U.S. patent application number 16/950755 was filed with the patent office on 2021-03-04 for single-chain cd27-receptor agonist proteins.
The applicant listed for this patent is Apogenix AG. Invention is credited to Christian GIEFFERS, Oliver HILL, Tim SCHNYDER, Meinolf THIEMANN.
Application Number | 20210061880 16/950755 |
Document ID | / |
Family ID | 1000005222086 |
Filed Date | 2021-03-04 |
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United States Patent
Application |
20210061880 |
Kind Code |
A1 |
GIEFFERS; Christian ; et
al. |
March 4, 2021 |
SINGLE-CHAIN CD27-RECEPTOR AGONIST PROTEINS
Abstract
Provided herein are specific CD27 receptor agonist proteins,
nucleic acids encoding the same, and methods of treating a subject
having a CD27L-associated disease or disorder. The CD27 receptor
agonist proteins provided herein comprise three soluble CD27L
domains an and Fc fragment. The CD27 receptor agonist proteins are
substantially non-aggregating and suitable for therapeutic,
diagnostic and/or research applications.
Inventors: |
GIEFFERS; Christian;
(Dossenheim, DE) ; HILL; Oliver; (Neckarsteinach,
DE) ; THIEMANN; Meinolf; (Schriesheim, DE) ;
SCHNYDER; Tim; (Igersheim, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Apogenix AG |
Heidelberg |
|
DE |
|
|
Family ID: |
1000005222086 |
Appl. No.: |
16/950755 |
Filed: |
November 17, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15956964 |
Apr 19, 2018 |
10844108 |
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16950755 |
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PCT/EP2016/075579 |
Oct 24, 2016 |
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15956964 |
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62245689 |
Oct 23, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2818 20130101;
C07K 16/2809 20130101; C07K 2319/74 20130101; C07K 2319/30
20130101; C07K 14/70575 20130101; A61K 38/00 20130101; C07K 2319/35
20130101; C07K 2319/32 20130101; C07K 2319/00 20130101; C07K
2319/22 20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; C07K 16/28 20060101 C07K016/28 |
Claims
1. A nucleic acid molecule encoding a cluster of differentiation 27
(CD27) receptor agonist protein, whereby said protein is a
single-chain fusion polypeptide comprising: (i) a first soluble
CD27L (CD27 ligand) domain, (ii) a first peptide linker having 3 to
8 amino acids, (iii) a second soluble CD27L domain, (iv) a second
peptide linker having 3 to 8 amino acids, (v) a third soluble CD27L
domain, (vi) a hinge-linker selected from the group consisting of
SEQ ID NOs: 16 and 19-24, and (vii) an antibody Fc fragment
consisting of the amino acid sequence of SEQ ID NO: 13 or amino
acids 1-217 of SEQ ID NO: 13; wherein each of the soluble CD27L
domains (i), (iii), and (v) comprises amino acids Asp56-Pro193 of
SEQ ID NO: 1.
2. The nucleic acid molecule of claim 1, wherein each of the
soluble CD27L domains (i), (iii), and (v) consists of amino acids
Glu51-Pro193 or Asp56-Pro193 of SEQ ID NO: 1.
3. The nucleic acid molecule of claim 1, wherein each of the
soluble CD27L domains (i), (iii), and (v) consists of amino acids
Glu51-Pro193 or Asp56-Pro193 of SEQ ID NO: 1, with one or more of
the soluble CD27L domains (i), (iii), and (v) having a mutation at
the amino acid position Glu51, Trp55, Asn63, Arg83, Arg122, Arg138,
Arg144, His123, His124, His148, Asn170, Arg179, or Asp182 of SEQ ID
NO: 1.
4. The nucleic acid molecule of claim 1, in operative linkage with
an expression control sequence.
5. An expression vector comprising the nucleic acid molecule of
claim 4.
6. A cell or a non-human organism transformed or transfected with
the nucleic acid molecule of claim 4.
7. The nucleic acid molecule of claim 1, comprising SEQ ID NO:
37.
8. The nucleic acid molecule of claim 1, comprising nucleotides 35
to 2146 of SEQ ID NO: 37.
9. The nucleic acid molecule of claim 1, comprising nucleotides 95
to 2146 of SEQ ID NO: 37.
10. An expression vector comprising the nucleic acid molecule of
claim 8, in operative linkage with an expression control
sequence.
11. An expression vector comprising the nucleic acid molecule of
claim 9, in operative linkage with an expression control
sequence.
12. A cell comprising the nucleic acid molecule of claim 8.
13. A cell comprising the nucleic acid molecule of claim 9.
14. The cell according to claim 12, which is a eukaryotic cell.
15. The cell according to claim 14, wherein the cell is a mammalian
cell.
16. The cell according to claim 15, wherein the cell is a Chinese
Hamster Ovary (CHO) cell.
17. The cell according to claim 13, which is a eukaryotic cell.
18. The cell according to claim 17, wherein the cell is a mammalian
cell.
19. The cell according to claim 18, wherein the cell is a Chinese
Hamster Ovary (CHO) cell.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 15/956,964, filed Apr. 19, 2018; which is a continuation of
PCT/EP2016/075579, filed Oct. 24, 2016; which claims priority to
U.S. Provisional Application No. 62/245,689, filed Oct. 23, 2015.
The contents of the above applications are incorporated herein by
reference in their entirety.
REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM
[0002] The Sequence Listing is concurrently submitted herewith with
the specification as an ASCII formatted text file via EFS-Web with
a file name of Sequence_Listing.txt with a creation date of Apr.
13, 2018, and a size of 128 kilobytes. The Sequence Listing filed
via EFS-Web is part of the specification and is hereby incorporated
in its entirety by reference herein.
FIELD OF THE INVENTION
[0003] The present invention provides specific CD27 receptor
agonist proteins comprising three soluble CD27L domains and an Fc
fragment, nucleic acid molecules encoding the CD27 receptor agonist
proteins, and uses thereof. The CD27 receptor agonist proteins are
substantially non-aggregating and suitable for therapeutic,
diagnostic and/or research applications.
BACKGROUND OF THE INVENTION
[0004] It is known that trimerization of TNF superfamily (TNFSF)
cytokines is required for efficient receptor binding and
activation. Trimeric complexes of TNF superfamily cytokines,
however, are difficult to prepare from recombinant monomeric units.
WO 01/49866 and WO 02/09055 disclose recombinant fusion proteins
comprising a TNF cytokine and a multimerization component,
particularly a protein from the C1q protein family or a collectin.
A disadvantage of these fusion proteins is, however, that the
trimerization domain usually has a large molecular weight and/or
that the trimerization is rather inefficient.
[0005] Schneider et al. (J Exp Med 187 (1989), 1205-1213) describe
that trimers of TNF cytokines are stabilized by N-terminally
positioned stabilization motifs. In CD95L, the stabilization of the
receptor binding domain trimer is presumably caused by N-terminal
amino acid domains which are located near the cytoplasmic
membrane.
[0006] Shiraishi et al. (Biochem Biophys Res Commun 322 (2004),
197-202) describe that the receptor binding domain of CD95L may be
stabilized by N-terminally positioned artificial .alpha.-helical
coiled-coil (leucine zipper) motifs. It was found, however, that
the orientation of the polypeptide chains to each other, e.g.
parallel or antiparallel orientation, can hardly be predicted.
Further, the optimal number of heptad-repeats in the coiled-coil
zipper motif are difficult to determine. In addition, coiled-coil
structures have the tendency to form macromolecular aggregates
after alteration of pH and/or ionic strength.
[0007] WO 01/25277 relates to single-chain oligomeric polypeptides
which bind to an extracellular ligand binding domain of a cellular
receptor, wherein the polypeptide comprises at least three receptor
binding sites of which at least one is capable of binding to a
ligand binding domain of the cellular receptor and at least one is
incapable of effectively binding to a ligand binding domain of the
cellular receptor, whereby the single-chain oligomeric polypeptides
are capable of binding to the receptor, but incapable of activating
the receptor. For example, the monomers are derived from cytokine
ligands of the TNF family, particularly from TNF-.alpha..
[0008] WO 2005/103077 discloses single-chain fusion polypeptides
comprising at least three monomers of a TNF family ligand member
and at least two peptide linkers that link the monomers of the TNF
ligand family members to one another. Recent experiments, however,
have shown that these single-chain fusion polypeptides show
undesired aggregation.
[0009] WO 2010/010051 discloses single-chain fusion polypeptides
comprising three soluble TNF family cytokine domains and at least
two peptide linkers. The described fusion polypeptides are
substantially non-aggregating.
[0010] Recent studies have shown that the in vivo agonistic
activity of the anti-CD27-mAb currently explored in the clinic is
dependent on Fc-gamma-R crosslinking [He, L. Z., N. Prostak, L. J.
Thomas, L. Vitale, J. Weidlick, A. Crocker, C. D. Pilsmaker, S. M.
Round, A. Tutt, M. J. Glennie, H. Marsh and T. Keler (2013).
"Agonist anti-human CD27 monoclonal antibody induces T cell
activation and tumor immunity in human CD27-transgenic mice." J
Immunol 191(8): 4174-4183]
[0011] There is a need in the art for novel CD27 receptor agonists
that exhibit high biological activity independent of Fc-gamma-R
based crosslinking in vivo, high stability, and allow for efficient
recombinant manufacturing.
SUMMARY OF THE INVENTION
[0012] The present invention provides specific CD27 receptor
agonist proteins that mimic the CD27:CD27L interaction in vivo,
exhibit low proteolytic degradation and a shorter in vivo half-life
as compared to agonistic monoclonal antibodies.
[0013] The CD27 receptor agonist proteins of the instant invention
generally comprise:(i) a first soluble CD27L cytokine domain; (ii)
a first peptide linker; (iii) a second soluble CD27L domain; (iv) a
second peptide linker; (v) a third soluble CD27L domain; (vi) a
third peptide linker (e.g., a hinge-linker) and (vii) an antibody
Fc fragment.
[0014] In one embodiment, the antibody Fc fragment (vii) is located
N terminal to the first CD27L domain (i) and/or C-terminal to the
third CD27L domain (v). In another embodiment the antibody Fc
fragment is located C-terminally to the third CD27L domain (v). In
one embodiment, the polypeptide is substantially non-aggregating.
In another embodiment, the second and/or third soluble CD27L domain
is an N-terminally shortened domain which optionally comprises
amino acid sequence mutations.
[0015] In one embodiment, at least one of the soluble CD27L
domains, particularly at least one of the soluble CD27L domains
(iii) and (v), is a soluble CD27L domain with an N-terminal
sequence which starts at amino acid Glu51 or Asp56 of human CD27L
and wherein Glu51 may be replaced by a neutral amino acid, e.g.,
Ser or Gly. In another embodiment, at least one of the soluble
CD27L domains, particularly at least one of the soluble CD27L
domains (iii) and (v), is a soluble CD27L domain with an N-terminal
sequences selected from (a) Glu51-Asp56 and (b) (Gly/Ser)51-Glu56.
In one embodiment, the soluble CD27L domain ends with amino acid
Pro193 of human CD27L and/or optionally comprises one or more
mutation at positions W55, N63, R83, R122, R138, R144, H123, H124,
H148, N170, R179, D182, E183. In one embodiment, the soluble CD27L
domains (i), (iii) and (v) comprise amino acids Glu51-Pro193 of
human CD27L according to SEQ ID NO: 1.
[0016] In one embodiment, at least one of the soluble CD27L
domains, particularly at least the soluble CD27L domains (i), is a
soluble CD27L domain with an N-terminal sequence which starts at
amino acid Glu51 and wherein Glu51 may be replaced by Gln. In one
embodiment, the first and second peptide linkers (ii) and (iv)
independently have a length of 3-8 amino acids, particularly a
length of 3, 4, 5, 6, 7, or 8 amino acids, and preferably are
glycine/serine linkers, optionally comprising an asparagine residue
which may be glycosylated. In one embodiment, the first and the
second peptide linkers (ii) and (iv) consist of the amino acid
sequence according to SEQ ID NO:2. In another embodiment, the
polypeptide additionally comprises an N-terminal signal peptide
domain, e.g., of SEQ ID NO: 17, which may comprise a protease
cleavage site, and/or which additionally comprises a C-terminal
element which may comprise and/or connect to a
recognition/purification domain, e.g., a Strep-tag attached to a
serine linker according to SEQ ID NO: 18.
[0017] In one embodiment, the antibody Fc fragment (vii) is fused
to the soluble CD27L domain (i) and/or (v) via a hinge-linker,
preferably of SEQ ID NO: 16. In another embodiment, the antibody Fc
fragment (vii) consists of the amino acid sequence as shown in SEQ
ID NO: 13 or 14.
[0018] In one embodiment, the single-chain fusion polypeptide of
the present invention comprises the amino acid sequence selected
from the group consisting of SEQ ID NO: 15, 25-35 and 43-47.
[0019] In one embodiment, the present invention provides a CD27
receptor agonist protein comprising a dimer of two single-chain
fusion polypeptides each having the amino acid sequence set forth
in SEQ ID NO: 27. In one embodiment, the two polypeptides are
covalently linked through three interchain disulfide bonds formed
between cysteine residues 457, 463, and 466 of each
polypeptide.
[0020] In one embodiment, one or more of the asparagine residues at
positions 149 and 300 of the mature polypeptide(s) SEQ ID NO: 27,
28, 29, 30, 34 or 35 are N-glycosylated. In another embodiment, the
asparagine residues at positions 149 and 300 of the polypeptide(s)
are both N-glycosylated.
[0021] In another embodiment, only the asparagine residue at
position 149 of the mature polypeptides SEQ ID NO: 31, 32, 43 or 47
is glycosylated as the asparagine 300 is not present in those
proteins.
[0022] In another embodiment, only the asparagine residue at
position145 of mature polypeptide SEQ ID NO: 33 is
glycosylated.
[0023] In another embodiment, one or more of the asparagine residue
at position144 and 290 of mature polypeptide of SEQ ID NO: 44 or 46
are N-glycosylated.
[0024] In another embodiment, only the asparagine residue at
position144 of the mature polypeptide(s) of SEQ ID NO: 45 is
N-glycosylated.
[0025] In another embodiment, the polypeptide(s) are further
post-translationally modified. In another embodiment, the
post-translational modification comprises the N-terminal glutamine
of the E51Q mutein modified to pyroglutamate.
DESCRIPTION OF THE FIGURES
[0026] FIG. 1 Domain structure of a single-chain fusion polypeptide
comprising three CD27L domains. I., II., Ill. Soluble CD27L
domains.
[0027] FIG. 2 Schematic picture representing the general structure
of CD27L. .box-solid..box-solid..box-solid. Cell membrane,
N-terminus located within the cell, [0028] 1. anti-parallel
.beta.-fold of receptor-binding domain (RBD), [0029] 2. interface
of RBD and cell membrane, [0030] 3. protease cleavage site.
[0031] FIG. 3 Single-chain fusion polypeptide comprising an
additional Fab antibody fragment.
[0032] FIG. 4 Dimerization of two C-terminally fused scFc fusion
polypeptides via three disulfide bridges.
[0033] FIG. 5 Analytical size exclusion chromatography of PROTEIN A
(SEQ ID NO: 15) and PROTEIN X (SEQ ID NO: 38) performed on a 1260
Infinity HPLC system using a Tosoh TSKgelG3000SWxl column. The
column was loaded with protein at a concentration of 0.6 mg/ml in a
total volume of 20 .mu.l. The flow rate was set to 0.5 ml/min. One
observes a single main peak at 16.39 min for PROTEIN A (Part B) and
18.91 min for PROTEIN X (Part A).
[0034] FIG. 6 Schematic representation of the hexavalent single
chain CD27 receptor agonist fusion protein of the invention.
CH2-Carbohydrates (5) present on the inner surface areas normally
shield the CH2-subdomain sterically (2) from proteases during "open
Fc-conformation transits" wherein hinge-interchain disulfide bonds
(4) are reduced and the covalent interchain linkage is disrupted.
This enables CH2-dissociation and exposure of the inner surface
areas and the upper hinge lysine K223 (6) towards proteases. Dimer
assoziation in the "open stage" remains intact due to the high
affinity of the CH3 domains (3) to each other. [0035] (1)
scCD27L-RBD; (2) CH2 domain; (3) CH3 domain; (4) Hinge-Cysteines
(left side: oxidized to disulfidbridges; right side reduced stage
with free thiols); (5) CH2-Carbohydrates attached to N297 position
(EU-numbering); (6) Upper Hinge Lysine (K223)
[0036] FIG. 7 SDS-PAGE results of PROTEIN A under non-reducing and
reducing conditions. 465 ng of PROTEIN A were loaded on an SDS-PAGE
4-12% Bis-Tris gel under non-reducing (A) or reducing (B)
conditions containing DTT as reducing agent. Gels were run at 170V
for 60 min and were subsequently stained using a silver-stain
protocol. One observes a molecular weight difference between the
main bands in A and B of about 80-100 kDa. As this is about half
the molecular weight as observed for the main band in A, this
indicates that the homodimer in A is covalently linked by disulfide
bridges. The bonds are lost under reducing conditions in B
[0037] FIG. 8 Elution fractions from affinity chromatography of
PROTEIN X along with column load and flow-through samples were
loaded on an SDS-PAGE 4-12% Bis-Tris gel under reducing (lanes 2-8)
or non-reducing (lanes 10-16) conditions. DTT was used as reducing
agent. Gels were run at 170V for 60 min and were subsequently
stained using a silver-stain protocol. Single bands of PROTEIN X
can be seen in lanes 4-6 and 12-14 indicating that all protein
elutes from the column in fractions 1 to 3. [0038] Shown is: Lane 1
and 9: marker/lane 17: empty/lane 2-8 and 10-16: Protein X with:
(2) reduced column load; (3) reduced column flow-through; (4)
reduced elution fraction 1; (5) reduced elution fraction 2; (6)
reduced elution fraction 3; (7) reduced elution fraction 4; (8)
reduced elution fraction 5; (10) non reduced column load; (11) non
reduced column flow-through; (12) non-reduced elution fraction 1;
(13) non-reduced elution fraction 2; (14) non-reduced elution
fraction 3; (15) non-reduced elution fraction 4; (16) non-reduced
elution fraction 5
[0039] FIG. 9 Effect of PROTEIN A on subcutaneous syngeneic colon
carcinoma model MC38-CEA female in female C57B1/6N mice. Shown is
wet tumor weight at necropsy. PROTEIN A, administered at either 1
mg/kg (Group 2) or 10 mg/kg (Group 3) is displayed versus it's
corresponding vehicle control PBS (Group 1). Data are displayed as
means.+-.SEM. P-values calculated compared to the vehicle control
(group 1) using the Mann Whitney test.
[0040] FIG. 10 Effect of PROTEIN A on subcutaneous syngeneic colon
carcinoma model MC38-CEA female in female C57B1/6N mice. Figure
depicts anti-tumor efficacy (tumor volume) at necropsy. PROTEIN A,
administered at either 1 mg/kg (Group 2) or 10 mg/kg (Group 3) is
displayed versus it's corresponding vehicle control PBS (Group 1).
Data are displayed as means.+-.SEM. P-values calculated compared to
the vehicle control (group 1) using the Mann Whitney test.
[0041] FIG. 11 Effect of PROTEIN A on subcutaneous syngeneic colon
carcinoma model CT26 female in female BALB/c mice. Figure depicts
wet tumor weight at necropsy (end of study). PROTEIN A,
administered at either 1 mg/kg (Group 2) or 10 mg/kg (Group 3) is
displayed versus it's corresponding vehicle control PBS (Group 1).
Data are displayed as means.+-.SEM. P-values calculated compared to
the vehicle control (group 1) using the Mann Whitney test
[0042] FIG. 12 Effect of PROTEIN A on subcutaneous syngeneic colon
carcinoma model CT26 female in female BALB/c mice. Figure depicts
tumor volume at the end of the study on day 21 (day 10) of. PROTEIN
A, administered at either 1 mg/kg (Group 2) or 10 mg/kg (Group 3)
is displayed versus it's corresponding vehicle control PBS (Group
1). Data are displayed as means.+-.SEM. P-values calculated
compared to the vehicle control (group 1) using the Mann Whitney
test
DETAILED DESCRIPTION OF THE INVENTION
[0043] The present invention provides a single-chain fusion
polypeptide comprising at least three soluble CD27L domains
connected by two peptide linkers and N-terminally and/or
C-terminally an antibody-derived dimerization domain. The inventors
have discovered that dimerization of the two single-chain fusion
polypeptides through the dimerization domain results in a
hexavalent CD27 receptor agonist, which provides high biological
activity and good stability.
[0044] Preferably, the single-chain fusion polypeptide is
non-aggregating. The term "non-aggregating" refers to a monomer
content of the preparation of .gtoreq.50%, preferably .gtoreq.70%
and more preferably .gtoreq.90%. The ratio of monomer content to
aggregate content may be determined by examining the amount of
aggregate formation using size-exclusion chromatography (SEC). The
stability concerning aggregation may be determined by SEC after
defined time periods, e.g. from a few to several days, to weeks and
months under different storage conditions, e.g. at 4.degree. C. or
25.degree. C. For the fusion protein, in order to be classified as
substantially non-aggregating, it is preferred that the "monomer"
content is as defined above after a time period of several days,
e.g. 10 days, more preferably after several weeks, e.g. 2, 3 or 4
weeks, and most preferably after several months, e.g. 2 or 3 months
of storage at 4.degree. C., or 25.degree. C. With regard to the
definition of "monomer" in the case of FC-fusion proteins, the
assembly of two polypeptide chains is driven by the FC-part and the
functional unit of the resulting assembled protein consists of two
chains. This unit is defined as "monomer" in the case of Fc-fusion
proteins regardless of being a dimerized single-chain fusion
polypeptide.
[0045] The single-chain fusion polypeptide may comprise additional
domains which may be located at the N- and/or C-termini thereof.
Examples for additional fusion domains are e.g. an N-terminal
signal peptide domain which may comprise a protease cleave site or
a C-terminal element which may comprise and/or connect to a
recognition/purification domain. According to a preferred
embodiment, the fusion polypeptide comprises a Strep-tag at its
C-terminus that is fused via a linker. An exemplary Strep-tag
including a short serine linker is shown in SEQ ID NO: 18.
[0046] The CD27 receptor agonist protein of the present invention
comprises three soluble domains derived from CD27L. Preferably,
those soluble domains are derived from a mammalian, particularly
human CD27L including allelic variants and/or derivatives thereof.
The soluble domains comprise the extracellular portion of CD27L
including the receptor binding domain without membrane located
domains. Like other proteins of the TNF superfamily, CD27L is
anchored to the membrane via an N -terminal portion of 15-30 amino
acids, the so-called stalk-region. The stalk region contributes to
trimerization and provides a certain distance to the cell membrane.
However, the stalk region is not part of the receptor binding
domain (RBD).
[0047] Importantly, the RBD is characterized by a particular
localization of its N- and C-terminal amino acids. Said amino acids
are immediately adjacent and are located centrally to the axis of
the trimer. The first N-terminal amino acids of the RBD form an
anti-parallel beta-strand with the C-terminal amino acids of the
RBD (FIG. 2).
[0048] Thus, the anti-parallel beta-strand of the RBD forms an
interface with the cell membrane, which is connected to and
anchored within the cell membrane via the amino acids of the stalk
region. It is highly preferred that the soluble CD27L domains of
the CD27 receptor agonist protein comprise a receptor binding
domain of the CD27L lacking any amino acids from the stalk region.
Otherwise, a long linker connecting the C-terminus of one of the
soluble domains with the N -terminus of the next soluble domain
would be required to compensate for the N-terminal stalk-region of
the next soluble domain, which might result in instability and/or
formation of aggregates.
[0049] A further advantage of such soluble domains is that the
N-terminal amino acids of the RBD are not accessible for any
anti-drug antibodies. Preferably, the single-chain fusion
polypeptide consisting of (i) a first soluble CD27L cytokine
domain; (ii) a first peptide linker; (iii) a second soluble CD27L
domain; (iv) a second peptide linker; (v) a third soluble CD27L
domain is capable of forming an ordered structure mimicking the
trimeric organization of its natural counterpart thereby comprising
at least one functional binding site for the respective CD27L
receptor. The single-chain fusion polypeptide comprising components
(i)-(v) is therefore also termed
single-chain-CD27L-receptor-binding-domain (scCD27L-RBD).
[0050] The CD27 receptor agonist protein comprises three functional
CD27 receptor binding sites, i.e. amino acid sequences capable of
forming a complex with a CD27 receptor . Thus, the soluble domains
are capable of binding to the corresponding CD27 receptor . In one
embodiment, at least one of the soluble domains is capable of
receptor activation, whereby apoptotic and/or proliferative
activity may be affected. In a further embodiment, one or more of
the soluble domains are selected as not being capable of receptor
activation.
[0051] The soluble CD27L domain may be derived from human CD27L as
shown in SEQ ID NO: 1. Preferably, the soluble CD27L domains are
derived from human CD27L, particularly starting from amino acids 51
or 56 and comprise particularly amino acids 51-193 or 56-193 of SEQ
ID NO: 1. Optionally, amino acid Glu51 of SEQ ID NO: 1 may be
replaced by a non-charged amino acid, e.g. Ser or Gly or is
replaced by Glutamine.
TABLE-US-00001 TABLE 1 Sequence of Wild-Type Human CD27L Protein
SEQ ID NO Sequence 1
MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLES
LGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIY
MVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQ
RLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP
[0052] As indicated above, the soluble CD27L domains may comprise
the wild-type sequences as indicated in SEQ ID NO: 1. It should be
noted, however, that it is possible to introduce mutations in one
or more of these soluble domains, e.g. mutations which alter (e.g.
increase or decrease) the binding properties of the soluble
domains. In one embodiment, soluble domains that cannot bind to the
corresponding cytokine receptor can be selected.
[0053] In a further embodiment of the invention, the soluble CD27L
domain (i) comprises a mutant of CD27L or a receptor binding domain
thereof resulting in reduced affinity and/or reduced activation of
CD27 receptor.
[0054] CD27L-Muteins Affecting Receptor Binding and/or Activity
[0055] The mutant may be generated by any technique known by a
skilled person. The substitution may affect at least one amino acid
of CD27L, e.g., human CD27L (e.g., SEQ ID NO: 1) or a receptor
binding domain thereof as described herein. Preferred substitutions
in this regard affect at least one of the following amino acids of
human CD27L of SEQ ID NO: 1: R83, R122, R138, R144, H123, H124,
H148, R179, D182, E183. In a preferred embodiment R138 and/or R179
are mutated to S or D.
[0056] The amino acid substitution(s) may affect the binding and/or
activity of CD27L, e.g., human CD27L, to or on either the CD27
binding or the CD27 induced signaling. The binding and/or activity
of the CD27 may be affected positively, i.e., stronger, more
selective or more specific binding and/or more activation of the
receptor. Alternatively, the binding and/or activity of the CD27
may be affected negatively, i.e., weaker, less selective or less
specific binding and/or less or no activation of the receptor. Thus
one embodiment is a CD27 receptor agonist protein as described
herein wherein at least one of the soluble domains comprises a
mutant of CD27L or a receptor binding domain thereof which binds
and/or activates CD27 to a lesser extent than the
wildtype-CD27L.
[0057] Further examples of mutants of CD27L, which show reduced
CD27L induced receptor aggregation/and or reduced signaling are
R144N and D182S.
[0058] CD27L-Muteins with Enhanced Stability/Solubility
[0059] One embodiment is a CD27 receptor agonist protein as
described herein, wherein at least one artificial N-glycosylation
consensus site is introduced into the sequence area defined by
T172-F185 of human CD27L (SEQ ID NO:1) resulting in reduced
receptor aggregation/and or reduced signaling. Examples of mutants
of CD27L resulting in an artificial N-glycosylation consensus site
in this region is D182S.
[0060] In a further embodiment of the invention, one or more of the
soluble CD27L domains (i), (iii), and (v) may comprise a mutant of
CD27L or a receptor binding domain thereof resulting in reduced
self-aggregation and/or prolonged in vivo stability.
[0061] Preferred substitutions in this regard are S117N, T119N,
S137N and R144N. The mutation(s) of each CD27L domain may be the
same or different.
[0062] The single-chain fusion molecule of the present invention
comprises three soluble CD27L domains, namely components (i), (iii)
and (v). The stability of a single-chain CD27L fusion polypeptide
against aggregation is enhanced, if the second and/or third soluble
CD27L domain is an N-terminally shortened domain which optionally
comprises amino acid sequence mutations. Thus, preferably, both the
second and the third soluble CD27L domain are N-terminally
shortened domains which optionally comprise amino acid sequence
mutations in the N-terminal regions, preferably within the first
five amino acids of the N-terminus of the soluble CD27L domain.
These mutations may comprise replacement of basic amino acids, by
neutral amino acids, particularly serine or glycine.
[0063] In contrast thereto, the selection of the first soluble
CD27L domain is not as critical. Here, a soluble domain having a
full-length N-terminal sequence may be used. It should be noted,
however, that also the first soluble CD27L domain may have an
N-terminally shortened and optionally mutated sequence.
[0064] In a further preferred embodiment of the present invention,
the soluble CD27L domains (i), (iii) and (v) are soluble human
CD27L domains. The first soluble CD27L domain (i) may be selected
from native, shortened and/or mutated sequences. Thus, the first
soluble CD27L domain (i) has an N-terminal sequence which may start
at amino acid Glu51 or Asp56 of human CD27L, and wherein Glu51 may
be replaced by a neutral amino acid, e.g. by Ser or Gly or by Gln
to enable pyroglutamate formation during expression. The second and
third soluble CD27L domains (iii) and (v) have a shortened
N-terminal sequence which preferably starts with amino acid Ser52
or Gly54 of human CD27L (SEQ ID NO:1) and wherein Glu51 may be
replaced by another amino acid, e.g. Ser or Gly.
[0065] Preferably, the N-terminal sequence of the soluble CD27L
domains (iii) and (v) is selected from:
[0066] (a) Glu51-Asp56
[0067] (b) (Gly/Ser)51-Asp56.
[0068] The soluble CD27L domain preferably ends with amino acid
P193 of human CD27L. In certain embodiments, the CD27L domain may
comprise internal mutations as described above.
[0069] Components (ii) and (iv) of the CD27 receptor agonist
protein are peptide linker elements located between components (i)
and (iii) or (iii) and (v), respectively. The flexible linker
elements have a length of 3-8 amino acids, particularly a length of
3, 4, 5, 6, 7, or 8 amino acids. The linker elements are preferably
glycine/serine linkers, i.e. peptide linkers substantially
consisting of the amino acids glycine and serine. In cases in in
which the soluble cytokine domain starts with S or G (N-terminus),
the linker ends before this S or G.
[0070] It should be noted that linker (ii) and linker (iv) do not
need to be of the same length. In order to decrease potential
immunogenicity, it may be preferred to use shorter linkers. In
addition it turned out that shorter linkers lead to single chain
molecules with reduced tendency to form aggregates. Whereas linkers
that are substantially longer than the ones disclosed here may
exhibit unfavorable aggregations properties.
[0071] If desired, the linker may comprise an asparagine residue
which may form a glycosylate site Asn-Xaa-Ser. In certain
embodiments, one of the linkers, e.g. linker (ii) or linker (iv)
comprises a glycosylation site. In other embodiments, both linkers
(iv) comprise glycosylation sites. In order to increase the
solubility of the CD27L agonist proteins and/or in order to reduce
the potential immunogenicity, it may be preferred that linker (ii)
or linker (iv) or both comprise a glycosylation site.
[0072] Preferred linker sequences are shown in Table 2. A preferred
linker is GSGSGNGS (SEQ ID NO: 2).
TABLE-US-00002 TABLE 2 Example Linker Sequences SEQ ID NO Sequence
2 GSGSGNGS 3 GSGSGSGS 4 GGSGSGSG 5 GGSGSG 6 GGSG 7 GGSGNGSG 8
GGNGSGSG 9 GGNGSG 10 GSGSGS 11 GSGS 12 GSG
[0073] The CD27 receptor agonist protein additionally comprises an
antibody Fc fragment domain which may be located N-terminal to the
first CD27L domain (i) and/or C-terminal to the third CD27L domain
(v). Preferably, the antibody Fc fragment domain comprises a
reduced capability to interact with Fc-gamma-R receptors in vivo.
Preferably, the antibody Fc fragment domain comprises or consists
of an amino acid sequence as shown in SEQ ID NO: 13 or 14 (see
Table 3). Sequence ID NO: 13 has N297S mutation compared to
wildtype human IGG1-Fc and does not bind to Fc-gamma-R receptors.
Sequence ID NO: 14 is a glycosylated (N297 wildtype) human IGG1 Fc
mutein with reduced Fc-gamma-R binding capability.
TABLE-US-00003 TABLE 3 Examples of Fc Fragment Domains SEQ ID NO
Sequence 13 PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 14
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV
SNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0074] Number of Glycosylation Sites and In Vivo Stability
[0075] The total number of glycosylation sites and the individual
position of the carbohydrates in three dimensions impacts the
in-vivo stability of CD27 receptor agonist proteins.
[0076] Further, carbohydrate recognition depends on local density
of the terminal saccharides, the branching of the carbohydrate tree
and the relative position of the carbohydrates to each other
matter.
[0077] Further, partially degraded carbohydrates reduce the in vivo
half-life of CD27 receptor agonist proteins through lectin-driven
mechanisms. By reducing the total number of glycosylation sites on
the molecule, the resulting compound is less accessible to these
mechanisms, increasing half-life.
[0078] Depletion of antibody CH2-domain carbohydrates is necessary
in order to avoid Fc-receptor based crosslinking in vivo and
potential CD27L-receptor superclustering-based toxicity. Also,
unwanted Fc-driven mechanisms like ADCC could lead to toxic
events.
[0079] Accordingly, in one embodiment, the overall number of
glycosylation sites on the CD27 receptor agonist proteins of the
instant invention is reduced through the depletion of CH2
glycosylation sites, particularly the N-glycosylation site,
resulting in CD27 receptor agonist proteins comprising N297S
equivalent mutations of SEQ ID NO: 15 (PROTEIN A) (according to the
EU numbering system) creating aglycosl-CH2 domains. In another
embodiment of the invention, one or more of the soluble CD27L
domains (i), (iii), and (v) may comprise a N63 and/or N170
exchanged to aspartate, serine or glycine resulting in CD27
receptor agonistic fusion proteins with a reduced number of
glycosylation sites. In a preferred embodiment, the N63[D,S,G] and
N170[D,S,G] mutations are restricted to the soluble CD27L domains
(iii) and (v) of the agonistic CD27 receptor agonistic fusion
proteins of the present invention.
[0080] CH2-Domain Destabilization is Compensated by an Additional
Hinge-Cysteine
[0081] CH2 (Heavy chain constant domain 2)-glycosylation present on
the inner surface areas normally shields the subdomain from
proteases during "open Fc-conformation transits" wherein
hinge-interchain disulfide bonds are reduced and the covalent
interchain linkage is disrupted (FIG. 6). This enables
CH2-dissociation and exposure of the inner surface area towards
proteases. CD27 receptor agonist proteins comprising an Fc-domain
with a N297S equivalent mutation of SEQ ID NO: 15 (PROTEIN A)
(according to the EU numbering system) creates an aglycosylated-CH2
and are therefore likely to be subject to protease digestion and
less stable than equivalent structures with wild-type CH2
glycosylation. This would impact the compound's stability during
USP/DSP/storage, where host cell proteases are present and have
long-term access to the structure. Accordingly, in certain
embodiments, the CD27 receptor agonist lacks CH2 glycosylation
sites, but comprises glycosylation sites in the linker sequences of
each polypeptide chain (e.g., GSGSGNGS, SEQ ID NO: 2).
[0082] According to a preferred embodiment of the invention, the
antibody Fc fragment domain is fused via a hinge-linker element.
The hinge-linker element has a length of 10-30 amino acids,
particularly a length of 15-25 amino acids, e.g. 22 amino acids.
The term "hinge-linker" includes any linker long enough to allow
the domains attached by the hinge-linker element to attain a
biologically active confirmation. The hinge-linker element
preferably comprises the hinge-region sequence of an
immunoglobulin, herein referred to as "Ig hinge-region". The term
"Ig hinge-region" means any polypeptide comprising an amino acid
sequence that shares sequence identity or similarity with a portion
of a naturally occurring Ig hinge-region sequence which includes
one or more cysteine residues, e.g., two cysteine residues, at
which the disulfide bonds link the two heavy chains of the
immunoglobulin.
[0083] Derivatives and analogues of the hinge-region can be
obtained by mutations. A derivative or analogue as referred to
herein is a polypeptide comprising an amino acid sequence that
shares sequence identity or similarity with the full length
sequence of the wild type (or naturally occurring protein) except
that it has one or more amino acid sequence differences
attributable to a deletion, insertion and/or substitution.
[0084] The number of molecules with open Fc-conformation in an
individual CD27 receptor agonist protein depends on the number of
interchain-disulfide bonds present in the hinge region.
Accordingly, in one embodiment a third cysteine (C225 according to
the EU numbering system) was introduced into the hinge region of
the CD27 receptor agonist proteins of the instant invention in
order to ameliorate the effect of depleting the CH2-glycosites.
[0085] Exchange of a Lysine to Glycine in the Hinge Region Results
in Enhanced Proteolytic Stability
[0086] In one embodiment, the CD27 receptor agonist proteins of the
invention additionally comprise a mutation of the upper-hinge
lysine (K223, according to the EU numbering system) to a glycine to
reduce proteolytic processing at this site, thereby enhancing the
overall stability of the fusion protein. Combining aforementioned
introduction of a third cysteine (C225, according to the EU
numbering system) with the aforementioned lysine to glycine
mutation (K223G, according to the EU numbering system) within the
hinge region results in an overall stabilized CD27 receptor agonist
protein of the instant invention.
[0087] A particularly preferred hinge-linker element including the
aforementioned cysteine (C225) and the lysine to glycine mutation
(K223G) comprises or consists of the amino acid sequence as shown
in SEQ ID NO: 16 (Table 4).
[0088] Endogenous Cysteines Interfere with Hinge-Disulfide
Formation
[0089] The interchain-disulfide connectivity of the hinge region
stabilizing the homodimer of the hexavalent CD27 receptor agonist
protein is also affected by the free thiol groups of the CD27L
subsequences. Free thiol groups can be created through reduction of
surface exposed disulfide-bridges, e.g. by reduction of the
C115-C151 disulfide of CD27L. This also leads to the aforementioned
open FC-conformation due to self-reduction of the hinge
disulfide-bridges of the structure by the endogenous free thiols of
the preparation at high protein concentrations. In consequence,
single-chain CD27L-FC fusion proteins comprising free thiols are
expected to be less stable during manufacture and storage, when
longtime exposure to oxygen and proteases occurs.
[0090] Therefore, to enable manufacture of a hexavalent CD27
receptor agonist at technical scale, the C115 and C151 residues are
preferably mutated simultaneously to a different amino acid (e.g.
S, A or G).
[0091] The CD27 receptor agonist protein may additionally comprise
an N-terminal signal peptide domain, which allows processing, e.g.
extracellular secretion, in a suitable host cell. Preferably, the
N-terminal signal peptide domain comprises a protease cleavage
site, e.g. a signal peptidase cleavage site and thus may be removed
after or during expression to obtain the mature protein. A
particularly preferred N-terminal signal peptide domain comprises
the amino acid sequence as shown in SEQ ID NO: 17 (Table 4).
[0092] Further, the CD27 receptor agonist protein may additionally
comprise a C-terminal element, having a length of e.g. 1-50,
preferably 10-30 amino acids which may include or connect to a
recognition/purification domain, e.g. a FLAG domain, a Strep-tag or
Strep-tag II domain and/or a poly-His domain. According to a
preferred embodiment, the fusion polypeptide comprises a Strep-tag
fused to the C-terminus via a short serine linker as shown in SEQ
ID NO: 18 (Table 4).
[0093] Preferred hinge-linker elements (SEQ ID NO: 16, 19-24), a
preferred N-terminal signal peptide domain (SEQ ID NO: 17) and
serine linker-strep tag (SEQ ID NO: 18) are shown in Table 4.
TABLE-US-00004 TABLE 4 Exemplary domains and linkers SEQ ID NO
Sequence 16 GSSSSSSSSGSCDKTHTCPPC 17 METDTLLVFVLLVWVPAGNG 18
SSSSSSAWSHPQFEK 19 GSSSSSSSGSCDKTHTCPPC 20 GSSSSSSGSCDKTHTCPPC 21
GSSSSSGSCDKTHTCPPC 22 GSSSGSCDKTHTCPPC 23 GSSSGSCDKTHTCPPCGS 24
GSSSGSCDKTHTCPPCGSGS
[0094] Utilizing the hinge linkers (SEQ ID NO: 16 and 19-24) to
fuse receptor binding modules of the invention to one of the
preferred Fc domains (Seq ID NO: 13 and 14) allows for formation of
covalently linked dimers of single chain receptor binding
polypetides. Thus, one embodiment of the present invention provides
a CD27 receptor agonist protein comprising a dimer of two
single-chain fusion polypeptides each having the amino acid
sequence set forth in SEQ ID NO: 27, wherein the two polypeptides
are covalently linked through three interchain disulfide bonds
formed between cysteine residues 457, 463, and 466 of each
polypeptide. As non-limiting example, further polypeptide dimers of
the same kind can be formed by covalently linking of cysteins at
positions (457, 463 and 456) of SEQ ID Nos: 28, 29, 30 and 35.
[0095] It is obvious for a person skilled in the art that all of
the non-limiting examples of CD27 receptor agonist proteins of
table 5 will allow for the formation of dimers. Further embodiments
of the invention are therefore covalently linked dimers of two
single-chain fusion proteins. For instance dimers of SEQ ID NOs:
31, 43, 47 linked at cysteine positions 453, 459 and 462, or dimers
of polypeptides of SEQ ID NO: 32 (linked at positions 450, 456 and
459), or dimers of polypeptides of SEQ ID NO:33 (linked at
positions 436, 442, 445), or dimers of polypeptides of SEQ ID NO:34
(linked at positions 454, 460 and 463), or dimers of polypeptides
of SEQ ID NO:49 (linked at positions 438, 444 and 447), or dimers
of polypeptides of SEQ ID NOs:44 or 46 (linked at positions 442,
448 and 451).
[0096] In one embodiment of the invention, the fusion polypeptide
comprises three soluble CD27L domains fused by peptide linker
elements of SEQ ID NO: 2. The first soluble CD27L domain (i)
consists of amino acids 51-193 of human CD27L according to SEQ ID
NO: 1 and the soluble CD27L domains (iii) and (v) consist of amino
acids 51-193 of human CD27L according to SEQ ID NO: 1.
[0097] Preferred Configuration CD27L-Fc
[0098] Additionally, the fusion polypeptide comprises an antibody
Fc fragment domain according to SEQ ID NO: 13 that is fused
C-terminally to the soluble CD27L domain (v) via a hinge-linker
according to SEQ ID NO: 16. The inventors surprisingly found that
this particular fusion polypeptide provides improved biological
activity as compared to bivalent agonistic anti-CD27-mAB and has a
prolonged stability as compared to similar fusion proteins
comprising a lysine in position 223 and a N297S mutation in the CH2
domain (according to the EU numbering). The amino acid sequence of
an exemplary embodiment of a CD27 receptor agonist protein of the
invention is set forth in SEQ ID NO: 27.
[0099] Further, the fusion polypeptide may comprise an N-terminal
signal peptide domain e.g. according to SEQ ID NO: 17. A specific
example of a CD27 receptor agonist protein of the invention is
shown in SEQ ID NO: 25.
[0100] According to another preferred embodiment, the fusion
polypeptide may additionally comprise a C-terminal Strep-tag that
is fused to the polypeptide of the invention via a short serine
linker as shown in SEQ ID NO: 18. According to this aspect of the
invention, the Fc fragment preferably consists of the amino acid
sequence as shown in SEQ ID NO: 13 or 14. Further, the Fc fragment
may consist of a shorter Fc fragment, for example including amino
acids 1-217 of SEQ ID NO: 13. Particularly preferred examples of
fusion polypeptides comprising a C-terminal Strep-tag are shown in
SEQ ID NO: 15 (PROTEIN A).
[0101] The exemplary CD27 receptor agonist proteins as shown in SEQ
ID NOs: 15,25, and 26, each comprises an N-terminal signal peptide
domain, at amino acids 1-20 of each sequence. In each case, the
mature protein starts with amino acid 21. Mature exemplary CD27
receptor agonist proteins (without a signal peptide) of the instant
invention are set forth in SEQ ID NO: 27-35 and 43-46. Exemplary
CD27 receptor agonist proteins described above are shown in Table
5.
[0102] According to one embodiment of the invention, the
single-chain CD27L fusion polypeptide domain comprises three
soluble CD27L domains fused by peptide linker elements of SEQ ID
NO: 2. The soluble CD27L domains (i), (iii) and (v) each consists
of amino acids 51-193 of human CD27L according to SEQ ID NO: 1
optionally with the soluble domain (i) comprising the E51Q
mutation. The single-chain-CD27L polypeptide comprising
aforementioned CD27L E51Q mutein in domain (i) is shown in SEQ ID:
36 (Table 5B), which is well suited to generate fusion proteins
with additional domains fused to either N-or C-terminal end with
enhanced stability compared to wild type.
[0103] According to another embodiment of the invention, the
single-chain CD27L fusion polypeptide domain comprises three
soluble CD27L domains fused by peptide linker elements of SEQ ID
NO: 2. The soluble CD27L domains (i), (iii) and (v) each consists
of amino acids 51-193 of human CD27L according to SEQ ID NO: 1
optionally with the soluble domain (i), (iii) and (v) comprising
the N63D and/or N170D mutation.
[0104] Exemplarily, a single-chain-CD27L polypeptide comprising
aforementioned CD27L N63D mutation in domain (i), (ii) and (v) is
shown in SEQ ID: 39 (Table 5B). In a preferred embodiment, the
linker (iv) of SEQ ID: 39 is exchanged to SEQ ID NO: 11 resulting
in SEQ ID: 40 (Table 5B).
[0105] According to still another embodiment of the invention, the
single-chain CD27L fusion polypeptide domain comprises three
soluble CD27L domains fused by peptide linker elements of SEQ ID
NO: 2. The soluble CD27L domains (i), (iii) and (v) each consists
of amino acids 56-193 of human CD27L according to SEQ ID NO: 1
optionally with the soluble domain (i), (iii) and (v) comprising
the N63D and/or N170D mutation. Exemplarily, a single-chain-CD27L
polypeptide a comprising aforementioned CD27L N63D mutation in
domain (i), (ii) and (v) is shown in SEQ ID: 41 (Table 5B).). In a
preferred embodiment, the linker (iv) of SEQ ID: 41 is exchanged to
SEQ ID NO: 11 resulting in SEQ ID: 42 (Table 5B).
[0106] In a preferred embodiment, an antibody Fc fragment domain
according to SEQ ID NO: 13 is fused C-terminally to the soluble
CD27L domain (v) of SEQ ID:36 via a hinge linker according to SEQ
ID NO:16 16. A specific example of a CD27 receptor agonist protein
of the invention comprising the E51Q mutein in domain (i), the
hinge linker of SEQ ID NO: 16 and an antibody Fc fragment according
to SEQ ID NO: 13 is shown in SEQ ID NO:30 (Table 5):
[0107] The CD27 receptor agonist as set forth in SEQ ID NO: 27 27
has a reduced total number of glycosylation sites (the N297S
mutation in the CH2 region providing an aglycosylated CH2 domain,
according to the EU numbering system), an increased number of
inter-chain disulfide bonds in the hinge region, and the mutation
of an upper-hinge lysine to a glycine (K223G, according to the EU
numbering system). These alterations provide a decrease in
potential degradation and CD27L receptor superclustering (along
with concomitant toxicity).
[0108] The CD27 receptor agonist as set forth in SEQ ID NO: 47
comprises the same layout as SEQ ID NO: 27 but with the second
peptide linker (iv) shortened thereby reducing protomer
dissociation and enhancing the proteins stability towards
proteases.
[0109] The CD27 receptor agonist as set forth in SEQ ID NO: 30
comprises the same layout as SEQ ID NO: 27 but with the E51Q
mutation in the soluble CD27L domains (i) thereby enabling
formation of pyroglutamate leading to protection of the N-terminus
against aminopeptidases and subsequently enhancing the overall
stability of the protein during manufacture and storage. The CD27
receptor agonist as set forth in SEQ ID NO: 31 comprises the same
layout as SEQ ID NO: 30 but with the second peptide linker (iv)
shortened, thereby reducing protomer dissociation and enhancing the
proteins stability towards proteases. The CD27 receptor agonist as
set forth in SEQ ID NO: 32 comprises the same layout as SEQ ID NO:
31 but with the third peptide linker (vi) shortened to reduce the
interdomain distance between the soluble CD27L domain (v) and the
Fc-domain (Vii) thereby enhancing the proteins stability towards
proteases. The CD27 receptor agonist as set forth in SEQ ID NO: 33
comprises:(i) a first soluble CD27L cytokine domain comprising
amino acids 55-193 from SEQ ID NO: 1 with the W55Q mutation; (ii) a
first peptide linker being SEQ ID NO: 2; (iii) a second soluble
CD27L domain comprising amino acids 55-193 from SEQ ID NO: 1; (iv)
a second peptide linker with SEQ ID NO: 11; (v) a third soluble
CD27L domain comprising amino acids 55-193 from SEQ ID NO: 1; (vi)
a third peptide linker with SEQ ID NO: 21 and (vii) an antibody Fc
fragment with SEQ ID NO: 13. The CD27 receptor agonist as set forth
in SEQ ID NO: 34 comprises the same layout as SEQ ID NO: 30 but but
with the third peptide linker (vi) shortened to reduce the
interdomain distance between the soluble CD27L domain (v) and the
Fc-domain (vii) thereby enhancing the proteins stability towards
proteases.
[0110] The CD27 receptor agonist as set forth in SEQ ID NO: 35
comprises the same layout as SEQ ID NO: 27 but with the N63D
mutation in the soluble CD27L domains (i), (iii) and (v) in order
to reduce the total number of N-linked carbohydrates on the
proteins surface thereby reducing carbohydrate driven in vivo
elimination of the compound. The CD27 receptor agonists as set
forth in SEQ ID NO: 43 combines the N63D mutation strategy
presented in SEQ ID 35 with a shorter linker (iv). As the shorter
linker (SEQ ID 11) lacks a glycosylation consensus sequence, the
total number of N-linked carbohydrates is further reduced.
Additional specific CD27 receptor agonist fusion proteins of the
invention with a reduced number of N-linked carbohydrates based on
the N63D mutation and are set forth in SEQ ID NO: 44 and SEQ ID NO:
45 (table 5). In SEQ ID 46, each of the soluble CD27L domains (i),
(iii) and (v) comprise the N63D and the N170D mutation depleting
further N-linked carbohydrates from CD27 receptor agonist fusion
protein
TABLE-US-00005 TABLE 5 Exemplary CD27 receptor agonist Proteins SEQ
ID NO Sequence 25
METDTLLVFVLLVWVPAGNGESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHG PROTEIN
A PELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLS
without Strep
FHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFEGVQWVRPGSGSGNGSESL
GWDVAELQLNHTGPQQDPRLYWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHIQVT
LAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCT
NLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLNHTGPQQDPRLYWQ
GGPALGRSELHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICS
PASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVR
PGSSSSSSSSGSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQK SLSLSPGK
15 METDTLLVFVLLVWVPAGNGESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHG
PROTEIN A
PELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLS CD27L-wt
+ FHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFEGVQWVRPGSGSGNGSESL SEQ13
(FC) + GWDVAELQLNHTGPQQDPRLYWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHIQVT
Signal + LAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCT
Strep NLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLNHTGPQQDPRLYWQ
GGPALGRSELHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICS
PASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVR
PGSSSSSSSSGSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQK
SLSLSPGSSSSSSAWSHPQFEK 26
METDTLLVFVLLVWVPAGNGESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHG CD27L-wt
+ PELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLS SEQ14
(FC) FHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFEGVQWVRPGSGSGNGSESL
GWDVAELQLNHTGPQQDPRLYWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHIQVT
LAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCT
NLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLNHTGPQQDPRLYWQ
GGPALGRSELHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICS
PASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVR
PGSSSSSSSSGSCDKTHTCPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKS LSLSPGK
27 ESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHI
CD27L-wt +
QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDT SEQ13
(FC) LCTNLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLNHTGPQQDPRL No
Signal YWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVG
No Strep ICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQ
No Glyco WVRPGSGSGNGSESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQL
RIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIA
SQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRPGSSSSSSSSGSCDKTHTCP
PCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 28
ESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHI CD27L-wt
+ QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDT SEQ13
(FC) LCTNLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLNHTGPQQDPRL No
Signal + YWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVG
StrepTag ICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQ
No Glyco WVRPGSGSGNGSESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQL
RIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIA
SQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRPGSSSSSSSSGSCDKTHTCP
PCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGSSSSSSAWSHPQF EK 29
ESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHI CD27L-wt
+ QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDT SEQ14
(FC) LCTNLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLNHTGPQQDPRL No
Signal YWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVG
No strep ICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQ
Glyco FC WVRPGSGSGNGSESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQL
RIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIA
SQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRPGSSSSSSSSGSCDKTHTCP
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 30
QSLGWDVAELQLnHTGPQQDPRLYWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHI Same as
27 QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDT with
E51Q in LCTnLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLnHTGPQQDPRL
module1 YWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVG
ICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTnLTGTLLPSRNTDETFFGVQ
WVRPGSGSGNGSESLGWDVAELQLnHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQL
RIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIA
SQRLTPLARGDTLCTnLTGTLLPSRNTDETFFGVQWVRPGSSSSSSSSGSCDKTHTCP
PCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 31
QSLGWDVAELQLnHTGPQQDPRLYWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHI Same as
30 QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDT With
E51Q LCTnLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLnHTGPQQDPRL
With L1 8 mer
YWQGGPALGRSELHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVG glyco
ICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTnLTGTLLPSRNTDETFFGVQ L2: 4
mer WVRPGSGSESLGWDVAELQLnHTGPQQDPRLYWQGGPALGRSELHGPELDKGQLRIHR
deglyco DGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRL
TPLARGDTLCTnLTGTLLPSRNTDETFFGVQWVRPGSSSSSSSSGSCDKTHTCPPCPA
PELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 32
QSLGWDVAELQLnHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHI Same as
31, QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDT
shortened
LCTnLTGTLLPSRNTDETFFGVQWVRPgsgsgngsESLGWDVAELQLnHTGPQQDPRL hinge
YWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVG
ICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTnLTGTLLPSRNTDETFFGVQ
WVRPgsgsESLGWDVAELQLnHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHR
DGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRL
TPLARGDTLCTnLTGTLLPSRNTDETFFGVQWVRPgsssssgsCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 33
QDVAELQLnHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVH N-terminal
IQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTP shortened
LARGDTLCTnLTGILLPSRNTDETFFGVQWVRPgsgsgngsDVAELQLnHTGP modules
QQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTA
SRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTnLTG
TLLPSRNTDETFFGVQWVRPgsgsDVAELQLnHTGPQQDPRLYWQGGPALGRS
FLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPAS
RSISLLRLSFHQGCTIASQRLTPLARGDTLCTnLTGILLPSRNTDETFFGVQW
VRPgsssssgsCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVIC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK 34
QSLGWDVAELQLnHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGI
YMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQ
RLTPLARGDTLCTnLTGILLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVA
ELQLnHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVT
LAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARG
DTLCTnLTGILLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLnHTG
PQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTT
ASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTnLT
GILLPSRNTDETFFGVQWVRPGSSSSSGSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
SSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 35
ESLGWDVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGI (SEQ39 + SEQ
YMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQ 16 + SEQ13)
RLTPLARGDTLCINLIGILLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVA
ELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVT
LAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARG
DTLCINLIGILLPSRNTDETFEGVQWVRPGSGSGNGSESLGWDVAELQLDHIG
PQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTT
ASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLT
GILLPSRNTDETFFGVQWVRPGSSSSSSSSGSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRIPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 43
ESLGWDVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHI
PROTEIN-C
QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDT (SEQ40 +
SEQ LCTNLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLDHTGPQQDPRL 16 +
SEQ13) YWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVG
ICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQ
WVRPGSGSESLGWDVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHR
DGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRL
TPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRPGSSSSSSSSGSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 44
DVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHI (SEQ41 +
QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPL SEQ16 +
ARGDTLCINLIGILLPSRNTDETFFGVQWVRPGSGSGNGSDVAELQLDHTGPQ SEQ13)
QDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTAS
RHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCINLIGT
LLPSRNTDETFFGVQWVRPGSGSGNGSDVAELQLDHTGPQQDPRLYWQGGPAL
GRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICS
PASRSISLLRLSFHQGCTIASQRLTPLARGDTLCINLIGILLPSRNTDETFFG
VQWVRPGSSSSSSSSGSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45
DVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHI (SEQ41 +
QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPL SEQ16 +
ARGDTLCINLIGILLPSRNTDETFFGVQWVRPGSGSGNGSDVAELQLDHTGPQ SEQ13)
QDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTAS
RHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCINLIGT
LLPSRNTDETFFGVQWVRPGSGSDVAELQLDHTGPQQDPRLYWQGGPALGRSF
LHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASR
SISLLRLSFHQGCTIASQRLTPLARGDTLCINLIGILLPSRNTDETFFGVQWV
RPGSSSSSSSSGSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK 46
DVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHI (SEQ44 +
QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPL N170D)
ARGDTLCIDLTGILLPSRNTDETFFGVQWVRPGSGSGNGSDVAELQLDHTGPQ
QDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTAS
RHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCIDLIGT
LLPSRNTDETFFGVQWVRPGSGSGNGSDVAELQLDHTGPQQDPRLYWQGGPAL
GRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICS
PASRSISLLRLSFHQGCTIASQRLTPLARGDTLCIDLTGILLPSRNTDETFFG
VQWVRPGSSSSSSSSGSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 47
ESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGI PROTEIN-B
YMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQ (Same as
RLTPLARGDTLCINLIGILLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVA SEQ31,
ELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVT without E51Q
LAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARG in module
(i)) DTLCINLIGILLPSRNTDETFFGVQWVRPGSGSESLGWDVAELQLNHTGPQQD
PRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRH
HPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCINLIGILL
PSRNTDETFFGVQWVRPGSSSSSSSSGSCDKTHTCPPCPAPELLGGPSVFLFP
PKPKDILMISRIPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYS
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
TABLE-US-00006 TABLE 5B Exemplary scCD27L-RBD modules 36
QSLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHI E51Q in
M1 QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDT
LCTNLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLNHTGPQQDPRL
YWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVG
ICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQ
WVRPGSGSGNGSESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQL
RIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIA
SQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRPG 39
ESLGWDVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHI 51-193
(i) (iii)
QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDT (v) with
N63D LCTNLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLDHTGPQQDPRL
(ii) 8 mer
YWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVG (iv) 8
mer ICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQ
WVRPGSGSGNGSESLGWDVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQL
RIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIA
SQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP 40
ESLGWDVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHI 51-193
(i) (iii)
QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDT (v) with
N63D LCTNLTGTLLPSRNTDETFFGVQWVRPGSGSGNGSESLGWDVAELQLDHTGPQQDPRL
(ii) 8 mer
YWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVG (iv): 4
mer ICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQ
WVRPGSGSESLGWDVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHR
DGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRL
TPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP 41
DVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLA 56-193
(i) (iii)
ICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNL (v) with
N63D TGTLLPSRNTDETFFGVQWVRPGSGSGNGSDVAELQLDHTGPQQDPRLYWQGGPALGR
(ii) 8 mer
SFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSIS (iv) L2:
8 mer LLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRPGSGSGN
GSDVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVT
LAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCT
NLTGTLLPSRNTDETFFGVQWVRP 42
DVAELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLA 56-193
(i) (iii)
ICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNL (v) with
N63D TGTLLPSRNTDETFFGVQWVRPGSGSGNGSDVAELQLDHTGPQQDPRLYWQGGPALGR
(ii) 8 mer
SFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSIS (iv) 4
mer LLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRPGSGSDV
AELQLDHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAIC
SSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTG
TLLPSRNTDETFFGVQWVRP
[0111] Furthermore, it has to be noted that the scCD27L-RBD module
(SEQ ID NO: 36 and 39-42) are well suited to generate fusion
proteins with additional domains fused to either N- or C-terminal
end employing the linkers described in Table 2 (SEQ ID NO:
2-12).
[0112] Above presented embodiments of the CD27 receptor agonist
proteins of the invention either address stability influencing
construction principles or aggregation resistance of soluble
receptor agonist proteins of the invention or modulate receptor
binding and activity of the receptor agonist proteins.
[0113] A further important property for describing suitability of a
substance as an active agent in medical preparations is its
pharmacokinetic profile (PK profile) Pharmacokinetics is the study
of drug disposition in the body and focuses on the changes in drug
plasma concentration. For any given drug and dose, the plasma
concentration will vary depending on the processes of absorption,
distribution and elimination. The time dependent decline of plasma
drug concentration and its final elimination from the body mainly
depends on biotransformation and excretion of the drug and is
generally measured as in vivo half-life time (Pharmacology, 4th
Edition; Elesevier 2013).
[0114] Understanding the course of events that make up the immune
response against a pathogen or a tumor allows to determine
advantageous PK profiles of the CD27 receptor agonist proteins of
the invention. The immune response against a pathogen or indeed a
tumor carrying antigens can be divided into several phases. Each
phase shows a characteristic duration and events usually take place
in specialized tissues. In particular, the priming phase describes
early events in an immune response when lymphocytes are being
presented with tumor-associated antigens in secondary lymphoid
organs. In order to recognize antigens through their T cell or B
cell receptor, T cells and B cells, respectively, need to form
cell-cell conjugates with antigen-presenting cells (APC). In case
of successful antigen-recognition, lymphocytes are also being
presented with co-stimulatory molecules such as CD27L by the APC.
As both presentation of antigen and co-stimulatory molecules occurs
at the interface of the APC/lymphocyte conjugate, this interaction
is rather short lived as the conjugate falls apart after several
minutes or very few hours. Following antigen recognition and
co-stimulation with molecules such as CD27L lymphocytes become
activated and enter the expansion phase during which they
proliferate in order to mount an immune response against the
tumor.
[0115] In light of the short physical interaction of APCs and
lymphocytes in secondary lymphoid organs, one could speculate that
the co-stimulatory signal elicited by recombinant biologics
targeting the CD27 pathway is desired to be short-lived. In fact,
long exposition to co-stimulatory signals might push lymphocytes
into a hyper-activated state possibly leading to systemic toxic
effects. Consequently, a favorable PK profile for biologics
targeting co-stimulatory pathways of the immune system would show a
comparably short terminal half-life in the range of hours or
possibly one day. This would be in contrast to antibodies targeting
the same pathways, which usually show a terminal half-life of
multiple days or even more than one week. In summary, biologics
activating co-stimulatory pathways of the immune system having a
half-life in the range of several hours are closer to the natural
ligand in term of their temporal activity in comparison to
stimulating antibodies. This could also make a positive
contribution to possible toxicity effects observed during the
treatment with some immune-stimulating antibodies.
[0116] Thus, in a further embodiment the CD27 receptor agonist
proteins of the invention have a short terminal half live such as
less than 4 days, less than three days, less than two days, less
than one day.
[0117] A further aspect of the present invention relates to a
nucleic acid molecule encoding a CD27 receptor agonist protein as
described herein. The nucleic acid molecule may be a DNA molecule,
e.g. a double-stranded or single- stranded DNA molecule, or an RNA
molecule. The nucleic acid molecule may encode the CD27 receptor
agonist protein or a precursor thereof, e.g. a pro- or pre-proform
of the CD27 receptor agonist protein which may comprise a signal
sequence or other heterologous amino acid portions for secretion or
purification which are preferably located at the N- and/or
C-terminus of the CD27 receptor agonist protein. The heterologous
amino acid portions may be linked to the first and/or second domain
via a protease cleavage site, e.g. a Factor X3, thrombin or IgA
protease cleavage site. A specific example of a nucleic acid
sequence of the invention is shown in Table 6 as SEQ ID NO: 37.
This nucleic acid molecule comprises the open reading frame
encoding the fusion polypeptide of SEQ ID NO: 25.
TABLE-US-00007 TABLE 6 Nucleic Acid Sequence of Exemplary CD27
receptor agonist Protein SEQ ID NO Sequence 37
AAGCTTTAGGGATAACAGGGTAATAGCCGCCACCATGGAGACTGACACCCTGCTGGTGTTCG
TGCTGCTGGTCTGGGTGCCTGCAGGAAATGGAGAGAGCCTGGGATGGGATGTGGCCGAACTC
CAGCTGAACCACACAGGCCCTCAGCAAGACCCTAGGCTCTACTGGCAGGGCGGCCCTGCTCT
GGGAAGGAGCTTTCTGCATGGCCCTGAACTGGATAAAGGCCAACTGCGTATTCATCGGGATG
GCATTTACATGGTCCATATCCAGGTGACCCTCGCCATCTGCTCCAGCACCACCGCTAGCAGG
CATCATCCCACCACCCTGGCCGTGGGCATTTGTTCCCCTGCCAGCCGGTCCATCTCCCTGCT
GAGGCTGAGCTTTCATCAGGGCTGCACCATCGCCTCCCAAAGGCTGACCCCTCTGGCCAGGG
GCGATACACTGTGTACCAATCTGACCGGCACCCTGCTCCCTAGCAGGAACACCGATGAAACC
TTTTTCGGAGTGCAGTGGGTGCGGCCTGGTTCCGGAAGCGGCAATGGCTCCGAAAGCCTCGG
CTGGGACGTGGCCGAGCTCCAACTGAACCACACCGGCCCTCAACAAGATCCTCGGCTCTATT
GGCAAGGCGGACCTGCTCTCGGCCGGAGCTTCCTGCATGGCCCTGAGCTGGACAAGGGCCAG
CTGCGTATTCATCGGGATGGAATCTATATGGTGCACATCCAAGTGACACTGGCCATTTGCAG
CAGCACCACCGCTAGCCGGCACCATCCTACCACCCTGGCTGTGGGCATCTGTTCCCCCGCTA
GCCGGTCCATCTCCCTGCTGAGGCTGAGCTTCCACCAGGGCTGTACCATCGCCAGCCAGAGG
CTGACCCCTCTGGCTAGGGGCGACACCCTGTGTACCAACCTGACCGGAACCCTGCTGCCTAG
CAGGAATACCGATGAGACCTTCTTCGGAGTGCAATGGGTGAGGCCTGGCTCTGGTTCTGGTA
ACGGTTCTGAGAGCCTCGGCTGGGACGTCGCTGAACTGCAGCTGAATCACACAGGCCCCCAG
CAGGACCCTAGGCTGTACTGGCAGGGAGGCCCTGCTCTCGGAAGGAGCTTTCTGCACGGCCC
TGAACTGGATAAGGGACAGCTCCGTATTCATCGGGATGGCATCTACATGGTGCATATCCAGG
TCACCCTGGCCATCTGCAGCTCCACCACCGCCTCCAGGCACCACCCTACCACCCTGGCTGTG
GGCATCTGCTCCCCTGCCTCCCGGAGCATCAGCCTGCTGAGGCTGTCCTTCCACCAAGGCTG
CACCATCGCTAGCCAAAGGCTGACCCCTCTGGCTAGGGGCGATACCCTGTGCACCAACCTGA
CCGGAACCCTGCTGCCTTCCCGGAACACCGACGAGACCTTTTTCGGCGTGCAGTGGGTCAGG
CCCGGATCctcgagTTCATCGTCCTCATCCGGCTCATGTGATAAGACCCACACCTGCCCTCC
CTGTCCTGCCCCTGAGCTGCTGGGCGGACCTTCTGTGTTCCTGTTCCCCCCCAAGCCTAAGG
ACACCCTGATGATCTCCAGGACCCCTGAGGTGACCTGTGTGGTGGTGGACGTGTCTCACGAA
GATCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTCCACAACGCCAAGACCAA
GCCTAGGGAGGAGCAGTACAGCTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACC
AGGATTGGCTGAACGGAAAGGAGTATAAGTGTAAGGTCTCCAACAAGGCCCTGCCTGCCCCC
ATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAGCCTCAGGTGTACACCCTGCC
TCCTAGCAGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCT
ACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAACAACTACAAGACC
ACCCCTCCTGTGCTGGACTCTGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAA
GTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATC
ACTACACCCAGAAGTCCCTGTCTCTGAGTCCGGGCAAGTAATAggcgcgcc
[0118] The nucleic acid molecule may be operatively linked to an
expression control sequence, e.g. an expression control sequence
which allows expression of the nucleic acid molecule in a desired
host cell. The nucleic acid molecule may be located on a vector,
e.g. a plasmid, a bacteriophage, a viral vector, a chromosomal
integration vector, etc. Examples of suitable expression control
sequences and vectors are described for example by Sambrook et al.
(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, and Ausubel et al. (1989), Current Protocols in Molecular
Biology, John Wiley & Sons or more recent editions thereof.
[0119] Various expression vector/host cell systems may be used to
express the nucleic acid sequences encoding the CD27 receptor
agonist proteins of the present invention. Suitable host cells
include, but are not limited to, prokaryotic cells such as
bacteria, e.g. E. coli, eukaryotic host cells such as yeast cells,
insect cells, plant cells or animal cells, preferably mammalian
cells and, more preferably, human cells. Further, the invention
relates to a non-human organism transformed or transfected with a
nucleic acid molecule as described above. Such transgenic organisms
may be generated by known methods of genetic transfer including
homologous recombination.
[0120] A further aspect of the present invention relates to a
pharmaceutical or diagnostic composition comprising as the active
agent at least one CD27 receptor agonist protein, a respective
nucleic acid encoding therefore, or a transformed or transfected
cell, all as described herein.
[0121] In another aspect, the present invention provides a
pharmaceutical composition comprising a CD27 receptor agonist
protein disclosed herein and one or more pharmaceutically
acceptable carriers, diluents, excipients, and/or adjuvants.
[0122] In another aspect, the present invention provides a nucleic
acid molecule encoding the CD27 receptor agonist protein. In
another embodiment, the present invention provides an expression
vector comprising the nucleic acid molecule. In another embodiment,
the present invention provides a cell comprising the nucleic acid
molecule. In a further embodiment, the cell is a eukaryotic cell.
In another embodiment, the cell is a mammalian cell. In another
embodiment, the cell is a Chinese Hamster Ovary (CHO) cell. In
other embodiments, the cell is selected from the group consisting
of CHO-DBX11, CHO-DG44, CHO-S, and CHO-K1 cells. In other
embodiments, the cell is selected from the group consisting of
Vero, BHK, HeLa, COS, MDCK, HEK-293, NIH-3T3, W138, BT483, Hs578T,
HTB2, BT20, T47D, NSO, CRL7030, HsS78Bst, PER.C6, SP2/0-Agl4, and
hybridoma cells.
[0123] In another aspect, the present invention provides a method
of treating a subject having a CD27L-associated disease or
disorder, the method comprising administering to the subject an
effective amount of the CD27 receptor agonist protein. In one
embodiment, the CD27 receptor agonist protein is administered
alone. In another embodiment, the CD27 receptor agonist protein is
administered before, concurrently, or after the administration of a
second agent. In another embodiment, the disease or disorder is
selected from the group consisting of: tumors, infectious diseases,
inflammatory diseases, metabolic diseases, autoimmune disorders,
degenerative diseases, apoptosis-associated diseases, and
transplant rejections. In one embodiment, the tumors are solid
tumors. In one embodiment, the tumors arise from the group of
cancers consisting of sarcoma, esophageal cancer, and gastric
cancer. In another embodiment, the tumors arise from Ewing's
sarcoma or fibrosarcoma. In another embodiment, the tumors arise
from the group of cancers consisting of Non-Small Cell Lung
Carcinoma (NSCLC), pancreatic cancer, colorectal cancer, breast
cancer, ovarian cancer, head and neck cancers, and Small Cell Lung
Cancer (SCLC). In another embodiment, the tumors are lymphatic
tumors. In one embodiment, the tumors are hematologic tumors.
[0124] In another embodiment, the tumors arise from non-Hodgkin's
lymphoma, leukemia, acute lymphoblastic leukemia (ALL), acute
myeloid leukemia (AML), B cell lymphoma, Burkitt's lymphoma,
chronic myelocytic leukemia (CML), chronic lymphocytic leukemia
(CLL), or hairy cell leukemia. In another embodiment, the
autoimmune disorders are rheumatoid diseases, arthritic diseases,
or rheumatoid and arthritic diseases. In a further embodiment, the
disease or disorder is rheumatoid arthritis. In another embodiment,
the degenerative disease is a neurodegenerative disease. In a
further embodiment, the neurodegenerative disease is multiple
sclerosis.
[0125] In one embodiment, the second agent is a chemotherapeutic,
radiotherapeutic, or biological agent. In one embodiment, the
second agent is selected from the group consisting of Duvelisib,
Ibrutinib, Navitoclax, and Venetoclax. In another embodiment, the
second agent is an apoptotic agent. In one embodiment, the
apoptotic second agent is selected from the group consisting of
Bortezomib, Azacitidine, Dasatinib, and Gefitinib. In a particular
embodiment, the pharmaceutical compositions disclosed herein are
administered to a patient by intravenous or subcutaneous
administration. In other embodiments, the disclosed pharmaceutical
compositions are administered to a patient byoral, parenteral,
intramuscular, intrarticular, intrabronchial, intraabdominal,
intracapsular, intracartilaginous, intracavitary, intracelial,
intracerebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial,
intraosteal, intrapelvic, intrapericardiac, intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal,
buccal, sublingual, intranasal, or transdermal administration.
[0126] In one embodiment, the CD27 receptor agonist protein is
administered as a single bolus. In another embodiment, CD27
receptor agonist protein may be administered over several divided
doses. The CD27 receptor agonist protein can be administered at
about 0.1-100 mg/kg. In one embodiment, the CD27 receptor agonist
protein can be administered at a dosage selected from the group
consisting of: about 0.1-0.5, 0.1-1, 0.1-10, 0.1-20, 0.1-50,
0.1-75, 1-10, 1-15, 1-7.5, 1.25-15, 1.25-7.5, 2.5-7.5, 2.5-15,
5-15, 5-7.5,1-20, 1-50, 7-75, 1-100, 5-10, 5-15, 5-20, 5-25, 5-50,
5-75, 10-20, 10-50, 10-75, and 10-100 mg/kg. In other embodiments,
the CD27 receptor agonist protein is present in pharmaceutical
compositions at about 0.1-100 mg/ml. In one embodiment, the CD27
receptor agonist protein is present in pharmaceutical compositions
at an amount selected from the group consisting of: about 0.1-0.5,
0.1-1, 0.1-10, 0.1-20, 0.1-50, 0.1-75, 1-10, 1-20, 1-50, 1-75,
1-100, 5-10, 5-15, 5-20, 5-25, 5-50, 5-75, 10-20, 10-50, 10-75, or
10-100 mg/ml. In other embodiments, a therapeutically effective
amount of CD27 receptor agonist protein is administered to a
subject. In another embodiment, a prophylactically effective amount
of CD27 receptor agonist protein is administered to a subject.
[0127] The term "CD27L-associated disease or disorder" as used
herein is any disease or disorder which may be ameliorated by
administering an effective amount of a CD27 receptor agonist to a
subject in need thereof. At least one CD27 receptor agonist
protein, respective nucleic acid encoding therefore, or transformed
or transfected cell, all as described herein may be used in
therapy, e.g., in the prophylaxis and/or treatment of disorders
caused by, associated with and/or accompanied by dysfunction of
CD27L, particularly proliferative disorders, such as tumors, e.g.
solid or lymphatic tumors; infectious diseases; inflammatory
diseases; metabolic diseases; autoimmune disorders, e.g. rheumatoid
and/or arthritic diseases; degenerative diseases, e.g.
neurodegenerative diseases such as multiple sclerosis;
apoptosis-associated diseases or transplant rejections.
[0128] The term "dysfunction of CD27L" as used herein is to be
understood as any function or expression of CD27L that deviates
from the normal function or expression of CD27L, e.g.,
overexpression of the CD27L gene or protein, reduced or abolished
expression of the CD27L gene or protein compared to the normal
physiological expression level of CD27L, increased activity of
CD27L, reduced or abolished activity of CD27L, increased binding of
CD27L to any binding partners, e.g., to a receptor, particularly a
CD27L receptor or another cytokine molecule, reduced or abolished
binding to any binding partner, e.g. to a receptor, particularly a
CD27L receptor or another cytokine molecule, compared to the normal
physiological activity or binding of CD27L.
[0129] In various embodiments, a method is provided for treating a
human subject suffering from a disorder which can be treated by
targeting CD27 receptors comprising administering to the human
subject a CD27 receptor agonist protein disclosed herein such that
the effect on the activity of the target, or targets, in the human
subject is agonistic, one or more symptoms is alleviated, and/or
treatment is achieved. The CD27 receptor agonist proteins provided
herein can be used to treat humans suffering from primary and
metastatic cancers, including carcinomas of breast, colon, rectum,
lung (e.g., small cell lung cancer "SCLC" and non- small cell lung
cancer "NSCLC"), oropharynx, hypopharynx, esophagus, stomach,
pancreas, liver, gallbladder and bile ducts, small intestine,
urinary tract (including kidney, bladder and urothelium), female
genital tract (including cervix, uterus, and ovaries as well as
choriocarcinoma and gestational trophoblastic disease), male
genital tract (including prostate, seminal vesicles, testes and
germ cell tumors), endocrine glands (including the thyroid,
adrenal, and pituitary glands), and skin, as well as hemangiomas,
melanomas, sarcomas (including those arising from bone and soft
tissues as well as Kaposi's sarcoma), tumors of the brain, nerves,
eyes, and meninges (including astrocytomas, gliomas, glioblastomas,
retinoblastomas, neuromas, neuroblastomas, Schwannomas, and
meningiomas), tumors arising from hematopoietic malignancies, acute
leukemia, acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), B cell lymphoma, Burkitt's lymphoma, chronic
myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL),
hairy cell leukemia, Hodgkin's and non-Hodgkin's lymphomas, DLBCL,
follicular lymphomas, hematopoietic malignancies, Kaposi's sarcoma,
malignant lymphoma, malignant histiocytosis, malignant melanoma,
multiple myeloma, paraneoplastic syndrome/hypercalcemia of
malignancy, or solid tumors.
[0130] A pharmaceutical composition comprising a CD27 receptor
agonist protein disclosed herein and a pharmaceutically acceptable
carrier is provided. In some embodiments, the pharmaceutical
composition comprises at least one additional therapeutic agent for
treating a disorder. For example, the additional agent may be a
therapeutic agent, a chemotherapeutic agent; an imaging agent, a
cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor
(including but not limited to a KDR and a TIE-2 inhibitor), a
co-stimulation molecule modulator or an immune checkpoint inhibitor
(including but not limited to anti-B7.1, anti-B7.2, anti-B7.3,
anti-B7.4, anti-CD28, anti-B7RP1, CTLA4-Ig, anti-CTLA-4, anti-PD-1,
anti-PD-L1, anti-PD-L2, anti-ICOS, anti-LAG-3, anti-Tim3,
anti-VISTA, anti-HVEM, anti-BTLA, LIGHT fusion protein, anti-CD137,
anti-CD137L, anti-OX40, anti-OX40L, anti-CD70, anti-CD27,
anti-GAL9, anti-A2AR, anti-KIR, anti-IDO-1, anti-CD20), a dendritic
cell/antigen-presenting cell modulator (including but not limited
to anti-CD40 antibody, anti-CD40L, anti-DC-SIGN, anti-Dectin-1,
anti-CD301, anti-CD303, anti-CD123, anti-CD207, anti-DNGR1,
anti-CD205, anti-DCIR, anti-CD206, anti-ILT7), a modulator for
Toll-like receptors (including but not limited to anti-TLR-1,
anti-TLR-2, anti-TLR-3, anti-TLR-4, anti-TLR-4, anti-TLR-5,
anti-TLR-6, anti-TLR-7, anti-TLR-8, anti-TLR-9), an adhesion
molecule blocker (including but not limited to an anti-LFA-1
antibody, an anti-E/L selectin antibody, a small molecule
inhibitor), an anti-cytokine antibody or functional fragment
thereof (including but not limited to an anti-IL-18, an anti-TNF,
or an anti-IL-6/cytokine receptor antibody), a bispecific
redirected T cell or NK cell cytotoxicity (including but not
limited to a BiTE.RTM.), a chimeric T cell receptor (CAR-T) based
therapy, a T cell receptor (TCR)-based therapy, a therapeutic
cancer vaccine, methotrexate, cyclosporin, rapamycin, FK506, a
detectable label or reporter, a TNF antagonist, an anti-rheumatic,
a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug
(NSAID), an analgesic, an anesthetic, a sedative, a local
anesthetic, a neuromuscular blocker, an antimicrobial, an
antipsoriatic, a corticosteriod, an anabolic steroid, an
erythropoietin, an immunization, an immunoglobulin, an
immunosuppressive, a growth hormone, a hormone replacement drug, a
radiopharmaceutical, an antidepressant, an antipsychotic, a
stimulant, an asthma medication, a beta agonist, an inhaled
steroid, an epinephrine or analog, a cytokine, or a cytokine
antagonist.
[0131] In an embodiment, a method of treating a cancer or in the
prevention or inhibition of metastases from the tumors described
herein, the CD27 receptor agonist protein(s) can be used alone or
in combination with one or more additional agents, e.g., a
chemotherapeutic, radiotherapy, or biological agent. In some
embodiments, the agent can include the following:13-cis-Retinoic
Acid; 2-CdA; 2-Chlorodeoxyadenosine; 5-Azacitidine; 5-Fluorouracil;
5-FU; 6-Mercaptopurine; 6-MP; 6-TG; 6-Thioguanine; Abraxane;
Accutane.RTM.; Actinomycin-D; Adriamycin.RTM.; Adrucil.RTM.;
Afinitor.RTM.; Agrylin.RTM.; Ala-Cort.RTM.; Aldesleukin;
Alemtuzumab; ALIMTA; Alitretinoin; Alkaban-AQ.RTM.; Alkeran.RTM.;
All-transretinoic Acid; Alpha Interferon; Altretamine;
Amethopterin; Amifostine; Aminoglutethimide; Anagrelide;
Anandron.RTM.; Anastrozole; Arabinosylcytosine; Ara-C Aranesp.RTM.;
Aredia.RTM.; Arimidex.RTM.; Aromasin.RTM.; Arranon.RTM.; Arsenic
Trioxide; Arzerra.TM.; Asparaginase; ATRA; Avastin.RTM.;
Azacitidine; BCG; BCNU; Bendamustine; Bevacizumab; Bexarotene;
BEXXAR.RTM.; Bicalutamide; BiCNU; Blenoxane.RTM.; Bleomycin;
Bortezomib; Busulfan; Busulfex.RTM.; C225; Calcium Leucovorin;
Campath.RTM.; Camptosar.RTM.; Camptothecin-11; Capecitabine
Carac.TM.; Carboplatin; Carmustine; Carmustine Wafer; Casodex.RTM.;
CC-5013; CCI-779; CCNU; CDDP; CeeNU; Cerubidine.RTM.; Cetuximab;
Chlorambucil; Cisplatin; Citrovorum Factor; Cladribine; Cortisone;
Cosmegen.RTM.; CPT-11; Cyclophosphamide; Cytadren.RTM.; Cytarabine;
Cytarabine Liposomal; Cytosar-U.RTM.; Cytoxan.RTM.; Dacarbazine;
Dacogen; Dactinomycin; Darbepoetin Alfa; Dasatinib; Daunomycin;
Daunorubicin; Daunorubicin Hydrochloride; Daunorubicin Liposomal;
DaunoXome.RTM.; Decadron; Decitabine; Delta-Cortef.RTM.;
Deltasone.RTM.; Denileukin; Diftitox; DepoCyt.TM.; Dexamethasone;
Dexamethasone Acetate; Dexamethasone Sodium Phosphate; Dexasone;
Dexrazoxane; DHAD; DIC; Diodex; Docetaxel; Doxil.RTM.; Doxorubicin;
Doxorubicin Liposomal; Droxia.TM.; DTIC; DTIC-Dome.RTM.;
Duralone.RTM.; Duvelisib; Efudex.RTM.; Eligard.TM.; Ellence.TM.;
Eloxatin.TM.; Elspar.RTM.; Emcyt.RTM.; Epirubicin; Epoetin Alfa;
Erbitux; Erlotinib; Erwinia L-asparaginase; Estramustine; Ethyol
Etopophos.RTM.; Etoposide; Etoposide Phosphate; Eulexin.RTM.;
Everolimus; Evista.RTM.; Exemestane; Fareston.RTM.; Faslodex.RTM.;
Femara.RTM.; Filgrastim; Floxuridine; Fludara.RTM.; Fludarabine;
Fluoroplex.RTM.; Fluorouracil; Fluorouracil (cream);
Fluoxymesterone; Flutamide; Folinic Acid; FUDR.RTM.; Fulvestrant;
Gefitinib; Gemcitabine; Gemtuzumab ozogamicin; Gemzar; Gleevec.TM.;
Gliadel.RTM. Wafer; GM-CSF; Goserelin; Granulocyte-Colony
Stimulating Factor (G-CSF); Granulocyte Macrophage Colony
Stimulating Factor (G-MCSF); Halotestin.RTM.; Herceptin.RTM.;
Hexadrol; Hexalen.RTM.; Hexamethylmelamine; HMM; Hycamtin.RTM.;
Hydrea.RTM.; Hydrocort Acetate.RTM.; Hydrocortisone; Hydrocortisone
Sodium Phosphate; Hydrocortisone Sodium Succinate; Hydrocortone
Phosphate; Hydroxyurea; Ibrutinib; Ibritumomab; Ibritumomab
Tiuxetan; Idamycin.RTM.; Idarubicin Ifex.RTM.; Interferon-alpha;
Interferon-alpha-2b (PEG Conjugate); Ifosfamide; Interleukin-11
(IL-11); Interleukin-2 (IL-2); Imatinib mesylate; Imidazole
Carboxamide; Intron A.RTM.; ipilimumab, Iressa.RTM.; Irinotecan;
Isotretinoin; Ixabepilone; Ixempra.TM.; KADCYCLA.RTM.; Kidrolase
(t) Lanacort.RTM.; Lapatinib; L-asparaginase; LCR; Lenalidomide;
Letrozole; Leucovorin; Leukeran; Leukine.TM.; Leuprolide;
Leurocristine; Leustatin.TM.; Lirilumab; Liposomal Ara-C; Liquid
Pred.RTM.; Lomustine; L-PAM; L-Sarcolysin; Lupron.RTM.; Lupron
Depot.RTM.; Matulane.RTM.; Maxidex; Mechlorethamine;
Mechlorethamine Hydrochloride; Medralone.RTM.; Medrol.RTM.;
Megace.RTM.; Megestrol; Megestrol Acetate; MEK inhibitors;
Melphalan; Mercaptopurine; Mesna; Mesnex.TM.; Methotrexate;
Methotrexate Sodium; Methylprednisolone; Meticorten.RTM.;
Mitomycin; Mitomycin-C; Mitoxantrone M-Prednisol.RTM.; MTC; MTX;
Mustargen.RTM.; Mustine; Mutamycin.RTM.; Myleran.RTM.; Mylocel.TM.;
Mylotarg.RTM.; Navitoclax; Navelbine.RTM.; Nelarabine; Neosar.RTM.;
Neulasta.TM.; Neumega.RTM.; Neupogen.RTM.; Nexavar.RTM.;
Nilandron.RTM.; Nilotinib; Nilutamide; Nipent.RTM.; Nitrogen
Mustard Novaldex.RTM.; Nivolumab; Novantrone.RTM.; Nplate;
Octreotide; Octreotide acetate; Ofatumumab; Oncospar.RTM.;
Oncovin.RTM.; Ontak.RTM.; Onxal.TM.; Oprelvekin; Orapred.RTM.;
Orasone.RTM.; Oxaliplatin; Paclitaxel; Paclitaxel Protein-bound;
Pamidronate; Panitumumab; Panretin.RTM.; Paraplatin.RTM.;
Pazopanib; Pediapred.RTM.; PEG Interferon; Pegaspargase;
Pegfilgrastim; PEG-INTRON.TM.; PEG-L-asparaginase; PEMETREXED;
Pembrolizumab; Pentostatin; Pertuzumab; Phenylalanine Mustard;
Pidilizumab; Platinol.RTM.; Platinol-AQ.RTM.; Prednisolone;
Prednisone; Prelone.RTM.; Procarbazine; PROCRIT.RTM.;
Proleukin.RTM.; Prolifeprospan 20 with Carmustine Implant;
Purinethol.RTM.; BRAF inhibitors; Raloxifene; Revlimid.RTM.;
Rheumatrex.RTM.; Rituxan.RTM.; Rituximab; Roferon-A.RTM.;
Romiplostim; Rubex.RTM.; Rubidomycin hydrochloride;
Sandostatin.RTM.; Sandostatin LAR.RTM.; Sargramostim;
Solu-Cortef.RTM.; Solu-Medrol.RTM.; Sorafenib; SPRYCEL.TM.;
STI-571; STIVAGRA.TM., Streptozocin; SU11248; Sunitinib;
Sutent.RTM.; Tamoxifen Tarceva.RTM.; Targretin.RTM.; Tasigna.RTM.;
Taxol.RTM.; Taxotere.RTM.; Temodar.RTM.; Temozolomide Temsirolimus;
Teniposide; TESPA; Thalidomide; Thalomid.RTM.; TheraCys.RTM.;
Thioguanine; Thioguanine Tabloid.RTM.; Thiophosphoamide;
Thioplex.RTM.; Thiotepa; TICE.RTM.; Toposar.RTM.; Topotecan;
Toremifene; Torisel.RTM.; Tositumomab; Trastuzumab; Treanda.RTM.;
Tremelimumab; Tretinoin; Trexall.TM.; Trisenox.RTM.; TSPA;
TYKERB.RTM.; Urelumab; VCR; Vectibix.TM.; Velban.RTM.;
Velcade.RTM.; Venetoclax; VePesid.RTM.; Vesanoid.RTM.; Viadur.TM.;
Vidaza.RTM.; Vinblastine; Vinblastine Sulfate; Vincasar Pfs.RTM.;
Vincristine; Vinorelbine; Vinorelbine tartrate; VLB; VM-26;
Vorinostat; Votrient; VP-16; Vumon.RTM.; Xeloda.RTM.; Zanosar.RTM.;
Zevalin.TM.; Zinecard.RTM.; Zoladex.RTM.; Zoledronic acid; Zolinza;
or Zometa.RTM., and/or any other agent not specifically listed here
that target similar pathways.
[0132] When two or more substances or principles are to be used as
part of a combined treatment regimen, they can be administered via
the same route of administration or via different routes of
administration, at essentially the same time or at different times
(e.g. essentially simultaneously, consecutively, or according to an
alternating regime). When the substances or principles are to be
administered simultaneously via the same route of administration,
they may be administered as different pharmaceutical formulations
or compositions or part of a combined pharmaceutical formulation or
composition, as will be clear to the skilled person.
[0133] Also, when two or more active substances or principles are
to be used as part of a combined treatment regimen, each of the
substances or principles may be administered in the same amount and
according to the same regimen as used when the compound or
principle is used on its own, and such combined use may or may not
lead to a synergistic effect. However, when the combined use of the
two or more active substances or principles leads to a synergistic
effect, it may also be possible to reduce the amount of one, more
than one, or all of the substances or principles to be
administered, while still achieving the desired therapeutic action.
This may, e.g., be useful for avoiding, limiting or reducing any
unwanted side-effects that are associated with the use of one or
more of the substances or principles when they are used in their
usual amounts, while still obtaining the desired pharmaceutical or
therapeutic effect.
[0134] The effectiveness of the treatment regimen used according to
the invention may be determined and/or followed in any manner known
per se for the disease or disorder involved, as will be clear to
the clinician. The clinician will also be able, where appropriate
and on a case-by-case basis, to change or modify a particular
treatment regimen, so as to achieve the desired therapeutic effect,
to avoid, limit or reduce unwanted side-effects, and/or to achieve
an appropriate balance between achieving the desired therapeutic
effect on the one hand and avoiding, limiting or reducing undesired
side effects on the other hand.
[0135] Generally, the treatment regimen will be followed until the
desired therapeutic effect is achieved and/or for as long as the
desired therapeutic effect is to be maintained. Again, this can be
determined by the clinician.
[0136] In various embodiments, pharmaceutical compositions
comprising one or more CD27 receptor agonist proteins, either alone
or in combination with prophylactic agents, therapeutic agents,
and/or pharmaceutically acceptable carriers are provided herein. In
various embodiments, non-limiting examples of the uses of the
pharmaceutical compositions disclosed herein include diagnosing,
detecting, and/or monitoring a disorder, preventing, treating,
managing, and/or ameliorating a disorder or one or more symptoms
thereof, and/or in research. The formulation of pharmaceutical
compositions, either alone or in combination with prophylactic
agents, therapeutic agents, and/or pharmaceutically acceptable
carriers, are known to one skilled in the art (US Patent
Publication No. 20090311253 A1).
[0137] As used herein, the phrase "effective amount" means an
amount of CD27L agonist protein that results in a detectable
improvement (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more from baseline) in
one or more parameters associated with a dysfunction of CD27L or
with a CD27L-associated disease or disorder.
[0138] Methods of administering a therapeutic agent provided herein
include, but are not limited to, oral administration, parenteral
administration (e.g., intradermal, intramuscular, intraperitoneal,
intravenous and subcutaneous), epidural administration,
intratumoral administration, mucosal administration (e.g.,
intranasal and oral routes) and pulmonary administration (e.g.,
aerosolized compounds administered with an inhaler or nebulizer).
The formulation of pharmaceutical compositions for specific routes
of administration, and the materials and techniques necessary for
the various methods of administration are available and known to
one skilled in the art (US Patent Publication No. 20090311253
A1).
[0139] In various embodiments, dosage regimens may be adjusted to
provide for an optimum desired response (e.g., a therapeutic or
prophylactic response). For example, a single bolus may be
administered, several divided doses may be administered over time
or the dose may be proportionally reduced or increased as indicated
by the exigencies of the therapeutic situation. In some
embodiments, parenteral compositions are formulated in dosage unit
form for ease of administration and uniformity of dosage. The term
"dosage unit form" refers to physically discrete units suited as
unitary dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier.
[0140] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a CD27 receptor agonist
protein provided herein is about 0.1-100 mg/kg, (e.g., about
0.1-0.5, 0.1-1, 0.1-10, 0.1-20, 0.1-50, 0.1-75, 1-10, 1-15, 1-7.5,
1.25-15, 1.25-7.5, 2.5-7.5, 2.5-15, 5-15, 5-7.5,1-20, 1-50, 7-75,
1-100, 5-10, 5-15, 5-20, 5-25, 5-50, 5-75, 10-20, 10-50, 10-75, or
10-100 mg/kg, or any concentration in between). In some
embodiments, the CD27 receptor agonist protein is present in a
pharmaceutical composition at a therapeutically effective
concentration, e.g., a concentration of about 0.1-100 mg/ml (e.g.,
about 0.1-0.5, 0.1-1, 0.1-10, 0.1-20, 0.1-50, 0.1-75, 1-10, 1-20,
1-50, 1-75, 1-100, 5-10, 5-15, 5-20, 5-25, 5-50, 5-75, 10-20,
10-50, 10-75, or 10-100 mg/ml, or any concentration in between).
Note that dosage values may vary with the type and/or severity of
the condition to be alleviated. It is to be further understood that
for any particular subject, specific dosage regimens may be
adjusted over time according to the individual need and/or the
professional judgment of the person administering or supervising
the administration of the compositions, and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed composition.
EXAMPLES
Example 1
Manufacture of a CD27 Receptor Agonist Protein
[0141] 1.1 Polypeptide Structure
[0142] A) Amino acids Met1-Gly20 [0143] Ig-Kappa-signal peptide,
assumed signal peptidase cleavage site after amino acid Gly 20.
[0144] B) Amino acids Glu21-Pro163 [0145] First soluble cytokine
domain of the human CD27L ligand (CD27L, amino acid 51-193 of SEQ
ID NO: 1).
[0146] C) Amino acids GIy164-Ser 171 [0147] First peptide linker
element of SEQ ID NO: 2.
[0148] D) Amino acids Glu172-Pro314 [0149] Second soluble cytokine
domain of the human CD27L ligand (CD27L, amino acids 51-193 of SEQ
ID NO: 1).
[0150] E) Amino acids GIy315-Ser322. [0151] Second peptide linker
element of SEQ ID NO: 2.
[0152] F) Amino acids Glu323-Pro465 [0153] Third soluble cytokine
domain of the human CD27L ligand (CD27L, amino acids 51-193 of SEQ
ID NO: 1).
[0154] G) Amino acids Gly466-Cys486 [0155] Hinge-linker element of
SEQ ID NO: 16.
[0156] H) Amino acids Pro487-Lys704 [0157] Antibody Fc fragment
domain of SEQ ID NO: 13.
[0158] The above CD27 receptor agonist protein is shown in SEQ ID
NO: 25.
[0159] The indicated linkers may be replaced by other preferred
linkers, e.g. as shown in SEQ ID NOs: 3-12.
[0160] The indicated Hinge-linker element may be replaced by other
preferred Hinge-linkers, e.g. as shown in SEQ ID NOs: 19-24.
[0161] It should be noted that the first and second peptide linkers
do not need to be identical.
[0162] The signal peptide sequence (A) may be replaced by any other
suitable, e.g. mammalian signal peptide sequence.
[0163] 1.2 Gene Cassette Encoding the Polypeptide
[0164] The synthetic gene may be optimized in view of its codon
usage for the expression in suitable host cells, e.g. insect cells
or mammalian cells. A preferred nucleic acid sequence is shown in
SEQ ID NO: 37.
Example 2
Expression and Purification
[0165] 2.1 Cloning, Expression and Purification of Fusion
Polypeptides
[0166] The aforementioned fusion proteins are expressed
recombinantly in two different eukaryotic host cells employing the
methods described below:
[0167] Method for Small Scale Expression of of CD27 Receptor
Agonist Fusion Proteins:
[0168] For initial analysis of aforementioned CD27 receptor agonist
fusion proteins, Hek293 cells grown in DMEM+GlutaMAX (GibCo)
supplemented with 10% FBS, 100 units/ml Penicillin and 100 [mu]g/ml
Streptomycin are transiently transfected with a plasmid containing
an expression cassette for a fusion polypeptide and an appropriate
selection marker, e.g. a functional expression cassette comprising
a blasticidine, puromycin or hygromycin resistence gene. In those
cases, where a plurality of polypeptide chains is necessary to
achieve the final product, the expression cassettes will be either
combined on one plasmid or positioned on different plasm ids during
the transfection. Cell culture supernatant containing recombinant
fusion polypeptide will be harvested three days post transfection
and clarified by centrifugation at 300.times.g followed by
filtration through a 0.22 .mu.m sterile filter.
[0169] Method for Large Scale Expression and Purification of CD27
Receptor Agonist Fusion Proteins
[0170] For larger scale expression of CD27 receptor agonist fusion
proteins to be used in vivo, synthetic DNA cassettes encoding the
aforementioned proteins is inserted into eukaryotic expression
vectors comprising appropriate selection markers (e.g. a functional
expression cassette comprising a blasticidin, puromycin or
hygromycin resistance gene) and genetic elements suitable to
enhance the number of transcriptionally active insertion sites
within the host cells genome. The sequence verified expression
vectors are introduced by electroporation into suspension adapted
Chinese Hamster Ovary cells (CHO-S, Invitrogen). Appropriate
selection pressure will be applied three days post-transfection to
the transfected cells. Surviving cells carrying the vector derived
resistance gene(s) are recovered by subsequent cultivation under
selection pressure. Upon stable growth of the selected cell pools
in chemically defined medium (PowerCHO2-CD, Lonza) at 37.degree. C.
and 7% CO2 atmosphere in an orbital shaker incubator (100 rpm, 50
mm shaking throw), the individual supernatants are analyzed by
ELISA-assays detecting the aforementioned proteins and the cell
pools with the highest specific productivity which were expanded in
shake flasks prior to protein production (orbital shaker, 100 rpm,
shaking throw 50 mm).
[0171] For lab-scale protein production, individual cell pools are
cultured for 7-12 days in chemically defined medium (PowerCHO2-CD,
Lonza) at 37.degree. C. and 7% CO2 atmosphere in a Wave bioreactor
20/50 EHT (GE-Healthcare). The basal medium is PowerCHO2-CD
supplemented with 4 mM Glutamax. Wave culture is started with a
viable cell concentration of 0.3 to 0.4.times.10e6 cells/ml and the
following settings (for a five- or ten liter bag): shaking
frequency 18 rpm, shaking ankle 7.degree., gas current 0.2-0.3
L/min, 7% CO2, 36.5.degree. C. During the Wave run, the cell
culture is fed twice with PowerFeed A (Lonza), usually on day 2
(20% feed) and day 5 (30% feed). After the second feed, shaking
frequency is increased to 22rpm, as well as the shaking ankle to
8.degree..
[0172] The bioreactor is usually harvested in between day 7 to day
12 when the cell viability drops below 80%. First, the culture
supernatant is clarified using a manual depth filtration system
(Millipore Millistak Pod, MCOHC 0.054 m.sup.2). For Strep-tagged
proteins, Avidin is added to a final concentration of 0.5 mg/L.
Finally, the culture supernatant containing the CD27 receptor
agonist fusion protein is sterile filtered using a bottle top
filter (0.22 .mu.m, PES, Corning) and stored at 2-8.degree. C.
until further processing.
[0173] For affinity purification Streptactin Sepharose is packed to
a column (gel bed 2 ml), equilibrated with 15 ml buffer W (100 mM
Tris-HCl, 150 mM NaCl, pH 8.0) or PBS pH 7.4 and the cell culture
supernatant is applied to the column with a flow rate of approx. 4
ml/min. Subsequently, the column is washed with 15 ml buffer W and
bound polypeptide is eluted stepwise by addition of 7.times.1 ml
buffer E (100 mM Tris HCl, 150 mM NaCl, 2.5 mM Desthiobiotin, pH
8.0). Alternately, PBS pH 7.4 containing 2.5 mM Desthiobiotin can
be used for this step.
[0174] Alternatively to the Streptactin Sepharose based method, the
affinity purification is performed employing a column with
immobilized Protein-A as affinity ligand and an Akta chromatography
system (GE-Healthcare). A solid phase material with high affinity
for the FC-domain of the fusion protein was chosen: MABSelect
Sure.TM. (GE Healthcare). Briefly, the clarified cell culture
supernatant is loaded on a HiTrap MabSelectSure column (CV=5 ml)
equilibrated in wash-buffer-1 (20 mM Pi, 95 mM NaCl, pH7.2) not
exceeding a load of 10 mg fusion protein per ml column-bed. The
column is washed with ten column-volumes (10CV) of aforementioned
equilibration buffer followed by four column-volumes (4CV) of
wash-buffer-2 (20 mM Pi, 95 mM NaCl, pH 8.0) to deplete host-cell
protein and host-cell DNA. The column is then eluted with elution
buffer (20 mM Pi, 95 mM NaCl, pH 3.5) and the eluate is collected
in up to ten fractions with each fraction having a volume equal to
column-bed volume (5 ml). Each fraction is neutralized with an
equal volume of aforementioned wash buffer 2. The linear velocity
is set to 150 cm/h and kept constant during the aforementioned
affinity chromatography method. The protein amount of the eluate
fractions is quantitated and peak fractions are concentrated by
ultrafiltration and further purified by size exclusion
chromatography (SEC).
[0175] Analytical size exclusion chromatography of PROTEIN A (SEQ
ID NO: 15) and the trivalent control protein PROTEIN X (SEQ ID NO:
38) is shown in FIG. 5. For preparation of the control protein
please refer to Example 4. The SEC was performed on a 1260 Infinity
HPLC system using a Tosoh TSKgelG3000SWxlcolumn. The column was
loaded with protein at a concentration of 0.6 mg/ml in a total
volume of 20 .mu.l. The flow rate was set to 0.5 ml/min. One
observes a single main peak at 16.39 min for PROTEIN A (FIG. 5:
Part B) and 18.91 min for PROTEIN X (FIG. 5 Part A). By using an
internal molecular weight standard (BioRad SEC Standard) one can
intrapolate the molecular weight of PROTEIN A and PROTEIN X from
respective retention times. Consequently, PROTEIN X has an apparent
molecular weight of 80.4 kDa and PROTEIN A shows a molecular weight
of 201.8 kDA. These values are in line with theoretically expected
values derived from the amino acid sequence.
[0176] Employing the aforementioned methods, recombinant CD27
receptor agonist fusion protein (PROTEIN-A, SEQ ID NO: 15) was
expressed in CHO-S cells and purified employing affinity
chromatography and subsequent SEC-based polishing.
[0177] The chromatogram of an analytical SEC of hexavalent
scCD27L-RBD-FC (PROTEIN-A, SEQ ID NO: 15) fusion protein is shown
in FIG. 5 (Part B). The chromatogram of an analytical SEC of
trivalent control protein (PROTEIN X SEQ ID NO: 38) is shown in
FIG. 5 (Part A).
[0178] For determination of the apparent molecular weight of
purified fusion polypeptide under native conditions a Superdex 200
column was loaded with standard proteins of known molecular weight.
Based on the elution volume of the standard proteins a calibration
curve was plotted and the apparent molecular weight of purified
fusion polypeptide was determined. The FC-domain comprising CD27
receptor agonist fusion proteins typically eluted from the
Superdex200 columns with an apparent molecular weight of approx.
160-180 kDa confirming the homodimerisation of the mature CD27
receptor agonist fusion polypeptide by the Fc domain.
Example 3
SDS-PAGE Results of Dimer Proteins Expressed from Protein A
[0179] To determine if the homodimer of Protein A is covalently
linked, sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) experiments were performed under reducing and
non-reducing conditions. The size of the main band under reducing
conditions is only about half of the size as observed under
non-reducing conditions. This indicates that the homodimer is
covalently linked by disulfide bridges (see also FIG. 7).
Example 4
Trivalent Control Protein
[0180] To compare the relative binding between hexavalent CD27
receptor agonist fusion proteins and the, trivalent CD27 stabilized
with bacteriophage RB69-FOLDON, PROTEIN X (SEQ ID NO: 38) was
expressed in CHO-S cells and purified as described in the former
section. The SEC-purified protein is served as control in the
following Examples. The sequence of PROTEIN X (SEQ ID NO: 38) is
shown in Table 7. Amino-acids 1-20 of PROTEIN X represent the
signal peptide and the mature proteins starts with amino acid
Glu51. This protein consists of three identical polypeptides each
comprising one soluble CD27L domain (E51-P193 of SEQ ID NO: 1);
this assembly stabilized by the trimerization domain of
bacteriophage RB69 fibritin fused with a flexible linker to the
C-terminus of CD27L.
TABLE-US-00008 TABLE 7 Trivalent control protein including a signal
peptide SEQ ID NO Sequence 38
METDTLLVFVLLVWVPAGNGESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSF (Protein X)
LHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSI
SLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRPGS
GSSGSSGSSGSGYIEDAPSDGKEYVRKDGAWVELPTASGPSSSSSSAWSHPQFEK
Example 5
CD27 Receptor Agonist Combined with TCR Activation Activates Murine
T-Cells
[0181] To assess the T cell activation capability of the CD27
receptor agonist protein, T cells are purified from mouse spleens
by negative selection using magnetic beads. Cells are labeled with
CFSE and incubated with or without varying amounts of the CD27
receptor agonist protein and combined with an anti-mouse CD3
antibody for 2-5 days at 37.degree. C. Data on CFSE dilution as a
means to measure cell division is acquired on a flow cytometer.
IFN.gamma. production is measured by an ELISA assay using cell
culture supernatants and an anti-mouse IFN.gamma. antibody for
capture.
[0182] One expects to observe a clear augmentation of IFN.gamma.
secretion by both CD4+ and CD8+ T cells when the CD27 receptor
agonist protein is present in the T cell cultures along with the
anti-mouse CD3 antibody. As well as higher IFN.gamma. production
one expects to see more T cells to be driven into cell cycle by
measuring CFSE dilution using flow cytometry. This would
demonstrate a co-stimulatory effect of the CD27 receptor agonist
protein in the context of T cell activation.
Example 6
In Vivo Binding of CD27 Receptor Agonist Protein to Mouse Immune
Cells and the Effect on Circulating Lymphocytes
[0183] To assess the binding of the CD27 receptor agonist protein
to immune cells in vivo, mice are treated with or without a single
i.v. injection of the CD27 receptor agonist protein at varying
concentrations. Animals are followed for up to 20 days and blood
samples are collected daily starting on the day of injection. Blood
samples are incubated with a fluorescent anti-human Fc antibody on
ice and red blood cells are subsequently lysed using red blood cell
lysis buffer. Samples are then analyzed on a flow cytometer. Total
lymphocytes from blood are identified based on their side and
forward scatter profile and stained populations from injected mice
are compared to cells from untreated control animals.
[0184] One expects the CD27 receptor agonist protein to bind to the
surface of circulating lymphocytes expressing the target receptor
CD27. The binding is likely to decline over time due to
target-mediated drug disposition, which potentially occurs via
internalization of agonist/receptor complexes.
[0185] In addition, the effect of the CD27 receptor agonist protein
on circulating lymphocyte populations is assessed. For that
purpose, blood samples from mice, which receive a single i.v.
injection of the CD27 receptor agonist protein at varying
concentrations are obtained daily over the course of 20 days
starting on the day of injection (see above). After red blood cell
lysis, cells are stained with fluorescent antibodies directed
against immune cell subsets such as B cells, CD4+ T cells, CD8+ T
cells or NK cells. Stained samples are analyzed on a flow
cytometer.
[0186] One expects to observe no significant changes in the number
of circulating immune cell subsets over the course of the treatment
period.
Example 7
CD27 Receptor Agonist Protein Enhances Antigen-Specific CD8+ T-Cell
Proliferation and Activation as well as Pentamer Staining on Mouse
Peripheral Blood Cells and Splenocytes
[0187] Mice are intravenously injected with 1-10 mg of chicken
ovalbumin in combination with varying amounts of the CD27 receptor
agonist protein. Anti-mouse CD27 and an irrelevant human IgG1
antibody are included as positive and negative controls,
respectively. The CD27 receptor agonist protein is co-injected with
ovalbumin on day 0 and an additional amount of CD27 receptor
agonist protein alone on day 1. Peripheral blood and spleen cells
are harvested on days 7-10. Splenocytes and whole-blood are used
for staining. After Fc-receptor blocking, cells are stained, at
room temperature for 30 min to 1 h, with fluorescently labelled H-2
Kb/SIINFEKL, a tetrameric complex of mouse MHC class I complexed
with the peptide T cell epitope from ovalbumin, and additionally
with fluorescent antibodies detecting mouse CD8 and mouse CD27.
Samples are subsequently treated with red blood cell lysis buffer
to eliminate red blood cells, washed and fixed. Cells are analyzed
on a flow cytometer counting the number of cells within the CD8+
and CD27+ T cell compartment, which recognize the SIINFEKL peptide
in the context of MHC class I and which are thus antigen
specific.
[0188] One would expect to observe a supplementary effect elicited
by the CD27 receptor agonist protein in a sense that the agonist
enhances the expansion of antigen-specific CD8+ T cells in the
context of an immune response. This would demonstrate a clear
co-stimulatory effect exerted by the CD27 receptor agonist
protein.
Example 8
Determination of the In Vitro Stability of CD27 Receptor Agonist
Proteins by Limited Protease Digestion
[0189] All CD27 receptor agonist proteins to be investigated will
be expressed and purified as hexavalent Fc-Fusion protein as
described in Example 1. The set will include CD27 receptor agonist
proteins comprising the N297S mutation [according to the EU
numbering system] in the CH2-domain and a hinge region that enables
the formation of three disulfide bridges and additionally lack the
upper hinge lysine [K223, according to the EU numbering system]
which is mutated to glycine [K223G]. In a limited protease
digestion assay, the aforementioned CD27 receptor agonist proteins
comprising the N297S mutation and the K223G mutation simultaneously
in context of a three-disulfide enabling hinge will be compared to
CD27 receptor agonist proteins comprising the N297S mutation but
have the K223 wildtype present either in the context of a two
disulfide or three disulfide enabling hinge region.
[0190] In addition, CD27 receptor agonist proteins with the second
linker element (iv) reduced to 4 amino-acids and the shortened
hinge element (vi) will be investigated (e.g. SEQ ID NO: 32 and
34). Both engineering strategies (N297S combined with K223G
mutation in context of a three-disulfide enabling hinge region) and
shortage of linker elements (iv and vi) have a potential impact on
the stability of the respective molecules.
[0191] The stability of different CD27 agonistic proteins of the
present invention can be addressed by limited protease digestion in
vitro. For this analysis, the aforementioned CD27 receptor agonist
proteins are incubated with low concentrations of proteases (e.g.
Trypsin, V8 protease) at different temperatures (e.g. 4.degree. C.,
25.degree. C., 37.degree. C.) for different amounts of time.
Quantification of specific proteolytic fragments and their
appearance over time can be subsequently measured by different
methods, like SDS-PAGE, analytical SEC or analytical
Mass-Spectrometry methods known in the art (e.g
Nano-RP-HPLC-ESI-MSMS). As the investigated proteins have most of
their sequences in common, the faster appearance and enlarged
quantities of specific proteolytic fragments from individual
proteins over time can then be used to judge their relative
stability and rank them to each other. With regard to
protease-based decoy kinetics of the aforementioned CD27 receptor
agonist proteins investigated, the following order regarding their
proteolytic stability is to be expected:
[0192] The CD27 receptor agonist proteins comprising the N297S and
the K223G and the three-disulfide enabling hinge region
simultaneously have a prolonged stability as compared to the CD27
receptor agonist proteins comprising the N297S and wildtype K223 in
the hinge region. The CD27 receptor agonist proteins comprising the
SEQ ID NO: 21 as hinge linker have a prolonged stability as
compared to CD27 receptor agonist proteins comprising the SEQ ID
NO: 16 as hinge linker element.
Example 9
Half-Life Determination
[0193] Molecule PROTEIN A is made up of two polypeptides covalently
linked by three interchain disulfide bonds and comprises the K223G
mutation in the hinge linker as well as the N297S mutation the Fc
region (according to the EU numbering), resulting in aglycosylation
of the CH2 domain. The purified PROTEIN-A was tested on the
half-life in mice.
[0194] Female CD1 mice were administered with 1.0 mg/kg of PROTEIN
A as a single intravenous bolus injection. Whole blood was
collected before application (pre-dose), and up to 312 hours after
test item administration. Serum was prepared and samples were
stored at -80.degree. C. until determination of serum
concentrations.
[0195] Quantitation of the PROTEIN A concentrations in mouse serum
was performed with an ELISA-assay detecting the CD27 agonist shown
in Table 8. Plates were coated with CD27-Fc. CD27-Ligand constructs
specifically binding to its receptor CD27 were then detected via
their Strep-Tag employing StrepTactin-HRP. ELISA assays were
carried out using reference PROTEIN A as calibration and control
samples. The measured data of the standard concentrations were used
to create calibration curves using a 5-parameter fit. This enabled
the determination of the unknown PROTEIN A concentrations in the
respective mouse serum samples.
[0196] Pharmacokinetic parameters were calculated using the mean
serum concentrations and the pharmacokinetic evaluation program PK
Solutions Version 2.0 for non-compartmental pharmacokinetic data
analysis (Summit Research Services, Montrose, Colo.). PK Solutions
is an automated, Excel-based application, which computes
pharmacokinetic parameters from concentration-time data obtained
from analysis of e.g. biological samples following intravenous or
extra-vascular routes of administration. PK Solutions calculates
results without presuming any specific compartmental model.
[0197] The results from the pharmacokinetics evaluation are
summarized in Table 8.
TABLE-US-00009 TABLE 8 Results of the exploratory PK study in
CD1-mice: single intravenous dose of 1 mg/kg of PROTEIN A. PROTEIN
A t.sub.max (h) 0.083 C.sub.max (.mu.g/ml) 9.63 t.sub.last (h) 24
C.sub.last (.mu.g/ml) 0.288 t.sub.1/2 E (h) 10.42 t.sub.1/2 E (d)
0.43 AUC.sub.0-t (.mu.g*h/ml) 33 AUC.sub.0-inf (.mu.g*h/ml) 38
[0198] The results show that PROTEIN A has a surprisingly short
terminal half-life of 10.42 hours in mice. This short half-life
constitutes a favorable therapeutic option since a short
co-stimulatory stimulus with CD27 receptor agonist proteins is
desirable.
Example 10
Stability/Aggregation Test
[0199] The contents of monomers and aggregates are determined by
analytical SEC as described in Example 2. For this particular
purpose the analysis is performed in buffers containing
physiological salt concentrations at physiological pH (e.g. 0.9%
NaCl, pH 7.4; PBS pH 7.4). A typical aggregation analysis is done
on a Superdex200 column (GE Healthcare). This column separates
proteins in the range between 10 to 800 kDa.
[0200] For determination of the apparent molecular weight of
purified fusion polypeptide under native conditions a Superdex 200
column is loaded with standard proteins of known molecular weight.
Based on the elution volume of the standard proteins a calibration
curve is plotted and the apparent molecular weight of purified
fusion proteins of unknown molecular weight is calculated based on
the elution volume.
[0201] SEC analysis of soluble, non-aggregated protein typically
shows a distinct single protein peak at a defined elution volume
(measured at OD at 280 nm or at OD 214 nm). This elution volume
corresponds to the apparent native molecular weight of the
particular protein. With regard to the definition of "monomer" in
the case of FC-fusion proteins, the assembly of two
polypeptide-chains is driven by the FC-part of the protein and the
functional unit is a protein consisting of two chains. This unit
that contains two FC-linked polypeptide chains is defined as
"monomer" in the case of Fc-fusion proteins regardless of being a
dimerized single-chain fusion polypeptide.
[0202] If protein aggregation occurs, the SEC analysis shows
additional protein peaks with lower retention volumes. Protein
oligomers potentially serve as aggregation seeds and a high content
of oligomers potentially leads to aggregation of the protein.
Oligomers of large molecular weight and aggregates elute in the
void volume of the Superdex200 column and cannot be analyzed by SEC
with respect to their native molecular weight.
[0203] Purified preparations of CD27 receptor agonist fusion
proteins should preferably contain only defined monomeric protein
and only a very low amount of oligomeric protein. The degree of
aggregation/oligomerization of a particular CD27 receptor agonist
fusion protein preparation is determined on basis of the SEC
analysis by calculating the peak areas of the OD280 diagram for the
defined monomer and the oligomer/aggregate fraction, respectively.
Based on the total peak area the percentage of defined monomer
protein is calculated as follows:
monomer content [%]=[Peak area monomer protein]/[Total peak
area].times.100)
Example 11
Determination of the Equilibrium Binding Constants for Tri-and
Hexavalent CD27 Receptor Ligand Constructs by QCM Analysis
[0204] The equilibrium binding constants (K.sub.D) of trivalent and
hexavalent PROTEIN X and PROTEIN A are calculated based on kinetic
binding data (k.sub.on and k.sub.off) that are determined with an
automated biosensor system (Attana A100). The A100 allows to
investigate molecular interactions in real-time based on the Quartz
Crystal Microbalance (QCM) technique.
[0205] For this purpose the human CD27 receptor is immobilized to
the surface of a carboxyl-activated QCM-chip. Subsequently the tri-
or hexavalent PROTEIN X or PROTEIN A, respectively, is used as an
analyte at different concentrations (e.g. 0.5, 1, 2, 5, and 10
.mu.g/ml) for analyzing the kinetic binding data for
ligand-receptor binding (k.sub.on) and dissociation (k.sub.off).
The analysis is done in real time and the respective K.sub.D can be
calculated: K.sub.D=k.sub.off/k.sub.on
[0206] The QCM analysis shows that the trivalent PROTEIN X binds to
the respective immobilized CD27 receptor with a K.sub.D in the low
nM-range with an expected K.sub.D of 1-100 nm. However, hexavalent
constructs of PROTEIN A show a higher binding affinity in the
pM-range towards the respective immobilized CD27 receptor with an
expected K.sub.D of 1-1000 pM. A common characteristic of the
kinetic binding data (k.sub.on and k.sub.off) is that the
hexavalent constructs show faster k.sub.on in comparison to the
trivalent constructs. In addition, slower dissociation (k.sub.off)
is commonly observed for the hexavalent ligands if compared to the
trivalent ligand.
Example 12
T Cell Proliferation Assay
[0207] Primary, human T cells were isolated from fresh buffy coat
preparations using negative selection and magnetic beads. Cells
were loaded with the dye CFSE and were seeded into 24-well plates
at 2.times.10e6 cells per well. T cells were incubated with an
anti-human CD3 antibody (clone HIT3a, 1 .mu.g/ml), anti-human CD28
antibody (clone CD28.2, 5 .mu.g/ml) and varying amounts of the
CD27L agonist (Protein A), 10-1000 ng/ml) or simply left in medium
as control. All cells were assessed for CFSE fluorescence on a
guava easyCyte flow cytometer after 6 days of incubation at
37.degree. C.
[0208] It was observed (Table 9) that cells only incubated with the
anti-CD3 and anti-CD28 antibodies loose CFSE fluorescence (GeoMean)
compared to the medium control indicating cell division thereby
diluting the CFSE dye. Importantly, this effect was even stronger
and concentration-dependent when cells were also incubated with the
CD27L agonist (Protein A). Using the GeoMean values one can derive
a percentage for cells driven into proliferation and it is clear
that cells incubated with the CD27L (Protein A) agonist
proliferated stronger than cells only being incubated with anti-CD3
and anti-CD28 antibodies or being left in medium alone.
TABLE-US-00010 TABLE 9 Protein A dependent T Cell Proliferation
Assay % of GeoMean proliferating (All Stimulation cells events)
Medium 0.39 559.94 a-CD3 + a-CD28 21.57 292.85 a-CD3 + a-CD28 +
CD27L 10 ng/ml 35.64 180.9 a-CD3 + a-CD28 + CD27L 100 ng/ml 44.87
140.92 a-CD3 + a-CD28 + CD27L 1000 ng/ml 50.03 137.17
Example 13
CD27 Agonist Binding Assay
[0209] Primary, human T cells were isolated from fresh buffy coat
preparations using negative selection and magnetic beads. Cells
were seeded into 24-well plates at 2.times.10e6 cells per well. T
cells were incubated with an anti-human CD3 antibody (clone HIT3a,
1 .mu.g/ml), anti-human CD28 antibody (clone CD28.2, 5 .mu.g/ml)
and varying amounts of Protein A (CD27L, 10-1000 ng/ml) or simply
left in medium as control. After 3 days at 37.degree. C. cells were
fluorescently labeled with anti-human CD27 and anti-human CD4 or
anti-human CD8 antibodies. CD27 fluorescence was assessed on a
guava easyCyte flow cytometer within CD4+ and CD8+ T cell
populations.
[0210] When comparing (Table 10) T cells incubated with anti-CD3
and anti-CD28 antibodies to control cells left in medium alone, one
observes a lower fluorescent signal for CD27 indicating an
activation-induced downregulation of the receptor. Importantly,
this effect was even stronger and dose-dependent, when cells were
co-incubated with the CD27 agonist (Protein A), which indicates a
supplementary effect caused by the CD27 agonist (Protein A). As the
agonist mimics the receptor-binding domain of the natural CD27
ligand (CD70), it is likely that the lower surface expression of
CD27 is due to receptor internalization upon binding of the CD27
agonist (Protein A). These results clearly suggest a binding of the
CD27 agonist (Protein A) to its receptor in vitro.
TABLE-US-00011 TABLE 10 CD27 agonist binding assay % of CD2 % of
CD27 positive positive Stimulation cells CD4 cells CD8 Medium 70.04
70.14 a-CD3 + a-CD28 55.82 45.8 a-CD3 + a-CD28 + CD27L 1 ng/ml
51.98 38.41 a-CD3 + a-CD28 + CD27L 10 ng/ml 42.86 21.43 a-CD3 +
a-CD28 + CD27L 100 ng/ml 9.43 5.34
Example 14
Antitumor Efficacy of PROTEIN A in Subcutaneous Syngeneic Colon
Carcinoma MC38-CEA in Female C57Bl/6N Mice
[0211] Material and Methods
[0212] For the evaluation of the anti-tumor efficacy of PROTEIN A
in the subcutaneously implanted syngeneic colon carcinoma model
MC38-CEA, the study consisted in 3 experimental groups each
containing 12 female C57Bl/6N mice 5-6 weeks of age.
[0213] All animals were implanted subcutaneously with 1.times.106
MCE38-CEA tumor cells in PBS in to the left flank of the animals. 8
days after tumor implantation when primary tumors reach a volume of
24.5-106.25 mm3, 36 tumor bearing animals were randomized into 3
groups (n=12). On the same day, treatment with 10 ml/kg vehicle
control (PBS), 1 mg/kg and 10 mg/kg test compound PROTEIN A was
initiated. Animals of all groups were treated intravenously (i.v)
twice weekly on days 8, 12, 15 and 19. The study was terminated 24
hours (day 20) after last administration on day 19, animals
sacrificed and a necropsy performed. At necropsy, animals were
weight and anaesthetized by isoflurane. Blood samples were
collected via retro bulbar vein puncture for preparation of serum.
Thereafter, animals were killed by cervical dislocation, primary
tumors were collected and wet weights and tumor volumes determined.
Additionally, also spleens tissues were collected for analysis.
[0214] Results
[0215] The mean animal body weight of all study groups either
remain stable or slightly increased during the course of the study.
No major body weight losses could be observed.
[0216] PROTEIN A showed a dose-dependent inhibition of the primary
tumor growth. Whereas tumor growth inhibition of PROTEIN A at dose
of 1 mg/kg (25.8%) was noticeable although not statistically
significant. APG 1293 at dose of 10 mg/kg produced a statistically
significant tumor growth inhibition (48.2%) as measured in vivo on
day 20. During necropsy, primary tumors were excised and tumor
volumes and wet tumor weights determined. Wet tumor weight of high
dose group (10 mg/kg) was significantly (P=0,0295) reduced compared
to vehicle (FIG. 9). And tumor volume of high dose group was also
significantly reduced compared to vehicle group. (FIG. 10).
[0217] Conclusion
[0218] PROTEIN A showed an in vivo dose-dependent anti-tumoral
efficacy response in the subcutaneous syngeneic MC38-CEA colon
carcinoma model in female C57Bl/6N mice.
Example 15
Antitumor Efficacy of PROTEIN A in Subcutaneous Syngeneic Colon
Carcinoma CT26 in Female BALB/c Mice
[0219] Material and Methods
[0220] The anti-tumor efficacy of PROTEIN A was evaluated in a
subcutaneously implanted syngeneic colon carcinoma CT26 in female
BALB/c mice, the study consisted in 3 experimental groups each
containing 10 females 5-6 weeks of age BALB/c mice. All animals
were implanted subcutaneously with 5.0.times.105 CT26 tumor cells
in cell culture media (RPMI w/o Phenol red) in to the right flank
of the animals. On day 0 (11 days after tumor implantation), when
primary tumors reach a volume of 23.5 mm3 to 132.7 mm3, 30 tumor
bearing animals were randomized into 3 groups (n=10). On the same
day, treatment with 10 ml/kg vehicle control (Group 1, PBS), 1
mg/kg (Group 2) and 10 mg/kg (Group3) test compound PROTEIN A was
initiated. Animals of all groups were treated intravenously (i.v)
twice weekly on days 11 (day 0), 15 (day 4) and 18 (day 7). The
study was terminated on day 21 (day 10) 72 hours after last
administration on day 18, animals sacrificed and a necropsy
performed. At necropsy, animals were weight and anaesthetized by
isoflurane. Blood samples were collected for preparation of serum.
Thereafter, animals were killed by cervical dislocation, primary
tumors were collected and wet weights determined. Additionally,
also spleens tissues were collected for analysis.
[0221] Results
[0222] The mean animal body weight of all study groups either
remain stable or slightly increased during the course of the study
(FIG. 1). It was not influenced by treatment with PROTEIN A.
[0223] PROTEIN A induced tumor growth inhibition compared to
vehicle control (PBS) in a subcutaneously implanted syngeneic colon
carcinoma CT26 model in female BALB/c at any dose tested (1 mg/kg
and 10 mg/kg). The effect of treatment with 1 mg/kg and 10 mg/kg
PROTEIN A on estimated tumor volume was comparable and significant
after 2nd PROTEIN A administration day 15 (day 4) onwards. PROTEIN
A at dose of 1 mg/kg and 10 mg/kg produced a statistically
significant tumor growth inhibition effect 84.7% (P<0.001) and
73.1% (P<0.001) respectively as measured in vivo on day 20.
[0224] During necropsy, primary tumors were excised and tumor
volumes and wet tumor weights determined. Wet tumor weight of low
dose group (1 mg/kg) was significantly (P=0,0175) reduced compared
to vehicle (FIG. 12). And tumor volume of high and low dose groupe
were also significantly reduced (P=0,0005 and P=0,0002) compared to
vehicle group. (FIG. 11).
[0225] Conclusion
[0226] PROTEIN A showed a highly significant tumor growth
inhibition effect compared to vehicle control (PBS) in a
subcutaneously implanted syngeneic colon carcinoma CT26 model in
female BALB/c at the tested doses of 1 mg/kg and 10 mg/kg.
Example 16
CD27 Receptor Agonist Protein Enhances Murine Antigen-Specific CD8
Positive T Cell Clonal Expansion In Vivo
[0227] T cells were isolated from the spleens and lymph nodes of
"donor" OT-1 mice using a gentleMACS Octo Dissociator (Miltenyi
Biotec). Cells were resuspended in PBS and injected intravenously
in "recipient" C57Bl/6 mice. One day later ("day 0"), mice were
injected intraperitoneally with 5 mg of chicken ovalbumin (OVA
protein) and intravenously with hexavalent Protein A (0.1, 1 or 10
mg/kg), trimeric CD27 ligand (Protein X) (10 mg/kg) or vehicle
control. At various time points, serial blood collection was
performed. Spleens were also collected at the final time point.
[0228] Blood and spleen samples were lysed and stained with
specific antibodies and Kb/OVA tetramer (H-2 Kb/SIINFEKL--specific
for OT-1 cells, Biozol--MBL) and analyzed by flow cytometry with a
BD Biosciences FACSCelesta BVR12. The Kb/OVA tetramer is a complex
of mouse MHC class I plus the OVA peptide that binds specifically
to the T cell receptor (TCR) of CD8 positive OT-1 T cells as well
as any other OVA-specific CD8 positive T cells. Data analysis was
performed with FlowJo 10.1 software (FlowJo, LLC). A minimum of ten
thousand CD8+ T cells were recorded and examined per sample and
there were three replicate animals per group. The percentage of
Kb/OVA tetramer positive cells (OT-1 cells) as a percentage of
total CD8 positive cells (plus average deviation) is presented in
Table 11. As one would expect, the hexavalent CD27 receptor agonist
but not the trimeric agonist enhanced the antigen-specific clonal
expansion of the CD8 positive OT-1 T cells. This demonstrates a
clear co-stimulatory effect exerted by the CD27 receptor agonist
protein.
TABLE-US-00012 TABLE 11 OVA-specific CD8 positive OT-1 T cell
clonal expansion following treatment with Protein A Treatment (n =
3 per group) Time of blood Protein Protein Protein A Trimeric
sampling A A 0.1 ligand post treatment PBS 10 mg/kg 1 mg/kg mg/kg
10 mg/kg OT-1 as a percent of total CD8 + T cells-average
(deviation) Day 06 1% (0%) 30% (4%) 31% (3%) 4% (2%) 1% (0%) Day 09
1% (0%) 10% (1%) 5% (1%) 2% (1%) 1% (0%) Day 13 1% (1%) 6% (1%) 4%
(1%) 2% (3%) 1% (0%)
Example 17
Half-Life of the CD27 Receptor Agonist is Correlated to the Total
Number of N-Linked Carbohydrates
[0229] Molecule PROTEIN A is made up of two polypeptides covalently
linked by three interchain disulfide bonds and comprises the K223G
mutation in the hinge linker as well as the N297S mutation the Fc
region (according to the EU numbering), resulting in a
glycosylation of the CH2 domain. PROTEIN B has the same Fc-domain
layout like PROTEIN A, but with linker element (iv) shorter and
lacking N-linked glycosylation consensus site. PROTEIN-B is
represented by SEQ ID NO: 47, but carries a C-terminal Streptag.
PROTEIN C has the same layout as PROTEIN B but comprising in each
of the soluble CD27L domains (i), (iii) and (v) the N63D mutation.
PROTEIN-C is represented by SEQ ID NO: 43. PROTEIN D has the same
layout as PROTEIN C comprising in each of the soluble CD27L domains
(i), (iii) and (v) the N63D mutation, but with the N-terminal
shortened soluble CD27L domains. PROTEIN-D is represented by SEQ ID
NO: 45.
[0230] Therefore, as the mature proteins consists of two covalently
linked polypeptides PROTEIN A comprises 16 N-linked carbohydrates,
PROTEIN B comprises 14 N-linked carbohydrates and PROTEIN C and
PROTEIN D both comprise 8 N-linked carbohydrates in total. The
purified PROTEIN-A, -B, -C and -D were tested regarding their
half-life in mice.
[0231] Female CD1 mice were administered with 10 mg/kg of PROTEIN A
or -B or -C or -D as a single intravenous bolus injection. Whole
blood was collected before application (pre-dose), and up to 312
hours after test item administration. Serum was prepared and
samples were stored at -80.degree. C. until determination of serum
concentrations.
[0232] Quantitation of the PROTEIN N-B/-C or -D concentrations in
mouse serum was performed with an ELISA-assay detecting the CD27
agonists shown in table 8. Plates were coated with CD27-Fc.
CD27-Ligand constructs specifically binding to its receptor CD27
were then detected via their Strep-Tag employing StrepTactin-HRP.
ELISA assays were carried out using reference PROTEIN A, -B, -C or
-D as calibration and control samples. The measured data of the
standard concentrations were used to create calibration curves
using a 5-parameter fit. This enabled the determination of the
unknown PROTEIN A, -B, -C or -D concentrations in the respective
mouse serum samples.
[0233] Pharmacokinetic parameters were calculated using the mean
serum concentrations and the pharmacokinetic evaluation program PK
Solutions Version 2.0 for non-compartmental pharmacokinetic data
analysis (Summit Research Services, Montrose, Colo.). PK Solutions
is an automated, Excel-based application, which computes
pharmacokinetic parameters from concentration-time data obtained
from analysis of e.g. biological samples following intravenous or
extra-vascular routes of administration. PK Solutions calculates
results without presuming any specific compartmental model.
[0234] The results from the pharmacokinetics evaluation are
summarized in Table 12.
TABLE-US-00013 TABLE 12 Results of the exploratory PK study in
CD1-mice: single intravenous dose of 10 mg/kg of PROTEIN A, -B -C
and -D. PROTEIN A PROTEIN B PROTEIN C PROTEIN D (16 N-linked (14
N-linked (8 N-linked (8 N-linked carbo- carbo- carbo- carbo-
hydrates) hydrates) hydrates) hydrates) t.sub.max (h) 0.083 0.083
0.083 0.083 C.sub.max 150 158 125 149 (.mu.g/ml) AUC.sub.0-t 557
190.1 191.4 203.9 (.mu.g*h/ml) AUC.sub.0-inf 576.8 201.9 220.8
243.8 (.mu.g*h/ml) Vd (ml/kg) 350.8 1904.5 1741.5 1847.6 Cl (ml/h)
16.755 49.526 45.294 41.022 t.sub.1/2 E (h) 14.5 20.3 26.6 31.2
[0235] The results show that PROTEIN A, -B, -C and -D have
different half lifes of 14.5, 20.3 26.6 and 31.2 hours in mice. The
half-life is inversely correlated to the total number of N-linked
carbohydrates. The CD27 receptor agonist (PROTEIN D) with 8
N-linked carbohydrates but comprising the N-terminal shortened
CD27L domains (i), (iii) and (v) confirms the data obtained with
PROTEIN C. The short half-lifes observed constitute a favorable
therapeutic option since a short co-stimulatory stimulus with CD27
receptor agonist proteins is desirable.
Sequence CWU 1
1
471193PRTHomo sapiensCD27 ligand (wt) 1Met Pro Glu Glu Gly Ser Gly
Cys Ser Val Arg Arg Arg Pro Tyr Gly1 5 10 15Cys Val Leu Arg Ala Ala
Leu Val Pro Leu Val Ala Gly Leu Val Ile 20 25 30Cys Leu Val Val Cys
Ile Gln Arg Phe Ala Gln Ala Gln Gln Gln Leu 35 40 45Pro Leu Glu Ser
Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His 50 55 60Thr Gly Pro
Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala65 70 75 80Leu
Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu 85 90
95Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu
100 105 110Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr
Thr Leu 115 120 125Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile
Ser Leu Leu Arg 130 135 140Leu Ser Phe His Gln Gly Cys Thr Ile Ala
Ser Gln Arg Leu Thr Pro145 150 155 160Leu Ala Arg Gly Asp Thr Leu
Cys Thr Asn Leu Thr Gly Thr Leu Leu 165 170 175Pro Ser Arg Asn Thr
Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg 180 185
190Pro28PRTArtificial Sequencelinker 2Gly Ser Gly Ser Gly Asn Gly
Ser1 538PRTArtificial Sequencelinker 3Gly Ser Gly Ser Gly Ser Gly
Ser1 548PRTArtificial Sequencelinker 4Gly Gly Ser Gly Ser Gly Ser
Gly1 556PRTArtificial Sequencelinker 5Gly Gly Ser Gly Ser Gly1
564PRTArtificial Sequencelinker 6Gly Gly Ser Gly178PRTArtificial
Sequencelinker 7Gly Gly Ser Gly Asn Gly Ser Gly1 588PRTArtificial
Sequencelinker 8Gly Gly Asn Gly Ser Gly Ser Gly1 596PRTArtificial
Sequencelinker 9Gly Gly Asn Gly Ser Gly1 5106PRTArtificial
Sequencelinker 10Gly Ser Gly Ser Gly Ser1 5114PRTArtificial
Sequencelinker 11Gly Ser Gly Ser1123PRTArtificial Sequencelinker
12Gly Ser Gly113218PRTArtificial Sequencehuman IGG1 Fc N297S 13Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro1 5 10
15Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
20 25 30Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp 35 40 45Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 50 55 60Glu Gln Tyr Ser Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu65 70 75 80His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 85 90 95Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 100 105 110Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 115 120 125Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 130 135 140Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn145 150 155 160Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 165 170
175Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
180 185 190Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 195 200 205Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210
21514217PRTArtificial Sequencehuman IGG1 Fc (wt) 14Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 85 90 95Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200
205Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 21515718PRTArtificial
SequencePROTEIN A (CD27L fused to deglyco Fc) 15Met Glu Thr Asp Thr
Leu Leu Val Phe Val Leu Leu Val Trp Val Pro1 5 10 15Ala Gly Asn Gly
Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu 20 25 30Asn His Thr
Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly 35 40 45Pro Ala
Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly 50 55 60Gln
Leu Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val65 70 75
80Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr
85 90 95Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser
Leu 100 105 110Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser
Gln Arg Leu 115 120 125Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr
Asn Leu Thr Gly Thr 130 135 140Leu Leu Pro Ser Arg Asn Thr Asp Glu
Thr Phe Phe Gly Val Gln Trp145 150 155 160Val Arg Pro Gly Ser Gly
Ser Gly Asn Gly Ser Glu Ser Leu Gly Trp 165 170 175Asp Val Ala Glu
Leu Gln Leu Asn His Thr Gly Pro Gln Gln Asp Pro 180 185 190Arg Leu
Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His 195 200
205Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile
210 215 220Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser Ser
Thr Thr225 230 235 240Ala Ser Arg His His Pro Thr Thr Leu Ala Val
Gly Ile Cys Ser Pro 245 250 255Ala Ser Arg Ser Ile Ser Leu Leu Arg
Leu Ser Phe His Gln Gly Cys 260 265 270Thr Ile Ala Ser Gln Arg Leu
Thr Pro Leu Ala Arg Gly Asp Thr Leu 275 280 285Cys Thr Asn Leu Thr
Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu 290 295 300Thr Phe Phe
Gly Val Gln Trp Val Arg Pro Gly Ser Gly Ser Gly Asn305 310 315
320Gly Ser Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His
325 330 335Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly
Pro Ala 340 345 350Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp
Lys Gly Gln Leu 355 360 365Arg Ile His Arg Asp Gly Ile Tyr Met Val
His Ile Gln Val Thr Leu 370 375 380Ala Ile Cys Ser Ser Thr Thr Ala
Ser Arg His His Pro Thr Thr Leu385 390 395 400Ala Val Gly Ile Cys
Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg 405 410 415Leu Ser Phe
His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro 420 425 430Leu
Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu 435 440
445Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg
450 455 460Pro Gly Ser Ser Ser Ser Ser Ser Ser Ser Gly Ser Cys Asp
Lys Thr465 470 475 480His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser 485 490 495Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 500 505 510Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 515 520 525Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 530 535 540Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Ser Ser Thr Tyr Arg Val Val545 550 555
560Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
565 570 575Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr 580 585 590Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 595 600 605Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys 610 615 620Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser625 630 635 640Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 645 650 655Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 660 665 670Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 675 680
685Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser
690 695 700Ser Ser Ser Ser Ser Ala Trp Ser His Pro Gln Phe Glu
Lys705 710 7151621PRTArtificial Sequencehinge linker 16Gly Ser Ser
Ser Ser Ser Ser Ser Ser Gly Ser Cys Asp Lys Thr His1 5 10 15Thr Cys
Pro Pro Cys 201720PRTArtificial Sequenceuniversal signal peptide
17Met Glu Thr Asp Thr Leu Leu Val Phe Val Leu Leu Val Trp Val Pro1
5 10 15Ala Gly Asn Gly 201815PRTArtificial Sequenceserine linker
with strep tag 18Ser Ser Ser Ser Ser Ser Ala Trp Ser His Pro Gln
Phe Glu Lys1 5 10 151920PRTArtificial Sequencehinge linker 19Gly
Ser Ser Ser Ser Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr1 5 10
15Cys Pro Pro Cys 202019PRTArtificial Sequencehinge linker 20Gly
Ser Ser Ser Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys1 5 10
15Pro Pro Cys2118PRTArtificial Sequencehinge linker 21Gly Ser Ser
Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro1 5 10 15Pro
Cys2216PRTArtificial Sequencehinge linker 22Gly Ser Ser Ser Gly Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys1 5 10 152318PRTArtificial
Sequencehinge linker 23Gly Ser Ser Ser Gly Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys1 5 10 15Gly Ser2420PRTArtificial Sequencehinge
linker 24Gly Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys1 5 10 15Gly Ser Gly Ser 2025704PRTArtificial
SequencePROTEIN A - no strep tag 25Met Glu Thr Asp Thr Leu Leu Val
Phe Val Leu Leu Val Trp Val Pro1 5 10 15Ala Gly Asn Gly Glu Ser Leu
Gly Trp Asp Val Ala Glu Leu Gln Leu 20 25 30Asn His Thr Gly Pro Gln
Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly 35 40 45Pro Ala Leu Gly Arg
Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly 50 55 60Gln Leu Arg Ile
His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val65 70 75 80Thr Leu
Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr 85 90 95Thr
Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu 100 105
110Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu
115 120 125Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr
Gly Thr 130 135 140Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe
Gly Val Gln Trp145 150 155 160Val Arg Pro Gly Ser Gly Ser Gly Asn
Gly Ser Glu Ser Leu Gly Trp 165 170 175Asp Val Ala Glu Leu Gln Leu
Asn His Thr Gly Pro Gln Gln Asp Pro 180 185 190Arg Leu Tyr Trp Gln
Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His 195 200 205Gly Pro Glu
Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile 210 215 220Tyr
Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr Thr225 230
235 240Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly Ile Cys Ser
Pro 245 250 255Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe His
Gln Gly Cys 260 265 270Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala
Arg Gly Asp Thr Leu 275 280 285Cys Thr Asn Leu Thr Gly Thr Leu Leu
Pro Ser Arg Asn Thr Asp Glu 290 295 300Thr Phe Phe Gly Val Gln Trp
Val Arg Pro Gly Ser Gly Ser Gly Asn305 310 315 320Gly Ser Glu Ser
Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His 325 330 335Thr Gly
Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala 340 345
350Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu
355 360 365Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val
Thr Leu 370 375 380Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His
Pro Thr Thr Leu385 390 395 400Ala Val Gly Ile Cys Ser Pro Ala Ser
Arg Ser Ile Ser Leu Leu Arg 405 410 415Leu Ser Phe His Gln Gly Cys
Thr Ile Ala Ser Gln Arg Leu Thr Pro 420 425 430Leu Ala Arg Gly Asp
Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu 435 440 445Pro Ser Arg
Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg 450 455 460Pro
Gly Ser Ser Ser Ser Ser Ser Ser Ser Gly Ser Cys Asp Lys Thr465 470
475 480His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser 485 490 495Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg 500 505 510Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro 515 520 525Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 530 535 540Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ser Ser Thr Tyr Arg Val Val545 550 555 560Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 565 570 575Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 580 585
590Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
595 600 605Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys 610 615 620Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser625 630 635 640Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp 645 650 655Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 660 665 670Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 675 680 685Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 690 695
70026703PRTArtificial SequenceCD27L-wt fused to SEQ_14 26Met Glu
Thr Asp Thr Leu Leu Val Phe Val Leu Leu Val Trp Val Pro1 5 10 15Ala
Gly Asn Gly Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu 20 25
30Asn His Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly
35 40 45Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys
Gly 50 55
60Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val65
70 75 80Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro
Thr 85 90 95Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile
Ser Leu 100 105 110Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala
Ser Gln Arg Leu 115 120 125Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys
Thr Asn Leu Thr Gly Thr 130 135 140Leu Leu Pro Ser Arg Asn Thr Asp
Glu Thr Phe Phe Gly Val Gln Trp145 150 155 160Val Arg Pro Gly Ser
Gly Ser Gly Asn Gly Ser Glu Ser Leu Gly Trp 165 170 175Asp Val Ala
Glu Leu Gln Leu Asn His Thr Gly Pro Gln Gln Asp Pro 180 185 190Arg
Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His 195 200
205Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile
210 215 220Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser Ser
Thr Thr225 230 235 240Ala Ser Arg His His Pro Thr Thr Leu Ala Val
Gly Ile Cys Ser Pro 245 250 255Ala Ser Arg Ser Ile Ser Leu Leu Arg
Leu Ser Phe His Gln Gly Cys 260 265 270Thr Ile Ala Ser Gln Arg Leu
Thr Pro Leu Ala Arg Gly Asp Thr Leu 275 280 285Cys Thr Asn Leu Thr
Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu 290 295 300Thr Phe Phe
Gly Val Gln Trp Val Arg Pro Gly Ser Gly Ser Gly Asn305 310 315
320Gly Ser Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His
325 330 335Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly
Pro Ala 340 345 350Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp
Lys Gly Gln Leu 355 360 365Arg Ile His Arg Asp Gly Ile Tyr Met Val
His Ile Gln Val Thr Leu 370 375 380Ala Ile Cys Ser Ser Thr Thr Ala
Ser Arg His His Pro Thr Thr Leu385 390 395 400Ala Val Gly Ile Cys
Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg 405 410 415Leu Ser Phe
His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro 420 425 430Leu
Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu 435 440
445Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg
450 455 460Pro Gly Ser Ser Ser Ser Ser Ser Ser Ser Gly Ser Cys Asp
Lys Thr465 470 475 480His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly Pro Ser Val 485 490 495Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 500 505 510Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu 515 520 525Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 530 535 540Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser545 550 555
560Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
565 570 575Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
Thr Ile 580 585 590Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 595 600 605Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu 610 615 620Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn625 630 635 640Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 645 650 655Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 660 665 670Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 675 680
685His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 690
695 70027684PRTArtificial SequenceCD27L-wt fused to SEQ_13 (no
signal peptide, no strep tag) 27Glu Ser Leu Gly Trp Asp Val Ala Glu
Leu Gln Leu Asn His Thr Gly1 5 10 15Pro Gln Gln Asp Pro Arg Leu Tyr
Trp Gln Gly Gly Pro Ala Leu Gly 20 25 30Arg Ser Phe Leu His Gly Pro
Glu Leu Asp Lys Gly Gln Leu Arg Ile 35 40 45His Arg Asp Gly Ile Tyr
Met Val His Ile Gln Val Thr Leu Ala Ile 50 55 60Cys Ser Ser Thr Thr
Ala Ser Arg His His Pro Thr Thr Leu Ala Val65 70 75 80Gly Ile Cys
Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser 85 90 95Phe His
Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala 100 105
110Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser
115 120 125Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg
Pro Gly 130 135 140Ser Gly Ser Gly Asn Gly Ser Glu Ser Leu Gly Trp
Asp Val Ala Glu145 150 155 160Leu Gln Leu Asn His Thr Gly Pro Gln
Gln Asp Pro Arg Leu Tyr Trp 165 170 175Gln Gly Gly Pro Ala Leu Gly
Arg Ser Phe Leu His Gly Pro Glu Leu 180 185 190Asp Lys Gly Gln Leu
Arg Ile His Arg Asp Gly Ile Tyr Met Val His 195 200 205Ile Gln Val
Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His 210 215 220His
Pro Thr Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser225 230
235 240Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala
Ser 245 250 255Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys
Thr Asn Leu 260 265 270Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp
Glu Thr Phe Phe Gly 275 280 285Val Gln Trp Val Arg Pro Gly Ser Gly
Ser Gly Asn Gly Ser Glu Ser 290 295 300Leu Gly Trp Asp Val Ala Glu
Leu Gln Leu Asn His Thr Gly Pro Gln305 310 315 320Gln Asp Pro Arg
Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser 325 330 335Phe Leu
His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg 340 345
350Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser
355 360 365Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val
Gly Ile 370 375 380Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg
Leu Ser Phe His385 390 395 400Gln Gly Cys Thr Ile Ala Ser Gln Arg
Leu Thr Pro Leu Ala Arg Gly 405 410 415Asp Thr Leu Cys Thr Asn Leu
Thr Gly Thr Leu Leu Pro Ser Arg Asn 420 425 430Thr Asp Glu Thr Phe
Phe Gly Val Gln Trp Val Arg Pro Gly Ser Ser 435 440 445Ser Ser Ser
Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro 450 455 460Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe465 470
475 480Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val 485 490 495Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe 500 505 510Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro 515 520 525Arg Glu Glu Gln Tyr Ser Ser Thr Tyr
Arg Val Val Ser Val Leu Thr 530 535 540Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val545 550 555 560Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 565 570 575Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 580 585
590Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
595 600 605Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro 610 615 620Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser625 630 635 640Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln 645 650 655Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His 660 665 670Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 675 68028698PRTArtificial
SequenceCD27L-wt fused to SEQ_13 (incl. strep tag) 28Glu Ser Leu
Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His Thr Gly1 5 10 15Pro Gln
Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly 20 25 30Arg
Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile 35 40
45His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile
50 55 60Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala
Val65 70 75 80Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu
Arg Leu Ser 85 90 95Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu
Thr Pro Leu Ala 100 105 110Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr
Gly Thr Leu Leu Pro Ser 115 120 125Arg Asn Thr Asp Glu Thr Phe Phe
Gly Val Gln Trp Val Arg Pro Gly 130 135 140Ser Gly Ser Gly Asn Gly
Ser Glu Ser Leu Gly Trp Asp Val Ala Glu145 150 155 160Leu Gln Leu
Asn His Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp 165 170 175Gln
Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu 180 185
190Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met Val His
195 200 205Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser
Arg His 210 215 220His Pro Thr Thr Leu Ala Val Gly Ile Cys Ser Pro
Ala Ser Arg Ser225 230 235 240Ile Ser Leu Leu Arg Leu Ser Phe His
Gln Gly Cys Thr Ile Ala Ser 245 250 255Gln Arg Leu Thr Pro Leu Ala
Arg Gly Asp Thr Leu Cys Thr Asn Leu 260 265 270Thr Gly Thr Leu Leu
Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly 275 280 285Val Gln Trp
Val Arg Pro Gly Ser Gly Ser Gly Asn Gly Ser Glu Ser 290 295 300Leu
Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His Thr Gly Pro Gln305 310
315 320Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg
Ser 325 330 335Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg
Ile His Arg 340 345 350Asp Gly Ile Tyr Met Val His Ile Gln Val Thr
Leu Ala Ile Cys Ser 355 360 365Ser Thr Thr Ala Ser Arg His His Pro
Thr Thr Leu Ala Val Gly Ile 370 375 380Cys Ser Pro Ala Ser Arg Ser
Ile Ser Leu Leu Arg Leu Ser Phe His385 390 395 400Gln Gly Cys Thr
Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly 405 410 415Asp Thr
Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn 420 425
430Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly Ser Ser
435 440 445Ser Ser Ser Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr
Cys Pro 450 455 460Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe465 470 475 480Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val 485 490 495Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe 500 505 510Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 515 520 525Arg Glu Glu
Gln Tyr Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr 530 535 540Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val545 550
555 560Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala 565 570 575Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg 580 585 590Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly 595 600 605Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro 610 615 620Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser625 630 635 640Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 645 650 655Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 660 665
670Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Ser Ser Ser Ser
675 680 685Ser Ala Trp Ser His Pro Gln Phe Glu Lys 690
69529683PRTArtificial SequenceCD27L-wt fused to SEQ_14 (no signal
peptide, no strep tag) 29Glu Ser Leu Gly Trp Asp Val Ala Glu Leu
Gln Leu Asn His Thr Gly1 5 10 15Pro Gln Gln Asp Pro Arg Leu Tyr Trp
Gln Gly Gly Pro Ala Leu Gly 20 25 30Arg Ser Phe Leu His Gly Pro Glu
Leu Asp Lys Gly Gln Leu Arg Ile 35 40 45His Arg Asp Gly Ile Tyr Met
Val His Ile Gln Val Thr Leu Ala Ile 50 55 60Cys Ser Ser Thr Thr Ala
Ser Arg His His Pro Thr Thr Leu Ala Val65 70 75 80Gly Ile Cys Ser
Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser 85 90 95Phe His Gln
Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala 100 105 110Arg
Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser 115 120
125Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly
130 135 140Ser Gly Ser Gly Asn Gly Ser Glu Ser Leu Gly Trp Asp Val
Ala Glu145 150 155 160Leu Gln Leu Asn His Thr Gly Pro Gln Gln Asp
Pro Arg Leu Tyr Trp 165 170 175Gln Gly Gly Pro Ala Leu Gly Arg Ser
Phe Leu His Gly Pro Glu Leu 180 185 190Asp Lys Gly Gln Leu Arg Ile
His Arg Asp Gly Ile Tyr Met Val His 195 200 205Ile Gln Val Thr Leu
Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His 210 215 220His Pro Thr
Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser225 230 235
240Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser
245 250 255Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr
Asn Leu 260 265 270Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu
Thr Phe Phe Gly 275 280 285Val Gln Trp Val Arg Pro Gly Ser Gly Ser
Gly Asn Gly Ser Glu Ser 290 295 300Leu Gly Trp Asp Val Ala Glu Leu
Gln Leu Asn His Thr Gly Pro Gln305 310 315 320Gln Asp Pro Arg Leu
Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser 325 330 335Phe Leu His
Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg 340 345 350Asp
Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser 355 360
365Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly Ile
370 375 380Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser
Phe His385 390 395 400Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr
Pro Leu Ala Arg Gly 405 410 415Asp Thr Leu Cys Thr Asn Leu
Thr Gly Thr Leu Leu Pro Ser Arg Asn 420 425 430Thr Asp Glu Thr Phe
Phe Gly Val Gln Trp Val Arg Pro Gly Ser Ser 435 440 445Ser Ser Ser
Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro 450 455 460Pro
Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro465 470
475 480Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr 485 490 495Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn 500 505 510Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg 515 520 525Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val 530 535 540Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser545 550 555 560Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 565 570 575Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 580 585
590Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
595 600 605Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu 610 615 620Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe625 630 635 640Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly 645 650 655Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr 660 665 670Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 675 68030684PRTArtificial SequenceSEQ_27
with AA exchange (E51Q of SEQ_1) 30Gln Ser Leu Gly Trp Asp Val Ala
Glu Leu Gln Leu Asn His Thr Gly1 5 10 15Pro Gln Gln Asp Pro Arg Leu
Tyr Trp Gln Gly Gly Pro Ala Leu Gly 20 25 30Arg Ser Phe Leu His Gly
Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile 35 40 45His Arg Asp Gly Ile
Tyr Met Val His Ile Gln Val Thr Leu Ala Ile 50 55 60Cys Ser Ser Thr
Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val65 70 75 80Gly Ile
Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser 85 90 95Phe
His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala 100 105
110Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser
115 120 125Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg
Pro Gly 130 135 140Ser Gly Ser Gly Asn Gly Ser Glu Ser Leu Gly Trp
Asp Val Ala Glu145 150 155 160Leu Gln Leu Asn His Thr Gly Pro Gln
Gln Asp Pro Arg Leu Tyr Trp 165 170 175Gln Gly Gly Pro Ala Leu Gly
Arg Ser Phe Leu His Gly Pro Glu Leu 180 185 190Asp Lys Gly Gln Leu
Arg Ile His Arg Asp Gly Ile Tyr Met Val His 195 200 205Ile Gln Val
Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His 210 215 220His
Pro Thr Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser225 230
235 240Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala
Ser 245 250 255Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys
Thr Asn Leu 260 265 270Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp
Glu Thr Phe Phe Gly 275 280 285Val Gln Trp Val Arg Pro Gly Ser Gly
Ser Gly Asn Gly Ser Glu Ser 290 295 300Leu Gly Trp Asp Val Ala Glu
Leu Gln Leu Asn His Thr Gly Pro Gln305 310 315 320Gln Asp Pro Arg
Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser 325 330 335Phe Leu
His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg 340 345
350Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser
355 360 365Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val
Gly Ile 370 375 380Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg
Leu Ser Phe His385 390 395 400Gln Gly Cys Thr Ile Ala Ser Gln Arg
Leu Thr Pro Leu Ala Arg Gly 405 410 415Asp Thr Leu Cys Thr Asn Leu
Thr Gly Thr Leu Leu Pro Ser Arg Asn 420 425 430Thr Asp Glu Thr Phe
Phe Gly Val Gln Trp Val Arg Pro Gly Ser Ser 435 440 445Ser Ser Ser
Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro 450 455 460Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe465 470
475 480Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val 485 490 495Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe 500 505 510Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro 515 520 525Arg Glu Glu Gln Tyr Ser Ser Thr Tyr
Arg Val Val Ser Val Leu Thr 530 535 540Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val545 550 555 560Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 565 570 575Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 580 585
590Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
595 600 605Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro 610 615 620Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser625 630 635 640Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln 645 650 655Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His 660 665 670Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 675 68031680PRTArtificial
SequenceSeq_30 with altered linker sequences 31Gln Ser Leu Gly Trp
Asp Val Ala Glu Leu Gln Leu Asn His Thr Gly1 5 10 15Pro Gln Gln Asp
Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly 20 25 30Arg Ser Phe
Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile 35 40 45His Arg
Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile 50 55 60Cys
Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val65 70 75
80Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser
85 90 95Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu
Ala 100 105 110Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu
Leu Pro Ser 115 120 125Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln
Trp Val Arg Pro Gly 130 135 140Ser Gly Ser Gly Asn Gly Ser Glu Ser
Leu Gly Trp Asp Val Ala Glu145 150 155 160Leu Gln Leu Asn His Thr
Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp 165 170 175Gln Gly Gly Pro
Ala Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu 180 185 190Asp Lys
Gly Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met Val His 195 200
205Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His
210 215 220His Pro Thr Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser
Arg Ser225 230 235 240Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly
Cys Thr Ile Ala Ser 245 250 255Gln Arg Leu Thr Pro Leu Ala Arg Gly
Asp Thr Leu Cys Thr Asn Leu 260 265 270Thr Gly Thr Leu Leu Pro Ser
Arg Asn Thr Asp Glu Thr Phe Phe Gly 275 280 285Val Gln Trp Val Arg
Pro Gly Ser Gly Ser Glu Ser Leu Gly Trp Asp 290 295 300Val Ala Glu
Leu Gln Leu Asn His Thr Gly Pro Gln Gln Asp Pro Arg305 310 315
320Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His Gly
325 330 335Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly
Ile Tyr 340 345 350Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser
Ser Thr Thr Ala 355 360 365Ser Arg His His Pro Thr Thr Leu Ala Val
Gly Ile Cys Ser Pro Ala 370 375 380Ser Arg Ser Ile Ser Leu Leu Arg
Leu Ser Phe His Gln Gly Cys Thr385 390 395 400Ile Ala Ser Gln Arg
Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys 405 410 415Thr Asn Leu
Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr 420 425 430Phe
Phe Gly Val Gln Trp Val Arg Pro Gly Ser Ser Ser Ser Ser Ser 435 440
445Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
450 455 460Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro465 470 475 480Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val 485 490 495Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val 500 505 510Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln 515 520 525Tyr Ser Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln 530 535 540Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala545 550 555
560Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
565 570 575Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr 580 585 590Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser 595 600 605Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr 610 615 620Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr625 630 635 640Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 645 650 655Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 660 665 670Ser
Leu Ser Leu Ser Pro Gly Lys 675 68032677PRTArtificial
SequenceSeq_31 with shorter hinge linker 32Gln Ser Leu Gly Trp Asp
Val Ala Glu Leu Gln Leu Asn His Thr Gly1 5 10 15Pro Gln Gln Asp Pro
Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly 20 25 30Arg Ser Phe Leu
His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile 35 40 45His Arg Asp
Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile 50 55 60Cys Ser
Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val65 70 75
80Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser
85 90 95Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu
Ala 100 105 110Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu
Leu Pro Ser 115 120 125Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln
Trp Val Arg Pro Gly 130 135 140Ser Gly Ser Gly Asn Gly Ser Glu Ser
Leu Gly Trp Asp Val Ala Glu145 150 155 160Leu Gln Leu Asn His Thr
Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp 165 170 175Gln Gly Gly Pro
Ala Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu 180 185 190Asp Lys
Gly Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met Val His 195 200
205Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His
210 215 220His Pro Thr Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser
Arg Ser225 230 235 240Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly
Cys Thr Ile Ala Ser 245 250 255Gln Arg Leu Thr Pro Leu Ala Arg Gly
Asp Thr Leu Cys Thr Asn Leu 260 265 270Thr Gly Thr Leu Leu Pro Ser
Arg Asn Thr Asp Glu Thr Phe Phe Gly 275 280 285Val Gln Trp Val Arg
Pro Gly Ser Gly Ser Glu Ser Leu Gly Trp Asp 290 295 300Val Ala Glu
Leu Gln Leu Asn His Thr Gly Pro Gln Gln Asp Pro Arg305 310 315
320Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His Gly
325 330 335Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly
Ile Tyr 340 345 350Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser
Ser Thr Thr Ala 355 360 365Ser Arg His His Pro Thr Thr Leu Ala Val
Gly Ile Cys Ser Pro Ala 370 375 380Ser Arg Ser Ile Ser Leu Leu Arg
Leu Ser Phe His Gln Gly Cys Thr385 390 395 400Ile Ala Ser Gln Arg
Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys 405 410 415Thr Asn Leu
Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr 420 425 430Phe
Phe Gly Val Gln Trp Val Arg Pro Gly Ser Ser Ser Ser Ser Gly 435 440
445Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
450 455 460Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr465 470 475 480Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 485 490 495Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 500 505 510Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Ser Ser 515 520 525Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 530 535 540Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala545 550 555
560Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
565 570 575Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 580 585 590Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala 595 600 605Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr 610 615 620Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu625 630 635 640Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 645 650 655Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 660 665 670Leu
Ser Pro Gly Lys 67533663PRTArtificial Sequenceexample with
N-terminal shortened RBD modules 33Gln Asp Val Ala Glu Leu Gln Leu
Asn His Thr Gly Pro Gln Gln Asp1 5 10 15Pro Arg Leu Tyr Trp Gln Gly
Gly Pro Ala Leu Gly Arg Ser Phe Leu 20 25 30His Gly Pro Glu Leu Asp
Lys Gly Gln Leu Arg Ile His Arg Asp Gly 35 40 45Ile Tyr Met Val His
Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr 50 55 60Thr Ala Ser Arg
His His Pro Thr Thr Leu Ala Val Gly Ile Cys Ser65 70 75 80Pro Ala
Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly 85 90 95Cys
Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr 100 105
110Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp
115 120 125Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly Ser Gly
Ser Gly
130 135 140Asn Gly Ser Asp Val Ala Glu Leu Gln Leu Asn His Thr Gly
Pro Gln145 150 155 160Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro
Ala Leu Gly Arg Ser 165 170 175Phe Leu His Gly Pro Glu Leu Asp Lys
Gly Gln Leu Arg Ile His Arg 180 185 190Asp Gly Ile Tyr Met Val His
Ile Gln Val Thr Leu Ala Ile Cys Ser 195 200 205Ser Thr Thr Ala Ser
Arg His His Pro Thr Thr Leu Ala Val Gly Ile 210 215 220Cys Ser Pro
Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe His225 230 235
240Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly
245 250 255Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser
Arg Asn 260 265 270Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg
Pro Gly Ser Gly 275 280 285Ser Asp Val Ala Glu Leu Gln Leu Asn His
Thr Gly Pro Gln Gln Asp 290 295 300Pro Arg Leu Tyr Trp Gln Gly Gly
Pro Ala Leu Gly Arg Ser Phe Leu305 310 315 320His Gly Pro Glu Leu
Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly 325 330 335Ile Tyr Met
Val His Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr 340 345 350Thr
Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly Ile Cys Ser 355 360
365Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly
370 375 380Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly
Asp Thr385 390 395 400Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro
Ser Arg Asn Thr Asp 405 410 415Glu Thr Phe Phe Gly Val Gln Trp Val
Arg Pro Gly Ser Ser Ser Ser 420 425 430Ser Gly Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro 435 440 445Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 450 455 460Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val465 470 475
480Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
485 490 495Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr 500 505 510Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 515 520 525Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu 530 535 540Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg545 550 555 560Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 565 570 575Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 580 585 590Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 595 600
605Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
610 615 620Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser625 630 635 640Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser 645 650 655Leu Ser Leu Ser Pro Gly Lys
66034681PRTArtificial Sequenceexample 34Gln Ser Leu Gly Trp Asp Val
Ala Glu Leu Gln Leu Asn His Thr Gly1 5 10 15Pro Gln Gln Asp Pro Arg
Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly 20 25 30Arg Ser Phe Leu His
Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile 35 40 45His Arg Asp Gly
Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile 50 55 60Cys Ser Ser
Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val65 70 75 80Gly
Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser 85 90
95Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala
100 105 110Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu
Pro Ser 115 120 125Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp
Val Arg Pro Gly 130 135 140Ser Gly Ser Gly Asn Gly Ser Glu Ser Leu
Gly Trp Asp Val Ala Glu145 150 155 160Leu Gln Leu Asn His Thr Gly
Pro Gln Gln Asp Pro Arg Leu Tyr Trp 165 170 175Gln Gly Gly Pro Ala
Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu 180 185 190Asp Lys Gly
Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met Val His 195 200 205Ile
Gln Val Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His 210 215
220His Pro Thr Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg
Ser225 230 235 240Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys
Thr Ile Ala Ser 245 250 255Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp
Thr Leu Cys Thr Asn Leu 260 265 270Thr Gly Thr Leu Leu Pro Ser Arg
Asn Thr Asp Glu Thr Phe Phe Gly 275 280 285Val Gln Trp Val Arg Pro
Gly Ser Gly Ser Gly Asn Gly Ser Glu Ser 290 295 300Leu Gly Trp Asp
Val Ala Glu Leu Gln Leu Asn His Thr Gly Pro Gln305 310 315 320Gln
Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser 325 330
335Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg
340 345 350Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile
Cys Ser 355 360 365Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu
Ala Val Gly Ile 370 375 380Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu
Leu Arg Leu Ser Phe His385 390 395 400Gln Gly Cys Thr Ile Ala Ser
Gln Arg Leu Thr Pro Leu Ala Arg Gly 405 410 415Asp Thr Leu Cys Thr
Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn 420 425 430Thr Asp Glu
Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly Ser Ser 435 440 445Ser
Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 450 455
460Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys465 470 475 480Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 485 490 495Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr 500 505 510Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu 515 520 525Gln Tyr Ser Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 530 535 540Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys545 550 555 560Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 565 570
575Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
580 585 590Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 595 600 605Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn 610 615 620Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu625 630 635 640Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 645 650 655Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 660 665 670Lys Ser Leu
Ser Leu Ser Pro Gly Lys 675 68035684PRTArtificial
SequenceSEQ39+SEQ16+SEQ13 35Glu Ser Leu Gly Trp Asp Val Ala Glu Leu
Gln Leu Asp His Thr Gly1 5 10 15Pro Gln Gln Asp Pro Arg Leu Tyr Trp
Gln Gly Gly Pro Ala Leu Gly 20 25 30Arg Ser Phe Leu His Gly Pro Glu
Leu Asp Lys Gly Gln Leu Arg Ile 35 40 45His Arg Asp Gly Ile Tyr Met
Val His Ile Gln Val Thr Leu Ala Ile 50 55 60Cys Ser Ser Thr Thr Ala
Ser Arg His His Pro Thr Thr Leu Ala Val65 70 75 80Gly Ile Cys Ser
Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser 85 90 95Phe His Gln
Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala 100 105 110Arg
Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser 115 120
125Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly
130 135 140Ser Gly Ser Gly Asn Gly Ser Glu Ser Leu Gly Trp Asp Val
Ala Glu145 150 155 160Leu Gln Leu Asp His Thr Gly Pro Gln Gln Asp
Pro Arg Leu Tyr Trp 165 170 175Gln Gly Gly Pro Ala Leu Gly Arg Ser
Phe Leu His Gly Pro Glu Leu 180 185 190Asp Lys Gly Gln Leu Arg Ile
His Arg Asp Gly Ile Tyr Met Val His 195 200 205Ile Gln Val Thr Leu
Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His 210 215 220His Pro Thr
Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser225 230 235
240Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser
245 250 255Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr
Asn Leu 260 265 270Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu
Thr Phe Phe Gly 275 280 285Val Gln Trp Val Arg Pro Gly Ser Gly Ser
Gly Asn Gly Ser Glu Ser 290 295 300Leu Gly Trp Asp Val Ala Glu Leu
Gln Leu Asp His Thr Gly Pro Gln305 310 315 320Gln Asp Pro Arg Leu
Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser 325 330 335Phe Leu His
Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg 340 345 350Asp
Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser 355 360
365Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly Ile
370 375 380Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser
Phe His385 390 395 400Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr
Pro Leu Ala Arg Gly 405 410 415Asp Thr Leu Cys Thr Asn Leu Thr Gly
Thr Leu Leu Pro Ser Arg Asn 420 425 430Thr Asp Glu Thr Phe Phe Gly
Val Gln Trp Val Arg Pro Gly Ser Ser 435 440 445Ser Ser Ser Ser Ser
Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro 450 455 460Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe465 470 475
480Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
485 490 495Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 500 505 510Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 515 520 525Arg Glu Glu Gln Tyr Ser Ser Thr Tyr Arg
Val Val Ser Val Leu Thr 530 535 540Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val545 550 555 560Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 565 570 575Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 580 585 590Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 595 600
605Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
610 615 620Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser625 630 635 640Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 645 650 655Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His 660 665 670Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 675 68036446PRTArtificial SequenceExemplary
scCD27L-RBD module 36Gln Ser Leu Gly Trp Asp Val Ala Glu Leu Gln
Leu Asn His Thr Gly1 5 10 15Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln
Gly Gly Pro Ala Leu Gly 20 25 30Arg Ser Phe Leu His Gly Pro Glu Leu
Asp Lys Gly Gln Leu Arg Ile 35 40 45His Arg Asp Gly Ile Tyr Met Val
His Ile Gln Val Thr Leu Ala Ile 50 55 60Cys Ser Ser Thr Thr Ala Ser
Arg His His Pro Thr Thr Leu Ala Val65 70 75 80Gly Ile Cys Ser Pro
Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser 85 90 95Phe His Gln Gly
Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala 100 105 110Arg Gly
Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser 115 120
125Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly
130 135 140Ser Gly Ser Gly Asn Gly Ser Glu Ser Leu Gly Trp Asp Val
Ala Glu145 150 155 160Leu Gln Leu Asn His Thr Gly Pro Gln Gln Asp
Pro Arg Leu Tyr Trp 165 170 175Gln Gly Gly Pro Ala Leu Gly Arg Ser
Phe Leu His Gly Pro Glu Leu 180 185 190Asp Lys Gly Gln Leu Arg Ile
His Arg Asp Gly Ile Tyr Met Val His 195 200 205Ile Gln Val Thr Leu
Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His 210 215 220His Pro Thr
Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser225 230 235
240Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser
245 250 255Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr
Asn Leu 260 265 270Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu
Thr Phe Phe Gly 275 280 285Val Gln Trp Val Arg Pro Gly Ser Gly Ser
Gly Asn Gly Ser Glu Ser 290 295 300Leu Gly Trp Asp Val Ala Glu Leu
Gln Leu Asn His Thr Gly Pro Gln305 310 315 320Gln Asp Pro Arg Leu
Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser 325 330 335Phe Leu His
Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg 340 345 350Asp
Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser 355 360
365Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly Ile
370 375 380Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser
Phe His385 390 395 400Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr
Pro Leu Ala Arg Gly 405 410 415Asp Thr Leu Cys Thr Asn Leu Thr Gly
Thr Leu Leu Pro Ser Arg Asn 420 425 430Thr Asp Glu Thr Phe Phe Gly
Val Gln Trp Val Arg Pro Gly 435 440 445372159DNAArtificial
SequenceDNA sequence encoding SEQ_25 37aagctttagg gataacaggg
taatagccgc caccatggag actgacaccc tgctggtgtt 60cgtgctgctg gtctgggtgc
ctgcaggaaa tggagagagc ctgggatggg atgtggccga 120actccagctg
aaccacacag gccctcagca agaccctagg ctctactggc agggcggccc
180tgctctggga aggagctttc tgcatggccc tgaactggat aaaggccaac
tgcgtattca 240tcgggatggc atttacatgg tccatatcca ggtgaccctc
gccatctgct ccagcaccac 300cgctagcagg catcatccca ccaccctggc
cgtgggcatt tgttcccctg ccagccggtc 360catctccctg ctgaggctga
gctttcatca gggctgcacc atcgcctccc aaaggctgac 420ccctctggcc
aggggcgata cactgtgtac caatctgacc ggcaccctgc tccctagcag
480gaacaccgat gaaacctttt tcggagtgca gtgggtgcgg cctggttccg
gaagcggcaa 540tggctccgaa agcctcggct gggacgtggc cgagctccaa
ctgaaccaca ccggccctca 600acaagatcct cggctctatt ggcaaggcgg
acctgctctc ggccggagct tcctgcatgg 660ccctgagctg
gacaagggcc agctgcgtat tcatcgggat ggaatctata tggtgcacat
720ccaagtgaca ctggccattt gcagcagcac caccgctagc cggcaccatc
ctaccaccct 780ggctgtgggc atctgttccc ccgctagccg gtccatctcc
ctgctgaggc tgagcttcca 840ccagggctgt accatcgcca gccagaggct
gacccctctg gctaggggcg acaccctgtg 900taccaacctg accggaaccc
tgctgcctag caggaatacc gatgagacct tcttcggagt 960gcaatgggtg
aggcctggct ctggttctgg taacggttct gagagcctcg gctgggacgt
1020cgctgaactg cagctgaatc acacaggccc ccagcaggac cctaggctgt
actggcaggg 1080aggccctgct ctcggaagga gctttctgca cggccctgaa
ctggataagg gacagctccg 1140tattcatcgg gatggcatct acatggtgca
tatccaggtc accctggcca tctgcagctc 1200caccaccgcc tccaggcacc
accctaccac cctggctgtg ggcatctgct cccctgcctc 1260ccggagcatc
agcctgctga ggctgtcctt ccaccaaggc tgcaccatcg ctagccaaag
1320gctgacccct ctggctaggg gcgataccct gtgcaccaac ctgaccggaa
ccctgctgcc 1380ttcccggaac accgacgaga cctttttcgg cgtgcagtgg
gtcaggcccg gatcctcgag 1440ttcatcgtcc tcatccggct catgtgataa
gacccacacc tgccctccct gtcctgcccc 1500tgagctgctg ggcggacctt
ctgtgttcct gttccccccc aagcctaagg acaccctgat 1560gatctccagg
acccctgagg tgacctgtgt ggtggtggac gtgtctcacg aagatcccga
1620ggtgaagttc aactggtacg tggacggcgt ggaggtccac aacgccaaga
ccaagcctag 1680ggaggagcag tacagctcca cctaccgggt ggtgtctgtg
ctgaccgtgc tgcaccagga 1740ttggctgaac ggaaaggagt ataagtgtaa
ggtctccaac aaggccctgc ctgcccccat 1800cgagaaaacc atctccaagg
ccaagggcca gcctcgggag cctcaggtgt acaccctgcc 1860tcctagcagg
gaggagatga ccaagaacca ggtgtccctg acctgtctgg tgaagggctt
1920ctacccttcc gatatcgccg tggagtggga gtctaatggc cagcccgaga
acaactacaa 1980gaccacccct cctgtgctgg actctgacgg ctccttcttc
ctgtactcca agctgaccgt 2040ggacaagtcc agatggcagc agggcaacgt
gttctcctgc tccgtgatgc acgaggccct 2100gcacaatcac tacacccaga
agtccctgtc tctgagtccg ggcaagtaat aggcgcgcc 215938220PRTArtificial
SequenceCD27L RBD fused to RB69-Foldon 38Met Glu Thr Asp Thr Leu
Leu Val Phe Val Leu Leu Val Trp Val Pro1 5 10 15Ala Gly Asn Gly Glu
Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu 20 25 30Asn His Thr Gly
Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly 35 40 45Pro Ala Leu
Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly 50 55 60Gln Leu
Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val65 70 75
80Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr
85 90 95Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser
Leu 100 105 110Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser
Gln Arg Leu 115 120 125Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr
Asn Leu Thr Gly Thr 130 135 140Leu Leu Pro Ser Arg Asn Thr Asp Glu
Thr Phe Phe Gly Val Gln Trp145 150 155 160Val Arg Pro Gly Ser Gly
Ser Ser Gly Ser Ser Gly Ser Ser Gly Ser 165 170 175Gly Tyr Ile Glu
Asp Ala Pro Ser Asp Gly Lys Phe Tyr Val Arg Lys 180 185 190Asp Gly
Ala Trp Val Glu Leu Pro Thr Ala Ser Gly Pro Ser Ser Ser 195 200
205Ser Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys 210 215
22039445PRTArtificial Sequenceexemplary scCD27L-RBD module 39Glu
Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asp His Thr Gly1 5 10
15Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly
20 25 30Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg
Ile 35 40 45His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu
Ala Ile 50 55 60Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr
Leu Ala Val65 70 75 80Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser
Leu Leu Arg Leu Ser 85 90 95Phe His Gln Gly Cys Thr Ile Ala Ser Gln
Arg Leu Thr Pro Leu Ala 100 105 110Arg Gly Asp Thr Leu Cys Thr Asn
Leu Thr Gly Thr Leu Leu Pro Ser 115 120 125Arg Asn Thr Asp Glu Thr
Phe Phe Gly Val Gln Trp Val Arg Pro Gly 130 135 140Ser Gly Ser Gly
Asn Gly Ser Glu Ser Leu Gly Trp Asp Val Ala Glu145 150 155 160Leu
Gln Leu Asp His Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp 165 170
175Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu
180 185 190Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met
Val His 195 200 205Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr Thr
Ala Ser Arg His 210 215 220His Pro Thr Thr Leu Ala Val Gly Ile Cys
Ser Pro Ala Ser Arg Ser225 230 235 240Ile Ser Leu Leu Arg Leu Ser
Phe His Gln Gly Cys Thr Ile Ala Ser 245 250 255Gln Arg Leu Thr Pro
Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu 260 265 270Thr Gly Thr
Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly 275 280 285Val
Gln Trp Val Arg Pro Gly Ser Gly Ser Gly Asn Gly Ser Glu Ser 290 295
300Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asp His Thr Gly Pro
Gln305 310 315 320Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala
Leu Gly Arg Ser 325 330 335Phe Leu His Gly Pro Glu Leu Asp Lys Gly
Gln Leu Arg Ile His Arg 340 345 350Asp Gly Ile Tyr Met Val His Ile
Gln Val Thr Leu Ala Ile Cys Ser 355 360 365Ser Thr Thr Ala Ser Arg
His His Pro Thr Thr Leu Ala Val Gly Ile 370 375 380Cys Ser Pro Ala
Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe His385 390 395 400Gln
Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly 405 410
415Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn
420 425 430Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro 435
440 44540441PRTArtificial Sequenceexemplary scCD27L-RBD module
40Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asp His Thr Gly1
5 10 15Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu
Gly 20 25 30Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu
Arg Ile 35 40 45His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr
Leu Ala Ile 50 55 60Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr
Thr Leu Ala Val65 70 75 80Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile
Ser Leu Leu Arg Leu Ser 85 90 95Phe His Gln Gly Cys Thr Ile Ala Ser
Gln Arg Leu Thr Pro Leu Ala 100 105 110Arg Gly Asp Thr Leu Cys Thr
Asn Leu Thr Gly Thr Leu Leu Pro Ser 115 120 125Arg Asn Thr Asp Glu
Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly 130 135 140Ser Gly Ser
Gly Asn Gly Ser Glu Ser Leu Gly Trp Asp Val Ala Glu145 150 155
160Leu Gln Leu Asp His Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp
165 170 175Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro
Glu Leu 180 185 190Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile
Tyr Met Val His 195 200 205Ile Gln Val Thr Leu Ala Ile Cys Ser Ser
Thr Thr Ala Ser Arg His 210 215 220His Pro Thr Thr Leu Ala Val Gly
Ile Cys Ser Pro Ala Ser Arg Ser225 230 235 240Ile Ser Leu Leu Arg
Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser 245 250 255Gln Arg Leu
Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu 260 265 270Thr
Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly 275 280
285Val Gln Trp Val Arg Pro Gly Ser Gly Ser Glu Ser Leu Gly Trp Asp
290 295 300Val Ala Glu Leu Gln Leu Asp His Thr Gly Pro Gln Gln Asp
Pro Arg305 310 315 320Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg
Ser Phe Leu His Gly 325 330 335Pro Glu Leu Asp Lys Gly Gln Leu Arg
Ile His Arg Asp Gly Ile Tyr 340 345 350Met Val His Ile Gln Val Thr
Leu Ala Ile Cys Ser Ser Thr Thr Ala 355 360 365Ser Arg His His Pro
Thr Thr Leu Ala Val Gly Ile Cys Ser Pro Ala 370 375 380Ser Arg Ser
Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys Thr385 390 395
400Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys
405 410 415Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp
Glu Thr 420 425 430Phe Phe Gly Val Gln Trp Val Arg Pro 435
44041430PRTArtificial Sequenceexemplary scCD27L-RBD module 41Asp
Val Ala Glu Leu Gln Leu Asp His Thr Gly Pro Gln Gln Asp Pro1 5 10
15Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His
20 25 30Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly
Ile 35 40 45Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser Ser
Thr Thr 50 55 60Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly Ile
Cys Ser Pro65 70 75 80Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser
Phe His Gln Gly Cys 85 90 95Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu
Ala Arg Gly Asp Thr Leu 100 105 110Cys Thr Asn Leu Thr Gly Thr Leu
Leu Pro Ser Arg Asn Thr Asp Glu 115 120 125Thr Phe Phe Gly Val Gln
Trp Val Arg Pro Gly Ser Gly Ser Gly Asn 130 135 140Gly Ser Asp Val
Ala Glu Leu Gln Leu Asp His Thr Gly Pro Gln Gln145 150 155 160Asp
Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe 165 170
175Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp
180 185 190Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys
Ser Ser 195 200 205Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala
Val Gly Ile Cys 210 215 220Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu
Arg Leu Ser Phe His Gln225 230 235 240Gly Cys Thr Ile Ala Ser Gln
Arg Leu Thr Pro Leu Ala Arg Gly Asp 245 250 255Thr Leu Cys Thr Asn
Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr 260 265 270Asp Glu Thr
Phe Phe Gly Val Gln Trp Val Arg Pro Gly Ser Gly Ser 275 280 285Gly
Asn Gly Ser Asp Val Ala Glu Leu Gln Leu Asp His Thr Gly Pro 290 295
300Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly
Arg305 310 315 320Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln
Leu Arg Ile His 325 330 335Arg Asp Gly Ile Tyr Met Val His Ile Gln
Val Thr Leu Ala Ile Cys 340 345 350Ser Ser Thr Thr Ala Ser Arg His
His Pro Thr Thr Leu Ala Val Gly 355 360 365Ile Cys Ser Pro Ala Ser
Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe 370 375 380His Gln Gly Cys
Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg385 390 395 400Gly
Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg 405 410
415Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro 420 425
43042426PRTArtificial Sequenceexemplary scCD27L-RBD module 42Asp
Val Ala Glu Leu Gln Leu Asp His Thr Gly Pro Gln Gln Asp Pro1 5 10
15Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His
20 25 30Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly
Ile 35 40 45Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser Ser
Thr Thr 50 55 60Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly Ile
Cys Ser Pro65 70 75 80Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser
Phe His Gln Gly Cys 85 90 95Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu
Ala Arg Gly Asp Thr Leu 100 105 110Cys Thr Asn Leu Thr Gly Thr Leu
Leu Pro Ser Arg Asn Thr Asp Glu 115 120 125Thr Phe Phe Gly Val Gln
Trp Val Arg Pro Gly Ser Gly Ser Gly Asn 130 135 140Gly Ser Asp Val
Ala Glu Leu Gln Leu Asp His Thr Gly Pro Gln Gln145 150 155 160Asp
Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe 165 170
175Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp
180 185 190Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys
Ser Ser 195 200 205Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala
Val Gly Ile Cys 210 215 220Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu
Arg Leu Ser Phe His Gln225 230 235 240Gly Cys Thr Ile Ala Ser Gln
Arg Leu Thr Pro Leu Ala Arg Gly Asp 245 250 255Thr Leu Cys Thr Asn
Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr 260 265 270Asp Glu Thr
Phe Phe Gly Val Gln Trp Val Arg Pro Gly Ser Gly Ser 275 280 285Asp
Val Ala Glu Leu Gln Leu Asp His Thr Gly Pro Gln Gln Asp Pro 290 295
300Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu
His305 310 315 320Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His
Arg Asp Gly Ile 325 330 335Tyr Met Val His Ile Gln Val Thr Leu Ala
Ile Cys Ser Ser Thr Thr 340 345 350Ala Ser Arg His His Pro Thr Thr
Leu Ala Val Gly Ile Cys Ser Pro 355 360 365Ala Ser Arg Ser Ile Ser
Leu Leu Arg Leu Ser Phe His Gln Gly Cys 370 375 380Thr Ile Ala Ser
Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu385 390 395 400Cys
Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu 405 410
415Thr Phe Phe Gly Val Gln Trp Val Arg Pro 420
42543680PRTArtificial SequenceSEQ40+SEQ16+SEQ13 43Glu Ser Leu Gly
Trp Asp Val Ala Glu Leu Gln Leu Asp His Thr Gly1 5 10 15Pro Gln Gln
Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly 20 25 30Arg Ser
Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile 35 40 45His
Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile 50 55
60Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val65
70 75 80Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu
Ser 85 90 95Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro
Leu Ala 100 105 110Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr
Leu Leu Pro Ser 115 120 125Arg Asn Thr Asp Glu Thr Phe Phe Gly Val
Gln Trp Val Arg Pro Gly 130 135 140Ser Gly Ser Gly Asn Gly Ser Glu
Ser Leu Gly Trp Asp Val Ala Glu145 150 155 160Leu Gln Leu Asp His
Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp 165 170 175Gln Gly Gly
Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu 180 185 190Asp
Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met Val His 195 200
205Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His
210
215 220His Pro Thr Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg
Ser225 230 235 240Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys
Thr Ile Ala Ser 245 250 255Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp
Thr Leu Cys Thr Asn Leu 260 265 270Thr Gly Thr Leu Leu Pro Ser Arg
Asn Thr Asp Glu Thr Phe Phe Gly 275 280 285Val Gln Trp Val Arg Pro
Gly Ser Gly Ser Glu Ser Leu Gly Trp Asp 290 295 300Val Ala Glu Leu
Gln Leu Asp His Thr Gly Pro Gln Gln Asp Pro Arg305 310 315 320Leu
Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His Gly 325 330
335Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile Tyr
340 345 350Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr
Thr Ala 355 360 365Ser Arg His His Pro Thr Thr Leu Ala Val Gly Ile
Cys Ser Pro Ala 370 375 380Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser
Phe His Gln Gly Cys Thr385 390 395 400Ile Ala Ser Gln Arg Leu Thr
Pro Leu Ala Arg Gly Asp Thr Leu Cys 405 410 415Thr Asn Leu Thr Gly
Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr 420 425 430Phe Phe Gly
Val Gln Trp Val Arg Pro Gly Ser Ser Ser Ser Ser Ser 435 440 445Ser
Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 450 455
460Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro465 470 475 480Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val 485 490 495Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val 500 505 510Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln 515 520 525Tyr Ser Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln 530 535 540Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala545 550 555 560Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 565 570
575Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
580 585 590Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser 595 600 605Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr 610 615 620Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr625 630 635 640Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe 645 650 655Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 660 665 670Ser Leu Ser
Leu Ser Pro Gly Lys 675 68044669PRTArtificial SequenceSeq41 +Seq16
+Seq13 44Asp Val Ala Glu Leu Gln Leu Asp His Thr Gly Pro Gln Gln
Asp Pro1 5 10 15Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser
Phe Leu His 20 25 30Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His
Arg Asp Gly Ile 35 40 45Tyr Met Val His Ile Gln Val Thr Leu Ala Ile
Cys Ser Ser Thr Thr 50 55 60Ala Ser Arg His His Pro Thr Thr Leu Ala
Val Gly Ile Cys Ser Pro65 70 75 80Ala Ser Arg Ser Ile Ser Leu Leu
Arg Leu Ser Phe His Gln Gly Cys 85 90 95Thr Ile Ala Ser Gln Arg Leu
Thr Pro Leu Ala Arg Gly Asp Thr Leu 100 105 110Cys Thr Asn Leu Thr
Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu 115 120 125Thr Phe Phe
Gly Val Gln Trp Val Arg Pro Gly Ser Gly Ser Gly Asn 130 135 140Gly
Ser Asp Val Ala Glu Leu Gln Leu Asp His Thr Gly Pro Gln Gln145 150
155 160Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser
Phe 165 170 175Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile
His Arg Asp 180 185 190Gly Ile Tyr Met Val His Ile Gln Val Thr Leu
Ala Ile Cys Ser Ser 195 200 205Thr Thr Ala Ser Arg His His Pro Thr
Thr Leu Ala Val Gly Ile Cys 210 215 220Ser Pro Ala Ser Arg Ser Ile
Ser Leu Leu Arg Leu Ser Phe His Gln225 230 235 240Gly Cys Thr Ile
Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp 245 250 255Thr Leu
Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr 260 265
270Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly Ser Gly Ser
275 280 285Gly Asn Gly Ser Asp Val Ala Glu Leu Gln Leu Asp His Thr
Gly Pro 290 295 300Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro
Ala Leu Gly Arg305 310 315 320Ser Phe Leu His Gly Pro Glu Leu Asp
Lys Gly Gln Leu Arg Ile His 325 330 335Arg Asp Gly Ile Tyr Met Val
His Ile Gln Val Thr Leu Ala Ile Cys 340 345 350Ser Ser Thr Thr Ala
Ser Arg His His Pro Thr Thr Leu Ala Val Gly 355 360 365Ile Cys Ser
Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe 370 375 380His
Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg385 390
395 400Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser
Arg 405 410 415Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg
Pro Gly Ser 420 425 430Ser Ser Ser Ser Ser Ser Ser Gly Ser Cys Asp
Lys Thr His Thr Cys 435 440 445Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu 450 455 460Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu465 470 475 480Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 485 490 495Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 500 505
510Pro Arg Glu Glu Gln Tyr Ser Ser Thr Tyr Arg Val Val Ser Val Leu
515 520 525Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys 530 535 540Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys545 550 555 560Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser 565 570 575Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys 580 585 590Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 595 600 605Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 610 615 620Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln625 630
635 640Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn 645 650 655His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 66545665PRTArtificial SequenceSeq41 + Seq16 + Seq13 45Asp Val
Ala Glu Leu Gln Leu Asp His Thr Gly Pro Gln Gln Asp Pro1 5 10 15Arg
Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His 20 25
30Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile
35 40 45Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr
Thr 50 55 60Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly Ile Cys
Ser Pro65 70 75 80Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe
His Gln Gly Cys 85 90 95Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala
Arg Gly Asp Thr Leu 100 105 110Cys Thr Asn Leu Thr Gly Thr Leu Leu
Pro Ser Arg Asn Thr Asp Glu 115 120 125Thr Phe Phe Gly Val Gln Trp
Val Arg Pro Gly Ser Gly Ser Gly Asn 130 135 140Gly Ser Asp Val Ala
Glu Leu Gln Leu Asp His Thr Gly Pro Gln Gln145 150 155 160Asp Pro
Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe 165 170
175Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp
180 185 190Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys
Ser Ser 195 200 205Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala
Val Gly Ile Cys 210 215 220Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu
Arg Leu Ser Phe His Gln225 230 235 240Gly Cys Thr Ile Ala Ser Gln
Arg Leu Thr Pro Leu Ala Arg Gly Asp 245 250 255Thr Leu Cys Thr Asn
Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr 260 265 270Asp Glu Thr
Phe Phe Gly Val Gln Trp Val Arg Pro Gly Ser Gly Ser 275 280 285Asp
Val Ala Glu Leu Gln Leu Asp His Thr Gly Pro Gln Gln Asp Pro 290 295
300Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu
His305 310 315 320Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His
Arg Asp Gly Ile 325 330 335Tyr Met Val His Ile Gln Val Thr Leu Ala
Ile Cys Ser Ser Thr Thr 340 345 350Ala Ser Arg His His Pro Thr Thr
Leu Ala Val Gly Ile Cys Ser Pro 355 360 365Ala Ser Arg Ser Ile Ser
Leu Leu Arg Leu Ser Phe His Gln Gly Cys 370 375 380Thr Ile Ala Ser
Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu385 390 395 400Cys
Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu 405 410
415Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly Ser Ser Ser Ser Ser
420 425 430Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro 435 440 445Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys 450 455 460Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val465 470 475 480Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 485 490 495Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 500 505 510Gln Tyr Ser
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 515 520 525Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 530 535
540Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln545 550 555 560Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met 565 570 575Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro 580 585 590Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn 595 600 605Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 610 615 620Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val625 630 635 640Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 645 650
655Lys Ser Leu Ser Leu Ser Pro Gly Lys 660 66546669PRTArtificial
SequenceSeq44 with N170D mutein 46Asp Val Ala Glu Leu Gln Leu Asp
His Thr Gly Pro Gln Gln Asp Pro1 5 10 15Arg Leu Tyr Trp Gln Gly Gly
Pro Ala Leu Gly Arg Ser Phe Leu His 20 25 30Gly Pro Glu Leu Asp Lys
Gly Gln Leu Arg Ile His Arg Asp Gly Ile 35 40 45Tyr Met Val His Ile
Gln Val Thr Leu Ala Ile Cys Ser Ser Thr Thr 50 55 60Ala Ser Arg His
His Pro Thr Thr Leu Ala Val Gly Ile Cys Ser Pro65 70 75 80Ala Ser
Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys 85 90 95Thr
Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu 100 105
110Cys Thr Asp Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu
115 120 125Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly Ser Gly Ser
Gly Asn 130 135 140Gly Ser Asp Val Ala Glu Leu Gln Leu Asp His Thr
Gly Pro Gln Gln145 150 155 160Asp Pro Arg Leu Tyr Trp Gln Gly Gly
Pro Ala Leu Gly Arg Ser Phe 165 170 175Leu His Gly Pro Glu Leu Asp
Lys Gly Gln Leu Arg Ile His Arg Asp 180 185 190Gly Ile Tyr Met Val
His Ile Gln Val Thr Leu Ala Ile Cys Ser Ser 195 200 205Thr Thr Ala
Ser Arg His His Pro Thr Thr Leu Ala Val Gly Ile Cys 210 215 220Ser
Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe His Gln225 230
235 240Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly
Asp 245 250 255Thr Leu Cys Thr Asp Leu Thr Gly Thr Leu Leu Pro Ser
Arg Asn Thr 260 265 270Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg
Pro Gly Ser Gly Ser 275 280 285Gly Asn Gly Ser Asp Val Ala Glu Leu
Gln Leu Asp His Thr Gly Pro 290 295 300Gln Gln Asp Pro Arg Leu Tyr
Trp Gln Gly Gly Pro Ala Leu Gly Arg305 310 315 320Ser Phe Leu His
Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His 325 330 335Arg Asp
Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys 340 345
350Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly
355 360 365Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu
Ser Phe 370 375 380His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr
Pro Leu Ala Arg385 390 395 400Gly Asp Thr Leu Cys Thr Asp Leu Thr
Gly Thr Leu Leu Pro Ser Arg 405 410 415Asn Thr Asp Glu Thr Phe Phe
Gly Val Gln Trp Val Arg Pro Gly Ser 420 425 430Ser Ser Ser Ser Ser
Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys 435 440 445Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 450 455 460Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu465 470
475 480Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys 485 490 495Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys 500 505 510Pro Arg Glu Glu Gln Tyr Ser Ser Thr Tyr Arg
Val Val Ser Val Leu 515 520 525Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys 530 535 540Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys545 550 555 560Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 565 570 575Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 580 585
590Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
595 600 605Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly 610 615 620Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln625 630 635 640Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 645 650
655His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 660
66547680PRTArtificial SequenceSeq31 without E51Q mutation in module
(i) 47Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His Thr
Gly1 5 10 15Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala
Leu Gly 20 25 30Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln
Leu Arg Ile 35 40 45His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val
Thr Leu Ala Ile 50 55 60Cys Ser Ser Thr Thr Ala Ser Arg His His Pro
Thr Thr Leu Ala Val65 70 75 80Gly Ile Cys Ser Pro Ala Ser Arg Ser
Ile Ser Leu Leu Arg Leu Ser 85 90 95Phe His Gln Gly Cys Thr Ile Ala
Ser Gln Arg Leu Thr Pro Leu Ala 100 105 110Arg Gly Asp Thr Leu Cys
Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser 115 120 125Arg Asn Thr Asp
Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly 130 135 140Ser Gly
Ser Gly Asn Gly Ser Glu Ser Leu Gly Trp Asp Val Ala Glu145 150 155
160Leu Gln Leu Asn His Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp
165 170 175Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro
Glu Leu 180 185 190Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile
Tyr Met Val His 195 200 205Ile Gln Val Thr Leu Ala Ile Cys Ser Ser
Thr Thr Ala Ser Arg His 210 215 220His Pro Thr Thr Leu Ala Val Gly
Ile Cys Ser Pro Ala Ser Arg Ser225 230 235 240Ile Ser Leu Leu Arg
Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser 245 250 255Gln Arg Leu
Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu 260 265 270Thr
Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly 275 280
285Val Gln Trp Val Arg Pro Gly Ser Gly Ser Glu Ser Leu Gly Trp Asp
290 295 300Val Ala Glu Leu Gln Leu Asn His Thr Gly Pro Gln Gln Asp
Pro Arg305 310 315 320Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg
Ser Phe Leu His Gly 325 330 335Pro Glu Leu Asp Lys Gly Gln Leu Arg
Ile His Arg Asp Gly Ile Tyr 340 345 350Met Val His Ile Gln Val Thr
Leu Ala Ile Cys Ser Ser Thr Thr Ala 355 360 365Ser Arg His His Pro
Thr Thr Leu Ala Val Gly Ile Cys Ser Pro Ala 370 375 380Ser Arg Ser
Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys Thr385 390 395
400Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys
405 410 415Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp
Glu Thr 420 425 430Phe Phe Gly Val Gln Trp Val Arg Pro Gly Ser Ser
Ser Ser Ser Ser 435 440 445Ser Ser Gly Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala 450 455 460Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro465 470 475 480Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 485 490 495Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 500 505 510Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 515 520
525Tyr Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
530 535 540Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala545 550 555 560Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro 565 570 575Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr 580 585 590Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser 595 600 605Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 610 615 620Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr625 630 635
640Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
645 650 655Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys 660 665 670Ser Leu Ser Leu Ser Pro Gly Lys 675 680
* * * * *