AU2020383176B2 - Fusion protein including modified interleukin-7 and TGF beta receptor II and use thereof - Google Patents
Fusion protein including modified interleukin-7 and TGF beta receptor II and use thereof Download PDFInfo
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- AU2020383176B2 AU2020383176B2 AU2020383176A AU2020383176A AU2020383176B2 AU 2020383176 B2 AU2020383176 B2 AU 2020383176B2 AU 2020383176 A AU2020383176 A AU 2020383176A AU 2020383176 A AU2020383176 A AU 2020383176A AU 2020383176 B2 AU2020383176 B2 AU 2020383176B2
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Classifications
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/52—Cytokines; Lymphokines; Interferons
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Abstract
The present invention relates to a fusion protein including modified interleukin-7 and TGF beta receptor II and use thereof. The fusion protein has a high production yield and can effectively inhibit cancer, and thus can be usefully utilized in the treatment of cancer or infectious diseases.
Description
[Invention Title]
FUSION PROTEIN COMPRISING MODIFIED INTERLEUKIN-7 AND TGF
This application claims priority to and the benefit of Korean Patent
Application No. 10-2019-0146805, filed on November 15, 2019, the disclosure of
which is incorporated herein by reference in its entirety.
[Technical Field]
The present invention relates to a fusion protein including modified
interleukin-7 and TGF-beta receptor II and use thereof.
[Background Art]
TGF beta receptor II (transforming growth factor beta receptor II, TBRII) is a
membrane protein of 70 to 80 kDa encoded by the TGFBR2 gene in humans. TBRII
forms a heterodimer with TBRI (TGF beta receptor I), and by binding with TGF-a to
transmit intracellular signals, it regulates the transcription of genes associated with cell
proliferation. TBRII consists of a C-terminal protein kinase domain and an N
terminal ecto domain. The ecto domain is a domain of a membrane protein that
extends into the extracellular space, and it forms a folded structure including a single
helix stabilized by 9 beta chains and 6 disulfide bonds in the strand.
In addition, TGF-a, which is known as a ligand of TBRII, is an
immunosuppressive cytokine that is overexpressed in cancer cells, and it is known as
one of the mechanisms of immune evasion of cancer cells, such as inhibiting the
proliferation of T cells due to TGF-a secreted from cancer cells and the like.
Recently, studies on the decoy receptor of TGF-a including a fragment of
TBRII have been actively conducted, but there is a problem in that the intracellular
activity and stability of the fragment of TBRII are poor.
Meanwhile, interleukin-7 (IL-7) is a cytokine that promotes an immune
response via B cells and T cells, and it particularly plays an important role in the
adaptive immune system. Specifically, IL-7 activates immune functions through the
survival and differentiation of T cells and B cells, the survival of lymphoid cells and
the promotion of natural killer cells (NK cells), and it is particularly important for the
development of T cells and B cells. It acts as a cofactor of the V(D)J rearrangement
of the pre-pro-B cell growth-stimulating factor and T cell receptor beta (TCRP) by
binding to the hepatocyte growth factor (HGF) (Muegge K, 1993, Science 261 (5117):
93-5).
However, when the recombinant IL-7 is produced for medical use, there are
problems in that a large amount of impurities is produced compared to general
recombinant proteins, it is easy to denature, and mass production is not easy.
Any discussion of the prior art throughout the specification should in no way
be considered as an admission that such prior art is widely known or forms part of the
common general knowledge in the field.
[Disclosure]
It is an object of the present invention to overcome or ameliorate at least one
of the disadvantages of the prior art, or to provide a useful alternative.
The present invention relates to a fusion protein including modified
interleukin-7 (IL-7) and TGF beta receptor II (TBRII).
The present invention also relates to a nucleic acid molecule which is isolated
and encodes the fusion protein.
The present invention also relates to an expression vector including the nucleic
acid molecule.
The present invention also relates to a host cell including the expression vector.
The present invention also relates to a pharmaceutical composition for
preventing or treating cancer or infectious disease, including the fusion protein as an
active ingredient.
The present invention also relates to the use of a fusion protein including
modified IL-7 and TBRII to produce a pharmaceutical preparation having a
prophylactic or therapeutic effect on cancer or infectious disease.
The present invention also relates to a method for preventing or treating cancer
or infectious disease, including the fusion protein as an active ingredient.
[Summary]
Unless the context clearly requires otherwise, throughout the description and
the claims, the words "comprise", "comprising", and the like are to be construed in an
inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the
sense of "including, but not limited to".
In a first aspect, the present invention provides a fusion protein, comprising modified interleukin 7 (IL-7) and TGF beta receptor II (transforming growth factor beta receptor II, TBRII).
In a second aspect, the present invention provides a nucleic acid molecule
which is isolated and encodes the fusion protein according to the first aspect.
In a third aspect, the present invention provides an expression vector,
comprising the nucleic acid molecule according to the second aspect.
In a fourth aspect, the present invention provides a host cell, comprising the
expression vector of the third aspect.
In addition, the present invention provides a fusion protein including modified
interleukin-7 (IL-7) and TGF beta receptor II (TBRII).
In addition, the present invention provides a nucleic acid molecule which is
isolated and encodes the fusion protein.
In addition, the present invention provides an expression vector including the
nucleic acid molecule.
In addition, the present invention provides a host cell including the expression
vector.
In addition, the present invention provides a pharmaceutical composition for
3a preventing or treating cancer or infectious disease, including the fusion protein as an active ingredient.
In addition, the present invention provides the use of a fusion protein including
modified IL-7 and TBRII to produce a pharmaceutical preparation having a
prophylactic or therapeutic effect on cancer or infectious disease.
In addition, the present invention provides a method for preventing or treating
cancer or infectious disease, including the fusion protein as an active ingredient.
[Advantageous Effects]
Since the fusion protein including modified interleukin-7 (IL-7) and TGF beta
receptor II (TBRII) according to the present invention has a high production yield and
can effectively inhibit cancer, it can be widely used in the treatment of cancer or
infectious diseases.
[Description of Drawings]
FIG. 1 shows the gene constructs of sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII.
FIG. 2 is a result of measuring the production amounts of the sTBRII-hyFc
IL7 and IL7-hyFc-sTBRII fusion proteins through batch culture.
FIG. 3 is a result of measuring the in vivo activity of the sTBRII-hyFc-IL7
fusion protein according to the dosage, and (a) is a result of measuring the number of
CD8+ T cells after administering the sTBRII-hyFc-IL7 fusion protein to a mouse
animal model, (b) is a result of measuring the increase rate of CD8+ T cells on Day 7
of administration, (c) is a result of measuring the number of CD4+ T cells, (d) is a
result of measuring the number of CD4+CD25+Foxp3+Treg cells, (e) is a result of
measuring the number of neutrophils, and (f) is a result of measuring the number of
NK cells (o: PBS; m: sTBRII-hyFc-IL7, 10 mpk; A: sTBRII-hyFc-IL7, 30 mpk; V:
sTBRII-hyFc-IL7, 100 mpk).
FIG. 4 is a result of measuring the in vivo activity of the sTBRII-hyFc-IL7
fusion protein according to the administration route, and (a) is a result of measuring
the number of lymphocytes after administering the sTBRII-hyFc-IL7 fusion protein to
a mouse animal model, and (b) is a result of measuring the increase rate of immune
cells according to intravenous administration and subcutaneous administration on Day
7 of administration.
FIG. 5 is a result of analyzing the in vivo activity of the sTBRII-hyFc-IL7 and
IL-7-hyFc-sTBRII fusion proteins, and (a) is a result of measuring the number of
CD8+ T cells, after administering the sTBRII-hyFc-IL7 fusion protein to a mouse
tumor model, (b) is a result of measuring the increase rate of CD8+ T cells on Day 7
of administration, (c) is a result of measuring the number of CD8+ T cells after
administering the IL-7-hyFc-sTBRII fusion protein to a mouse tumor model, and (d)
is a result of measuring the increase rate of CD8+ T cells on Day 7 of administration.
FIG. 6 is a result of measuring the concentration of TGF beta in serum, after
administering the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins to mouse
tumor models.
FIG. 7 is a result of measuring tumor volume, after administering the sTBRII
hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins to mouse tumor models.
FIG. 8 is a result showing (a) the cell proliferation diagram and (b) the
standard curve thereof, after treating 2E8 cell lines with the sTBRII-hyFc-IL7 and IL
7-hyFc-sTBRII fusion proteins.
FIG. 9 is a result showing the standard curve of luminance of the SBE reporter,
after treating the SMAD Signaling Pathway SBE Reporter-HEK293 cell lines with the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins.
FIG. 10 is a result of comparative analysis of the simultaneous binding
strengths of TGF-13 and IL-7Ra by each fusion protein, and after binding to TGF-P
(1t ligand), it is a result of measuring the binding strengths of (a) the sTBRII-hyFc
IL7 fusion protein and (b) the IL-7-hyFc-sTBRII fusion protein, according to the
concentration of IL-7Ra (2 "ligandd.
FIG. 11 is a result of comparative analysis of the simultaneous binding
strengths of TGF- 1 and IL-7Ra by each fusion protein, and after binding to IL-7Ra
(1t ligand), it is a result of measuring the binding strengths of (a) the sTBRII-hyFc
IL7 fusion protein and (b) the IL-7-hyFc-sTBRII fusion protein, according to the
concentration of TGF-P1 ( 2 "ligandd.
[Best Mode]
Hereinafter, the present invention will be described in detail.
One aspect of the present invention provides a fusion protein including
modified interleukin-7 (IL-7) and TGF beta receptor II (transforming growth factor
beta receptor II, TBRII).
The modified IL-7 may have the following structure:
A-IL-7;
In this case, A is an oligopeptide which consists of 1 to 10 amino acid residues,
and the modified IL-7 is IL-7 or a polypeptide having similar activity.
As used herein, the term "IL-7 or a polypeptide having similar activity" refers
to a polypeptide or protein having the same or similar sequence and activity as IL-7.
The IL-7 may include an IL-7 protein or a fragment thereof. In this case, IL
7 may be derived from humans, rats, mice, monkeys, cattle or sheep.
Specifically, human IL-7 may have the amino acid sequence of SEQ ID NO:
1 (Genbank Accession No. P13232); rat IL-7 may have the amino acid sequence
disclosed in Genbank Accession No. P56478; mouse IL-7 may have the amino acid
sequence disclosed in Genbank Accession No. P10168; monkey IL-7 may have the
amino acid sequence disclosed in Genbank Accession No. NP_001279008; bovine IL
7 may have the amino acid sequence disclosed in Genbank Accession No. P26895; and
sheep IL-7 may have the amino acid sequence disclosed in Genbank Accession No.
Q28540.
The IL-7 may be a polypeptide which consists of the amino acid sequence of
SEQIDNO:1. In addition, the modified IL-7 may have about 70%,75%, 80%,85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% or more homology to the
sequence of SEQ ID NO: 1.
In addition, the IL-7 protein or fragment thereof may include variously
modified proteins or peptides, that is, variants. The modification may be performed
through a method of substituting, deleting or adding one or more proteins to wild-type
IL-7, which does not alter the function of IL-7. These various proteins or peptides
may have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
or 99% or more homology to the wild-type protein.
Conventionally, substitution of wild-type amino acid residues may be
performed by alanine or by conservative amino acid substitutions that do not affect or
weaken the charge of the entire protein, that is, polarity or hydrophobicity.
Table 1 below may be referenced for conservative amino acid substitutions.
[Table 1]
Basic Arginine (Arg, R)
Lysine (Lys, K) Histidine (His, H)
Acidic Glutamic acid (Glu, E) Aspartic acid (Asp, D)
Uncharged polar Glutamine (Gln, 0) Asparagine (Asn, N) Serine (Ser, S) Threonine (Thr, T) Tyrosine (Tyr, Y)
Non-polar Phenylalanine (Phe, F) Tryptophan (Trp, W) Cysteine (Cys, C) Glycine (Gly, G) Alanine (Ala, A) Valine (Val, V) Proline (Pro, P) Methionine (Met, M) Leucine (Leu, L) Norleucine Isoleucine
For each amino acid, additional conservative substitutions include
"homologs" of the amino acids. In this case, the term "homolog" refers to an amino
acid in which a methylene group (CH 2) is inserted into the side chain of the beta
position of the side chain of the amino acid. Examples of such "homologs" include,
but are not limited to, homophenylalanine, homoarginine, homoserine and the like.
As used herein, the term "IL-7 protein" is also used as a concept including "IL-7
protein and fragments thereof." The terms "protein", "polypeptide" and "peptide"
may be used as interchangeable concepts, unless otherwise specified.
In the structure of the modified IL-7, A may be directly linked to the N
terminus of the IL-7 or linked through a linker. The A may be linked to the N
terminus of IL-7. The A is characterized in that it includes 1 to 10 amino acids, and the amino acid may be selected from the group consisting of methionine, glycine and combinations thereof.
Methionine and glycine do not induce an immune response in the body.
Protein therapeutics produced from E. coli necessarily include methionine at the N
terminus, but no immune side effects have been reported therefrom. In addition,
glycine is widely used in a GS linker, but it does not induce an immune response even
in commercially available products such as Dulaglutide (Cell Biophys. 1993 Jan-Jun:
22(1-3): 189- 224).
According to an exemplary embodiment, the A may be an oligopeptide
including 1 to 10 amino acids selected from the group consisting of methionine (Met,
M), glycine (Gly, G) and combinations thereof. Preferably, it may be an oligopeptide
including 2 to 10 amino acids, more preferably, an oligopeptide including 3 to 10
amino acids, but is not limited thereto. Specifically, the A may have one N-terminal
sequence selected from the group consisting of methionine, glycine, methionine
methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine
methionine-methionine, methionine-methionine-glycine, methionine-glycine
methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine
methionine-glycine, glycine-glycine-methionine, glycine-glycine-glycine,
methionine-methionine-methionine-methionine, methionine-glycine-methionine
methionine, methionine-glycine-glycine-methionine, methionine-glycine-glycine
glycine, methionine-glycine-methionine-glycine, glycine-methionine-methionine
methionine, glycine-methionine-glycine-glycine, glycine-glycine-glycine-glycine,
methionine-methionine-methionine-methionine-methionine, methionine-methionine
glycine-methionine-methionine, methionine-methionine-glycine-glycine-methionine,
methionine-glycine-methionine-methionine-glycine, methionine-methionine methionine-methionine-glycine, glycine-glycine-glycine-glycine-glycine, glycine glycine-methionine-methionine-methionine, glycine-glycine-glycine-methionine glycine, methionine-glycine-methionine-glycine-methionine-glycine, methionine methionine-methionine-glycine-glycine-glycine, methionine-methionine-glycine glycine-methionine-methionine, glycine-glycine-methionine-methionine-glycine glycine, methionine-glycine-methionine-glycine-methionine-glycine-methionine glycine, methionine-methionine-methionine-methionine-glycine-glycine-glycine glycine, methionine-methionine-glycine-glycine-methionine-methionine-glycine glycine, methionine-methionine-methionine-methionine-glycine-glycine-glycine glycine, methionine-glycine-methionine-glycine-methionine-glycine-methionine glycine-methionine-glycine or methionine-methionine-methionine-methionine methionine-glycine-glycine-glycine-glycine-glycin.
The modified IL-7 may consist of the amino acid sequence of SEQ ID NO: 2
or SEQ ID NO: 3.
As used herein, the terms "TBRII" or "TGF beta receptor II", unless otherwise
indicated, refer to any wild-type TBRII obtained from any vertebrae animal sources,
including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).
The human TBRII may consist of the amino acid sequence of SEQ ID NO: 4.
Specifically, the TBRII may be an extracellular domain of TBRII. The extracellular
domain of TBRII may have the 2 4th to 1 5 9th amino acid sequence of human TBRII
(SEQ ID NO: 4), and the extracellular domain of TBRII may consist of the amino acid
sequence of SEQ ID NO: 5.
As used herein, the term "sTBRII" means soluble TBRII, and it may be an
extracellular domain of human TBRII.
The modified IL-7 and TBRII may be linked by an immunoglobulin Fc
domain.
The Fc domain may be a wild type or variant. The Fc domain variant may
be an Fc domain of a modified immunoglobulin. In this case, the Fc domain of the
modified immunoglobulin may have a modified binding strength to the Fc receptor
and/or complement such that the antibody-dependent cellular cytotoxicity (ADCC) or
complement-dependent cytotoxicity (CDC) is weakened. The modified
immunoglobulin may be selected from the group consisting of IgGI, IgG2, IgG3, IgG4,
IgAl, IgA2, IgD, IgE and combinations thereof. In particular, the Fc domain of the
modified immunoglobulin may include a hinge region, a CH2 domain and a CH3
domain in the direction from the N-terminus to the C-terminus. In this case, the hinge
region may include a human IgD hinge region, the CH2 domain may include a portion
of the amino acid residue of the CH2 domain of human IgD and a portion of the amino
acid residue of the CH2 domain of human IgG4, and the CH3 domain may include a
portion of the amino acid residue of the CH3 domain of human IgG4. The hinge
region may be an IgG1 hinge region, which may include the amino acid sequence of
SEQ ID NO: 6.
As used herein, the terms "Fc domain", "Fc fragment" or "Fc" refer to proteins
that include heavy chain constant region 2 (CH2) and heavy chain constant region 3
(CH3) of an immunoglobulin, but do not include the variable regions of heavy and
light chains thereof and the light chain constant region 1 (CLi). It may further
include a hinge region of the heavy chain constant region. Hybrid Fc or hybrid Fc
fragment is also referred to herein as "hFc" or "hyFc".
As used herein, the term "Fc domain variant" means that some amino acids in the Fc domain are substituted or are prepared by combining different types of Fc domains. The Fc domain variant may prevent cleavage at the hinge region.
Specifically, the 1 4 4 th amino acid and/or the 1 4 5 th amino acid of SEQ ID NO: 9 may
be modified. Preferably, the 14 4 th aminoacid K of SEQ ID NO: 9 may be substituted
with G or S (K144G, K144S), and the 1 4 5th amino acid E may be substituted with G
or S (E145G, E145S).
In addition, the Fc domain or Fc domain variant of the modified
immunoglobulin may be represented by the following Formula (I):
[Formula (I)]
N'-(Z1)p-Y-Z2-Z3-Z4-C'
In the above formula,
N' is the N-terminus of the polypeptide, and C' is the C-terminus of the
polypeptide;
p is an integer of 0 or 1; and
ZI is an amino acid sequence having 5 to 9 consecutive amino acid residues
in the N-terminal direction from position 98 of the amino acid residues at positions 90
to 98 of SEQ ID NO: 7,
wherein Y is an amino acid sequence having 5 to 64 consecutive amino acid
residues in the N-terminal direction from position 162 of the amino acid residues at
positions 99 to 162 of SEQ ID NO: 7,
wherein Z2 is an amino acid sequence having 4 to 37 consecutive amino acid
residues in the C-terminal direction from position 163 of the amino acid residues at
positions 163 to 199 of SEQ ID NO: 7,
wherein Z3 is an amino acid sequence having 71 to 106 consecutive amino
acid residues in the N-terminal direction from position 220 of the amino acid residues at positions 115 to 220 of SEQ ID NO: 8, and wherein Z4 is an amino acid sequence having 80 to 107 amino acid sequences of in the C-terminal direction from position 221 of the amino acid residues at positions
221 to 327 of SEQ ID NO: 8.
In addition, the Fc fragment of the present invention may be a natural-type
sugar chain, an increased sugar chain compared to the natural type, a reduced sugar
chain compared to the natural type, or a form in which the sugar chain has been
removed. The immunoglobulin Fc sugar chain may be modified by conventional
methods such as chemical methods, enzymatic methods, genetic engineering methods
using microorganisms and the like. Removal of the sugar chain from the Fc fragment
sharply decreases the binding affinity of the primary complement components C1 to
CIq, leads to a decrease or loss of ADCC or CDC, and thereby does not induce an
unnecessary immune response in vivo. In this respect, the immunoglobulin Fc
fragment in the form of deglycosylated or aglycosylated sugar chain may be more
suitable for the purposes of the present invention as a drug carrier. As used herein,
the term "deglycosylation" means that sugars are enzymatically removed from an Fc
fragment. In addition, the term "aglycosylation" means that the Fc fragment is
produced in an unglycosylated form by prokaryotes, preferably, E. coli.
In addition, the Fc domain of the modified immunoglobulin may include the
amino acid sequence of SEQ ID NO: 9 (hyFc), SEQ ID NO: 10 (hyFcM1), SEQ ID
NO: 11 (hyFcM2), SEQ ID NO: 12 (hyFcM3) or SEQ ID NO: 13 (hyFcM4).
According to the present invention, the Fc domain of the modified
immunoglobulin may be described in U.S. Patent No. 7,867,491, and the production
of the Fc domain of the modified immunoglobulin may be performed with reference to the description in U.S. Patent No. 7,867,491.
In addition, the fusion protein may be one in which the TBRII, Fc domain and
modified IL-7 are sequentially linked in the direction from the N-terminus to the C
terminus. This fusion protein maybe referred to as "sTBRII-hyFc-IL7."
In the case of the sTBRII-hyFc-IL7 fusion protein, a first linker may be further
included between the TBRII and the Fc domain. The first linker may consist of 20
to 60 contiguous amino acids, 25 to 50 contiguous amino acids, or 30 to 40 amino
acids. In an embodiment, the first linker may consist of20 amino acids. In addition,
the first linker may include (G4S)n (here, n is an integer of 1 to 5). Preferably, the
first linker may consist of the amino acid sequence of SEQ ID NO: 14.
In addition, a second linker may be further included between the Fc domain
and the modified IL-7. The second linker may consist of 1 to 30 contiguous amino
acids, 3 to 20 contiguous amino acids, or 4 to 16 amino acids. In addition, the second
linker may include (SG3)n (here, n is an integer of 1 to 5). In an exemplary
embodiment, the second linker may consist of the amino acid sequence of SEQ ID NO:
15.
Therefore, the sTBRII-hyFc-IL7 fusion protein may have the following
structure.
N' - TBRII - (L1)p - Fc domain - (L2)q - A-IL-7 - C'
In the above,
N' is the N-terminus, and C' is the C-terminus,
Li is the first linker, and L2 is the second linker, and
p and q are integers of 0 or 1.
The sTBRII-hyFc-IL7 fusion protein may consist of the amino acid sequence of SEQ ID NO: 17. The fusion protein may have 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more homology to the amino acid
sequence of SEQ ID NO: 17.
In addition, the fusion protein may be one in which the modified IL-7, Fc
domain or TBRII is sequentially linked in the direction from the N-terminus to the C
terminus. This fusion protein may be referred to as "IL7-hyFc-sTBRII".
In the case of the IL7-hyFc-sTBRII fusion protein, a first linker may be further
included between the modified IL-7 and the Fc domain. The first linker may consist
of 20 to 60 contiguous amino acids, 25 to 50 contiguous amino acids, or 30 to 40 amino
acids. In an embodiment, the first linker may consist of20 amino acids. In addition,
the first linker may include (G4S)n (here, n is an integer of 1 to 5). Preferably, the
first linker may consist of the amino acid sequence of SEQ ID NO: 14.
In addition, a third linker may be further included between the Fc domain and
TBRII. The third linker may consist of 1 to 30 contiguous amino acids, 3 to 20
contiguous amino acids, or 4 to 16 amino acids. In an exemplary embodiment, the
third linker may consist of the amino acid sequence of SEQ ID NO: 16.
Therefore, the IL7-hyFc-sTBRII fusion protein may have the following
structure.
N' - A-IL-7 - (L1)p - Fc domain - (L3)r - TBRII - C'
In the above,
N' is the N-terminus, and C' is the C-terminus,
Li is thefirst linker, and L2 is the second linker, and
p and r are integers of 0 or 1.
The IL7-hyFc-sTBRII fusion protein may consist of the amino acid sequence
of SEQ ID NO: 18. The fusion protein may have 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more homology to the amino acid
sequence of SEQ ID NO: 18.
In addition, the present invention provides a nucleic acid molecule encoding
the fusion protein.
In addition, the nucleic acid molecule may additionally include a signal
sequence (or signal peptide) or a leader sequence.
As used herein, the term "signal sequence (or signal peptide)" refers to a short
peptide which is present at the N-terminus of a newly synthesized protein classified
into the secretory pathway. Signal sequences that are useful in the present invention
include antibody 1418 (Gillies et al., JImmunol Meth 1989 125:191-202), antibody
light chain signal sequences, for example, MOPC141 antibody heavy chain signal
sequence (Sakano et al., Nature 1980 286: 676-683) and other signal sequences known
in the art (e.g., refer to Watson et al., Nucleic Acid Research 1984 12:5145-5164).
The characteristics of the signal peptide are well known in the art, and it is
generally known to include 16 to 30 amino acid residues, and it may contain more or
less amino acid residues. A typical signal peptide consists of three regions: a basic
N-terminal region, a central hydrophobic region and a more polar C-terminal region.
The central hydrophobic region includes 4 to 12 hydrophobic residues that
anchor the signal sequence through the membrane lipid bilayer during migration of
immature polypeptides. After initiation, the signal sequence is cleaved within the
lumen of the ER by cellular enzymes commonly known as signal peptidases. In this
case, the signal sequence may be a tissue plasma activation (tPa), HSV gDs or a secretion signal sequence of growth hormone. Preferably, the secretion signal sequence used in higher eukaryotic cells including mammals and the like may be used, and more preferably, the tPa sequence (SEQ ID NO: 19) or the amino acid sequence of SEQ ID NO: 20 may be used. In addition, the signal sequence of the present invention may be used by substituting codons with high expression frequency in host cells.
In addition, the present invention provides an expression vector including the
nucleic acid molecule.
As used herein, the term "vector" is understood as a nucleic acid means
including a nucleotide sequence that may be introduced into a host cell, recombined
and inserted into the host cell genome, or spontaneously replicated as an episome.
The vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors,
viral vectors and analogs thereof. Examples of viral vectors include, but are not
limited to, retrovirus, adenovirus and adeno-associated virus.
In the present invention, useful expression vectors may be RcCMV
(Invitrogen, Carlsbad) or variants thereof. Useful expression vectors may include a
human CMV (cytomegalovirus) promoter to promote the continuous transcription of
a gene of interest in mammalian cells, and a polyadenylation signal sequence of bovine
growth hormone to increase the stable level of RNA after transcription. In an
embodiment of the present invention, the expression vector is pAD15, which is a
modified vector of RcCMV.
As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells
into which a recombinant expression vector may be introduced.
In the present invention, an appropriate host cell may be transformed or transfected with the DNA sequence of the present invention, and it may be used for the expression and/or secretion of a protein of interest. Currently preferred host cells that can be used in the present invention include immortal hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, CapT cells (human amniotic fluid derived cells) and COS cells.
As used herein, the terms "transformation" and "transfection" mean the
introduction of a nucleic acid (e.g., a vector) into a cell by a number of techniques
known in the art.
In addition, the present invention provides a pharmaceutical composition for
preventing or treating cancer or infectious disease, including the fusion protein
including modified IL-7 and TBRII as an active ingredient.
The cancer may be selected from the group consisting of stomach cancer, liver
cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer,
pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid
leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary
gland cancer and lymphoma, but is not limited thereto.
The infectious disease may be selected from the group consisting of hepatitis
B, hepatitis C, human papilloma virus infection, cytomegalovirus infection, viral
respiratory disease and influenza, but is not limited thereto.
In the composition for treating or preventing cancer or infectious disease
according to the present invention, the active ingredient may be included in an arbitrary
amount (effective amount) according to the use, formulation, purpose of combination
and the like, as long as the active ingredient may exhibit an anti-cancer activity or
therapeutic effect on infectious disease, and a typical effective amount will be determined within the range of 0.001 wt.% to 20.0 wt.% based on the total weight of the composition. Herein, the term "effective amount" refers to the amount of an active ingredient capable of inducing an anti-cancer effect or a therapeutic effect on infectious disease. Such an effective amount may be determined empirically within the range of ordinary skill in the art.
In this case, the pharmaceutical composition may further include a
pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be
any carrier as long as it is a non-toxic substance suitable for delivery into a patient.
Distilled water, alcohols, fats, waxes and inert solids may be included as carriers.
Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in
the pharmaceutical composition.
Specifically, the pharmaceutical composition may be prepared in a parenteral
formulation according to the administration route by a conventional method known in
the art, including a pharmaceutically acceptable carrier in addition to the active
ingredient. Herein, the term "pharmaceutically acceptable" means that it does not
inhibit the activity of an active ingredient and does not have toxicity beyond what the
application (prescription) target may adapt to.
When the pharmaceutical composition is prepared in a parenteral formulation,
it may be formulated in the form of an injection, a transdermal administration, a nasal
inhalation and a suppository according to methods known in the art together with a
suitable carrier. When formulated as an injection, sterile water, ethanol, polyols such
as glycerol or propylene glycol, or mixtures thereof may be used as suitable carriers,
and preferably, Ringer's solution, phosphate buffered saline (PBS) or sterile water for
injection containing triethanolamine, an isotonic solution such as 5% dextrose or the
like may be used. Regarding the formulation of a pharmaceutical composition, it is known in the art, and specifically, reference may be made to the literature [Remington's
PharmaceuticalSciences (19th ed., 1995)]. The above literature is considered as part
of the present specification.
The preferred dosage of the pharmaceutical composition may be in the range
of 0.01 pg/kg to 10 g/kg per day, or 0.01 mg/kg to 1 g/kg per day depending on the
patient's condition, weight, gender, age, patient severity and route of administration.
Administration may be performed once a day or divided into several times. Such
dosages should not be construed as limiting the scope of the invention in any aspect.
Subjects to which the pharmaceutical composition may be applied (prescribed)
are mammals and humans, and particularly, it is preferably humans. In addition to
the active ingredient, the pharmaceutical composition of the present application may
further include any known compound or natural extract, which has already been
verified for safety and has an anti-cancer activity or therapeutic effect on infectious
disease in order to increase and reinforce an anti-cancer activity.
In addition, the present invention provides the use of a fusion protein including
modified IL-7 and TBRII to produce a pharmaceutical preparation having a
prophylactic or therapeutic effect on cancer or infectious disease.
In addition, the present invention provides a method for preventing or treating
cancer or infectious disease, including a fusion protein including modified IL-7 and
TBRII as an active ingredient.
It is preferable to apply the therapeutically effective amount differently
according to various factors including the specific composition including the type and
degree of the reaction to be achieved, whether other agents are used in some cases, the
subject's age, weight, general health status, gender and diet, administration time, administration route and the secretion rate of the composition, the treatment period, drugs used with or concurrently with the specific composition, and similar factors well known in the field of medicine. Therefore, it is preferable to determine the effective amount of a composition suitable for the purposes of the present invention in consideration of the above-described factors.
The subject is applicable to any mammal, and the mammal includes humans
and primates, as well as livestock such as cattle, pigs, sheep, horses, dogs, cats and the
like.
[Modes of the Invention]
Hereinafter, the present invention will be described in more detail through
examples. These examples are for describing the present invention more specifically,
and the scope of the present invention is not limited to these examples.
Example 1. Preparation of sTBRII-hyFc-IL7 and IL7-hyF-sTBRII
fusion proteins
In order to prepare a fusion protein in which human-derived IL-7 and soluble
TGF beta receptor II (sTBRII) are linked, the inventors of the present invention fused
IL-7 or sTBRII to the N-terminus of an Fc domain, and prepared a gene construct in
the form where IL-7 or sTBRII was fused to the C-terminus of an Fc domain. In the
present invention, a fusion protein in which the TBRII, Fc domain and IL-7 are
sequentially linked in the direction from the N-terminus to the C-terminus is expressed
as "sTBRII-hyFc-IL7", and a fusion protein in which the IL-7, Fc domain or TBRII is
sequentially linked in the direction from the N-terminus to the C-terminus is expressed
as "IL7-hyFc-sTBRII."
First, in the preparation of an sTBRII-hyFc-IL7 gene construct, by using only
the extracellular domain of 24 to 159 amino acids in the known amino acid sequence
of TGF beta receptor II (Accession Number: NP003233.4), sTBRII was fused to the
N-terminus of the Fc domain, and a gene construct was prepared by fusing IL-7 to the
C-terminus of the Fc domain using a known amino acid sequence (Accession Number:
NP000871.1) (FIG. 1).
Further, in the preparation of the IL7-hyFc-sTBRII gene construct, a gene
construct was prepared by respectively fusing IL-7 and sTBRII to the N-terminus and
C-terminus of the Fc domain using the same sequence as above (FIG. 1).
The IL-7, sTBRII and Fc domain were prepared as sub-vectors by
synthesizing each gene at Cosmo Genetech, and Golden Gateway assembly was
performed to prepare the synthesized three gene segments into one gene segment to
obtain an expression vector with the pGP30 vector.
The expression vector was transfected into CHO cells (suspension-adapted
Chinese Hamster Ovary cells) adapted for suspension culture with the Neon
Transfection system (Invitrogen, MPKI1096), and a highly productive cell line was
selected through HT selection and amplification of Methotrexate (Sigma, M8407).
In addition, single cells were secured through limiting dilution cloning to obtain
production cell lines which produce the sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII
fusion proteins.
Each production cell line was subjected to batch culture by 80 mL each in a
250 mL Erlenmeyer flask (Corning, 431144) by using the Hycell CHO medium
(Hyclone, SH30949.02) to confirm the culture productivity. As a result, it was
confirmed that the IL7-hyFc-sTBRII fusion protein was produced in an amount of 0.6
g/L, and the sTBRII-hyFc-IL7 fusion protein was produced in an amount of 0.95 g/L
(FIG. 2).
Example 2. In vivo activity analysis of sTBRII-hyFc-1L7 fusion protein
according to dosage
The inventors of the present invention conducted an experiment to confirm
the pharmacodynamics profile of the fusion protein according to intravenous
administration. Briefly, after intravenous administration of the sTBRII-hyFc-IL7
fusion protein at 10, 30 and 100 mg/kg (mpk) to a C57BL/6 (B6)mouse animal model,
changes of immune cells in the blood over time were analyzed. In addition, blood
was collected through retro-orbital bleeding on Days 3, 7, 10, 14, 17 and 21 after
administration of the sTBRII-hyFc-IL7 fusion protein. Each value was calculated by
confirming the expression rate of the cell surface indicator and the composition ratio
of each cell in the blood through flow cytometry, and multiplying these by the value
measured using a complete blood count (CBC).
As a result, in mice to which the sTBRII-hyFc-IL7 fusion protein was
administered, the number of CD8' T cells, which are the major target cells of IL-7,
increased in a concentration-dependent manner, and the peak point was shown on Day
7 of administration (FIG. 3a). In addition, it was confirmed that the increase rate of
CD8' T cells on Day 7 of administration increased by 6.22, 12.45 and 35.04 times at
concentrations of 10, 30 and 100 mpk, respectively, compared to the control group
(PBS treatment) (FIG. 3b).
In addition, in mice to which the sTBRII-hyFc-IL7 fusion protein was
administered, CD4' T cells and CD4+CD25+Foxp3 + Treg cells, which are the target
cells of IL-7, also showed a tendency to increase (FIGS. 3c and 3d). However, in
neutrophils and NK cells other than the target cells, no changes were observed according to the administration (FIGS. 3e and 3f).
Example 3. In vivo activity analysis of sTBRII-hyFc-1L7 fusion protein
according to the administration route
The inventors of the present invention conducted an experiment to measure
the in vivo activity of the sTBRII-hyFc-IL7 fusion protein according to the
administration route. Briefly, after intravenous injection (i.v.) or subcutaneous
injection (s.c.) of the sTBRII-hyFc-IL7 fusion protein (10 mpk) to a C57BL/6 (B6)
mouse animal model, changes of immune cells in the blood over time were analyzed.
Blood was collected through retro-orbital bleeding on Days 0, 4, 7, 11 and 14 after
administration of the sTBRII-hyFc-IL7 fusion protein. Each value was measured by
using a complete blood count (CBC).
As a result, it was confirmed that the number of immune cells (lymphocytes)
was increased in mice to which the sTBRII-hyFc-IL7 fusion protein was administered,
compared to the control group (PBS treatment) on Day 7 of administration (FIG. 4a).
Specifically, it was confirmed that the number of immune cells increased by about 1.51
times compared to the control group in the intravenous administration group on Day 7
of administration, and the number of immune cells increased by about 3.04 times
compared to the control group in the subcutaneous administration group (FIG 4b).
Example 4. In vivo activity analysis of sTBRII-hyFc-1L7 and IL-7-hyFc
sTBRII fusion proteins in mouse tumor models
The inventors of the present invention conducted an experiment to analyze the
in vivo activity of the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in
mouse tumor models. Briefly, a mouse tumor model was prepared by subcutaneously injecting 1 x 105 MC38 colon cancer cell lines into a C57BL/6 (B6) mouse animal model. Afterwards, on Day 6 of tumor formation, each fusion protein was administered subcutaneously (s.c.) at 10 mpk or 20 mpk, and then changes of immune cells in the blood were analyzed by using flow cytometry and a complete blood count
(CBC). Blood was collected through retro-orbital bleeding on Days 0, 3, 7, 11, 15
and 18 after administration of each fusion protein.
As a result, in tumor mice to which the sTBRII-hyFc-IL7 fusion protein was
administered, the number of CD8' T cells increased in a concentration-dependent
manner, and the peak point was shown on Day 7 of administration (FIG. 5a). In
addition, it was confirmed that the increase rate of CD8' T cells on Day 7 of
administration increased by 11.08 and 21.82 times at concentrations of 10and20mpk,
respectively, compared to the control group (PBS treatment) (FIG. 5b).
In addition, in tumor mice to which the IL-7-hyFc-sTBRII fusion protein was
administered, the number of CD8' T cells increased in a concentration-dependent
manner, and the peak point was shown on Day 7 of administration (FIG. 5a). In
addition, it was confirmed that the increase rate of CD8' T cells on Day 7 of
administration increased by 18.74 and 29.56 times at concentrations of 10 and 20 mpk,
respectively, compared to the control group (PBS treatment) (FIG. 5b).
From the above results, it was confirmed that both of the sTBRII-hyFc-IL7
and IL-7-hyFc-sTBRII fusion proteins have an activity of increasing the proliferation
of target cells such as CD8' T cells in a concentration-dependent manner.
Example 5. Analysis of TGF beta inhibitory activity of sTBRII-hyFc-1L7
and IL-7-hyFc-sTBRII fusion proteins in mouse tumor models
The inventors of the present invention conducted an experiment to analyze the inhibitory activity of the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins against TGF beta in mouse tumor models. Briefly, a mouse tumor model was prepared by subcutaneously injecting 1 X105 MC38 colon cancer cell lines into a
C57BL/6 (B6) mouse animal model. Afterwards, on Day 6 of tumor formation, each
fusion protein was administered subcutaneously (s.c.) at 20 mpk. Blood was
collected through retro-orbital bleeding at 2, 6, 24, 48, 72 and 168 hours after
administration of each fusion protein, and serum was isolated from the collected blood
samples. The concentration of TGF beta present in the serum was measured by using
the Mouse TGF-beta 1 DuoSet ELISA Kit (R&D systems, catalog# DY1679-05).
As a result, in tumor mice to which the sTBRII-hyFc-IL7 fusion protein was
administered, TGF beta in serum was inhibited for about 48 hours after administration,
and was detected again 72 hours after administration, and 168 hours after
administration, it was confirmed that it recovered to a level similar to that of the control
group (PBS treatment) (FIG. 6). In addition, in tumor mice to which the IL7-hyFc
sTBRII fusion protein was administered, TGF beta in serum was inhibited for about
72 hours after administration, and it was confirmed that it recovered to a level similar
to that of the control group (PBS treatment) 168 hours after administration (FIG. 6).
From the above results, it was confirmed that the sTBRII-hyFc-IL7 and IL-7
hyFc-sTBRII fusion proteins have an effect of inhibiting TGF beta in serum for 48 to
72 hours through subcutaneous administration.
Example 6. Analysis of anti-cancer activity of sTBRII-hyFc-1L7 and IL
7-hyFc-sTBRII fusion proteins in mouse tumor models
The inventors of the present invention conducted an experiment to confirm
the anti-cancer activity of the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in mouse tumor models. Briefly, a mouse tumor model was prepared by subcutaneously injecting 1 x 105 MC38 colon cancer cell lines into a C57BL/6 (B6) mouse animal model. Afterwards, on Day 6 of tumor formation, each fusion protein was administered subcutaneously (s.c.) at 20 mpk. Changes in tumor volume were measured at intervals of 2 to 3 days after administration. The tumor volume was calculated using the following formula.
Tumor volume = {(long axis length) x (short axis length) 2 } /2
In addition, the tumor growth inhibition rate (% tumor growth inhibition) was
calculated using the following formula.
% Tumor growth inhibition (% TGI) = {(MTVcontrol - MTVtreated)/
MTVcontrol} x 100 (* MTV = median tumor volume)
As a result, it was confirmed that in mice to which the sTBRII-hyFc-IL7 and
IL-7-hyFc-sTBRII fusion proteins were administered, the tumor volume was
significantly reduced compared to the control group (PBS treatment) (FIG. 7). In
particular, in mice to which the sTBRII-hyFc-IL7 fusion protein was administered, on
Day 20 after transplantation of the MC38 colorectal cancer cell line, the tumor growth
inhibition rate was 61.79% compared to the control group (FIG. 7a), and in mice to
which the IL7-hyFc-sTBRII fusion protein was administered, the tumor growth
inhibition rate was 83.14% compared to the control group (FIG. 7B).
From the above results, it was confirmed that the sTBRII-hyFc-IL7 and IL-7
hyFc-sTBRII fusion proteins have remarkable anti-cancer efficacy through
subcutaneousadministration.
Example 7. In vitro IL-7 bioactivity analysis of sTBRII-hyFc-1L7 and IL
7-hyFc-sTBRII fusion proteins
In order to measure the biological activity of IL-7 constituting the sTBRII
hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in vitro, the inventors of the present
invention conducted an experiment by using a 2E8 cell line (ATCC TIB-239TM
Mouse B lymphocyte cell line) that proliferates dependently on IL-7. Since human
derived IL-7 has cross-reactivity between species with mice, human-derived IL-7 may
induce proliferation of the 2E8 cell line, which is a mouse cell line.
Briefly, after thawing the 2E8 cell line that had been stored frozen, stabilized
cells were prepared by subculture at least three times in a T-75 flask. The 2E8 cell
line was cultured by using an IMDM (ATCC® 3 0 - 2 0 0 5 TM) medium including mouse
IL-7 (Cell Signaling, 5217SC) and FBS (Hyclone, SH30084.03). Afterwards, the
cultured 2E8 cell line was suspended in the IMDM medium including no mouse IL-7
to create a starvation state for IL-7, and then it was dispensed into a 96-well plate at 1
x 105 cells/well. Afterwards, the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion
proteins were treated on the cells in a concentration gradient sequentially diluted
starting from 3 nM by 1/3, respectively, and it was cultured for 3 days in an incubator
at 37°C and 5% CO2.
Cell proliferation was quantified using the CellTiter 96 AQueous One Solution
Assay (Promega, G3581). The cells were treated with the MTS reagent and cultured
for 4 hours in an incubator at 37°C and 5% CO2, and then, absorbance at a wavelength
of 490 nm was measured using an ELISA plate reader. By using the GraphPad
Prism program (GraphPad Software), the standard curve of absorbance and the EC5 o
(50% effective concentration) values of the two fusion proteins were calculated based
thereon.
As a result, the EC5 o of the sTBRII-hyFc-IL7 fusion protein was measured to
be 52.52 pM, and the EC5 o of the IL7-hyFc-sTBRII fusion protein was measured to be
56.21 pM (FIG. 8 and Table 2).
[Table 2]
EC5 o (pM) sTBRII-hyFc-IL-7 IL-7-hyFc-sTBRII
52.52 56.21
Example 8. In vitro analysis of TGF beta inhibitory activity of sTBRII
hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins
In order to measure the biological activity of sTBRII constituting the sTBRII
hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins, the inventors of the present invention
conducted an experiment using the SMAD Signaling Pathway SBE Reporter-HEK293
cell line (BPS Bioscience, 60653), which can measure the sub-transmission signals
induced by TGF beta as luminescence signals.
Briefly, the SMAD Signaling Pathway SBE Reporter-HEK293 cell line was
cultured in an incubator at 37°C and 5% C02 by using Growth Medium lB (BPS
Bioscience, 79531) including Geneticin (Invitrogen, 11811031). Afterwards, the
cultured SMAD Signaling Pathway SBE Reporter-HEK293 cell line was suspended in
Assay Medium 1B (BPS Bioscience, 79617-2) and dispensed into a white clear-bottom
96-well microplate at 3.5 x 10 4 cells/well. Afterwards, the sTBRII-hyFc-IL7 and IL
7-hyFc-sTBRII fusion proteins were treated on the cells in a concentration gradient
sequentially diluted starting from 50 nM by 1/3, respectively, and after treating the
cells together with 20 ng/mL of TGF beta (BPS Bioscience, 90900-1) at the same time,
the cells were cultured for 18 hours in an incubator at 37°C and 5% Co 2. The cells for which the culture was completed were treated with ONE-Step TM Luciferase reagent and cultured by shaking the plate at room temperature for 30 minutes. Afterwards, the degree of luminescence of the SBE reporter was measured with the Plate-reading
Luminometer (TECAN SPARK 10M). By using the GraphPad Prism* program
(GraphPad Software), the luminance standard curve of the SBE reporter and the IC5 0
values of the two fusion proteins were calculated.
As a result, in the SMAD Signaling Pathway SBE Reporter-HEK293 cell line,
the IC 5 0 value of the sTBRII-hyFc-IL-7 of the fusion protein was measured to be 1.874
nM, and the IC5 0 value of the IL-7-hyFc -sTBRII fusion protein was measured to be
0.4148 nM (FIG. 9 and Table 3).
[Table 3]
ICso (nM) sTBRII-hyFc-IL-7 IL-7-hyFc-sTBRII 1.874 0.4148
Example 9. Analysis of simultaneous binding strengths of sTBRII-hyFc
IL7 and IL-7-hyFc-sTBRII fusion proteins
The inventors of the present invention conducted biolayer interferometry (BLI)
to compare and analyze the simultaneous binding strengths of sTBRII-hyFc-IL7 and
IL-7-hyFc-sTBRII for IL-7Ra (CD127) and TGF- 1. Experiments were performed
by using the Amine Reactive Second-Generation Biosensor (AR2G) and the AR2G
Reagent Kit.
After placing each fusion protein into the samples in the order of recombinant
human TGF- 1 protein (2 ptg/mL) (immobilized ligand (lt ligand)) - fusion protein
(1,000 nM) - recombinant human IL-7Ra protein (1, 2, 4, 8 pg/mL) ( 2 "ligandd, or in the order of recombinant human IL-7Ra protein (2 ptg/mL) (immobilized ligand (1t ligand)) - fusion protein (1,000 nM) - recombinant human TGF- 1 protein (0.25, 0.5,
1, 2 pg/mL) ( 2 " ligandd, the simultaneous binding and binding of the fusion protein
bound to the 1" ligand according to the concentration of the 2 ligand were compared
and analyzed.
As a result of confirming the binding of each fusion protein to the immobilized
I" ligand (TGF-P 1, IL-7R a), the association was well performed over time, and the
binding rates of both fusion proteins were similar. In addition, it was confirmed that
the binding with TGF- 1 was relatively better than the binding with IL-7Ra.
Afterwards, after binding to TGF- 1 (1t ligand), the binding in the fusion
protein according to the concentration of IL-7R a (2 "ligand) was determined, and as
a result, the binding increased in a concentration-dependent manner in both fusion
proteins. (FIG. 10). In addition, after binding to IL-7Ra (St ligand), the binding in
the fusion protein according to the concentration of TGF-P1 ( 2 d ligand) was
determined, and as a result, the binding increased in a concentration-dependent manner
in both fusion proteins (FIG. 11).
From the above results, it was confirmed that the sTBRII-hyFc-IL-7 and IL
7-hyFc-sTBRII fusion proteins are capable of simultaneous binding with the target
proteins TGF-13 and IL-7Ra.
Claims (16)
- [CLAIMS][Claim 1]A fusion protein, comprising modified interleukin 7 (IL-7) and TGF betareceptor II (transforming growth factor beta receptor II, TBRII).
- [Claim 2]The fusion protein of claim 1, wherein the modified IL-7 has the followingstructure:A - IL-7,wherein A is an oligopeptide which consists of 1 to 10 amino acid residues,andwherein the modified IL-7 is IL-7 or a polypeptide having a similar activity.
- [Claim 3]The fusion protein of claim 2, wherein the IL-7 consists of the amino acidsequence of SEQ ID NO: 1; and/orwherein the A is linked to the N-terminus of IL-7; and/orwherein the A is selected from the group consisting of methionine, glycine,methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine,methionine-methionine-methionine, methionine-methionine-glycine, methionineglycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine,glycine-methionine-glycine, glycine-glycine-methionine, glycine-glycine-glycine,methionine-methionine-methionine-methionine, methionine-glycine-methioninemethionine, methionine-glycine-glycine-methionine, methionine-glycine-glycine glycine, methionine-glycine-methionine-glycine, glycine-methionine-methionine methionine, glycine-methionine-glycine-glycine, glycine-glycine-glycine-glycine, methionine-methionine-methionine-methionine-methionine, methionine-methionine glycine-methionine-methionine, methionine-methionine-glycine-glycine-methionine, methionine-glycine-methionine-methionine-glycine, methionine-methionine methionine-methionine-glycine, glycine-glycine-glycine-glycine-glycine, glycine glycine-methionine-methionine-methionine, glycine-glycine-glycine-methionine glycine, methionine-glycine-methionine-glycine-methionine-glycine, methionine methionine-methionine-glycine-glycine-glycine, methionine-methionine-glycine glycine-methionine-methionine, glycine-glycine-methionine-methionine-glycine glycine, methionine-glycine-methionine-glycine-methionine-glycine-methionine glycine, methionine-methionine-methionine-methionine-glycine-glycine-glycine glycine, methionine-methionine-glycine-glycine-methionine-methionine-glycine glycine, methionine-methionine-methionine-methionine-glycine-glycine-glycine glycine, methionine-glycine-methionine-glycine-methionine-glycine-methionine glycine-methionine-glycine or methionine-methionine-methionine-methionine methionine-glycine-glycine-glycine-glycine-glycine.
- [Claim 4]The fusion protein of any one of claims 1 to 3, wherein the TBRII consists ofthe amino acid sequence of SEQ ID NO: 4; and/orwherein the TBRII is an extracellular domain of TBRII.
- [Claim 5]The fusion protein of claim 4, wherein the extracellular domain of TBRII consists of the amino acid sequence of SEQ ID NO: 5.
- [Claim 6]The fusion protein of any one of claims 1 to 5, wherein the modified IL-7 andTBRII are linked by an immunoglobulin Fc domain.
- [Claim 7]The fusion protein of claim 6, wherein the Fc domain is a wild type or variant;and/orwherein the TBRII, Fc domain and modified IL-7 are linked in the direction from theN-terminus to the C-terminus.
- [Claim 8]The fusion protein of claim 6 or claim 7, wherein a first linker is furtherincluded between TBRII and the Fc domain; and/orwherein a second linker is further included between the Fc domain and modified IL-7.
- [Claim 9]The fusion protein of claim 8, wherein the first linker consists of the aminoacid sequence of SEQ ID NO: 14; and/orwherein the second linker consists of the amino acid sequence of SEQ ID NO: 15.
- [Claim 10]The fusion protein of claim 6, wherein the modified IL-7, Fc domain andTBRII are linked in the direction from the N-terminus to the C-terminus.
- [Claim 11]The fusion protein of claim 10, wherein a first linker is further includedbetween the modified IL-7 and Fc domain; and/orwherein a third linker is further included between the Fc domain and TBRII.
- [Claim 12]The fusion protein of claim 11, wherein the first linker consists of the aminoacid sequence of SEQ ID NO: 14; and/orwherein the third linker consists of the amino acid sequence of SEQ ID NO: 16.
- [Claim 13]A nucleic acid molecule which is isolated and encodes the fusion proteinaccording to any one of claims 1 to 12.
- [Claim 14]The nucleic acid molecule of claim 13, wherein the nucleic acid moleculefurther includes a signal sequence or a leader sequence.
- [Claim 15]An expression vector, comprising the nucleic acid molecule according toclaim 13 or 14.
- [Claim 16]A host cell, comprising the expression vector of claim 15.Genexine, Inc. Postech Research and Business Development Foundation Patent Attorneys for the Applicant/Nominated PersonSPRUSON&FERGUSON
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| WO2022086988A1 (en) * | 2020-10-20 | 2022-04-28 | Cura Therapeutics, Inc. | Multi-functional and multi-valent interleukin-tgf-beta receptor fusion polypeptides |
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| US20020004037A1 (en) * | 1998-06-16 | 2002-01-10 | Victor Koteliansky | Variant type II TGF-beta receptor fusion proteins and methods |
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| EP1699822B1 (en) * | 2003-12-30 | 2008-04-23 | MERCK PATENT GmbH | Il-7 fusion proteins with antibody portions, their preparation and their use |
| EP1746161A1 (en) * | 2005-07-20 | 2007-01-24 | Cytheris | Glycosylated IL-7, preparation and uses |
| MA43135A (en) * | 2015-10-30 | 2018-09-05 | Aleta Biotherapeutics Inc | COMPOSITIONS AND METHODS FOR THE TREATMENT OF CANCER |
| CN108697765A (en) * | 2015-12-04 | 2018-10-23 | 格纳西尼有限公司 | The pharmaceutical composition that IL-7 fusion proteins are merged containing immunoglobulin FC for preventing or treating disease caused by human papilloma virus |
| WO2017134305A1 (en) * | 2016-02-05 | 2017-08-10 | Orionis Biosciences Nv | Bispecific signaling agents and uses thereof |
| KR20180133198A (en) * | 2016-05-04 | 2018-12-13 | 암젠 인크 | Interleukin-2 mutein for proliferation of T-regulatory cells |
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| US20230257438A1 (en) * | 2020-06-30 | 2023-08-17 | Gi Innovation, Inc. | Fusion protein comprising anti-lag-3 antibody and il-2, and use thereof |
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| US20020004037A1 (en) * | 1998-06-16 | 2002-01-10 | Victor Koteliansky | Variant type II TGF-beta receptor fusion proteins and methods |
| KR20160146584A (en) * | 2015-06-11 | 2016-12-21 | 주식회사 제넥신 | Modified interleukin-7 protein and uses thereof |
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