WO2015054958A1

WO2015054958A1 - Anti-cd20-flex bifunctional fusion protein, and preparation method and use thereof

Info

Publication number: WO2015054958A1
Application number: PCT/CN2013/090277
Authority: WO
Inventors: 钱卫珠
Original assignee: 上海张江生物技术有限公司; 上海海思太科药业有限公司
Priority date: 2013-10-17
Filing date: 2013-12-24
Publication date: 2015-04-23
Also published as: CN104558194B; CN104558194A; AU2013403112B2; AU2013403112A1

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 dual function fusion protein, preparation method thereof and use thereof

The present invention is in the field of biotechnology, and more particularly, the present invention discloses a class of anti-CD20 bifunctional fusion proteins, methods for their preparation, and their use in the preparation of antibody tumor drugs. The anti-CD20 bifunctional fusion protein disclosed in the present invention has a CD20-Flex bifunctional fusion protein similar to the structure and function of the whole antibody, and can bind to both CD20 and Flt3 ligand. Background technique

Tumors, especially malignant tumors, are diseases that seriously endanger human health in the world today, ranking second among all diseases. Moreover, in recent years, its incidence has shown a clear upward trend. Malignant tumors have poor therapeutic effects, high metastatic rate, and poor prognosis. At present, conventional treatment methods such as radiotherapy, chemotherapy and surgery have alleviated the pain and prolonged the survival time to a large extent, but these methods have great limitations, and the curative effect is difficult to further improve.

Antibody-targeted drugs have the advantages of good specificity, small side effects, and long half-life cycle, and have been widely used in the clinical treatment of tumors. Antibody drugs have achieved encouraging therapeutic effects in clinical treatment. However, due to the heterogeneity and complexity of tumorigenesis and development process, antibody targeting drugs of a single target only specifically target a target protein to exert killing, blocking and other effects, and it is difficult to kill tumor cells more effectively. Invigorate the body's immunity and overcome tumor recurrence. Therefore, the current field of antibody research is mainly focused on improving the affinity of antibodies, discovering new antibody targets, and preparing multi-targeted antibodies. The use of computer-aided design and library technology to improve antibody affinity has been well established, and the use of proteomics techniques in conjunction with mass spectrometry to discover new drug targets has also been achieved. Today's methods have enabled rapid preparation of antibodies with affinity to InM, and new drug targets are constantly being tested in preclinical and clinical trials. However, the current method for preparing multivalent antibodies is mainly to transform the variable region of the antibody into a single-stranded antibody. The multivalent antibody obtained by this method has low general antibody affinity and the therapeutic effect is not as expected. Recently, the DVD antibody has a structure in which the heavy chain variable region and the light chain variable region of the two antibodies are respectively fused to form HV1-linker-HV2 and LV1-linker-LV2. Although the DVD antibody overcomes the disadvantage of low affinity of the single-chain antibody, due to the excessive molecular weight and large changes in the structure of the antibody, the expression level thereof is not high; moreover, when the molecular weight of the target protein is large or large on the cell membrane, In the case of a complex, it affects the binding of the DVD antibody to the dual target.

Rituximab (rituximab) is a monoclonal antibody against CD20, which is sold as rituximab. In 1997, it was approved by the US FDA for the treatment of B-cell non-Hodgkin's lymphoma, follicular lymphoma, and in combination with CHOP for the treatment of other B-cell lymphomas such as diffuse large B-cell lymphoma.

Although in-depth study of the mechanism of antibody treatment of tumors, it is currently believed that antibodies can significantly kill tumor cells and inhibit tumor growth through CDC, ADCC and induced cell death, but more and more evidence indicates antibody-induced body-specific immunity. Killing plays an important role in overcoming tumor recurrence.

The Flt3 ligand extracellular domain (Flex) not only increases the number of DCs produced by the bone marrow, but also allows the DC function to mature, giving the latter a stronger antigen presentation and anti-tumor effect. More importantly, Flex also It can release mature DC from the bone marrow into the peripheral tissues. In vivo experiments confirmed that the application of Flex increased the DC in the spleen, bone marrow, lymph nodes and liver of the mice in a time-dependent manner. Therefore, it is a hot spot for antibody research to prepare a class of antibodies that are effective in stimulating tumor-specific immunity while killing. Summary of the invention

In order to solve the above problems, the inventors of the present invention conducted long-term research and constructed a CD20-Flex bifunctional fusion protein having a similar whole antibody structure and function by genetic engineering using a large number of experiments, and the bifunctional fusion protein can be used. It binds to CD20 and has the function of a Flt3 ligand.

The invention discloses:

A CD20-Flex bifunctional fusion protein having a similar overall antibody structure and function, which is characterized in that it binds to both the CD20 antigen and the Flt3 ligand.

2. The above dual function fusion protein consists of three peptide chains, respectively

Flex-CL-Hinge-CH2-CH3 (SEQ ID NO: 18) rituximab heavy chain knob mutant (SEQ ID

NO : 16 ) rituximab light chain (SEQ ID NO: 10).

3. An isolated nucleotide molecule encoding the above three peptide chains, having SEQ ID NO: 17,

The nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 9.

A vector comprising the above-described nucleic acid molecule and an expression control sequence operably linked to the sequence of the nucleic acid molecule, wherein the vector may be pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), One of pDHF.

5. The above vector is pcDNA3.1(+) or pcDNA3.1/ZEO(+).

A host cell comprising the above vector, which is a eukaryotic cell.

7. The above host cell is a mammalian cell.

8. The above host cell is a CHO cell. 9. A preparation of the above CD20-Flex bifunctional fusion protein, the method comprising: a) separately cloning the CD20 antibody rituximab variable region and the Flt3 ligand extramembrane region; b) constructing a knob mutant in the antibody Fc region: T366W, S354C; hole mutants: T366S, L368A, Y407V and Y394C;

c) fusion of the Flt3 ligand extracellular domain with the antibody light chain constant region to construct a Flex-CL fusion fragment; d) fusion of the rituximab heavy chain variable region with the knob mutant, Flex-CL and the hole mutant, Expression vector;

e) The two expression vectors constructed are transfected with an expression vector containing the rituximab light chain gene for expression, isolation and purification.

10. A composition comprising the above CD20-Flex bifunctional fusion protein, and a pharmaceutically acceptable carrier.

11. Use of the above CD20-Flex dual function fusion protein for the preparation of antibody tumor drugs.

12. Use of the above composition for the preparation of an antitumor drug.

13. Any of the above uses, including the use of other anti-tumor drugs.

It is an object of the present invention to provide a class of CD20-Flex bifunctional fusion proteins having a similar overall antibody structure and function, which binds to both CD20 and Flt3 ligands. These antibodies retain both the structure and size of similar antibodies, have similar affinity and half-life to the parent antibody, and retain the CD20 antibody's ability to kill tumor-like CDCs, ADCCs, and induce cell death, combined with Flt3 ligands. The proliferation and maturation of DC and NK cells are induced near the tumor, which stimulates tumor-specific immunity and overcomes tumor recurrence.

The invention has a deep understanding of the structure of the antibody. On the basis of this, a fusion protein which retains the structure and function of the similar antibody and has the function of Flt3 ligand is established, and its structure is shown in Fig. 1. In order to demonstrate that the method has a better therapeutic effect than the combination of the rituximab antibody and the Flex-Ig, the present invention conducts an experiment of the subsequent anti-tumor effect of the protein. In principle, the method of the present invention can be widely applied to various antibodies and Flex to construct a bifunctional fusion protein, and accelerate the development of a biologically and medically meaningful fusion protein. At the same time, the method of constructing a bifunctional fusion protein by combining the variable region of the antibody with Flex provides a new idea for the design of the bifunctional fusion protein.

In the present invention, any suitable vector may be used, which may be one of pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR, and the expression vector includes a suitable transcriptional and translational regulatory sequence. Fusion DNA sequences.

A mammalian or insect host cell culture system can be used for expression of the bifunctional fusion protein of the invention, Cells such as COS, CHO, NSO, sf and sf21 can be used in the present invention.

A usable host cell is a prokaryotic cell containing the above vector, which may be one of DH5a, BL21(DE3), TGI.

The bifunctional fusion protein having the structural and functional advantages of the antibody disclosed in the present invention is prepared by culturing the above host cell under expression conditions to express a bifunctional fusion protein, and isolating or purifying the bifunctional fusion protein.

The bifunctional fusion protein disclosed in the present invention can be isolated and purified by affinity chromatography. According to the characteristics of the affinity column utilized, conventional methods such as high salt buffer, pH change, etc. can be used for elution and binding. And a bifunctional fusion protein on the column.

Using the above method, the bifunctional fusion protein can be purified into a substantially homogeneous substance, for example in

SDS-PAGE electrophoresis is a single band.

According to a preferred embodiment of the invention, a bifunctional fusion protein comprises an antigen binding region of rituximab and an extracellular region of a Flt3 ligand.

A bifunctional fusion protein has the structural and functional advantages of an antibody, and the preparation method comprises the following steps: 1) cloning the rib line of the rituximab antibody heavy chain variable region and the Flt3 ligand extracellular region;

2) fused the gene of the extracellular domain of the Flt3 ligand to the constant region of the antibody light chain to construct a Flex-CL fusion fragment;

3) Construction of the knob mutant of the antibody Fc region: T366W, S354C; hole mutant: T366S, L368A, Y407V and Y394C;

4) fused the rituximab heavy chain variable region to the knob mutant, and the Flex-CL and the hole mutant were fused to the expression vector;

5) The above expression vector is co-transduced with the rituximab antibody light chain, and the bifunctional fusion protein is obtained by separation and purification.

The above constructed intact heavy and light chain genes were separately loaded into the eukaryotic expression vector pcDNA3.1(+) (product of Invitrogen). The above plasmids were transfected together with CHO-K1 cells (ATCC) by liposome method, and cell clones stably expressing the bifunctional fusion protein were screened using a selection medium containing 60 (^g/ml G418. Using a Protein A column, Affinity chromatography was purified from the supernatant of the cell culture to obtain a bifunctional fusion protein.

The above bifunctional fusion protein or preparation is used in the preparation of an anticancer drug, and is also used in combination with other antitumor drugs.

The present invention discloses the above bifunctional fusion protein, which can be combined with pharmaceutically acceptable excipients. The composition of the preparation thus exerts a more stable therapeutic effect, and these preparations can ensure the conformational integrity of the amino acid core sequence of the bifunctional fusion protein disclosed in the present invention, while also protecting the polyfunctional group of the protein from degradation (including but not limited to aggregation, Deamination or oxidation). Usually, for liquid preparations, usually

Storage at 2 ° C - 8 ° C for at least one year, for lyophilized preparations, stable at 30 ° C for at least six months. The preparation may be a suspension, a water needle, a lyophilized preparation, etc., which is commonly used in the pharmaceutical field, preferably a water needle or a lyophilized preparation, and is pharmaceutically acceptable for the aqueous needle or lyophilized preparation of the above bifunctional fusion protein disclosed in the present invention. Excipients include one or a combination of a surfactant, a solution stabilizer, an isotonicity adjusting agent and a buffer, wherein the surfactant comprises a nonionic surfactant such as a polyoxyethylene sorbitan fatty acid ester (Tween 20 or 80). ); Poloxamer (eg poloxamer 188); Triton; alkyl with twelve sulfate (SDS); sodium lauryl sulfate; alkyl with fourteen, linoleyl or octadecyl alkyl with sarcosine; Pluronics; MONAQUAT ^TM et al., which was added The amount should minimize the granulation tendency of the bifunctional fusion protein. The solution stabilizer can be a saccharide, including reducing sugars and non-reducing saccharides, amino acids including monosodium or histidine glutamate, and alcohols including triols. , a solution of a higher sugar alcohol, propylene glycol, polyethylene glycol or a combination thereof, the solution stabilizer is added in an amount such that the final formed formulation remains stable for a period of time that is considered stable by those skilled in the art. , Isotonicity adjusting agent may be sodium chloride, mannitol, one buffer may be TRIS, histidine buffer, a phosphate buffer one.

The above preparation is a composition comprising a bifunctional fusion protein, and the antitumor effect is remarkable after administration to an animal including a human. Specifically, it is effective for the prevention and/or treatment of tumors and can be used as an antitumor drug.

The bifunctional fusion protein and the composition thereof in the present invention are administered to an animal including a human, and the dosage is different depending on the age and weight of the patient, the disease characteristics and severity, and the administration route, and can be referred to an animal experiment. The results and various conditions, the total dose can not exceed a certain range. Specifically, the dose for intravenous injection is 0.1 to 3000 mg/day.

The anti-tumor drug referred to in the present invention refers to a drug having an inhibitory and/or therapeutic tumor, which may include a delay accompanying the development of symptoms associated with tumor growth and/or a decrease in the severity of these symptoms, and further includes an accompanying tumor growth accompanying The relief of symptoms and the prevention of other symptoms also reduce or prevent metastasis.

The bifunctional fusion protein and the composition thereof disclosed by the invention can also be administered in combination with other antitumor drugs for the treatment of tumors, and the antitumor drugs include 1. cytotoxic drugs (1) acting on the chemical structure of DNA Drugs: sputum agents such as nitrogen mustard, nitrosours, methanesulfonates; platinum compounds such as cisplatin, carboplatin and oxalic acid; mitomycin (MMC); (2) affecting nucleic acid synthesis Drugs: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta, etc; thymidine synthase inhibitors such as fluorouracil (5FU, FT-207, capecitabine), etc.; purine nucleoside synthesis Enzyme inhibitors such as 6-mercaptopurine (6-MP) and 6-TG Etc.; nucleotide reductase inhibitors such as hydroxyurea (HU); DNA polymerase inhibitors such as cytarabine (Ara-C) and Gemz (Gemz); (3) drugs acting on nucleic acid transcription: selection Drugs that act on DNA templates and inhibit DNA-dependent RNA polymerase, thereby inhibiting RNA synthesis, such as actinomycin 1), daunorubicin, doxorubicin, epirubicin, aclarithromycin, and phosporin (4) Drugs mainly acting on tubulin synthesis: paclitaxel, taxotere, vinblastine, vinorelbine, podophyllum, homoharringtonine; (5) other cytotoxic drugs: asparagine The enzyme mainly inhibits the synthesis of protein; 2. Hormone antiestrogens: tamoxifen, droloxifene, exemestane, etc.; aromatase inhibitors: aminoglutethimide, lantron, letrozole, ruined, etc. Antiandrogen: Fluoramide RH-LH agonist/antagonist: Norred, enadine, etc.; 3. Biological response modifier: mainly inhibits tumor interferon through the body's immune function; interleukin-2; thymosin Class; 4, monoclonal antibody: MabThera; Cetuximab (C225); Trastuzumab Bevacizumab (Avastin); 5, other drugs with unknown mechanisms and pending further research; cell differentiation inducers such as retinoids; apoptosis inducers. The bifunctional fusion proteins and compositions thereof disclosed herein can be administered in combination with one or a combination of the above-described antitumor drugs. DRAWINGS

Figure 1. Schematic diagram of the structure of the CD20-Flex BiFP bifunctional fusion protein.

Figure 2. Rituximab heavy chain knob mutant structure map.

Figure 3. Flex-CL-Hinge-CH2-CH3 block diagram.

Figure 4. Affinity of CD20-Flex BiFP for CD20.

Figure 5. CDC killing activity of CD20-Flex BiFP.

Figure 6. ADCC killing activity of CD20-Flex BiFP.

Figure 7. Induction of apoptotic activity of CD20-Flex BiFP.

Figure 8. Tumor protection activity in mice of CD20-Flex BiFP. detailed description

Example 1. Cloning of the heavy chain variable region gene of rituximab antibody

The rituximab heavy chain variable region gene was cloned by PCR using the rituximab antibody heavy chain as a template. The reaction conditions were: 95 ° C for 15 minutes; 94 ° C for 50 seconds, 58 ° C for 50 seconds, 72 ° C for 50 seconds, 30 cycles; The PCR product rituximab HV was obtained at 72 ° C for 10 minutes. Antibody signal peptide amino acid sequence MGWSCIILFLVATATGVHS. The correct cloned fragment was obtained by sequencing. SEQ ID NO: 2 shows the amino acid sequence of the rituximab heavy chain variable region, the nucleotide sequence of which is SEQ ID NO: 1. Example 2. Cloning of the Flt3 ligand variable region gene

Human lymphocytes were isolated using lymphocyte separation solution (product of Dingguo Biotechnology Development Co., Ltd.), and total RNA was extracted using Trizol Trial IJ (Invitrogen), according to the literature (Cloned human and mouse kappa immunoglobulin constant and J region genes conserve homology In functional segments. Hieter PA, Max EE, Seidman JG, Maizel JV Jr, Leder P. Cell. 1980 Nov;22( l Pt 1): 197-207.) The nucleotide sequence of a human immunoglobulin C gamma 1 gene. Ellison JW, Berson BJ, Hood LE. Nucleic Acids Res. 1982 Jul 10; 10(13):4071 -9.) The Flt3 ligand extracellular domain gene was amplified by RT-PCR. The PCR product was purified by agarose gel electrophoresis and cloned into pGEM-T vector. After sequencing, it was confirmed that the correct clone was obtained. SEQ ID NO: 4 shows the Flex amino acid sequence, the nucleotide sequence of which is SEQ ID NO: 3 o Example 3. Cloning of antibody light chain constant region, Fc region

Separation of healthy human lymphocytes with lymphocyte separation solution (Dingguo Biotechnology Development Co., Ltd.)

Trizol tried IJ (Invitrogen) to extract total RNA, according to the literature (Cloned human and mouse kappa immunoglobulin constant and J region genes conserve homology in functional segments. Hieter PA, Max EE, Seidman JG, Maizel JV Jr, Leder P. Cell 1980 Nov;22( l Pt 1): 197-207.) The nucleotide sequence of a human immunoglobulin C gamma 1 gene. Ellison JW, Berson BJ, Hood LE. Nucleic Acids Res. 1982 Jul 10; (13): 4071 -9.) The antibody light chain constant region and the Fc region gene were amplified by RT-PCR reaction. The PCR product was purified by agarose gel electrophoresis and cloned into pGEM-T vector. After sequencing, it was confirmed that the correct clone was obtained. SEQ ID NO: 6 shows a CL amino acid sequence having the nucleotide sequence of SEQ ID NO: 5; SEQ ID NO: 8 shows an Fc amino acid sequence having the nucleotide sequence of SEQ ID NO: 7. Example 4. Antibody Fc region Construction of knob mutant

The Fc region of the antibody obtained in Example 3 was introduced into the mutation point by the method of overlap PC: T366W, S354C (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 US A. 201 1 ; 108(27): 1 1 187-1 1 192.). PCR product by agar It was purified by sugar gel electrophoresis and cloned into pGEM-T vector. After sequencing, it was confirmed that the correct clone was obtained. SEQ ID NO: 12 shows the Fc-knob amino acid sequence, the nucleotide sequence of which is SEQ ID NO: 11. Example 5. Construction of antibody Fc region hole mutant

The Fc region of the antibody obtained in Example 3 was introduced into the mutation point by overlap PCR: T366S,

L368A, Y407V and Y394C (Schaefer WW, egula 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 by agarose gel electrophoresis and cloned into pGEM-T vector. After sequencing, it was confirmed that the correct clone was obtained. SEQ ID NO: 14 shows the Fc-hole amino acid sequence having the nucleotide sequence of SEQ ID NO: 13. Example 6. Construction of rituximab heavy chain mutant

Using the rituximab heavy chain variable region obtained in Example 1 and the Fc region knob mutant obtained in Example 4 as a template, the rituximab heavy chain variable region was fused with the Fc region knob mutant by overlap PCR, and then loaded. The expression vector was introduced into the Fc constant region of the Rituximab heavy chain to introduce the double mutation S354C and T366W to construct the Rituximab heavy chain knob mutant (Fig. 2). SEQ ID NO: 16 shows the amino acid sequence of the rituximab heavy chain knob mutant having the nucleotide sequence of SEQ ID NO: 15. Example 7. Construction of Flex Mutants

The Flex obtained in Example 2, the CL obtained in Example 3 and the Fc region hole mutant obtained in Example 5 were used as templates, and the Flex was fragment-fused with CL and Fc region holes by the method of overlap PC, and then loaded into an expression vector. , Building Flex-CL-Hinge-CH2-CH3 (Figure 3). SEQ ID NO: 18 shows the amino acid sequence of Flex-CL-Hinge-CH2-CH3 having the nucleotide sequence of SEQ ID NO: 17. Example 8. Expression and purification of CD20-Flex dual function fusion protein

3 x l0 ⁵ CHO-K1 cells (ATCC CRL-9618) were seeded in 3.5 cm tissue culture dishes, and transfected when the cells were cultured to 90%-95% confluence: plasmid lO g rituximab heavy chain knob mutant, Flex- CL-Hinge-CH2-CH3 and 4 g rituximab light 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 Lipofectamine 2000 eagent (product of Invitrogen) was dissolved in 500 μl serum-free DMEM medium, and allowed to stand at room temperature for 5 minutes. The above two liquids were mixed and incubated at room temperature for 20 minutes to form a DNA-liposome complex with 3 ml of serum-free. The DMEM medium was replaced with the serum-containing medium in the culture dish, and then the formed DNA-liposome complex was added to the plate, and ₂ ml of DMEM complete medium containing 10% serum was added after 4 hours of incubation in a CO ₂ incubator. , continue to culture in a CO ₂ incubator. After transfection for 24 h, cells were transfected with 60 (^g/ml G418 selection medium to screen for resistant clones. Cell culture supernatants were screened by ELISA for high expression clones: goat anti-human IgG (Fc) coated on ELISA plate, 4 After overnight at °C, block with 2% BSA-PBS for 2 h at 37 ° C, add the resistant clone culture supernatant or standard (Human myeloma IgG 1, κ) to be tested, incubate at 37 ° C for 2 h, add HRP-羊The anti-human IgG (K) was subjected to a binding reaction, incubated at 37 ° C for 1 h, added with TMB at 37 ° C for 5 min, and finally terminated with H ₂ SO _{4 to} measure the A _{45 ()} value. The serum-free medium was expanded and the bifunctional fusion protein was isolated and purified using a Protein A affinity column (product of GE). The purified antibody was dialyzed against PBS, and finally the concentration of the purified antibody was quantitatively determined by ultraviolet absorption method. Fusion protein affinity detection

The affinity of all fusion proteins was determined by radioimmunoassay [Cragg MS, Morgan SM, Chan HT, Morgan BP, Filatov AV, Johnson PW, French, 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 the iodobead method. The 125 iodine-conjugated fusion protein was incubated with Daudi (ATCC CCL-213) and 8266 cells for 2 hours at 37 °C. The cell-bound and free iodine-labeled antibodies were separated by centrifugation and the radioactivity of 125 iodine-labeled antibodies bound to Daudi cells was detected (see Figure 4). The fusion protein affinity constant was determined by fitting the titration binding curve to the Hill equation curve. The affinity of the CD20-Flex dual-function fusion protein for CD20 and Flt3 is similar to that of the parental rituximab and Flt3 ligands reported in the literature [ Title: Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Author: eff, ME Carrier, K.; Chambers, KS (...) 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.]. Among them, the affinity value of CD20 protein was 4.82 ± 0.25 nM; the affinity for Flt3 was 230 ± 40 pmol/L. Experimental Example 2. CDC Functional Detection of CD20-Flex Fusion Protein

Raji and Daudi cells were incubated in the phenol-free medium at 37 °C with the fusion protein for 1 hour, then incubated with 10% normal human serum for 37 hours in the medium, and then tested for CDC using the standard LDH kit (Promega). active. As shown in Figure 5, the fusion protein CD20-Flex BiFP has similar CDC killing activity as rituximab. Experimental Example 3. ADCC Functional Detection of CD20-Flex Fusion Protein

Daudi cells (ATCC CCL-213) were cultured at different fusion protein concentrations in a ratio of E:T to 25:1 (37 °, 4 hours), and then ADCC activity was measured using a standard LDH kit (Promega). As shown in Figure 6, the fusion protein CD20-Flex BiFP has ADCC killing activity similar to that of rituximab. Experimental Example 4. Detection of Apoptosis Function of CD20-Flex Fusion Protein

Ramos cells were incubated with 10 g/ml of CD20-Flex fusion protein and various concentrations of caspase inhibitor, while 20 g/ml of goat anti-human F(ab') ₂ fragment (Southern Biotechnology) was added. After 16 hours, the apoptotic cells were flow-stained with the Annexin V-Fluos kit (BD). As shown in Figure 7, after cross-linking of the CD20-Flex fusion protein secondary antibody, a similar degree of cell death after cross-linking with rituximab was induced, and the induced cell death exhibited a dose-dependent caspase inhibitor. Experimental Example 5. In vivo tumor formation assay of CD20-Flex fusion protein in mice

5 x 10 ⁶ Daudi or A20-CD20 cells were injected into the tail of BALB/c mice, 0, 4 and

The mice were treated with a 200 μ _§ fusion protein in the tail vein for 8 days. On day 42, the fusion protein-treated mice without tumors were inoculated subcutaneously with lxl O ⁴ A20-CD20 or Α20 cells on the other side. The tumor size of the tumor-bearing mice was observed and the therapeutic effect was evaluated. As shown in Figure 8, the CD20-Flex fusion protein not only has a similar killing effect on rituximab to the first vaccinated tumor, but also exhibits long-term tumor protective activity against the re-vaccinated tumor.

Claims

Rights request

I. A CD20-Flex bifunctional fusion protein with a structure and function similar to that of a whole antibody, characterized by being able to bind to the CD20 antigen and having the function of a Flt3 ligand.

2. The bifunctional fusion protein of claim 1, consisting of three peptide chains, respectively

Flex-CL-Hinge-CH2-CH3 (SEQ ID NO: 18) rituximab heavy chain knob mutant (SEQ ID

NO: 16) rituximab light chain (SEQ ID NO: 10).

3. An isolated nucleotide molecule encoding the three peptide chains described in claim 2 and having the nucleotide sequences described in SEQ ID NO: 17, SEQ ID NO: 15, and SEQ ID NO: 9.

4. A vector containing the nucleic acid molecule of claim 3 and an expression control sequence operatively connected to the sequence of the nucleic acid molecule, wherein the vector can be pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+ ), one of pDHF.

5. The vector of claim 4 is pcDNA3.1(+) or pcDNA3.1/ZEO(+).

6. A host cell containing the vector of claim 5, which is a eukaryotic cell.

7. The host cell of claim 6 is a mammalian cell.

8. The host cell of claim 7 is a CHO cell.

9. A method of preparing the CD20-Flex bifunctional fusion protein according to claim 1, the method comprising: a) cloning the CD20 antibody rituximab variable region and the Flt3 ligand extramembrane region respectively;

b) Construct knob mutants in the Fc region of the antibody: T366W, S354C; hole mutants: T366S, L368A, Y407V and Y394C;

c) Fuse the extramembrane region of Flt3 ligand with the constant region of the antibody light chain to construct the Flex-CL fusion fragment; d) Fuse the variable region of the rituximab heavy chain with the knob mutant, and fuse Flex-CL with the hole mutant, respectively, and load Expression vector;

e) Co-transfect the two constructed expression vectors with the expression vector containing the rituximab light chain gene for expression, separation and purification.

10. A composition containing the CD20-Flex bifunctional fusion protein of claim 1 and a pharmaceutically acceptable carrier.

II. Use of the CD20-Flex bifunctional fusion protein according to claim 1 in the preparation of antibody tumor drugs.

12. The use of the composition according to claim 10 in the preparation of anti-tumor drugs.

13. The use of any one of claims 11 and 12, further including combined use with other anti-tumor drugs.