U.S. patent application number 16/138331 was filed with the patent office on 2019-03-28 for chimeric polypeptides and uses thereof.
The applicant listed for this patent is Kite Pharma, Inc.. Invention is credited to Stuart A. Sievers, Jed J.W. Wiltzius.
Application Number | 20190092818 16/138331 |
Document ID | / |
Family ID | 63878804 |
Filed Date | 2019-03-28 |
United States Patent
Application |
20190092818 |
Kind Code |
A1 |
Wiltzius; Jed J.W. ; et
al. |
March 28, 2019 |
CHIMERIC POLYPEPTIDES AND USES THEREOF
Abstract
The invention provides novel peptides (e.g., linkers) and
polypeptide compositions comprising the linkers (e.g., fusion
proteins) and methods of using the polypeptide compositions.
Peptides (e.g., linkers) are useful as tags and for engineering
fusion proteins (e.g., antigen binding molecules, scFv).
Polypeptide linkers described herein facilitate flexibility of
linked peptides allowing for proper folding, conformation and
reduced immunogenicity.
Inventors: |
Wiltzius; Jed J.W.; (Santa
Monica, CA) ; Sievers; Stuart A.; (Santa Monica,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kite Pharma, Inc. |
Santa Monica |
CA |
US |
|
|
Family ID: |
63878804 |
Appl. No.: |
16/138331 |
Filed: |
September 21, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62562223 |
Sep 22, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/5156 20130101;
C07K 7/08 20130101; C07K 2319/40 20130101; A61K 2039/5158 20130101;
C07K 7/06 20130101; A61P 35/02 20180101; C12N 15/62 20130101; C07K
2317/32 20130101; C07K 16/2803 20130101; C07K 2317/34 20130101;
A61K 39/0011 20130101; A61K 39/001112 20180801; C07K 4/00 20130101;
C07K 16/44 20130101; C07K 14/003 20130101; C07K 2317/622 20130101;
C12N 15/85 20130101 |
International
Class: |
C07K 14/00 20060101
C07K014/00; C12N 15/85 20060101 C12N015/85; C12N 15/62 20060101
C12N015/62; C07K 4/00 20060101 C07K004/00; C07K 7/06 20060101
C07K007/06; C07K 7/08 20060101 C07K007/08 |
Claims
1. A peptide comprising 6-20 amino acids and a consensus sequence
BPXXXZ, wherein each X is independently a glycine (G) or serine
(S), B is a positively charged amino acid and Z is glycine (G) or a
negatively charged amino acid and wherein the peptide is not
GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1), GSGKPGSGEG (SEQ ID NO: 13),
GKPGSGEG (SEQ ID NO: 14), or SGKPGSGE (SEQ ID NO: 15).
2. (canceled)
3. The peptide of claim 1, wherein B is lysine (K) or arginine
(R).
4-5. (canceled)
6. The peptide of claim 1, wherein Z is a negatively charged amino
acid selected from glutamic acid (.epsilon.) or aspartic acid
(D).
7. (canceled)
8. The peptide of claim 1, wherein the consensus sequence is
GKPGSGE (SEQ ID NO: 5), GKPGSGG (SEQ ID NO: 6) or GSGKPGSGEGG (SEQ
ID NO: 31).
9-10. (canceled)
11. The peptide of claim 1, wherein the peptide comprises an amino
acid sequence of GGGSGKPGSGEGGGS (SEQ ID NO: 7).
12. The peptide of claim 1, wherein the peptide comprises an amino
acid sequence of GGGSGKPGSGEGGGGS (SEQ ID NO: 8).
13. The peptide of claim 1, wherein the peptide comprises an amino
acid sequence of GGGGSGKPGSGGGGS (SEQ ID NO: 9).
14. The peptide of claim 1, wherein the peptide comprises an amino
acid sequence of GGGGSGKPGSGEGGS (SEQ ID NO: 10).
15. The peptide of claim 1, wherein the peptide comprises an amino
acid sequence of GGGGSGKPGSGEGGGS (SEQ ID NO: 11).
16. The peptide of claim 1, wherein the peptide comprises an amino
acid sequence of GGGGSGKPGSGEGGGGS (SEQ ID NO: 12).
17. The peptide of claim 1, wherein the peptide comprises an amino
acid sequence of STSGSGKPGSGEGST (SEQ ID NO: 17).
18. A peptide comprising 8-20 amino acids and an amino acid
sequence at least 80% identical to any one of GGGSGKPGSGEGGGS (SEQ
ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO:8), GGGGSGKPGSGGGGS (SEQ ID
NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10), GGGGSGKPGSGEGGGS (SEQ ID
NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12), STSGSGKPGSGEGST (SEQ ID
NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID
NO: 19), or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).
19. A peptide comprising 8-20 amino acids and an amino acid
sequence that contains at least six (6) identical amino acids out
of ten (10) contiguous amino acids found in any one of
GGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO: 8),
GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),
GGGGSGKPGSGEGGGS (SEQ ID NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO:
12), STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO:
18) GGGGGSGGGGSGGGGS (SEQ ID NO: 19), or GGGGSGGGGSGGGGGS (SEQ ID
NO: 20).
20. A peptide comprising the amino acid sequence GGGGSGGGGSGGGGSG
(SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID NO: 19), or
GGGGSGGGGSGGGGGS (SEQ ID NO: 20).
21. A fusion protein comprising: a) a first polypeptide; and b) a
peptide according to claim 1.
22. (canceled)
23. The fusion protein of claim 21, wherein the fusion protein is
an antigen binding molecule comprising a light chain variable
domain and a heavy chain variable domain.
24-25. (canceled)
26. A polynucleotide encoding a peptide according to claim 1.
27. A polynucleotide encoding the fusion protein of claim 21.
28. An expression vector comprising a polynucleotide of claim
27.
29. A recombinant cell comprising a polynucleotide of claim 27.
30. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/562,223, filed Sep. 22, 2017, which is
incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 20, 2018, is named KPI-005US1_ST25.txt and is 8,626 bytes
in size.
TECHNICAL FIELD
[0003] The present disclosure is directed to novel peptides and
polypeptide compositions comprising such peptides (e.g., linkers,
chimeric polypeptides, antigen biding molecules) and methods of
using and preparing the same.
BACKGROUND
[0004] Antigen binding molecules, including antibodies, are used in
immunotherapy and solid phase-based applications such as
biosensors, affinity chromatography, and immunoassays. These
antibodies and antigen binding molecules gain their utility by
virtue of their ability to specifically bind their targets.
[0005] Fusion proteins may require linker sequences, which are
often peptide-based when employed in biotechnological and
biotherapeutic applications, which may serve a range of
scientifically-relevant applications. For example, a linker may be
used as a spacer moiety in order to impart a desired structural
and/or functional property to a larger molecule. In another
example, a linker may impart little or no structural or functional
properties to a larger molecule, but may be used simply as a
distinguishing feature (e.g., a "marker" or "biomarker" or "tag"),
uniquely identifying a larger molecule. In still another example, a
linker may be used to impart a recognizable feature that may serve
as a binding site for an antibody directed against a larger
molecule comprising the peptide sequence.
SUMMARY
[0006] The present invention provides, among other things, novel
peptide sequences which allow for the proper expression, folding,
identification and activity of a fusion protein. The novel peptides
described herein, may be used for connecting domains within a
fusion protein (e.g., scFvs) facilitating flexibility of the
individual peptide domains. ScFvs comprise the binding domain of
most CAR constructs. A scFv comprises IgG variable light and heavy
chains and a flexible peptides (e.g., linker) connecting these two
domains. A linker must be long enough to connect the domains into a
single protein construct. Further, it is desirable that a linker or
tag construct not be a potential cause of immunogenicity.
[0007] Commonly used linkers include repeats of
glycine-glycine-glycine-glycine-serine (G4S) (SEQ ID NO: 32) due to
their intrinsic flexibility and simplicity of side chains, which
may be less immunogenic in therapeutic applications. The 18-residue
Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1), was first
described by Whitlow et al. in 1993. The peptides described herein,
may also be used as a peptide tag. In some embodiments, the fusion
protein comprises a polypeptide fused to a peptide (e.g., a linker)
described herein.
[0008] Novel chimeric polypeptides described herein comprising a
consensus sequence BPXXXZ combine desirable attributes (e.g.,
flexibility and reduced immunogenicity) suitable for incorporation
into fusion proteins useful for therapeutic intervention.
[0009] In one aspect, the present invention provides a peptide
comprising 6-20 amino acids and a consensus sequence BPXXXZ,
wherein X is a glycine (G) or serine (S), B is a positively charged
amino acid and Z is glycine (G) or a negatively charged amino acid.
In one embodiment, the present invention provides a peptide,
wherein the consensus sequence is KPGSGE (SEQ ID NO: 4). In another
embodiment, the consensus sequence is GKPGSGE (SEQ ID NO: 5) or
GKPGSGG (SEQ ID NO: 6).
[0010] In one aspect, the present invention provides a peptide
comprising 6-20 amino acids and a consensus sequence BPXXXZ,
wherein X is a glycine (G) or serine (S), B is a positively charged
amino acid and Z is glycine (G) or a negatively charged amino acid,
and wherein the peptide is not GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1),
GSGKPGSGEG (SEQ ID NO: 13), GKPGSGEG (SEQ ID NO: 14), or SGKPGSGE
(SEQ ID NO: 15).
[0011] In one aspect, the present invention provides a peptide
comprising 8-20 amino acids and a consensus sequence XBPXXXZX,
wherein each X is independently a glycine (G) or serine (S), B is a
positively charged amino acid and Z is glycine (G) or a negatively
charged amino acid and P is proline.
[0012] In one aspect, the present invention provides a peptide
comprising 8-20 amino acids and a consensus sequence XBPXXXZX,
wherein each X is independently a glycine (G) or serine (S), B is a
positively charged amino acid and Z is glycine (G) or a negatively
charged amino acid and P is proline, and wherein the peptide is not
GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1), GSGKPGSGEG (SEQ ID NO: 13),
GKPGSGEG (SEQ ID NO: 14), or SGKPGSGE (SEQ ID NO: 15).
[0013] In some embodiments, the present invention provides a
peptide comprising 8-20 amino acids and a consensus sequence
XBPXXXZX, wherein each X is independently a glycine (G) or serine
(S), B is lysine (K) or arginine (R), and Z is glycine (G) or a
negatively charged amino acid, and P is proline.
[0014] In some embodiments, the present invention provides a
peptide comprising 8-20 amino acids and a consensus sequence
XBPXXXZX, wherein X is independently a glycine (G) or serine (S), B
is lysine (K), and Z is glycine (G) or a negatively charged amino
acid, and P is proline.
[0015] In some embodiments, the present invention provides a
peptide comprising 8-20 amino acids and a consensus sequence
XBPXXXZX, wherein each X is independently a glycine (G) or serine
(S), B is a positively charged amino acid, and Z is glycine (G) and
P is proline.
[0016] In some embodiments, Z is a negatively charged amino acid
selected from glutamic acid (E) or aspartic acid (D). In some
embodiments, Z is glutamic acid (E).
[0017] In some embodiments, the present invention provides a
peptide, wherein the consensus sequence is GKPGSGE (SEQ ID NO: 5)
or GKPGSGG (SEQ ID NO: 6). In some embodiments, the consensus
sequence is GKPGSGE (SEQ ID NO: 5). In some embodiments, the
peptide comprises the consensus sequence GSGKPGSGEGG (SEQ ID NO:
31).
[0018] In some embodiments, the peptide comprises one or more
repeats of the consensus sequence. In some embodiments, the repeats
are contiguous. In some embodiments, the peptide repeats are
separated by 1-4 amino acids. In some embodiments, the peptide is
xxxGKPGSGExxxGKPGSGExxx (SEQ ID NO: 3), wherein X is a glycine (G)
or serine (S).
[0019] In some embodiments, the peptide is not GSTSGSGKPGSGEGSTKG
(SEQ ID NO: 1), GSGKPGSGEG (SEQ ID NO: 13), GKPGSGEG (SEQ ID NO:
14), or SGKPGSGE (SEQ ID NO: 15). In certain embodiments, the
peptide is not GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1). In certain
embodiments, the linker is not GSGKPGSGEG (SEQ ID NO: 13). In
certain embodiments, the peptide is not GKPGSGEG (SEQ ID NO: 14).
In certain embodiments, the peptide is not SGKPGSGE (SEQ ID NO:
15).
[0020] In some embodiments, the peptide comprises 6-20 amino acids.
In some embodiments, the peptide comprises 10-20 amino acids. In
some embodiments, the peptide comprises 14-19 amino acids. In some
embodiments, the peptide comprises 15-17 amino acids. In some
embodiments, the peptide comprises 15-16 amino acids. In some
embodiments, the peptide comprises 16 amino acids.
[0021] In some embodiments, the peptide comprises an amino acid
sequence of GGGSGKPGSGEGGGS (SEQ ID NO: 7). In some embodiments,
the peptide comprises an amino acid sequence of GGGSGKPGSGEGGGGS
(SEQ ID NO: 8). In some embodiments, the peptide comprises an amino
acid sequence of GGGGSGKPGSGGGGS (SEQ ID NO: 9). In some
embodiments, the peptide comprises an amino acid sequence of
GGGGSGKPGSGEGGS (SEQ ID NO: 10). In some embodiments, the peptide
comprises an amino acid sequence of GGGGSGKPGSGEGGGS (SEQ ID NO:
11). In some embodiments, the peptide comprises an amino acid
sequence of GGGGSGKPGSGEGGGGS (SEQ ID NO: 12). In some embodiments,
the peptide comprises an amino acid sequence of STSGSGKPGSGEGST
(SEQ ID NO: 17). In some embodiments, the peptide comprises an
amino acid sequence of GGGGSGGGGSGGGGSG (SEQ ID NO: 18). In some
embodiments, the peptide comprises an amino acid sequence of
GGGGGSGGGGSGGGGS (SEQ ID NO: 19). In some embodiments, the peptide
comprises an amino acid sequence of GGGGSGGGGSGGGGGS (SEQ ID NO:
20).
[0022] In one aspect, the present invention provides a peptide
comprising 8-20 amino acids and an amino acid sequence at least 80%
identical to any one of GGGSGKPGSGEGGGS (SEQ ID NO: 7),
GGGSGKPGSGEGGGGS (SEQ ID NO: 8), GGGGSGKPGSGGGGS (SEQ ID NO: 9),
GGGGSGKPGSGEGGS (SEQ ID NO: 10), GGGGSGKPGSGEGGGS (SEQ ID NO: 11),
GGGGSGKPGSGEGGGGS (SEQ ID NO: 12), STSGSGKPGSGEGST (SEQ ID NO: 17),
GGGGSGGGGSGGGGSG (SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID NO: 19),
or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).
[0023] In some embodiments, the peptide comprises an amino acid
sequence that is at least 90% identical to any one of
GGGSGKPGSGEGGGS (SEQ ID NO:7), GGGSGKPGSGEGGGGS (SEQ ID NO:8),
GGGGSGKPGSGGGGS (SEQ ID NO:9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),
GGGGSGKPGSGEGGGS (SEQ ID NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO:
12), STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO:
18), GGGGGSGGGGSGGGGS (SEQ ID NO: 19), or GGGGSGGGGSGGGGGS (SEQ ID
NO: 20).
[0024] In one aspect, the present invention provides a peptide
comprising 8-20 amino acids and an amino acid sequence that
contains at least six (6) identical amino acids out of ten (10)
contiguous amino acids found in any one of GGGSGKPGSGEGGGS (SEQ ID
NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO: 8), GGGGSGKPGSGGGGS (SEQ ID
NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10), GGGGSGKPGSGEGGGS (SEQ ID
NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12), STSGSGKPGSGEGST (SEQ ID
NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID
NO: 19), or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).
[0025] In some embodiments, the peptide amino acid sequence
contains at least seven (7), at least eight (8) or at least nine
(9) identical amino acids out of ten (10) contiguous amino acids
found in any one of GGGSGKPGSGEGGGS (SEQ ID NO: 7),
GGGSGKPGSGEGGGGS (SEQ ID NO: 8), GGGGSGKPGSGGGGS (SEQ ID NO: 9),
GGGGSGKPGSGEGGS (SEQ ID NO: 10), GGGGSGKPGSGEGGGS (SEQ ID NO: 11),
GGGGSGKPGSGEGGGGS (SEQ ID NO: 12), STSGSGKPGSGEGST (SEQ ID NO: 17),
GGGGSGGGGSGGGGSG (SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID NO: 19),
or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).
[0026] In some embodiments, the peptide comprises an amino acid
sequence of GGGGSGGGGSGGGGSG (SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ
ID NO: 19), or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).
[0027] In one aspect, the present invention provides a fusion
protein comprising a first polypeptide; a second polypeptide; and a
peptide linker as described herein. In some aspects, the fusion
protein is an antigen binding molecule. In some embodiments, the
antigen binding molecule is a scFv. In some embodiments, the first
polypeptide is a light chain variable domain and the second
polypeptide is a heavy chain variable domain. In some embodiments,
the fusion protein is a chimeric antigen receptor.
[0028] In one aspect, the present invention provides a
polynucleotide encoding a peptide (e.g., linker, tag) as described
herein. In some embodiments, the present invention provides a
polynucleotide encoding a fusion protein as described herein.
[0029] In one aspect, the present invention provides an expression
vector comprising a polynucleotide encoding a peptide (e.g., a
linker or fusion protein) as described herein. In some embodiments,
the present invention provides a recombinant cell comprising a
polynucleotide encoding a peptide (e.g., a linker or fusion
protein) as described herein. In some embodiments, the recombinant
cell comprises an expression vector comprising a polynucleotide
encoding a peptide (e.g., a linker or fusion protein) as described
herein.
[0030] Any aspect or embodiment described herein may be combined
with any other aspect or embodiment as disclosed herein. While the
present invention has been described in conjunction with the
detailed description thereof, the foregoing description is intended
to illustrate and not limit the scope of the present invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
[0031] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. All other
published references, dictionaries, documents, manuscripts and
scientific literature cited herein are hereby incorporated by
reference.
[0032] Other features and advantages of the invention will be
apparent from the Drawings and the following Detailed Description,
including the Examples, and the claims.
BRIEF DESCRIPTION OF THE DRAWING
[0033] The above and further features will be more clearly
appreciated from the following detailed description when taken in
conjunction with the accompanying drawings. The drawings however,
are for illustration purposes only, not for limitation.
[0034] FIG. 1 shows an amino acid sequence alignment of exemplary
peptide (e.g., linker) sequences.
[0035] FIG. 2 shows a bar graph of results of an epitope mapping
ELISA experiment of peptides comprising SEQ ID NOs 1, 21-25, 1 and
26-30, respectively.
[0036] FIG. 3 shows a bar graph of the results of antibody binding
profiles of polypeptide linkers comprising SEQ ID Nos 32, 9-11 and
17, respectively.
[0037] FIG. 4 is a series of plots showing the results of flow
cytometry experiments performed using cells presenting a chimeric
antigen receptor (CAR) comprising the peptide KL2 (SEQ ID NO: 10),
KL3 (SEQ ID NO: 11), KL4 (SEQ ID NO: 7), KL5 (SEQ ID NO: 12), KL6
(SEQ ID NO: 8), and G4S2 (SEQ ID NO: 18).
DEFINITIONS
[0038] In order for the present invention to be more readily
understood, certain terms are first defined below. Additional
definitions for the following terms and other terms are set forth
throughout the Specification.
[0039] As used in this Specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise.
[0040] Unless specifically stated or obvious from context, as used
herein, the term "or" is understood to be inclusive and covers both
"or" and "and."
[0041] The term "and/or" where used herein is to be taken as
specific disclosure of each of the two specified features or
components with or without the other. Thus, the term "and/or" as
used in a phrase such as "A and/or B" herein is intended to include
A and B, A or B, A (alone), and B (alone). Likewise, the term
"and/or" as used in a phrase such as "A, B, and/or C" is intended
to encompass each of the following aspects: A, B, and C; A, B, or
C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B
(alone); and C (alone).
[0042] The terms "e.g.," and "i.e." as used herein, are used merely
by way of example, without limitation intended, and should not be
construed as referring only those items explicitly enumerated in
the specification.
[0043] The terms "or more", "at least", "more than", and the like,
e.g., "at least one" are understood to include but not be limited
to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,
127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,
140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300,
400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more
than the stated value. Also included is any greater number or
fraction in between.
[0044] Conversely, the term "no more than" includes each value less
than the stated value. For example, "no more than 100 nucleotides"
includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87,
86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70,
69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53,
52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36,
35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and
0 nucleotides. Also included is any lesser number or fraction in
between.
[0045] The terms "plurality", "at least two", "two or more", "at
least second", and the like, are understood to include but not
limited to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200,
300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or
more. Also included is any greater number or fraction in
between.
[0046] Throughout the specification the word "comprising," or
variations such as "comprises" or "comprising," will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps. It is understood that wherever aspects are described herein
with the language "comprising," otherwise analogous aspects
described in terms of "consisting of" and/or "consisting
essentially of" are also provided.
[0047] Unless specifically stated or evident from context, as used
herein, the term "about" refers to a value or composition that is
within an acceptable error range for the particular value or
composition as determined by one of ordinary skill in the art,
which will depend in part on how the value or composition is
measured or determined, i.e., the limitations of the measurement
system. For example, "about" or "comprising essentially of" may
mean within one or more than one standard deviation per the
practice in the art. "About" or "comprising essentially of" may
mean a range of up to 10% (i.e., .+-.10%). Thus, "about" may be
understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,
0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated
value. For example, about 5 mg may include any amount between 4.5
mg and 5.5 mg. Furthermore, particularly with respect to biological
systems or processes, the terms may mean up to an order of
magnitude or up to 5-fold of a value. When particular values or
compositions are provided in the instant disclosure, unless
otherwise stated, the meaning of "about" or "comprising essentially
of" should be assumed to be within an acceptable error range for
that particular value or composition.
[0048] As described herein, any concentration range, percentage
range, ratio range or integer range is to be understood to be
inclusive of the value of any integer within the recited range and,
when appropriate, fractions thereof (such as one-tenth and
one-hundredth of an integer), unless otherwise indicated.
[0049] Units, prefixes, and symbols used herein are provided using
their Systeme International de Unites (SI) accepted form. Numeric
ranges are inclusive of the numbers defining the range.
[0050] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure is related. For
example, Juo, "The Concise Dictionary of Biomedicine and Molecular
Biology", 2nd ed., (2001), CRC Press; "The Dictionary of Cell &
Molecular Biology", 5th ed., (2013), Academic Press; and "The
Oxford Dictionary Of Biochemistry And Molecular Biology", Cammack
et al. eds., 2nd ed, (2006), Oxford University Press, provide those
of skill in the art with a general dictionary for many of the terms
used in this disclosure.
[0051] "Administering" refers to the physical introduction of an
agent to a subject, using any of the various methods and delivery
systems known to those skilled in the art. Exemplary routes of
administration for the formulations disclosed herein include
intravenous, intramuscular, subcutaneous, intraperitoneal, spinal
or other parenteral routes of administration, for example by
injection or infusion. The phrase "parenteral administration" as
used herein means modes of administration other than enteral and
topical administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intra-arterial,
intrathecal, intralymphatic, intralesional, intracapsular,
intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular, subarachnoid, intraspinal, epidural and intrasternal
injection and infusion, as well as in vivo electroporation. In some
embodiments, the formulation is administered via a non-parenteral
route, e.g., orally. Other non-parenteral routes include a topical,
epidermal, or mucosal route of administration, for example,
intranasally, vaginally, rectally, sublingually or topically.
Administering may also be performed, for example, once, a plurality
of times, and/or over one or more extended periods.
[0052] As used herein, an antigen binding molecule, an antibody, or
an antigen binding molecule thereof "cross-competes" with a
reference antibody or an antigen binding molecule thereof if the
interaction between an antigen and the first binding molecule, an
antibody, or an antigen binding molecule thereof blocks, limits,
inhibits, or otherwise reduces the ability of the reference binding
molecule, reference antibody, or an antigen binding molecule
thereof to interact with the antigen. Cross competition may be
complete, e.g., binding of the binding molecule to the antigen
completely blocks the ability of the reference binding molecule to
bind the antigen, or it may be partial, e.g., binding of the
binding molecule to the antigen reduces the ability of the
reference binding molecule to bind the antigen. In certain
embodiments, an antigen binding molecule that cross-competes with a
reference antigen binding molecule binds the same or an overlapping
epitope as the reference antigen binding molecule. In other
embodiments, the antigen binding molecule that cross-competes with
a reference antigen binding molecule binds a different epitope as
the reference antigen binding molecule. Numerous types of
competitive binding assays may be used to determine if one antigen
binding molecule competes with another, for example: solid phase
direct or indirect radioimmunoassay (RIA); solid phase direct or
indirect enzyme immunoassay (EIA); sandwich competition assay
(Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase
direct biotin-avidin EIA (Kirkland et al., 1986, J. Immunol.
137:3614-3619); solid phase direct labeled assay, solid phase
direct labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A
Laboratory Manual, Cold Spring Harbor Press); solid phase direct
label RIA using 1-125 label (Morel et al., 1988, Molec. Immunol.
25:7-15), solid phase direct biotin-avidin EIA (Cheung, et al.,
1990, Virology 176:546-552), and direct labeled RIA (Moldenhauer et
al., 1990, Scand. J. Immunol. 32:77-82).
[0053] An "antigen" refers to any molecule that provokes an immune
response or is capable of being bound by an antibody or an antigen
binding molecule. The immune response may involve either antibody
production, or the activation of specific immunologically-competent
cells, or both. A person of skill in the art would readily
understand that any macromolecule, including virtually all proteins
or peptides, could serve as an antigen. An antigen may be
endogenously expressed, i.e. expressed by genomic DNA, or may be
recombinantly expressed. An antigen may be specific to a certain
tissue, such as a cancer cell, or it may be broadly expressed. In
addition, fragments of larger molecules may act as antigens. In one
embodiment, antigens are tumor antigens.
[0054] The term "allogeneic" refers to any material derived from
one individual, which is then introduced to another individual of
the same species, e.g., allogeneic T cell transplantation.
[0055] The terms "transduction" and "transduced" refer to the
process whereby foreign DNA is introduced into a cell via viral
vector (see Jones et al., "Genetics: principles and analysis,"
Boston: Jones & Bartlett Publ. (1998)). In some embodiments,
the vector is a retroviral vector, a DNA vector, a RNA vector, an
adenoviral vector, a baculoviral vector, an Epstein Barr viral
vector, a papovaviral vector, a vaccinia viral vector, a herpes
simplex viral vector, an adenovirus associated vector, a lentiviral
vector, or any combination thereof.
[0056] A "cancer" refers to a broad group of various diseases
characterized by the uncontrolled growth of abnormal cells in the
body. Unregulated cell division and growth results in the formation
of malignant tumors that invade neighboring tissues and may
metastasize to distant parts of the body through the lymphatic
system or bloodstream. A "cancer" or "cancer tissue" may include a
tumor. Examples of cancers that may be treated by the methods of
the present invention include, but are not limited to, cancers of
the immune system including lymphoma, leukemia, myeloma, and other
leukocyte malignancies. In some embodiments, the methods of the
present invention may be used to reduce the tumor size of a tumor
derived from, for example, bone cancer, pancreatic cancer, skin
cancer, cancer of the head or neck, cutaneous or intraocular
malignant melanoma, uterine cancer, ovarian cancer, rectal cancer,
cancer of the anal region, stomach cancer, testicular cancer,
uterine cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, multiple myeloma, Hodgkin's Disease,
non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell
lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular
lymphoma (FL), transformed follicular lymphoma, splenic marginal
zone lymphoma (SMZL), cancer of the esophagus, cancer of the small
intestine, cancer of the endocrine system, cancer of the thyroid
gland, cancer of the parathyroid gland, cancer of the adrenal
gland, sarcoma of soft tissue, cancer of the urethra, cancer of the
penis, chronic or acute leukemia, acute myeloid leukemia, chronic
myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non
T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of
childhood, lymphocytic lymphoma, cancer of the bladder, cancer of
the kidney or ureter, carcinoma of the renal pelvis, neoplasm of
the central nervous system (CNS), primary CNS lymphoma, tumor
angiogenesis, spinal axis tumor, brain stem glioma, pituitary
adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer,
T-cell lymphoma, environmentally induced cancers including those
induced by asbestos, other B cell malignancies, and combinations of
said cancers. In one particular embodiment, the cancer is multiple
myeloma. The particular cancer may be responsive to chemo- or
radiation therapy or the cancer may be refractory. A refractor
cancer refers to a cancer that is not amendable to surgical
intervention and the cancer is either initially unresponsive to
chemo- or radiation therapy or the cancer becomes unresponsive over
time.
[0057] An "anti-tumor effect" as used herein, refers to a
biological effect that may present as a decrease in tumor volume, a
decrease in the number of tumor cells, a decrease in tumor cell
proliferation, a decrease in the number of metastases, an increase
in overall or progression-free survival, an increase in life
expectancy, or amelioration of various physiological symptoms
associated with the tumor. An anti-tumor effect may also refer to
the prevention of the occurrence of a tumor, e.g., a vaccine.
[0058] A "cytokine," as used herein, refers to a non-antibody
protein that is released by one cell in response to contact with a
specific antigen, wherein the cytokine interacts with a second cell
to mediate a response in the second cell. A cytokine may be
endogenously expressed by a cell or administered to a subject.
Cytokines may be released by immune cells, including macrophages, B
cells, T cells, and mast cells to propagate an immune response.
Cytokines may induce various responses in the recipient cell.
Cytokines may include homeostatic cytokines, chemokines,
pro-inflammatory cytokines, effectors, and acute-phase proteins.
For example, homeostatic cytokines, including interleukin (IL) 7
and IL-15, promote immune cell survival and proliferation, and
pro-inflammatory cytokines may promote an inflammatory response.
Examples of homeostatic cytokines include, but are not limited to,
IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and
interferon (IFN) gamma. Examples of pro-inflammatory cytokines
include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a,
tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth
factor (FGF) 2, granulocyte macrophage colony-stimulating factor
(GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1),
soluble vascular adhesion molecule 1 (sVCAM-1), vascular
endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental
growth factor (PLGF). Examples of effectors include, but are not
limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and
perforin. Examples of acute phase-proteins include, but are not
limited to, C-reactive protein (CRP) and serum amyloid A (SAA).
[0059] "Chemokines" are a type of cytokine that mediates cell
chemotaxis, or directional movement. Examples of chemokines
include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3,
macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic
protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein
1.alpha. (MIP-1.alpha., MIP-1a), MIP-1.beta. (MIP-1b),
gamma-induced protein 10 (IP-10), and thymus and activation
regulated chemokine (TARC or CCL17).
[0060] A "therapeutically effective amount," "effective dose,"
"effective amount," or "therapeutically effective dosage" of a
therapeutic agent, e.g., engineered CAR T cells, is any amount
that, when used alone or in combination with another therapeutic
agent, protects a subject against the onset of a disease or
promotes disease regression evidenced by a decrease in severity of
disease symptoms, an increase in frequency and duration of disease
symptom-free periods, or a prevention of impairment or disability
due to the disease affliction. The ability of a therapeutic agent
to promote disease regression may be evaluated using a variety of
methods known to the skilled practitioner, such as in human
subjects during clinical trials, in animal model systems predictive
of efficacy in humans, or by assaying the activity of the agent in
in vitro assays.
[0061] The term "lymphocyte" as used herein includes natural killer
(NK) cells, T cells, or B cells. NK cells are a type of cytotoxic
(cell toxic) lymphocyte that represent a major component of the
inherent immune system. NK cells reject tumors and cells infected
by viruses. It works through the process of apoptosis or programmed
cell death. They were termed "natural killers" because they do not
require activation in order to kill cells. T-cells play a major
role in cell-mediated-immunity (no antibody involvement). Its
T-cell receptors (TCR) differentiate themselves from other
lymphocyte types. The thymus, a specialized organ of the immune
system, is primarily responsible for the T cell's maturation. There
are six types of T-cells, namely: Helper T-cells (e.g., CD4+
cells), Cytotoxic T-cells (also known as TC, cytotoxic T
lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cells or
killer T cell), Memory T-cells ((i) stem memory TSCM cells, like
naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+(L-selectin),
CD27+, CD28+ and IL-7Ra+, but they also express large amounts of
CD95, IL-2R3, CXCR3, and LFA-1, and show numerous functional
attributes distinctive of memory cells); (ii) central memory TCM
cells express L-selectin and the CCR7, they secrete IL-2, but not
IFN.gamma. or IL-4, and (iii) effector memory TEM cells, however,
do not express L-selectin or CCR7 but produce effector cytokines
like IFN.gamma. and IL-4), Regulatory T-cells (Tregs, suppressor T
cells, or CD4+CD25+ regulatory T cells), Natural Killer T-cells
(NKT) and Gamma Delta T-cells. B-cells, on the other hand, play a
principal role in humoral immunity (with antibody involvement).
They make antibodies and antigens, perform the role of
antigen-presenting cells (APCs), and turn into memory B-cells after
activation by antigen interaction. In mammals, immature B-cells are
formed in the bone marrow.
[0062] The term "genetically engineered", "engineered", or
"modified" refers to a method of modifying a cell, including, but
not limited to, creating a deficiency in a gene by deleting a
coding or non-coding region or a portion thereof or by antisense
technology, or increasing expression of a protein introducing a
coding region or a portion thereof. In some embodiments, the cell
that is modified is a stem cell (e.g., hematopoietic stem cell
(HSC), embryonic stem cell (ES), induced pluripotent stem (iPS)
cell), lymphocyte (e.g., a T cell), which may be obtained either
from a patient or a donor. The cell may be modified to express an
exogenous construct, such as, e.g., a pre-TCR alpha protein, a
chimeric antigen receptor (CAR) or a T cell receptor (TCR), which
may be incorporated into the cell's genome.
[0063] An "immune response" refers to the action of a cell of the
immune system (for example, T lymphocytes, B lymphocytes, natural
killer (NK) cells, macrophages, eosinophils, mast cells, dendritic
cells and neutrophils) and soluble macromolecules produced by any
of these cells or the liver (including Abs, cytokines, and
complement) that results in selective targeting, binding to, damage
to, destruction of, and/or elimination from a vertebrate's body of
invading pathogens, cells or tissues infected with pathogens,
cancerous or other abnormal cells, or, in cases of autoimmunity or
pathological inflammation, normal human cells or tissues.
[0064] The term "immunotherapy" refers to the treatment of a
subject afflicted with, or at risk of contracting or suffering a
recurrence of, a disease by a method comprising inducing,
enhancing, suppressing, or otherwise modifying an immune response.
Examples of immunotherapy include, but are not limited to, T cell
therapies. T cell therapy may include adoptive T cell therapy,
tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell
therapy, engineered autologous cell therapy (eACT.TM.), and
allogeneic T cell transplantation. However, one of skill in the art
would recognize that the conditioning methods disclosed herein
would enhance the effectiveness of any transplanted T cell therapy.
Examples of T cell therapies are described in U.S. Patent
Publication Nos. 2014/0154228 and 2002/0006409, U.S. Pat. No.
5,728,388, and International Publication No. WO 2008/081035.
[0065] The T cells of the immunotherapy may come from any source
known in the art.
[0066] For example, T cells may be differentiated in vitro from a
hematopoietic stem cell population; induced pluripotent stem cells
(iPS), embryonic stem cells (ES), or T cells may be obtained from a
subject. T cells may be obtained from, e.g., peripheral blood
mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord
blood, thymus tissue, tissue from a site of infection, ascites,
pleural effusion, spleen tissue, and tumors. In addition, the T
cells may be derived from one or more T cell lines available in the
art. T cells may also be obtained from a unit of blood collected
from a subject using any number of techniques known to the skilled
artisan, such as FICOLL.TM. separation and/or apheresis. Additional
methods of isolating T cells for a T cell therapy are disclosed in
U.S. Patent Publication No. 2013/0287748, which is herein
incorporated by references in its entirety.
[0067] The term "engineered Autologous Cell Therapy," which may be
abbreviated as "eACT.TM.," also known as adoptive cell transfer, is
a process by which a patient's own T cells are collected and
subsequently genetically altered to recognize and target one or
more antigens expressed on the cell surface of one or more specific
tumor cells or malignancies. A "patient" as used herein includes
any human who is afflicted with a cancer (e.g., a lymphoma or a
leukemia). The terms "subject" and "patient" are used
interchangeably herein.
[0068] As used herein, the term "in vitro cell" refers to any cell,
which is cultured ex vivo. In particular, an in vitro cell may
include a T cell.
[0069] The terms "peptide," "polypeptide," and "protein" are used
interchangeably, and refer to a compound comprised of amino acid
residues covalently linked by peptide bonds. A protein or peptide
contains at least two amino acids, and no limitation is placed on
the maximum number of amino acids that may comprise a protein or
peptide's sequence. As used herein, peptides of the present
invention may function as a linker (e.g., joining two peptides or
polypeptides). As used herein, peptides of the present invention
may function as a biomarker or tag. The terms peptide, tag, or
linker are used interchangeably. Polypeptides include any peptide
or protein comprising two or more amino acids joined to each other
by peptide bonds. As used herein, the term refers to both short
chains, which also commonly are referred to in the art as peptides,
oligopeptides and oligomers, for example, and to longer chains,
which generally are referred to in the art as proteins, of which
there are many types. "Polypeptides" include, for example,
biologically active fragments, substantially homologous
polypeptides, oligopeptides, homodimers, heterodimers, variants of
polypeptides, modified polypeptides, derivatives, analogs, fusion
proteins, among others. The polypeptides include natural peptides,
recombinant peptides, synthetic peptides, or a combination
thereof.
[0070] "Stimulation," as used herein, refers to a primary response
induced by binding of a stimulatory molecule with its cognate
ligand, wherein the binding mediates a signal transduction event. A
"stimulatory molecule" is a molecule on a T cell, e.g., the T cell
receptor (TCR)/CD3 complex, which specifically binds with a cognate
stimulatory ligand present on an antigen present cell. A
"stimulatory ligand" is a ligand that when present on an antigen
presenting cell (e.g., an APC, a dendritic cell, a B-cell, and the
like) may specifically bind with a stimulatory molecule on a T
cell, thereby mediating a primary response by the T cell,
including, but not limited to, activation, initiation of an immune
response, proliferation, and the like. Stimulatory ligands include,
but are not limited to, an anti-CD3 antibody, an MHC Class I
molecule loaded with a peptide, a superagonist anti-CD2 antibody,
and a superagonist anti-CD28 antibody.
[0071] A "costimulatory signal," as used herein, refers to a
signal, which in combination with a primary signal, such as TCR/CD3
ligation, leads to a T cell response, such as, but not limited to,
proliferation and/or upregulation or down regulation of key
molecules.
[0072] A "costimulatory ligand" as used herein, includes a molecule
on an antigen presenting cell that specifically binds a cognate
co-stimulatory molecule on a T cell. Binding of the costimulatory
ligand provides a signal that mediates a T cell response,
including, but not limited to, proliferation, activation,
differentiation, and the like. A costimulatory ligand induces a
signal that is in addition to the primary signal provided by a
stimulatory molecule, for instance, by binding of a T cell receptor
(TCR)/CD3 complex with a major histocompatibility complex (MHC)
molecule loaded with peptide. A co-stimulatory ligand may include,
but is not limited to, 3/TR6, 4-1BB ligand, agonist or antibody
that binds Toll ligand receptor, B7-1 (CD80), B7-2 (CD86), CD30
ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM),
human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like
transcript (ILT) 3, inducible costimulatory ligand (ICOS-L),
intercellular adhesion molecule (ICAM), ligand that specifically
binds with B7-H3, lymphotoxin beta receptor, MHC class I
chain-related protein A (MICA), MHC class I chain-related protein B
(MICB), OX40 ligand, PD-L2, or programmed death (PD) L1. A
co-stimulatory ligand includes, without limitation, an antibody
that specifically binds with a co-stimulatory molecule present on a
T cell, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28,
CD30, CD40, CD7, ICOS, ligand that specifically binds with CD83,
lymphocyte function-associated antigen-1 (LFA-1), natural killer
cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor
superfamily member 14 (TNFSF14 or LIGHT).
[0073] A "costimulatory molecule" is a cognate binding partner on a
T cell that specifically binds with a costimulatory ligand, thereby
mediating a costimulatory response by the T cell, such as, but not
limited to, proliferation. Costimulatory molecules include, but are
not limited to, A "costimulatory molecule" is a cognate binding
partner on a T cell that specifically binds with a costimulatory
ligand, thereby mediating a costimulatory response by the T cell,
such as, but not limited to, proliferation. Costimulatory molecules
include, but are not limited to, 4-1BB/CD137, B7-H3, BAFFR, BLAME
(SLAMF8), BTLA, CD 33, CD 45, CD100 (SEMA4D), CD103, CD134, CD137,
CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247,
CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha; beta; delta; epsilon;
gamma; zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5,
CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8alpha, CD8beta,
CD9, CD96 (Tactile), CD1-1a, CD1-1b, CD1-1c, CD1-1d, CDS, CEACAM1,
CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM
(LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta,
IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD,
ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT,
LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily
member 14; TNFSF14), LTBR, Ly9 (CD229), lymphocyte
function-associated antigen-1 (LFA-1 (CD11a/CD18), MHC class I
molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40,
PAG/Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocytic
activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244;
2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll
ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or fragments,
truncations, or combinations thereof.
[0074] The terms "reducing" and "decreasing" are used
interchangeably herein, and indicate any change that is less than
the original. "Reducing" and "decreasing" are relative terms,
requiring a comparison between pre- and post-measurements
"Reducing" and "decreasing" include complete depletions.
[0075] "Treatment" or "treating" of a subject refers to any type of
intervention or process performed on, or the administration of an
active agent to, the subject with the objective of reversing,
alleviating, ameliorating, inhibiting, slowing down or preventing
the onset, progression, development, severity, or recurrence of a
symptom, complication or condition, or biochemical indicia
associated with a disease. In one embodiment, "treatment" or
"treating" includes a partial remission. In another embodiment,
"treatment" or "treating" includes a complete remission.
[0076] To calculate percent identity, the sequences being compared
are typically aligned in a way that gives the largest match between
the sequences. One example of a computer program that may be used
to determine percent identity is the GCG program package, which
includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387;
Genetics Computer Group, University of Wisconsin, Madison, Wis.).
The computer algorithm GAP is used to align the two polypeptides or
polynucleotides for which the percent sequence identity is to be
determined. The sequences are aligned for optimal matching of their
respective amino acid or nucleotide (the "matched span," as
determined by the algorithm.) In certain embodiments, a standard
comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein
Sequence and Structure 5:345-352 for the PAM 250 comparison matrix;
Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919
for the BLOSUM 62 comparison matrix) is also used by the
algorithm.
[0077] Various aspects of the invention are described in further
detail in the following subsections.
DETAILED DESCRIPTION
[0078] Compositions are described herein that provide a means to
make (e.g., design, engineer) chimeric or fusion polypeptides. A
peptide (e.g., linker) sequence as described herein allows for the
proper expression, folding and activity of a fusion protein. The
present invention provides, among other things, novel polypeptides
(e.g., linkers) and fusion proteins comprising the same. In some
embodiments, the present invention provides polynucleotide
compositions encoding a peptide (e.g., linker, tag) or fusion
protein described herein. In some embodiments, the present
invention provides an expression vector comprising the
polynucleotide encoding a peptide (e.g., linker, tag) or fusion
protein. In other embodiments, the present invention provides a
cell comprising the polynucleotide and/or the expression vector
encoding a peptide (e.g., linker, tag) or fusion protein. Described
herein are novel compositions comprising peptide linkers,
polypeptide compositions comprising polypeptides joined by the
peptide linkers and related polynucleotides, vectors, cells and
pharmaceutical compositions. In some embodiments, the peptide
(e.g., linker, tag) is fused to one or more polypeptides. Described
linker sequences operably join two peptides/polypeptides of
interest such that the expression and activity (e.g., antigen
binding) of the polypeptides connected by the linkers are durable
and optimal. The peptide linker or tag may be fused at the
C-terminus, N-terminus, or anywhere within the polypeptide to
achieve the desired function.
Peptide Linkers
[0079] Novel chimeric polypeptide linkers described herein
comprising a consensus sequence XYPXXXZX combine desirable
attributes suitable for incorporation into fusion proteins useful
for therapeutic intervention. In one aspect, the present invention
provides a linker comprising 8-20 amino acids and a consensus
sequence XYPXXXZX, wherein X is a glycine (G) or serine (S), B is a
positively charged amino acid and Z is glycine (G) or a negatively
charged amino acid. The inventors have discovered that both the
spacing and charge of the of the amino acid residues in the
consensus sequence contribute to functionality of the linker in
addition to antibody recognition of the linker sequence.
[0080] In one aspect, the present invention provides a linker
comprising 6-20 amino acids and a consensus sequence BPXXXZ,
wherein X is a glycine (G) or serine (S), B is lysine (K) or
arginine (R), and Z is glycine (G) or a negatively charged amino
acid, and P is proline.
[0081] In some embodiments, the present invention provides a linker
comprising 8-20 amino acids and a consensus sequence XBPXXXZX,
wherein X is a glycine (G) or serine (S), B is lysine (K) or
arginine (R), and Z is glycine (G) or a negatively charged amino
acid, and P is proline.
[0082] In some embodiments, the present invention provides a linker
comprising 8-20 amino acids and a consensus sequence XBPXXXZX,
wherein X is a glycine (G) or serine (S), B is lysine (K), and Z is
glycine (G) or a negatively charged amino acid, and P is
proline.
[0083] In some embodiments, the present invention provides a linker
comprising 8-20 amino acids and a consensus sequence XBPXXXZX,
wherein X is a Glycine (G) or serine (S), B is a positively charged
amino acid, and Z is glycine (G), and P is proline.
[0084] In some embodiments, Z is a negatively charged amino acid
selected from glutamic acid (E) or aspartic acid (D). In some
embodiments, Z is glutamic acid (E).
[0085] In some embodiments, the present invention provides a
linker, wherein the consensus sequence is GKPGSGE (SEQ ID NO: 5) or
GKPGSGG (SEQ ID NO: 6). In some embodiments, the consensus sequence
is GKPGSGE (SEQ ID NO: 5).
[0086] In some embodiments, the peptide comprises an amino acid
sequence of GGGGSGGGGSGGGGSG (SEQ ID NO: 18).
[0087] The linker peptide sequence may be of any appropriate length
to connect one or more proteins of interest and is preferably
designed to be sufficiently flexible so as to allow the proper
folding and/or function and/or activity of one or both of the
peptides it connects. Thus, the linker peptide may have a length of
no more than 10, no more than 11, no more than 12, no more than 13,
no more than 14, no more than 15, no more than 16, no more than 17,
no more than 18, no more than 19, or no more than 20 amino acids.
In some embodiments, the linker peptide may have a length of at
least 3, at least 4, at least 5, at least 6, at least 7, at least
8, at least 9, at least 10, at least 11, at least 12, at least 13,
at least 14, at least 15, at least 16, at least 17, at least 18, at
least 19, or at least 20 amino acids. In some embodiments, the
linker comprises at least 7 and no more than 20 amino acids, at
least 7 and no more than 19 amino acids, at least 7 and no more
than 18 amino acids, at least 7 and no more than 17 amino acids, at
least 7 and no more than 16 amino acids, at least 7 and no more 15
amino acids, at least 7 and no more than 14 amino acids, at least 7
and no more than 13 amino acids, at least 7 and no more than 12
amino acids or at least 7 and no more than 11 amino acids. In
certain embodiments, the linker comprises 15-17 amino acids, and in
particular embodiments, comprises 16 amino acids. In some
embodiments, the linker comprises 10-20 amino acids. In some
embodiments, the linker comprises 14-19 amino acids. In some
embodiments, the linker comprises 15-17 amino acids. In some
embodiments, the linker comprises 15-16 amino acids. In some
embodiments, the linker comprises 16 amino acids. In some
embodiments, the linker comprises 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20 amino acids.
[0088] As used herein, a "conservative amino acid substitution" is
one in which the amino acid residue is replaced with an amino acid
residue having a similar side chain. Families of amino acid
residues having side chains have been defined in the art. These
families include amino acids with basic or positively charged side
chains (e.g., lysine, arginine, histidine), acidic or negatively
charged side chains (e.g., aspartic acid, glutamic acid), uncharged
polar side chains (e.g., glycine, asparagine, glutamine, serine,
threonine, tyrosine, cysteine, tryptophan), nonpolar side chains
(e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine), beta-branched side chains (e.g.,
threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine). In certain
embodiments, one or more amino acid residues within a polypeptide
linker, fusion protein, CDR(s) or within a framework region(s) of
an antibody or antigen binding molecule provided herein (or
fragment thereof) may be replaced with an amino acid residue with a
similar side chain.
[0089] Conservative amino acid substitutions, which are encompassed
by the present disclosure, may encompass non-naturally occurring
amino acid residues, which are typically incorporated by chemical
peptide synthesis rather than by synthesis in biological systems.
These include peptidomimetics and other reversed or inverted forms
of amino acid moieties. Naturally occurring residues may be divided
into classes based on common side chain properties: [0090]
hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; [0091] neutral
hydrophilic: Cys, Ser, Thr, Asn, Gln; [0092] acidic (negatively
charged): Asp, Glu; [0093] basic (negatively charged): His, Lys,
Arg; [0094] residues that influence chain orientation: Gly, Pro;
and [0095] aromatic: Trp, Tyr, Phe.
[0096] Non-conservative substitutions may involve the exchange of a
member of one of these classes for a member from another class.
Such substituted residues may be introduced, for example, into
regions of a human antibody that are homologous with non-human
antibodies, or into the non-homologous regions of the molecule.
Exemplary conservative amino acid substitutions are set forth in
Table A below.
TABLE-US-00001 TABLE A Original Residues Exemplary Substitutions
Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln Asp Glu Cys Ser, Ala
Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu, Val,
Met, Ala, Phe, Norleucine Leu Norleucine, Ile, Val, Met, Ala, Phe
Lys Arg, 1,4 Diamino-butyric acid, Gln, Asn Met Leu, Phe, Ile Phe
Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr,
Phe Tyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala,
Norleucine
[0097] In some embodiments, the linker comprises an amino acid
sequence of GGGSGKPGSGEGGGS (SEQ ID NO: 7). In some embodiments,
the linker comprises an amino acid sequence of GGGSGKPGSGEGGGGS
(SEQ ID NO: 8). In some embodiments, the linker comprises an amino
acid sequence of GGGGSGKPGSGGGGS (SEQ ID NO: 9). In some
embodiments, the linker comprises an amino acid sequence of
GGGGSGKPGSGEGGS (SEQ ID NO: 10). In some embodiments, the linker
comprises an amino acid sequence of GGGGSGKPGSGEGGGS (SEQ ID NO:
11). In some embodiments, the linker comprises an amino acid
sequence of GGGGSGKPGSGEGGGGS (SEQ ID NO: 12). In some embodiments,
the linker comprises an amino acid sequence of STSGSGKPGSGEGST (SEQ
ID NO: 17). In some embodiments, the peptide comprises an amino
acid sequence of GGGGSGGGGSGGGGSG (SEQ ID NO: 18). In some
embodiments, the peptide comprises an amino acid sequence of
GGGGGSGGGGSGGGGS (SEQ ID NO: 19). In some embodiments, the peptide
comprises an amino acid sequence of GGGGSGGGGSGGGGGS (SEQ ID NO:
20).
[0098] In one aspect, the present invention provides a linker
comprising 6-20 amino acids and an amino acid sequence at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of
GGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO:8),
GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),
GGGGSGKPGSGEGGGS (SEQ ID NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO:
12), STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO:
18), GGGGGSGGGGSGGGGS (SEQ ID NO: 19) or GGGGSGGGGSGGGGGS (SEQ ID
NO: 20).
[0099] In some embodiments, the linker amino acid sequence contains
at least five (5), six (6), seven (7), eight (8) or at least nine
(9) identical amino acids out of ten (10) contiguous amino acids
found in any one of GGGSGKPGSGEGGGS (SEQ ID NO: 7),
GGGSGKPGSGEGGGGS (SEQ ID NO: 8), GGGGSGKPGSGGGGS (SEQ ID NO: 9),
GGGGSGKPGSGEGGS (SEQ ID NO: 10), GGGGSGKPGSGEGGGS (SEQ ID NO: 11),
GGGGSGKPGSGEGGGGS (SEQ ID NO: 12), STSGSGKPGSGEGST (SEQ ID NO: 17),
GGGGSGGGGSGGGGSG (SEQ ID NO: 18), or GGGGGSGGGGSGGGGS (SEQ ID NO:
19) or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).
[0100] In one aspect, the present invention provides a linker
comprising 8-20 amino acids and an amino acid sequence that
contains at least six (6) identical amino acids out of ten (10)
contiguous amino acids found in any one of GGGSGKPGSGEGGGS (SEQ ID
NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO: 8), GGGGSGKPGSGGGGS (SEQ ID
NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10), GGGGSGKPGSGEGGGS (SEQ ID
NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12), STSGSGKPGSGEGST (SEQ ID
NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID
NO: 19) or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).
[0101] In some embodiments, the linker amino acid sequence contains
at least seven (7), at least eight (8) or at least nine (9)
identical amino acids out of ten (10) contiguous amino acids found
in any one of GGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ
ID NO: 8), GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID
NO: 10), GGGGSGKPGSGEGGGS (SEQ ID NO: 11), or GGGGSGKPGSGEGGGGS
(SEQ ID NO: 12), STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG
(SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID NO: 19) or
GGGGSGGGGSGGGGGS (SEQ ID NO: 20).
Fusion Protein
[0102] In one aspect, the present invention provides a fusion
protein comprising a first polypeptide; a second polypeptide; and a
linker as described herein. Polypeptide composition and
polynucleotides encoding the polypeptide compositions are described
herein, in which the polypeptide compositions comprise a first and
second peptide/polypeptide, connected by a linker sequence
disclosed herein. The inventors have surprisingly found that a
linker according to the present invention provides both optimal
flexibility of the first and second peptide and length to avoid
steric hindrance and allow correct folding.
[0103] Polypeptide compositions produced in this manner are
commonly referred to a fusion or chimeric protein/polypeptides and
typically are made by the expression (e.g., transcription,
translation) of nucleic acid sequences encoding the polypeptide
compositions, in the appropriate system. Means by which to make
fusion and/or chimeric polypeptides are well-known in the art (see
for example, Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Springs Harbor Laboratory, 1992) New York which is
incorporated by reference herein in its entirety).
[0104] In the polypeptide compositions described herein, the two
polypeptides (e.g., a first polypeptide and a second polypeptide)
may be recombinantly joined by any of the linker polypeptides
described above, with the linker disposed between the two
polypeptides. For example, in certain embodiments, the polypeptides
or compositions comprise a first and a second polypeptide
recombinantly joined by a linker comprising GGGSGKPGSGEGGGS (SEQ ID
NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO: 8), GGGGSGKPGSGGGGS (SEQ ID
NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10), GGGGSGKPGSGEGGGS (SEQ ID
NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12), STSGSGKPGSGEGST (SEQ ID
NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID
NO: 19) or GGGGSGGGGSGGGGGS (SEQ ID NO: 20). The two polypeptides
may be any amino acid sequences including full-length proteins,
protein fragments or portions, functional protein fragments or
portions, functional protein domains and the like, of either two
different proteins or the same protein.
[0105] As used herein, the term "polypeptide" or "peptide" refers a
polymer of amino acid residues typically joined exclusively by
peptide bonds, that may be produced naturally (e.g., isolated,
essentially purified or purified) or synthetically (e.g., by
chemical synthesis). A polypeptide produced by expression of a
non-host DNA molecule is a "heterologous" peptide or polypeptide.
An "amino acid residue" comprising the polypeptide may be a natural
or non-natural amino acid residue linked by peptide bonds and/or
bonds different from peptide bonds. The amino acid residues may be
in D-configuration or L-configuration. In some aspects, the
polypeptides referred to herein are proteins, peptides or fragments
thereof produced by the expression of recombinant nucleic acid. In
some embodiments, the polypeptide compositions described herein
comprise two polypeptides connected by a linker sequence.
[0106] As used herein, "functional fragment" or "portion" is
intended to refer to less than the entire mature or native protein
which is sufficient to retain one or more of the desired biological
activities of the mature or native protein (e.g., sufficient to
retain a therapeutic or ameliorative biological activity with
respect to a disorder to be treated). Thus, amino acid sequences or
polypeptides may be modified, for example, polypeptide sequences
into which amino acids have been inserted, deleted and/or
substituted in such a manner that the modifications do not
substantially interfere with the polypeptide's ability to encode a
functional agent.
[0107] The linker or polypeptide linker described herein refers to
a peptide sequence designed to connect (e.g., join, link) two
protein sequences, wherein the linker peptide sequence is typically
not disposed between the two protein sequences in nature. In the
context of the present invention, the phrase "linked" or "joined"
or "connected" generally refers to a functional linkage between two
contiguous or adjacent amino acid sequences to produce a
polypeptide that generally does not exist in nature. In certain
embodiments, linkage may be used to refer to a covalent linkage of,
for example, the amino acid sequences of a first polypeptide and
the second polypeptide (e.g., antibody heavy chain and light
chain). Generally, linked proteins are contiguous or adjacent to
one another and retain their respective operability and function
when joined. Peptides comprising the chimeric polypeptides
disclosed herein are linked by means of an interposed peptide
linker comprising one or more amino acids. Such linkers may provide
desirable flexibility to permit the desired expression, activity
and/or conformational positioning of the chimeric polypeptide. A
typical amino acid linker is generally designed to be flexible or
to interpose a structure, such as an alpha-helix, between the two
protein moieties. A linker may be fused to the N-terminus or
C-terminus of a polypeptide, or inserted internally.
[0108] In a polypeptide composition comprising a linker, the 5' end
(e.g., terminus) of the linker peptide sequence (e.g., amino acid
sequence) is adjacent to and covalently linked to the 3' end of one
protein sequence (first peptide) (e.g., full-length protein or
protein domain, fragment or variant) and, further, the 3' end of
the linker amino acid sequence is adjacent to and covalently linked
to the 5' end of another protein sequence (second peptide).
Antigen Binding Molecules
[0109] In some aspects, the fusion protein is an antigen binding
molecule. In some embodiments, the first polypeptide is a light
chain variable domain and the second polypeptide is a heavy chain
variable domain. In some embodiments, the use of a linker as
described herein to join an antibody heavy chain and light chain
variable region, provides the benefit of permitting optimal
flexibility and length to avoid steric hindrance and allow correct
folding of the antigen binding domains. Proper conformation of the
first and second peptides is essential for antigen recognition and
binding.
[0110] As used herein, the terms "variable region" or "variable
domain" are used interchangeably and mean a portion of an antibody,
generally, a portion of a light or heavy chain, typically the
amino-terminal end of the antibody, and comprising about 100-130
amino acids in the heavy chain and about 90 to 115 amino acids in
the light chain, which differ extensively in sequence among
antibodies and are used in the binding and specificity of a
particular antibody for a particular antigen. The variability in
sequence is concentrated in those regions called complementarity
determining regions (CDRs) while the more highly conserved regions
in the variable domain are called framework regions (FR). The CDRs
of the light and heavy chains are primarily responsible for the
interaction and specificity of the antibody with antigen.
[0111] In certain embodiments, the variable region of an antigen
binding molecule is a human variable region. In further
embodiments, the variable region comprises rodent, human or murine
CDRs and human framework regions (FRs). In further embodiments, the
variable region is a primate (e.g., a non-human primate) variable
region. In yet further embodiments, the variable region is a rabbit
variable region. In other embodiments, the variable region
comprises human CDRs and non-human (e.g., rabbit, murine, rat or
non-human primate) framework regions (FRs). In other embodiments,
the variable region comprises non-human (e.g., rabbit, murine, rat
or non-human primate) CDRs and human framework regions (FRs).
[0112] The terms "VH," "VH domain" and "VH chain" are used
interchangeably and mean the heavy chain variable region of an
antigen binding molecule, antibody or an antigen binding fragment
thereof. As used herein, the term "heavy chain" when used in
reference to an antibody may refer to any distinct type, e.g.,
alpha (.alpha.), delta (.delta.), epsilon (.epsilon.), gamma
(.gamma.) and mu (.mu.), based on the amino acid sequence of the
constant domain, which give rise to IgA, IgD, IgE, IgG and IgM
classes of antibodies, respectively, including subclasses of IgG,
e.g., IgG1, IgG2, IgG3 and IgG4.
[0113] The terms "VL," "VL domain" and "VL chain" are used
interchangeably and mean the light chain variable region of an
antigen binding molecule, antibody or an antigen binding fragment
thereof. As used herein, the term "light chain" when used in
reference to an antibody may refer to any distinct type, e.g.,
kappa (.kappa.) or lambda (.lamda.) based on the amino acid
sequence of the constant domains. Light chain amino acid sequences
are well known in the art. In specific embodiments, the light chain
is a human light chain.
[0114] An "antigen binding molecule," "antigen binding portion," or
"antibody fragment" refers to any molecule that comprises the
antigen binding parts (e.g., CDRs) of the antibody from which the
molecule is derived. An antigen binding molecule may include the
antigenic complementarity determining regions (CDRs). Examples of
antibody fragments include, but are not limited to, Fab, Fab',
F(ab').sub.2, and Fv fragments, dAb, linear antibodies, scFv
antibodies, and multispecific antibodies formed from antigen
binding molecules. Peptibodies (i.e., Fc fusion molecules
comprising peptide binding domains) are another example of suitable
antigen binding molecules. In some embodiments, the antigen binding
molecule binds to an antigen on a tumor cell. In some embodiments,
the antigen binding molecule binds to an antigen on a cell involved
in a hyperproliferative disease or to a viral or bacterial antigen.
In further embodiments, the antigen binding molecule is an antibody
or fragment thereof, including one or more of the complementarity
determining regions (CDRs) thereof. In further embodiments, the
antigen binding molecule is a single chain variable fragment
(scFv).
[0115] As used herein, the terms "single-chain antibody" and
"single chain fragment variable (scFv)" are used interchangeably
and mean an antigen binding molecule in which a VL and a VH region
are joined via a linker to form a continuous protein chain wherein
the linker is long enough to allow the protein chain to fold back
on itself and form a monovalent antigen binding site (see, e.g.,
Bird et al., (1988) Science 242:423-26 and Huston et al., (1988)
Proc. Natl. Acad. Sci. U.S.A. 85:5879-83 (1988). FMC63 (Nicholson
et al., (1997) Mol. Immunol. 34: (16-17) 1157-65) is a specific
example of a scFv, and is specific for CD19.
[0116] In some embodiments, the antigen binding molecule is a
scFv.
[0117] In some embodiments, the present invention provides antigen
binding molecules, including scFv, that comprise a consensus
sequences BPXXXZ, XBPXXXZX or exemplary linker sequence as
described herein (e.g., KPGSGE (SEQ ID NO: 4), GKPGSGE (SEQ ID NO:
5), GKPGSGG (SEQ ID NO: 6), GGGSGKPGSGEGGGS (SEQ ID NO: 7),
GGGSGKPGSGEGGGGS (SEQ ID NO: 8), GGGGSGKPGSGGGGS (SEQ ID NO: 9),
GGGGSGKPGSGEGGS (SEQ ID NO: 10), GGGGSGKPGSGEGGGS (SEQ ID NO: 11),
GGGGSGKPGSGEGGGGS (SEQ ID NO: 12), STSGSGKPGSGEGST (SEQ ID NO: 17),
GGGGSGGGGSGGGGSG (SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID NO: 19)
or GGGGSGGGGSGGGGGS (SEQ ID NO: 20). In some embodiments, the
molecules comprising these sequences and cells presenting such
molecules, polynucleotides encoding the antigen binding molecules
are also provided, and form an aspect of the instant
disclosure.
[0118] As used herein, the term "binding affinity" means the
strength of the sum total of non-covalent interactions between a
single binding site of a molecule (e.g., an antigen binding
molecule such as an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y may
generally be represented by the dissociation constant (Kd).
Affinity may be measured and/or expressed in a number of ways known
in the art, including, but not limited to, equilibrium dissociation
constant (Kd), and equilibrium association constant (Ka). The Kd is
calculated from the quotient of koff/kon, whereas Ka is calculated
from the quotient of kon/koff. kon refers to the association rate
constant of, e.g., an antibody to an antigen, and koff refers to
the dissociation of, e.g., an antibody to an antigen. The kon and
koff may be determined by standard techniques known to one of
ordinary skill in the art, such as BIAcore.RTM. or KinExA or
surface plasmon resonance.
[0119] In certain embodiments, an antigen binding molecule
comprises a single chain, wherein the heavy chain variable region
and the light chain variable region are connected by a linker as
described herein, to form a scFv (e.g., an antigen binding molecule
of instant disclosure). In some embodiments, the VH is located at
the N terminus of the linker and the VL is located at the C
terminus of the linker. In other embodiments, the VL is located at
the N terminus of the linker and the VH is located at the C
terminus of the linker. In some embodiments, the linker comprises
at least about 5, at least about 8, at least about 9, at least
about 10, at least about 11, at least about 12, at least about 13,
at least about 14, at least about 15, at least about 16, at least
about 17, at least about 18, at least about 19, at least about 20,
at least about 25, at least about 30, at least about 35, at least
about 40, at least about 45, at least about 50, at least about 60,
at least about 70, at least about 80, at least about 90, or at
least about 100 amino acids. In some embodiments, the linker
comprises between about 8 amino acids and about 18 amino acids
(e.g., 16 amino acids).
[0120] Chimeric Antigen Receptors
[0121] An antigen binding molecule may form a component of a CAR or
TCR, and may serve to direct the CAR or TCR to recognize a target
of interest. As used herein, in the context of a CAR or TCR, an
antigen binding molecule means any component of a CAR or TCR that
directs the CAR or TCR to a desired target and associates with that
target. In specific embodiments, an antigen binding molecule
component of a CAR or TCR comprises a scFv comprising a heavy and
light chain variable region joined by a linker described herein.
The heavy and light variable regions may be derived from the same
antibody or two different antibodies. Antigen binding molecules
used in a CAR or TCR may be derived from an antibody known or
suspect to bind to a target of interest.
[0122] T cells may be engineered to express, for example, a
chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR
positive (CAR+) T cells are engineered to express a CAR. CARs may
comprise, e.g., an extracellular single chain variable fragment
(scFv) with specificity for a particular tumor antigen, which is
directly or indirectly linked to an intracellular signaling part
comprising at least one costimulatory domain, which is directly or
indirectly linked to at least one activating domain; the components
may be arranged in any order. The costimulatory domain may be
derived from, e.g., CD28 or 4-1BB, and the activating domain may be
derived from, e.g., any form of CD3-zeta. In certain embodiments,
the CAR is designed to have two, three, four, or more costimulatory
domains. A CAR scFv may be designed to target, for example, CD19,
which is a transmembrane protein expressed by cells in the B cell
lineage, including all normal B cells, and B cell malignances such
as NHL, CLL, and non-T cell ALL. In some embodiments, a CAR is
engineered such that the costimulatory domain is expressed as a
separate polypeptide chain. Examples of CAR T cell therapies and
constructs are described in U.S. Patent Publication Nos.
2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708, which
are incorporated by reference in their entirety for any
purpose.
[0123] An antigen binding molecule of the instant disclosure may
also be a fully human monoclonal antibody, from which a scFv may be
generated, which may then form a component of a CAR or TCR provided
herein. Fully human monoclonal antibodies may be generated by any
number of techniques with which those having ordinary skill in the
art will be familiar. Such methods include, but are not limited to,
Epstein Barr Virus (EBV) transformation of human peripheral blood
cells (e.g., containing B lymphocytes), in vitro immunization of
human B-cells, fusion of spleen cells from immunized transgenic
mice carrying inserted human immunoglobulin genes, isolation from
human immunoglobulin V region phage libraries, or other procedures
as known in the art and based on the disclosure herein.
[0124] Procedures have been developed for generating human
monoclonal antibodies in non-human animals. For example, mice in
which one or more endogenous immunoglobulin genes have been
inactivated by various means have been prepared. Human
immunoglobulin genes have been introduced into the mice to replace
the inactivated mouse genes. In this technique, elements of the
human heavy and light chain locus are introduced into strains of
mice derived from embryonic stem cell lines that contain targeted
disruptions of the endogenous heavy chain and light chain loci (see
also Bruggemann et al., (1997) Curr. Opin. Biotechnol.
8:455-58).
[0125] It will further be appreciated that where desired, the
various domains and regions described herein may be expressed in a
separate chain from the antigen binding molecule (e.g., scFv) and
activating domains, in so-called "trans" configuration. Thus, in
one embodiment an activating domain may be expressed on one chain,
while the antigen binding molecule, and/or an extracellular domain,
and/or a transmembrane domain and/or a costimulatory domain
(depending on the desired construction of the CAR or TCR) may be
expressed on a separate chain.
[0126] Additionally, the N to C-terminal, or extracellular to
intracellular, order of the components of a CAR of the instant
disclosure may be varied as desired. The antigen binding molecule
(the scFv) will be extracellular in order to be associated with the
target antigen, and may include a leader or signal peptide at the N
terminal end of the scFv that is most distal to the cell
membrane.
Polynucleotides
[0127] In one aspect, the present invention provides a
polynucleotide encoding a linker as described herein. In some
embodiments, the present invention provides a polynucleotide
encoding a fusion protein as described herein. The instant
disclosure is also directed to polynucleotides encoding antibodies
and antigen binding molecules, such as a scFv, that comprising a
linker as described herein, molecules comprising this sequence and
cells presenting such molecules.
Expression Vectors
[0128] In one aspect, the present invention provides an expression
vector comprising a polynucleotide encoding a linker or fusion
protein as described herein. In certain aspects, provided herein
are vectors comprising a polynucleotide of the instant disclosure.
In some embodiments, the instant disclosure is directed to a vector
or a set of vectors comprising a polynucleotide encoding a linker,
or fusion protein, as described herein. In other embodiments, the
instant disclosure is directed to a vector or a set of vectors
comprising a polynucleotide encoding an antibody or an antigen
binding molecule thereof, as disclosed herein.
[0129] Any vector known in the art may be suitable for the instant
disclosure. In some embodiments, the vector is a viral vector. In
some embodiments, the vector is a retroviral vector, a DNA vector,
a murine leukemia virus vector, an SFG vector, a plasmid, a RNA
vector, an adenoviral vector, a baculoviral vector, an Epstein Barr
viral vector, a papovaviral vector, a vaccinia viral vector, a
herpes simplex viral vector, an adenovirus associated vector (AAV),
a lentiviral vector, or any combination thereof. In some
embodiments of the instant disclosure one, two or more vectors may
be employed.
Recombinant Cells
[0130] In some embodiments, the present invention provides a
recombinant cell comprising a polynucleotide encoding a linker or
fusion protein as described herein. In some embodiments, the
recombinant cell comprises an expression vector comprising a
polynucleotide encoding a linker or fusion protein as described
herein. In some aspects, provided herein are cells comprising a
polynucleotide or a vector of the instant disclosure. In some
embodiments, the instant disclosure is directed to host cells, such
as in vitro cells, comprising a polynucleotide encoding a linker or
fusion protein, as described herein. In some embodiments, the
instant disclosure is directed to host cells, e.g., in vitro cells,
comprising a polynucleotide encoding an antibody or an antigen
binding molecule thereof, as disclosed herein.
[0131] Suitable host cells may be derived from a variety of
organisms, including, but not limited to, mammals, plants, birds
(e.g., avian systems), insects, yeast, and bacteria. In some
embodiments, host cells are mammalian cells. Any mammalian cell
susceptible to cell culture, and to expression of polypeptides, may
be utilized in accordance with the present invention as a host
cell. Non-limiting examples of mammalian cells that may be used in
accordance with the present invention include human embryonic
kidney 293 cells (HEK293), HeLa cells; BALB/c mouse myeloma line
(NSO/1, ECACC No: 85110503); human retinoblasts (PER.C6 (CruCell,
Leiden, The Netherlands)); monkey kidney CV1 line transformed by
SV40 (COS-7, ATCC CRL 1651); human fibrosarcomacell line (e.g.,
HT-1080); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.,
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc.
Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,
Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1
ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC
CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2);
canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells
(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75);
human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT
060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.
Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; a human hepatoma
line (Hep G2), human cell line CAP and AGE1.HN, and Glycotope's
panel.
[0132] Non-limiting examples of host cells suitable for the present
invention include cells and cell lines derived from Pichia
pastoris, Pichia methanolica, Pichia angusta, Schizosacccharomyces
pombe, Saccharomyces cerevisiae, and Yarrowia lipolytica for yeast;
Sodoptera frugiperda, Trichoplusis ni, Drosophila melangoster and
Manduca sexta for insects; and Escherichia coli, Salmonella
typhimurium, Bacillus subtilis, Bacillus lichenifonnis, Bacteroides
fragilis, Clostridia perfringens, Clostridia difficile for
bacteria; and Xenopus Laevis from amphibian.
[0133] Additionally, any number of available hybridoma cell lines
may be utilized in accordance with the present invention. One
skilled in the art will appreciate that hybridoma cell lines might
have different nutrition requirements and/or might require
different culture conditions for optimal growth and polypeptide or
protein expression, and will be able to modify conditions as
needed.
EXAMPLES
[0134] While certain compounds, compositions and methods of the
present invention have been described with specificity in
accordance with certain embodiments, the following examples serve
only to illustrate the compounds of the invention and are not
intended to limit the same.
Example 1: Epitope Mapping to Identify Linker Consensus
Sequence
[0135] The specific binding of antibody Clone 8 and 16 raised
against a CAR comprising the linker sequence of GSTSGSGKPGSGEGSTKG
(SEQ ID NO: 1), were used for epitope mapping ELISA experiments of
the full length SEQ ID NO: 1 and variants truncated on either the
N- or C-terminus and containing either a biotin moiety on the
N-terminus, or a lysine residue with a biotin moiety on the
C-terminus (SEQ ID NOs: 21-30).
[0136] The antibodies were captured in 96-well plate format using
plates pre-coated with Protein G (Pierce). The plates were washed
6.times. in PBST buffer followed by incubation with target
peptides. An additional 6.times. wash was performed with PBST and
the antibodies were further incubated with streptavidin-HRP. Upon a
final 6.times. wash in PBST, signal was detected and quantified via
enhanced chemiluminescense kit (ECL, from GE Healthcare) and a
Varioskan Flash plate reader (Thermo Fisher). The results of
epitope mapping ELISA experiments, shown in FIG. 2 demonstrate that
although both antibodies bind to the full length 18mer (SEQ ID NO:
1), Clone 8 specifically binds to the 7mer subsequence GKPGSGE (SEQ
ID NO: 5) and Clone 16 specifically binds to the 5mer subsequence
KPGSG (SEQ ID NO: 16). Taken together, these data were used to
generate a consensus sequence based on the minimal binding epitopes
for clone 8 and 16.
Example 2: Antibody Binding Profile of Exemplary Linker
Sequences
[0137] The specific binding of a panel of antibodies raised against
a CAR comprising the linker sequence of GSTSGSGKPGSGEGSTKG (SEQ ID
NO: 1), the linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 2), or the
or the anti-CD19 scFv clone FMC63 were used to determine the
antibody binding profile of exemplary linker sequences from the
KIP-1, KIP-4, and KIP-3 antibodies respectively, as described
herein. Also included in this assay were peptides comprising linker
sequences KL1 (SEQ ID NO: 9), KL2 (SEQ ID NO: 10), KL3 (SEQ ID NO:
11), and the truncated Whitlow linker (SEQ ID NO: 17) described in
FIG. 1. The antibodies were captured in 96-well plate format using
plates pre-coated with Protein G (Pierce). The plates were washed
6.times. in PBST buffer followed by incubation with target
peptides. An additional 6.times. wash was performed with PBST and
the antibodies were further incubated with streptavidin-HRP. Upon a
final 6.times. wash in PBST, signal was detected and quantified via
enhanced chemiluminescense kit (ECL, from GE Healthcare) and a
Varioskan Flash plate reader (Thermo Fisher). The results of the
antibody profile ELISA experiments, shown in FIG. 3 demonstrate the
breadth of antibody binding of linkers according to the present
invention.
Example 3: Flow Cytometry Results of CAR Expressing Cells
Comprising Chimeric Linkers
[0138] CAR T cells were assayed via flow cytometry using Protein L
as a control to confirm the expression of each CAR construct
comprising the linker sequences SEQ ID NO: 1 (FMC63 WT), or the SEQ
ID NO: 2 (FMC63 G4S). These results confirm expression of the CAR
constructs on the surface of T cells. As shown in FIG. 4, CAR T
cells were produced in the context of scFv FMC63 and 24C1 scFv. KL2
(SEQ ID NO: 10), KL3 (SEQ ID NO: 11), KL4 (SEQ ID NO: 7), KL5 (SEQ
ID NO: 12), KL6 (SEQ ID NO: 8), and G4S2 (SEQ ID NO: 18) linkers
were used to link the VL and VH domains of the scFv.
SEQUENCES AND SEQ ID NOs
[0139] The instant disclosure comprises a number of nucleic acid
and polypeptide sequences. For convenience, Table B below
correlates each sequence with its appropriate SEQ ID NO.
TABLE-US-00002 TABLE B SEQ ID NO Sequence SEQ ID NO: 1
GSTSGSGKPGSGEGSTKG SEQ ID NO: 2 GGGGSGGGGSGGGGS SEQ ID NO: 3
xxxGKPGSGExxxGKPGSGExxx SEQ ID NO: 4 KPGSGE SEQ ID NO: 5 GKPGSGE
SEQ ID NO: 6 GKPGSGG SEQ ID NO: 7 GGGSGKPGSGEGGGS SEQ ID NO: 8
GGGSGKPGSGEGGGGS SEQ ID NO: 9 GGGGSGKPGSGGGGS SEQ ID NO: 10
GGGGSGKPGSGEGGS SEQ ID NO: 11 GGGGSGKPGSGEGGGS SEQ ID NO: 12
GGGGSGKPGSGEGGGGS SEQ ID NO: 13 GSGKPGSGEG SEQ ID NO: 14 GKPGSGEG
SEQ ID NO: 15 SGKPGSGE SEQ ID NO: 16 KPGSG SEQ ID NO: 17
STSGSGKPGSGEGST SEQ ID NO: 18 GGGGSGGGGSGGGGSG SEQ ID NO: 19
GGGGGSGGGGSGGGGS SEQ ID NO: 20 GGGGSGGGGSGGGGGS SEQ ID NO: 21
GSTSGSGKPGSGEGST SEQ ID NO: 22 GSTSGSGKPGSGEG SEQ ID NO: 23
GSTSGSGKPGSGE SEQ ID NO: 24 GSTSGSGKPGSG SEQ ID NO: 25 GSTSGSGKPG
SEQ ID NO: 26 GSGKPGSGEGSTKG SEQ ID NO: 27 SGKPGSGEGSTKG SEQ ID NO:
28 GKPGSGEGSTKG SEQ ID NO: 29 KPGSGEGSTKG SEQ ID NO: 30 PGSGEGSTKG
SEQ ID NO: 31 GSGKPGSGEGG SEQ ID NO: 32 GGGGS
EQUIVALENTS
[0140] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
following claims.
[0141] Use of ordinal terms such as "first," "second," "third,"
etc., in the claims to modify a claim element does not by itself
connote any priority, precedence, or order of one claim element
over another or the temporal order in which acts of a method are
performed, but are used merely as labels to distinguish one claim
element having a certain name from another element having a same
name (but for use of the ordinal term) to distinguish the claim
elements.
[0142] The articles "a" and "an" as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to include the plural referents.
Claims or descriptions that include "or" between one or more
members of a group are considered satisfied if one, more than one,
or all of the group members are present in, employed in, or
otherwise relevant to a given product or process unless indicated
to the contrary or otherwise evident from the context. The
invention includes embodiments in which exactly one member of the
group is present in, employed in, or otherwise relevant to a given
product or process. The invention also includes embodiments in
which more than one, or the entire group members are present in,
employed in, or otherwise relevant to a given product or process.
Furthermore, it is to be understood that the invention encompasses
all variations, combinations, and permutations in which one or more
limitations, elements, clauses, descriptive terms, etc., from one
or more of the listed claims is introduced into another claim
dependent on the same base claim (or, as relevant, any other claim)
unless otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise. Where elements are presented as lists, (e.g., in
Markush group or similar format) it is to be understood that each
subgroup of the elements is also disclosed, and any element(s) may
be removed from the group. It should be understood that, in
general, where the invention, or aspects of the invention, is/are
referred to as comprising particular elements, features, etc.,
certain embodiments of the invention or aspects of the invention
consist, or consist essentially of, such elements, features, etc.
For purposes of simplicity those embodiments have not in every case
been specifically set forth in so many words herein. It should also
be understood that any embodiment or aspect of the invention may be
explicitly excluded from the claims, regardless of whether the
specific exclusion is recited in the specification. The
publications, websites and other reference materials referenced
herein to describe the background of the invention and to provide
additional detail regarding its practice are hereby incorporated by
reference.
Sequence CWU 1
1
32118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly
Glu Gly Ser Thr 1 5 10 15 Lys Gly 215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(3)Gly or SerMOD_RES(11)..(13)Gly or
SerMOD_RES(21)..(23)Gly or Ser 3Xaa Xaa Xaa Gly Lys Pro Gly Ser Gly
Glu Xaa Xaa Xaa Gly Lys Pro 1 5 10 15 Gly Ser Gly Glu Xaa Xaa Xaa
20 46PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Lys Pro Gly Ser Gly Glu 1 5 57PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Gly
Lys Pro Gly Ser Gly Glu 1 5 67PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 6Gly Lys Pro Gly Ser Gly Gly
1 5 715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Gly Gly Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly
Gly Gly Ser 1 5 10 15 816PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Gly Gly Gly Ser Gly Lys Pro
Gly Ser Gly Glu Gly Gly Gly Gly Ser 1 5 10 15 915PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Gly
Gly Gly Gly Ser Gly Lys Pro Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
1015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Gly Gly Gly Gly Ser Gly Lys Pro Gly Ser Gly Glu
Gly Gly Ser 1 5 10 15 1116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Gly Gly Gly Gly Ser Gly Lys
Pro Gly Ser Gly Glu Gly Gly Gly Ser 1 5 10 15 1217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Gly
Gly Gly Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Gly Gly Gly 1 5 10
15 Ser 1310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly 1 5 10
148PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Gly Lys Pro Gly Ser Gly Glu Gly 1 5
158PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Ser Gly Lys Pro Gly Ser Gly Glu 1 5
165PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Lys Pro Gly Ser Gly 1 5 1715PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Ser
Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15
1816PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 1 5 10 15 1916PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 19Gly Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 2016PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser 1 5 10
15 2116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly
Glu Gly Ser Thr 1 5 10 15 2214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 22Gly Ser Thr Ser Gly Ser Gly
Lys Pro Gly Ser Gly Glu Gly 1 5 10 2313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Gly
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu 1 5 10
2412PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly
1 5 10 2510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly 1 5 10
2614PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
Lys Gly 1 5 10 2713PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 27Ser Gly Lys Pro Gly Ser Gly Glu Gly
Ser Thr Lys Gly 1 5 10 2812PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 28Gly Lys Pro Gly Ser Gly Glu
Gly Ser Thr Lys Gly 1 5 10 2911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 29Lys Pro Gly Ser Gly Glu Gly
Ser Thr Lys Gly 1 5 10 3010PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 30Pro Gly Ser Gly Glu Gly Ser
Thr Lys Gly 1 5 10 3111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 31Gly Ser Gly Lys Pro Gly Ser
Gly Glu Gly Gly 1 5 10 325PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 32Gly Gly Gly Gly Ser 1 5
* * * * *