9Met Trp Phe Leu Thr Thr Leu Leu
Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val
Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr
Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser
Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro
Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu
Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln
Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys
Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu
Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser
Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val
Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn Ala Ser
Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr Leu Ser
Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230
235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser
Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val
Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu
Gln Leu Pro Thr Pro 275 280 285Val Trp Phe His Val Leu Phe Tyr Leu
Ala Val Gly Ile Met Phe Leu 290 295 300Val Asn Thr Val Leu Trp Val
Thr Ile Arg Lys Glu Leu Lys Arg Lys305 310 315 320Lys Lys Trp Asp
Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys 325 330 335Val Ile
Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys 340 345
350Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys
355 360 365Glu Pro Gln Gly Ala Thr 37010374PRTArtificialPolypeptide
10Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln1
5 10 15Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val
Ser 20 25 30Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu
His Leu 35 40 45Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr
Ala Thr Gln 50 55 60Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser
Val Asn Asp Ser65 70 75 80Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser
Gly Arg Ser Asp Pro Ile 85 90 95Gln Leu Glu Ile His Arg Gly Trp Leu
Leu Leu Gln Val Ser Ser Arg 100 105 110Val Phe Thr Glu Gly Glu Pro
Leu Ala Leu Arg Cys His Ala Trp Lys 115 120 125Asp Lys Leu Val Tyr
Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140Lys Phe Phe
His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile145 150 155
160Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr
165 170 175Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro
Ala Pro 180 185 190Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu
Gly Asn Leu Val 195 200 205Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu
Gln Arg Pro Gly Leu Gln 210 215 220Leu Tyr Phe Ser Phe Tyr Met Gly
Ser Lys Thr Leu Arg Gly Arg Asn225 230 235 240Thr Ser Ser Glu Tyr
Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255Leu Tyr Trp
Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270Ser
Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280
285Val Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu
290 295 300Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys
Arg Lys305 310 315 320Lys Lys Trp Asn Leu Glu Ile Ser Leu Asp Ser
Gly His Glu Lys Lys 325 330 335Val Ile Ser Ser Leu Gln Glu Asp Arg
His Leu Glu Glu Glu Leu Lys 340 345 350Cys Gln Glu Gln Lys Glu Glu
Gln Leu Gln Glu Gly Val His Arg Lys 355 360 365Glu Pro Gln Gly Ala
Thr 37011335PRTArtificialPolypeptide 11Met Trp Phe Leu Thr Thr Leu
Leu Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala
Val Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu
Thr Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser
Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr
Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly
Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90
95Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg
100 105 110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala
Trp Lys 115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn
Gly Lys Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr
Ile Leu Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His
Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile
Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn
Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr
Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215
220Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg
Asn225 230 235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg
Glu Asp Ser Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp
Gly Asn Val Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val
Leu Gly Phe Phe Pro Pro Gly Tyr 275 280 285Gln Val Ser Phe Cys Leu
Val Met Val Leu Leu Phe Ala Val Asp Thr 290 295 300Gly Leu Tyr Phe
Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp305 310 315 320Trp
Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 325 330
33512530PRTArtificialPolypeptide 12Met Trp Phe Leu Thr Thr Leu Leu
Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val
Ile Ser Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr
Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser
Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro
Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu
Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln
Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys
Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu
Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser
Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val
Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn Ala Ser
Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr Leu Ser
Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230
235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser
Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val
Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Phe
Phe Pro Pro Gly Tyr 275 280 285Gln Val Ser Phe Cys Leu Val Met Val
Leu Leu Phe Ala Val Asp Thr 290 295 300Gly Leu Tyr Phe Ser Val Lys
Thr Asn Ile Arg Ser Ser Thr Arg Asp305 310 315 320Trp Lys Asp His
Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys Arg 325 330 335Ser Lys
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 340 345
350Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
355 360 365Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys
Leu Leu 370 375 380Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
Thr Thr Gln Glu385 390 395 400Glu Asp Gly Cys Ser Cys Arg Phe Pro
Glu Glu Glu Glu Gly Gly Cys 405 410 415Glu Leu Arg Val Lys Phe Ser
Arg Ser Ala Asp Ala Pro Ala Tyr Gln 420 425 430Gln Gly Gln Asn Gln
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 435 440 445Glu Tyr Asp
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 450 455 460Gly
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu465 470
475 480Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
Gly 485 490 495Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
Gly Leu Ser 500 505 510Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
Met Gln Ala Leu Pro 515 520 525Pro Arg
53013460PRTArtificialPolypeptide 13Met Trp Phe Leu Thr Thr Leu Leu
Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val
Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr
Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser
Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro
Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu
Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln
Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys
Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu
Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser
Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val
Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn Ala Ser
Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr Leu Ser
Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230
235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser
Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val
Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Phe
Phe Pro Pro Gly Tyr 275 280 285Gln Val Ser Phe Cys Leu Val Met Val
Leu Leu Phe Ala Val Asp Thr 290 295 300Gly Leu Tyr Phe Ser Val Lys
Thr Asn Ile Arg Ser Ser Thr Arg Asp305 310 315 320Trp Lys Asp His
Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys Arg 325 330 335Ser Lys
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 340 345
350Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
355 360 365Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys
Leu Leu 370 375 380Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
Thr Thr Gln Glu385 390 395 400Glu Asp Gly Cys Ser Cys Arg Phe Pro
Glu Glu Glu Glu Gly Gly Cys 405 410 415Glu Leu Arg Leu Lys Ile Gln
Val Arg Lys Ala Ala Ile Thr Ser Tyr 420 425 430Glu Lys Ser Asp Gly
Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu 435 440 445Thr Tyr Glu
Thr Leu Lys His Glu Lys Pro Pro Gln 450 455
46014440PRTArtificialPolypeptide 14Met Trp Phe Leu Thr Thr Leu Leu
Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val
Ile Thr Leu Gln Pro Pro
Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu
Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn
Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser
Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu Tyr Arg Cys Gln Arg Gly
Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln Leu Glu Ile His Arg Gly
Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105 110Val Phe Thr Glu Gly
Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys 115 120 125Asp Lys Leu
Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140Lys
Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile145 150
155 160Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg
Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe
Pro Ala Pro 180 185 190Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu
Glu Gly Asn Leu Val 195 200 205Thr Leu Ser Cys Glu Thr Lys Leu Leu
Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu Tyr Phe Ser Phe Tyr Met
Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230 235 240Thr Ser Ser Glu
Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255Leu Tyr
Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265
270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Phe Phe Pro Pro Gly Tyr
275 280 285Gln Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val
Asp Thr 290 295 300Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser
Ser Thr Arg Asp305 310 315 320Trp Lys Asp His Lys Phe Lys Trp Arg
Lys Asp Pro Gln Asp Lys Arg 325 330 335Ser Lys Arg Ser Arg Leu Leu
His Ser Asp Tyr Met Asn Met Thr Pro 340 345 350Arg Arg Pro Gly Pro
Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 355 360 365Arg Asp Phe
Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu 370 375 380Tyr
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu385 390
395 400Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
Cys 405 410 415Glu Leu Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp
Gly Lys Val 420 425 430Tyr Ile Asn Met Pro Gly Arg Gly 435
44015467PRTArtificialPolypeptide 15Met Trp Phe Leu Thr Thr Leu Leu
Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val
Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr
Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser
Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro
Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu
Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln
Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys
Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu
Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser
Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val
Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn Ala Ser
Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr Leu Ser
Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230
235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser
Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val
Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Phe
Phe Pro Pro Gly Tyr 275 280 285Gln Val Ser Phe Cys Leu Val Met Val
Leu Leu Phe Ala Val Asp Thr 290 295 300Gly Leu Tyr Phe Ser Val Lys
Thr Asn Ile Arg Ser Ser Thr Arg Asp305 310 315 320Trp Lys Asp His
Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys Arg 325 330 335Ser Lys
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 340 345
350Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
355 360 365Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys
Leu Leu 370 375 380Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
Thr Thr Gln Glu385 390 395 400Glu Asp Gly Cys Ser Cys Arg Phe Pro
Glu Glu Glu Glu Gly Gly Cys 405 410 415Glu Leu Gly Arg Leu Val Pro
Arg Gly Arg Gly Ala Ala Glu Ala Ala 420 425 430Thr Arg Lys Gln Arg
Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu 435 440 445Gln Gly Gln
Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro 450 455 460Tyr
Tyr Lys46516510PRTArtificialPolypeptide 16Met Trp Phe Leu Thr Thr
Leu Leu Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys
Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu
Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser
Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser
Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75
80Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile
85 90 95Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser
Arg 100 105 110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His
Ala Trp Lys 115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg
Asn Gly Lys Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu
Thr Ile Leu Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr
His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly
Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu
Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200
205Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln
210 215 220Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly
Arg Asn225 230 235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg
Arg Glu Asp Ser Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu
Asp Gly Asn Val Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln
Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285Phe Trp Val Leu Val
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 290 295 300Leu Val Thr
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser305 310 315
320Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
325 330 335Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
Phe Ala 340 345 350Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu
Tyr Ile Phe Lys 355 360 365Gln Pro Phe Met Arg Pro Val Gln Thr Thr
Gln Glu Glu Asp Gly Cys 370 375 380Ser Cys Arg Phe Pro Glu Glu Glu
Glu Gly Gly Cys Glu Leu Arg Val385 390 395 400Lys Phe Ser Arg Ser
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 405 410 415Gln Leu Tyr
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 420 425 430Leu
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 435 440
445Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
450 455 460Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
Arg Arg465 470 475 480Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
Ser Thr Ala Thr Lys 485 490 495Asp Thr Tyr Asp Ala Leu His Met Gln
Ala Leu Pro Pro Arg 500 505 51017343PRTArtificialPolypeptide 17Met
Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln1 5 10
15Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser
20 25 30Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His
Leu 35 40 45Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala
Thr Gln 50 55 60Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val
Asn Asp Ser65 70 75 80Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly
Arg Ser Asp Pro Ile 85 90 95Gln Leu Glu Ile His Arg Gly Trp Leu Leu
Leu Gln Val Ser Ser Arg 100 105 110Val Phe Thr Glu Gly Glu Pro Leu
Ala Leu Arg Cys His Ala Trp Lys 115 120 125Asp Lys Leu Val Tyr Asn
Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140Lys Phe Phe His
Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile145 150 155 160Ser
His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170
175Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro
180 185 190Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn
Leu Val 195 200 205Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg
Pro Gly Leu Gln 210 215 220Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys
Thr Leu Arg Gly Arg Asn225 230 235 240Thr Ser Ser Glu Tyr Gln Ile
Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255Leu Tyr Trp Cys Glu
Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270Ser Pro Glu
Leu Glu Leu Gln Val Leu Gly Ala Pro Arg Glu Lys Tyr 275 280 285Trp
Leu Gln Phe Phe Ile Pro Leu Leu Val Val Ile Leu Phe Ala Val 290 295
300Asp Thr Gly Leu Phe Ile Ser Thr Gln Gln Gln Val Thr Phe Leu
Leu305 310 315 320Lys Ile Lys Arg Thr Arg Lys Gly Phe Arg Leu Leu
Asn Pro His Pro 325 330 335Lys Pro Asn Pro Lys Asn Asn
34018335PRTArtificialPolypeptide 18Met Trp Phe Leu Thr Thr Leu Leu
Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val
Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr
Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser
Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro
Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu
Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln
Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys
Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu
Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser
Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val
Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn Ala Ser
Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr Leu Ser
Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230
235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser
Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val
Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Phe
Phe Pro Pro Gly Tyr 275 280 285Gln Val Ser Phe Cys Leu Val Met Val
Leu Leu Phe Ala Val Asp Thr 290 295 300Gly Leu Tyr Phe Ser Val Lys
Thr Asn Ile Arg Gly Ala Gly Arg Asp305 310 315 320Trp Lys Asp His
Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 325 330
33519561PRTArtificialPolypeptide 19Met Trp Phe Leu Thr Thr Leu Leu
Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val
Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr
Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser
Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro
Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu
Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln
Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys
Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu
Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser
Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val
Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn Ala Ser
Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr Leu Ser
Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230
235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser
Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val
Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Ser
Asn Leu Phe Val Ala 275 280 285Ser Trp Ile Ala Val Met Ile Ile Phe
Arg Ile Gly Met Ala Val Ala 290 295 300Ile Phe Cys Cys Phe Phe Phe
Pro Ser Gly Gly Ser Gly Gly Gly Ser305 310 315 320Gly Trp Arg Arg
Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys 325 330 335Glu Phe
Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg 340 345
350Asn His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr
355 360 365Ser Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu
Pro Ala 370
375 380Tyr Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser
Arg385 390 395 400Lys Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile
Tyr Glu Val Ile 405 410 415Gly Lys Ser Gln Pro Lys Ala Gln Asn Pro
Ala Arg Leu Ser Arg Lys 420 425 430Glu Leu Glu Asn Phe Asp Val Tyr
Ser Gly Gly Ser Gly Gly Gly Ser 435 440 445Gly Arg Val Lys Phe Ser
Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln 450 455 460Gly Gln Asn Gln
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu465 470 475 480Tyr
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 485 490
495Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
500 505 510Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
Gly Glu 515 520 525Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
Gly Leu Ser Thr 530 535 540Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
Met Gln Ala Leu Pro Pro545 550 555
560Arg20575PRTArtificialPolypeptide 20Met Trp Phe Leu Thr Thr Leu
Leu Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala
Val Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu
Thr Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser
Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr
Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly
Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90
95Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg
100 105 110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala
Trp Lys 115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn
Gly Lys Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr
Ile Leu Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His
Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile
Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn
Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr
Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215
220Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg
Asn225 230 235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg
Glu Asp Ser Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp
Gly Asn Val Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val
Leu Gly Phe Phe Pro Pro Gly Tyr 275 280 285Gln Val Ser Phe Cys Leu
Val Met Val Leu Leu Phe Ala Val Asp Thr 290 295 300Gly Leu Tyr Phe
Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp305 310 315 320Trp
Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys Trp 325 330
335Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys Glu Phe
340 345 350Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg
Asn His 355 360 365Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr
Ile Tyr Ser Met 370 375 380Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser
Gln Glu Pro Ala Tyr Thr385 390 395 400Leu Tyr Ser Leu Ile Gln Pro
Ser Arg Lys Ser Gly Ser Arg Lys Arg 405 410 415Asn His Ser Pro Ser
Phe Asn Ser Thr Ile Tyr Glu Val Ile Gly Lys 420 425 430Ser Gln Pro
Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys Glu Leu 435 440 445Glu
Asn Phe Asp Val Tyr Ser Gly Gly Ser Gly Gly Gly Ser Gly Arg 450 455
460Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
Gln465 470 475 480Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
Glu Glu Tyr Asp 485 490 495Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
Glu Met Gly Gly Lys Pro 500 505 510Arg Arg Lys Asn Pro Gln Glu Gly
Leu Tyr Asn Glu Leu Gln Lys Asp 515 520 525Lys Met Ala Glu Ala Tyr
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 530 535 540Arg Gly Lys Gly
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr545 550 555 560Lys
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 565 570
57521505PRTArtificialPolypeptide 21Met Trp Phe Leu Thr Thr Leu Leu
Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val
Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr
Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser
Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro
Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu
Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln
Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys
Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu
Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser
Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val
Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn Ala Ser
Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr Leu Ser
Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230
235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser
Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val
Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Phe
Phe Pro Pro Gly Tyr 275 280 285Gln Val Ser Phe Cys Leu Val Met Val
Leu Leu Phe Ala Val Asp Thr 290 295 300Gly Leu Tyr Phe Ser Val Lys
Thr Asn Ile Arg Ser Ser Thr Arg Asp305 310 315 320Trp Lys Asp His
Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys Trp 325 330 335Arg Arg
Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys Glu Phe 340 345
350Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg Asn His
355 360 365Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr
Ser Met 370 375 380Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu
Pro Ala Tyr Thr385 390 395 400Leu Tyr Ser Leu Ile Gln Pro Ser Arg
Lys Ser Gly Ser Arg Lys Arg 405 410 415Asn His Ser Pro Ser Phe Asn
Ser Thr Ile Tyr Glu Val Ile Gly Lys 420 425 430Ser Gln Pro Lys Ala
Gln Asn Pro Ala Arg Leu Ser Arg Lys Glu Leu 435 440 445Glu Asn Phe
Asp Val Tyr Ser Gly Gly Ser Gly Gly Gly Ser Gly Arg 450 455 460Leu
Lys Ile Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser465 470
475 480Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr
Glu 485 490 495Thr Leu Lys His Glu Lys Pro Pro Gln 500
50522485PRTArtificialPolypeptide 22Met Trp Phe Leu Thr Thr Leu Leu
Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val
Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr
Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser
Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro
Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu
Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln
Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys
Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu
Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser
Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val
Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn Ala Ser
Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr Leu Ser
Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230
235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser
Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val
Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Phe
Phe Pro Pro Gly Tyr 275 280 285Gln Val Ser Phe Cys Leu Val Met Val
Leu Leu Phe Ala Val Asp Thr 290 295 300Gly Leu Tyr Phe Ser Val Lys
Thr Asn Ile Arg Ser Ser Thr Arg Asp305 310 315 320Trp Lys Asp His
Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys Trp 325 330 335Arg Arg
Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys Glu Phe 340 345
350Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg Asn His
355 360 365Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr
Ser Met 370 375 380Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu
Pro Ala Tyr Thr385 390 395 400Leu Tyr Ser Leu Ile Gln Pro Ser Arg
Lys Ser Gly Ser Arg Lys Arg 405 410 415Asn His Ser Pro Ser Phe Asn
Ser Thr Ile Tyr Glu Val Ile Gly Lys 420 425 430Ser Gln Pro Lys Ala
Gln Asn Pro Ala Arg Leu Ser Arg Lys Glu Leu 435 440 445Glu Asn Phe
Asp Val Tyr Ser Gly Gly Ser Gly Gly Gly Ser Gly Ala 450 455 460Arg
Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val Tyr Ile Asn465 470
475 480Met Pro Gly Arg Gly 48523512PRTArtificialPolypeptide 23Met
Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln1 5 10
15Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser
20 25 30Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His
Leu 35 40 45Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala
Thr Gln 50 55 60Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val
Asn Asp Ser65 70 75 80Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly
Arg Ser Asp Pro Ile 85 90 95Gln Leu Glu Ile His Arg Gly Trp Leu Leu
Leu Gln Val Ser Ser Arg 100 105 110Val Phe Thr Glu Gly Glu Pro Leu
Ala Leu Arg Cys His Ala Trp Lys 115 120 125Asp Lys Leu Val Tyr Asn
Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140Lys Phe Phe His
Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile145 150 155 160Ser
His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170
175Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro
180 185 190Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn
Leu Val 195 200 205Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg
Pro Gly Leu Gln 210 215 220Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys
Thr Leu Arg Gly Arg Asn225 230 235 240Thr Ser Ser Glu Tyr Gln Ile
Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255Leu Tyr Trp Cys Glu
Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270Ser Pro Glu
Leu Glu Leu Gln Val Leu Gly Phe Phe Pro Pro Gly Tyr 275 280 285Gln
Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr 290 295
300Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg
Asp305 310 315 320Trp Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro
Gln Asp Lys Trp 325 330 335Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu
Thr Ser Pro Lys Glu Phe 340 345 350Leu Thr Ile Tyr Glu Asp Val Lys
Asp Leu Lys Thr Arg Arg Asn His 355 360 365Glu Gln Glu Gln Thr Phe
Pro Gly Gly Gly Ser Thr Ile Tyr Ser Met 370 375 380Ile Gln Ser Gln
Ser Ser Ala Pro Thr Ser Gln Glu Pro Ala Tyr Thr385 390 395 400Leu
Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser Arg Lys Arg 405 410
415Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu Val Ile Gly Lys
420 425 430Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys
Glu Leu 435 440 445Glu Asn Phe Asp Val Tyr Ser Gly Gly Ser Gly Gly
Gly Ser Gly Gly 450 455 460Arg Leu Val Pro Arg Gly Arg Gly Ala Ala
Glu Ala Ala Thr Arg Lys465 470 475 480Gln Arg Ile Thr Glu Thr Glu
Ser Pro Tyr Gln Glu Leu Gln Gly Gln 485 490 495Arg Ser Asp Val Tyr
Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys 500 505
51024615PRTArtificialPolypeptide 24Met Trp Phe Leu Thr Thr Leu Leu
Leu Trp Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val
Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr
Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser
Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro
Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu
Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln
Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105
110Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys
115 120 125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys
Ala Phe 130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu
Lys Thr Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser
Gly Met Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val
Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185
190Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val
195 200 205Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly
Leu Gln 210 215 220Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu
Arg Gly Arg Asn225 230 235 240Thr Ser Ser Glu Tyr Gln Ile Leu Thr
Ala Arg Arg Glu Asp Ser Gly 245 250 255Leu Tyr Trp Cys Glu Ala Ala
Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270Ser Pro Glu Leu Glu
Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285Val Trp Phe
His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu 290 295 300Val
Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys305 310
315 320Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys
Lys 325 330 335Val Ile Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu
Glu Leu Lys 340 345 350Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu
Gly Val His Arg Lys 355 360 365Glu Pro Gln Gly Ala Thr Gly Trp Arg
Arg Lys Arg Lys Glu Lys Gln 370 375 380Ser Glu Thr Ser Pro Lys Glu
Phe Leu Thr Ile Tyr Glu Asp Val Lys385 390 395 400Asp Leu Lys Thr
Arg Arg Asn His Glu Gln Glu Gln Thr Phe Pro Gly 405 410 415Gly Gly
Ser Thr Ile Tyr Ser Met Ile Gln Ser Gln Ser Ser Ala Pro 420 425
430Thr Ser Gln Glu Pro Ala Tyr Thr Leu Tyr Ser Leu Ile Gln Pro Ser
435 440 445Arg Lys Ser Gly Ser Arg Lys Arg Asn His Ser Pro Ser Phe
Asn Ser 450 455 460Thr Ile Tyr Glu Val Ile Gly Lys Ser Gln Pro Lys
Ala Gln Asn Pro465 470 475 480Ala Arg Leu Ser Arg Lys Glu Leu Glu
Asn Phe Asp Val Tyr Ser Gly 485 490 495Gly Ser Gly Gly Gly Ser Gly
Arg Val Lys Phe Ser Arg Ser Ala Asp 500 505 510Ala Pro Ala Tyr Lys
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 515 520 525Leu Gly Arg
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 530 535 540Asp
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly545 550
555 560Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
Glu 565 570 575Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
Asp Gly Leu 580 585 590Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
Tyr Asp Ala Leu His 595 600 605Met Gln Ala Leu Pro Pro Arg 610
61525248PRTCanis lupus familiaris 25Met Trp Gln Leu Val Ser Ser Thr
Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Thr Gln Ala Asp Val Pro
Lys Ala Val Val Val Leu Glu Pro Lys 20 25 30Trp Asn Arg Val Leu Thr
Met Asp Ser Val Thr Leu Lys Cys Gln Gly 35 40 45Asp His Leu Leu Arg
Asp Asn Tyr Thr Trp Leu His Asn Gly Arg Pro 50 55 60Ile Ser Asn Gln
Ile Ser Thr Tyr Ile Ile Lys Asn Ala Ser Ile Lys65 70 75 80Asn Ser
Gly Glu Tyr Arg Cys Gln Thr Asp Gln Ser Lys Leu Ser Asp 85 90 95Pro
Val Gln Leu Glu Val His Thr Gly Trp Leu Leu Leu Gln Val Pro 100 105
110Arg Leu Val Phe Gln Glu Gly Glu Leu Ile Gln Leu Lys Cys His Ser
115 120 125Trp Lys Asn Thr Pro Val Arg Asn Val Gln Tyr Phe Gln Asn
Gly Arg 130 135 140Gly Lys Lys Phe Phe Tyr Asn Asn Ser Glu Tyr His
Ile Pro Ala Ala145 150 155 160Thr Ser Glu His Asn Gly Ser Tyr Phe
Cys Arg Gly Ile Ile Gly Lys 165 170 175Lys Asn Glu Ser Ser Glu Ala
Val Asn Ile Ile Ile Gln Gly Ser Ser 180 185 190Leu Pro Ser Thr Ser
Leu Leu Leu Ser His Trp Pro Gln Ile Pro Phe 195 200 205Ser Leu Val
Met Ala Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe 210 215 220Ala
Val Gln Arg Asp Leu Arg Ser Ser Met Gly Asn Leu Lys Asn Ser225 230
235 240Lys Val Ile Trp Ser Gln Gly Ser 24526372PRTCanis lupus
familiaris 26Met Trp Leu Leu Thr Val Leu Leu Leu Trp Val Pro Ala
Gly Ala Gln1 5 10 15Thr Asp Pro Val Lys Ala Val Ile Thr Leu Gln Pro
Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Ser Val Thr Leu Trp Cys
Glu Gly Pro His Leu 35 40 45Pro Gly Asp Ser Ser Thr Gln Trp Phe Leu
Asn Gly Thr Ala Thr Gln 50 55 60Thr Leu Thr Pro Arg Tyr Arg Ile Ala
Ala Ala Ser Val Asn Asp Asn65 70 75 80Gly Glu Tyr Arg Cys Gln Thr
Gly Leu Ser Val Leu Ser Asp Pro Ile 85 90 95Gln Leu Gly Ile His Arg
Asp Trp Leu Ile Leu Gln Val Ser Gly Arg 100 105 110Val Phe Thr Glu
Gly Glu Pro Leu Thr Leu Arg Cys His Gly Trp Asn 115 120 125Asn Lys
Leu Val Tyr Asn Val Leu Phe Tyr Gln Asn Gly Thr Val Leu 130 135
140Lys Phe Ser Pro Gln Asn Ser Glu Phe Thr Ile Leu Lys Thr Thr
Leu145 150 155 160His His Asn Gly Ile Tyr His Cys Ser Ala Met Gly
Lys His Arg Tyr 165 170 175Glu Ser Ala Gly Val Ser Ile Thr Ile Lys
Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Lys Ala Ser Leu Ser Ser
Pro Ile Leu Glu Gly His Val Val 195 200 205Asn Leu Ser Cys Glu Thr
Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu Tyr Phe Ser
Phe Tyr Met Gly Ser Lys Thr Leu Leu Ser Arg Asn225 230 235 240Thr
Ser Ser Glu Tyr Gln Ile Leu Thr Ala Lys Lys Glu Asp Ser Gly 245 250
255Leu Tyr Trp Cys Glu Ala Thr Thr Glu Asp Gly Asn Val Val Lys Arg
260 265 270Ser Pro Glu Leu Glu Leu Gln Val Val Gly Pro Gln Thr Leu
Thr Pro 275 280 285Val Trp Phe His Val Leu Phe Tyr Val Ala Met Gly
Met Ile Phe Leu 290 295 300Val Asp Thr Ile Phe Cys Met Ile Ile His
Lys Glu Leu Gln Arg Lys305 310 315 320Lys Lys Trp Asn Leu Glu Ile
Ser Leu Tyr Ser Gly Leu Glu Lys Arg 325 330 335Val Asp Ser Tyr Leu
Gln Lys Glu Arg Asp Leu Glu Glu Pro Lys Tyr 340 345 350Gln Glu Leu
Glu Gln Leu Gln Glu Lys Thr Pro Gln Lys Pro Pro Glu 355 360 365Gly
Glu Gln Gln 37027287PRTCanis lupus familiaris 27Met Trp Leu Leu Thr
Val Leu Leu Leu Trp Val Pro Ala Gly Ala Gln1 5 10 15Thr Asp Trp Leu
Ile Leu Gln Val Ser Gly Arg Val Phe Thr Glu Gly 20 25 30Glu Pro Leu
Thr Leu Arg Cys His Gly Trp Asn Asn Lys Leu Val Tyr 35 40 45Asn Val
Leu Phe Tyr Gln Asn Gly Thr Val Leu Lys Phe Ser Pro Gln 50 55 60Asn
Ser Glu Phe Thr Ile Leu Lys Thr Thr Leu His His Asn Gly Ile65 70 75
80Tyr His Cys Ser Ala Met Gly Lys His Arg Tyr Glu Ser Ala Gly Val
85 90 95Ser Ile Thr Ile Lys Glu Leu Phe Pro Ala Pro Val Leu Lys Ala
Ser 100 105 110Leu Ser Ser Pro Ile Leu Glu Gly His Val Val Asn Leu
Ser Cys Glu 115 120 125Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln
Leu Tyr Phe Ser Phe 130 135 140Tyr Met Gly Ser Lys Thr Leu Leu Ser
Arg Asn Thr Ser Ser Glu Tyr145 150 155 160Gln Ile Leu Thr Ala Lys
Lys Glu Asp Ser Gly Leu Tyr Trp Cys Glu 165 170 175Ala Thr Thr Glu
Asp Gly Asn Val Val Lys Arg Ser Pro Glu Leu Glu 180 185 190Leu Gln
Val Val Gly Pro Gln Thr Leu Thr Pro Val Trp Phe His Val 195 200
205Leu Phe Tyr Val Ala Met Gly Met Ile Phe Leu Val Asp Thr Ile Phe
210 215 220Cys Met Ile Ile His Lys Glu Leu Gln Arg Lys Lys Lys Trp
Asn Leu225 230 235 240Glu Ile Ser Leu Tyr Ser Gly Leu Glu Lys Arg
Val Asp Ser Tyr Leu 245 250 255Gln Lys Glu Arg Asp Leu Glu Glu Pro
Lys Tyr Gln Glu Leu Glu Gln 260 265 270Leu Gln Glu Lys Thr Pro Gln
Lys Pro Pro Glu Gly Glu Gln Gln 275 280 2852834DNAArtificialPrimer
28cgggaattcg gagacaacat gtggttcttg acaa 342942DNAArtificialPrimer
29ttggtaccca ggtggaaaga agccaagcac ttgaagctcc aa
423042DNAArtificialPrimer 30ttggagcttc aagtgcttgg cttctttcca
cctgggtacc aa 423133DNAArtificialPrimer 31ccggaattct catttgtctt
gagggtcctt tct 333234DNAArtificialPrimer 32cgggaattcg gagacaacat
gtggttcttg acaa 343333DNAArtificialPrimer 33ccggaattct catttgtctt
gagggtcctt tct 33
* * * * *