Human interferon-beta variant conjugated immunocytokine and method for preparing same

Abstract

The present invention relates to an immunocytokine in which a human interferon-beta variant is conjugated to an antibody or a fragment thereof, and a method for preparing the same. The human interferon-beta variant has superior activity or functions compared with natural interferon-beta, and the productivity of the immunocytokine according to the present invention is excellent. In addition, the immunocytokine according to the present invention can be favorably used as a target therapeutic agent for multiple sclerosis or cancer since the immunocytokine express both of functions of the interferon-beta and characteristics of the antibody binding to a specific antigen.

Claims

1. An immunocytokine fusion protein comprising: (a) a human interferon-beta variant comprising a peptide defined by SEQ ID NO: 2; and (b) trastuzumab or an antigen-binding fragment thereof that is linked to the human interferon-beta variant, wherein the human interferon-beta variant has human interferon-beta activity and comprises an N-linked glycan.

2. The immunocytokine fusion protein of claim 1, wherein the human interferon-beta variant is linked to the trastuzumab or antigen-binding fragment thereof via a peptide linker.

3. The immunocytokine fusion protein of claim 2, wherein the peptide linker comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 11.

4. The immunocytokine fusion protein of claim 1, wherein the amino acid sequence of the human interferon-beta variant polypeptide is located at a heavy chain C-terminus, a light chain C-terminus, or each of heavy and light chain C-termini of the amino acid sequence of the antibody or fragment thereof.

5. The immunocytokine fusion protein of claim 1, wherein the immunocytokine comprises an amino acid sequence of SEQ ID NO: 12 and 17.

6. A polynucleotide encoding the immunocytokine fusion protein of claim 1.

7. A vector comprising the polynucleotide of claim 6.

8. A host cell transfected with the vector of claim 7.

9. A method for preparing an immunocytokine fusion protein, the method comprising: (a) providing the host cell of claim 8; (b) culturing the provided cell; and (c) preparing an immunocytokine fusion protein by collecting the immunocytokine fusion protein from the cell or a culture medium.

10. A method for increasing a yield of target-specific human interferon-beta, the method comprising: (a) cloning a polynucleotide encoding an immunocytokine fusion protein of claim 1 into an expression vector; (b) introducing the expression vector into a host cell; (c) culturing the host cell; and (d) collecting the immunocytokine fusion protein from the cell or a culture medium.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the results of western blot analysis of the expression levels of the immunocytokines produced in host cells according to the present invention (1: culture medium, 2: B12 heavy chain-natural interferon, 3: B12 heavy chain-interferon variant, 4: B12 light chain-natural interferon, 5: B12 light chain-interferon variant).

(2) FIG. 2 is a schematic diagram showing the immunocytokine with the human interferon-beta variant according to the present invention.

(3) FIG. 3 is a schematic diagram showing a procedure of constructing pRBLX2-INF by inserting a gene nucleotide sequence of heavy chain-linker-interferon into pRBLX2 vector (left) and a procedure of constructing pRBLX2-CAF by inserting a gene nucleotide sequence of heavy chain-linker-interferon-beta variant into pRBLX2 vector (right).

(4) FIG. 4 shows SDS-PAGE results of the expression of the immunocytokine with the human interferon-beta variant according to the present invention (right) and the immunocytokine with human interferon-beta (left). Here, the heavy and light chains of each case are indicated by (Lane 1 is for a marker).

(5) FIG. 5 shows western blot results of the protein expression of the immunocytokine with a human interferon-beta variant according to the present invention (Lane 2) and the immunocytokine with control human interferon (Lane 1) using anti-human IgG antibody (left) and anti-interferon antibody (right), respectively.

(6) FIG. 6 shows BCA assay results of the expression levels of the immunocytokines produced in host cells (ACC #1: B12 heavy chain-natural interferon, ACC #2: B12 heavy chain-interferon variant, ACC #6: B12 light chain-natural interferon, ACC #7: B12 light chain-interferon variant).

(7) FIG. 7 shows the results of STAT-1 phosphorylation, indicating the interferon activity of the immunocytokine in which the human interferon-beta variant was conjugated to B12 antibody according to the present invention.

(8) FIG. 8 shows the results wherein cells were treated with the immunocytokine, in which the human interferon-beta variant was conjugated with B12 antibody according to the present invention, for 24 hours, and then the interferon-beta activity of the immunocytokine was investigated through cytotoxicity (Carbiferon: the human interferon-beta variant, B12: B12 antibody, ACC #2: immunocytokine in which the human interferon-beta variant was conjugated with B12 antibody).

(9) FIG. 9 shows the results wherein cells were treated with the immunocytokine in which the human interferon-beta variant is conjugated with B12 antibody according to the present invention for 48 hours, and then the interferon-beta activity of the immunocytokine was investigated through cytotoxicity (Carbiferon: the human interferon-beta variant, B12: B12 antibody, ACC #2: immunocytokine in which the human interferon-beta variant was conjugated with B12 antibody).

(10) FIG. 10 shows schematic diagrams of immunocytokines produced by linking a rigid helical linker to ERBB2 (Herceptin) antibody (A) and c-MET antibody (B) and then conjugating the human interferon-beta variant thereto, respectively.

MODE FOR CARRYING OUT THE INVENTION

(11) Hereinafter, the present invention will be described in detail.

(12) However, the following Examples are merely for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

(13) Vector Cloning and Host Cell Transfection

(14) For the cloning of an immunocytokine in which an interferon-beta variant is conjugated with an antibody heavy chain (ACC #2) and an immunocytokine in which an interferon-beta variant is conjugated with an antibody light chain (ACC #7), B12 sequence was used. The human interferon-beta variant sequences were inserted into the heavy chain and light chain of the B12 sequence using a linker, respectively, followed by synthesis using a vector. The synthesized genes were digested with respective proper restriction enzymes, and ligated to the IgG expression vector, followed by a sequencing process, thereby finally constructing vectors expressing ACC #2 and ACC #7. upon completion of the cloning, the ACC #2 and ACC #7 vectors were respectively extracted in large quantities through transformation, and then used for transfection.

(15) CHO-S cells were subcultured for at least 5 passages at a density of 310.sup.5 cells/ml to be prepared for transfection. When the survival rate of the cells was maintained at 90% or higher after the subculture, the cells were seeded at a density of 510.sup.5 cells/mL to be prepared for transfection. The survival rate (>95%) and cell density (110.sup.6 cells/mL) were monitored at 24 h after the cell seeding, and 50 g of DNA was transfected into CHO-S cells, which were cultured in a 30-mL culture medium, using a transfection solvent.

Example 2

(16) Confirmation of Immunocytokine Expression in Host Cells

(17) 48 hours after cell transfection, the expression levels of ACC #2 and ACC #7 were determined by concentration measurement (BCA assay) and western-blot assay.

(18) For BCA assay, reagent A (containing sodium carbonate, bicinchoninic acid, and the like) and reagent B (containing 4% cupric sulfate) were prepared at a ratio of 50:1, and mixed with the standard solution (BSA solution, 0-2000 ug/ml) and a sample (10 uL of sample and 200 uL of reagent). The resultant solution was incubated at 37 C. for 30 minutes, and then the absorbance was determined at 562 nm for concentration calculation. The curve obtained based on the standard solution was used for the concentration calculation.

(19) Western-blot testing was conducted as described below. First, each of the cultured media was collected, and loaded on 10% SDS PAGE gel. The loaded gel was transferred onto PVDF membrane, which was then blocked with 5% BSA solution, and then probed with primary and secondary antibodies. After completion of washing with TBST solution, the membrane was imaged on a film. The image of the film was developed with developer and fixer.

(20) The results indicated that the expression levels of the immunocytokines in which the human interferon-beta variants were conjugated to B12 heavy and light chains were higher than those of the immunocytokine in which the natural human interferons were conjugated to B12 heavy and light chain (FIG. 1).

Example 3

(21) Preparation of Immunocytokines

(22) The linker represented by SEQ ID NO: 5 was inserted into a heavy chain region of an antibody, and interferon-beta or an interferon-beta variant was conjugated thereto. FIG. 2 is a schematic diagram showing a structure of an immunocytokine with a human interferon-beta variant.

(23) The linker represented by SEQ ID NO: 5 and interferon-beta or interferon-beta variant were cloned into a heavy chain of an antibody. Thereafter, restriction enzymes AvrII (CCTAGG) cleavage site and Bstz17I (GTATAC) cleavage site were inserted into the 3-terminus and the 5-terminus of the whole gene, respectively, thereby ensuring a final gene of the heavy chain. In addition, restriction enzymes EcoRV (GATATC) cleavage site and Pad (TTAATTAA) cleavage site were inserted into the 3-terminus and the 5-terminus of a light chain of the antibody, respectively, thereby ensuring a final gene of the light chain. FIG. 3 shows a schematic diagram of the production procedures.

Example 4

(24) Confirmation of Immunocytokine Expression

(25) For confirmation of the expression of an immunocytokine with human interferon-beta and an immunocytokine with a human interferon-beta variant, 50 g of pRBLX2-INF or pRBLX2-CAF vector was transfected into CHO-S cells, and the expression was induced while the cells were cultured for 7 days. After 7 days, the culture liquid was collected, and then centrifuged (8000 rpm, 10 minutes) to remove cells. A small amount of the culture liquid with cells removed was taken, mixed with 5 sample buffer, and boiled at 100 for 10 minutes, thereby inducing sufficient protein denaturation. The prepared sample was loaded onto a Tricine SDS-PAGE gel together with a marker, and subjected to electrophoresis at a voltage of 130 V for 1 hour and 30 minutes. Thereafter, the gel was carefully separated, immersed in a Coomassie blue staining solution, and then shaken for 30 minutes for staining. After the staining, the gel was transferred into a de-staining buffer, and then de-stained with shaking for 30 minutes. The de-staining was repeated three times.

(26) For clearer comparison of the expression levels, western blotting was performed using anti-interferon-beta antibody and anti-human IgG-HRP. After Tricine SDS-PAGE was performed by the same method as above, the gel was carefully separated, and placed on 3M paper, and then a polyvinylidene difluoride (PVDF) membrane was disposed thereon, and again covered with 3M paper. Thereafter, the resultant structure was immersed in 1 transfer buffer and proteins were transferred at a voltage of 100 V for 70 minutes. The membrane was blocked at room temperature for 1 hour and 30 minutes by adding 5% Tris-buffered saline-Tween 20 (TBS-T, 0.1% Tween 20). The PVDF membrane was washed twice with TBS-T, and then immersed in TBS-T. The anti-interferon-beta antibody was prepared by dilution in TBS-T at 1:1000, while the anti-human IgG-HRP antibody was prepared by dilution in TBS-T at 1:3000. The membrane was immersed in the antibody dilution, followed by reaction at room temperature for 2 hours with shaking. After the completion of this procedure, the resulting product was washed three times with TBS-T for 10 minutes, and then allowed to react at room temperature for 1 hour by adding a secondary antibody conjugated with horseradish peroxidase (HRP). After washing was again conducted, bands were identified using an enhanced chemiluminescence (ECL, Intron) reagent. The intensities of the bands were determined by using C-DiGit (LI-COR, USA).

(27) As a result, as shown in FIG. 4, a light chain was observed at the site of 25 KDa, while an immunocytokine with interferon-beta or an immunocytokine complex with a human interferon-beta variant was observed between 70 KDa and 100 KDa.

(28) In FIG. 5, Lane 1 indicates an immunocytokine with human interferon-beta, and Lane 2 indicates an immunocytokine with human interferon-beta variant. The Tricine-SDS PAGE and western blotting results confirmed that the expression level of the immunocytokine with the human interferon-beta variant was higher than that of the immunocytokine with human interferon-beta. In addition, for exact comparison of the expression levels, each culture liquid was measured by Cedex Bio (Roche, USA). The results confirmed that the immunocytokine with human interferon-beta showed a concentration below the measurement range (10 mg/L or less), indicating a low level of expression, whereas the immunocytokine with the human interferon-beta variant showed a concentration of about 32 mg/L, indicating a 3-fold increase in the level of expression.

Example 5

(29) Confirmation of Interferon Activity of Immunocytokine Through pSTAT-1 Phosphorylation

(30) For confirmation of the interferon function of an immunocytokine in which a human interferon-beta variant is conjugated with B12 antibody according to the present invention, the STAT-1 phosphorylation depending on the treatment with either interferon or an antibody-interferon conjugate was examined.

(31) 310.sup.5 OVCAR-3 cells were dispensed in each well of a 6-well plate, and cultured for 24 hours at 37.5 C. and 5% CO.sub.2. After 24 hours, the cell culture liquid was removed, and a human interferon-beta variant (Carbiferon) was diluted to a concentration of 600 ng/mL and an immunocytokine in which a human interferon-beta variant was conjugated with B12 antibody (ACC) was diluted to a concentration of 600 ng/mL or 1800 ng/mL in the culture liquid, followed by treatment for 1 hour. Thereafter, the plate was collected, and each well was washed three times with PBS, treated with 100 L of RIPA buffer containing a protease inhibitor and a phosphatase inhibitor, and placed on ice for 30 minutes to dissolve the cells. The dissolved cells were placed in a 1.5-mL tube, and centrifuged at 13,000 rpm at 4 C. and then only the supernatant (lysate) was taken, and collected in a new tube. The protein concentration of the lysate was quantified by BCA assay, and then 30 g of the lysate was taken, mixed with 5 sample buffer, and boiled at 100 C. for 10 minutes to induce sufficient protein denaturation. The prepared sample was loaded onto a 10% SDS-PAGE gel with a marker, and was allowed to fall at 70 V for 30 minutes and 120 V for 1 hour. Thereafter, the gel was carefully separated, and placed on 3M paper, and then a polyvinylidene difluoride (PVDF) membrane was disposed thereon, and again covered with 3M paper. Thereafter, the resultant structure was immersed in transfer buffer, followed by protein transfer at 100 V for 90 minutes. The membrane was blocked in Tris-buffered saline-Tween 20 (TBS-T, 0.1% Tween 20) containing 5% BSA for 1 hour and 30 minutes, and then the anti-p-STAT1 antibody was prepared by dilution in TBS-T at 1:1000 and the anti-GAPDH antibody was prepared by dilution in TBS-T at 1:3000. The membrane was immersed in the antibody dilution, followed by reaction with shaking at room temperature for 2 hours. After this procedure, the resulting product was washed three times with TBS-T for 10 minutes, and then a horseradish peroxidase (HRP)-conjugated secondary antibody was added thereto, followed by reaction at room temperature for 1 hour. After washing was again conducted, bands were treated with an enhanced chemiluminescence (ECL, Intron) reagent, followed by film development.

(32) The results confirmed that both human interferon-beta (Carbiferon) and immunocytokine treated groups showed pSTAT-1 phosphorylation, indicating that the interferon-beta activity of the immunocytokine in which the human interferon-beta variant (Carbiferon) was conjugated with B12 antibody maintained intact (FIG. 7).

Example 6

(33) Confirmation of Interferon Activity of Immunocytokine Through Cytotoxicity Test

(34) For confirmation of the interferon function of an immunocytokine in which a human interferon-beta variant is conjugated with B12 antibody according to the present invention, the cytotoxicity depending on the treatment with interferon or an antibody-interferon conjugate was examined.

(35) For examination of cytotoxicity, 110.sup.4 OVCAR-3 cells were dispensed in each well of a 96-well plate, and cultured for 24 hours at 37.5 C. and 5% CO.sub.2. After 24 hours, the cell culture liquid was removed, and the cells were treated with the human interferon-beta variant (Carbiferon), B12 antibody, and the immunocytokine in 10-10000 ng/mL, respectively, followed by culture for 24 hours or 48 hours. After the culture for 24 hours or 48 hours, the culture liquid was removed, and PBS washing was conducted two times. WST reagent was mixed with the culture liquid at 1:10, and each well was treated with 10 uL of the mixture, and left at 37.5 C. and 5% CO.sub.2 for 2 hours, and then the absorbance was determined at a wavelength of 430 nm.

(36) The results confirmed that the cell group treated with only B12 antibody showed no cytotoxicity, whereas the cell groups treated with the human interferon-beta variant or the immunocytokine showed cytotoxicity in a concentration-dependent manner, indicating that the human interferon-beta variant still exhibited interferon activity even in a form of the immunocytokine (FIGS. 8 and 9).

Example 7

(37) Production of Immunocytokines in which Antibody Heavy Chain is Conjugated with Interferon-Beta Variant

(38) Immunocytokines in which, besides B12 antibody, ERBB2 (Herceptin) antibody and c-MET antibody were conjugated to an interferon-beta variant, respectively, were prepared as follows.

(39) As shown in FIG. 10, a rigid helical linker was linked to a heavy chain region of ERBB2 (Herceptin) antibody and c-MET antibody, respectively. Thereafter, a human interferon-beta variant was conjugated thereto, thereby producing expression cassettes expressing an anti-c-Met immunocytokine (A) and an anti-ERBB2 immunocytokine (B), respectively.

(40) These immunocytokines were cloned into pRBLX2 vectors, respectively, and then each vector was transfected into CHO-S cells, followed by culture for 7 days, thereby inducing expression. The transfection, culture, and the collection of expressed products were conducted as described in Example 4.

(41) When comparing, using CHO-S cells, the expression level between the immuno-cytokine in which the human interferon-beta was conjugated to c-Met antibody or ERBB2 antibody and the immunocytokine in which the human interferon-beta variant was conjugated to the same, it was confirmed that the expression level of the immunocytokine with the human interferon-beta variant was higher than the expression level of the immunocytokine with human interferon-beta, indicating that the immunocytokine with the human interferon-beta variant possesses an excellent interferon activity in comparison with the immunocytokine with human interferon-beta.

(42) As described above, it was verified that the human interferon-beta variant according to the present invention is very favorably expressed in comparison with wild-type interferon-beta.

INDUSTRIAL APPLICABILITY

(43) The immunocytokines with human interferon-beta variants according to the present invention can be used as a target therapeutic agent for a disease (such as multiple sclerosis or cancer) in that the immunocytokines are excellent in both the interferon activity and the characteristics of antibody recognizing a specific antigen, together with their significantly higher production efficiency in comparison with the immunocytokines with natural interferon-beta, leading to their highly industrial applicability.