WO2015149708A1 - New recombinant bifunctional fusion protein, preparation method therefor and use thereof - Google Patents

Abstract

Disclosed are a recombinant bifunctional fusion protein, the preparation method therefor and the use thereof. Specifically, disclosed is a fusion protein having an element A comprising TGF-β receptor extracellular domain, an element B comprising VEGFR1 second extracellular domain D2 and an immunoglobulin element C that are connected in series. The protein can simultaneously bind to two kinds of ligands: VEGF and TGF-β1, and inhibit the biological activity of the ligands. Also provided is the use of the new recombinant bifunctional fusion protein in treating diseases.

Description

Novel recombinant bifunctional fusion protein and preparation method and use thereof Technical field

The invention relates to the field of biomedicine. More specifically, the present invention relates to a novel recombinant bifunctional fusion protein, a process for its preparation and use.

Background technique

Diseases such as tumors and liver fibrosis take tens of millions of lives every year, causing hundreds of millions of dollars in economic losses. The binding of TGF-β and VEGFR receptors on the surface of cell membrane and the corresponding ligands is an important cause of the development and development of such diseases. Therefore, the development of corresponding protein ligand drugs has a good application prospect.

Currently known protein drugs include growth factors, hormone proteins, enzyme proteins, cytokines, interferons, erythropoietin, and fusion proteins. In addition to the fusion protein, other protein drugs are homogeneous proteins, that is, contain only one protein component. Existing fusion protein drugs (such as Avastin and Aflibercept) are composed of the extracellular domain of the receptor protein and the human IgG Fc segment. Although they contain two or more protein components, they still Only one function is to block the binding of one cell membrane endogenous receptor to the corresponding ligand.

Although genetically recombinant bifunctional fusion proteins have been reported, their compositions and corresponding targets vary. Bifunctional fusion protein drugs which have hitherto been able to bind to both TGF-β1 and VEGF and simultaneously block the biological activities induced by these two targets have not been reported. Therefore, the development of such novel bifunctional fusion protein drugs is of great significance for prolonging the survival time of patients, improving the quality of life of patients and reducing mortality.

Summary of the invention

The object of the present invention is to provide a novel recombinant bifunctional fusion protein, a preparation method and use thereof.

In a first aspect of the invention, there is provided a fusion protein comprising the following elements fused together:

(i) an optional signal peptide at the N-terminus;

(ii) a first protein component;

(iii) a second protein component;

(iv) an immunoglobulin element linked to the first protein element and/or the second protein element,

Wherein the signal peptide is operably linked to the fusion element consisting of (ii), (iii) and (iv);

And the first protein element is a TGF-β receptor extramembrane region protein element; the second protein element is a protein element comprising a second extramembranous region D2 of the vascular endothelial growth factor receptor VEGFR1.

In another preferred embodiment, "operably linked" means that the signal peptide can direct expression or transmembrane transfer (localization) of the fusion element.

In another preferred embodiment, the fusion protein has a structure selected from the group consisting of:

(1) Structure of Formula Ia or Formula Ib:

D-A-B-C (Ia), or

D-B-A-C (Ib)

(2) The structure described in Formula IIa or IIb:

D-A-C-B (IIa), or

D-B-C-A (IIb)

among them,

A is a TGF-β receptor extracellular domain protein element;

B is a protein element comprising a second extramembranous region D2 of the vascular endothelial growth factor receptor VEGFR1;

C is an immunoglobulin element;

D is an optional signal peptide sequence;

"-" means a peptide bond or a peptide linker to which the above elements are attached.

In another preferred embodiment, the element A is selected from the group consisting of TβRI, TβRII, TβRIII.

In another preferred embodiment, the element A is a protein element of the TβRII extramembranous region.

In another preferred embodiment, the element A has the amino acid sequence shown in positions 24-186 of SEQ ID NO.: 1.

In another preferred embodiment, the element B has the amino acid sequence (core sequence) shown at positions 190-282 of SEQ ID NO.: 1 and is 94, 95, 96, 97, 98, 99, or 100 in length. Amino acids.

In another preferred embodiment, the amino acid sequence flanking the amino acid sequence (core sequence) shown in positions 190-282 of SEQ ID NO.: 1 in element B is derived from the second extramembranous region of native VEGFR1, respectively. Amino acid sequence on both sides of D2 (Domain 2,).

In another preferred embodiment, the D2 has a flanking sequence, and the flanking sequence comprises:

a first flanking sequence at the amino terminus of D2; and/or a second flanking sequence at the carboxy terminus of D2.

In another preferred embodiment, the first flanking sequence consists of 1-5 amino acid residues.

In another preferred embodiment, the second flanking sequence consists of 1-2 amino acid residues.

In another preferred embodiment, the first and second flanking sequences are derived from the amino acid sequences flanking the second extramembranous region D2 (positions 190-282 of SEQ ID NO.: 1) of native VEGFR1, respectively.

In another preferred embodiment, the first flanking sequence is SDTGR.

In another preferred embodiment, the second flanking sequence is NT.

In another preferred embodiment, the element C is an Fc fragment of human immunoglobulin IgG1.

In another preferred embodiment, the peptide linker is 0-10 amino acids in length, preferably 0-5 amino acids.

In another preferred embodiment, the fusion protein further comprises a signal peptide element D.

In another preferred embodiment, the amino acid sequence of the signal peptide element D is shown as positions 1-23 of SEQ ID NO: 1.

In another preferred embodiment, the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 1.

In another preferred embodiment, the fusion protein does not contain a signal peptide and the structural formula is selected from the group consisting of:

(1) The structure of the formula Ia' or formula Ib':

A-B-C (Ia'), or

B-A-C (Ib')

(2) Structure of Formula IIa' or Formula IIb':

A-C-B (IIa'), or

B-C-A (IIb')

In the formula, A, B, C and "-" are as defined above.

In another preferred embodiment, the fusion protein has the following functions:

a) binding activity to VEGF EC ₅₀ is 0.6-2 nM;

b) binding activity to TGF-β1 EC ₅₀ is 1.5-2.5 nM;

c) can simultaneously bind to both VEGF and TGF-β1 ligands;

d) blocking VEGF-induced angiogenesis in vitro or in vivo;

e) inhibits migration and invasion of tumor cells induced by TGF-β1.

In a second aspect of the invention, there is provided a protein dimer consisting of two fusion proteins according to any one of the first aspects of the invention.

In another preferred embodiment, the dimer has a structure selected from the group consisting of:

(1) Structures of Formula Ia-1 or Formula Ib-1:

(2) The structure described in Formula IIa-1 or Formula IIb-1:

among them,

A is a TGF-β receptor extracellular domain protein element;

B is a protein element comprising a second extramembranous region D2 of VEGFR;

C is an immunoglobulin element;

D is an optional signal peptide sequence;

"-" means a peptide bond or a peptide linker connecting the above elements;

"‖" means a disulfide bond.

In another preferred embodiment, the fusion protein does not contain a signal peptide and has a structure selected from the group consisting of:

(1) Structures of Formula Ia-1' or Formula Ib-1':

(2) Structures of Formula IIa-1' or Formula IIb-1':

In the formula, A, B, C, "-" and "‖" are as defined above.

In a third aspect of the invention, an isolated polynucleotide is provided, the polynucleotide encoding according to The fusion protein of the first aspect of the invention.

According to a fourth aspect of the invention, a vector comprising the polynucleotide of the third aspect of the invention is provided.

According to a fifth aspect of the invention, there is provided a host cell comprising the vector of the fourth aspect of the invention or the polynucleotide of the third aspect of the invention.

In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell (eg, CHO cell, NSO cell, 293 cell).

According to a sixth aspect of the invention, a method of producing a protein comprising the steps of:

(1) cultivating the host cell of claim 8 under conditions suitable for expression, thereby expressing the fusion protein of claim 1;

(2) isolating the fusion protein or a dimer formed from the fusion protein.

In a seventh aspect of the invention, a pharmaceutical composition is provided, the composition comprising:

a fusion protein according to the first aspect of the invention and/or a protein dimer according to the second aspect of the invention, and

A pharmaceutically acceptable carrier.

In an eighth aspect of the invention, there is provided a use of a fusion protein according to the first aspect of the invention and/or a protein dimer according to the second aspect of the invention for the preparation of a medicament for the treatment of a disease.

In another preferred embodiment, the disease is a disease associated with TGF-β and VEGF.

In another preferred embodiment, the disease is selected from the group consisting of: tumor, liver fibrosis.

In another preferred embodiment, the tumor comprises: a colorectal cancer tumor, a lung cancer tumor, a liver cancer tumor, a breast cancer tumor, a gastric cancer tumor, and a pancreatic cancer tumor.

In a ninth aspect of the invention, a method of inhibiting a disease associated with TGF-β and VEGF, comprising the step of administering the fusion protein of the first aspect to a subject in need thereof.

In another preferred embodiment, the fusion protein is administered as a monomer and/or a dimer.

In another preferred embodiment, the object is a human.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure. It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 is a schematic diagram showing the molecular structure of a recombinant fusion protein TβRII-D2-Fc, which shows that TβRII-D2-Fc contains three components, the extramembranous end of TGF-β type receptor (TβRII), VEGF receptor 1 Membrane The second region of the outer end (VEGFR1-D2), and the Fc fragment of human IgG1.

Figure 2A shows the nucleotide sequence of TβRII-D2-Fc, in which 69 nucleotides of red are signal peptide coding sequences, 483 nucleotides of blue are coding sequences of the outer end of TβRII membrane, 300 reddish The nucleotide is the coding sequence of VEGFR1-D2; the first 6 of the 702 nucleotides in black are EcoRI cleavage sites, and the last 696 nucleotides are coding sequences of the human IgG1 Fc fragment.

Figure 2B shows the amino acid sequence of TβRII-D2-Fc, in which the red 23 amino acids are signal peptides, the blue 161 amino acids are TβRII membrane outer ends, the light red 100 amino acids are VEGFR1-D2, and the black 234 The amino acids are the EcoRI site (2 amino acids "EF") and the human IgG1 Fc fragment (232 amino acids).

Fig. 3A shows the SDS-PAGE electrophoresis pattern of each fusion protein. In the figure, the R lane is an electrophoresis band under reducing conditions, and the NR lane is an electrophoresis band under non-reducing conditions, from TβRII- The electropherogram of D2-Fc shows that under reducing conditions, the molecular size of TβRII-D2-Fc is about 80-90 kDa, and under non-reducing conditions is greater than 170 kDa, which is larger than the theoretical value (57 kDa, 120 kDa) because there are many molecules. Glycosylation site, suggesting that the protein is glycosylated; from the electropherogram of D2-TβRII-Fc, it can be seen that the size is slightly larger than TβRII-D2-Fc under both reducing and non-reducing conditions, which is The change in structural composition results in a change in spatial structure; from the electropherogram of TβRII-Fc-D2, it can be seen that its size is similar to that of TβRII-D2-Fc, which is 80-90 kDa under reducing conditions and greater than 170 kDa under non-reducing conditions.

Figure 3B shows the HPLC analysis of the TβRII-D2-Fc protein. It can be seen from the figure that the TβRII-D2-Fc protein has a high purity and a polymer content of less than 2%.

Figure 4A shows the results of the TGF-β1 binding activity test of each fusion protein and the target. As can be seen from the figure, the fusion proteins of the three different combinations have the binding activity to TGF-β1, wherein the activity of TβRII-D2-Fc Preferably, D2-TβRII-Fc is less active and TβRII-Fc-D2 is the weakest. The EC _{50 of the} three proteins were: TβRII-D2-Fc = 1.52 nM; D2-TβRII-Fc = 2.14 nM; TβRII-Fc-D2 = 2.50 nM. The positive control protein TβRII-Fc also had good TGF-β1 binding activity with an EC ₅₀ of 2.30 nM. It should be emphasized that at the same concentration, the binding activity of TβRII-D2-Fc to the target is about 34% higher than that of TβRII-Fc. Both D2-Fc and the negative control protein IgG-Fc were inactive.

Figure 4B shows the results of the test for binding activity of each fusion protein to the target VEGF-165. As can be seen from the figure, the fusion proteins of the three different combinations have the binding activity to VEGF-165, wherein D2-TβRII-Fc has the best activity, TβRII-Fc-D2 has the second activity, and TβRII-D2-Fc. Relatively weak. The EC _{50 of the} three proteins were: TβRII-D2-Fc=0.60nM; D2-TβRII-Fc=0.11nM; TβRII-Fc-D2=0.16nM; positive control protein D2-Fc had good VEGF-165 binding. The activity had an EC ₅₀ of 0.14 nM; neither TβRII-Fc nor the negative control IgG-Fc had activity.

As can be seen by comparing FIG. 4A and FIG. 4B, the fusion protein of the present invention has excellent simultaneous binding to the target TGF-β1 and target VEGF activity.

Figure 5 shows that TβRII-D2-Fc inhibits TGF-β1-induced tumor cell invasion, Figure A is a control (no additional addition of TβRII-D2-Fc and TGF-β1 in the medium), and Figure B adds TGF- Β1 10ng/ml, TGF-β1 10ng/ml, TβRII-D2-Fc 1μg/ml was added to Figure C, TGF-β110ng/ml and TβRII-D2-Fc 10μg/ml were added to Figure D, added in Figure E. TGF-β1 10 ng/ml, TβRII-D2-Fc 50 μg/ml, and Figure F added TGF-β1 10 ng/ml and hIgG 50 μg/ml. As can be seen from Fig. 5, TGF-β1 significantly induced the invasion of tumor cells from the upper layer of the chamber to the lower layer (Invasion), but after the addition of TβRII-D2-Fc, Tumor cell invasion was significantly inhibited and the inhibitory effect was dose dependent. The negative control protein hIgG did not inhibit TGF-β1-induced tumor cell invasion (Invasion). The results of this experiment indicate that TβRII-D2-Fc binds to TGF-β1 and blocks the binding of TGF-β1 to the TGF-β receptor on the tumor cell membrane, thereby inhibiting downstream signaling and ultimately inhibiting tumor cell invasion. .

Figure 6 shows that TβRII-D2-Fc blocks vascular endothelial cell tubular formation induced by VEGF. Panel A is a control (only VEGF-165 is added to the medium, no additional TβRII-D2-Fc is added), and Figure B is added with VEGF-165 20 ng/ml, TβRII-D2-Fc 20 μg/ml, and Figure C is added with VEGF- 165 20 ng/ml, TβRII-D2-Fc 50 μg/ml, Figure D was added with VEGF-165 20 ng/ml, TβRII-D2-Fc 100 μg/ml, and Figure E was added with VEGF-165 20 ng/ml and hIgG 100 μg/ml. It can be seen from Fig. 6 that VEGF-165 induces tubular formation of vascular endothelial cells (HUVEC), but if TβRII-D2-Fc is simultaneously added, the tubular formation of HUVEC cells can be significantly inhibited, and the inhibitory effect is dose-dependent. The negative control protein hIgG could not inhibit the vascular formation of HUVEC cells induced by VEGF-165.

Figure 7A shows the inhibitory effect of the fusion protein of the present invention on the growth of mouse breast cancer cells (4T1). It can be seen from the figure that if the tumor cells are not treated after subcutaneous inoculation, the tumor volume grows to 600 mm ³ (mm ³ ) after 20 days, and after treatment with the fusion protein, the two doses (5 mg/kg, 10 mg/kg) Both TβRII-D2-Fc and high dose (10 mg/kg) D2-Fc significantly inhibited tumor growth, and the tumor volume after 20 days was only half that of the negative control group, which was 300 mm ³ (mm ³ ). TβRII-Fc has a certain inhibitory effect, but it is not significant.

Figure 7B shows the inhibitory effect of the fusion protein of the present invention on the metastasis of mouse breast cancer cells (4T1). As can be seen from the figure, both doses of TβRII-D2-Fc significantly inhibited tumor metastasis to the lungs, and the inhibition rate was 60-70% compared with the negative control group. TβRII-Fc inhibited tumor metastasis by 61%, while D2-Fc inhibition was only 50%.

detailed description

The inventors have for the first time established a genetic engineering technology platform through extensive and in-depth research, which can be used to produce recombinant bifunctional fusion protein drugs, such as TβRII-D2-Fc. The present invention has been completed on this basis.

Taking TβRII-D2-Fc as an example, it has the following functions: 1) can bind to both VEGF and TGF-β1 ligands; 2) block VEGF-induced in vitro or in vivo angiogenesis; 3) inhibit TGF -β1 induced migration and invasion of tumor cells.

The experiments confirmed that the TβRII-D2-Fc of the present invention not only has the binding activity to VEGF and TGF-β1 at the same time, but has an EC ₅₀ of 0.60 and 1.53, respectively, and can treat certain diseases synergistically and more effectively, especially such as tumors and old age. Eye macular degeneration, liver fibrosis and other diseases.

VEGFR and its extramembrane region

The VEGFR protein belongs to the receptor tyrosine kinase superfamily and is a membrane mosaic protein. The extramembranous portion of VEGFR has approximately 750 amino acid residues and consists of seven Ig domains that are structurally similar to immunoglobulins. Upon binding to its corresponding ligand, VEGFR proteins can induce a range of different biological functional responses based on their corresponding receptor properties. VEGFR proteins of the invention include: VEGFR1 (Flt-1), VEGFR2 (KDR/FLk-1), VEGFR3 (Flt-4), or a combination thereof.

In the present invention, VEGFR1 (Flt-1) is preferred, and preferably native VEGFR1 is wild type.

D2 of the present invention refers to the second extramembranous region (Domain 2) of VEGFR1 (Flt-1). A representative D2 sequence is position 190-282 of SEQ ID NO.:1.

TGF-β receptor and its extramembranous region

The TGF-beta receptor is a single transmembrane protein receptor with serine/threonine protein kinase activity in the intracellular region, which functions as a heterodimer. There are currently 1 to 5 types of TGF-β receptors, and there are 3 types of TGF-β glycoprotein receptors (ie, type I, type II, and type III receptors) on the surface of various human cells. Type and type II receptors act as signal transduction. Type I and type II receptors are single transmembrane serine/threonine protein kinase receptors. The extracellular zone is shorter and the cytoplasmic zone is longer. The extracellular domain is rich in cysteine. The cytoplasmic domain contains a serine/threonine protein kinase domain. When combined with its corresponding ligand, TGF-β protein induces a range of different biological functional responses based on its corresponding receptor properties.

The TGF-beta receptor protein of the present invention comprises: TβRI, TβRII, TβRIII or a combination thereof. In the present invention, TβRII is preferred, and preferably the natural TβRII is wild type.

A preferred class of TGF-beta receptor extramembranous regions of the invention refers to the extramembranous region of TβRII. A representative extracellular domain sequence of TβRII is position 24-184 of SEQ ID NO.:1.

Immunoglobulin G element

In the present invention, a suitable immunoglobulin G element is not particularly limited and may be an immunoglobulin element derived from a human or other mammal, or a mutant and a derivative thereof. An element of human immunoglobulin is preferred.

Human immunoglobulin G includes four subclasses: IgG1, IgG2, IgG3, IgG4. The protein structures of these four subclasses have great similarities, with four regions: one variable region (VH) and three constant regions (CH1, CH2, CH3). The Fc fragment consists of two constant regions (CH2-CH3) with a disulfide bond in the CH2 region such that the two Fc fragment monomers constitute a covalently bound homodimer. Under normal physiological conditions, the concentration of IgG in human plasma is highest in IgG1, IgG2 is second, and IgG3 and IgG4 concentrations are lower.

A preferred G element is a human IgGl Fc fragment, or a mutant or derivative thereof.

Bifunctional fusion protein and preparation thereof

In the present invention, "recombinant bifunctional fusion protein", "protein of the invention", "fusion protein of the invention", "bifunctional fusion protein" are used interchangeably and mean having the structure of formula Ia or Ib, or IIIa or The structure described in IIIb, that is, a fusion protein comprising a protein element comprising a TGF-β receptor extramembrane region protein element, a second extramembranous region D2 of VEGFR, and an immunoglobulin element. A representative example is TβRII-D2-Fc. The protein of the invention may be a monomer or a multimer (e.g., a dimer) formed from a monomer. Furthermore, it is to be understood that the term also encompasses active fragments and derivatives of fusion proteins.

As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is isolated and purified, as separated from other substances present in the natural state.

As used herein, "isolated recombinant fusion protein" means that the recombinant fusion protein is substantially free of natural Other related proteins, lipids, sugars or other substances. One skilled in the art can purify recombinant fusion proteins using standard protein purification techniques. Substantially pure proteins produce a single major band on a non-reducing polyacrylamide gel.

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

The present invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby.

As used herein, the term "primer" refers to a generic term for a oligodeoxynucleotide that, in pairing with a template, can be used to synthesize a DNA strand complementary to a template under the action of a DNA polymerase. The primer may be native RNA, DNA, or any form of natural nucleotide. The primer may even be a non-natural nucleotide such as LNA or ZNA. The primer is "substantially" (or "substantially") complementary to a particular sequence on a strand on the template. The primer must be sufficiently complementary to a strand on the template to initiate extension, but the sequence of the primer need not be fully complementary to the sequence of the template. For example, a sequence that is not complementary to the template is added to the 5' end of the primer complementary to the template at the 3' end, and such primer is still substantially complementary to the template. As long as there are sufficiently long primers to bind well to the template, the non-fully complementary primers can also form a primer-template complex with the template for amplification.

The full length nucleotide sequence of the element of the fusion protein of the present invention (e.g., VEGFR1D2 or TβRII extramembranous region) or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or a synthetic method. For PCR amplification, primers can be designed according to published nucleotide sequences, particularly open reading frame sequences, and used as commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art. The template is amplified to obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.

Once the relevant sequences are obtained, the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short. Usually, a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.

The invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered using the vector or fusion protein coding sequences of the invention, and methods of producing the proteins of the invention by recombinant techniques.

The polynucleotide sequences of the present invention can be utilized to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally there are the following steps:

(1) using the polynucleotide (or variant) encoding the protein of the present invention, or containing the polynucleoside An acid recombinant expression vector transforms or transduces a suitable host cell;

(2) a host cell cultured in a suitable medium;

(3). Separating and purifying the protein from the culture medium or the cells.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences of the proteins of the invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.

The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS, or 293 cells, and the like.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl ₂ method, and the procedures used are well known in the art. Another method is to use MgCl _2. Conversion can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The cultivation is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to the appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.

The protein in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

antibody

In the present invention, "antibody" and "ligand" are used interchangeably and refer to polyclonal antibodies and monoclonal antibodies, particularly monoclonal antibodies, which are specific for the protein of the present invention. Here, "specificity" means that the antibody can bind to the protein of the present invention or a fragment thereof, respectively. Preferably, those antibodies which bind to a protein or fragment of the invention but which do not recognize and bind to other non-related antigen molecules. Antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art.

The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single chain Fv molecules; Or chimeric antibodies.

Peptide linker

The invention provides a bifunctional fusion protein which optionally contains a peptide linker. The size and complexity of the peptide linker may affect the activity of the protein. In general, the peptide linker should be of sufficient length and flexibility to ensure that the two proteins attached have sufficient freedom in space to perform their function. At the same time, the effect of the formation of an alpha helix or a beta sheet in the peptide linker on the stability of the fusion protein is avoided.

The length of the linker peptide is generally from 0 to 10 amino acids, preferably from 0 to 5 amino acids.

Pharmaceutical composition and method of administration

The invention also provides a composition comprising an effective amount of a fusion protein of the invention, and a pharmaceutically acceptable carrier. In general, the fusion proteins of the invention may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8.

As used herein, the term "effective amount" or "effective amount" refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals, such as from 0.001 to 99% by weight; preferably 0.01-95 wt%; more preferably, 0.1-90 wt%.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a fusion protein of the invention and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.

The effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the fusion protein of the invention such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc. In general, when the fusion protein of the present invention is administered at a dose of about 5 mg to 20 mg/kg of animal body weight per day (preferably 5 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.

The fusion protein of the present invention is particularly suitable for the treatment of diseases in which VEGF and TGF-β1 are excessively secreted, or diseases characterized by abnormal vascular proliferation and invasion of tumor cells. Representative diseases include, but are not limited to, tumors, wet macular degeneration, or liver fibers.

The fusion protein of the present invention and its dimer or multimer mainly include the following advantages:

1) It can bind to both VEGF and TGF-β1 ligands, and has strong binding activity to VEGF and TGF-β1, and the binding activity EC ₅₀ can reach 0.60 nM and 1.53 nM, respectively;

2) blocking VEGF-induced angiogenesis in vitro or in vivo;

3) inhibiting the migration and invasion of tumor cells induced by TGF-β1;

4) can block liver fibrosis induced by TGF-β1 and VEGF;

5) It can block the immunosuppressive activity of TGF-β, thereby enhancing immune function.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. Experimental methods in which the specific conditions are not indicated in the following examples are generally carried out according to the conditions described in conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight.

Example 1

Construction of TβRII-D2-Fc expression vector

The TβRII extramembrane region gene coding sequence consists of 483 nucleotides, as shown at positions 70-552 of SEQ ID NO.: 2. The VEGFR1-D2 gene coding sequence consists of 300 nucleotides, as shown at positions 553-852 of SEQ ID NO.: 2, including 279 nucleotides of the D2 coding sequence, 15 nucleotides of the upstream flanking sequence, The downstream flanking sequence is 6 nucleotides. A signal peptide coding sequence derived from TβRII (i.e., positions 1-69 in SEQ ID NO.: 2) was added to the 5' end of the TβRII extramembrane region to constitute 852 nucleotides.

At the amino terminus of the 852 nucleotides, a "HindIII" gene cloning site was added, and an "EcoRI" gene cloning site was added to the carboxy terminus to form a gene fragment containing 873 nucleotides.

The synthesized product (synthesized by Nanjing Kingsray Biotech Co., Ltd.) was digested with HindIII/EcoRI and cloned into the pHB-Fc plasmid vector to form a pHB-TbRII-D2-Fc protein expression vector. The pHB-Fc plasmid vector was prepared by using the pcDNA/HA-FLAG (Accession #: FJ524378) vector as the starting plasmid, and the endozyme EcoRI was followed by the Fc sequence of human IgG1, which was preceded by the endonuclease HindIII. The human cytomegalovirus (HCMV) promoter sequence (Accession#: X17403) was followed by the ampicillin resistance gene and the Chinese hamster glutamine synthetase gene (Accession #: X03495) in front of the HCMV promoter. After the sequence design, the company will be commissioned by Shanghai Jierui Biological Engineering Co., Ltd. for synthesis and transformation.

The sequence SEQ ID NO: 2 is the nucleotide sequence encoding the recombinant bifunctional fusion protein, as shown in Figure 2A. The full length is 1554 bp, of which 1-69 bp is the signal peptide coding sequence, 70-552 bp is the TβRII extramembrane region coding sequence, 553-852 bp is the VEGFR1D2 coding sequence, 853-858EcoRI cleavage site GAATTC, and 859-1554 bp is the Fc fragment. TGA is the termination password.

Figure 1 is a schematic diagram showing the molecular structure of the recombinant bifunctional fusion protein TβRII-D2-Fc. This schematic diagram is for illustrative purposes only and does not represent the specific actual structure of the bifunctional fusion protein of the invention.

The sequence SEQ ID NO: 1 is the amino acid sequence encoding the recombinant bifunctional fusion protein, as shown in Figure 2B. Full length 518 amino acids. Among them, 1-23 amino acids are signal peptides, 24-184 amino acids are TβRII extramembrane regions, 185-284 are VEGFR1D2 fragments containing flanking sequences (underlined), and 285-286 are EcoRI cleavage sites 2 The amino acids, amino acids 287-518 are Fc fragments.

Example 2

Expression of TβRII-D2-Fc, D2-TβRII-Fc, TβRII-Fc-D2

The host cell used for protein expression was CHO-K1 cells (Cat# CCL-61) purchased from ATCC. The cells were acclimated into CHO-K1 cells that were cultured in suspension in serum-free medium (EX-CELLTM 302) after a series of domestication steps.

Using this cell, plasmid pHB-TβRII-D2-Fc, pHB-D2-TβRII-Fc, and pHB-TβRII-Fc-D2 were separately transferred into cells by electroporation. Specifically, the cells in the logarithmic growth phase were collected under aseptic conditions, centrifuged (1200 rpm x 5 min), resuspended in complete medium, and the cell density was adjusted to 1 x 10 ⁷ cells/ml. Transfer 350 ul of cell suspension to a 0.4 cm electric rotor and pulse once under set electrical conditions (voltage range 200 to 350 V, generally 260 V, time 20 ms or so). Add 10–30 ug of plasmid DNA to an electric rotor containing cells, mix gently, and place the electric rotor into the electro-rotator to pass the pulse. Take out the electric rotor, let stand for 5 minutes, add 0.6 ml of cell culture medium, mix it, suck it out, transfer it to the culture dish, and put it into the incubator. Protein expression was examined after 24-48 hours. If protein expression is successful, the gene is successfully transferred. At this time, the cells are diluted with the medium and then transferred to 20 96-well cell culture plates with 3000-5000 cells per well. The cells are screened through a series of pressures (glutamine synthetase inhibitors) to finally screen for cell lines that are highly expressed.

In the production of the protein, cells of the cell line with high expression of the protein were inoculated into a cell reactor containing 3 liters of EX-CELLTM 302 medium at a cell density of 3 x 10 ⁵ cells/ml, and culture conditions were 37 ° C, 5% CO 2 . The cells are tested by pH, glucose, glutamine, etc. during the cultivation process, and supplemented with nutrients according to various indicators. When the cell density reached 5-6 x 10 ⁶ cells/ml, the culture temperature was lowered from 37 ° C to 33 ° C, and the culture was continued until the cell viability reached 60-70%. The harvested cell culture supernatant was concentrated by ultrafiltration and purified by Protein A affinity chromatography. The purified protein was quantitatively determined by the Lowry method (refer to the 2010 edition of the Chinese Pharmacopoeia), and the protein quantification standard was bovine serum albumin (batch number 140619-201120, China National Institute for Drug Control). The size of the produced protein by SDS-PAGE analysis is basically consistent with the theoretical value, and the endotoxin content is lower than the standard requirement.

By protein electrophoresis (SDS-PAGE) analysis, it was found that under reducing conditions, TβRII-D2-Fc was at the position of ~80kDa (monomer), and under non-reducing conditions, it was larger than 170kDa (dimer). The protein size is similar to TβRII-D2-Fc. In addition, the measured molecular weight of the recombinant protein of the three structural combinations (Fig. 3) suggests that the recombinant protein has a certain degree of glycosylation (the theoretical molecular weight of the dimer is 114 kDa).

HPLC analysis of protein purity was greater than 98%.

Example 3

Detection of binding activity of TβRII-D2-Fc to target (VEGF and TGF-β1)

The binding properties of the fusion protein to the target (VEGF and TGF-β1) were determined by enzyme-linked immunosorbent assay (ELISA). Specific steps are as follows:

TGF-β1 (Cat: 10804-HNAH, Sino biological Inc.) and VEGF-165 (Cat: 11066-HNAH, Sino biological) were coated with CBS (Sigma-Aldrich Co., Product code: 1001329288 C3041-100CAP). Inc.) were diluted to 500ng / ml, was added to the ELISA plates take 100ul (Nunc ^TM, Cat: 442404) per well 50ng. The coated plates were placed in a refrigerator at 4 ° C overnight. The test was washed once with 0.05% PBS-T and then with 3% skim milk for 1 hour at room temperature. Two-fold serial dilutions of TbRII-D2-Fc protein (100 nM, 50 nM, 25 nM, up to 0.0244 nM) were added to the coated plates at 100 ul per well. After incubating for one hour at room temperature, the sample was discarded, washed 5 times with 0.05% PBS-T, and then 100 ul of diluted (1:20000) HRP-Rabbit Anti-Human IgG Fc (Luoyang Aotong, Cat#: C030222) was added. Incubate for one hour at room temperature, wash the washing solution 5 times, add HRP substrate, and darken the color for 10-20 minutes, then stop the color reaction with 2N H ₂ SO ₄ and read the 0D450 value on the microplate reader.

The results showed (Table 1) that TβRII-D2-Fc had binding activities to TGF-β1 (Fig. 4A) and VEGF-A (Fig. 4B), respectively, and the corresponding EC ₅₀ reached 1.53 nM and 0.6 nM, respectively. The other two structurally combined proteins also have strong binding activity to the two targets, with EC _{50 of} about 2.5 nM (TGF-β1) and 0.16 nM (VEGF-165), respectively.

Table 1 Binding activity of fusion protein to target EC ₅₀ (nM)

	TβRII-D2-Fc	D2-TβRII-Fc	TβRII-Fc-D2	D2-Fc	TβRII-Fc
						TGF-β1	1.53	2.14	2.50	Inactive	2.33
VEGF-A	0.60	0.11	0.16	0.14	Inactive

Example 4

TβRII-D2-Fc inhibits tumor cell invasion induced by TGF-β1

Tumor cell invasion experiments were performed using a 24-well cell chamber plate. The method comprises the following steps: adding a medium containing TGF-β1 to the chamber, adding tumor cells (PC-3) and a certain concentration of TβRII-D2-Fc to the upper layer of the filter, and incubating for 24 hours in the incubator, filtering with crystal violet The membrane was stained and the density of cells at the bottom of the photographed filter was observed.

The invasion of TGF-β1-induced tumor cells by TβRII-D2-Fc was analyzed using PC-3 cells. The specific procedure was as follows: A Matrigel-containing cell sieve was placed in a 24-well culture plate containing cell culture medium (BD Bioscience), and the culture solution contained 10 ng/ml of TGF-β1. Add 1×10 ⁵ PC-3 cells to the cell sieve, then add different concentrations of TβRII-D2-Fc and hIgG to the corresponding sieve according to the grouping requirements, and place the 24-well cell culture plate in the cell culture incubator. The medium was cultured at 30 ° C under 5% CO ₂ for 24 hours. Remove the sieve, fix the filter with 4% paraformaldehyde for 15 minutes, stain 0.5% crystal violet dye solution for 15 minutes, wash off the excess dye solution after dyeing, and wipe off the cells that have not passed through the upper layer of the membrane with a cotton swab. Photograph the invading cells under a microscope. There are 5 different fields of view in the upper, lower, left and right of each film.

As shown in Figure 5, TβRII-D2-Fc significantly inhibited the invasion of PC-3 cells from the upper layer to the lower layer of the chamber, and the inhibitory effect was dose-dependent.

Example 5

TβRII-D2-Fc blocks vascular endothelial cell tubular formation induced by VEGF

The HUVEC cells were adjusted to a concentration of 3 × 10 ⁵ /ml, and the cells were added to a 96-well culture plate containing Matrigel at 50 ul per well. The prepared culture medium containing VEGF (20 ng/ml) and various concentrations of TβRII-D2-Fc (20, 50, 100 ug/ml) was then added to the culture plate at 50 ul per well. The culture plates were placed in an incubator and photographed and archived under different microscopes at different time points (0h, 2h, 4h, 6h, 8h, 24h).

The results showed that HUVEC cells formed a vascular pattern under the microscope when cultured in a gel in the presence of VEGF, similar to the formation of blood vessels in vivo. This experiment is often used to verify the effects of a drug on angiogenesis. Using this experiment, the inventors analyzed the effect of TβRII-D2-Fc on angiogenesis in vitro. The results indicate that (Fig. 6), TβRII-D2-Fc can significantly inhibit the tubular formation of HUVEC cells.

Example 6

TβRII-D2-Fc inhibits tumor growth and tumor metastasis

The following components were mixed using conventional techniques to prepare a final concentration of 1 wt% recombinant protein solution, which was formulated as follows:

Recombinant protein 10mg

Saline added to 10ml

Adjust the pH to 6.8-7.1.

1x10 ⁵ mouse breast cancer cells (4T1) were subcutaneously injected into the mammary gland of normal female Balb/c mice, and were randomly divided into 5 groups on the second day. The first group was intraperitoneally injected with 5 mg/kg TβRII-D2-Fc and the second group. 10 mg/kg TβRII-D2-Fc was intraperitoneally injected, 10 mg/kg D2-Fc (control) was injected intraperitoneally, and 10 mg/kg TβRII-Fc (control) was injected intraperitoneally in the fourth group twice a week for 6 times. The fifth group was a negative control and PBS was injected intraperitoneally. Tumor volume was measured three times a week. On the 21st day after the treatment, the mice were sacrificed, the tumors were harvested, the lungs were taken, and the lung metastases were observed under a microscope.

The results showed that TβRII-D2-Fc significantly inhibited tumor growth at both doses (Fig. 7A), and the inhibition rates were all greater than 50%. The tumor inhibition rate of D2-Fc is also very good, reaching ~55%, while the tumor inhibition rate of TβRII-Fc is only 26%.

In the lung metastasis (Fig. 7B), the negative control group had an average of 11.4 metastases, and the TβRII-D2-Fc treatment group had 4.2 (5 mg/kg) and 3.56 (10 mg/kg), respectively. Although TβRII-Fc could not effectively inhibit tumor growth, it significantly inhibited tumor metastasis in the lungs (4.4 of metastases). Although D2-Fc inhibited tumor growth well, the inhibition of tumor metastasis was significantly weaker than that of TβRII-D2-Fc and D2-Fc.

result

The bifunctional fusion protein TβRII-D2-Fc has a stronger synergistic inhibitory effect on tumor growth and tumor metastasis, while a fusion protein targeting only one target can only have a significant inhibitory effect, that is, it can only inhibit tumor growth. (blocking VEGF), either only inhibit tumor metastasis (blocking TGF-β1).

Furthermore, the inhibitory effect of the experimental group of 10 mg/kg TβRII-D2-Fc was also significantly better than that of the experimental group of 5 mg/kg D2-Fc+5 mg/kg TβRII-Fc.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (12)

1. A fusion protein characterized in that the fusion protein comprises the following elements fused together:

   (i) an optional signal peptide at the N-terminus;

   (ii) a first protein component;

   (iii) a second protein component;

   (iv) an immunoglobulin element linked to the first protein element and/or the second protein element,

   Wherein the signal peptide is operably linked to the fusion element consisting of (ii), (iii) and (iv);

   And the first protein element is a TGF-β receptor extramembrane region protein element; the second protein element is a protein element comprising a second extramembranous region D2 of the vascular endothelial growth factor receptor VEGFR1.
2. The fusion protein according to claim 1, wherein said fusion protein has a structure selected from the group consisting of:

   (1) Structure of Formula Ia or Formula Ib:

   D-A-B-C (Ia), or

   D-B-A-C (Ib)

   (2) The structure described in Formula IIa or IIb:

   D-A-C-B (IIa), or

   D-B-C-A (IIb)

   among them,

   A is a TGF-β receptor extracellular domain protein element;

   B is a protein element comprising a second extramembranous region D2 of the vascular endothelial growth factor receptor VEGFR1;

   C is an immunoglobulin element;

   D is an optional signal peptide sequence;

   "-" means a peptide bond or a peptide linker to which the above elements are attached.
3. The fusion protein according to claim 1, wherein the fusion protein has the following functions:

   a) binding activity to VEGF EC ₅₀ is 0.6-2 nM;

   b) binding activity to TGF-β1 EC ₅₀ is 1.5-2.5 nM;

   c) can simultaneously bind to both VEGF and TGF-β1 ligands;

   d) blocking VEGF-induced angiogenesis in vitro or in vivo;

   e) inhibits migration and invasion of tumor cells induced by TGF-β1.
4. A protein dimer characterized in that the dimer consists of two fusion proteins according to any one of claims 1-3.
5. The dimer of claim 4 wherein said dimer has a structure selected from the group consisting of:

   (1) Structures of Formula Ia-1 or Formula Ib-1:

   

   (2) The structure described in Formula IIa-1 or Formula IIb-1:

   

   among them,

   A is a TGF-β receptor extracellular domain protein element;

   B is a protein element comprising a second extramembranous region D2 of VEGFR;

   C is an immunoglobulin element;

   D is an optional signal peptide sequence;

   "-" means a peptide bond or a peptide linker connecting the above elements;

   "‖" means a disulfide bond.
6. An isolated polynucleotide, characterized in that the polynucleotide encodes the fusion protein of claim 1.
7. A vector comprising the polynucleotide of claim 6.
8. A host cell comprising the vector of claim 7 or a polynucleotide in which the polynucleotide of claim 6 is integrated.
9. A method of producing a protein, characterized in that it comprises the steps of:

   (1) cultivating the host cell of claim 8 under conditions suitable for expression, thereby expressing the fusion protein of claim 1;

   (2) isolating the fusion protein or a dimer formed from the fusion protein.
10. A pharmaceutical composition, characterized in that the composition comprises:

    The fusion protein of claim 1 and/or the protein dimer of claim 4, and

    A pharmaceutically acceptable carrier.
11. Use of the fusion protein of claim 1 and/or the protein dimer of claim 4 for the preparation of a medicament for the treatment of a disease.
12. A method of inhibiting a disease associated with TGF-β and VEGF, comprising the step of administering the fusion protein of claim 1 to a subject in need thereof.

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