KR20120129834A - Novel bi-specific monoclonal antibody with advanced anti-cancer activity and pharmaceutical composition for treating cancer comprising the same - Google Patents

Abstract

PURPOSE: A novel bispecific monoclonal antibody and a composition for cancer treatment including the same with improved anticancer activities are provided to express improved antibody dependent cellular cytotoxicity. CONSTITUTION: A novel bispecific monoclonal antibody comprises an antibody which combines with the cancer cell surface antigen and a domain which combines with immunocyte surface antigen. A domain which binds with the immunocyte surface antigen is connected with linker at the C-terminal of Fc domain in antibody which binds with the cancer cell surface antigen. The antibody bonded with the cancer cell surface antigen is in IgG form. The domain bonded with the immunocyte surface antigen is in scFv(Single-chain variable fragment) form. The linker is peptide. The linker peptide has the sequence number 4(SEQ ID NO:4).

Description

Novel bi-specific monoclonal antibody with advanced anti-cancer activity and pharmaceutical composition for treating cancer comprising the same}

The present invention relates to a novel bispecific monoclonal antibody having improved anticancer activity and a composition for treating cancer comprising the same, and more particularly, to a antibody binding to a cancer cell surface antigen and a domain to bind an immune cell surface antigen. And a bispecific monoclonal antibody having a domain linking an immune cell surface antigen to a C-terminus of an Fc region of an antibody binding to a cancer cell surface antigen with a linker, and a composition for treating cancer comprising the antibody as an active ingredient. will be.

Representative cancer cell killing function of the anti-cancer antibody is antibody dependent cell cytotoxicity (ADCC), and the antibody selectively bound to the surface of the cancer cell, by binding to the Fc part and the Fc receptor present on the surface of the immune cell It plays a role in gathering immune cells around cancer cells. Activated immune cells around cancer cells secrete various cytokines (cytokine) to kill cancer cells. Due to this apoptosis process, the binding capacity of the antibody to the Fc receptor may be an important factor in determining the degree of antibody-dependent cytotoxicity.

The bispecific monoclonal antibody for anticancer therapy is composed of two domains, and is composed of a domain capable of binding to a specific antigen on the surface of cancer cells and a domain capable of binding to an Fc receptor on the surface of immune cells. The purpose of the early development of bispecific monoclonal antibodies was to obtain binding to Fc receptors that are stronger than the Fc portion of normal antibodies in the form of immunoglobulin (IgG). The binding force of IgG 1 to the Fc receptor present on the surface of Natural Killer cell (NK cell) was about 1000 nM (dissociation constant Kd value), using a domain exhibiting Fc receptor binding ability 10 times higher. Bispecific monoclonal antibodies were prepared. In addition to Fab'-type antibody fragments, the binding domain used scFv as well as IgG-specific bispecific monoclonal antibodies. Diabodies, tandem diabodies, tandem scFv, and the like have been recently developed as methods for simultaneously expressing two binding domains (Current Opinion in Drug Discovery & Development, 2009, 12 (2): 276-283, mAbs, 2009, 1 (6): 1-9).

2B1 antibody, developed by Chiron, USA, is a form of mouse IgG that can bind to natural killer cell surface antigen (CD16) and cancer cell surface antigen (Her2). Development was discontinued due to side effects such as cytokine storms (Cancer Res. 1995, 55: 4586-4593, Immunology Letters, 2008, 116: 126-140, Cancer Immunol. Immunother. 1996, 42: 141-150). Meanwhile, C6.5 / NM3E2 is a tandem scFv that can bind to natural killer cell surface antigen (CD16) and cancer cell surface antigen (Her2), and has problems such as low expression rate and low antigen affinity for CD16 (Molecular Immunology , 1999, 36: 433-446, J. of Immunology, 2001, 166: 6112-6117). In addition, HRS-3 / A9, developed by Biotest, Germany, is a chemical method of Fab 'binding to the surface antigen (CD16) of natural killer cells and Fab' binding to the cancer cell surface antigen (Her2). In linked form, the antibody was discontinued in phase 3 clinical trials due to its low in vivo potency and side effects such as cytokine seizures. In addition, linking two Fab's using a chemical linker has shown problems of heterogeneous composition and low production yield (Cancer Immunol. Immunother. 1999, 48: 9-21, Immunology Letters, 2008, 116: 126-140, Advanced Drug Delivery Reviews, 2003, 55: 171-197). AFM-13, developed by Affimed, Germany, is a CD19 / CD16 tandem diabody (US20050089519) with a low affinity for surface antigens (CD16) of natural killer cells, as low as 850 nM (dissociation constant Kd). Side (US20050089519).

Comparing the ADCC of bispecific monoclonal antibodies targeting Her2, a cancer cell surface antigen, 2B1 is about 2 times higher than Herceptin (Roche / Genentec), and C6B1D2-NM3E2 in tandem scFv form is about 5 times lower than Herceptin The result was (Journal of Immunology, 2001, 166: 6112-6117). In the improved PK of C6B1D2-NM3E2, the effect of ADCC in the form of a minibody connecting one or two Anti-Her2 scFv was about 100 times less than that of Herceptin (JBC, 2006, 279 (52). (53907-53914). Compared with Herceptin, an IgG-type control antibody, the superior effect of the dual antibody could not be confirmed.

Even in the case of HRS-3 / A9, there was no significant increase in ADCC in the CD30 + cells L540 compared with the control antibody HRS-3. Rather, when A9, an anti-CD16 antibody, was added together with HRS-3, ADCC was reduced (US5643759).

In addition, the bispecific scFv (HLA class II X CD89) form using Neutrophil recruiting showed an increased ADCC effect than the anti-HLA class II (B lymphoid cell) IgG1 form, which is an IgG form of the antibody, but the EC50 value was 2.5. It has been increased by a factor of two, so that no significant improvement can be obtained (Journal of Immunology, 2010, 184: 1210-1217).

Therefore, no bispecific monoclonal antibody has been reported that can show superior ADCC improvement compared to IgG-type control antibody regardless of the type of cancer cell surface antigen. . In other words, a large amount of administration must be administered to obtain a desired therapeutic effect with a low ADCC improvement effect, and when a large amount of bispecific monoclonal antibody is administered, unnecessary immune cell activation occurs and side effects may occur.

Expression of the tandem scFv form (HLA class II X CD89) was expressed in E. coli as insoluble in the inclusion body and did not undergo refolding, and CHO cells showed very low expression levels of ˜5 μg / L. Insect cells (SF21) confirmed the expression of about 60 ~ 100㎍ / L very unsuitable for commercialization (Journal of Immunology, 2010, 184: 1210-1217). HRS-3 / A9 is a form of CD30 / CD16 diFab in which two Fabs are linked by a chemical linker, which cannot be used commercially due to the low productivity of about 100 mg based on 1 L of culture (Clinical Cancer research, 2001, 7: 1873). -1881). Therefore, there is a need for a novel form of bispecific monoclonal antibody capable of ensuring high expression levels and productivity with genetic recombination technology.

Therefore, the present inventors have tried to develop a bispecific antibody that can ensure high expression and productivity, and exhibit improved ADCC effect. As a result, the scFv that is selective for the surface source of natural killer cells (CD16) is selected as the carboxyl of the anticancer antibody. A novel form of bispecific monoclonal antibody fused with a peptide linker at the end was prepared, and the increased CD16 binding ability and the improved ADCC effect were confirmed, and high expression and productivity were secured to complete the present invention.

One object of the present invention is to provide a bispecific monoclonal antibody comprising an antibody binding to a cancer cell surface antigen and a domain binding to an immune cell surface antigen.

Another object of the present invention is to provide a method for producing the bispecific monoclonal antibody.

Still another object of the present invention is to provide a composition for treating cancer, comprising the bispecific monoclonal antibody as an active ingredient and a pharmaceutically acceptable carrier.

As one embodiment for achieving the above object, the present invention comprises an antibody that binds to a cancer cell surface antigen and a domain that binds to an immune cell surface antigen, and is immune to the C-terminus of the Fc region of an antibody that binds to a cancer cell surface antigen. Provided are bispecific monoclonal antibodies in which domains that bind cell surface antigens are linked by linkers.

The antibody binding to the cancer cell surface antigen is not particularly limited, but it is preferable to use a monospecific monoclonal antibody in the form of IgG, and the domain binding to the immune cell surface antigen is not particularly limited. However, scFv -chain variable fragment. The linker is not particularly limited, but is preferably composed of the amino acid sequence of SEQ ID NO: 4 as a peptide linker.

As used herein, the term "bispecific monoclonal antibody" refers to a monoclonal antibody capable of binding to two different antigens, and does not exist naturally, and refers to a form produced by genetic engineering. For the purposes of the present invention, the bispecific monoclonal antibody may simultaneously bind to immune cell surface antigens and cancer cell surface antigens.

As used herein, the term "antibody" includes immunoglobulin molecules that are immunologically reactive with specific antigens, and include both polyclonal and monoclonal antibodies. The term also includes forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies). For the purposes of the present invention, the antibody refers to a bispecific monoclonal antibody that is a heterologous antibody, and in the case of immunoglobulin (IgG) form, which is a disadvantage of the existing improved anti-cancer therapeutic antibody, the Fc portion of IgG is variously immunized. In order to prevent interfering targeting around cancer cells by binding to various Fc receptors present on the surface of the cell, domains that bind immune cell surface antigens to the Fc region may bind to only the desired immune cells. To be. In the present invention, the term domain can be used interchangeably with antibody fragments.

As used herein, the term “antibody fragment” includes antigen-binding forms of an antibody, including fragments having antigen-binding ability, eg, Fab ′, F (ab ′) 2, Fab, Fv and rIgG. The term also refers to a recombinant single chain Fv fragment (scFv) and includes bivalent or bispecific molecules, diabodies, triabodies and tetrabodies.

As used herein, the term "immune cell" includes, but is not limited to, immune cells that act on antibody-dependent cytotoxic mechanisms, and may preferably be natural killer cells or T cells.

As used herein, the term "immune cell surface antigen" includes, but is not limited to, antigens present on the surface of natural killer cells or T cells, preferably CD3, CD16, CD32, CD64, or CD89. The present invention may comprise an antibody fragment comprising a domain that binds to the immunocell surface antigens. According to one embodiment of the present invention, bispecific monoclonal antibodies were prepared using CD16 (FIG. 1).

As used herein, the term "cancer cell surface antigen" includes, without limitation, antigens present on the surface of cancer cells, but may preferably be CD19, CD20, Her2, Epithelial cell adhesion molecule (EpCAM) or epidermal growth factor receptor (EGFR). . The dissociation constant (K _D value) for the immune cell or cancer cell surface antigen of the bispecific monoclonal antibody of the present invention is preferably, but not limited to, 100 nM or less. The anti-cancer therapeutic improved antibody under development improved the antigen affinity for immune cells by modifying the Fc amino acid sequence or the sugar chain structure, in which case the affinity for CD16 was reported to be up to 50 nM. When using immune cells as T cells (MT101), the affinity for CD3 was reported to be about 100 nM. According to one embodiment of the present invention, bispecific monoclonal antibodies were prepared using CD20 or Her2.

As used herein, the term “scFv” refers to the minimum antibody fragment having a complete antigen-recognition and antigen-binding site, comprising the V _H and V _L domains of an antibody, wherein the domain is present in a single polypeptide chain.

In addition, the term “monoclonal antibody” in the present invention means an antibody molecule of a single molecular composition obtained from substantially the same antibody population, which monoclonal antibody exhibits single binding specificity and affinity for a particular epitope.

As used herein, the term "cytotoxicity" refers to a phenomenon in which an immune cell or the like kills its own cells infected with a virus or a parasitic organism, and in the present invention, mainly antibody-dependent cell-mediated cytotoxicity (ADCC). Means. Antibody-dependent cytotoxicity refers to a mechanism in which macrophages or leukocytes recognize an antibody bound to an antigen and destroy the antigen. Antibody-dependent cytotoxic mechanism is the action of T cells that are too large to be ingested by phagocytosis, such as parasites or alien solidified tumors, and directly recognize and attack the antigen as the antibody covers the surface of the antigen. It plays an important role in the case of being inhibited. In the present invention, the ADCC mechanism produces an anticancer effect using an immune response using a cancer cell surface protein as an antigen.

Much research and practical use of anticancer drugs have been conducted to treat cancer using antibodies that recognize cancer cell-specific proteins as antigens. While IgG-type anticancer drugs such as Herceptin and Rituximab exist, bispecific antibodies that recognize both cancer cell surface antigens and immune cell surface antigens have not been successful. Specifically, existing bispecific antibodies have shown problems such as heterogeneous composition, low expression rate, low antigen affinity, and ADCC effect not improved compared to simple IgG products. When a large amount is administered to obtain a desired therapeutic effect with a low ADCC improvement effect, unnecessary immune cells are activated, resulting in side effects such as a cytokine attack.

Accordingly, the bispecific antibody of the present invention is a linker in which a domain that binds an immune cell surface antigen to the C-terminus of the Fc region of the antibody that binds to an IgG type cancer cell surface antigen is linked with a linker, which is higher than a conventional bispecific antibody. Expression problems, productivity and improved ADCC effect solved this problem. First, the bispecific antibody of the present invention has a homogeneous composition as a monoclonal antibody, and as shown in Examples 1-2, the CD20 / CD16 tandem scFv shows a low expression rate as known in the art and has not been obtained and other experiments have been performed. In comparison, the bispecific antibodies of the present invention showed improved expression and yield. In addition, by binding the scFv to the C-terminal was confirmed to have a high antigen binding capacity (Fig. 2 and 3) and superior ADCC effect according to the constitutive features that do not inhibit the binding capacity of the cancer cell surface antigen recognition site (Fig. 4).

According to an embodiment of the present invention, the present inventors prepared bispecific monoclonal antibodies in the form of CD20 / CD16 IgG-scFv and Her2 / CD16 IgG-scFv in IgG-scFv form (Examples 1-1 and 1-3). As a result of measuring the binding force with CD16, an immune cell surface antigen, ELISA and BIAcore method, it was confirmed that the binding force is higher than the control anticancer antibody and Fc engineering antibody (Example 2 and Table 1). In addition, the antibody-dependent cytotoxicity (ADCC) effect was analyzed using a cell (Daudi) overexpressed cancer cell surface antigen CD20. As a result, the control anticancer antibody of the bispecific monoclonal antibody of the present invention did not exhibit ADCC effect, while the other control Fc engineering anticancer antibody began to develop antibody-dependent cytotoxicity from 1 ng / mL, The monoclonal antibody started to develop antibody-dependent cytotoxicity from 0.1ng / mL, confirming the occurrence of up to 80% antibody-dependent cytotoxicity in the 10ng / mL group (Fig. 4). Accordingly, it was confirmed that the bispecific monoclonal antibody of the present invention can be used as a model of a new bispecific monoclonal antibody with improved ADCC effect.

Bispecific monoclonal antibody of the present invention is in the form of a double-antibody IgG-scFv, scFv is a domain that binds to the surface antigen of immune cells, characterized in that the antibody that binds to the surface antigen of the cancer cell is linked with a peptide linker, A typical schematic diagram of this configuration is shown in FIG. 1.

In another aspect, the present invention provides a method for preparing the bispecific monoclonal antibody.

Antibodies of the invention can be produced using methods known in the art, such as phage display methods or yeast cell surface expression systems. Antibodies of the invention may be derived from any animal, including mammals, birds, and the like, including humans.

Preferably, the method for producing a bispecific monoclonal antibody of the present invention comprises the steps of: (a) obtaining an antibody binding to a cancer cell surface antigen and a polynucleotide encoding a domain binding to an immune cell surface antigen, respectively; (b) linking the 5'-terminus of the domain that binds the immune cell surface antigen to the 3'-terminus of the antibody that binds the cancer cell surface antigen to obtain a gene of a bispecific monoclonal antibody; (c) cloning the gene of the obtained bispecific monoclonal antibody to obtain a bispecific monoclonal antibody expression vector; (d) introducing the obtained expression vector into a host cell to obtain a transformant; And, (e) culturing the transformant and recovering the bispecific monoclonal antibody therefrom.

More preferably, the steps (a) and (b) comprise (i) a gene encoding the heavy and light chains of the antibody binding to the cancer cell surface antigen and the heavy chain variable region and the light chain variable region of the antibody binding to the immune cell surface antigen. Amplifying a gene encoding the gene in the expression vector; (ii) linking the heavy chain variable region and the light chain variable region of the antibody that binds the immune cell surface antigen with a linker; And (iii) linking the heavy chain region 3′-terminus of the antibody binding to the cancer cell surface antigen with the immune cell scFv prepared in (ii) with a linker. In addition, a bispecific monoclonal antibody can be prepared by cloning the bound gene in another expression vector through the above steps. The linker of step (ii) may be a polynucleotide encoding (GGGGS) n peptides of various lengths, and preferably may be SEQ ID NO: 1. The linker of step (iii) may be a polynucleotide encoding (GGGGS) n peptides of various lengths, preferably SEQ ID NO: 3.

According to an embodiment of the present invention, amplifying a gene encoding the heavy and light chains of an antibody that recognizes Her2 or CD20 as a cancer cell surface antigen and an antibody that recognizes CD16 as an immune cell surface antigen in an expression vector, an immune cell surface antigen And linking the heavy chain variable region and the light chain variable region of the cancer cell surface antigen with the linker peptide (SEQ ID NO: 1), connecting the heavy chain region 3'-terminus of the cancer cell surface antigen and the immune cell scFv with the linker peptide (SEQ ID NO: 3). Cloning the genes linked to another expression vector through the above steps, and expressing and purifying the animal cells in the form of bispecific monoclonal antibodies CD20 / CD16 IgG-scFv and Her2 / CD16 IgG-scFv. Was prepared. In the present invention, half-life in mouse plasma can be identified for pharmacokinetics studies of bispecific monoclonal antibodies.

According to one embodiment of the present invention, to test whether the bispecific monoclonal antibody has a cancer cell inhibitory effect in vivo, efficacy experiments were conducted in anti-cancer model mice. In the case of CD20 / CD16 bispecific monoclonal antibodies, Rituxan was used as a reference drug. In the case of Her2 / CD16 bispecific monoclonal antibody, Herceptin was used as a reference drug. As a result, the tumor size was reduced compared to the vehicle-administered group after 4 days of antibody administration, and the increase in tumor size was significantly reduced compared to the vehicle-administered group at day 12.

In another aspect, the present invention provides a composition for treating cancer, comprising the bispecific monoclonal antibody as an active ingredient and a pharmaceutically acceptable carrier.

As used herein, the term "cancer" includes, without limitation, esophageal cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer Prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, or multiple myeloma hematoma cancer.

The bispecific monoclonal antibody of the present invention was confirmed to have an antibody-dependent cytotoxicity (ADCC) effect, which is a function of an antibody that kills cancer cells superior to conventional control or Fc engineering anticancer antibodies. It was confirmed that it can be used (Fig. 4). In addition, the anticancer treatment effect was confirmed in xenograft mouse anticancer model administered with Daudi cells, which is a lymphoma, which supports that the composition containing the bispecific monoclonal antibody of the present invention can be used as a cancer treatment composition.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water suitable for the living body such as saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added.

Compositions comprising the bispecific monoclonal antibodies according to the present invention, respectively, oral formulations, external preparations, suppositories or sterile injections of powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols according to conventional methods It can be formulated and used in the form of a solution.

In another aspect, the present invention provides a method of treating cancer using the bispecific monoclonal antibody.

The method may be a method of treating cancer comprising administering the pharmaceutical composition to a subject in need thereof, wherein the bispecific monoclonal antibodies, carriers, and types of cancer used are as described above. same.

The composition may be administered in single or multiple amounts in a pharmaceutically effective amount. In this case, the composition may be administered in the form of a liquid, powder, aerosol, capsule, enteric skin tablets or capsules or suppositories. Routes of administration include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endothelial administration, oral administration, topical administration, intranasal administration, pulmonary administration, rectal administration, and the like. However, upon oral administration, since the peptide is digested, the oral composition must be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell. In addition, the anticancer composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The composition comprising the bispecific monoclonal antibody of the present invention is administered in a pharmaceutically effective amount. “Pharmaceutically effective amount” means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, wherein an effective dose level is the severity of the disease, the activity of the drug, the age of the patient, Body weight, health, sex, sensitivity to the drug of the patient, time of administration of the composition of the invention used, route of administration and rate of release, duration of treatment, elements including drugs used or combined with the composition of the invention used and other medical fields Can be determined according to well-known factors.

Using a cancer therapeutic composition comprising the bispecific monoclonal antibody of the present invention, a powerful anti-cancer antibody is attracted by a domain capable of binding to an immune cell surface antigen, thereby controlling a control anticancer antibody or a reported Fc engineered (Fc engineering). It can be used for more effective chemotherapy because it can show increased antibody-dependent cytotoxicity than antibodies.

1 is a schematic diagram showing the structure of bispecific anticancer antibody (Rituxan-CD16).
Figure 2 shows the anticancer antibody (Rituxan), bispecific anticancer antibody (Rituxan-CD16) and Fc engineering anticancer antibody (Rituxan-Xc) against human immune cell surface antigens by Enzyme Immunoassay (ELISA, Enzyme Linked ImmunoSorbent Assay) It is a graph showing the result of measuring the binding force.
FIG. 3 shows control anticancer antibody (Rituxan), bispecific anticancer antibody (Rituxan-CD16) and Fc engineering anticancer antibody (Rituxan-Xc) against mouse immune cell surface antigens by enzyme immunoassay (ELISA, Enzyme Linked ImmunoSorbent Assay) It is a graph showing the result of measuring the binding force.
Figure 4 is a graph showing the results of experiments on the degree of antibody-dependent cytotoxicity (ADCC) between bispecific anticancer antibody (Rituxan-CD16), control antibody (Rituxan), Fc engineering anticancer antibody (Rituxan-Xc).
5 is a graph showing the results of experiments of pharmacokinetics between bispecific anticancer antibody (Rituxan-CD16), control antibody (Rituxan), Fc engineering anticancer antibody (Rituxan-Xc).
Figure 6 is a graph showing the results of experiments in the anticancer effect between anti-cancer antibody (Rituxan-CD16), control antibody (Rituxan), Fc engineering anticancer antibody (Rituxan-Xc) in anti-cancer model animals.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  One: Bispecificity  Preparation of Monoclonal Antibodies

Example  1-1: CD20 Of CD16 IgG - scFv  Produce

Genes encoding the heavy and light chains of CD20 antibodies for the purpose of producing CD20 / CD16 IgG-scFv in the CHO-S mammalian cell line used genes of rituximab antibody, genes encoding heavy and light chains of CD16 antibody. The sequence of anti-CD16 scFv used in C6.5 / NM3E2 antibody was used (Molecular Immunology, 1999, 36: 433-446).

Expression of nucleic acids encoding CD20 / CD16 IgG-scFv fusion polypeptides can be carried out by conventional methods in prokaryotic or eukaryotic cells, and eukaryotic host-vector systems include a wide range of vectors for many different species. A representative vector for amplifying DNA is a pcDNA 3.1 vector, and again, a vector capable of expressing an antibody protein encoding an antibody polypeptide in a CHO-S mammalian cell line through gene recombination was obtained.

To design genes that can express CD20 / CD16 IgG-scFv, Rituximab was set to IgG type and CD16 antibody to scFv type. The heavy and light chain variable regions of the CD16 antibody are linked to the linker peptide GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 2), and the heavy chain region C-terminus of Rituximab and the CD 16 scFv antibody are linked using the linker peptide GGGGSGGGGSGGGGS (SEQ ID NO: 4). (SEQ ID NO: 6).

To express the CD20 / CD16 IgG-scFv protein, a gene capable of decoding CD16 scFv linked to the rituximab heavy chain region was cloned into an expression vector, and another gene capable of decoding the rituximab light chain region was obtained. The expression vector was cloned.

For antibody production, floating animal cells transfected with the gene (bispecific anti-cancer antibody, IgG-scFv form) using a polymer that improves the efficiency of intracellular gene transfer were collected in a 500 mL Erlenmeyer flask (Corning Costa). Incubated at 200 mL per dog. 1 L of mixed solution of RPMI (Invitrogen Corp.) supplemented with ultra low IgG fetal bovine serum (Invitrogen Corp.) and hypercellular medium was used to produce recombinant protein in a cell culture (pure) for 4 days. It was. Cell cultures were obtained and centrifuged to separate supernatants containing suspended cells and secreted recombinant protein and filtered once with a 0.22 μm vacuum filter device (Millipore, Maryland, USA).

For antibody purification, the CD20 / CD16 IgG-ScFv antibody was purified from the culture using a recombinant Protein-A Sepharose column (Hitrap MabSelect Sure, 5 mL, GE healthcare). The filtered culture medium was loaded onto a recombinant protein-A Sepharose column. The column was washed with 20 mM column volume with 50 mM Tris-Cl (pH 7.2), 100 mM NaCl, and impurities were washed with 50 mM Na-citrate (pH 5.0) at 20 column volume. The antibody was eluted with 50 mM Na-citrate 100 mM NaCl, pH 3.5, and neutralized with 1M Tris-HCl, pH 8.0. The fractions were analyzed by SDS-PAGE and the positive fractions were pooled and concentrated with a centrifuge concentrator (Amicon Ultra, 30,000 MWCO, Millipore, Bedford, MD). Using the same centrifugal concentrator, 50-fold volume of PBS, pH 7.4 was added and concentrated to perform buffer exchange.

Finally, the antibody was sterile filtered with a syringe filter of 0.22 μm pore diameter, and the absorbance (A280) was measured to determine antibody concentration. 1 is a schematic diagram showing the structure of bispecific anticancer antibody (Rituxan-CD16). As shown in Figure 1, it was confirmed that a novel bispecific anti-cancer antibody in the form of scFv bound to the antibody terminal of the IgG form.

Example  1-2: CD20 Of CD16 tandem scFv  Produce

Genes encoding the heavy and light chains of CD20 antibodies were used for the production of CD20 / CD16 tandem scFv in CHO-S mammalian cell lines using genes of rituximab antibody, and genes encoding heavy and light chains of CD16 antibody. , The sequence of anti-CD16 scFv used in the C6.5 / NM3E2 antibody was used (Molecular Immunology, 1999, 36: 433-446).

To design genes capable of expressing CD20 / CD16 tandem scFv, CD20 antibody A gene encoding the heavy and light chain variable regions of rituximab and a gene sequence encoding the heavy and light chain variable regions of the CD16 antibody were obtained, and the heavy and light chain variable regions of the CD20 and CD16 antibodies were linked. The linker peptide was designed in the same manner, and the linker peptide of SEQ ID NO: 2 was used. As a linker peptide connecting the CD20 scFv type gene and the CD16 scFv type gene, a linker of SEQ ID NO: 4 was used. A gene (SEQ ID NO: 7) that can be encoded to express the CD20 / CD16 tandem scFv protein was cloned into the expression vector.

For antibody production, a suspension of animal cells transfected with the gene (bispecific anticancer antibody, tandem diabody form) using a polymer that enhances the efficiency of intracellular gene transfer was used in a 500 mL Erlenmeyer flask (Corning Costa). Incubated at 200 mL per dog. 1 L of mixed solution of RPMI (Invitrogen Corp.) supplemented with ultra low IgG fetal bovine serum (Invitrogen Corp.) and hypercellular medium was used to produce recombinant protein in a cell culture (pure) for 4 days. It was. Cell cultures were obtained and centrifuged to separate supernatants containing suspended cells and secreted recombinant protein and filtered once with a 0.22 μm vacuum filter device (Millipore, Bedford, MD).

Histidine-bound antibody protein was purified from the culture medium using a histidine purification column (GE healthcare). Cultures were loaded after equilibrating a histidine purification column (GE healthcare) of a size appropriate for the amount of human antibody in the medium. The column was washed thoroughly with PBS, the antibody was eluted with a buffer containing 0.5M imidazole, and then buffer exchanged to PBS, pH 7.4 through a dialysis cassette (Thermo scientific).

Finally, the antibody was sterile filtered with a syringe filter of 0.22 um pore diameter and concentrated with Amicon Ultra (Millipore, Maryland, USA). In the process, CD16 / CD16 tandem scFv did not proceed with CD16 binding and ADCC experiments because the precipitates were unstable and the yield was very low.

Example  1-3: Her2 Of CD16 IgG - scFv  Produce

Genes encoding the heavy and light chains of the Her2 antibody for the purpose of producing Her2 / CD16 IgG-scFv in the CHO-S mammalian cell line used the genes of Trastuzumab antibody, and the heavy and light chains of the CD16 antibody. As the gene encoding, the sequence of the anti-CD16 scFv used in the C6.5 / NM3E2 antibody was used (Molecular Immunology, 1999, 36: 433-446).

To design genes capable of expressing Her2 / CD16 IgG-scFv, trastuzumab was set to IgG type and CD16 antibody to scFv type. The linker peptide that connects the heavy and light chain variable regions of the CD16 antibody is linked to GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 2). (4)) (SEQ ID NO: 10).

To express the Her2 / CD16 IgG-scFv protein, a gene capable of decoding CD16 scFv linked to the trastuzumab heavy chain region was cloned into an expression vector, and another gene capable of decoding the trastuzumab light chain region was obtained. The expression vector was cloned.

For antibody production, 1 bottle of floating animal cells transfected with the gene (bispecific anticancer antibody, immunoglobulin form) using a polymer that improves the efficiency of intracellular gene transfer in a 500 mL culture flask (Corning Costa) Incubated at 200 mL per sugar. 1 L of mixed solution of RPMI (Invitrogen Corporation) supplemented with ultra low IgG fetal bovine serum (Invitrogen Corporation) and a supercellular medium was used to produce recombinant protein in a cell culture (pure) for 4 days. It was. Cell cultures were obtained and centrifuged to separate supernatants containing suspended cells and secreted recombinant protein and filtered once with a 0.22 μm vacuum filter device (Millipore, Maryland, USA). Her2 / CD16 IgG-scFv cultures were purified using recombinant Protein-A Sepharose column (Hitrap MabSelect Sure, 5 mL, GE healthcare). The filtered culture medium was loaded onto a recombinant protein-A Sepharose column. The column was washed with 20 mM column volume with 50 mM Tris-Cl (pH 7.2), 100 mM NaCl, and impurities were washed with 50 mM Na-citrate (pH 5.0) at 20 column volume. The antibody was eluted with 50 mM Na-citrate 100 mM NaCl, pH 3.5, and neutralized with 1M Tris-HCl, pH 8.0. After the first purification, only the antibody in which the anti-CD16 domain was normally expressed was selected using Hitrap. The column was equilibrated with neutral pH buffer and eluted using the imidazole concentration difference.

Example  1-4: Fc engineering  Form anti - CD20  Antibody production

Amino acid modifications were engineered in the Fc region of the Rituximab IgG antibody, a CD20 antibody, to enhance affinity for the Fc receptor, CD16 (FcrRIII). By a known method, serine (S) 239 was replaced with aspartic acid (D) in the CH2 region of the Fc region of IgG1 (S239D), and isoleucine (I) 332 was replaced with glutamic acid (E) (I332E). (US2008 / 0260731). Genes substituted with S239D and I332E of the CH2 region of the rituximab heavy chain region (SEQ ID NO: 11) were cloned into the expression vector, and the rituximab light chain region was cloned into another expression vector.

For the production of antibodies, 1 bottle of 500 ml culture conical flask (Corning Costa) was used for floating animal cells transfected with the gene (Fc engineering anticancer antibody, immunoglobulin form) using a polymer that enhances the efficiency of intracellular gene transfer. Incubated at 200 mL per sugar. 1 L of mixed solution of RPMI (Invitrogen Corp.) supplemented with ultra low IgG fetal bovine serum (Invitrogen Corp.) and hypercellular medium was used to produce recombinant protein in a cell culture (pure) for 4 days. It was. Cell cultures were obtained and centrifuged to separate supernatants containing suspended cells and secreted recombinant protein and filtered once with a 0.22 μm vacuum filter device (Millipore, Maryland, USA). The prepared supernatant was passed through a recombinant protein-A Sepharose column equilibrated with equilibration buffer (50 mM Tris-HCl, pH 7.2, 100 mM NaCl). The antibody bound to the column was eluted with elution buffer (50 mM Na-citrate (pH 3.5), 100 mM NaCl) solution, then neutralized with 2 M Tris-HCl (pH 9.0) and PBS (phosphate buffered saline, pH 7.4, Invitrogen) buffer. The purified antibody was electrophoresed on Bis-Tris 4-12% gradient SDS-polyacrylamide gel (NuPAGE gel, Invitrogen) under non-reducing and reducing conditions. As a result, about 160 kDa in the non-reduced state, about 55 kDa heavy chain and about 25 kDa light chain were observed in the reduced state.

Example  2: CD16  Antigen binding

Example  2-1: ELISA  Human using method CD16  Antigen binding

Color reaction through mediators that bind to a specific site of the antibody after treatment of anticancer antibody, bispecific and Fc engineering anticancer antibodies to human CD16 coated on 96 well plate (Nunc) Was measured using a microplate reader (molecular device). Final binding degree between the antigen and the antibody was determined based on the measured values (FIG. 2).

FIG. 2 shows the binding capacity of control anticancer antibody (Rituxan), bispecific anticancer antibody (Rituxan-CD16) and Fc engineering anticancer antibody (Rituxan-Xc) to immune cell surface antigens by Enzyme Linked ImmunoSorbent Assay (ELISA) As a graph showing the results of the measurement, each antibody was performed at 12 concentration intervals (including 0 μg / ml, duplicates), starting with up to 100 μg / ml. As shown in FIG. 2, the final binding strength between the two proteins was found to be in the order of the control anticancer antibody <Fc engineering anticancer antibody << bispecific anticancer antibody.

These results support that the bispecific antibody of the present invention can improve the binding capacity of the human immune cell surface antigen, and eventually improve the ADCC effect.

Example  2-2: ELISA  Mouse using method CD16  Antigen binding

Color reaction through mediators that bind to specific regions of the antibody after treatment of anticancer antibody, bispecific and Fc engineering anticancer antibodies to mouse immune cell surface antigen (mouse CD16) coated on 96 well plate (Nunc) Was measured using a microplate reader (molecular device). Final binding degree between the antigen and the antibody was determined based on the measured values (FIG. 3).

Figure 3 shows the binding capacity of the control anticancer antibody (Rituxan), bispecific anticancer antibody (Rituxan-CD16) and Fc engineering anticancer antibody (Rituxan-Xc) to immune cell surface antigens by enzyme immunoassay (ELISA, Enzyme Linked ImmunoSorbent Assay) As a graph showing the results of the measurement, at this time, each antibody was carried out in 8 concentration intervals (including 0 nM, duplicates) starting with the highest 200 nM. As shown in FIG. 3, the final binding strength between the two proteins was found to be in the order of the control anticancer antibody <Fc engineering anticancer antibody << bispecific anticancer antibody.

These results are consistent with the binding ability of the three antibodies to human CD16, which supports that the bispecific antibody of the present invention may improve the binding ability of the mouse immune cell surface antigens, which in turn may improve the ADCC effect. .

Example  2-3: BIAcore  Human using method CD16  Antigen binding

The degree of binding was determined using the equilibrium dissociation constants calculated by real-time monitoring of binding and dissociation between human immune cell surface antigen, anticancer antibody, bispecific and Fc engineering anticancer antibodies with BIACORE (GE healthcare).

Control anti-cancer antibodies estimated equilibrium dissociation constant of 10 ^- For the present experiments can not be measured ⁶ has anti-cancer bispecific antibody with Fc engineering anti-cancer antibodies, respectively 7.5 * ^10-9, An equilibrium dissociation constant of 1.0 * 10 ^-7 was observed. The equilibrium dissociation constants measured by real-time binding and dissociation monitoring of the two proteins are in the order of control anticancer antibody (estimated)> Fc engineering anticancer antibody >> bispecific anticancer antibody.

 Comparison of Equilibrium Dissociation Constants between Anticancer Antibodies sample Coupling constant Dissociation constant Parallel dissociation constant Anticancer Antibody - - - Bispecific Anticancer Antibody 4.5 * 10 ⁴ 3.4 * 10 ^-4 7.5 * 10 ^-9 Fc engineering anticancer antibody 3.4 * 10 ⁵ 3.4 * 10 ^-2 1.0 * 10 ^-7

Example  3: In vitro ADCC  Confirmation of Antibody-Dependent Cytotoxicity by Assay

Daudi (ATCC, CCL-213) cells overexpressed with CD20 were dispensed to 1.5 x 10 ⁶ cells in a culture vessel, and then placed in a medium containing Calcein AM diluted to a final concentration of 3 u M for 30 minutes. The reaction was carried out in a 37 ° C. incubator. After 30 minutes, the reaction solution was washed twice with PBS. After washing, 3 mL of medium was added to suspend and the suspension was dispensed 50 [mu] l per well, and then 75 x 10 ⁴ cells per well of human PBMC cells were dispensed into each well dispensed with the Daudi cells. A 20 μl volume of control anticancer antibody (Rituxan) and bispecific anticancer antibody (Rituxan-CD16), and Fc engineering anticancer antibody (Rituxan-Xc) diluted to desired concentrations were added to each well, followed by gentle Daudi cells and human PBMC cells. After mixing, the reaction was carried out for 4 hours at 37 ℃ incubator. After the reaction, the culture plate was centrifuged and only the supernatant was recovered to measure the amount of calcein-AM released (FIG. 4).

4 is a graph showing the results of experiments on the degree of antibody-dependent cytotoxicity (ADCC) development between bispecific anticancer antibody (Rituxan-CD16), control anticancer antibody (Rituxan) and Fc engineering anticancer antibody (Rituxan-Xc). The ratio of (Daudi) cells and effector cells (human PBMC) was 1:50, and the amount of calcein-AM (Calcein-AM) indicating antibody-dependent cytotoxicity generated by antibody treatment was determined. As shown in FIG. 4, when the treatment concentration of each antibody was treated from 0.001 ng / mL to 10 ng / mL, the control anticancer antibody (Rituxan) showed almost no antibody-dependent cytotoxicity in the entire treatment concentration range. In the bispecific anticancer antibody (Rituxan-CD16), antibody-dependent cytotoxicity began to develop from 0.1 ng / mL, and up to 80% of the antibody-dependent cytotoxicity was observed in the 10 ng / mL treatment group.

Example  4: Pharmacokinetics  ( Pharmacokinetics ) analysis

Each BALB / c mouse (7 weeks old, female) was intraperitoneally injected with 5 mg / kg of bispecific monoclonal antibody, control anticancer antibody or Fc engineering anticancer antibody, which is CD20 / CD16 IgG-scFv prepared in Example 1-1. Administered. Blood was collected from the tails of mice immediately before and 30 minutes, 1, 3, 6, 12, 24, 36, 48, 72. 96, 120 hours later. The collected blood was measured for concentration of bispecific monoclonal antibody in plasma by ELISA method. The results of blood half-life (T1 / 2), Cmax, and Tmax were derived from the non-compartment model analysis of the WinNolin program (Phoenix ^TM WinNolinVersion 6.1, Pharsight Corporation, USA). (Table 2). As a result of measuring the blood half-life of the bispecific anticancer antibody, the blood half-life of the control anticancer antibody was slightly decreased (about 248 hours), but it was confirmed to be about 216 hours which was increased by about 100 hours than the Fc engineering anticancer antibody (FIG. 5).

Example  5: Anticancer Effect Confirmation Experiment in Anticancer Model Animals

Daudi cells (ATCC, CCL-213TM) were cultured in a 37-degree incubator using medium containing FBS in RPMI1640 (Gibco). For animal administration, a cell mixture prepared with 1x10 ⁷ Daudi cells and Matrigel (BD, 356237) was implanted in the subcutaneous right side of SCID mice (9 weeks old, female). After transplantation, bispecific anticancer antibodies were administered intraperitoneally twice a week at the time point when the tumor size stabilized (about 400 mm ³ ). As the drug was administered, the decrease in tumor size was observed from 4 days after the administration of the vehicle.However, in the tumor size measured on the 12th day of the administration, the Fc engineering anticancer antibody, the control anticancer antibody and the bispecific anticancer antibody have similar drug suppression effects. Was generated (FIG. 6).

<110> HANWHA CHEMICAL CORPORATION <120> Novel bi-specific monoclonal antibody with advanced anti-cancer activity and pharmaceutical composition for treating cancer comprising the same <130> PA120394KR <150> KR 10-2011-0046885 <151> 2011-05-18 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> linker peptide <400> 1 ggtggagggg gttcaggagg gggaggatct ggtggcggcg gatccggggg agggggatcc 60                                                                           60 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 2 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly   1 5 10 15 Gly Gly Gly Ser              20 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> linker peptide <400> 3 ggtggaggtg gcagcggtgg tggcggcagt ggcggtggcg gctcc 45 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 4 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 <210> 5 <211> 2193 <212> DNA <213> Artificial Sequence <220> <223> CD20IgG-CD16scFv <400> 5 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcgt gcactcccag 60 gtgcagctgc agcagcccgg cgccgagctg gtgaagcccg gcgcctccgt gaagatgtcc 120 tgcaaggcct ccggctacac cttcacctcc tacaacatgc actgggtgaa gcagaccccc 180 ggccggggcc tggagtggat cggcgccatc taccccggca acggcgacac ctcctacaac 240 cagaagttca agggcaaggc caccctgacc gccgacaagt cctcctccac cgcctacatg 300 cagctgtcct ccctgacctc cgaggactcc gccgtgtact actgcgcccg gtccacctac 360 tacggcggcg actggtactt caacgtgtgg ggcgccggca ccaccgtgac cgtgtccgcc 420 gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc cacctccggc 480 ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 540 tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagtcctcc 600 ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg cacccagacc 660 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggccgagccc 720 aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct gctgggcggc 780 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc ccggaccccc 840 gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa gttcaactgg 900 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac 960 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 1020 gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa gaccatctcc 1080 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgcccccctc ccgggacgag 1140 ctgaccaaga accaggtgtc cctgacctgc ctggtgaagg gcttctaccc ctccgacatc 1200 gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac cccccccgtg 1260 ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 1320 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 1380 cagaagtccc tgtccctgtc ccccggcaag ggtggaggtg gcagcggtgg tggcggcagt 1440 ggcggtggcg gctcctcctc cgagctgacc caggaccccg ccgtgtccgt ggccctgggc 1500 cagaccgtgc ggatcacctg ccagggcgac tccctgcggt cctactacgc ctcctggtac 1560 cagcagaagc ccggccaggc ccccgtgctg gtgatctacg gcaagaacaa ccggccctcc 1620 ggcatccccg accggttctc cggctcctcc tccggcaaca ccgcctccct gaccatcacc 1680 ggcgcccagg ccgaggacga ggccgactac tactgcaact cccgggactc ctccggcaac 1740 cacgtggtgt tcggcggcgg caccaagctg accgtgggcg gtggaggggg ttcaggaggg 1800 ggaggatctg gtggcggcgg atccggggga gggggatccg aggtgcagct ggtggagtcc 1860 ggcggcggcg tggtgcggcc cggcggctcc ctgcggctgt cctgcgccgc ctccggcttc 1920 accttcgacg actacggcat gtcctgggtg cggcaggccc ccggcaaggg cctggagtgg 1980 gtgtccggca tcaactggaa cggcggctcc accggctacg ccgactccgt gaagggccgg 2040 ttcaccatct cccgggacaa cgccaagaac tccctgtacc tgcagatgaa ctccctgcgg 2100 gccgaggaca ccgccgtgta ctactgcgcc cggggccggt ccctgctgtt cgactactgg 2160 ggccagggca ccctggtgac cgtgtcccgg tga 2193 <210> 6 <211> 730 <212> PRT <213> Artificial Sequence <220> <223> CD20IgG-CD16scFv <400> 6 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys              20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe          35 40 45 Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu      50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn  65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser                  85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn         115 120 125 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys     130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro                 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr             180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val         195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn     210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu                 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp             260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp         275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly     290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp                 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro             340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu         355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn     370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr                 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys             420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys         435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu     450 455 460 Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 465 470 475 480 Gly Gly Gly Gly Ser Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser                 485 490 495 Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu             500 505 510 Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro         515 520 525 Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp     530 535 540 Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr 545 550 555 560 Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp                 565 570 575 Ser Ser Gly Asn His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val             580 585 590 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser         595 600 605 Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val     610 615 620 Val Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 625 630 635 640 Thr Phe Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys                 645 650 655 Gly Leu Glu Trp Val Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly             660 665 670 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala         675 680 685 Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr     690 695 700 Ala Val Tyr Tyr Cys Ala Arg Gly Arg Ser Leu Leu Phe Asp Tyr Trp 705 710 715 720 Gly Gln Gly Thr Leu Val Thr Val Ser Arg                 725 730 <210> 7 <211> 1680 <212> DNA <213> Artificial Sequence <220> <223> CD20-CD16 tandem scFv <400> 7 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcgt gcactcccag 60 gtgcagctgc agcagcccgg cgccgagctg gtgaagcccg gcgcctccgt gaagatgtcc 120 tgcaaggcct ccggctacac cttcacctcc tacaacatgc actgggtgaa gcagaccccc 180 ggccggggcc tggagtggat cggcgccatc taccccggca acggcgacac ctcctacaac 240 cagaagttca agggcaaggc caccctgacc gccgacaagt cctcctccac cgcctacatg 300 cagctgtcct ccctgacctc cgaggactcc gccgtgtact actgcgcccg gtccacctac 360 tacggcggcg actggtactt caacgtgtgg ggcgccggca ccaccgtgac cgtgtccgcc 420 ggtggagggg gttcaggagg gggaggatct ggtggcggcg gatccggggg agggggatcc 480 cagatcgtgc tgtcccagtc ccccgccatc ctgtccgcct cccccggcga gaaggtgacc 540 atgacctgcc gggcctcctc ctccgtgtcc tacatccact ggttccagca gaagcccggc 600 tcctccccca agccctggat ctacgccacc tccaacctgg cctccggcgt gcccgtgcgg 660 ttctccggct ccggctccgg cacctcctac tccctgacca tctcccgggt ggaggccgag 720 gacgccgcca cctactactg ccagcagtgg acctccaacc cccccacctt cggcggcggc 780 accaagctgg agatcaagcg gtcctccggt ggagggggtt caggaggggg aggatctggt 840 ggcggcggat cgggaggttc cgaggtgcag ctggtggagt ccggcggcgg cgtggtgcgg 900 cccggcggct ccctgcggct gtcctgcgcc gcctccggct tcaccttcga cgactacggc 960 atgtcctggg tgcggcaggc ccccggcaag ggcctggagt gggtgtccgg catcaactgg 1020 aacggcggct ccaccggcta cgccgactcc gtgaagggcc ggttcaccat ctcccgggac 1080 aacgccaaga actccctgta cctgcagatg aactccctgc gggccgagga caccgccgtg 1140 tactactgcg cccggggccg gtccctgctg ttcgactact ggggccaggg caccctggtg 1200 accgtgtccc ggggtggagg gggttcagga gggggaggat ctggtggcgg cggatccggg 1260 ggagggggat cctcctccga gctgacccag gaccccgccg tgtccgtggc cctgggccag 1320 accgtgcgga tcacctgcca gggcgactcc ctgcggtcct actacgcctc ctggtaccag 1380 cagaagcccg gccaggcccc cgtgctggtg atctacggca agaacaaccg gccctccggc 1440 atccccgacc ggttctccgg ctcctcctcc ggcaacaccg cctccctgac catcaccggc 1500 gcccaggccg aggacgaggc cgactactac tgcaactccc gggactcctc cggcaaccac 1560 gtggtgttcg gcggcggcac caagctgacc gtgggcctgg tgccgcgcgg cagcgtcgac 1620 gagcagaagc tgatctccga ggaggacctg catcatcatc atcatcatca tcattagtga 1680                                                                         1680 <210> 8 <211> 558 <212> PRT <213> Artificial Sequence <220> <223> CD20-CD16 tandem scFv <400> 8 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys              20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe          35 40 45 Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu      50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn  65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser                  85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn         115 120 125 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly                 165 170 175 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile             180 185 190 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr         195 200 205 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser     210 215 220 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 225 230 235 240 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr                 245 250 255 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ser Ser Gly Gly Gly             260 265 270 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Glu         275 280 285 Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly Ser     290 295 300 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Gly 305 310 315 320 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser                 325 330 335 Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val Lys             340 345 350 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu         355 360 365 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala     370 375 380 Arg Gly Arg Ser Leu Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 385 390 395 400 Thr Val Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly                 405 410 415 Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Glu Leu Thr Gln Asp Pro             420 425 430 Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly         435 440 445 Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly     450 455 460 Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly 465 470 475 480 Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu                 485 490 495 Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn             500 505 510 Ser Arg Asp Ser Ser Gly Asn His Val Val Phe Gly Gly Gly Thr Lys         515 520 525 Leu Thr Val Gly Leu Val Pro Arg Gly Ser Val Asp Glu Gln Lys Leu     530 535 540 Ile Ser Glu Glu Asp Leu His His His His His His His His His 545 550 555 <210> 9 <211> 2190 <212> DNA <213> Artificial Sequence <220> <223> Her2IgG-CD16scFv <400> 9 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcgt gcactccgag 60 gtgcagctgg tggagtccgg cggcggcctg gtgcagcccg gcggctccct gcggctgtcc 120 tgcgccgcct ccggcttcaa catcaaggac acctacatcc actgggtgcg gcaggccccc 180 ggcaagggcc tggagtgggt ggcccggatc taccccacca acggctacac ccggtacgcc 240 gactccgtga agggccggtt caccatctcc gccgacacct ccaagaacac cgcctacctg 300 cagatgaact ccctgcgggc cgaggacacc gccgtgtact actgctcccg gtggggcggc 360 gacggcttct acgccatgga ctactggggc cagggcaccc tggtgaccgt gtcctccgcc 420 tccaccaagg gcccctccgt gttccccctg gccccctcct ccaagtccac ctccggcggc 480 accgccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 540 aactccggcg ccctgacctc cggcgtgcac accttccccg ccgtgctgca gtcctccggc 600 ctgtactccc tgtcctccgt ggtgaccgtg ccctcctcct ccctgggcac ccagacctac 660 atctgcaacg tgaaccacaa gccctccaac accaaggtgg acaagaaggt ggagcccaag 720 tcctgcgaca agacccacac ctgccccccc tgccccgccc ccgagctgct gggcggcccc 780 tccgtgttcc tgttcccccc caagcccaag gacaccctga tgatctcccg gacccccgag 840 gtgacctgcg tggtggtgga cgtgtcccac gaggaccccg aggtgaagtt caactggtac 900 gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gtacaactcc 960 acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 1020 tacaagtgca aggtgtccaa caaggccctg cccgccccca tcgagaagac catctccaag 1080 gccaagggcc agccccggga gccccaggtg tacaccctgc ccccctcccg ggaggagatg 1140 accaagaacc aggtgtccct gacctgcctg gtgaagggct tctacccctc cgacatcgcc 1200 gtggagtggg agtccaacgg ccagcccgag aacaactaca agaccacccc ccccgtgctg 1260 gactccgacg gctccttctt cctgtactcc aagctgaccg tggacaagtc ccggtggcag 1320 cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1380 aagtccctgt ccctgtcccc cggcaagggt ggaggtggca gcggtggtgg cggcagtggc 1440 ggtggcggct cctcctccga gctgacccag gaccccgccg tgtccgtggc cctgggccag 1500 accgtgcgga tcacctgcca gggcgactcc ctgcggtcct actacgcctc ctggtaccag 1560 cagaagcccg gccaggcccc cgtgctggtg atctacggca agaacaaccg gccctccggc 1620 atccccgacc ggttctccgg ctcctcctcc ggcaacaccg cctccctgac catcaccggc 1680 gcccaggccg aggacgaggc cgactactac tgcaactccc gggactcctc cggcaaccac 1740 gtggtgttcg gcggcggcac caagctgacc gtgggcggtg gagggggttc aggaggggga 1800 ggatctggtg gcggcggatc cgggggaggg ggatccgagg tgcagctggt ggagtccggc 1860 ggcggcgtgg tgcggcccgg cggctccctg cggctgtcct gcgccgcctc cggcttcacc 1920 ttcgacgact acggcatgtc ctgggtgcgg caggcccccg gcaagggcct ggagtgggtg 1980 tccggcatca actggaacgg cggctccacc ggctacgccg actccgtgaa gggccggttc 2040 accatctccc gggacaacgc caagaactcc ctgtacctgc agatgaactc cctgcgggcc 2100 gaggacaccg ccgtgtacta ctgcgcccgg ggccggtccc tgctgttcga ctactggggc 2160 cagggcaccc tggtgaccgt gtcccggtga 2190 <210> 10 <211> 729 <212> PRT <213> Artificial Sequence <220> <223> Her2IgG-CD16scFv <400> 10 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln              20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile          35 40 45 Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu      50 55 60 Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala  65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn                  85 90 95 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val     210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu                 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr             260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Asp Val         275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val     290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu                 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala             340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro         355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln     370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr                 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu             420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser         435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser     450 455 460 Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 465 470 475 480 Gly Gly Gly Ser Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val                 485 490 495 Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg             500 505 510 Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val         515 520 525 Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg     530 535 540 Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly 545 550 555 560 Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser                 565 570 575 Ser Gly Asn His Val Val Phe Gly Gly Thr Lys Leu Thr Val Gly             580 585 590 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly         595 600 605 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val     610 615 620 Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 625 630 635 640 Phe Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly                 645 650 655 Leu Glu Trp Val Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr             660 665 670 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys         675 680 685 Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala     690 695 700 Val Tyr Tyr Cys Ala Arg Gly Arg Ser Leu Leu Phe Asp Tyr Trp Gly 705 710 715 720 Gln Gly Thr Leu Val Thr Val Ser Arg                 725 <210> 11 <211> 1413 <212> DNA <213> Artificial Sequence <220> <223> Fc engineering CD20 <400> 11 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcgt gcactcccag 60 gtgcagctgc agcagcccgg cgccgagctg gtgaagcccg gcgcctccgt gaagatgtcc 120 tgcaaggcct ccggctacac cttcacctcc tacaacatgc actgggtgaa gcagaccccc 180 ggccggggcc tggagtggat cggcgccatc taccccggca acggcgacac ctcctacaac 240 cagaagttca agggcaaggc caccctgacc gccgacaagt cctcctccac cgcctacatg 300 cagctgtcct ccctgacctc cgaggactcc gccgtgtact actgcgcccg gtccacctac 360 tacggcggcg actggtactt caacgtgtgg ggcgccggca ccaccgtgac cgtgtccgcc 420 gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc cacctccggc 480 ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 540 tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagtcctcc 600 ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg cacccagacc 660 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggccgagccc 720 aagtcctgcg acaagaccca cacctgccct ccctgccccg ctcccgagct gctgggcgga 780 cccgacgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc ccggaccccc 840 gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa gttcaactgg 900 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac 960 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 1020 gagtacaagt gcaaggtgtc caacaaggct ctgcccgctc ccgaggagaa gaccatctcc 1080 aaggctaagg gacagcctcg ggagcctcag gtgtacaccc tgcccccctc ccgggacgag 1140 ctgaccaaga accaggtgtc cctgacctgc ctggtgaagg gcttctaccc ctccgacatc 1200 gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac cccccccgtg 1260 ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 1320 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 1380 cagaagtccc tgtccctgtc ccccggcaag tga 1413 <210> 12 <211> 470 <212> PRT <213> Artificial Sequence <220> <223> Fc engineering CD20 <400> 12 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys              20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe          35 40 45 Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu      50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn  65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser                  85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn         115 120 125 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys     130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro                 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr             180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val         195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn     210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu                 245 250 255 Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp             260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp         275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly     290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp                 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro             340 345 350 Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu         355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn     370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr                 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys             420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys         435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu     450 455 460 Ser Leu Ser Pro Gly Lys 465 470

Claims (13)

Bispecificity comprising an antibody binding to a cancer cell surface antigen and a domain that binds an immune cell surface antigen, wherein a domain that binds the immune cell surface antigen to the C-terminus of the Fc region of the antibody that binds the cancer cell surface antigen is linked by a linker Monoclonal antibodies.
The method of claim 1,
The antibody that binds to the cancer cell surface antigen is a bispecific monoclonal antibody of the IgG form.
The method of claim 1,
The domain that binds to the immune cell surface antigen is a bispecific monoclonal antibody in the form of a scFv (Single-chain variable fragment).
The method of claim 1,
The linker is a peptide, bispecific monoclonal antibody.
5. The method of claim 4,
The linker peptide is SEQ ID NO: 4, bispecific monoclonal antibody.
The method of claim 1,
The immune cell is a natural killer cell (Natural Killer cell) or T-specific bispecific monoclonal antibody.
The method of claim 1,
The immune cell surface antigen is a bispecific monoclonal antibody selected from the group consisting of CD3, CD16, CD32, CD64 and CD89.
The method of claim 1,
The cancer cell surface antigen is a bispecific monoclonal antibody selected from the group consisting of CD19, CD20, Her2, Epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR).
The method of claim 1,
Dissociation Constants for Immune Cell Surface Antigens (K _D) bispecific monoclonal antibody having an affinity with a value of 100 nM or less.
(a) obtaining an antibody that binds a cancer cell surface antigen and a polynucleotide encoding a domain that binds an immune cell surface antigen, respectively;
(b) linking the 5'-terminus of the domain that binds the immune cell surface antigen to the 3'-terminus of the Fc region of the antibody that binds the cancer cell surface antigen to obtain a gene of a bispecific monoclonal antibody;
(c) cloning the gene of the obtained bispecific monoclonal antibody to obtain a bispecific monoclonal antibody expression vector;
(d) introducing the obtained expression vector into a host cell to obtain a transformant; And
(e) culturing the transformant, and recovering the bispecific monoclonal antibody therefrom, the method of producing the bispecific monoclonal antibody of any one of claims 1 to 9.
The method of claim 10, wherein the steps (a) and (b)
(i) amplifying in a expression vector a gene encoding the heavy and light chains of the antibody binding to the cancer cell surface antigen and the gene encoding the heavy and light chain variable regions of the antibody binding to the immune cell surface antigen;
(ii) linking the heavy chain variable region and the light chain variable region of the antibody that binds the immune cell surface antigen with a linker; And
(iii) linking the heavy chain region 3′-terminus of the antibody that binds the cancer cell surface antigen and the immune cell scFv prepared in (ii) with a linker, a method for producing a bispecific monoclonal antibody.
A cancer treatment composition comprising the bispecific monoclonal antibody of any one of claims 1 to 9 and a pharmaceutically acceptable carrier.
The method of claim 12,
The cancer is esophageal cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer A cancer treatment composition selected from the group consisting of connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, and multiple myeloma blood cancer.

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