U.S. patent application number 16/898122 was filed with the patent office on 2020-10-22 for single-chain light 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 | 20200331979 16/898122 |
Document ID | / |
Family ID | 1000004928852 |
Filed Date | 2020-10-22 |
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United States Patent
Application |
20200331979 |
Kind Code |
A1 |
GIEFFERS; Christian ; et
al. |
October 22, 2020 |
SINGLE-CHAIN LIGHT RECEPTOR AGONIST PROTEINS
Abstract
Provided herein are specific LIGHT receptor agonist proteins,
nucleic acids encoding the same, and methods of treating a subject
having a LIGHT-associated disease or disorder. The LIGHT receptor
agonist proteins provided herein comprise three soluble LIGHT
domains and an Fc fragment. The LIGHT 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: |
1000004928852 |
Appl. No.: |
16/898122 |
Filed: |
June 10, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15958707 |
Apr 20, 2018 |
10683332 |
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16898122 |
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PCT/EP2016/075536 |
Oct 24, 2016 |
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15958707 |
|
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62245943 |
Oct 23, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/52 20130101;
C07K 2319/02 20130101; C07K 2319/30 20130101; A61P 35/00
20180101 |
International
Class: |
C07K 14/52 20060101
C07K014/52; A61P 35/00 20060101 A61P035/00 |
Claims
1. A LIGHT receptor agonist protein comprising a single-chain
fusion polypeptide comprising: (i) a first soluble LIGHT cytokine
domain, (ii) a first peptide linker, (iii) a second soluble LIGHT
cytokine domain, (iv) a second peptide linker, and (v) a third
soluble LIGHT cytokine domain, (vi) a hinge-linker, and (vii) an
antibody Fc fragment, wherein the antibody Fc fragment (vii)
consists of the amino acid sequence as shown in SEQ ID NO: 13 or
amino acids 1-217 of SEQ ID NO: 13; wherein the soluble LIGHT
cytokine domains (i), (iii) and (v) each consists of amino acids
E91-V240, or N93-V240, or P94-V240 of human LIGHT cytokine
according to SEQ ID NO: 1.
2. The LIGHT receptor agonist protein of claim 1, wherein the
antibody Fc fragment (vii) is fused to the C-terminal end of the
third LIGHT cytokine domain (v) via the hinge-linker (vi).
3. The LIGHT receptor agonist protein of claim 1, wherein the
soluble LIGHT cytokine domains (i), (iii) and (v) each consists of
amino acids P94-V240 of human LIGHT cytokine according to SEQ ID
NO: 1.
4. The LIGHT receptor agonist protein of claim 1, wherein the
soluble LIGHT cytokine domain (i) consists of amino acids E91-V240
of SEQ ID:1, and wherein the soluble LIGHT cytokine domains (iii)
and (v) consist of either amino acid N93-V240 or P94-V240.
5. The LIGHT receptor agonist protein of claim 1, which is
substantially non-aggregating.
6. The LIGHT receptor agonist protein of claim 1, wherein the first
and second peptide linkers (ii) and (iv) independently consist of
glycine and serine, or consist of glycine, serine and asparagine,
wherein the asparagine is optionally glycosylated.
7. The LIGHT receptor agonist protein of claim 6, wherein the first
and the second peptide linkers (ii) and (iv) comprise the amino
acid sequence according to SEQ ID NO: 2.
8. The LIGHT receptor agonist protein of claim 1, wherein the hinge
linker has one of the sequences of SEQ ID NOs: 16 and 19-24.
9. The LIGHT receptor agonist protein of claim 3, wherein the hinge
linker has one of the sequences of SEQ ID NOs: 16 and 19-24.
10. The LIGHT receptor agonist protein of claim 4, wherein the
hinge linker has one of the sequences of SEQ ID NOs: 16 and
19-24.
11. The LIGHT receptor agonist of claim 1, comprising two of the
single-chain fusion polypeptides.
12. The LIGHT receptor agonist protein of claim 11, wherein the
polypeptide(s) are further post-translationally modified.
13. The LIGHT receptor agonist protein of claim 12, wherein the
post-translational modification comprises modification of the
N-terminal glutamine to pyroglutamate.
14. A pharmaceutical or diagnostic composition comprising the LIGHT
receptor agonist protein of claim 1 and a pharmaceutically
acceptable carriers, diluents, excipients, and/or adjuvants.
15. A LIGHT receptor agonist protein comprising a single-chain
fusion polypeptide comprising: (i) a first soluble LIGHT cytokine
domain, (ii) a first peptide linker, (iii) a second soluble LIGHT
cytokine domain, (iv) a second peptide linker, and (v) a third
soluble LIGHT cytokine domain, (vi) a hinge-linker, and (vii) an
antibody Fc fragment, wherein the antibody Fc fragment (vii)
consists of the amino acid sequence as shown in SEQ ID NO: 13 or
amino acids 1-217 of SEQ ID NO: 13; wherein the soluble LIGHT
cytokine domains (i), (iii) and (v) each independently consists of
amino acids E91-V240, or N93-V240, or P94-V240 of human LIGHT
cytokine according to SEQ ID NO: 1, having no mutation or having
one or two mutations at position selected from the group consisting
of: N93, N102, E115, T116, Q117, L118, G119, L120, C154, R172,
Y173, E175, E176, E178, C187, R228, D229.
16. The LIGHT receptor agonist protein of claim 15, wherein N93 is
replaced by glycine or serine in at least one of the soluble LIGHT
cytokine domains.
17. The LIGHT receptor agonist protein of claim 15, wherein P94 is
replaced by glycine or serine in at least one of the soluble LIGHT
cytokine domains.
18. The LIGHT receptor agonist protein of claim 15, wherein E91 is
replaced by glutamine, glycine, or serine.
19. The LIGHT receptor agonist protein of claim 15, wherein the
first and second peptide linkers (ii) and (iv) independently
consist of glycine and serine, or consist of glycine, serine and
asparagine, wherein the asparagine is optionally glycosylated.
20. The LIGHT receptor agonist protein of claim 15, wherein the
first and the second peptide linkers (ii) and (iv) consist of the
amino acid sequence according to SEQ ID NO: 2.
21. The LIGHT receptor agonist protein of claim 15, wherein the
hinge linker has one of the sequences of SEQ ID NOs: 16 and 19-24.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/958,707, filed Apr. 20, 2018, which is a continuation of
PCT/EP2016/075536, filed Oct. 24, 2016; which claims priority to
U.S. Provisional Application No. 62/245,943, 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.
19, 2018, and a size of 95.8 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 LIGHT receptor
agonist proteins comprising three soluble LIGHT domains and an Fc
fragment, nucleic acid molecules encoding the LIGHT receptor
agonist proteins, and uses thereof. The LIGHT 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 Clq 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 (1988), 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] There is a need in the art for novel LIGHT receptor agonists
that exhibit high biological activity independent of Fc-gamma-R
based crosslinking in vivo, high stability, and allow for efficient
recombinant manufacturing. Additionally, there is need in the art
for enabling technologies to create human LIGHT-receptor selective
biologics as human LIGHT has at least three interaction partners in
vivo: LT-beta-R, DcR3 and HVEM.
SUMMARY OF THE INVENTION
[0011] The present invention provides specific LIGHT receptor
agonist proteins that mimic the LIGHT-receptor(s):LIGHT interaction
in vivo, exhibit low proteolytic degradation and a shorter in vivo
half-life as compared to agonistic monoclonal antibodies.
[0012] The LIGHT receptor agonist proteins of the instant invention
generally comprise: (i) a first soluble LIGHT cytokine domain; (ii)
a first peptide linker; (iii) a second soluble LIGHT domain; (iv) a
second peptide linker; (v) a third soluble LIGHT domain; (vi) a
third peptide linker (e.g., a hinge-linker) and (vii) an antibody
Fc fragment.
[0013] In one embodiment, the antibody Fc fragment (vii) is located
N terminal to the first LIGHT domain (i) and/or C-terminal to the
third LIGHT domain (v). In another embodiment the antibody Fc
fragment is located C-terminally to the third LIGHT domain (v). In
one embodiment, the polypeptide is substantially non-aggregating.
In another embodiment, the second and/or third soluble LIGHT domain
is an N-terminally shortened domain which optionally comprises
amino acid sequence mutations.
[0014] In one embodiment, at least one of the soluble LIGHT
domains, particularly at least one of the soluble LIGHT domains
(iii) and (v), is a soluble LIGHT domain with an N-terminal
sequence which starts at amino acid Glu91 or Asn93 or Pro94 of
human LIGHT and wherein Glu91 or Asn93 or Pro94 may be replaced by
a neutral amino acid, e.g., Ser or Gly. In another embodiment, at
least one of the soluble LIGHT domains, particularly at least one
of the soluble LIGHT domains (iii) and (v), is a soluble LIGHT
domain with an N-terminal sequences selected from (a) Asn93-Ala95
and (b) (Gly/Ser)93-Ala95. In one embodiment, the soluble LIGHT
domain ends with amino acid Val240 of human LIGHT and/or optionally
comprises one or more mutation at positions: N93, N102, E115, T116,
Q117, L118, G119, L120, C154, R172, Y173, E175, E176, E178, C187,
R228, D229. In one embodiment, the soluble LIGHT domains (i), (iii)
and (v) comprise amino acids Glu91-Val240 of human LIGHT according
to SEQ ID NO: 01.
[0015] In one embodiment, at least one of the soluble LIGHT
domains, particularly at least the soluble LIGHT domains (i), is a
soluble LIGHT domain with an N-terminal sequence which starts at
amino acid Glu91 and wherein Glu91 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.
[0016] In one embodiment, the antibody Fc fragment (vii) is fused
to the soluble LIGHT 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.
[0017] 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:
[0018] 15, and 25-32.
[0019] In one embodiment, the present invention provides a LIGHT
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 468, 474, and 477 of each
polypeptide.
[0020] In one embodiment the asparagine residue at position 156 of
the mature polypeptide(s) SEQ ID NO: 27, 28, 29, 30, and 32 is
N-glycosylated.
[0021] In another embodiment, the asparagine residues at positions
156 and 312 of the polypeptide SEQ ID NO: 31 are both
N-glycosylated.
[0022] 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 E91Q mutein modified to pyroglutamate.
DESCRIPTION OF THE FIGURES
[0023] FIG. 1 Domain structure of a single-chain fusion polypeptide
comprising three LIGHT domains. I., II., III. Soluble LIGHT
domains.
[0024] FIG. 2 Schematic picture representing the general structure
of LIGHT. [0025] .box-solid..box-solid..box-solid.Cell membrane,
N-terminus located within the cell, [0026] 1. anti-parallel
.beta.-fold of receptor-binding domain (RBD), [0027] 2. interface
of RBD and cell membrane, [0028] 3. protease cleavage site.
[0029] FIG. 3 Single-chain fusion polypeptide comprising an
additional Fab antibody fragment.
[0030] FIG. 4 Dimerization of two C-terminally fused scFc fusion
polypeptides via three disulfide bridges.
[0031] FIG. 5 Schematic representation of the hexavalent single
chain LIGHT 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
association in the "open stage" remains intact due to the high
affinity of the CH3 domains (3) to each other. [0032] (1)
scLIGHT-RBD; (2) CH2 domain; (3) CH3 domain; (4) Hinge-Cysteines
(left side: oxidized to disulfide bridges; right side reduced stage
with free thiols); (5) CH2-Carbohydrates attached to N297 position
(EU-numbering); (6) Upper Hinge Lysine (K223).
[0033] FIG. 6 Analytical size exclusion chromatography of PROTEIN A
(A) and a PROTEIN B (B) performed on a 1260 Infinity HPLC system
using a Tosoh TSKgelG3000SWxl column. The column was loaded with
protein at a concentration of 0.94 mg/ml (A) or 0.77 mg/ml (B) 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 16.239 (A) and 15.842 min (B).
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention provides a single-chain fusion
polypeptide comprising at least three soluble LIGHT 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 LIGHT receptor agonist, which provides high biological
activity and good stability.
[0035] Preferably, the single-chain fusion polypeptide is
non-aggregating. The term "non-aggregating" refers to a monomer
content of the preparation of 50%, preferably 70% and more
preferably 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.
[0036] 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.
[0037] The LIGHT receptor agonist protein of the present invention
comprises three soluble domains derived from LIGHT. Preferably,
those soluble domains are derived from a mammalian, particularly
human LIGHT including allelic variants and/or derivatives thereof.
The soluble domains comprise the extracellular portion of LIGHT
including the receptor binding domain without membrane located
domains. Like other proteins of the TNF superfamily, LIGHT 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).
[0038] 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).
[0039] 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 LIGHT domains of
the LIGHT receptor agonist protein comprise a receptor binding
domain of the LIGHT 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.
[0040] 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 LIGHT cytokine
domain; (ii) a first peptide linker; (iii) a second soluble LIGHT
domain; (iv) a second peptide linker; (v) a third soluble LIGHT
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 LIGHT
receptor. The single-chain fusion polypeptide comprising components
(i)-(v) is therefore also termed
single-chain-LIGHT-receptor-binding-domain (scLIGHT-RBD).
[0041] The LIGHT receptor agonist protein comprises three
functional LIGHT-receptor binding sites, i.e. amino acid sequences
capable of forming a complex with a LIGHT-receptor. Thus, the
soluble domains are capable of binding to the corresponding
LIGHT-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.
[0042] The soluble LIGHT domain may be derived from human LIGHT as
shown in SEQ ID NO: 1. Preferably, the soluble LIGHT domains are
derived from human LIGHT, particularly starting from amino acids 91
or 94 and comprise particularly amino acids 91-240 or 94-240 of SEQ
ID NO: 1. Optionally, amino acid Glu91 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 LIGHT Protein
SEQ ID NO Sequence 1 MEESVVRPSVFVVDGQTDIPFTRLGRSHRRQSCSVARVGLG
LLLLLMGAGLAVQGWFLLQLHWRLGEMVIRLPDGPAGSWEQ
LIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFL
RGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITH
GLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGG
VVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMV
[0043] As indicated above, the soluble LIGHT 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.
[0044] In a further embodiment of the invention, the soluble LIGHT
domain (i) comprises a mutant of LIGHT or a receptor binding domain
thereof resulting in reduced affinity and/or reduced activation of
LIGHT-receptor.
[0045] LIGHT-Muteins Affecting Receptor Binding and/or Activity
[0046] The mutant may be generated by any technique known by a
skilled person. The substitution may affect at least one amino acid
of LIGHT, e.g., human LIGHT (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 LIGHT of SEQ ID NO: 1: E115, T116, Q117, L118, G119, L120,
R172, Y173, E175, E176, E178, R228, D229. In a preferred embodiment
Y173 is mutated to S, T, D, E, R or F.
[0047] Human LIGHT has at least three different
receptors/interaction partners in vivo, namely, LT-beta-R, DcR3 and
HVEM. The amino acid substitution(s) may affect the binding and/or
activity of LIGHT, e.g., human LIGHT, to or on either the
LIGHT-receptor(s) binding or the LIGHT-receptor(s) induced
signaling. The binding and/or activity of the LIGHT-receptor 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 LIGHT-receptor
may be affected negatively, i.e., weaker, less selective or less
specific binding and/or less or no activation of the receptor or
receptor(s).
[0048] Thus one embodiment is a LIGHT receptor agonist protein as
described herein wherein at least one of the soluble domains
comprises a mutant of LIGHT or a receptor binding domain thereof
which binds and/or activates LIGHT-receptor(s) to a lesser extent
than the wildtype-LIGHT.
[0049] The single-chain fusion molecule of the present invention
comprises three soluble LIGHT domains, namely components (i), (iii)
and (v). The stability of a single-chain LIGHT fusion polypeptide
against aggregation is enhanced, if the second and/or third soluble
LIGHT domain is an N-terminally shortened domain which optionally
comprises amino acid sequence mutations. Thus, preferably, both the
second and the third soluble LIGHT 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 LIGHT domain.
These mutations may comprise replacement of basic amino acids, by
neutral amino acids, particularly serine or glycine.
[0050] In contrast thereto, the selection of the first soluble
LIGHT 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 LIGHT domain may have an
N-terminally shortened and optionally mutated sequence.
[0051] In a further preferred embodiment of the present invention,
the soluble LIGHT domains (i), (iii) and (v) are soluble human
LIGHT domains. The first soluble LIGHT domain (i) may be selected
from native, shortened and/or mutated sequences. Thus, the first
soluble LIGHT domain (i) has an N-terminal sequence which may start
at amino acid Glu91 or Ala95 of human LIGHT, and wherein Glu91 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 LIGHT domains (iii) and (v) have a shortened
N-terminal sequence which preferably starts with amino acid Asn93
or Pro94 of human LIGHT (SEQ ID NO:1) and wherein Asn93 may be
replaced by another amino acid, e.g. Ser or Gly.
[0052] Preferably, the N-terminal sequence of the soluble LIGHT
domains (iii) and (v) is selected from:
(a) Asn93 or Pro94
(b) (Gly/Ser) 93.
[0053] The soluble LIGHT domain preferably ends with amino acid
V240 of human LIGHT. In certain embodiments, the LIGHT domain may
comprise internal mutations as described above.
[0054] Components (ii) and (iv) of the LIGHT 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 which
the soluble cytokine domain starts with S or G (N-terminus), the
linker ends before this S or G.
[0055] 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.
[0056] 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 LIGHT 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.
[0057] Preferred linker sequences are shown in Table 2. A preferred
linker is GSGSGNGS (SEQ ID NO: 2). Another preferred linker is GSGS
(SEQ ID NO:11).
TABLE-US-00002 TABLE 2 ExampleLinkerSequences 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
[0058] The LIGHT receptor agonist protein additionally comprises an
antibody Fc fragment domain which may be located N-terminal to the
first LIGHT domain (i) and/or C-terminal to the third LIGHT 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 PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNVVYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 14
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW
LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK
[0059] Number of Glycosylation Sites and In Vivo Stability
[0060] The total number of glycosylation sites and the individual
position of the carbohydrates in three dimensions impacts the
in-vivo stability of LIGHT receptor agonist proteins. 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.
[0061] Further, partially degraded carbohydrates reduce the in vivo
half-life of LIGHT receptor agonist proteins through lectin-driven
mechanisms. By reducing the total number of glycosylation sites
and/or their relative position on the molecule's surface, the
resulting compound is less accessible to these mechanisms,
increasing half-life. In a preferred embodiment, the first linker
(ii) is glycosylated and the second linker (iv) is not glycosylated
to avoid carbohydrate patterns in close proximity on the proteins
accessible surface. In a preferred embodiment, the linkers with
(SEQ ID NO: 2) and (SEQ ID NO:11) are combined in one scLIGHT-RBD
module.
[0062] Depletion of antibody CH2-domain carbohydrates is necessary
in order to avoid Fc-receptor based crosslinking in vivo and
potential LIGHT-receptor superclustering-based toxicity. Also,
unwanted Fc-driven mechanisms like ADCC could lead to toxic events.
Accordingly, in one embodiment, the overall number of glycosylation
sites on the LIGHT receptor agonist proteins of the instant
invention is reduced through the depletion of CH2 glycosylation
sites, particularly the N-glycosylation site, resulting in LIGHT
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 LIGHT domains (i), (iii), and
(v) may comprise a N102 exchanged to aspartate, serine or glycine
resulting in LIGHT receptor agonistic fusion proteins with a
further reduced number of glycosylation sites. In a preferred
embodiment, the N102 [D,S,G] mutation is restricted to the soluble
LIGHT domains (iii) and (v) of the agonistic LIGHT receptor
agonistic fusion proteins of the present invention.
[0063] CH2-Domain Destabilization is Compensated by an Additional
Hinge-Cysteine
[0064] 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. 5). This enables
CH2-dissociation and exposure of the inner surface area towards
proteases. LIGHT 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 LIGHT 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).
[0065] 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.
[0066] 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.
[0067] The number of molecules with open Fc-conformation in an
individual LIGHT 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 LIGHT receptor agonist proteins of the instant invention in
order to ameliorate the effect of depleting the CH2-glycosites.
[0068] Exchange of a lysine to glycine in the hinge region results
in enhanced proteolytic stability In one embodiment, the LIGHT
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 LIGHT receptor agonist protein of the instant
invention.
[0069] 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).
[0070] The LIGHT 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).
[0071] Further, the LIGHT 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).
[0072] 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
[0073] In one embodiment of the invention, the fusion polypeptide
comprises three soluble LIGHT domains fused by two different
peptide linker elements. The first linker element (ii) consists of
SEQ ID NO: 2. The second linker element (iv) consists of SEQ ID NO:
11. The first soluble LIGHT domain (i) consists of amino acids
91-240 of human LIGHT according to SEQ ID NO: 1 and the soluble
LIGHT domains (iii) and (v) consist of amino acids 94-240 of human
LIGHT according to SEQ ID NO: 1. The resulting scLIGHT-RBD sequence
module is shown in Table 5b SEQ ID NO: 36.
[0074] In another embodiment of the invention, the fusion
polypeptide comprises three soluble LIGHT domains fused by peptide
linker elements of SEQ ID NO: 2. The first soluble LIGHT domain (i)
consists of amino acids 91-240 of human LIGHT according to SEQ ID
NO: 1 and the soluble LIGHT domains (iii) and (v) consist of amino
acids 93-240 of human LIGHT according to SEQ ID NO: 1. The
resulting scLIGHT-RBD sequence module is shown in table 5b SEQ ID
NO: 39.
[0075] In another embodiment of the invention, the fusion
polypeptide comprises three soluble LIGHT domains fused by peptide
linker elements of SEQ ID NO: 2. The first soluble LIGHT domain (i)
consists of amino acids 91-240 of human LIGHT according to SEQ ID
NO: 1 and the soluble LIGHT domains (iii) and (v) consist of amino
acids 94-240 of human LIGHT according to SEQ ID NO: 1. The
resulting scLIGHT-RBD sequence module is shown in table 5b SEQ ID
NO: 40.
[0076] Preferred Configuration LIGHT-Fc
[0077] Additionally, the fusion polypeptide comprises an antibody
Fc fragment domain according to SEQ ID NO: 13 that is fused
C-terminally to the soluble LIGHT 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-LIGHT-receptor-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 LIGHT receptor agonist
protein of the invention is set forth in SEQ ID NO: 27.
[0078] 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 LIGHT receptor agonist protein of the invention is
shown in SEQ ID NO: 25.
[0079] 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.
[0080] 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).
[0081] The exemplary LIGHT 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
LIGHT receptor agonist proteins (without a signal peptide) of the
instant invention are set forth in SEQ ID NO: 27-35.
[0082] Exemplary LIGHT receptor agonist proteins described above
are shown in Table 5.
[0083] The LIGHT receptor agonist as set forth in SEQ ID NO: 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 interchain
disulfide bonds in the hinge region, and the mutation of an
upper-hinge lysine to a glycine (K223G, according to the EU
numbering system). Additionally, the second peptide linker (iv) is
shortened and the modules (iii) and (v) are N-terminal shortened,
thereby reducing all in all protomer dissociation and enhancing the
proteins stability towards proteases These alterations provide a
decrease in potential degradation and LIGHT receptor
superclustering (along with concomitant toxicity).
[0084] The LIGHT receptor agonist as set forth in SEQ ID NO: 30
comprises the same layout as SEQ ID NO: 27 but with the E91Q
mutation in the soluble LIGHT 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.
[0085] The LIGHT receptor agonist as set forth in SEQ ID NO:32
comprises the same layout as SEQ ID NO: 30 but with the third
peptide linker (vi) shortened to reduce the interdomain distance
between the soluble LIGHT domain (v) and the Fc-domain (Vii)
thereby enhancing the proteins stability towards proteases.
[0086] According to one embodiment of the invention, the
single-chain LIGHT fusion polypeptide domain comprises a
scLIGHT-RBD module as shown in SEQ ID NO: 39 optionally with the
soluble domain (i) comprising the E91Q mutation. A specific example
of a LIGHT receptor agonist protein of the invention comprising the
E91Q 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-US-00005 TABLE 5 Exemplary LIGHT receptor agonistProteins SEQ
ID NO Sequence 25
METDTLLVFVLLVWVPAGNGEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSY PROTEIN
A HDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSP
without CGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGS
Strep GNGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKV
QLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGG
VVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSPAAHLTGANSSLTGSGGPL
LWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRT
PRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLR
DGTRSYFGAFMVGSSSSSSSSGSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK 15
METDTLLVFVLLVWVPAGNGEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSY PROTEIN
A HDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSP SEQ36
+ CGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGS SEQ13
(FC) + GNGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKV
Signal + QLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGG
Strep VVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSPAAHLTGANSSLTGSGGPL
LWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRT
PRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLR
DGTRSYFGAFMVGSSSSSSSSGSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGSSSSSSAWSHPQFEK 26
METDTLLVFVLLVWVPAGNGEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSY SEQ36 +
HDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSP SEQ14
(FC) + CGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGS
Signal + GNGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKV
No Strep QLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGG
VVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSPAAHLTGANSSLTGSGGPL
LWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRT
PRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLR
DGTRSYFGAFMVGSSSSSSSSGSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK 27
EVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQ SEQ36 +
LGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGV SEQ13
(FC) + VHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSG
No Signal
GPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLY No Strep
KRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLV No Glyco
RLRDGTRSYFGAFMVGSGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGA
LVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRA
TSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSSSSSSS
SGSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 28
EVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQ SEQ36 +
LGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGV SEQ13
(FC) VHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSG No
Signal + GPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLY
StrepTag KRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLV
No Glyco RLRDGTRSYFGAFMVGSGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGA
LVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRA
TSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSSSSSSS
SGSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSS
SSSSAWSHPQFEK 29
EVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQ SEQ36 +
LGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGV SEQ14
(FC) VHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSG No
Signal GPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLY
No strep KRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLV
Glyco FC RLRDGTRSYFGAFMVGSGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGA
LVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRA
TSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSSSSSSS
SGSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 30
QVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQ Same as
27 LGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGV with
E91Q VHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSG in
GPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLY
KRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLV
RLRDGTRSYFGAFMVGSGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGA
LVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRA
TSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSSSSSSS
SGSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 31
QVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQ Protein
B LGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGV SEQ 39
VHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSNPAAHLTGANSSLTGS With L1
GGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGL 8 mer
YKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERL L2: 8
mer VRLRDGTRSYFGAFMVGSGSGNGSNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGL
SYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQ
SPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGS
SSSSSSSGSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 32
QVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQ Same as
30, LGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGV
shortened
VHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSG hinge
GPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLY
KRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLV
RLRDGTRSYFGAFMVGSGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGA
LVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRA
TSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVgsssssgs
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 33
EVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQ
Linker1+2
LGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGV 8 mer
VHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSG N93-
GPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLY
deletionin
KRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLV RBD
RLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSY module 2
HDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSP and 3
CGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSSS
SSSSSGSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 34
QVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQ SEQ33
with LGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGV
N-terminal
VHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSG amino
acid GPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLY
exchange KRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLV
E->Q RLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSY
HDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSP
CGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSSS
SSSSSGSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 35
EVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQ Seq33
with LGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGV
shorter VHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSG
hinge- GPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLY
linker KRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLV
RLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSY
HDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSP
CGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVgsss
ssgsCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP
IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
TABLE-US-00006 TABLE 5B Exemplary scLIGHT-RBD modules 36
EVNPAAHLTGANSSLIGSGGPLLWETQLGLAFLRGLSYHDGALVVIKAGYYYI L1 8 mer
YSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRV L2 4 mer
WWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSP
AAHLTGANSSLIGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKV
QLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDS
SFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSPAAHLTGAN
SSLIGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCP
LGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVH
LEAGEEVVVRVLDERLVRLRDGTRSYFGAFMV 39
EVNPAAHLTGANSSLIGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYI L1 8 mer
YSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRV L2 8 mer
WWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSN
PAAHLTGANSSLIGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSK
VQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWD
SSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSNPAA
HLTGANSSLIGSGGPLLWETQLGLAFLRGLSYHDGALVVIKAGYYYIYSKVQL
GGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSF
LGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMV 40
EVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQ L1 8 mer
LGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGV L2 8 mer
VHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSG N93
GPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLY Deleted
in KRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLV mod-2
and 3 RLRDGTRSYFGAFMVGSGSGNGSPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSY
HDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSP
CGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMV
[0087] Furthermore, it has to be noted that the scLIGHT-RBD modules
(SEQ ID NO: 36, 39 and SEQ ID NO: 40) 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).
[0088] Above presented embodiments of the LIGHT 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.
[0089] 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).
[0090] 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 LIGHT 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 LIGHT 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 LIGHT lymphocytes become
activated and enter the expansion phase during which they
proliferate in order to mount an immune response against the
tumor.
[0091] 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 LIGHT-RECEPTOR 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. Thus, in a
further embodiment, the LIGHT 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.
[0092] A further aspect of the present invention relates to a
nucleic acid molecule encoding a LIGHT 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 LIGHT receptor
agonist protein or a precursor thereof, e.g. a pro- or pre-proform
of the LIGHT 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 LIGHT 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 LIGHT
receptor agonist Protein SEQ ID NO Sequence 37
AAGCTTTAGGGATAACAGGGTAATAGCCGCCACCATGGAGACTGACACCCTGCTGG
TGTTCGTGCTGCTGGTCTGGGIGCCTGCAGGAAATGGAGAAGTGAACCCCGCCGCC
CATCTGACCGGCGCTAACAGCAGCCTGACAGGTTCTGGCGGACCCCTCCTGTGGGA
GACACAACTGGGCCTGGCCTTCCTGAGGGGCCTGAGCTACCATGATGGCGCCCTGG
TGGTGACCAAGGCCGGCTACTACTACATCTATTCCAAGGTCCAGCTCGGAGGCGTG
GGATGCCCTCTGGGACTGGCCTCCACCATCACCCACGGCCTGTACAAGCGGACCCC
TAGGTACCCCGAGGAACTGGAACTGCTCGTCTCCCAACAGAGCCCTTGCGGCAGGG
CTACCTCCTCCAGCAGGGTGTGGTGGGACTCCAGCTTCCTGGGAGGCGTCGTCCAC
CTGGAGGCTGGAGAAGAAGTGGTGGTGCGGGTCCTGGACGAAAGGCTGGTGAGGCT
CAGGGACGGCACCCGGTCCTACTTTGGAGCCTTTATGGTGGGCTCCGGATCTGGTA
ACGGCAGCCCCGCTGCTCATCTGACAGGCGCCAATAGCAGCCTGACAGGCAGCGGA
GGCCCTCTGCTGTGGGAAACACAGCTGGGCCTGGCCTTTCTGAGGGGCCTGTCCTA
TCACGATGGAGCCCTGGTGGTGACCAAAGCCGGCTATTACTATATCTACAGCAAGG
TGCAGCTGGGCGGAGTGGGATGTCCTCTGGGCCTGGCCTCCACCATCACACACGGA
CTGTATAAGCGGACACCTAGGTATCCCGAAGAGCTGGAGCTCCTGGTGTCCCAGCA
AAGCCCTTGTGGAAGGGCTACCTCCAGCAGCAGGGTCTGGTGGGACTCCTCCTTCC
TGGGCGGCGTGGTCCATCTGGAAGCTGGCGAGGAGGTGGTGGTGAGGGTCCTGGAT
GAGAGGCTGGTCAGGCTGAGGGATGGCACCCGGTCCTATTTTGGCGCTTTCATGGT
GGGCTCTGGTAGCCCTGCCGCCCACCTGACAGGAGCCAACAGCAGCCTGACAGGAA
GCGGCGGCCCTCTGCTGTGGGAGACCCAACTGGGCCTGGCCTTCCTGCGGGGCCTC
TCCTACCACGACGGCGCTCTGGTGGTGACCAAGGCCGGCTATTATTATATCTACTC
CAAAGTCCAGCTGGGAGGCGTCGGCTGTCCTCTCGGACTGGCTTCCACCATCACCC
ATGGCCTGTACAAAAGGACCCCTAGGTACCCCGAAGAGTTAGAACTGCTGGTCTCC
CAGCAGTCCCCTTGCGGAAGGGCCACAAGCAGCAGCCGGGTGTGGTGGGACTCCAG
CTTTCTGGGCGGAGTGGTGCACCTGGAAGCCGGAGAGGAGGTCGTGGTCAGGGTCC
TGGATGAAAGGCTGGTGCGGCTGAGGGATGGCACCAGGTCCTATTTCGGCGCCTTC
ATGGTCggatcctcgagTTCATCGTCCTCATCCGGCTCATGTGATAAGACCCACAC
CTGCCCTCCCTGTCCTGCCCCTGAGCTGCTGGGCGGACCTTCTGTGTTCCTGTTCC
CCCCCAAGCCTAAGGACACCCTGATGATCTCCAGGACCCCTGAGGTGACCTGTGTG
GTGGTGGACGTGTCTCACGAAGATCCCGAGGTGAAGTTCAACTGGTACGTGGACGG
CGTGGAGGTCCACAACGCCAAGACCAAGCCTAGGGAGGAGCAGTACAGCTCCACCT
ACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGAAAGGAG
TATAAGTGTAAGGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCTC
CAAGGCCAAGGGCCAGCCTCGGGAGCCTCAGGTGTACACCCTGCCTCCTAGCAGGG
AGGAGATGACCAAGAACCAGGIGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCT
TCCGATATCGCCGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAACAACTACAAGAC
CACCCCTCCTGTGCTGGACTCTGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCG
TGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAG
GCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGAGTCCGGGCAAGTAATA
ggcgcgcc
[0093] 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.
[0094] Various expression vector/host cell systems may be used to
express the nucleic acid sequences encoding the LIGHT 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.
[0095] A further aspect of the present invention relates to a
pharmaceutical or diagnostic composition comprising as the active
agent at least one LIGHT receptor agonist protein, a respective
nucleic acid encoding therefore, or a transformed or transfected
cell, all as described herein.
[0096] In another aspect, the present invention provides a
pharmaceutical composition comprising a LIGHT receptor agonist
protein disclosed herein and one or more pharmaceutically
acceptable carriers, diluents, excipients, and/or adjuvants. In
another aspect, the present invention provides a nucleic acid
molecule encoding the LIGHT 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-AgI4, and
hybridoma cells.
[0097] In another aspect, the present invention provides a method
of treating a subject having a LIGHT-associated disease or
disorder, the method comprising administering to the subject an
effective amount of the LIGHT receptor agonist protein. In one
embodiment, the LIGHT receptor agonist protein is administered
alone. In another embodiment, the LIGHT 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. 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.
[0098] 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, intraarticular, 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.
[0099] In one embodiment, the LIGHT receptor agonist protein is
administered as a single bolus. In another embodiment, LIGHT
receptor agonist protein may be administered over several divided
doses. The LIGHT receptor agonist protein can be administered at
about 0.1-100 mg/kg. In one embodiment, the LIGHT 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 LIGHT receptor agonist protein is present in pharmaceutical
compositions at about 0.1-100 mg/ml. In one embodiment, the LIGHT
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 LIGHT receptor agonist protein is administered to a
subject. In another embodiment, a prophylactically effective amount
of LIGHT receptor agonist protein is administered to a subject.
[0100] The term "LIGHT-associated disease or disorder" as used
herein is any disease or disorder which may be ameliorated by
administering an effective amount of a LIGHT receptor agonist to a
subject in need thereof. At least one LIGHT 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
LIGHT, 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.
[0101] The term "dysfunction of LIGHT" as used herein is to be
understood as any function or expression of LIGHT that deviates
from the normal function or expression of LIGHT, e.g.,
overexpression of the LIGHT gene or protein, reduced or abolished
expression of the LIGHT gene or protein compared to the normal
physiological expression level of LIGHT, increased activity of
LIGHT, reduced or abolished activity of LIGHT, increased binding of
LIGHT to any binding partners, e.g., to a receptor, particularly a
LIGHT receptor or another cytokine molecule, reduced or abolished
binding to any binding partner, e.g. to a receptor, particularly a
LIGHT receptor or another cytokine molecule, compared to the normal
physiological activity or binding of LIGHT.
[0102] In various embodiments, a method is provided for treating a
human subject suffering from a disorder which can be treated by
targeting LIGHT-receptors comprising administering to the human
subject a LIGHT 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 LIGHT 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.
[0103] A pharmaceutical composition comprising a LIGHT 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.
[0104] In an embodiment, a method of treating a cancer or in the
prevention or inhibition of metastases from the tumors described
herein, the LIGHT 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-Cod.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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] In various embodiments, pharmaceutical compositions
comprising one or more LIGHT receptor agonist proteins, either
alone or in combination with prophylactic agents, therapeutic
agents, and/or pharmaceutically acceptable carriers are provided
herein. In various embodiments, nonlimiting 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).
[0110] As used herein, the phrase "effective amount" means an
amount of LIGHT 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 LIGHT or
with a LIGHT-associated disease or disorder.
[0111] 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).
[0112] 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.
[0113] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a LIGHT 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 LIGHT 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 LIGHT Receptor Agonist Protein
[0114] A) Amino acids Met1-Gly20 [0115] Ig-Kappa-signal peptide,
assumed signal peptidase cleavage site after amino acid Gly 20.
[0116] B) Amino acids Glu21-Val170 [0117] First soluble cytokine
domain of the human LIGHT (LIGHT, amino acid 91-240 of SEQ ID NO:
1).
[0118] C) Amino acids GIy171-Ser 178 [0119] First peptide linker
element of SEQ ID NO: 2.
[0120] D) Amino acids Pro179-Val325 [0121] Second soluble cytokine
domain of the human LIGHT (LIGHT, amino acid 94-240 of SEQ ID NO:
1).
[0122] E) Amino acids GIy326-Ser329. [0123] Second peptide linker
element of SEQ ID NO: 11.
[0124] F) Amino acids Pro330-Val476 [0125] Third soluble cytokine
domain of the human LIGHT ligand (LIGHT, amino acid 94-240 of SEQ
ID NO: 1).
[0126] G) Amino acids Gly477-Cys497 [0127] Hinge-linker element of
SEQ ID NO: 16.
[0128] H) Amino acids Pro498-Lys715 [0129] Antibody Fc fragment
domain of SEQ ID NO: 13.
[0130] The above LIGHT receptor agonist protein is shown in SEQ ID
NO: 25 The indicated linkers may be replaced by other preferred
linkers, e.g. as shown in SEQ ID NOs: 3-12.
[0131] The indicated Hinge-linker element may be replaced by other
preferred Hinge-linkers, e.g. as shown in SEQ ID NOs: 19-24.
[0132] It should be noted that the first and second peptide linkers
do not need to be identical.
[0133] The signal peptide sequence (A) may be replaced by any other
suitable, e.g. mammalian signal peptide sequence.
[0134] 1.2 Gene Cassette Encoding the Polypeptide
[0135] 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
[0136] 2.1 Cloning, Expression and Purification of Fusion
Polypeptides
[0137] The aforementioned fusion proteins are expressed
recombinantly in two different eukaryotic host cells employing the
methods described below:
[0138] Method for Small Scale Expression of LIGHT Receptor Agonist
Fusion Proteins:
[0139] For initial analysis of aforementioned LIGHT 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 resistance 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.
[0140] Method for Large Scale Expression and Purification of LIGHT
Receptor Agonist Fusion Proteins
[0141] For larger scale expression of LIGHT 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).
[0142] 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 22 rpm, as well as the shaking ankle to
8.degree..
[0143] 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, MC0HC 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 LIGHT 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.
[0144] 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.
[0145] Alternative 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).
[0146] Employing the aforementioned methods, recombinant LIGHT
receptor agonist fusion proteins (PROTEIN-A, SEQ ID NO: 15 and
PROTEIN-B, SEQ ID NO: 31) were expressed in CHO-S cells and
purified employing affinity chromatography.
[0147] Analytical size exclusion chromatography of PROTEIN A with
asymmetric linker and shortened LIGHT domains (SEQ ID NO: 15) and
PROTEIN B with symmetric linkers both glycosylated and (Refers to
SEQ ID NO: 31) is shown in FIG. 6.
[0148] The SEC was performed on a 1260 Infinity HPLC system using a
Tosoh TSKgeIG3000SWxl column. The column was loaded with protein at
a concentration of 0.94 mg/ml (A) or 0.77 mg/ml (B) 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 16.24 min for PROTEIN A (FIG. 6,
Part A) and 15.84 min for PROTEIN B (FIG. 6, Part B). Consequently,
PROTEIN B has a higher apparent molecular weight due to the
glycosylated linker (iv). Both proteins were prepared by AFC only,
indicating due to the absence of aggregates high solubility.
Protein A By using an internal molecular weight standard (BioRad
SEC Standard) one can intrapolate the molecular weight of PROTEIN A
and PROTEIN B from respective retention times.
[0149] 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 LIGHT
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 LIGHT
receptor agonist fusion polypeptides by the Fc domain.
Example 3. Determination of Temperature Stability
[0150] The stability of PROTEIN A (scLIGHT-RBD-Fc) upon elevated
temperatures was assessed. Therefore, this protein was exposed for
10 min in an incubator for the indicated temperatures and then
cooled on ice. The binding activity towards its receptor HVEM was
then assessed employing the following ELISA assay:
Plates were coated with HVEM-Fc. PROTEIN A (heat treated and
non-treated control) was added to the plate and then detected via
its Strep-Tag employing StrepTactin-HRP. Binding activity of the
non-treated control was set as 100%. Values below 100% indicate a
loss in binding activity towards the receptor.
[0151] The results from the temperature stability evaluation for
PROTEIN A at concentration of 75 ng/ml are summarized in the
following
TABLE-US-00008 PROTEIN A (scLIGHT-RBD-Fc) Relative Binding to
Receptor [%] Control 100 50.degree. C. 87 60.degree. C. 91
70.degree. C. 95 80.degree. C. 91
[0152] Surprisingly, PROTEIN A (scLIGHT-RBD-Fc) is very stable upon
exposure to elevated temperatures. Even for an incubation at
80.degree. C. for 10 minutes, no relevant decrease in binding to
the receptor HVEM was observed.
Example 4. Trivalent Control Protein
[0153] To compare the relative binding between hexavalent LIGHT
receptor agonist fusion proteins and the, trivalent LIGHT-RBD
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 LIGHT domain (E91-V240 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 LIGHT.
TABLE-US-00009 TABLE 7 Trivalent control protein including a signal
peptide SEQ ID NO Sequence 38
METDTLLVFVLLVWVPAGNGEVNPAAHLTGANSSLTGSG (Protein X)
GPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYTYSKVQ
LGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPC
GRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVR
LRDGTRSYFGAFMVGSGSSGSSGSSGSGYIEDAPSDGKF
YVRKDGAWVELPTASGPSSSSSSAWSHPQFEK.
Example 5. Determination of the In Vitro Stability of LIGHT
Receptor Agonist Proteins by Limited Protease Digestion
[0154] All LIGHT 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 LIGHT 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 LIGHT receptor agonist proteins
comprising the N297S mutation and the K223G mutation simultaneously
in context of a three-disulfide enabling hinge will be compared to
LIGHT 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.
[0155] In addition, LIGHT receptor agonist proteins with the second
linker element (iv) reduced to 4 amino-acids and the shortened
hinge element (vi) will be investigated. 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.
[0156] The stability of different LIGHT receptor agonistic proteins
of the present invention can be addressed by limited protease
digestion in vitro. For this analysis, the aforementioned LIGHT
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 LIGHT receptor
agonist proteins investigated, the following order regarding their
proteolytic stability is to be expected:
[0157] The LIGHT 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 LIGHT
receptor agonist proteins comprising the N297S and wildtype K223 in
the hinge region. The LIGHT receptor agonist proteins comprising
the SEQ ID NO: 21 as hinge linker have a prolonged stability as
compared to LIGHT receptor agonist proteins comprising the SEQ ID
NO: 16 as hinge linker element.
Example 6. Half-Life Determination
[0158] 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.
[0159] 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.
[0160] Quantitation of the PROTEIN A concentrations in mouse serum
was performed with an ELISA-assay detecting the HVEM agonist shown
in Table 8. Plates were coated with HVEM-Fc. LIGHT constructs
specifically binding to its receptor HVEM 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.
[0161] 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.
[0162] The results from the pharmacokinetics evaluation are
summarized in Table 8.
TABLE-US-00010 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) 12.8 t.sub.last (h) 192
C.sub.last (.mu.g/ml) 0.141 t.sub.1/2 E (h) 36.47 t.sub.1/2 E (d)
1.52 AUC.sub.0-t (.mu.g*h/ml) 346 AUC.sub.0-inf (.mu.g*h/ml)
353
[0163] The results show that PROTEIN A has a surprisingly short
terminal half-life of 36.47 hours in mice. This short half-life
constitutes a favorable therapeutic option since a short
co-stimulatory stimulus with LIGHT receptor agonist proteins is
desirable.
Example 7: Stability/Aggregation Test
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] Purified preparations of LIGHT 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 LIGHT 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 8: Determination of the Equilibrium Binding Constants for
Tri- and Hexavalent LIGHT-Receptor Ligand Constructs by QCM
Analysis
[0169] 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.
[0170] For this purpose, the human LIGHT-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.
The QCM analysis shows that the trivalent PROTEIN X binds to the
respective immobilized LIGHT-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 LIGHT-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 9: Apoptosis Induction by Tri- and Hexavalent
LIGHT-Receptor Ligand Constructs in Human Breast and Colon
Carcinoma Cells
[0171] The human breast cancer cell line MDA-MB-231 and the human
colon cancer cell line HT-29 are incubated with PROTEIN X and
PROTEIN A at varying concentrations (between 0.1 ng/ml and 100
.mu.g/ml) in combination with varying amounts of IFN.gamma. (1-100
U/ml). Cells are incubated at 37.degree. C. in the presence of
IFN.gamma. and PROTEIN X or PROTEIN A for 24 h, 48 h or 72 h. At
each time point cells are harvested and processed for flow
cytometric analysis assessing the binding of Propidium Iodide (PI)
to double-stranded nucleic acids such as DNA and the upregulation
of Annexin V. Both binding of PI and Annexin V upregulation are
critical indicators for the induction of apoptotic cell death.
[0172] One expects to observe a supplementary effect exerted by
PROTEIN A and PROTEIN X in a sense that both PROTEIN A and PROTEIN
X will significantly increase the PI and Annexin V double-positive
cell populations when compared to cells incubated with IFN.gamma.
or PROTEIN A or PROTEIN X alone. This supplementary effect is
likely to hold true for both MDA-MB-231 and HT-29 cells, but can be
more significant in the case of HT-29 cells. This implies that
PROTEIN X and PROTEIN A can drive certain human cancer cell lines
into apoptosis in the presence of IFN.gamma..
Example 10: Human In Vitro T Cell Proliferation Assay
[0173] Total T cells (human) purified by negative selection and
magnetic beads (pan T cell isolation kit, Miltenyi Biotec) from the
peripheral blood of healthy donors and stained with CFSE
(CellTrace.TM. CFSE Cell Proliferation Kit, for flow cytometry,
ThermoFisher) and seeded into 24-well plates at 2.times.10e6 cells
per well. Cells were incubated at 37.degree. C. for 5 days with
media alone, soluble anti-CD3 antibody (clone OKT3 at 1 .mu.g/ml)
alone, anti-CD3 antibody plus anti-CD28 antibody (clone 28.2 at 1
.mu.g/ml) or anti-CD3 antibody plus mature Protein A (SEQ ID NO:27)
at 10, 100 or 1000 ng/ml, respectively.
[0174] On day 5, cells were washed and stained with DAPI (to
exclude dead cells) and specific antibodies. Expression of Forward
Scatter (FSC or size) and CFSE dilution (a measurement of
proliferation) was measured by flow cytometry with a Guava EasyCyte
12 Flow Cytometer (EMD Millipore). Data analysis was performed on a
minimum of ten thousand recorded events per sample with FlowJo 10.1
software (FlowJo, LLC). The percentage of responding cells was
determined by gating on Forward Scatter and CFSE using the media
control to determine proper gate location. Cells that had either
increased cell size or decreased CFSE levels were labeled as
responding cells. The individual data from two biological
replicates from one donor is shown in table "Quantification of T
cell activation" (below). These results are consistent with results
from additional donors.
[0175] This data clearly shows that treatment of human T cells in
vitro with Protein A enhances T cell activation and proliferation
as compared to antibody stimulation alone.
[0176] Quantification of T cell activation:
TABLE-US-00011 Human T cell activation following treatment with
Protein A in vitro % of cells responding Stimulation Sample 1
Sample 2 Media 3 3 anti-CD3 56 62 anti-CD3/28 87 85 anti-CD3 +
Protein A 10 ng/ml 69 58 anti-CD3 + Protein A 100 ng/ml 68 66
anti-CD3 + Protein A 1000 ng/ml 59 57
Sequence CWU 1
1
401240PRTHomo sapiensLIGHT ligand (wt) 1Met Glu Glu Ser Val Val Arg
Pro Ser Val Phe Val Val Asp Gly Gln1 5 10 15Thr Asp Ile Pro Phe Thr
Arg Leu Gly Arg Ser His Arg Arg Gln Ser 20 25 30Cys Ser Val Ala Arg
Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly 35 40 45Ala Gly Leu Ala
Val Gln Gly Trp Phe Leu Leu Gln Leu His Trp Arg 50 55 60Leu Gly Glu
Met Val Thr Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp65 70 75 80Glu
Gln Leu Ile Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala 85 90
95His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu
100 105 110Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu
Ser Tyr 115 120 125His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr
Tyr Tyr Ile Tyr 130 135 140Ser Lys Val Gln Leu Gly Gly Val Gly Cys
Pro Leu Gly Leu Ala Ser145 150 155 160Thr Ile Thr His Gly Leu Tyr
Lys Arg Thr Pro Arg Tyr Pro Glu Glu 165 170 175Leu Glu Leu Leu Val
Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser 180 185 190Ser Ser Arg
Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His 195 200 205Leu
Glu Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg Leu 210 215
220Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met
Val225 230 235 24028PRTArtificial 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
21515729PRTArtificial SequencePROTEIN A (LIGHT ligand 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 Val Asn Pro Ala Ala His Leu
Thr Gly Ala Asn 20 25 30Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu Leu
Trp Glu Thr Gln Leu 35 40 45Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr
His Asp Gly Ala Leu Val 50 55 60Val Thr Lys Ala Gly Tyr Tyr Tyr Ile
Tyr Ser Lys Val Gln Leu Gly65 70 75 80Gly Val Gly Cys Pro Leu Gly
Leu Ala Ser Thr Ile Thr His Gly Leu 85 90 95Tyr Lys Arg Thr Pro Arg
Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser 100 105 110Gln Gln Ser Pro
Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp 115 120 125Asp Ser
Ser Phe Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Glu 130 135
140Val Val Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg Asp
Gly145 150 155 160Thr Arg Ser Tyr Phe Gly Ala Phe Met Val Gly Ser
Gly Ser Gly Asn 165 170 175Gly Ser Pro Ala Ala His Leu Thr Gly Ala
Asn Ser Ser Leu Thr Gly 180 185 190Ser Gly Gly Pro Leu Leu Trp Glu
Thr Gln Leu Gly Leu Ala Phe Leu 195 200 205Arg Gly Leu Ser Tyr His
Asp Gly Ala Leu Val Val Thr Lys Ala Gly 210 215 220Tyr Tyr Tyr Ile
Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro225 230 235 240Leu
Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro 245 250
255Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys
260 265 270Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser Ser
Phe Leu 275 280 285Gly Gly Val Val His Leu Glu Ala Gly Glu Glu Val
Val Val Arg Val 290 295 300Leu Asp Glu Arg Leu Val Arg Leu Arg Asp
Gly Thr Arg Ser Tyr Phe305 310 315 320Gly Ala Phe Met Val Gly Ser
Gly Ser Pro Ala Ala His Leu Thr Gly 325 330 335Ala Asn Ser Ser Leu
Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr 340 345 350Gln Leu Gly
Leu Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala 355 360 365Leu
Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln 370 375
380Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr
His385 390 395 400Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu
Leu Glu Leu Leu 405 410 415Val Ser Gln Gln Ser Pro Cys Gly Arg Ala
Thr Ser Ser Ser Arg Val 420 425 430Trp Trp Asp Ser Ser Phe Leu Gly
Gly Val Val His Leu Glu Ala Gly 435 440 445Glu Glu Val Val Val Arg
Val Leu Asp Glu Arg Leu Val Arg Leu Arg 450 455 460Asp Gly Thr Arg
Ser Tyr Phe Gly Ala Phe Met Val Gly Ser Ser Ser465 470 475 480Ser
Ser Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro 485 490
495Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
500 505 510Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr 515 520 525Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn 530 535 540Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg545 550 555 560Glu Glu Gln Tyr Ser Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val 565 570 575Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 580 585 590Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 595 600 605Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 610 615
620Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe625 630 635 640Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu 645 650 655Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe 660 665 670Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly 675 680 685Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr 690 695 700Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Ser Ser Ser Ser Ser Ser705 710 715 720Ala
Trp Ser His Pro Gln Phe Glu Lys 7251621PRTArtificial 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 2025715PRTArtificial
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 Val Asn
Pro Ala Ala His Leu Thr Gly Ala Asn 20 25 30Ser Ser Leu Thr Gly Ser
Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu 35 40 45Gly Leu Ala Phe Leu
Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val 50 55 60Val Thr Lys Ala
Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly65 70 75 80Gly Val
Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu 85 90 95Tyr
Lys Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser 100 105
110Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp
115 120 125Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu Ala Gly
Glu Glu 130 135 140Val Val Val Arg Val Leu Asp Glu Arg Leu Val Arg
Leu Arg Asp Gly145 150 155 160Thr Arg Ser Tyr Phe Gly Ala Phe Met
Val Gly Ser Gly Ser Gly Asn 165 170 175Gly Ser Pro Ala Ala His Leu
Thr Gly Ala Asn Ser Ser Leu Thr Gly 180 185 190Ser Gly Gly Pro Leu
Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu 195 200 205Arg Gly Leu
Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala Gly 210 215 220Tyr
Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro225 230
235 240Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr
Pro 245 250 255Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln
Ser Pro Cys 260 265 270Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp
Asp Ser Ser Phe Leu 275 280 285Gly Gly Val Val His Leu Glu Ala Gly
Glu Glu Val Val Val Arg Val 290 295 300Leu Asp Glu Arg Leu Val Arg
Leu Arg Asp Gly Thr Arg Ser Tyr Phe305 310 315 320Gly Ala Phe Met
Val Gly Ser Gly Ser Pro Ala Ala His Leu Thr Gly 325 330 335Ala Asn
Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr 340 345
350Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala
355 360 365Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys
Val Gln 370 375 380Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser
Thr Ile Thr His385 390 395 400Gly Leu Tyr Lys Arg Thr Pro Arg Tyr
Pro Glu Glu Leu Glu Leu Leu 405 410 415Val Ser Gln Gln Ser Pro Cys
Gly Arg Ala Thr Ser Ser Ser Arg Val 420 425 430Trp Trp Asp Ser Ser
Phe Leu Gly Gly Val Val His Leu Glu Ala Gly 435 440 445Glu Glu Val
Val Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg 450 455 460Asp
Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val Gly Ser Ser Ser465 470
475 480Ser Ser Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro
Pro 485 490 495Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro 500 505 510Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr 515 520 525Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn 530 535 540Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg545 550 555 560Glu Glu Gln Tyr
Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 565 570 575Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 580 585
590Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
595 600 605Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu 610 615 620Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe625 630 635 640Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu 645 650 655Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 660 665 670Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 675 680 685Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 690 695 700Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys705 710
71526714PRTArtificial SequenceSEQ36 fused to wtFc (incl. signal
peptide; no strep tag) 26Met Glu Thr Asp Thr Leu Leu Val Phe Val
Leu Leu Val Trp Val Pro1 5 10 15Ala Gly Asn Gly Glu Val Asn Pro Ala
Ala His Leu Thr Gly Ala Asn 20 25 30Ser Ser Leu Thr Gly Ser Gly Gly
Pro Leu Leu Trp Glu Thr Gln Leu 35 40 45Gly Leu Ala Phe Leu Arg Gly
Leu Ser Tyr His Asp Gly Ala Leu Val 50 55 60Val Thr Lys Ala Gly Tyr
Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly65 70 75 80Gly Val Gly Cys
Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu 85 90 95Tyr Lys Arg
Thr Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser 100 105 110Gln
Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp 115 120
125Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Glu
130 135 140Val Val Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg
Asp Gly145 150 155 160Thr Arg Ser Tyr Phe Gly Ala Phe Met Val Gly
Ser Gly Ser Gly Asn 165 170 175Gly Ser Pro Ala Ala His Leu Thr Gly
Ala Asn Ser Ser Leu Thr Gly 180 185 190Ser Gly Gly Pro Leu Leu Trp
Glu Thr Gln Leu Gly Leu Ala Phe Leu 195 200 205Arg Gly Leu Ser Tyr
His Asp Gly Ala Leu Val Val Thr Lys Ala Gly 210 215 220Tyr Tyr Tyr
Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro225 230 235
240Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro
245 250 255Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln Ser
Pro Cys 260 265 270Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp Asp
Ser Ser Phe Leu 275 280 285Gly Gly Val Val His Leu Glu Ala Gly Glu
Glu Val Val Val Arg Val 290 295 300Leu Asp Glu Arg Leu Val Arg Leu
Arg Asp Gly Thr Arg Ser Tyr Phe305 310 315 320Gly Ala Phe Met Val
Gly Ser Gly Ser Pro Ala Ala His Leu Thr Gly 325 330 335Ala Asn Ser
Ser Leu Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr 340 345 350Gln
Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala 355 360
365Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln
370 375 380Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile
Thr His385 390 395 400Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu
Glu Leu Glu Leu Leu 405 410 415Val Ser Gln Gln Ser Pro Cys Gly Arg
Ala Thr Ser Ser Ser Arg Val 420 425 430Trp Trp Asp Ser Ser Phe Leu
Gly Gly Val Val His Leu Glu Ala Gly 435 440 445Glu Glu Val Val Val
Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg 450 455 460Asp Gly Thr
Arg Ser Tyr Phe Gly Ala Phe Met Val Gly Ser Ser Ser465 470 475
480Ser Ser Ser Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro
485 490 495Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro 500 505 510Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 515 520 525Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 530 535 540Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu545 550 555 560Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 565 570 575His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 580 585 590Lys
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 595 600
605Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
610 615 620Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr625 630 635 640Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 645 650 655Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 660 665 670Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 675 680 685Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 690 695 700Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys705 71027695PRTArtificial SequenceSEQ36
fused to deglyco-Fc (no strep tag) 27Glu Val Asn Pro Ala Ala His
Leu Thr Gly Ala Asn Ser Ser Leu Thr1 5 10 15Gly Ser Gly Gly Pro Leu
Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe 20 25 30Leu Arg Gly Leu Ser
Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala 35 40 45Gly Tyr Tyr Tyr
Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys 50 55 60Pro Leu Gly
Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr65 70 75 80Pro
Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro 85 90
95Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser Ser Phe
100 105 110Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Glu Val Val
Val Arg 115 120 125Val Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly
Thr Arg Ser Tyr 130 135 140Phe Gly Ala Phe Met Val Gly Ser Gly Ser
Gly Asn Gly Ser Pro Ala145 150 155 160Ala His Leu Thr Gly Ala Asn
Ser Ser Leu Thr Gly Ser Gly Gly Pro 165 170 175Leu Leu Trp Glu Thr
Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser 180 185 190Tyr His Asp
Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile 195 200 205Tyr
Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala 210 215
220Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro
Glu225 230 235 240Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys
Gly Arg Ala Thr 245 250 255Ser Ser Ser Arg Val Trp Trp Asp Ser Ser
Phe Leu Gly Gly Val Val 260 265 270His Leu Glu Ala Gly Glu Glu Val
Val Val Arg Val Leu Asp Glu Arg 275 280 285Leu Val Arg Leu Arg Asp
Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met 290 295 300Val Gly Ser Gly
Ser Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser305 310 315 320Leu
Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu 325 330
335Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr
340 345 350Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly
Gly Val 355 360 365Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr His
Gly Leu Tyr Lys 370 375 380Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu
Leu Leu Val Ser Gln Gln385 390 395 400Ser Pro Cys Gly Arg Ala Thr
Ser Ser Ser Arg Val Trp Trp Asp Ser 405 410 415Ser Phe Leu Gly Gly
Val Val His Leu Glu Ala Gly Glu Glu Val Val 420 425 430Val Arg Val
Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly Thr Arg 435 440 445Ser
Tyr Phe Gly Ala Phe Met Val Gly Ser Ser Ser Ser Ser Ser Ser 450 455
460Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro465 470 475 480Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys 485 490 495Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val 500 505 510Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp 515 520 525Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 530 535 540Ser Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp545 550 555 560Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 565 570
575Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
580 585 590Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys 595 600 605Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 610 615 620Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys625 630 635 640Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 645 650 655Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 660 665 670Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 675 680 685Leu
Ser Leu Ser Pro Gly Lys 690 69528709PRTArtificial SequenceSEQ36
fused to deglyco-Fc (incl. strep tag) 28Glu Val Asn Pro Ala Ala His
Leu Thr Gly Ala Asn Ser Ser Leu Thr1 5 10 15Gly Ser Gly Gly Pro Leu
Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe 20 25 30Leu Arg Gly Leu Ser
Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala 35 40 45Gly Tyr Tyr Tyr
Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys 50 55 60Pro Leu Gly
Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr65 70 75 80Pro
Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro 85 90
95Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser Ser Phe
100 105 110Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Glu Val Val
Val Arg 115 120 125Val Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly
Thr Arg Ser Tyr 130 135 140Phe Gly Ala Phe Met Val Gly Ser Gly Ser
Gly Asn Gly Ser Pro Ala145 150 155 160Ala His Leu Thr Gly Ala Asn
Ser Ser Leu Thr Gly Ser Gly Gly Pro 165 170 175Leu Leu Trp Glu Thr
Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser 180 185 190Tyr His Asp
Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile 195 200 205Tyr
Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala 210 215
220Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro
Glu225 230 235 240Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys
Gly Arg Ala Thr 245 250 255Ser Ser Ser Arg Val Trp Trp Asp Ser Ser
Phe Leu Gly Gly Val Val 260 265 270His Leu Glu Ala Gly Glu Glu Val
Val Val Arg Val Leu Asp Glu Arg 275 280 285Leu Val Arg Leu Arg Asp
Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met 290 295 300Val Gly Ser Gly
Ser Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser305 310 315 320Leu
Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu 325 330
335Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr
340 345 350Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly
Gly Val 355 360 365Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr His
Gly Leu Tyr Lys 370 375 380Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu
Leu Leu Val Ser Gln Gln385 390 395 400Ser Pro Cys Gly Arg Ala Thr
Ser Ser Ser Arg Val Trp Trp Asp Ser 405 410 415Ser Phe Leu Gly Gly
Val Val His Leu Glu Ala Gly Glu Glu Val Val 420 425 430Val Arg Val
Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly Thr Arg 435 440 445Ser
Tyr Phe Gly Ala Phe Met Val Gly Ser Ser Ser Ser Ser Ser Ser 450 455
460Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro465 470 475 480Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys 485 490 495Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val 500 505 510Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp 515 520 525Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 530 535 540Ser Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp545 550 555 560Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 565 570
575Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
580 585 590Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys 595 600 605Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 610 615 620Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys625 630 635 640Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 645 650 655Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 660 665 670Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 675 680 685Leu
Ser Leu Ser Pro Gly Ser Ser Ser Ser Ser Ser Ala Trp Ser His 690 695
700Pro Gln Phe Glu Lys70529694PRTArtificial SequenceSEQ36 fused to
wt-Fc (no signal peptide, no strep tag) 29Glu Val Asn Pro Ala Ala
His Leu Thr Gly Ala Asn Ser Ser Leu Thr1 5 10 15Gly Ser Gly Gly Pro
Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe 20 25 30Leu Arg Gly Leu
Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala 35 40 45Gly Tyr Tyr
Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys 50 55 60Pro Leu
Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr65 70 75
80Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro
85 90 95Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser Ser
Phe 100 105 110Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Glu Val
Val Val Arg 115 120 125Val Leu Asp Glu Arg Leu Val Arg Leu Arg Asp
Gly Thr Arg Ser Tyr 130 135 140Phe Gly Ala Phe Met Val Gly Ser Gly
Ser Gly Asn Gly Ser Pro Ala145 150 155 160Ala His Leu Thr Gly Ala
Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro 165 170 175Leu Leu Trp Glu
Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser 180 185 190Tyr His
Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile 195 200
205Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala
210 215 220Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr
Pro Glu225 230 235 240Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro
Cys Gly Arg Ala Thr 245 250 255Ser Ser Ser Arg Val Trp Trp Asp Ser
Ser Phe Leu Gly Gly Val Val 260 265 270His Leu Glu Ala Gly Glu Glu
Val Val Val Arg Val Leu Asp Glu Arg 275 280 285Leu Val Arg Leu Arg
Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met 290 295 300Val Gly Ser
Gly Ser Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser305 310
315 320Leu Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly
Leu 325 330 335Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu
Val Val Thr 340 345 350Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val
Gln Leu Gly Gly Val 355 360 365Gly Cys Pro Leu Gly Leu Ala Ser Thr
Ile Thr His Gly Leu Tyr Lys 370 375 380Arg Thr Pro Arg Tyr Pro Glu
Glu Leu Glu Leu Leu Val Ser Gln Gln385 390 395 400Ser Pro Cys Gly
Arg Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser 405 410 415Ser Phe
Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Glu Val Val 420 425
430Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly Thr Arg
435 440 445Ser Tyr Phe Gly Ala Phe Met Val Gly Ser Ser Ser Ser Ser
Ser Ser 450 455 460Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro465 470 475 480Pro Val Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 485 490 495Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 500 505 510Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 515 520 525Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 530 535 540Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp545 550
555 560Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro 565 570 575Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 580 585 590Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn 595 600 605Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 610 615 620Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr625 630 635 640Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 645 650 655Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 660 665
670Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
675 680 685Ser Leu Ser Pro Gly Lys 69030695PRTArtificial
SequenceSeq27 with altered first RBD module E1Q (equals E91Q in
Seq1) 30Gln Val Asn Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu
Thr1 5 10 15Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu
Ala Phe 20 25 30Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val
Thr Lys Ala 35 40 45Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly
Gly Val Gly Cys 50 55 60Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly
Leu Tyr Lys Arg Thr65 70 75 80Pro Arg Tyr Pro Glu Glu Leu Glu Leu
Leu Val Ser Gln Gln Ser Pro 85 90 95Cys Gly Arg Ala Thr Ser Ser Ser
Arg Val Trp Trp Asp Ser Ser Phe 100 105 110Leu Gly Gly Val Val His
Leu Glu Ala Gly Glu Glu Val Val Val Arg 115 120 125Val Leu Asp Glu
Arg Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr 130 135 140Phe Gly
Ala Phe Met Val Gly Ser Gly Ser Gly Asn Gly Ser Pro Ala145 150 155
160Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro
165 170 175Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly
Leu Ser 180 185 190Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala Gly
Tyr Tyr Tyr Ile 195 200 205Tyr Ser Lys Val Gln Leu Gly Gly Val Gly
Cys Pro Leu Gly Leu Ala 210 215 220Ser Thr Ile Thr His Gly Leu Tyr
Lys Arg Thr Pro Arg Tyr Pro Glu225 230 235 240Glu Leu Glu Leu Leu
Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr 245 250 255Ser Ser Ser
Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val 260 265 270His
Leu Glu Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg 275 280
285Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met
290 295 300Val Gly Ser Gly Ser Pro Ala Ala His Leu Thr Gly Ala Asn
Ser Ser305 310 315 320Leu Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu
Thr Gln Leu Gly Leu 325 330 335Ala Phe Leu Arg Gly Leu Ser Tyr His
Asp Gly Ala Leu Val Val Thr 340 345 350Lys Ala Gly Tyr Tyr Tyr Ile
Tyr Ser Lys Val Gln Leu Gly Gly Val 355 360 365Gly Cys Pro Leu Gly
Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys 370 375 380Arg Thr Pro
Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln385 390 395
400Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser
405 410 415Ser Phe Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Glu
Val Val 420 425 430Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg
Asp Gly Thr Arg 435 440 445Ser Tyr Phe Gly Ala Phe Met Val Gly Ser
Ser Ser Ser Ser Ser Ser 450 455 460Ser Gly Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro465 470 475 480Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 485 490 495Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 500 505 510Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 515 520
525Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
530 535 540Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp545 550 555 560Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu 565 570 575Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 580 585 590Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys 595 600 605Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 610 615 620Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys625 630 635
640Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
645 650 655Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 660 665 670Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 675 680 685Leu Ser Leu Ser Pro Gly Lys 690
69531701PRTArtificial SequenceSeq39 fused to human IGG1 Fc of Seq13
31Gln Val Asn Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr1
5 10 15Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala
Phe 20 25 30Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr
Lys Ala 35 40 45Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly
Val Gly Cys 50 55 60Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu
Tyr Lys Arg Thr65 70 75 80Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu
Val Ser Gln Gln Ser Pro 85 90 95Cys Gly Arg Ala Thr Ser Ser Ser Arg
Val Trp Trp Asp Ser Ser Phe 100 105 110Leu Gly Gly Val Val His Leu
Glu Ala Gly Glu Glu Val Val Val Arg 115 120 125Val Leu Asp Glu Arg
Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr 130 135 140Phe Gly Ala
Phe Met Val Gly Ser Gly Ser Gly Asn Gly Ser Asn Pro145 150 155
160Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly
165 170 175Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg
Gly Leu 180 185 190Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala
Gly Tyr Tyr Tyr 195 200 205Ile Tyr Ser Lys Val Gln Leu Gly Gly Val
Gly Cys Pro Leu Gly Leu 210 215 220Ala Ser Thr Ile Thr His Gly Leu
Tyr Lys Arg Thr Pro Arg Tyr Pro225 230 235 240Glu Glu Leu Glu Leu
Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala 245 250 255Thr Ser Ser
Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val 260 265 270Val
His Leu Glu Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu 275 280
285Arg Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe
290 295 300Met Val Gly Ser Gly Ser Gly Asn Gly Ser Asn Pro Ala Ala
His Leu305 310 315 320Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly
Gly Pro Leu Leu Trp 325 330 335Glu Thr Gln Leu Gly Leu Ala Phe Leu
Arg Gly Leu Ser Tyr His Asp 340 345 350Gly Ala Leu Val Val Thr Lys
Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys 355 360 365Val Gln Leu Gly Gly
Val Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile 370 375 380Thr His Gly
Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu385 390 395
400Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser
405 410 415Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His
Leu Glu 420 425 430Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu
Arg Leu Val Arg 435 440 445Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly
Ala Phe Met Val Gly Ser 450 455 460Ser Ser Ser Ser Ser Ser Ser Gly
Ser Cys Asp Lys Thr His Thr Cys465 470 475 480Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 485 490 495Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 500 505 510Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 515 520
525Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
530 535 540Pro Arg Glu Glu Gln Tyr Ser Ser Thr Tyr Arg Val Val Ser
Val Leu545 550 555 560Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys 565 570 575Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys 580 585 590Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser 595 600 605Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 610 615 620Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln625 630 635
640Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
645 650 655Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln 660 665 670Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn 675 680 685His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 690 695 70032692PRTArtificial SequenceSeq30 with
shorter hinge linker 32Gln Val Asn Pro Ala Ala His Leu Thr Gly Ala
Asn Ser Ser Leu Thr1 5 10 15Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr
Gln Leu Gly Leu Ala Phe 20 25 30Leu Arg Gly Leu Ser Tyr His Asp Gly
Ala Leu Val Val Thr Lys Ala 35 40 45Gly Tyr Tyr Tyr Ile Tyr Ser Lys
Val Gln Leu Gly Gly Val Gly Cys 50 55 60Pro Leu Gly Leu Ala Ser Thr
Ile Thr His Gly Leu Tyr Lys Arg Thr65 70 75 80Pro Arg Tyr Pro Glu
Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro 85 90 95Cys Gly Arg Ala
Thr Ser Ser Ser Arg Val Trp Trp Asp Ser Ser Phe 100 105 110Leu Gly
Gly Val Val His Leu Glu Ala Gly Glu Glu Val Val Val Arg 115 120
125Val Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr
130 135 140Phe Gly Ala Phe Met Val Gly Ser Gly Ser Gly Asn Gly Ser
Pro Ala145 150 155 160Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr
Gly Ser Gly Gly Pro 165 170 175Leu Leu Trp Glu Thr Gln Leu Gly Leu
Ala Phe Leu Arg Gly Leu Ser 180 185 190Tyr His Asp Gly Ala Leu Val
Val Thr Lys Ala Gly Tyr Tyr Tyr Ile 195 200 205Tyr Ser Lys Val Gln
Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala 210 215 220Ser Thr Ile
Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu225 230 235
240Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr
245 250 255Ser Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly
Val Val 260 265 270His Leu Glu Ala Gly Glu Glu Val Val Val Arg Val
Leu Asp Glu Arg 275 280 285Leu Val Arg Leu Arg Asp Gly Thr Arg Ser
Tyr Phe Gly Ala Phe Met 290 295 300Val Gly Ser Gly Ser Pro Ala Ala
His Leu Thr Gly Ala Asn Ser Ser305 310 315 320Leu Thr Gly Ser Gly
Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu 325 330 335Ala Phe Leu
Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr 340 345 350Lys
Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val 355 360
365Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys
370 375 380Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser
Gln Gln385 390 395 400Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg
Val Trp Trp Asp Ser 405 410 415Ser Phe Leu Gly Gly Val Val His Leu
Glu Ala Gly Glu Glu Val Val 420 425 430Val Arg Val Leu Asp Glu Arg
Leu Val Arg Leu Arg Asp Gly Thr Arg 435 440 445Ser Tyr Phe Gly Ala
Phe Met Val Gly Ser Ser Ser Ser Ser Gly Ser 450 455 460Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu465 470 475
480Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
485 490 495Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 500 505 510His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 515 520 525Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Ser Ser Thr 530 535 540Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn545 550 555 560Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 565 570 575Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 580 585 590Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 595 600
605Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
610 615 620Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro625 630 635 640Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr 645 650 655Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 660 665
670Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
675 680 685Ser Pro Gly Lys 69033699PRTArtificial
SequenceConfiguration with deletion of N93 in RBD 2 and 3 33Glu Val
Asn Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr1 5 10 15Gly
Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe 20 25
30Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala
35 40 45Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly
Cys 50 55 60Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys
Arg Thr65 70 75 80Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser
Gln Gln Ser Pro 85 90 95Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp
Trp Asp Ser Ser Phe 100 105 110Leu Gly Gly Val Val His Leu Glu Ala
Gly Glu Glu Val Val Val Arg 115 120 125Val Leu Asp Glu Arg Leu Val
Arg Leu Arg Asp Gly Thr Arg Ser Tyr 130 135 140Phe Gly Ala Phe Met
Val Gly Ser Gly Ser Gly Asn Gly Ser Pro Ala145 150 155 160Ala His
Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro 165 170
175Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser
180 185 190Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr
Tyr Ile 195 200 205Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro
Leu Gly Leu Ala 210 215 220Ser Thr Ile Thr His Gly Leu Tyr Lys Arg
Thr Pro Arg Tyr Pro Glu225 230 235 240Glu Leu Glu Leu Leu Val Ser
Gln Gln Ser Pro Cys Gly Arg Ala Thr 245 250 255Ser Ser Ser Arg Val
Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val 260 265 270His Leu Glu
Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg 275 280 285Leu
Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met 290 295
300Val Gly Ser Gly Ser Gly Asn Gly Ser Pro Ala Ala His Leu Thr
Gly305 310 315 320Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu
Leu Trp Glu Thr 325 330 335Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu
Ser Tyr His Asp Gly Ala 340 345 350Leu Val Val Thr Lys Ala Gly Tyr
Tyr Tyr Ile Tyr Ser Lys Val Gln 355 360 365Leu Gly Gly Val Gly Cys
Pro Leu Gly Leu Ala Ser Thr Ile Thr His 370 375 380Gly Leu Tyr Lys
Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu385 390 395 400Val
Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg Val 405 410
415Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu Ala Gly
420 425 430Glu Glu Val Val Val Arg Val Leu Asp Glu Arg Leu Val Arg
Leu Arg 435 440 445Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val
Gly Ser Ser Ser 450 455 460Ser Ser Ser Ser Ser Gly Ser Cys Asp Lys
Thr His Thr Cys Pro Pro465 470 475 480Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro 485 490 495Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 500 505 510Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 515 520 525Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 530 535
540Glu Glu Gln Tyr Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val545 550 555 560Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 565 570 575Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys 580 585 590Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu 595 600 605Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe 610 615 620Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu625 630 635 640Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 645 650
655Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
660 665 670Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr 675 680 685Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 690
69534699PRTArtificial SequenceConfiguration SEQ33 with N terminal
amino acid exchange E1Q 34Gln Val Asn Pro Ala Ala His Leu Thr Gly
Ala Asn Ser Ser Leu Thr1 5 10 15Gly Ser Gly Gly Pro Leu Leu Trp Glu
Thr Gln Leu Gly Leu Ala Phe 20 25 30Leu Arg Gly Leu Ser Tyr His Asp
Gly Ala Leu Val Val Thr Lys Ala 35 40 45Gly Tyr Tyr Tyr Ile Tyr Ser
Lys Val Gln Leu Gly Gly Val Gly Cys 50 55 60Pro Leu Gly Leu Ala Ser
Thr Ile Thr His Gly Leu Tyr Lys Arg Thr65 70 75 80Pro Arg Tyr Pro
Glu Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro 85 90 95Cys Gly Arg
Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser Ser Phe 100 105 110Leu
Gly Gly Val Val His Leu Glu Ala Gly Glu Glu Val Val Val Arg 115 120
125Val Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr
130 135 140Phe Gly Ala Phe Met Val Gly Ser Gly Ser Gly Asn Gly Ser
Pro Ala145 150 155 160Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr
Gly Ser Gly Gly Pro 165 170 175Leu Leu Trp Glu Thr Gln Leu Gly Leu
Ala Phe Leu Arg Gly Leu Ser 180 185 190Tyr His Asp Gly Ala Leu Val
Val Thr Lys Ala Gly Tyr Tyr Tyr Ile 195 200 205Tyr Ser Lys Val Gln
Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala 210 215 220Ser Thr Ile
Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu225 230 235
240Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr
245 250 255Ser Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly
Val Val 260 265 270His Leu Glu Ala Gly Glu Glu Val Val Val Arg Val
Leu Asp Glu Arg 275 280 285Leu Val Arg Leu Arg Asp Gly Thr Arg Ser
Tyr Phe Gly Ala Phe Met 290 295 300Val Gly Ser Gly Ser Gly Asn Gly
Ser Pro Ala Ala His Leu Thr Gly305 310 315 320Ala Asn Ser Ser Leu
Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr 325 330 335Gln Leu Gly
Leu Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala 340 345 350Leu
Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln 355 360
365Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr His
370 375 380Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu
Leu Leu385 390 395 400Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr
Ser Ser Ser Arg Val 405 410 415Trp Trp Asp Ser Ser Phe Leu Gly Gly
Val Val His Leu Glu Ala Gly 420 425 430Glu Glu Val Val Val Arg Val
Leu Asp Glu Arg Leu Val Arg Leu Arg 435 440 445Asp Gly Thr Arg Ser
Tyr Phe Gly Ala Phe Met Val Gly Ser Ser Ser 450 455 460Ser Ser Ser
Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro465 470 475
480Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
485 490 495Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr 500 505 510Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn 515 520 525Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg 530 535 540Glu Glu Gln Tyr Ser Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val545 550 555 560Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 565 570 575Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 580 585 590Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 595 600
605Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
610 615 620Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu625 630 635 640Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe 645 650 655Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly 660 665 670Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr 675 680 685Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 690 69535696PRTArtificial
SequenceConfiguration Seq33 with shorter hinge linker 35Glu Val Asn
Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr1 5 10 15Gly Ser
Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe 20 25 30Leu
Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala 35 40
45Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys
50 55 60Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys Arg
Thr65 70 75 80Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln
Gln Ser Pro 85 90 95Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp
Asp Ser Ser Phe 100 105 110Leu Gly Gly Val Val His Leu Glu Ala Gly
Glu Glu Val Val Val Arg 115 120 125Val Leu Asp Glu Arg Leu Val Arg
Leu Arg Asp Gly Thr Arg Ser Tyr 130 135 140Phe Gly Ala Phe Met Val
Gly Ser Gly Ser Gly Asn Gly Ser Pro Ala145 150 155 160Ala His Leu
Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro 165 170 175Leu
Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser 180 185
190Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile
195 200 205Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly
Leu Ala 210 215 220Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro
Arg Tyr Pro Glu225 230 235 240Glu Leu Glu Leu Leu Val Ser Gln Gln
Ser Pro Cys Gly Arg Ala Thr 245 250 255Ser Ser Ser Arg Val Trp Trp
Asp Ser Ser Phe Leu Gly Gly Val Val 260 265 270His Leu Glu Ala Gly
Glu Glu Val Val Val Arg Val Leu Asp Glu Arg 275 280 285Leu Val Arg
Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met 290 295 300Val
Gly Ser Gly Ser Gly Asn Gly Ser Pro Ala Ala His Leu Thr Gly305 310
315 320Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu
Thr 325 330 335Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr His
Asp Gly Ala 340 345 350Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile
Tyr Ser Lys Val Gln 355 360 365Leu Gly Gly Val Gly Cys Pro Leu Gly
Leu Ala Ser Thr Ile Thr His 370 375 380Gly Leu Tyr Lys Arg Thr Pro
Arg Tyr Pro Glu Glu Leu Glu Leu Leu385 390 395 400Val Ser Gln Gln
Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg Val 405 410 415Trp Trp
Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu Ala Gly 420 425
430Glu Glu Val Val Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg
435 440 445Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val Gly Ser
Ser Ser 450 455 460Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala465 470 475 480Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro 485 490 495Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val 500 505 510Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 515 520 525Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 530 535 540Tyr
Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln545 550
555 560Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala 565 570 575Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro 580 585 590Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr 595 600 605Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser 610 615 620Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr625 630 635 640Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 645 650 655Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 660 665
670Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
675 680 685Ser Leu Ser Leu Ser Pro Gly Lys 690
69536456PRTArtificial SequenceExemplary scLIGHT-RBD module 36Glu
Val Asn Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr1 5 10
15Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe
20 25 30Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys
Ala 35 40 45Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val
Gly Cys 50 55 60Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr
Lys Arg Thr65 70 75 80Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val
Ser Gln Gln Ser Pro 85 90 95Cys Gly Arg Ala Thr Ser Ser Ser Arg Val
Trp Trp Asp Ser Ser Phe 100 105 110Leu Gly Gly Val Val His Leu Glu
Ala Gly Glu Glu Val Val Val Arg 115 120 125Val Leu Asp Glu Arg Leu
Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr 130 135 140Phe Gly Ala Phe
Met Val Gly Ser Gly Ser Gly Asn Gly Ser Pro Ala145 150 155 160Ala
His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro 165 170
175Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser
180 185 190Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr
Tyr Ile 195 200 205Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro
Leu Gly Leu Ala 210 215 220Ser Thr Ile Thr His Gly Leu Tyr Lys Arg
Thr Pro Arg Tyr Pro Glu225 230 235 240Glu Leu Glu Leu Leu Val Ser
Gln Gln Ser Pro Cys Gly Arg Ala Thr 245 250 255Ser Ser Ser Arg Val
Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val 260 265 270His Leu Glu
Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg 275 280 285Leu
Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met 290 295
300Val Gly Ser Gly Ser Pro Ala Ala His Leu Thr Gly Ala Asn Ser
Ser305 310 315 320Leu Thr
Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu 325 330
335Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr
340 345 350Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly
Gly Val 355 360 365Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr His
Gly Leu Tyr Lys 370 375 380Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu
Leu Leu Val Ser Gln Gln385 390 395 400Ser Pro Cys Gly Arg Ala Thr
Ser Ser Ser Arg Val Trp Trp Asp Ser 405 410 415Ser Phe Leu Gly Gly
Val Val His Leu Glu Ala Gly Glu Glu Val Val 420 425 430Val Arg Val
Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly Thr Arg 435 440 445Ser
Tyr Phe Gly Ala Phe Met Val 450 455372192DNAArtificial SequenceDNA
sequence encoding PROTEIN A (Seq25) 37aagctttagg gataacaggg
taatagccgc caccatggag actgacaccc tgctggtgtt 60cgtgctgctg gtctgggtgc
ctgcaggaaa tggagaagtg aaccccgccg cccatctgac 120cggcgctaac
agcagcctga caggttctgg cggacccctc ctgtgggaga cacaactggg
180cctggccttc ctgaggggcc tgagctacca tgatggcgcc ctggtggtga
ccaaggccgg 240ctactactac atctattcca aggtccagct cggaggcgtg
ggatgccctc tgggactggc 300ctccaccatc acccacggcc tgtacaagcg
gacccctagg taccccgagg aactggaact 360gctcgtctcc caacagagcc
cttgcggcag ggctacctcc tccagcaggg tgtggtggga 420ctccagcttc
ctgggaggcg tcgtccacct ggaggctgga gaagaagtgg tggtgcgggt
480cctggacgaa aggctggtga ggctcaggga cggcacccgg tcctactttg
gagcctttat 540ggtgggctcc ggatctggta acggcagccc cgctgctcat
ctgacaggcg ccaatagcag 600cctgacaggc agcggaggcc ctctgctgtg
ggaaacacag ctgggcctgg cctttctgag 660gggcctgtcc tatcacgatg
gagccctggt ggtgaccaaa gccggctatt actatatcta 720cagcaaggtg
cagctgggcg gagtgggatg tcctctgggc ctggcctcca ccatcacaca
780cggactgtat aagcggacac ctaggtatcc cgaagagctg gagctcctgg
tgtcccagca 840aagcccttgt ggaagggcta cctccagcag cagggtctgg
tgggactcct ccttcctggg 900cggcgtggtc catctggaag ctggcgagga
ggtggtggtg agggtcctgg atgagaggct 960ggtcaggctg agggatggca
cccggtccta ttttggcgct ttcatggtgg gctctggtag 1020ccctgccgcc
cacctgacag gagccaacag cagcctgaca ggaagcggcg gccctctgct
1080gtgggagacc caactgggcc tggccttcct gcggggcctc tcctaccacg
acggcgctct 1140ggtggtgacc aaggccggct attattatat ctactccaaa
gtccagctgg gaggcgtcgg 1200ctgtcctctc ggactggctt ccaccatcac
ccatggcctg tacaaaagga cccctaggta 1260ccccgaagag ttagaactgc
tggtctccca gcagtcccct tgcggaaggg ccacaagcag 1320cagccgggtg
tggtgggact ccagctttct gggcggagtg gtgcacctgg aagccggaga
1380ggaggtcgtg gtcagggtcc tggatgaaag gctggtgcgg ctgagggatg
gcaccaggtc 1440ctatttcggc gccttcatgg tcggatcctc gagttcatcg
tcctcatccg gctcatgtga 1500taagacccac acctgccctc cctgtcctgc
ccctgagctg ctgggcggac cttctgtgtt 1560cctgttcccc cccaagccta
aggacaccct gatgatctcc aggacccctg aggtgacctg 1620tgtggtggtg
gacgtgtctc acgaagatcc cgaggtgaag ttcaactggt acgtggacgg
1680cgtggaggtc cacaacgcca agaccaagcc tagggaggag cagtacagct
ccacctaccg 1740ggtggtgtct gtgctgaccg tgctgcacca ggattggctg
aacggaaagg agtataagtg 1800taaggtctcc aacaaggccc tgcctgcccc
catcgagaaa accatctcca aggccaaggg 1860ccagcctcgg gagcctcagg
tgtacaccct gcctcctagc agggaggaga tgaccaagaa 1920ccaggtgtcc
ctgacctgtc tggtgaaggg cttctaccct tccgatatcg ccgtggagtg
1980ggagtctaat ggccagcccg agaacaacta caagaccacc cctcctgtgc
tggactctga 2040cggctccttc ttcctgtact ccaagctgac cgtggacaag
tccagatggc agcagggcaa 2100cgtgttctcc tgctccgtga tgcacgaggc
cctgcacaat cactacaccc agaagtccct 2160gtctctgagt ccgggcaagt
aataggcgcg cc 219238227PRTArtificial SequenceLIGHT-RBD fused to
RB69 FOLDON (PROTEIN X) 38Met Glu Thr Asp Thr Leu Leu Val Phe Val
Leu Leu Val Trp Val Pro1 5 10 15Ala Gly Asn Gly Glu Val Asn Pro Ala
Ala His Leu Thr Gly Ala Asn 20 25 30Ser Ser Leu Thr Gly Ser Gly Gly
Pro Leu Leu Trp Glu Thr Gln Leu 35 40 45Gly Leu Ala Phe Leu Arg Gly
Leu Ser Tyr His Asp Gly Ala Leu Val 50 55 60Val Thr Lys Ala Gly Tyr
Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly65 70 75 80Gly Val Gly Cys
Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu 85 90 95Tyr Lys Arg
Thr Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser 100 105 110Gln
Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp 115 120
125Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Glu
130 135 140Val Val Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg
Asp Gly145 150 155 160Thr Arg Ser Tyr Phe Gly Ala Phe Met Val Gly
Ser Gly Ser Ser Gly 165 170 175Ser Ser Gly Ser Ser Gly Ser Gly Tyr
Ile Glu Asp Ala Pro Ser Asp 180 185 190Gly Lys Phe Tyr Val Arg Lys
Asp Gly Ala Trp Val Glu Leu Pro Thr 195 200 205Ala Ser Gly Pro Ser
Ser Ser Ser Ser Ser Ala Trp Ser His Pro Gln 210 215 220Phe Glu
Lys22539462PRTArtificial SequenceExemplary scLIGHT-RBD module 39Glu
Val Asn Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr1 5 10
15Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe
20 25 30Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys
Ala 35 40 45Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val
Gly Cys 50 55 60Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr
Lys Arg Thr65 70 75 80Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val
Ser Gln Gln Ser Pro 85 90 95Cys Gly Arg Ala Thr Ser Ser Ser Arg Val
Trp Trp Asp Ser Ser Phe 100 105 110Leu Gly Gly Val Val His Leu Glu
Ala Gly Glu Glu Val Val Val Arg 115 120 125Val Leu Asp Glu Arg Leu
Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr 130 135 140Phe Gly Ala Phe
Met Val Gly Ser Gly Ser Gly Asn Gly Ser Asn Pro145 150 155 160Ala
Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly 165 170
175Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu
180 185 190Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr
Tyr Tyr 195 200 205Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys
Pro Leu Gly Leu 210 215 220Ala Ser Thr Ile Thr His Gly Leu Tyr Lys
Arg Thr Pro Arg Tyr Pro225 230 235 240Glu Glu Leu Glu Leu Leu Val
Ser Gln Gln Ser Pro Cys Gly Arg Ala 245 250 255Thr Ser Ser Ser Arg
Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val 260 265 270Val His Leu
Glu Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu 275 280 285Arg
Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe 290 295
300Met Val Gly Ser Gly Ser Gly Asn Gly Ser Asn Pro Ala Ala His
Leu305 310 315 320Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly
Pro Leu Leu Trp 325 330 335Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg
Gly Leu Ser Tyr His Asp 340 345 350Gly Ala Leu Val Val Thr Lys Ala
Gly Tyr Tyr Tyr Ile Tyr Ser Lys 355 360 365Val Gln Leu Gly Gly Val
Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile 370 375 380Thr His Gly Leu
Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu385 390 395 400Leu
Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser 405 410
415Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu
420 425 430Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg Leu
Val Arg 435 440 445Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe
Met Val 450 455 46040460PRTArtificial SequenceExemplary scLIGHT-RBD
module 40Glu Val Asn Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser
Leu Thr1 5 10 15Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly
Leu Ala Phe 20 25 30Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val
Val Thr Lys Ala 35 40 45Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu
Gly Gly Val Gly Cys 50 55 60Pro Leu Gly Leu Ala Ser Thr Ile Thr His
Gly Leu Tyr Lys Arg Thr65 70 75 80Pro Arg Tyr Pro Glu Glu Leu Glu
Leu Leu Val Ser Gln Gln Ser Pro 85 90 95Cys Gly Arg Ala Thr Ser Ser
Ser Arg Val Trp Trp Asp Ser Ser Phe 100 105 110Leu Gly Gly Val Val
His Leu Glu Ala Gly Glu Glu Val Val Val Arg 115 120 125Val Leu Asp
Glu Arg Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr 130 135 140Phe
Gly Ala Phe Met Val Gly Ser Gly Ser Gly Asn Gly Ser Pro Ala145 150
155 160Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly
Pro 165 170 175Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg
Gly Leu Ser 180 185 190Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala
Gly Tyr Tyr Tyr Ile 195 200 205Tyr Ser Lys Val Gln Leu Gly Gly Val
Gly Cys Pro Leu Gly Leu Ala 210 215 220Ser Thr Ile Thr His Gly Leu
Tyr Lys Arg Thr Pro Arg Tyr Pro Glu225 230 235 240Glu Leu Glu Leu
Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr 245 250 255Ser Ser
Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val 260 265
270His Leu Glu Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg
275 280 285Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala
Phe Met 290 295 300Val Gly Ser Gly Ser Gly Asn Gly Ser Pro Ala Ala
His Leu Thr Gly305 310 315 320Ala Asn Ser Ser Leu Thr Gly Ser Gly
Gly Pro Leu Leu Trp Glu Thr 325 330 335Gln Leu Gly Leu Ala Phe Leu
Arg Gly Leu Ser Tyr His Asp Gly Ala 340 345 350Leu Val Val Thr Lys
Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln 355 360 365Leu Gly Gly
Val Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr His 370 375 380Gly
Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu385 390
395 400Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg
Val 405 410 415Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His Leu
Glu Ala Gly 420 425 430Glu Glu Val Val Val Arg Val Leu Asp Glu Arg
Leu Val Arg Leu Arg 435 440 445Asp Gly Thr Arg Ser Tyr Phe Gly Ala
Phe Met Val 450 455 460
* * * * *