AU2013403112B2

AU2013403112B2 - Anti-CD20-Flex bifunctional fusion protein, and preparation method and use thereof

Info

Publication number: AU2013403112B2
Application number: AU2013403112A
Authority: AU
Inventors: Weizhu Qian
Original assignee: Shanghai Biomabs Pharmaceuticals Co Ltd
Current assignee: Shanghai Biomabs Pharmaceuticals Co Ltd
Priority date: 2013-10-17
Filing date: 2013-12-24
Publication date: 2017-05-04
Anticipated expiration: 2033-12-24
Also published as: CN104558194A; CN104558194B; AU2013403112A1; WO2015054958A1

Abstract

Disclosed are an anti-CD20-Flex bifunctional fusion protein, and preparation method and use thereof in the preparation of antitumor drugs. More particularly, disclosed are a bifunctional fusion protein CD20-Flex having a complete-antibody-like structure and function, and amino acid sequence, preparation method and use thereof in the preparation of the antitumor drugs. The bifunctional fusion protein CD20-Flex is able to combine with CD20 and also has the function of Flt3 ligand, thus effectively stimulating the tumor specificity immunity of the body while having lethality.

Description

ANTI-CD20-FLEX BIFUNCTIONAL FUSION PROTEIN, AND PREPARATION 2013403112 04 Apr 2017

METHOD AND USE THEREOF 5 Technical Field

The present invention belongs to the field of biotechnology. More specifically, the present invention discloses anti-CD20 bifunctional fusion proteins, their preparation method and use thereof in manufacture of an antibody drug for treating tumor. The anti-CD20 bifunctional fusion proteins disclosed in the present invention are CD20-Flex bifunctional 0 fusion protein having a structure and function similar to a complete antibody, which can not only bind to CD20 but also has a function of Flt3 ligand.

Technical Background

Tumor, especially malignant tumor, is a disease severely harmful to human health in the 5 present world, and it ranks second among all kinds of diseases. Recently the incidence of tumor is obviously increasing. For malignant tumor, its treatment result is unsatisfactory, and it has a high metastasis rate in the advanced stage and a bad prognosis in most cases. Currently, the conventional treatment methods clinically used, such as radiotherapy, chemotherapy and surgery, may greatly relieve pain and extend the survival time, but these 0 methods all have many limitations and their efficacies are difficult to be further improved.

Antibody targeted drugs are advantage in good specificity, less side effect and long half-life period, etc. Nowadays, they have been widely used in clinical treatment of tumor. Antibody drugs have produced promising treatment efficacy in clinical treatment. However, the antibody targeted drug against a single target merely specifically targets a target protein 25 to exert its killing and blocking functions due to heterogeneity and complexity of tumorigenesis and tumor development, it is difficult to be more efficient to kill tumor cell, activate organism immunity and overcome reoccurrence of tumor. Therefore, the current study on antibody mainly focuses on improvement of the antibody affinity, development of a new target for the antibody, and preparation of a multi-target antibody, etc. The method for 30 improving antibody affinity by using computer aided design and library technology has been very mature, and discovery of a new drug target by proteomics technique combined with mass spectrometric detection technology has also been realized. With the current method, the antibody having an affinity up to InM can be quickly prepared. New drug targets are also continuously subjected to pre-clinical and clinical experiments. However, the current method 35 for preparing a multivalent antibody mainly aims to modify the variable region of an -1- antibody to a single strand antibody. The multivalent antibody obtained by this method generally has a low antibody affinity and it does not achieve the expected efficacy. DVD antibody recently developed is formed by fusing the variable regions of heavy chain and light chain of two antibodies to form a structure of HVl-linker-HV2 or LV1-linker-LV2. Although 5 such a DVD antibody could overcome the defect of low affinity of a single strand antibody, its molecular weight is too large and its antibody structure changes too much, resulting in that its expression amount is not enough. Moreover, when the target protein has a large molecular weight or forms a large complex on the cell membrane, the binding of the DVD antibody to the double targets will be influenced. 2013403112 04 Apr 2017 0 Rituximab, with a trade name of Mabthera, is a chimeric monoclonal antibody against CD20. It was approved by FDA of USA in 1997 to be used for treating B cell non-Hodgkin's lymphoma and follicular lymphoma. Together with CHOP, it can be used to treat other B cell lymphomas, such as diffuse large B cell lymphoma.

As continuous and intensive study on the mechanism of treating tumor by antibody, 5 currently it is believed that antibody can obviously kill tumor cells and inhibit growth of tumor through CDC, ADCC and induction of cell apoptosis. However, more and more evidences demonstrated that organism-specific immune killing induced by antibody played an important role in overcoming reoccurrence of tumor.

Ectodomain of Flt3 ligand (Flex) can not only promote bone marrow to increase the 0 production amount of DC, but also facilitate maturation of DC function, which allow the cells to have stronger antigen presentation and anti-tumor functions. More importantly, Flex can release the mature DC from bone marrow into peripheral tissues. In vivo experiments demonstrated that DC in spleen, bone marrow, lymph node and liver of mouse was increased in a time-dependent manner after administration of Flex. Therefore, it is a study hotspot at 25 the present stage to prepare a kind of antibodies that exhibit a killing activity and can efficiently activate tumor-specific immunity of organism.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated 30 otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be 35 readily identifiable by a person skilled in the art and may assist in putting into practice the -2- invention as defined in the claims of this application. 2013403112 04 Apr 2017

Summary of Invention

To solve the above problems, and/or to at least provide the public with a useful choice, the present inventors construct a kind of CD20-Flex bifunctional fusion proteins having a 5 structure and function similar to a complete antibody by genetic engineering technology after a long-term research and a lot of experiments. The bifunctional fusion proteins can bind to CD20 and exhibit the function of Flt3 ligand.

The present invention provides the following contents: 1. A CD20-Flex bifunctional fusion protein having a structure and function similar to a 0 complete antibody, wherein the fusion can not only bind to CD20 protein but also has the function of Flt3 ligand; said bifunctional fusion protein, consisting of three peptide chains, which are said Flex-CL-Hinge-CH2-CH3 with an amino acid sequence set forth in SEQ ID NO: 18, knob mutant of the heavy chain of rituximab with an amino acid sequence set forth in SEQ ID NO: 5 16, and the light chain of rituximab with an amino acid sequence set forth in SEQ ID NO: 10. 2. An isolated nucleotide molecule encoding the above three peptide chains, respectively having a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO: 15 and SEQ ID NO: 9. 3. A vector containing the nucleic acid molecule of the invention as described above and 0 regulatory sequence(s) for expression operably linked to the sequence of the nucleic acid molecule, wherein the vector may be one vector selected from the group of pDRl, pcDNA3.1(+), pcDNA3.l/ZEO(+) and pDHFR. 4. The vector of the invention as described above, which is pcDNA3.1(+) or pcDNA3.1/ZEO(+). 25 5. A host cell containing the above-mentioned vector of the invention, which is a eukaryotic cell. 6. The host cell of the invention as described above, which is a mammal cell. 7. The host cell of the invention as described above, which is a CHO cell. 8. A method for preparing the above-mentioned CD20-Flex bifunctional fusion protein 30 of the invention, comprising: a) Cloning the variable region of the CD20 antibody rituximab and the ectodomain of the Flt3 ligand, respectively; b) Constructing a knob mutant, T366W and S354C, and a hole mutant, T366S, L368A, Y407V and Y394C, at the Fc region of the antibody, respectively; 35 c) Fusing the ectodomain of the Flt3 ligand with the constant region of the light chain of -3- the antibody to construct a Flex-CL fusion fragment; 2013403112 04 Apr 2017 d) Fusing the variable region of the heavy chain of rituximab to the knob mutant, and the Flex-CL to the hole mutant, respectively, and inserting into an expression vector, respectively; 5 e) Co-transfecting the two constructed expression vectors together with an expression vector containing the gene for the light chain of rituximab, expressing, isolating and purifying to obtain the bifunctional fusion protein. 9. A composition comprising the above-mentioned CD20-Flex bifunctional fusion protein of the invention, and a pharmaceutically acceptable carrier. 0 10. Use of the above-mentioned CD20-Flex bifunctional fusion protein of the invention, in the preparation of an antibody drug for treating tumor. 11. Use of the above-mentioned composition of the invention in the preparation of an anti-tumor drug. 12. Use of the invention as mentioned under either of items 10 and 11 above, further 5 comprising a combinational use with other anti-tumor drug(s). 13. A method of treating a tumor in a patient, the method comprising administering the CD20-Flex bifunctional fusion protein of item 1. 14. A method of treating a tumor in an animal, the method comprising administering the composition of item 9. 0 15. A method as mentioned under either of items 13 and 14 above, further comprising a combinational treatment with other anti-tumor drugs.

Description 25 The present disclosure is intended to provide a kind of CD20-Flex bifunctional fusion proteins having a structure and function similar to a complete antibody, which can not only bind to CD20 and but also exhibit the function of Flt3 ligand. These antibodies not only retain the structure and size of a similar antibody and exhibit an affinity and half life similar to the parent antibody, but also retain the function of killing tumor similar to CD20 antibody, 30 including CDC, ADCC and capability of inducing apoptosis. Additionally, they also have the function of the Flt3 ligand, including inducing proliferation and maturation of DC and NK cells around the tumor, activating tumor-specific immunization of an organism, and overcoming the recurrence of tumor.

Up deep understanding on the antibody structure, the present disclosure established a 35 fusion protein which not only retains the structure and function of a similar antibody but also -4- have the function of Flt3 ligand. The structure of the protein is shown in Fig. 1. To demonstrate that the present method produces an efficacy better than the combinational use of rituximab antibody and Flex-Ig, subsequent experiments using the protein for treating tumor were conducted in the present invention. Principally, the present method can be widely 5 used in construction of a bifunctional fusion protein by using various antibody to fuse with Flex, thus accelerating the research and development of a fusion protein having a biologically and medical sense. Meanwhile, the method for constructing a bifunctional fusion protein by combining the variable region of an antibody with Flex also provides a new idea for designing a bifunctional fusion protein for the later research. 2013403112 04 Apr 2017 0 In the present disclosure, any suitable vector can be used, which may be any of pDRl, pcDNA3.1(+), pcDNA3.1/ZEO(+) and pDHFR. The expression vector can include a fusion DNA sequence linking with suitable regulatory sequence(s) for transcription and translation.

Cell culture systems for mammal or insect host cells may be used for expression of the present bifunctional fusion protein. Specifically, COS, CHO, NSO, sf9 and sf21, etc., can be 5 used in the present disclosure.

Cells that can be used are prokaryotic cells, such as any of DH5a, BL21(DE3), and TGI.

The method for preparing the bifunctional fusion protein having a structure and function benefit of an antibody disclosed in the present invention includes culturing the above-mentioned host cell under conditions for expression to express the bifunctional fusion 0 protein, and isolating or purifying the bifunctional fusion protein.

Affinity chromatography may be used to isolate and purify the bifunctional fusion protein disclosed in the present application. According to the property of the used affinity column, conventional methods, such as using a high salt buffer and changing pH, could be used to elute the bifunctional fusion protein bound to the affinity column. 25 The bifunctional fusion protein may be purified to a basically uniform substance with the above methods. For example, it may be shown as a single band by SDS-PAGE electrophoresis.

According to one preferred embodiment of the present invention, a kind of bifunctional fusion proteins contain the antigen-binding region of rituximab and ectodomain of Flt3 30 ligand. A method for preparing a kind of bifunctional fusion proteins having a structure and function benefit of an antibody comprises the following steps: a) Cloning the genes of the variable region of the heavy chain of the CD20 antibody rituximab and the ectodomain of the Flt3 ligand, respectively; 35 b) Fusing the gene of the ectodomain of the Flt3 ligand with the constant region of the -5- light chain of the antibody to construct a Flex-CL fusion fragment; 2013403112 04 Apr 2017 c) Constructing a knob mutant, T366W and S354C, and a hole mutant, T366S, L368A, Y407V and Y394C, at the Fc region of the antibody, respectively; d) Fusing the variable region of the heavy chain of rituximab with the knob mutant, and 5 the Flex-CL with the hole mutant, respectively, and inserting into an expression vector, respectively; e) Co-transfecting the above constructed expression vectors together with the light chain of antibody rituximab, expressing, isolating and purifying to obtain the bifunctional fusion protein. 0 The gene of the complete heavy chain and the gene of the complete light chain, as constructed above, are inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen), respectively. These plasmids are together transfected into CHO-K1 cell (ATCC) by a liposome transfection method. Cell clones stably expressing the bifunctional fusion protein are screened by a selection culture medium containing 600pg/ml G418. The 5 bifunctional fusion protein is purified from the supernatant of the cell culture by affinity chromatography by using Protein A affinity column.

The above bifunctional fusion protein or formulation can be used in preparation of an anti-cancer drug. They can be used in combination with other anti-tumor drugs.

The above bifunctional fusion proteins disclosed in the present invention can be 0 formulated with a pharmaceutically acceptable auxiliary material to form drug formulation compositions, thereby stably exerting its efficacy. These formulations can ensure the conformational integrity of the core amino acid sequence of the bifunctional fusion protein disclosed in the present invention and at the same time protect the multiple functional groups of the protein from degrading (including but is not limited to agglomeration, deamination or 25 oxidation). In general, for a liquid formulation, it can be stored at 2°C to 8°C for at least one year. For a lyophilized formulation, it can be kept stable at 30°C for at least six months. As used herein, formulation can be the formulation conventionally used in the pharmaceutical field, such as suspension, solution for injection and lyophilized formulation, preferably solution for injection and lyophilized formulation. For the solution for injection and 30 lyophilized formulation of the above bifunctional fusion protein disclosed in the present invention, the pharmaceutically acceptable auxiliary material includes surfactant, stabilizer for solution, isotonic regulator or buffer, or combination thereof. Surfactant includes nonionic surfactant, for example, polyoxyethylene sorbitan fatty acid ester (Tween 20 or Tween 80), poloxamer, such as poloxamer 188, Triton, sodium lauryl sulfate (SDS), sodium 35 lauryl sulfate, tetradecyl, oleyl or octadecyl sarcosine, Pluronics, MONAQUAT™, etc. The -6- addition amount of surfactant should reduce the granulation trend of the bifunctional fusion protein as much as possible. The stabilizer for solution may be sugars, including reducing sugar and non-reducing sugar, amino acids, including monosodium glutamate or histidine, alcohols, including tribasic alcohol, advanced alditol, propylene glycol or polyethylene 5 glycol, or combination thereof. The addition amount of stabilizer for solution should ensure the finally formed formulation to be kept in a stable state within a time acceptable to the skilled artisan. Isotonic regulator can be sodium chloride or mannitol. Buffer may be TRIS, histidine buffer, or phosphate buffer. 2013403112 04 Apr 2017

The above-mentioned formulations are compositions containing the bifunctional fusion 0 protein. After administering to the animal, including human being, they produce an obvious anti-tumor effect. Specifically, they are effect in prevention and/or treatment of tumor, and thus can be used as an anti-tumor drug.

When administered to the animal, including human being, the dose of the bifunctional fusion protein and its composition as disclosed in the present invention will depend on the 5 age and weight of the patient, the property and severity of the disease, and the administration route. The dose may refer to the results obtained from the animal experiments and various conditions, and the total administration amount should not exceed a certain range. Specifically, the dose for intravenous injection is 0.1~3000mg per day.

As used herein, the term “anti-tumor drug” is meant to refer to a drug capable of 0 inhibiting and/or treating tumor, including delaying the development of symptoms associated with tumor growth and/or reduction of the severity of these symptoms. Further comprised is alleviation of symptoms associated with the growth of the existed tumor and prevention of occurrence of other symptoms, and reduction or prevention of metastasis.

The bifunctional fusion protein and its composition disclosed in the present invention 25 can be used in combination with other anti-tumor drugs to treat tumor. Such anti-tumor drug includes: 1. Cytotoxic drugs, including (1) drugs acting on the chemical structure of DNA, including alkylating agent such as nitrogen mustards, nitrosoureas, mesylates; platinum compounds such as cisplatin, 30 carboplatin and oxaliplatin, etc.; mitomycin (MMC); (2) drugs affecting synthesis of nucleic acid, including dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta, etc.; thymidine synthetase inhibitors such as fluorouracil (5FU, FT-207, capecitabine) etc.; purine nucleoside synthetase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG, etc.; nucleotide reductase inhibitors such as 35 hydroxyurea (HU) etc.; DNA polymerase inhibitors such as cytosine arabinoside (Ara-C) and -7- gemcitabine (Gemz) etc.; 2013403112 04 Apr 2017 (3) drugs acting on transcription of nucleic acid, including drugs selectively acting on DNA template to inhibit DNA-dependent RNA polymerase thereby inhibiting RNA synthesis, such as actinomycin D, daunorubicin, doxorubicin, epirubicin, aclacinomycin, mithramycin, 5 etc.; (4) drugs mainly acting on tubulin synthesis, including paclitaxel, docetaxel, vinblastine, vinorelbine, podophyllotoxin alkaloids, homoharringtonine; (5) Other cytotoxic drugs, including asparaginase which mainly inhibits synthesis of protein; 0 2. hormones, including antiestrogens such as tamoxifen, droloxifene, exemestane, etc.; aromatase inhibitors such as aminoglutethimide, formes-tane, letrozole, arimidex, etc.; antiandrogens such as flutamide; RH-LH agonist/antagonist such as zoladex, enatone, etc.; 3. biological response modifiers which inhibit tumor mainly through the immune function of organism, including interferon, interleukin-2, thymosins; 5 4. monoclonal antibody, including MabThera, Cetuximab (C225), Trastuzumab,

Bevacizumab (Avastin); and 5. others, including drugs with unknown mechanism and to be further studied, such as agents for inducing cell differentiation such as retinoids, agents for inducing cell apoptosis.

The bifunctional fusion protein and its composition disclosed in the present invention 0 can be used in combination with any of the above-mentioned anti-tumor drugs or combination thereof.

The term “comprising” as used in this specification means “consisting at least in part of’. When interpreting each statement in this specification that includes the term 25 “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner.

Descriptions on Drawings

Fig. 1: A structural representation of a CD20-Flex bifunctional fusion protein (BiFP). 30 Fig. 2: The structure of the knob mutant of the heavy chain of rituximab.

Fig. 3: The structure of the Flex-CL-Hinge-CH2-CH3.

Fig. 4: The affinity of the CD20-Flex BiFP to CD20.

Fig. 5: The CDC killing activity of the CD20-Flex BiFP.

Fig. 6: The ADCC killing activity of the CD20-Flex BiFP. 35 Fig. 7: The activity of inducing apoptosis of the CD20-Flex BiFP. -8-

Fig. 8: The protection of the CD20-Flex BiFP to the mouse with in vivo tumor. 2013403112 04 Apr 2017

Specific Mode for Carrying Out the Invention

Example 1: Cloning of the gene for the variable region of the heavy chain of rituximab 5 The gene of the variable region of the heavy chain of rituximab was cloned by PCR by using the heavy chain of rituximab as template. The reaction conditions included 95 °C for 15 minutes, 94°C for 50 seconds, 58°C for 50 seconds, 72°C for 50 seconds, totally 30 circles, 72°C for 10 minutes. Thus, the PCR product, rituximab HV, was obtained. The amino acid sequence for the signal sequence of the antibody was MGWSCIILFLVATATGVHS. The 0 cloning fragment having a correct sequence after sequencing was ready for use. The amino acid sequence of the variable region of heavy chain of rituximab was shown in SEQ ID NO: 2, and the nucleotide sequence was shown in SEQ ID NO: 1.

Example 2: Cloning of the gene for the variable region of Flt3 ligand 5 Lymphocytes were isolated from healthy human by using a lymphocyte separation liquid (Dingguo Biotechnology Development Co.). Total RNA was extracted by the Trizol reagent (Invitrogen). The gene of the ectodomain of Flt3 ligand was amplified by RT-PCR according to the methods described in Hieter PA, Max EE, Seidman JG, Maizel JV Jr, Leder P., Cloned human and mouse kappa immunoglobulin constant and J region genes conserve 0 homology in functional segments, Cell. 1980 Nov;22(l Pt 1):197-207, and Ellison JW, Berson BJ, Hood LE., The nucleotide sequence of a human immunoglobulin C gammal gene. Nucleic Acids Res., 1982 Jul 10; 10(13):4071-9. The PCR product was purified and recovered by an agar gel electrophoresis and then cloned into a pGEM-T vector. Clone with a correct sequence after sequencing was obtained. The amino acid sequence of Flex was shown 25 in SEQ ID NO: 4, and the nucleotide sequence was shown in SEQ ID NO: 3.

Example 3: Cloning of the constant region of the light chain and the Fc region of the antibody

Lymphocytes were isolated from healthy human by using a lymphocyte separation 30 liquid (Dingguo Biotechnology Development Co.). Total RNA was extracted by the Trizol reagent (Invitrogen). The genes of the constant region of the light chain and the Fc region of the antibody were amplified by RT-PCR according to the methods described in Hieter PA, Max EE, Seidman JG, Maizel JV Jr, Leder P., Cloned human and mouse kappa immunoglobulin constant and J region genes conserve homology in functional segments, Cell. 35 1980 Nov;22(l Pt 1):197-207, and Ellison JW, Berson BJ, Hood LE., The nucleotide -9- sequence of a human immunoglobulin C gammal gene. Nucleic Acids Res., 1982 Jul 10; 10(13):4071-9. The PCR product was purified and recovered by an agar gel electrophoresis and then cloned into a pGEM-T vector. Clone with a correct sequence after sequencing was obtained. The amino acid sequence of CL was shown in SEQ ID NO: 6, and 5 the nucleotide sequence was shown in SEQ ID NO: 5; and the amino acid sequence of Fc was shown in SEQ ID NO: 8, and the nucleotide sequence was shown in SEQ ID NO: 7. 2013403112 04 Apr 2017

Example 4: Construction of the knob mutant for the Fc region of the antibody Mutations, T366W and S354C, were introduced in the Fc region of the antibody 0 obtained in Example 3 by overlap PCR (Schaefer WW, Regula JTJ, Bahner MM, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proc Natl Acad Sci USA. 2011; 108(27): 11187—11192). The PCR product was purified and recovered by an agar gel electrophoresis and then cloned into a pGEM-T vector. Clone with a correct sequence after sequencing was obtained. The amino acid sequence of 5 the Fc-knob was shown SEQ ID NO: 12, and the nucleotide sequence was shown in SEQ ID NO: 11.

Example 5: Construction of the hole mutant for the Fc region of the antibody Mutations, T366S, L368A, Y407V and Y394C, were introduced in the Fc region of the 0 antibody obtained in Example 3 by overlap PCR (Schaefer WW, Regula JTJ, Bahner MM, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proc Natl Acad Sci USA. 2011; 108(27): 11187-11192). The PCR product was purified and recovered by an agar gel electrophoresis and then cloned into a pGEM-T vector. Clone with a correct sequence after sequencing was obtained. The amino acid 25 sequence of the Fc-hole was shown in SEQ ID NO: 14, and the nucleotide sequence was shown in SEQ ID NO: 13.

Example 6: Construction of the mutant for the heavy chain of rituximab The variable region of the heavy chain of rituximab was fused to the knob mutant of the 30 Fc region by overlap PCR by using the variable region of the heavy chain of rituximab obtained in Example 1 and the knob mutant of the Fc region obtained in Example 4 as templates and then inserted into an expression vector. Double mutations, S354C and T366W, were introduced into the constant region of the Fc of the heavy chain of rituximab to construct the knob mutant of the heavy chain of rituximab (Fig. 2). The amino acid sequence 35 of the knob mutant of the heavy chain of rituximab was shown in SEQ ID NO: 16 and the -10- nucleotide sequence was shown in SEQ ID NO: 15. 2013403112 04 Apr 2017

Example 7: Construction of Flex mutant

Flex was fused to the CL and the hole of the Fc region by overlap PCR by using the 5 Flex obtained in Example 2, CL obtained in Example 3 and the hole mutant of Fc region obtained in Example 5 as templates and then inserted into an expression vector to construct Flex-CL-Hinge-CH2-CH3 (Fig. 3). The amino acid sequence of the knob mutant of the heavy chain of Flex-CL-Hinge-CH2-CH3 was shown in SEQ ID NO: 18 and the nucleotide sequence was shown in SEQ ID NO: 17. 0

Example 8: Expression and purification of the CD20-Flex bifunctional fusion protein 3xl05 CHO-K1 cells (ATCC CRL-9618) were inoculated in 3.5cm tissue culture dish. Transfection was conducted after cells reached a 90-95% confluence. 10qg plasmids for knob mutants of rituximab heavy chain, Flex-CL-Hinge-CH2-CH3 and 4μg rituximab light 5 chain (Li B, Zhao L, Guo H, et al., Characterization of a rituximab variant with potent antitumor activity against rituximab-resistant B-cell lymphoma, Blood, 2009; 114(24):5007-5015), and 20μ1 Lipofectamine2000 Reagent (Invitrogen) were respectively dissolved in 500μ1 serum-free DMEM culture medium and stood at room temperature for 5 minutes. The above two solutions were mixed together and incubated at 0 room temperature for 20 minutes to allow formation of DNA-liposome complex. During the incubation, 3ml serum-free DMEM culture mediums were used to replace the serum-containing culture medium in the culture dish. The resultant DNA-liposome complexes were added into a plate and cultured in a CO2 incubator for 4 hours. And then 2ml DMEM complete culture mediums supplemented with 10% serum were added and further 25 cultured in the CO2 incubator. After transfection for 24 hours, cells were screen for resistant clones in a selection culture medium containing 600μg/ml G418. The cell culture supernatant was detected by ELISA to screen the clones having high expression. Goat anti-human IgG (Fc) was coated on the ELISA plate. The plate was placed under 4°C overnight and then blocked by 2% BSA-PBS at 37°C for 2 hours. The culture supernatant of the resistant clone 30 to be detected or standard (Human myeloma IgGl, k) was added and incubated at 37°C for 2 hours. HRP-goat anti-human IgG(K) was added to allow for a binding reaction and then incubated at 37°C for 1 hour. TMB was added at 37°C for reaction for 5 minutes. H2SO4 was finally added to stop the reaction. A450 was detected. The high-expressing clones obtained by the screening were subjected to amplification culture in a serum-free culture medium. The 35 bifunctional fusion protein was isolated and purified by Protein A affinity column (GE). The -11- purified antibody was dialyzed by PBS. The concentration of the antibody after purification was quantified by a UV absorption method. 2013403112 04 Apr 2017

Experiment 1: Detection on affinity of the fusion protein 5 The affinity of all fusion proteins were detected by radioimmunoassay (Cragg MS,

Morgan SM, Chan HT, Morgan BP, Filatov AV, Johnson PW, French RR, Glennie MJ (2003) Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts. Blood 101(3): 1045-1052). Briefly, the purified fusion protein was labeled by an iodobead method. The fusion protein labeled by 1251 was incubated with Daudi (ATCC CCL-213) and 0 8266 cells at 37°C for 2 hours. The antibodies bound to the cells were isolated from the free

antibodies labeled by iodine by centrifugation. The radioactivity of the antibodies bound to the Daudi cells and labeled by iodine was detected (Fig. 4). The affinity constant of the fusion protein was obtained by fitting the titration curve with the Hill equation curve. The affinities of the CD20-Flex bifunctional fusion protein to CD20 and to Flt3 were respectively 5 similar to those of the parent rituximab and Flt3 ligand as reported (Title: Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20, Author: Reff, Μ. E.; Carner, K.; Chambers, K. S. (...) Source: Blood, 1994, 83(2): 435-445; Turner AM, Lin NL, Issarachai S, Lyman SD, Broudy VC. FLT3 receptor expression on the surface of normal and malignant human hematopoietic cells, Blood, 1996;88(9):3383-3390). The affinity to the 0 CD20 protein was 4.82+0.25 nM, and the affinity to Flt3 was 230+40 pmol/L

Experiment 2: Detection on the CDC function of the CD20-Flex fusion protein

Raji and Daudi cells were cultured with the fusion protein at 37 °C in a phenol red-free culture medium for 1 hour. And then 10% normal human serum were added into the culture 25 medium and incubated at 37°C for 4 hours. CDC activity was detected by a standard LDH kit (Promega). As shown in Fig. 5, the CD20-Flex BiFP exhibited a CDC killing activity similar to rituximab.

Experiment 3: Detection on the ADCC function of the CD20-Flex fusion protein 30 Daudi cells (ATCC CCL-213) were cultured with the fusion protein in different protein concentrations in a E:T ratio of 25:1 (37°C, 4 hours). ADCC activity was detected by a standard LDH kit (Promega). As shown in Fig. 6, the CD20-Flex BiFP exhibited a ADCC killing activity similar to rituximab. 35 Experiment 4: Detection on apoptosis function of the CD20-Flex fusion protein -12-

Ramos cells and 10pg/ml CD20-Flex fusion protein were co-incubated with different concentrations of caspase inhibitors. 20pg/ml goat-anti-human F(ab)’2 fragment (Southern Biotechnology) was added in the meantime. 16 hours later, the dead cells were subjected to single-staining by flow cytometry by using Annexin V-Fluos kit (BD Company). As shown 5 in Fig. 7, after crosslinking with the secondary antibody, the CD20-Flex fusion protein could induce cell death to a degree similar to rituximab crosslinking to the secondary antibody. The induced cell death showed a dose-dependence on the caspase inhibitor. 2013403112 04 Apr 2017

Experiment 5: In vivo tumorigenicity assay in mouse by using the CD20-Flex fusion 0 protein 5xl06 Daudi or A20-CD20 cells were injected into the tail vein of BALB/c mice. The fusion protein was injected into the tail vein of the mice at day 0, 4 and 8. At day 42, the mice without tumor after treating by the fusion protein were inoculated subcutaneously by lx 104 A20-CD20 or A20 cells at another side. The size of the tumor in the tumor-bearing 5 mice was observed to evaluate the treatment effect. As shown in Fig. 8, the CD20-Flex fusion protein could not only kill the initially inoculated tumor similar to rituximab, but also exhibit a long-term protection activity to the tumor inoculated again. -13-

Claims

Claims

1. A CD20-Flex bifunctional fusion protein having a structure and function similar to a complete antibody, wherein the fusion protein can not only bind to CD20, but also has the function of Flt3 ligand; said bifunctional fusion protein consisting of three peptide chains, which are Flex-CL-Hinge-CH2-CH3 with an amino acid sequence set forth in SEQ ID NO: 18, knob mutant of the heavy chain of rituximab with an amino acid sequence set forth in SEQ ID NO: 16, and the light chain of rituximab with an amino acid sequence set forth in SEQ ID NO: 10.
2. An isolated nucleotide molecule encoding the three peptide chains as described in claim 1, respectively having a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO: 15 and SEQ ID NO: 9.
3. A vector containing the nucleic acid molecule of claim 2 and regulatory sequence(s) for expression operably linked to the sequence of the nucleic acid molecule, wherein the vector may be one vector selected from the group of pDRl, pcDNA3.1(+), pcDNA3.1/ZEO(+) and pDHFR.
4. The vector of claim 3, which is pcDNA3.1(+) or pcDNA3.1/ZEO(+).
5. A host cell containing the vector of claim 4, which is a eukaryotic cell.
6. The host cell of claim 5, which is a mammal cell.
7. The host cell of claim 6, which is a CHO cell.
8. A method for preparing the CD20-Flex bifunctional fusion protein of claim 1, comprising: a) Cloning the variable region of the CD20 antibody rituximab and the ectodomain of the Flt3 ligand, respectively; b) Constructing a knob mutant, T366W and S354C, and a hole mutant, T366S, L368A, Y407V and Y394C, at the Fc region of the antibody, respectively; c) Fusing the ectodomain of the Flt3 ligand with the constant region of the light chain of the antibody to construct a Flex-CL fusion fragment; d) Fusing the variable region of the heavy chain of rituximab to the knob mutant, and the Flex-CL with the hole mutant, respectively, and inserting into an expression vector, respectively; e) Co-transfecting the two constructed expression vectors together with an expression vector containing the gene for the light chain of rituximab, expressing, isolating and purifying to obtain the bifunctional fusion protein.
9. A composition comprising the CD20-Flex bifunctional fusion protein of claim 1 and a pharmaceutically acceptable carrier.
10. Use of the CD20-Flex bifunctional fusion protein of claim 1 in the preparation of an antibody drug for treating tumor.
11. Use of the composition of claim 9 in the preparation of an anti-tumor drug.
12. Use of claim 10 or 11, further comprising a combinational use with other anti-tumor drug(s).
13. A method of treating a tumor in a patient, the method comprising administering the CD20-Flex bifunctional fusion protein of claim 1.
14. A method of treating a tumor in an animal, the method comprising administering the composition of claim 9.
15. A method of claim 13 or claim 14, further comprising a combinational treatment with other anti-tumor drug(s).