TW200427698A - Preparation and application of anti-tumor bifunctional fusion proteins - Google Patents

Abstract

Provided herein is a chimeric protein, which chimeric protein comprises a F1t3 ligand, or a biologically active fragment thereof, and a proteinuous or peptidyl tumoricidal agent, and uses thereof, particularly in the treatment of malignancy.

Description

200427698 发明 Description of the invention: Related applications refer to this application before and after. This application is a Chinese application No. 03U9290.9 filed on June 13, 2003 and filed on January 30, 2003. 〇〇 (Title: Preparation and Application of Antitumor Bifunctional Fusion Protein), which is incorporated by reference in its entirety. Statements about federally sponsored research or development are not applicable. [Technical field to which the invention belongs] The present invention relates to the field of tumor immunology, and mainly relates to an anti-tumor bifunctional fusion protein and its nucleic acid sequence, and its preparation and application method for preparing anti-tumor drugs. [Prior art] Tumor immunotherapy involves the control of natural host defense mechanisms or the manipulation of immune agents to cause tumor regression. Immunotherapy is a recognized form of cardiopulmonary resentment, accompanied by traditional forms of surgical resection, radiation therapy, and chemotherapy for the treatment of malignant conditions. In fact, immunotherapy is often used as a "complementary therapy" 'in more common therapies, such as surgery and radiation. The motive force of this combination therapy lies in the shortcomings of the traditional form. For example, liver cancer, breast cancer, and lymphoma are the most common cancers in China. However, three points <-hepatocellular carcinoma patients have a tumor burden that is unsuitable for surgery at the time of diagnosis. More importantly, even though the surgical resection can be applied to such patients, the problem of long-distance, undetected micrometastasis is still not addressed by this therapy. Similarly, the traditional therapies of radiation therapy and chemotherapy have significant limitations. 89753.doc 200427698 is most prominent in the systemic suppression of hematopoietic and immune systems. Therefore, the toxic effects of radiation and chemotherapy can limit the efficacy of these therapies in situations where radical treatment is most needed-patients with a significant tumor burden at the time of diagnosis. Therefore, it is generally desirable to discover novel and effective strategies that will complement traditional therapies. Tumor immunotherapy can be achieved by administering antibodies specific to the tumor antigen. Although antibodies have typically been used as transporters of toxic moiety groups, recent studies have shown that monoclonal antibodies (mAb) that resist certain cell surface molecules, such as FAS, EGFR, and HER2, are triggered directly by triggering the cell decay pathway. Causes tumor cell death. See, for example, Shimizu and others,

Commun. 228 (2): 375-79 (1996). This suggests that modulating specific signaling pathways, especially those that cause tumor cell death, can provide a successful strategy for antibody-mediated tumor immunotherapy. At least one antibody using this strategy has been successful during clinical trials. Herceptin, a monoclonal antibody specific for human HER2, causes cell death in Her2 + tumor cells and has been successfully used in vivo for breast cancer in vivo treatment. See, for example, Burstein et al., 乂 C7, > z. Ozc (9 /. 21: 2889-95 (2003). However, one of the perceived limitations of this antibody therapy is that long-distance transfer may still escape this therapy Or the possibility that antigen-negative variants will develop, leading to a later recurrence of metastatic disease. Immunotherapy can also be achieved by eliciting an active anti-tumor immune response from the patient after administration of a tumor vaccine. Ideally, a tumor vaccine It is the transmission of immunogenic tumor antigens to appropriate antigen-presenting cells, resulting in the production of an effective and long-lasting anti-tumor immune response. Studies have shown that dendritic cells (DC), an antigen-presenting cell type, are effective in anti-tumor immune responses. Department plays a qualitative role 89753 200427698. See, for example, Zitvogel et al., J. and; λ MM · 1S1: 87-97 (1996); Choudhury et al., Muster Tour: 1133-42 (1997); and DiNicola Et al., Thin Yinghuo: # · Zhiying F | Fasheng: 265-73 (1998). DC stimulates individual CD4 + and CD8 + T cells to differentiate into T helper cells (Th) and cytotoxic T lymphocytes (CTL) ). DC can show high content Type I and Type II major tissue adaptation complex (MHC) antigens, costimulatory molecules, adhesion molecules, and secretion of high levels of IL-12, an effective cytokine in CTL differentiation and activation. See, for example, Banchereau et al. Human, Nature 392: 245-52 (1998); Banchereau et al .: 767-811 (2000). Because the antitumor response mediated by CTL is thought to produce long-term protection against tumor regrowth, DC seems to be The selected antigen presents cells for use in tumor immunotherapy. Although tumor vaccines clearly confer long-term protection against tumor metastatic neoplasms, and even subsequent tumor attacks, the clinical application of this knowledge is well established Difficult. See, for example, Fong et al., J Mus IE .: 245-73 (2000). First, it has proven difficult to reliably expand functional DCs in in vitro expansion proposals. Because this immunity must be MHC-limited Therefore, any in vitro DC used in the immunotherapy strategy must be the DC of the patient being treated. Second, the reproducible activation of DC in vivo has not yet been achieved. Third, there is no obvious The proposal has been established to allow activation of the desired DC population and antigen loading, meaning to be able to shed anti-tumor responders. In summary, activated DCs are selectively located at the expansion of tumor sites that are currently immunogenic to tumor antigens. This is a problem that remains unresolved. Therefore, although it is clear that immune molecules, such as tumor-specific antibodies and vaccines that induce immune responses, can affect tumor growth, it allows simultaneous reduction of tumor growth 89753 200427698 and produces a protective immune response A unified approach is still lacking. SUMMARY OF THE INVENTION Provided herein is a chimeric protein that allows simultaneous eradication of tumor cells and stimulates an effective anti-tumor immune response. To be clear, this age-bound protein contains at least two ingredients. The first component is the Flt3 ligand (FL) or its biologically active fragment. FL is an effective chemotropic molecule and activator of DC and other antitumor effectors such as NK cells. The second component is a tumor killer that causes cell death. Such agents can be ligands or tumor-specific antibodies, which directly cause cell decay, meaning direct initiation via a cellular withering response (such as Fas ligands), or tumor-specific antibodies, which indirectly mediate cell death. , ≫ Means that the relevant cells withered by cytokine deprivation (such as anti-EGFR antibodies). Therefore, this chimeric protein can reduce tumor burden by directly dying the cells of tumor cells, while also targeting and activating DC and other antitumor effectors, such as NK cells, to infiltrate tumor tissues. Thus, tumor antigens released by dying tumor cells can be processed and presented by FL-activated DC ', which then effectively acts as antigen-presenting cells for use in a specific anti-tumor immune response. Therefore, 'this chimeric protein simultaneously achieves direct and indirect tumor cell removal, and at the same time induces an effective active immune response against tumor cells, which will prevent recurrence of tumor growth. In one aspect, the invention is directed to a chimeric protein comprising a Flt3 ligand or a biologically active fragment thereof, and a proteinaceous or peptidic tumoricidal agent. In another aspect, the present invention is directed to an isolated nucleic acid that encodes a chimeric protein. The synthetic protein comprises a Flt3 ligand or a biologically active fragment thereof, 89753 200427698, and a proteinaceous or peptide-based tumoricidal agent. Recombinant cells comprising the nucleic acid, and methods for producing the chimeric protein using the nucleic acid are also provided. In yet another aspect, the present invention is directed to a pharmaceutical composition comprising an effective amount of a fusion protein comprising an F! T3 ligand and a proteinaceous or peptide-based tumoricidal agent, and a pharmaceutically acceptable carrier. Or excipients. In some specific embodiments of the present invention, the amino acid sequence of the chimeric protein, and the nucleotide sequence that encodes the chimeric protein include Figures 16-18, 20-22, 27-29, 35-37 And the order shown in 39-42. A plasmid containing these nucleotide sequences is deposited in _ and has a receiving number of _. In a further aspect, the present invention is directed to a combination comprising: a) an effective chimeric protein comprising a Flt3 ligand and a proteinaceous or peptide-based tumor killer; and b) an effective amount of anti-neoplastic tumor Agent. In yet another aspect, the present invention is directed to a method for treating neoplasms in a mammal, the method comprising administering to a mammal in need or desire such treatment an effective amount of a combination of the above-mentioned combinations. In another aspect, the present invention is directed to a kit comprising an effective amount of a chimeric protein comprising a Flt3 ligand and a proteinaceous or peptidic tumorigenic agent, and an indicator device for administering the chimeric protein. In one aspect, the present invention is directed to a method for treating neoplasms in mammals, which method comprises administering to a mammal in need or want such treatment an effective amount of a tumor comprising a Flt3 ligand and a proteinaceous or peptidic tumor Chimeric protein. In another aspect, the present invention is directed to a method for causing the decay of caspase-3 intermediary cells in a cell, which method comprises administering a cell that is effective or in need of such defamation 89753 -10- 200427698 The amount comprises a chimeric protein of a Flt3 ligand and a proteinaceous or peptidic tumoricidal agent. In yet another aspect, the invention describes a vaccine comprising an effective amount of a chimeric protein comprising a Flt3 ligand and a proteinaceous or peptidic tumorigenic agent, and an immune response enhancer. In another aspect, the present invention is directed to a method of defamating an anti-neoplastic immune response in a mammal, which method comprises administering to a mammal that requires or wants such defamation an effective amount of a vaccine disclosed herein. In yet another aspect, the present invention is directed to a method for generating tumor-specific lymphocytes, which method comprises administering to a mammal an effective amount of a chimeric protein comprising a ligand and a proteinaceous or peptide-based tumoricidal agent, in order to A tumor-specific lymphocyte is produced, and the tumor-specific lymphocyte that has been produced is recovered from the mammal. DETAILED DESCRIPTION OF THE INVENTION For the sake of clarity and not limitation, the detailed description of the present invention is divided into the following subsections. A. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. All patents, applications, published applications and other publications cited herein are hereby incorporated by reference in their entirety. If μ is mentioned in this paragraph, it is contrary to or not related to the meanings of the patents, applications, published applications, and other communiques, which are incorporated herein for reference. The proposed definitions take precedence over the definitions of 89753 -11-200427698, which are hereby incorporated by reference. As used herein, "π" or "a" means "'at least one (one)" or "one or more (multiple)". As used herein, "nucleic acid π" refers to deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA) in any form, including especially single-stranded, double-helixed, triple-helixed, linear and circular forms. It also includes multinucleated Nucleotides, oligonucleotides, chimeras of nucleic acids, and the like. The nucleic acids described herein can be composed of conventional deoxyribonucleotides and ribonucleotides, and are based on the bases adenosine and cytosine , Guanine, thymidine, and uracil nucleoside, or may be composed of analogs or derivatives of these bases. In addition, various other oligonuclear acid derivatives with non-conventional phosphodiester main darts, also Included in this article are, for example, phosphate triesters, polynuclear peptides (PNAs), methyl phosphonates, phosphorothioates, polynucleic acid primers, blocked nucleic acids (LNA), and the like. 'Composition' f refers to any mixture of two or more products or compounds. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof. "Π combinations as used herein" Means between two or more Any combination of these projects. Β. Chimeric protein comprising a Flt3 ligand and a tumoricidal agent, and a nucleic acid encoded by the same. On the one hand, the present invention is directed to a post-protein, and the post-protein comprises a Flt3 ligand or a biologically active fragment thereof, and a protein. Sexual or peptidyl tumorcide. This chimeric protein is preferably an isolated protein, meaning that it is not bound to other proteins, polypeptides or other molecules. In some embodiments, the embedded protein 89753-12-200427698 is a purified product of a recombinant host cell culture or a purified extract. Any suitable Flt3 ligand can be used in the compositions and methods provided herein. The term '' Flt3 ligand ', as used herein, refers to a type of polypeptide that binds and causes a message by the Flt3 receptor found in the primitive particle cells. It is also intended that the Flt3 ligand or a biologically active fragment thereof may comprise conservative amino acid substitutions that do not substantially alter its activity. Appropriate conservative substitutions of amino acids are known to those skilled in the art and can be made in general without altering the biological activity of the formed molecules. Those skilled in the art will understand that, in general, a single amino acid substitution in a non-essential region of a polypeptide will not substantially alter biological activity. See, eg, Watson et al., Molecular Biology of Genes, 4th edition, 1987, Benjamin / Cummings Publishing Company, p. 224. Such example substitutions are preferably made according to those proposed in Table 1 below: Table 1 Conservative substitutions of the original residues Ala (A) Gly; Ser Arg (R) Lys Asn (N) Gin; His Cys (C) Ser Gin (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gin lie (I) Leu; Val Leu (L) lie; Val Lys (K) Arg; Gin; Glu Met (M) ) Leu; Tyr; lie -13- 89753 200427698

Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr⑺ Trp; Phe Val (V) lie; Leu Other substitutions are also allowed, and can be replaced empirically or based on known conservative substitutions Decide

The Ht3 ligand is a type I transmembrane protein, which can be released as a soluble homodimer protein. See, for example, Lyman et al., Adaptation, Handbook of Cytokines (edited by Thomson et al., 4th edition (2003)). In a specific embodiment, the Flt3 ligand or a biological or active fragment thereof is a soluble Flt3 ligand. In a specific embodiment of the betel composition and method mentioned herein, the Flt3 ligand or its biologically active fragment is a mammalian Flt3-ligand, and more preferably a human Flt3-ligand. The human Flt3 ligand is 72% identical to mouse proteins under the amino acid level and preserves many characteristics of mouse proteins, including glycosylation sites, key cysteine residues, and splice junctions. Suitable Flt3 ligand proteins are included in Lyman et al., Ce //, 21 · 1157-67 (1993), Hannum et al., 2M: 364-67 (1996); US Patent 5,843,423; US Patent Application Serial Number: 200030113341 And those disclosed in 20030148516; and Gene Bank acceptance numbers NM001459, U2 9874, U03858 and U04806. This Rt3 ligand receptor, Flt3, is a member of the class III receptor tyrosine kinase (RTKIII) receptor family. In normal cells, the Flt3 line is expressed in immature hematopoietic cells, typically CD34 + cells, placenta, gonads, and brain. See, for example, Rosnet et al. S2: 1110-19 (1993); Small et al. ); And Rosnet et al., Shamelessly ID: 238-48 (1996). Ht3 is also highly expressed in hematological malignancies, including acute myeloid leukemia, B-precursor acute lymphoblastic leukemia, spinal dysplasia leukemia, T-cell acute lymphoblastic leukemia, and chronic myelogenous leukemia. The Flt3 receptor is stimulated by its ligand and activates the signal transduction pathway, which includes STAT5, phospholipid inositol 3'-kinase, PLC r, MAPK, SHC, SHP2, and SHIP. See, for example, Gilliand et al., CWr. // emato / · 殳: 274-81 (2002). Both cell membrane binding and soluble FL bind and activate the Flt3 receptor. In a specific embodiment, the Flt3 ligand or a biologically active fragment thereof stimulates the proliferation of blood stem or primitive particle cells. In a specific embodiment, the Flt3 ligand or a biologically active fragment thereof can stimulate the proliferation of cells selected from the group consisting of myeloid precursor cells, monocytes, macrophages, B-cells, trees Cells (DC) and natural killer (NK) cells. The Flt3 ligand is mainly expressed in the bone marrow environment by hematopoietic cells and other cells, including fibroblasts and B, T, and myeloid cell precursors. The Flt3 ligand is a growth factor for CD34 + primordial granulocytes, and will stimulate the growth and differentiation of dendritic cells and NK cells. For example, one study has shown that Flt3 has significant antitumor activity via NK cell activation mediators. Peron et al., Mi 6164-70 (1998).

The Flt3 ligand also promotes the maturation of DCs, making DCs more effective as antigen-presenting cells for tumor antigens. See, e.g., Fong et al., TTzer 9. (12): 1127-38 (2002). More importantly, when induced by the Flt3 ligand, mature DCs are released from the bone marrow to peripheral tissues, thus increasing the number of antigen-presenting cells that can be used to stimulate the immune response. However, the proliferative effect is effectively elicited by the Flt3 ligand 89753 -15- 200427698, which typically requires the presence of other hematopoietic growth factors and mesotheliocytes. Any biological fragment of FL can be used in the compositions and methods of the invention. The term "biological activity" as used herein refers to a derivative or fragment of FL that still substantially retains its function as a Flt3 stimulator. Typically, the Flt3 coordination system binds Flt3 to cells and stimulates Or multiple message transduction pathways, and cause cellular responses, such as proliferation. Under normal circumstances, the derivative or fragment retains at least 50% of its Flt3 stimulating activity. The derivative or fragment preferably retains at least 60% of its Flt3 stimulating activity. %, 70%, 80%, 90%, 95%, 99%, and 100%. Flt3 stimulating activity can be measured by any suitable method, including but not limited to determining the activation of signaling molecules, such as STAT5, PLC r, Or assess viability in vitro in Flt3-dependent cell lines. For example, the BAF / B03 cell line lacks the flt3 receptor and is IL-3 dependent. However, the BAF / B03 cell line is infected with Flt3 metastasis, making It is responsive to the proliferation induced by the Flt3 ligand. See Hatakeyama et al., Ce //, Zhan: 837-45 (1989). In a specific embodiment, the Flt3 ligand or a biologically active fragment thereof, Among chimeric proteins, Sequence identification code: the amino acid sequence of 2. In a specific embodiment, the Flt3 coordination system is bound to an antibody that specifically binds to the amino acid sequence proposed in the sequence identification code ... 2, and The Flt3 ligand system essentially retains its biological activity. Any appropriate Flt3 ligand-specific antibody can be used. In another embodiment, the Flt3 ligand contains an amino acid sequence, which is at least 80% The same as the sequence identification code: 2 amino acids 28 to 128. In yet another specific embodiment, the Flt3 ligand comprises 89753 -16- 200427698 an amino acid sequence selected from the sequence identification code. The amino acid residue 28_ad 'of 2 and the sequence identification code: 28_i82 of the amino acid residue of 2. In one particular embodiment, the rigid ligand includes the amino acid Mm of the sequence identification code: 2. In another specific embodiment, the Flt3 ligand contains at least one amino acid residue, and the Flt3 ligand has 1 to 40% identity to the amino acid sequence proposed in the sequence recognition ㉟: 2: "Sex" where the percentage of homosexuality is proposed for the sequence identifier: 2 The amino acid sequence of the same size amino acid sequence is determined sequentially, and the Flt3 coordination system substantially retains its biological activity. Any tumor killer or biologically active fragment thereof can be used in the methods and compositions provided herein As used herein, the term "tumor killer" refers to X kinds of agents that cause tumor cell death. The tumor killer is preferably proteinaceous or peptide-based. Cell death can be cell death, necrosis, etc. In a specific embodiment, the cell death is caused by the withering of the cells. The withering can be directly caused by a ligand that causes the withering of the signaling pathway, such as the Fas ligand, or indirectly, such as growth factors. deprivation. The term "cell dying" as used herein refers to the death of stylized cells of tumor cells, which will eventually cause condensation of chromatin and fragmentation of DNA. Any suitable method can be used to assess cell decay, including but not limited to flow cell count analysis, such as TUNEL analysis, agarose gel analysis, and caspase 3 activation. In another embodiment, the tumor killer of the chimeric protein is a naturally occurring antitumor agent. Such agents include receptor ligands ' which cause stasis or cell death in tumor cells. Examples of naturally occurring molecules such as ligands that cause cell death include alpha, Fas ^ D9 custom ligands, TRAIL, lymphotoxin (LT), TWEAK and TNF ligands 89753 -17- 200427698 others in the supergroup member. In a specific embodiment, the tumoricidal agent is selected from the group consisting of a Fas ligand, TNF, TRAIL, or a biologically active extracellular functional site thereof. See, for example, in another embodiment, the biologically active fragment of a tumoricidal agent retains at least 50% of its cellular withering activity. The derivative or fragment preferably retains at least 60%, 70%, 80%, 90%, 95%, 99%, and 100% of its cellular withering activity. In another specific embodiment, the tumor killer of the chimeric protein is an antibody that inhibits tumor proliferation, and in some cases causes cell death. Examples of such antibodies include growth factor receptors. For example, the epidermal growth factor receptor (EGFR) subgroup is composed of EGFR, HER2, HER3, and HER4, and its ideology is a transmembrane protein with tyrosine kinase activity. These proteins are highly expressed in many malignant conditions, including prostate cancer, colon cancer, breast cancer, pancreatic cancer, kidney cancer, ovarian cancer, and lung cancer. Specific anti-EGFR or anti-HER2 mAbs can block the binding of EGFR or HER2 to their ligands, and successively block the proliferation of tumors and send a signal pathway to inhibit tumor growth and directly or indirectly cause tumor cells; weekly. See, for example, C / z > z. Ca / zcer S · 1720-30 (2002); Brodowicz et al. 5r_ Qmcerii: 1764-70 (2001); Crombet-Ramos et al., / J. Cancer 101: 567 -75 (2002); Herbst et al., Expert Opin · Biol, Ther · L ·, Ί \ 9-32 (2QQ \). In yet another embodiment, the tumor killer of the chimeric protein is an antibody that binds to a tumor-specific or tumor-associated antigen that causes cell death. For example, p230 is a protein specifically expressed in human liver cancer, breast cancer, and melanoma cells. Its name is derived from the apparently specific 230KD band, which uses mAb SM5-1 and appears in Western blotting. See U.S. 89753-18-200427698 patent application serial number: 09 / 915,746. P230 is so specific that it can be used as a target gene in tumor immunotherapy. Cell death can be caused by binding P230 to its ligand or antibody. Some anti-SM5-1 antibody systems are described in Example 3. In a specific embodiment, the anti-system is an SM5-1 antibody, and the serial number of the co-pending application filed on November 26, 2003 (lawyer case directory number 54906-2000100; title: Linked to cancer) SM5-1 specific antibody and its use), which is incorporated by reference in its entirety. The humanized anti-SM5-1 antibody described herein is referred to as ReSM5-l in this co-pending application. In a specific embodiment, the tumoricidal agent is an antibody or a biologically active fragment thereof. As used herein, the term “ff antibody” refers to a complete antibody, a Fab fragment, a Fab ′ fragment, an F (aV) 2 fragment, an Fv fragment, a diabody, a single chain antibody, and a multispecific antibody formed from an antibody fragment. Molecules retain substantially all of their desired biological activity. Antibodies that can be used in the methods and compositions can be produced in cell cultures, in phages, or in a variety of animals including, but not limited to, cows, rabbits, goats, mice, rats, rats, cheeks, Guinea pig, sheep, dog, cat, monkey, chimpanzee, macaque. Therefore, the anti-systems useful in this method are mammalian antibodies. Plutonium technology can be used to isolate the original antibody or produce a variant with altered specificity or antibody avidity characteristics. This technique is routine and known in the art. In a specific embodiment, the resistance system is made by recombination methods known in the art. For example, recombinant antibodies can be made by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to sequentially transfer the DNA expressing at least one VL and one 89753-19-200427698 VH region in a host cell. Examples of recombinant methods of antibody production and manufacturing include Delves, antibody manufacturing: basic techniques (Wiley, 1997); Shephard et al., Monoclonal antibodies (Oxford University Press, 2000); and Goding, monoclonal antibodies · Principles And Practice (University Press, 1993). Antibodies that can be used in this method can be modified by recombinant methods to increase the greater efficacy of the antibody in the desired function of the vehicle. It is also intended to cover that antibodies can be modified by substitution using recombinant methods. These substitutions are typically conservative substitutions. For example, at least one amino acid in the constant region of an antibody may be replaced with a different residue. See, for example, U.S. Patent 5,624,821, U.S. Patent 6,194,551, Application No. WO 9958572; and Angal et al., Mo /, 30: 105-08 (1993). Modifications on amino acids include deletion, addition, and substitution of amino acids. In some cases, such changes are performed to reduce unwanted activity, such as complement-dependent cytotoxicity. The antibody may be a humanized antibody. The term `` humanized antibody '' as used herein refers to an antibody in which the amino acid sequence in the non-antigen-binding region has been changed so that the antibody more closely resembles a human antibody, but still retains it Primitive antigen specificity. Typically, the variable region is a species, such as an old mouse, and the constant region is of human origin. The antibody may be a chimeric antibody. As used herein, the term `` chimeric antibody '' refers to an antibody in which the amino acid sequence has been altered so that the antibody contains sequences from more than one mammal, but still retains the original antigenic specificity The term "single-chain variable fragment (ScFv)" as used herein refers to a genetically engineered antibody that includes an immunoglobulin variable heavy chain (VH) and a light chain (VL). Sex peptides are linked together 0 89753 -20- 200427698 The antibodies of the methods and compositions of the present invention are preferably single strains. The term "π monoclonal antibody '" as used herein refers to a single antibody made from a single B cell. The antibody may be a human antibody. The term "human antibody" as used herein refers to an antibody in which substantially the entire sequence of light and heavy chain sequences, including the complementarity determining regions (CDRs), is derived from a human gene. In a specific embodiment, the 'human monoclonal antibody system uses trioma virus technology, human cell technology (see' e.g., Kozbor et al., ⑽2 ^ also less 4; 72 (1983), EBV transformation technology (see 'e.g., Cole Et al., Monoclonal Antibody and Cancer Therapy 77_96 (1985)) or using phage display (see, for example, 222/581: 581 (1991)). In a particular embodiment, the human anti-system Produced in transgenic mice. Techniques for making such partially-to-complete human antibodies are known in the art, and any such technique can be used. According to a particularly preferred embodiment, the complete human antibody sequence is Manufactured in transgenic mice designed to express human heavy and light chain antibody genes. An example of the preparation of transgenic mice that produce human antibodies can be found in application number WO 02/43478. Obtained from transgenic mice that produce the desired antibodies B cells can then be fused to produce hybridoma cell lines for continuous production of antibodies. See, for example, U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,5 45,806; and Jakobovits, j (iv · 31 · · 33_42 (1998); Green et al., J. Me < i · 188 · · 483-495 (1998). In a specific embodiment, the resistance provided herein The system inhibits the proliferation of target tumor cells. Compared to the cell proliferation in the absence of antibodies or the presence of non-binding antibodies, if the anti-system inhibits cell proliferation, the antibody is inhibitory to the proliferative line. Proliferation can be quantified using any appropriate method. Typical Above, the proliferative system 89753 -21-200427698 was measured in vitro by evaluating the incorporation of radiolabeled nucleotides into DNA (eg 3H-thymidine). In a specific embodiment, the proliferative system emits light through ATP Measured, for example, CellTiter-GloTM Luminescent Cell Viability Assay (Promega). Therefore, antibodies can be specific or targeted for any molecule that modulates cell viability or cell growth. In a specific embodiment, anti- The system is selected from the group consisting of anti-p230 antibodies, anti-CD20 antibodies, anti-Her2 antibodies, anti-Her3 antibodies, anti-Her4 antibodies, anti-EGFR antibodies or biologically active fragments thereof. Specific examples of such antibodies are included in In the example paragraphs below, and as disclosed, for example, in U.S. Patent Nos. 5,677,171; 6,399,061; 6,458,356; 6,455,043; and 5,705,157. Chimerics comprising a 'Flt3 ligand or a biologically active fragment thereof, and a tumor killer Proteins can be linked by any suitable link. For example, the Flt3 ligand can be linked to a tumoricidal agent via a peptidyl linker, a cleavable linker, or the like. In a particular embodiment, the linker peptide is (Gly4Ser) 3. The chimeric proteins of the compositions and methods herein may include Flt3 ligands and a tumoricidal agent linked in any order. In a specific embodiment, the Flt3 coordination system is located at the N-terminus of the chimeric protein. In another embodiment, the Flt3 coordination system is located at the C-terminus of the chimeric protein. The chimeric protein may further include a peptidyl fragment at its C-terminus, which includes a peptidyl tag. Any appropriate marking can be used. For example, this tag may be a FLAG, HA, HA1, c-Myc, 6-His, AU1, EE, T7, 4A6, ε, B, gE, and Tyl tags (see Table 2). This tag can be used for purification of chimeric proteins. 89753 -22- 200427698 Table 2. Exemplary epitope tagging system epitope peptide sequence recognition antibody reference FLAG AspTyrLysAspAspAspLys 11 4E11 Prickett1 HA TyrProTyrAspValProAspTyrAla 12 12Ca5 Xie2 HA1 CysGlnAspLeuProGlyAsnAspAs GluS-Gerase-Single-Gerase His HisHisHisHisHisHis 15 BAbCO * AU1 AspThrTyrArgTyrlle 16 BAbCO EE GluTyrMetProMetGlu 17 anti -EE Tolbert4 T7 AlaSerMetThrGlyGlyGlnGlnMet GlyArg 18 Invitrogen Chen5 Tseng6 4A6 ,, SerPheProGlnPheLysProGlnGluIle 19 4A6 Rudiger7 ε LysGlyPheSerTyrPheGlyGluAsp LeuMetPro 20 anti -PKC ε Olah8 B GlnTyrProAlaLeuThr 21 D11, F10 Wang9 gE GlnArgGlnTyrGlyAspValPheLys GlyAsp 22 3B3 Grose10 Tyl GluValHisThrAsnGlnAspProLeu Asp 23 BB2, TYG5 Bastin11 1. Prickett et al., 7 (6): 580-584 (1989) 2. Xie et al., Endocrinology, \ 39 (ΙΏ: 4563-4567 (1998) 3. Nagelkerke et al. People, Gal. 18: 2694-2698 (1997) 4. Tolbert and Lameh, //: 113-119 (1998) 5. Chen and Katz, 25 (1): 22-24 (1998) 6. Tseng and Verma, Gene, 169: 287-288 (1996) 7. Rudiger ψ A, BioTechniques, 23 (1): 96-97 (1997) 8. Olah et al. 221: 94-102 (1994) 9. Wang et al., Gene, 169m: 53-58 (1996) 10. Grose, US Patent 5,710,248] Λ · Bastin et al., Mol. Biochem. Parasitology, ZZ_: 235 -239 (1996) Invitrogen, Sigma,

Santa Cruz Biotech -23-89753/0 ^ 0 In a specific embodiment, 疋 sequence identification code: 10 sequel "Human shells are included in the sequence identification code: 2 no. 22., ,, /, sequence 硪 other code ^ 4 , Sequence identification code: 18, sequence n code. 22 or sequence identification code: u sequence extinguishes, so another, Su Ning only the lack of amino acid sequence. Acid: its — mutual: facial hair stitches, Disc protein; the protein contains Flt3 ligand or its biological poem, human 1_ or peptidic tumor killer. In the specific implementation of your system is encoded by an isolated nucleic acid, the nucleic acid is included in the cis identification code : 1, sequence identification code: 9 Shun An ~ S σ in Shun Yue

、 / 、 Sequence code: 13, Sequence ID: 17 Sequence ID: 21 or Shun, then the version · 飞 Baishi Pilot sequence 4 code · 25 nucleotide sequence. Chimeric proteins encoding isolated nucleic acid vectors are also intended to be included. · This vector can further include the cousin is ^ / 匕 〇 to show the withering order, and can be used to link to the nucleic acid encoding the Flt3 ligand, and the proteinaceous or peptide-based tumor killer Γ makes the Flt3 ligand Any suitable dna structure, such as the peak compound, the mysterious pg /, the biological live film & can be used in the present invention. Such structures include, but are not limited to, the receipt number deleted at the gene bank.

order. Further intended for use in the present invention are the DNA sequences and the proteins formed in U.S. Patent No. 5,843,423, and U.S. Patent Application Serial No. 20-113341 and Cells. Any suitable DNA construct that encodes a tumoricidal agent or a biologically active fragment thereof can be used in the compositions and methods herein. The order of the examples includes those disclosed in the example paragraph below. Any suitable carrier can be used. Examples of asexual propagation and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in, for example, Pouwds et al., Asexual Propagation Vectors · Laboratory Manual (Latest Version of Elsevier). 89753 -24- 200427698 Table fa contains operably linked to cis > Shanren π Xiangtian transcription or translation regulation nucleotide sequence "Protein DNA sequence, sugar, disease and disease main force. He Shengzi Mammalian, micro-biology, early on ... A is the sequence "Examples include transcription promoters 'longitudinal or enhancer, _ eight nuclear spiny spine addiction 3 丨 technology ☆ μ,' mouth position, and control of transcription, transduction The proper sequence of initiation and termination. When each Yamada 'sequence is functionally related to the sequence of the septic protein DNA, the nucleotide and glycan I sequence is "operably linked".

Therefore, the 4 promoter nuclear #acid sequence controls the bile A sequence for the chimeric protein coding, and the promoter nuclear ^ acid sequence is operably linked to the DNA sequence for the chimeric protein coding. The ability to replicate in the desired host cell: ' often conferred by a complex source ' and a selection gene by which the transformant is identified ' V may additionally be incorporated into a performance vector. In addition, a sequence encoding a suitable message peptide that does not naturally bind to a Flt-3 ligand or a tumoricidal agent can be incorporated into a performance vector. For example, the DNA sequence for the message peptide (secretion leader) can be fused within the framework to the sequence used for the coding of the chimeric protein, so that this sequence is first translated into a fusion protein containing the message peptide. A functional message peptide in the intended host cell will enhance the extracellular secretion of the chimeric polypeptide. The message peptide can split the apposition of the polypeptide when the chimeric polypeptide is secreted from the cell. Mammalian or insect host cell culture systems can also be used to express recombinant chimeric polypeptides. The baculovirus system used to produce heterologous proteins in insect cells was reviewed by Luckow and Summers in Lan # / Jiehengshi · 47 (1988). Established cell lines of mammalian origin can also be used. Examples of suitable mammalian host cell lines include the COS-7 cell line of monkey kidney cells (ATCCCRL 1651) (Gluzman et al., Ce //, 22: 175, 1981), L cells, C127 -25- 89753 200427698 cells, 3T3 cells (ATCC CCL 163), Chinese cheeky rat ovary (CHO) cells, HeLa cells and BHK (ATCC CRL 10) cell lines, and CV-1 / EBNA-1 cell lines, derived from the African green monkey kidney cell line CVI (ATCC CCL 70), as described by McMahan et al. J. 1Q: 2821, 1991), and the NSO cell line (Galfre et al., Methods Enzymol. 71 ·· 3-46 (1981)). The mammalian host cell expression vector's transcription and translation control sequence can be excised from the viral genome. Common promoter sequences and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 virus genome, such as SV40 origin, early and late promoters: enhancers, splices, and polyadenylation sites. Additional genetic building blocks can be provided by β to express structural gene sequences in mammalian host cells . Viral early and late promoter lines are particularly useful because both are readily available as fragments from the viral genome and may also contain replicated viral sources. See, for example, Fiers et al. 273: 113 (1978). Smaller or larger SV40 fragments can also be used, provided that they contain a sequence of approximately 250 bp, extending from the HindIII position, towards the Bgll position, and the SV40 virus source at the replicated position. An example expression vector for use in mammalian host cells can be constructed as disclosed by Okayama and Berg, Mo / _ & // Wan / 〇 /. 3: 280 (1983). A useful system for the stable expression of mammalian cDNA in breast epithelial cells of C127 mice can be constructed substantially as described in Cosman et al. (Shi / · / mmwzo / · 23: 935, 1986). A useful high performance vector PMLSVN1 / N4, described by Cosman et al., Nature 312 ... 768, 1984, has been registered as ATCC 39890. Other useful mammalian expression vectors are described in EP-A-0367566 and U.S. Patent Application 89753 -26- 200427698 No. 07 / 701,415, both of which are incorporated herein by reference. These vectors can be derived from retroviruses. Instead of the natural message sequence, a different message sequence can be added, such as the message sequence described in US Pat. No. 4,965,195 for IL-7; described in Cosman et al., 312 ... 768 (1984) for IL-2 The body's message sequence; the IL-4 message peptide described in EP 367,566; the type IIL-I receptor message peptide described in US Patent 4,968,607; and the type IIIL-1 receptor message peptide described in EP 460,846. A method of making a chimeric protein is also intended to be encompassed by growing a recombinant cell containing a nucleic acid encoded by a chimeric protein, the chimeric protein comprising a Flt3 ligand or a biologically active fragment thereof and a tumoricidal agent, such that Several of the encoded chimeric proteins were expressed by the cells, and the expressed chimeric proteins were recovered. In a specific embodiment, the method further comprises isolating and / or purifying the recovered chimeric protein. The products of this method are further intended to be included. This chimeric protein can be purified to substantial homogeneity, and when analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), it is shown by a single protein mass spectrum band. For example, when an expression system that secretes recombinant proteins is used, the culture medium can be concentrated first and a commercially available protein concentration filter such as an Amicon or Millipore Pellicon ultrafiltration unit can be used. After this concentration step, the concentrate can be applied to a purification matrix, such as a gel filtration medium. Alternatively, an anion exchange resin such as a matrix or substrate having a pendant diethylaminoethyl (DEAE) group can be used. The matrix may be acrylamide, agarose, dextran, cellulose or other types commonly used for protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include a variety of insoluble matrices, including sulfopropyl or carboxymethyl. The sulfopropyl group is preferably 89753-27-200427698. Finally, one or more reverse-phase high-performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium (such as silicone with pendant methyl or other aliphatic groups) can be used for further purification合 protein. Some or all of the foregoing purification steps, in various combinations, are conventional and can be employed to provide a substantially homogeneous recombinant protein. An affinity column containing the ligand-binding functional site of the flt3 receptor can be used to purify the expressed affinity polypeptide with affinity. The postpeptide can be removed from the affinity column using conventional techniques, such as in a high-salt dissolution buffer and then dialyzed into a lower-salt buffer for use, or by changing the pH or other ingredients, depending on the used Depending on the affinity matrix. Alternatively, the affinity column may contain an antibody that will bind FL. A transformed yeast host cell can also be used to express the chimeric protein as a secreted polypeptide to simplify purification. Secreted recombinant polypeptides obtained from yeast host cell fermentation can be purified by methods similar to those disclosed by Urdal et al. (J. Chromatog. 296: 171, 1984). Recombinant cells comprising nucleic acids are also provided. In a specific embodiment, the cell is a eukaryotic cell. In a particular embodiment, the cells are CHO, COS or NSO cells. The chimeric protein and the nucleic acid encoding the chimeric protein can be prepared by any appropriate method, such as chemical synthesis, recombinant production, or a combination thereof. See, for example, Ausubel et al., A current proposal in molecular biology, John Wiley & Sons, Inc. (2.0), and Sambrook et al., Molecular Asexual Reproduction: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). In one example method, a nucleic acid that encodes a chimeric protein is prepared using regression PCR technology, such as disclosed in 89753-28-200427698 in P__u et al., Hulun ~ guan: Ka Si (i " 2). A pharmaceutical composition is intended to be included, which comprises a chimeric protein comprising a ligand or a biologically active fragment thereof and a proteinaceous or skin-based tumor killer: and a pharmaceutically acceptable carrier or excipient. For use, the Benfarm H composition 'can therefore be formulated in a customary manner using one or more physiologically acceptable carriers, including formulations and adjuvants, which facilitate the processing of the active compounds into pharmaceuticals On the preparation. These medical musical compositions can be manufactured in a manner known per se, for example, by conventional mixing, dissolving, M, sugar-coated tablet manufacturing, grinding, emulsifying, coating, capturing or drying. The appropriate formulation depends on the chosen route of administration. t When the drug is administered in liquid form, add a liquid carrier, such as water, petroleum, oils of animal or plant origin, such as Huafeng Oil Depot β-Metaplasty / from rough oil, soybean oil or sesame oil, or synthetic oils. The liquid form 4 of the pharmaceutical composition may further contain physiological salt water, dextrose or other sugar solutions' or glycols, such as ethylene glycol, propyl alcohol, or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains ⑽ to 90% by weight of the protein of the present invention, and preferably about 1 to about 28% of the protein of the present invention. In another aspect, the provided in this article is a combination of:) Effectively < a chimeric protein comprising a Flt3 ligand and a proteinaceous or peptide-based tumoricidal agent; and b) an effective amount of anti- Tumor agent. In a specific embodiment, the neoplastic agent is an agent that inhibits the growth of melanoma, breast cancer or hepatocellular carcinoma. Growth inhibition can occur by causing arrest or cell death in tumor cells. Exemplary anti-neoplastic agents include cytokines, ligands, anti-thorium, radionuclides, and chemotherapeutic agents. Such agents include mesothelin II _ 89753 -29- 200427698 2), interferon (IFN) TNF; photosensitizers, including xylalanin tetrasulfonic acid aluminum (III), hemocyanin and xylylene Blue pigments; radionuclides such as iodine-131 (1311), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), chromium-99m (99mTc), baht-186 (186Re) and Streptomycin-188 (1 8 8Re); chemotherapeutic agents, such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunorubicin, neocarcinostatin and carbochlorin Amine platinum; bacteria, plants and other toxins, such as diphtheria toxin, Pseudomonas exotoxin A, staphylococcal enterotoxin A, acacia-A toxin, ricin toxin A Natural ricin toxin A), TGF-α toxin, cytotoxins and lilies (phytotoxins) obtained from Chinese cobra (air snake); ribosomal inactivating proteins from plants, bacteria and fungi, such as Aspergillus constrictor (Ribosome-inactivating protein made from locally discarded tadpoles), saporin (ribosome-inactivating protein from E. stubble) and RNase; tyramine Kinase inhibitors; ly207702 (purine nuclear difluoride); microlipids containing antitumor agents (such as antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.); and other antibodies or antibody fragments , Such as F (ab). In one aspect, a kit is provided to perform the methods disclosed herein. Such kits contain in one or more containers an effective amount of a chimeric protein comprising a Flt3 ligand and a proteinaceous or peptidic tumorigenic agent, in a pharmaceutically acceptable form, and for administration of the post-protein The indicating device is intended to be included. In a specific embodiment, the kit further comprises an effective amount of an anti-neoplastic agent as disclosed above. The preferred medicinal form is a combination of sterile saline, dextrose solution or buffer solution or other pharmaceutically acceptable sterile fluid. Alternatively, the composition can be lyophilized or container-dried; in this case, the kit further comprises a pharmaceutically acceptable solution in the container, preferably sterile 89753-30-200427698 'to reconstitute the compound, A solution for the purpose of shooting with ,,,, and y / wang. For example, the medicine solution is saline and right-hand shake, 々, 、,,,, /, 疋 sugar> cereal solution. In another embodiment, the kit of the present invention / ~, 贯, a # 牛 v includes a needle or a syringe, and is preferably packaged in the form of a fungus for injection, injection, and & and / or a packaged alcohol pad. As appropriate, add instructions regarding the administration of the composition by exhaustion, _, or by the patient. As used in this article, the term "treatment of Gypsophila f" "Efficacy or effective amount" means that M. infestation is effective when administered alone or in combination with another therapeutic agent to cells, tissues or diseases. An amount that prevents or improves a tumor or tumor-associated disease state or tumor progression. A therapeutically effective dose is a compound that is sufficient to cause improvement in symptoms such as treatment, epidemic, prevention or amelioration of associated medical symptoms, and to increase the rate of treatment, healing, prevention or amelioration of such symptoms. When applied to an individual active ingredient administered separately, a therapeutically effective dose refers to that individual ingredient. When applied to a combination, a therapeutically effective dose refers to the combined amount of active ingredients that will cause a therapeutic effect, whether combined, continuously or simultaneously. c. A method using a chimeric protein comprising a Flt3 ligand and a tumoricidal agent. On the other hand, provided herein is a method that causes the dying of the intercellular cells of the aspartase-3 in the cell. This method This includes administering an effective amount of a chimeric protein comprising a Flt3 ligand and a proteinaceous or moonyl tumor killer to a cell in need or desire for such slander. In a specific embodiment, the cell is a mammalian cell. In a particular embodiment, the cells are mammalian neoplasm cells. In a specific embodiment, the cell line is contained in a mammal. The activation of caspase plays an important role in the withering changes of cells in the cell 89753 -31-200427698. See, for example, Budihardjo et al., D / w ·. Price 15: 269-90 (1999). The caspase family is a family of cysteine proteases, with a high degree of specificity, which means that for the division after aspartic acid and the recognition sequence of at least four amino acids, the absolute position of the division is the N-terminus. Requirements. See, for example, Grutter, Cwrr. Qp. So 10: 649-55 (2000). Caspase 3, also known as CPP32, YAMA, and apapain, is specific to the division of WEHD. It is used to split various substrates, such as lamins, PARP, DFF, and other substrates, and it is the downstream or actuator caspase. Existing in the cell as an inactive zymogen, caspase 3 is activated after being cleaved by caspase 9 and Apaf-1 (upstream caspase). This upstream caspase It is activated after the withering of extracellular cells due to binding of a ligand such as Fas ligand, TNF or TRAIL to its appropriate receptor. Caspase activation can be easily measured using methods known in the art. For examples, see, for example, Cell Withering: A Practical Approach (Studzinski, 1999). Caspase 3 is a member of the family of cysteine proteases that are important in cell death or programmed cell death. See, for example, Cmtter, CWr. Qpin. ISVrwcii / ra / Inst. 6 > IQ: 649-55 (2000); Budihardjo et al. Caspase 3 is present in the cell as a zymogen and is activated by proteolysis, typically by an "initiator" caspase, such as caspase-8, -9 or 10 . This active caspase-3 then splits other proteins, mainly involving the DNA repair process or the structural components of the cytoskeleton or nuclear skeleton, which contains the position of the recognition sequence DEVD after aspartic acid. Detection of caspase 3 activation is routine and is known in 89753 -32- 200427698 skills. For example, U.S. patents: 6,342,611; 6,391,575, 6,335,429 and U.S. patents, U.S. profit-seeking application serial number 20030186214. Therefore, the detection of caspase 3 activation can be performed by any method, which can be used in this article. The donor & Method for treating neoplasms with chimeric proteins of protein or its biologically active fragment and protein, insecticidal or peptide-based tumoricidal agents, this female, male and female 40,000 to include mammals in need or want such treatment Administer an effective amount of a chimeric protein as described in paragraph B. In a specific embodiment φ, I, birth, upper, middle 'tumors are melanoma, breast cancer or hepatocellular carcinoma. The method provided in this article is a method for manufacturing tumor-specific lymphocytes. The method includes administering to a mammal an effective amount of a protein comprising Flt3 ligand and protein bismuth— ,, or terpeptide. A chimeric protein of a tumor agent is used to produce, synthesize, lymphocytes, and recover the tumor-specific lymphocytes that have been produced from the mammal.一 ^ 里 之 # The combination of the chimeric protein disclosed in Youyou B and the anti-neoplastic agent disclosed in paragraph B 'to treat neoplastic tumors in mammals in which such treatment is needed or desired, is also intended to be covered Included. Any patient can be treated with the methods and compositions provided herein. Such patients are mammals, and Yu She, Che and Jia are awarded. In a particular embodiment, the disease is cancerous. The veterinary uses of the disclosed methods and compositions are also intended to be covered. ~: Patients treated by Ben Wan, including patients with adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma, or four cancer tumors. Tumors can be adrenal glands, bladder, bones, bone marrow, brain, breasts, uterine head, gallbladder, palate '89753 -33- 200427698 same intestine, heart, kidney, liver, lung, muscle, ovary, pancreas, thyroid Cancer of the parathyroid glands, penis, prostate, salivary glands, skin, spleen, testes, thymus, thyroid and uterus. Such tumors include, but are not limited to, neoplastic tumors of the middle frame nervous system: glioblastoma multiforme, astrocytoma, oligodendritic tumors, ependymal and choroid plexus tumors, pineal gland tumors, and neuronal tumors. , Neural tube blastoma, schwannomas, meningioma, meningeal flesh C 'eye neoplasms · basal cell carcinoma, squamous cell carcinoma, melanoma, rhabdomyosarcoma, retinoblastoma; neoplasm of endocrine glands : Pituitary neoplasms, thyroid neoplasms, adrenal cortex neoplasms, neuroendocrine system neoplasms, gastrointestinal and endocrine system neoplasms, gonadal neoplasms; head and neck neoplasms: head and neck Cancer, oral, pharyngeal, larynx, odontogenic tumors: breast neoplasms: large cell lung cancer, small cell lung cancer, non-small cell lung cancer chest. Stomach tumor, malignant mesothelioma, thymoma, primary germ cell tumor of the chest; neoplasm of the digestive tract · neoplasm of the esophagus, neoplasm of the stomach, neoplasm of the liver, neoplasm of the gallbladder, exocrine Pancreatic neoplasms, small intestine, appendix, and abdomen M < neoplasms, adenocarcinomas of the colon and rectum, neoplasms of the anus 丨 genitourinary tract < neoplasms: renal cell carcinoma, neoplasm of renal pelvis and ureter, bladder neoplasm Tumors, neoplasms of the urethra, neoplasms of the prostate, neoplasms of the penis, adenomas of the female genital organs: adenomas of the vulva and vagina, =: tumors, adenocarcinoma of the uterus, ovarian cancer, gynecological sarcoma; breast Neoplasms of the skin; neoplasms of the skin: & base cell carcinoma, squamous carcinoma, dermal fibrosarcoma, Merkei cell tumor; malignant melanoma; bone and soft tissue neoplasms: osteosarcoma, malignant fibrous histiocytoma , Chondrosarcoma, Ewing's sarcoma, proneural ectoderm tumor, angiosarcoma; neoplasms of the hematopoietic system: spinal cord development 89753 -34- 200427698 adverse symptoms cluster, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia HTLV- 1 with T-cell leukemia / lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin's disease, non-Hodgkm's lymphoma, mast cell leukemia; children's praise tumor Acute lymphoblastic leukemia, acute myeloid leukemia, neuroblastoma, bone tumors, rhabdomyosarcoma, lymphoma, kidney and liver tumors. As used herein, "inhibition" or "treatment" or "treatment" includes delaying the development of symptoms associated with uncontrolled tumor growth and / or reducing the severity of such symptoms that are about to or are expected to develop. These terms further include the improvement of existing uncontrolled or undesired or Teng Yu j | — Yue Wang and M ‘growth-related symptoms, prevention of other symptoms, and improvement or prevention of metabolic causes subordinate to the symptoms. ^, These terms indicate that beneficial results have been conferred on mammals with a malignant condition or the possibility of developing such a disease or symptoms.

In carrying out the methods of treatment or use provided herein, a therapeutically effective amount of a chimeric protein provided herein is administered to a mammal having symptoms to be treated. Suihe protein can be administered according to the method herein, whether it is used alone or in combination with other therapies, such as the use of other immune-enhancing factors (such as cytokines), chemotherapeutic agents, anti-neoplastic agents and the like. When a disc—or multiple bioactive agents—are co-administered, the post-proteins provided herein can be administered simultaneously or sequentially with the bioactive agent. In the case of successive administrations', the physician in charge will determine the appropriate order of administration of the protein of the invention and the combined use of the bioactive agent. Toxicity and therapeutic efficacy of such therapeutics can be determined in cell cultures or experimental animals using standard medical procedures, such as LD5. (Lethal dose to individual group) and called (in the individual group 89753 -35- 200427698 million red dose). The dose ratio between toxicity and therapeutic effect is 4 and the number, and it can be expressed as the ratio between LD5q_5g. Shows high therapeutic index (chimeric protein line is better. Data obtained from cell culture assays and animal death studies) 'τ is used to formulate a dose of Fanyuan for human identification. The dose of this compound is preferably Located within the range of circulating concentration, including ED5 with little or no toxicity. This dosage can be changed within this range, depending on the dosage form used and the route of administration used. The correct formula, route of administration and dose can be individualized The choice is based on the symptoms of the patient. See, for example, et al., Pharmacological Basics of Medicine (Latest Version). The interval and interval can be individually adjusted to provide the plasma content of the active substance, which is sufficient to maintain the desired / Oral effect or minimum effective concentration (MEC). MEC will vary for each compound but can be estimated from in vitro data; for example, using the tests described herein, a concentration of 50-90% of tumor proliferation must be achieved. Any appropriate route of administration Both can be used. The mode of administration is not particularly important. Dosage forms include tablets # 1, lozenges, sachets, dispersions, suspensions, solutions, capsules, stickers, etc. See also For example, Remington's Medical Sciences, Mack Publishing Company (Easton, PA), the latest version. In one specific embodiment, the mode of administration is intravenous bolus. The designated physician will usually determine the dosage of the antibody provided herein. Expected Dosage will vary based on individual patient < age, weight, and response. Formulation and administration of this method of protein 4 technology can be found in Remington's Medical Sciences, Mack Publishing Company (Easton, PA), latest edition. What is intended to cover The formulation and administration considerations of the chimeric proteins provided herein are similar to antibodies. Appropriate routes of administration may include, for example, oral, rectal, transmucosal or enteral administration; parenteral delivery, including 89753 -36- 200427698 intramuscular, Subcutaneous and intraosseous injections, as well as intrathecal, direct indoor, intravenous, intraperitoneal, intranasal or intraocular injections. The compounds used in pharmaceutical compositions or the methods of the present invention can be used Various conventional methods, such as oral ingestion, inhalation, topical application or skin, subcutaneous, intraperitoneal, parenteral, intraarterial or intravenous injection. Intravenous administration is better for patients. '' Alternatively, we can administer the post-proteins locally rather than systematically, for example, by direct injection of antibodies into tumors, often in storage or continuous release formulations. Furthermore, 'my people Synaptic protein f can be administered in the target drug delivery system 'for example in microlipids that have been covered with tissue-specific antibodies such as tumors as targets. Microlipids will be targeted on tumor tissues and will be selectively Absorption. When a therapeutically effective amount of the chimeric protein of the method herein is administered by intravenous, dermal or subcutaneous injection, the protein provided herein will be pyrogen-free, unacceptably acceptable 、、 治 ”/ 合 '夜 form. The preparation of this parenterally acceptable protein solution, such as Η, ^ ^ ω, ρη osmosis, foot comfort, etc., is within the technical scope of this page . For intravenous, cutaneous or subcutaneous injection is preferred ::: In addition to the protein of the present invention, it should contain an isotonic vehicle, 1 gasification training spray, Ringer's spray, dextrose injection, right Rotary M rat sodium > King ejaculate, lactic acidified, P 4 # ^ Wood Straw > King cumshot or such a technique 2 (other vehicles. The pharmaceutical composition of the present invention may also contain diazepam Agents, preservatives, buffers, anti-glucosamine 1 1 anti-lice agents or other additives known to those skilled in the art. For transmucosal administration, M " suitable permeants for the barrier to be penetrated ' This formula towel is generally known. This kind of permeabilizing agent is 89753 -37- 200427698 in the art. For administration by inhalation, a chimeric protein for use according to this method may be suitable. It is delivered as an aerosol spray obtained from a pressurized packaging or aerosol can, using a suitable propellant such as dichlorodifluoromethane, trichlorofluoro ^ a &, -iL tetrafluoroacetamidine, carbon dioxide or Other suitable gases. In the case of adding ::: glue, the dose can be determined by providing a door. Passing:… ten miles < amount. Capsules and cartridges 1 for inhalers or blowers 1 such as gelatin can be formulated to contain a powder mixture of the compound with a suitable powder base Xin or powder. Formulated to provide ^^ by injection, such as by bolus injection or continuous infusion. Injection = early agent "is presented 'for example in Ann, or in a multi-dose container with preservatives. These combinations Substances can take multiple forms, such as suspensions, solutions or emulsions, in oily or aqueous vehicles, and can be formulated in two formulations, such as suspensions, stabilizers and / or dispersants. For parenteral administration Pharmaceutical formulations include activated figures in a water-soluble form. In addition, 'suspensions of active compounds can be made in a suitable way = raw: shot suspensions. Suitable lipophilic solvents or vehicles include grease-proof surfaces, ... sesame oil' Or synthetic fatty acid brews, such as oleate, or microlipids. Aqueous injection suspensions can show the best of both worlds, such as dimethylcellulose, and flowers that increase the viscosity of the suspension. The solution may also contain appropriate stability to allow Preparation height Concentrated solution of medicament. Or: Add the compound in the final solubility form, before use, with a suitable vehicle: powdery pyrogenated water. W For example, sterile, free of embedding that can be used in the pharmaceutical compositions provided herein. The combined antibody amount is based on the nature of the symptoms treated by 89753 -38- 200427698. Sex, broadness, severity ', and after the patient has received previous treatments and blemishes, the physician in charge will determine the protein of the present invention. The amount of :: Taiwan? Individual patients. First, the doctor of your stomach will administer a low dose of the prescription = protein Λ, and observe the patient's response. A larger dose can be administered, person: Zhongbaibei, Until the most appropriate treatment effect is obtained for the patient, 'at this time the dose is not good', and no further increase. It is intended to cover that various pharmaceutical compositions used to implement Shiwanwanfa should contain about 0.01 micrograms per kilogram of body weight: 00: grams (preferably about 0.1 micrograms to about 10 milligrams, more preferably about 01 micrograms to about 1 microgram). * G) Chimeric protein of the present invention. When administered, the therapeutic composition for use in the present invention is, of course, a chimeric protein that is free of pyrogens, physiologically acceptable V-form is not the method, and may optionally be included in the composition as described above. The therapeutically useful medicament can be substituted or additionally administered simultaneously or successively with the present invention. Exemplary agents for chimeric protein N include anti-neoplastic agents, as disclosed in paragraph C above. The chimeric proteins provided one by one can be administered alone or in combination with other therapeutic modalities. For example, the method of treatment can further include the step of transmitting ionizing radiation to the cells in contact with the chimeric insect shellfish. When judged by measuring the survival of malignant cells, the ionizing radiation is transmitted at a dose sufficient to cause substantial ... The degree of cell killing caused is preferably qualitatively greater than the typical forms of radiation that are quoted by either the individual antibody or the individual ionizing radiation, including point rays, r rays, 0; particles and X-rays. . It can be transmitted from an external source, such as a x-ray machine or a 7-camera camera, or from a radionuclide from a patient in Syria, to a malignant tissue. It is also possible to use a method known in the art, 89753 -39- 200427698, using radionuclides. The use of ionizing light in the treatment of malignant conditions is described in, for example, S. Hellman, The Principles of Free Radical Therapy, in Cancer: Principles and Practice of Oncology 2 (V.T. DeVita, Jr. et al.) , 4th edition, 1993). Typically, a usable dose range is between about ^ and 500 cGy (meaning, about 1 to about 500 rads). In one aspect, provided herein is a vaccine comprising an effective amount of a chimeric protein comprising a K ^ ligand and a proteinaceous or peptidic tumorigenic agent, and an immune response enhancer. In another aspect, provided herein is a method of slaughtering an anti- 袅 C immune response in a mammal, the method comprising administering an effective amount of a vaccine to a mammal in need of or wanting such slander, which Contains an effective amount of a chimeric protein and immune response enhancer comprising a Flt3 ligand and a proteinaceous or peptidic tumorigenic agent. The term "immune response enhancer" as used herein refers to any agent that enhances or prolongs the immune response to a target antigen, such as a tumor antigen. The enhancement of the immune response can be a bonus or a boost. As used in this article, immune response, a stagnation system encompasses the response of B cells, τ_ cells, or both B_ and τ_ cells. Exemplary immune response enhancers include other fine tongues such as IL-12, iL-2, IFN-7, adjuvants, immunostimulating peptides, and the like. The immune response enhancer of the composition and method of the invention T can be simultaneously or sequentially Λ chimeric proteins, administered via the same route or different routes.妾 Seed epidemic can be carried out by conventional methods. For example, the immunogen can be used in a suitable Greek release J, such as saline or water, or a complete or incomplete adjuvant. Furthermore, the immunogen may or may not be bound to the carrier to make the protein immunogenic 89753 200427698

Sex. Examples of such carrier molecules include, but are not limited to, bovine serum albumin (SA), keyhole cyanocyanine (KLH), tetanus toxoid and the like. Immunogens can also be coupled with lipoproteins, administered as microlipids, or accompanied by an adjuvant. The immunogen can be administered by any route suitable for antibody production, such as intravenously, intraperitoneally, intramuscularly, subcutaneously. The immunogen can be administered at one time or at periodic intervals until an anti-tumor cell T-cell response or a significant titer of anti-tumor cell antibodies is produced. The presence of anti-tumor cell responses can be assessed before and after the immune response by measuring the frequency with which the precursor CTL (cytotoxic T-lymphocyte) resists tumor antigens. See, for example, coulie, p. Et al., ^ C 5Q · 289-97 (1992). Antibodies can be detected in serum using immunoassays known in the art. 2. The administration of the vaccine of the present invention can be aimed at either prevention or treatment. When provided as a precautionary measure, the chimeric protein is provided before any evidence of a malignant condition or before any symptoms. Prophylactic administration of post-proteins ^: Dairy animals are preferably used in humans to prevent or reduce malignant conditions. When

When the two treatments are provided, the inflammatory synthesizing protein is provided at the time of the onset of the disease (or shortly after) / at the time of any signs of the disease. Chimeric proteins are used to attenuate disease. Local administration to the only affected area can be achieved by means known in the art > each including but not limited to topical application, injection and implantation of porous devices:: Yes: Reconstituted cells expressing perfusate, porous Implantation of sexual devices: There are separate chimeric proteins or with immune response enhancers. ,: Peiwan was first evaluated in animal mode, initially in rodents, 7 in non-human primates, and finally in humans. Immune action 89753 -41-200427698 Preface "Safety by searching for immune effects on the general health of immunized animals (weight change, fever, appetite behavior, etc.) and searching for pathological changes during necropsy And measured. After initial testing in animals, cancer can be tested. Conventional methods are used to assess the patient's immune response to measure the efficiency of vaccines. See, for example, current proposals for immunology (latest version). Examples in which the T-lymphocytes can be isolated include, but are not limited to, peripheral blood, field cell lymphocytes (PBL), lymph nodes or tumor infiltrating lymphocytes (TIL). Such lymphocytes can be isolated from the individual to be treated or obtained from a supplier, and cultured in vitro by methods known in the art. See, for example, Kawakami Y. et al., / M / 〇 / said: μ5 · (1989). Lymphocytes can be cultured in culture using techniques known in the art. Viability was assessed by cone blue dye exclusion testing. Parameters that can be evaluated to determine the efficacy of these sensitized D lymphocytes include, but are not limited to, the production of immune cells or tumor regression in the mammal being treated. Use customary methods to evaluate these parameters. Such methods include cytotoxicity testing, mixed lymphocyte response, and cytokine production testing. Any conventional tumor pattern can be used to provide a test pattern for chimeric proteins. Tumor mouse recipients can be of any appropriate strain. The tumor may be allogeneic, allogeneic, or heterologous to the tumor. Recipients may be immune active or immune compromised in one or more immune-related functions, including, but not limited to, dish / dish, scid, and beige mice. In a specific embodiment, the recipient is a transgenic mouse. In a particular embodiment, the mouse is a Balb / c or C57BL / 6 mouse. Any suitable tumor source can be used in animal model experiments, including established cell lines, dissociated cells obtained from new tumor samples, and short-term 89753 -42- 200427698 tumor cell lines. Exemplary tumor cell lines include Renca cells, B16 melanoma cells, Hepal cells, BT-474 cells, Raji cells, QYC cells, D2F2 cells, 4T1 cells, and A20 cells. In at least one administration, the dosage of the chimeric protein ranges from 1 microgram / mouse to 1 mg / mouse. Antibodies can be administered by any appropriate route. In a specific embodiment, the dose of antibody is 100 micrograms / mouse twice a week. In a specific embodiment, the tumor is injected subcutaneously on day 0, and the volume of the primary tumor is measured at a specified time point using a caliper. Any appropriate control protein can be used. In one example, the control antibody is a purified Igh isotype control antibody that has been raised against the hapten dinitrophenyl. The broad scope of the present invention is best understood with reference to the following examples, which are not intended to limit the invention to specific embodiments. [Embodiments] D. Examples Example 1 The human Flt3 ligand extracellular region (hFLex) cDNA is completely synthesized: Since the Flt3 ligand is a type I transmembrane protein, the extracellular region is at the N-terminus and the N-terminus of FL The modification may adversely affect its biological activity. Therefore, we adopted a method for constructing tetravalent biospecific antibodies (see Figure 1A). See Column et al., Institute of Research 15 · 159-163 (1997). Typically, a tetravalent bispecific antibody system is constructed by fusing the DNA encoded by a single-chain antibody to the C-terminus of an antibody with a different specificity. In order to obtain a bifunctional fusion protein with high biological activity, our system has a fusion protein with FLex at the N-terminus and an antibody molecule at the C-terminus (see 89753 -43- 200427698 Figure 1B). First, this FLex gene was fused to the 5 'end of the human IgG1 cDNA (hinge plus CH2 plus CH3) to generate a FLex-Ig fusion gene. This hFLex-Ig fusion gene was then fused to the 5 'end of the single-chain antibody gene to generate a F! Ex-Ig-scFv fusion gene. / LFZex cDAM combination ... The cDNA sequence of the human FLt3 ligand gene, the gene bank database has the acceptance number U03858. Nucleic acids 84 to 161 encode the message peptide of the FLt3 ligand, and nucleotides 162 to 629 encode the extracellular region of the Flt3 ligand. Therefore, the size of the gene encoding both the message peptide of the Flt3 ligand and the extracellular region was 546 bp. The FLex gene was synthesized as described in Prodromou C et al., Protein Eng. 5 (8): 827-829. In short, the FLex cDNA was divided into 10 DNA fragments of about 75 bp. These fragments were designed using the following criteria: (1) each fragment overlaps with an adjacent fragment at a length of 20bp; (2) the size of the final fragment can be shorter than 75bp; and (3) the antisense strand is selected as the last Primers, and meaningful strands were selected as primers for all other fragments. The primers were then synthesized commercially (Shengong Biotechnology (Shanghai, China)). PCR was performed in a 50 microliter volume containing 85 nM of each primer, 1.5 mM MgCl2, 200 mM dNTP, and 2.5 units of PfUDNA polymerase. The proposed PCR cycle is: preculture (94 ° C, 5 minutes); 30 cycles of denaturation (94 ° C, 1 minute), tempering (56 ° C, 1 minute), and extension at 72 ° C. This extension time varies depending on the number of primers, where time is calculated using the following equation: Extension time (seconds) = number of primers X 6 (seconds)). This last extension was at 72 ° C for 5 minutes. The PCR reaction products were separated on a 1% agarose gel. The correct DNA fragment was 89753 -44- 200427698, which was gel purified and asexually propagated to the pGEM-T vector (Promega), and its sequence was confirmed. See Figure 2 (sequence identification codes: 1 and 2). This clonal breeding line is denoted as pGEM-T / hFlex. Example 2 Recognition of asexual reproduction of the constant region of human IgGl The natural human IgGl cDNA of 1416 bp encodes a 471 amino acid and a translation termination code. The constant region of IgGl was reproduced asexually by RT-PCR using human peripheral blood mononuclear cells (PBMC) by Ficoll-Hypaque density gradient centrifugation, and isolated from liver lipids of healthy volunteers. RNA was isolated from PBMC using TRIzol reagent (Gibco BRL). The cDNA of the IgG1 Fc fragment was obtained by single-step RT-PCR (Qiagen). The primers for RT-PCR are as follows: Fc is meaningful, 5'-gca etc gag ttt tac ccg gag aca ggg aga g-3 '; Fc is negative, 5'-gag ccc aaa tet tgt gac aaa ac-3'. RT-PCR products were separated on an agarose gel. The correct DNA fragment was gel-purified and asexually propagated to the pGEM-T carrier (Promega), and its sequence was confirmed. This clonal breeding line is represented as pGEM-T / IgFc. Example 3 Construction of SM5-1 chimeric antibody and humanized antibody L Asexual reproduction of SM5-1 heavy and light chain variable region genes in mice. RNA was isolated from SM5 using TRIzol reagent (Gibco BRL, Grand Island, NY). -1 (IgGl, / c) hybridoma cells (stored in ATCC with ATCC designated number HB-12588). The SM5-1 heavy and light variable region cDNA line uses the 5 'RACE system (Gibco BRL, Gaithersburg, MD), and asexually propagates from hybridoma cells according to the manufacturing instructions. The aggregated PCR products were analyzed by agarose gel electrophoresis (Figure 5). About 590bp 89753-45-200427698 specific heavy chain PCR fragment and about 530bp light chain fragment were individually purified by gel and cloned into pGEM-T vector (Promega, Madison, WI) for sequential determination. The DNA sequences of the heavy (SM, VH) and light (SM, VL) variable regions are individually sequence identifiers: 7 (Figure 6) and sequence identifiers: 9 (Figure 7). 2. The four vectors used in combination or the two vectors pAH4604 and PAG4622 were kindly provided by Professor SL Morrison (Department of Microbiology and Molecular Genetics, UCLA). See Coloma et al., 152: 89 (1992). Using the PCR method, EcoRV and Xbal positions were added to the 5 'terminus of the heavy chain variable region gene (VH), and Nhel positions were added to the 3f terminus. The PCR product was propagated asexually into the pGEM-T vector, and its sequence was confirmed. VH was excised by digestion with EcoRV and Nhel, and inserted into the EcoRV / Nhel position of the pAH4604 vector containing human r-1 constant region gene (CH). The resulting pAH4604-VH carrier system was split with Xbal and BamHI 'and asexually propagated the 3.3 kb fragment containing the post-combined rodent / human anti-body chain gene to the PDR vector to generate the chimeric heavy chain expression vector pDR-SMVHCH. The nucleotide and inferred amino acid sequence of the SM5-1 chimeric heavy chain (chSMVHCH) is shown in the sequence identification codes: 11 and 12 (Figure 8). Human / c-chain constant cDNA (CL) was obtained as a 0.3 kb PCR product derived from pAG4622. The pAG4622 line was kindly provided by Professor S. L. Morrison (Department of Microbiology and Molecular Genetics, UCLA). The light chain variable region gene of (VL) SM5-1 was fused to the 5 'end of CL by an overlapping PCR method. The resulting chimeric light chain gene (VLCL) contains a Hindlll position upstream of the start codon and an EcoRI position downstream of the stop codon, and then the chimeric light chain is propagated asexually to the pGEM-T vector gene, and the sequence confirm. The VLCL gene was excised by digestion with Hindlll and EcoRI, and ligated into a pDR vector to generate a chimeric light chain expression vector 89753 -46- 200427698 pDR-SMVLCL. The nucleotide and deduced amino acid sequences of the SM5-1 chimeric light chain (chSMVLCL) are shown in the sequence identification codes: 13 and 14 (Figure 9). The expression vectors pDR-SMVHCH and pDR-SMVLCL are shown in Fig. 10 and Fig. 11. 3. We must do everything we can. Choose the VH of the human antibody KOL as the backbone of the humanized heavy chain, and choose the VL of the human Bence-Jones protein REI as the humanized light chain. The light and heavy variable region genes of humanized antibodies were synthesized using the PCR method described in Example 1. The light and heavy chain expression vectors for humanized antibodies are constructed in the same manner as the chimeric antibodies described above. First, three CDRs from the SM5-1 light or heavy chain were directly grafted to the human antibody light or backbone region to generate a humanized recombination gene. Humanized VL and VH each propagate asexually to the expression vector, and then briefly co-express in COS cells. Transfected COS cells will produce humanized SM5-1 Ab. Humanized antibodies in the supernatant of COS cell cultures were quantified by ELISA, and the binding system of the antibodies to melanoma cells was determined by flow cell count analysis. Antigen-binding activity testing revealed that this antibody binds poorly to human hepatoma cell QYC, which indicates that some human FR residues must be altered to restore full binding activity. Important FR residues that may have an influence on binding activity are analyzed and back mutation detection is performed. A humanized anti-system showing the same antigen-binding activity as the unhumanized SM5-1 was obtained, and was called nhuSM'f. In competitive binding assays, huSM5-l antibodies show comparable antibody avidity, such as mouse SM5-1 antibody or chimeric SM5-1 antibody. The light and heavy chain expression vectors are represented as pDR-huSMVHCH and pDR-huSMVECL. The sequence of nucleotides and amino acids in the huSM's heavy and light variable regions are shown individually in sequence identification codes: 15 and 16 (Figure 12) and sequence identification codes: 17 and 18 (Figure 13). The sequence of nucleotides and amino acids of the heavy and light chains of huSM is 89753 -47- 200427698, which are shown individually in sequence identifiers: 19 and 20 (Figure 14) and sequence identifiers: 21 and 22 (Figure 15). 4. Table collections of the necessary techniques for ceremonies · Keep CHOdhfif cells in complete DMEM medium containing glycine, hypoxanthine, and thymidine (GHT) before transferring infection. Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was used to co-transfect appropriate light and heavy expression vectors into CHOdhfr- cells according to the manufacturing instructions. Transfected cells were then selected in DMEM medium without GHT and with a stepwise increase in MTX content up to 1.0. Select resistant clones and expand them for further analysis. The culture supernatant obtained from the cell asexual propagation line was detected by sandwich ELISA 'using goat anti-human IgG (Fc) (KPL) as capture antibody and goat anti-human / C-HRP (KPL) as detection Antibodies, analysis of fusion protein production. Purified human IgGl / c (Sigma) was used as a standard in the ELISA assay. The asexual lines that produce the highest amount of antibodies are selected and grown in serum-free medium. Recombinant antibodies were purified from serum-free culture supernatants by protein A affinity chromatography. 5. 和 和 力 友 #. The affinity (Kd) of chimeric and humanized antibodies is used

BlAcore (Pharmacia) assay, as described in Karlsson R. et al., C / Tmmwno /. 145: 229 (1991). The Kd values of the chimeric antibody and the humanized antibody are 3.78 × 10-9 and 9.31 × 1 (Γ9. These results show that the humanized SM5-1 antibody has satisfactory antibody affinity and can be used. Treatment in humans. Example 4 Construction of huSM / FL and chSM / FL bifunctional fusion proteins 89753 -48- 200427698 Three different fusion proteins were constructed for further study of their biological functions. A huSMVH / Fc / FL Construction · Human The Flt3 ligand extracellular region (hFLex) cDNA was obtained as a 500 bp PCR amplified fragment derived from pGEM-T / hFlex. The FLex gene was fused to the 3 'end of the huSM heavy chain gene using overlapping PCR. The resulting fusion gene was PCR The product contains the Hindlll position upstream of the start codon and the EcoRI position downstream of the stop codon. The fusion product is propagated asexually to the pGEM-T vector, and the sequence is confirmed. Nucleic acid and inferred amine of huSMVH / Fc / FL The amino acid sequence is shown in the sequence identifiers: 23 and 24 (Figure 16). The huSMVH / Fc / FL fusion gene was excised by digestion with Hindlll and EcoRI, and inserted into the Hindlll / EcoRI position of the pDR vector to generate fusion. Gene expression vector pDR-li uSMVH / Fc / FL. Appropriate light (pDR-huSMVLCL) and fusion gene (pDR-huSMFv / Fc / FL) expression vectors were co-transfected into CHOdhfi " cells using Lipofectamine 2000 reagent. Then, GHT and the DTX medium containing MTX content gradually increasing up to 1.0 // M, select transfected cells. Select drug-resistant clonal propagation lines and expand for further analysis. Will be obtained from cell clonal propagation lines Culture supernatants were analyzed for fusion protein production by sandwich ELISA using goat anti-human IgG (Fc) (KPL) as capture antibody and goat anti-human / c-HRP (KPL) as detection antibody. Purified human IgGl // c (Sigma) was used as the standard in the ELISA test. Asexual breeding lines that produced the highest amount of fusion protein were selected and grown in serum-free medium. The fusion protein line was through the protein A affinity layer 89753 -49- 200427698 Β · huSMFv / Fc / chain / FL construction · Human Flt3 ligand extracellular domain (hFLex) cDNA line derived from pGEM_T / 500bp PCR for hFlex .HFLex fragment obtained by overlapping the PCR-based gene, linked via gene, fused to the 3 'terminus of the heavy chain gene huSM. The amino acid sequence of the linker peptide is (Gly4Ser) 3 (sequence identification code: 6, in Figure 4). The final pcr product containing the Hindlll position upstream of the start codon and the EcoRI position downstream of the stop codon is propagated asexually to the pGEM-T vector (Promega), and the order is confirmed (in sequence ID: 25 and 26 (Shown in Figure 17). The huSMFv / Fc / link / FL fusion gene was excised by digestion with Hindlll and EcoRI and inserted into the Hindlll / EcoRI position of the pDR vector to generate a fusion gene expression vector pDR-huSVHv / Fc / link 7 fl. Appropriately light (pDR-huSMVLCL) and fusion gene (huSMVjj / Fc / link / FL) expression vectors were co-transfected into cHOdhfr-cells using a lipidamine 2000 reagent. Transfected cells were then selected in DMEM medium containing no GHT and gradually increasing in MTX content up to 10 / zM. The resistant clones were selected and expanded for further analysis. The culture supernatant obtained from the cell asexual propagation line was analyzed by sandwich ELISA using goat anti-human IgG (Fc) (KPL) as the capture antibody and goat anti-human (jq> l) as the detection antibody. Fusion protein production. Purified human IgG1 / ^ (Sigma) was used as the standard in the detection. Asexual lines that produce the highest amount of fusion protein are selected and grown in serum-free medium. The fused protein was purified from the serum-free culture supernatant by protein A affinity chromatography. Construction of C. FL / Fc / huSMFv. Extracellular region of human scratch 3 ligand plus news 89753 -50- 200427698

The peptide cDNA was obtained as a 550bp PCR amplified fragment derived from pGEM-T / hFlex. The hFLexPCR product contains a Hindlll position at the 5 'end, followed by a Kozak sequence to aid performance. Human IgG1 cDNA (hinge plus CH2 plus CH3) was amplified from pGEM-T / IgFc by PCR. We used the overlapping PCR method to fuse the FLex gene to the 5 'end of the human IgGl cDNA to generate the FL / Fc fusion gene (shown in Figure 3, sequence identification codes: 3 and 4). The huSM heavy chain variable region cDNA is linked to the gene using overlapping PCR

In the method, the 5 'terminus of the light chain variable region gene was fused to generate a huSM single chain antibody (ScFv) gene. The amino acid sequence of the linker peptide is (Gly4Sei03 (sequence identification code: 6). Then, the FL / Fc fusion gene is fused to the S-terminus of the huSM ScFv gene by overlapping PCR to generate a FL / Fc / huSMFv fusion Gene. This FL / Fc / huSMFv

The fusion gene PCR product contains a Hindlll position at the 5f end and an EcoRI position downstream of the stop codon. Then, the product was vegetatively propagated to the pGEM-T vector (Promega), and its sequence was confirmed (shown in sequence identification codes: 27 and 28 in Fig. 18). Then, the fusion gene was digested with Hindlll and EcoRI, and the fusion gene was excised and inserted into the Hindlll / EcoRI position of the pDR vector to generate a fusion gene expression vector pDR-FL / Fc / huSMFv. A schematic diagram of the FL / Fc / huSMFv fusion gene is shown in FIG. 19. -Transfection of the appropriate fusion gene expression vector (pDR-τ FL / Fc / huSMFv) into CHOdhf cells using Lipidamine 2000 reagent. Then, transfected cells were selected in DMEM medium containing no GHT and gradually increasing the MTX content up to 1.0 // M. Select resistant clones and expand for further analysis. The culture supernatant obtained from the cell asexual propagation line was analyzed for fusion by sandwich ELISA using goat anti-human IgG (Fc) as capture antibody, and mountain 89753 -51-200427698 sheep anti-human FLex as detection antibody. Protein production. Asexual lines that produce the highest amount of fusion protein are selected and grown in serum-free medium. Fusion proteins were purified from serum-free culture supernatants by protein A affinity chromatography. In the same way as the huSM / FL fusion protein described above, three different ChSM / FL fusion proteins were constructed, expressed and purified. The nucleotide and inferred amino acid sequences of chSMVH / Fc / FL, chSMVH / Fc / link / FL, FL / Fc / chSMFv are shown individually in sequence identifiers: 29 and 30 (Figure 20), sequence identifiers : 31 and 32 (Figure 21) and sequence identification codes: 33 and 34 (Figure 22). '' Example 5 Construction of CD20 / FL bifunctional fusion protein L Synthesis of variable region genes of anti-CD20 mAb 2B8. The variable region cDNA of anti-CD20 mouse monoclonal antibody 2B8 was used as described in Example 1 and revealed using Synthesized sequentially in US Patent 6,399,061. The PCR reaction products were separated on a 1% agarose gel. The correct DNA fragments were gel purified and cloned asexually into the pGEM-T vector (Promega), and their sequence was confirmed. The nucleotide and amino acid sequences of the heavy and light variable region 2138 are shown in sequence identifiers: 35 and 36 (Figure 23) and sequence identifiers: 37 and 38 (Figure 24). In this example, the correct asexual propagation lines for the 2B8 light and heavy chain vectors are individually designated as PGEM-T / CD20H and pGEM-T / CD20L. 2. Construction of a chimeric antibody expression vector. Using PCR, add EcoRV and Xbal positions to the 5 ′ terminus of the heavy chain variable region gene (VH), and Nhel positions to the 3 ′ terminus. The PCR product was asexually propagated into the pGEM-T vector, and its sequence was confirmed. VH was excised by digestion with EcoRV and Nhel, and inserted into the EcoRV / Nhel position of pAH4604 vector containing human r_1 constant region gene (CH) in 89753 -52- 200427698. The resulting pAH4604-VH vector was divided by Xbal and BamHI, and the 3.3 kb fragment containing the chimeric rodent / human antibody heavy chain gene was vegetatively propagated to the pDR vector to generate the chimeric heavy chain expression vector pDR-CD20VHCH. The nucleotide and amino acid sequences of the anti-CD20 chimeric heavy chain (CD20VHCH) are shown in the sequence identification codes: 39 and 40 (Figure 25). Human / c-chain constant cDNA (CL) was obtained as a 0.3 kb PCR product derived from pAG4622. The light chain variable region gene (VL) of 2B8 was fused to the 5f end of CL by an overlapping PCR method. The resulting chimeric light chain gene (vLCL) contains the Hindlll position upstream of the start codon and the EcoRI position downstream of the stop codon, and then the chimeric light chain is propagated asexually to the pGEM-T vector gene, and the sequence Confirmed. The VLCL gene was excised by digestion with Hindlll and EcoRI, and ligated to a pDR vector to generate a chimeric light chain expression vector. The nucleotide and amino acid sequences of the anti-CD20 chimeric light chain (CD20VLCL) are shown in sequence identifiers: 41 and 42 (Figure 26). 3. Construction of CD20VH / Fc / FL · Human Flt3 ligand extracellular region (hFLex) cDNA was obtained from a 500bp PCR amplified fragment derived from pGEM-T / hFlcx. The FLex gene was fused to the 3 'end of the 2B8 heavy chain gene by overlapping PCR. The resulting fusion gene PCR product contains a Hindlll position upstream of the start codon and an EcoRI position downstream of the stop codon. Then, the product was asexually propagated to the pGEM-T vector, and the sequence was confirmed. The nucleotide and corollary amino acid sequences of CD20 VH / Fc / FL are shown in sequence identifiers: 43 and 44 (Figure 27). The CD20VH / Fc / FL fusion gene was excised by digestion with Hindlll and EcoRI, and inserted into the Hindlll / EcoRI position of the pDR vector to generate a fusion gene expression vector 89753 -53- 200427698 pDR-CD20VH / Fc / FL. 4. Construction of CD20VH / Fc / M junction / FL. Human Flt3 ligand extracellular

Region (hFLex) cDNA was obtained as a 500 bp PCR amplification fragment derived from pGEM-T / hFlex. The hFLex gene is fused to the 3 'terminus of the 2B8 heavy chain gene through a contiguous gene by an overlapping PCR method. The amino acid sequence of the linked peptide is (Gly4 Ser) 3 (sequence identification code: 6). The final PCR product containing the Hindlll position upstream of the start codon and the EcoRI position downstream of the stop codon is propagated to the pGEM-T vector (Promega), and the sequence is confirmed (shown by sequence ID: 45 and 46) (In Figure 28). The CD20VH / Fc / link / FL fusion gene was excised by digestion with Hindlll and EcoRI and inserted into the Hindlll / EcoRI position of the pDR vector to generate a fusion gene expression vector pDR-CD20VH / link / FL.

5. Construction of FL / Fc / CD20Fv. The extracellular region of the human Flt3 ligand plus the message peptide cDNA was obtained from a 550 bp PCR amplified fragment derived from pGEM-T / hFlex. The hFLexPCR product contains Hindlll at the 5 'end, followed by a Kozak sequence to aid performance. Human IgG1 cDNA (hinge plus CH2 plus CH3) was amplified from pGEM-T / IgFc by PCR. We used the overlapping method PCR to fuse the FLex gene to the 5 'end of the human IgGl cDNA to generate the FL / Fc fusion gene.

The 2B8 heavy chain variable region cDNA is fused to the 5 'terminus of the light chain variable region gene via a ligation gene using an overlapping PCR method to generate a 2B8 single-chain antibody (ScFv) gene. The amino acid sequence of the linked peptide is (Gl% Ser) 3. Then, the FL / Fc fusion gene was fused to the 5 'end of the 2B8 ScFv gene by overlapping PCR to generate a FL / Fc / CD20Fv fusion gene. The FL / Fc / CD20Fv fusion gene PCH product contains a Hindlll position at the 5 'end and an EcoRI position 89753 -54- 200427698 downstream of the stop codon. Then, the product was vegetatively propagated to the pGEM-T vector (promega), and the sequence was confirmed (identified by sequence identification codes: 47 and 48 in Fig. 29). Then, the fusion gene was digested by Hindlll and EcoRI, and inserted into the Hindlll / EcoRI position of the pOR vector to generate a fusion gene expression vector pDR-FL / Fc / CD20Fv. A schematic diagram of the FL / Fc / CD20Fv fusion gene is shown in FIG. 30. 6. Construction of 2B8 chimeric light chain expression vector. The human / c chain constant cDNA (CL) was obtained from a 0.3 kb PCR product derived from pAG4622. PAG4622 was kindly provided by Professor SL Morrison (Department of Microbiology and Molecular Genetics, UCLA). The light chain variable region gene (VL) of SM5-1 was fused to the 5 'end of CL using an overlapping PCR method. The resulting chimeric light chain gene (VLCL) contains the position of Hindlll upstream of the start codon and the position of EcoRI downstream of the stop codon, and then the product is asexually propagated to the pGEM-T vector, and the sequence is confirmed. The VLCL gene was excised by digestion with Hindlll and EcoRI and ligated to the pDR vector to generate a chimeric light chain expression vector pDR-CDaiA ^ CL. 7. Fusion protein expression and purification. As described in Example 4, three different fusion proteins were expressed and purified. Example 6 Her2 / FL · Construction of a bifunctional fusion protein 1. Synthesis of variable region genes of anti-HER2 mAb rhuMAb HER2 · Variable humanized anti-HER2 antibody (rhuMAb HER2, Herceptin) The region cDNA was synthesized as described in Example 1 using the sequence disclosed in Carter et al., Proc Mzi / 89: 4285 (1992). The PCR products were separated on a 1% agarose gel. The correct DNA fragments were gel-purified and vegetatively propagated to the pGEM-T vector (Promega), and their sequence was confirmed. Anti-her2 89753 -55- 200427698 The sequence of nucleotides and amino acids in the heavy and light variable regions of the antibody are shown in the sequence identifiers: 49 and 50 (Figure 31) and sequence identifiers: 51 and 52 (Figure 32). In this example, the asexual propagation lines of rhuMAb HER2 light chain (VL) and heavy chain (VH) vectors are individually designated as pGEM-T / her2H and pGEM-T / her2L. 2 · Construction of chimeric antibody expression vector. Using PCR method,

The 5 'terminus of the heavy chain variable region gene (VH) was added to the EcoRV and Xbal positions, and the 3f terminus was added to the Nhel position. The PCR product was vegetatively propagated to the pGEM-T vector, and the sequence was confirmed. VH was excised by digestion with EcoRV and Nhel, and inserted into the EcoRV / Nhel position of the pAH4604 vector containing the human 7-1 constant region gene (CH). The resulting pAH46〇4-VH vector was divided by Xbal and BamHI, and '3.3 kb fragment containing the chimeric rodent / human antibody heavy chain gene was vegetatively propagated to the pDR vector to generate the chimeric heavy chain expression vector pDR- her2VHCH. The nucleotide and amino acid sequences of the anti-her2 humanized heavy chain (her2VHCH) are shown in the sequence identification codes: 53 and 54 (Figure 33). Human / c-chain constant cDNA (CL) was obtained as a 0.3 kb PCR product derived from pAG4622. The humanized light chain variable region gene (VL) was fused to the 5 'end of CL by an overlapping PCR method. The humanized light chain gene (VLCL) formed contains the position of Hindlll upstream of the start codon and the position of EcoRI downstream of the stop codon, and then the humanized light chain is propagated asexually to the pGEM-T vector gene. Confirmed. The VL CL gene was excised by digestion with Hindlll and EcoRI, and was linked to the pDR vector to generate a humanized light chain expression vector pDR-hedVL Cl. The sequence of the nuclear acid and amino acid of the anti-her2 humanized light chain (her2VLCL) is shown in the sequence identification codes: 55 and 56 (Figure 34). 3. Construction of Her2Fv / Fc / FL, human Flt3 ligand extracellular region (hFLex) 89753 -56- 200427698 cDNA was obtained from a 500bp PCR amplified fragment derived from pGEM-T / hFlex. The FLex gene was fused to the 3 'end of the rhuMAb HER2 heavy chain gene using an overlapping PCR method. The resulting fusion gene PCR product contains a Hindlll position upstream of the start codon and an EcoRI position downstream of the stop codon. The product was then vegetatively propagated to the pGEM-T vector, and its sequence was confirmed. The nucleotide and amino acid sequences of Her2 / Fv / Fc / FL are shown in the sequence identification codes: 57 and 58 (Figure 35). The Her2 / Fv / Fc / FL fusion gene was excised by digestion with Hindlll and EcoRI, and inserted into the Hindlll / EcoRI position of the pDR vector to generate a fusion gene expression vector pDR-Her2 / Fv / Fc / FL 〇4 · Valence ## The human Flt3 ligand extracellular region (hFLex) cDNA was obtained from a 500 bp PCR amplified fragment derived from pGEM-T / hFlex. The hFLex gene was fused to the 3 'terminus of the rhuMAb HER2 heavy chain gene via a ligation gene using an overlapping PCR method. The amino acid sequence of the linked peptide is (Gly4Ser) 3. The final PCR product contains the Hindlll position upstream of the start codon and the EcoRI position downstream of the stop codon. Then, the product was vegetatively propagated to the pGEM-T vector (Promega), and its sequence was confirmed (shown in FIG. 36 by sequence identification codes: 59 and 60). The Her2VH / Fv / Fc / FL fusion gene was excised by digestion with Hindlll and EcoRI, and inserted into the Hindlll / EcoRI position of the pDR vector to generate a fusion gene expression vector pDR-Her2VH / Fv / Fc / FL. 5. Construction of FL / Fc / HER2Fv. The extracellular region of the human Flt3 ligand plus the information peptide cDNA was obtained from a 550bp PCR amplified fragment derived from pGEM-T / hFlex. The hFLexPCR product contains Hindlll at the 5 'end, followed by a Kozak sequence to aid performance. Human IgG1 cDNA (hinge plus CH2 plus CH3) was amplified from pGEM-T / IgFc by PCR. We used the overlapping PCR method to fuse the FLex 89753-57-200427698 gene to the 5f end of the human IgGl cDNA to generate the FL / Fc fusion gene. The rhuMAb HER2 heavy chain variable region cdNA is fused to the 5f terminus of the light chain variable region gene using an overlapping PCR method using a linker gene to generate a rhuMAb HER2 single-chain antibody (ScFv) gene. The amino acid sequence of the linked peptide is (Gly4Ser) 3. Then, the FL / Fc fusion gene was fused to the 5 'end of the rhuMAb HER2 ScFv gene using an overlapping PCR method to generate a FL / Fc / HER2Fv fusion gene. The FL / Fc / HER2Fv fusion gene PCR product contains a Hindlll position at the 5 'end and an EcoRI position downstream of the stop codon. Then, the product was asexually propagated to the pGEM-T vector (Promega), and the sequence was confirmed (shown in FIG. 37 by sequence identification codes: 61 and 62). Then, the fusion gene was excised by digestion with Hindlll and EcoRI, and inserted into the Hindlll / EcoRI position of the pDR vector to generate a fusion gene expression vector pDR-FL / Fc / HER2Fv. A schematic diagram of the FL / Fc / HER2Fv fusion gene is shown in FIG. 38. 6. Construction of rhuMAb HER2 chimeric light chain expression vector. K & Chromadine cDNA (CL) was obtained from a 0.3 kb PCR product derived from pAG4622. The light chain variable region gene (VL) of SM5-1 was fused to the 5 * end of & by the overlapping PCR method. The resulting chimeric light chain gene (VLCL) contains a Hindlll position upstream of the start codon and an EcoRI position downstream of the stop codon. Then, the product was vegetatively propagated to the pGEM-T vector, and its sequence was confirmed (sequence identifiers: 55 and 56, in FIG. 34). The VLCL gene was excised by digestion with Hindlll and EcoRI, and ligated to a pDR vector that was split with the same restriction enzyme to generate a chimeric light chain expression vector pDR-HER2VL CL. 7. Fusion protein expression and purification. As described in Example 4, three different fusion proteins were expressed and purified. 89753 -58- 200427698 Example 7 Construction of hFL / Trail fusion protein / · Construction of hFLex / Trail fusion protein. The cDNA sequence of the human FLt3 ligand gene used has the gene bank acceptance number HSU37518. The extracellular functional site cDNA (aa residues 95-281) for human Trail was synthesized as described in Example 1. Then, the PCR reaction products were separated on a 1% agarose gel. The correct DNA fragment was gel-purified and asexually propagated to the pGEM-T carrier (Promega), and its sequence was confirmed. The asexual breeding line is represented as pGEM-T / hTrail. hFLex cDNA was obtained from a 550bp PCR amplified fragment derived from pGEM-T / hFlex (hFLex gene was fused to the 5 'end of the Trailex gene by a ligation gene via overlapping PCR (Pitti et al., J. Biol · Chem. 271: 12687-90 (1996)). The amino acid sequence of the linked peptide is (Gly4Ser) 3 (sequence identification code: 6). The PCR product of the fusion gene contains the Hindlll position upstream of the start codon and the EcoRI position downstream of the stop codon. Then, the product was asexually propagated to the pGEM-T vector (Promega), and its sequence was confirmed (shown in FIG. 39 with sequence identifiers: 63 and 64). The hFLex / Trailex fusion gene fragment was obtained by Hindlll and EcoRI Digested and excised, inserted into the Hindlll / EcoRI position of the pDR vector. A schematic diagram of the hFLex-Trailex fusion gene is shown in Figure 40. The appropriate pDR-hFLex / Trailex expression vector uses a lipidamine 2000 reagent (Gibco BRL) According to the manufacturing instructions, transfect into CHOdhfr · cells. Then select the transfected cells in the DMEM medium containing no GHT and gradually increasing the MTX content up to 1.0. Select drug-resistant asexual propagation lines, And expand For further analysis, the culture supernatant derived from the cell clonal propagation line 89753 -59- 200427698 'by sandwich ELISA, using goat anti-human Trailex as capture antibody and goat anti-human FLex-HRP as detection antibody, Analyze fusion protein production. Select clones that produce the highest amount of fusion protein and grow in serum-free medium. The hFLex / Trailex fusion protein is then fixed to agar by affinity (goat anti-human Trail antibody). Sugar-4B) chromatography, purified from the serum-free culture supernatant. 2. Construction of hFLex / IZ / Trailex fusion protein. The hFLex gene was passed through the DNA sequence encoded by the isoleucine zipper pair (IZ). Fusion to the 51-terminus of the Trailex gene by overlapping PCR. See Harbury et al., Science, / 9P3, 262: 1401 (1993). The PCR product of the fusion gene contains an upstream of the start codon.

Hindlll position and EcoRI position downstream of the stop codon. Then, the product was vegetatively propagated to the pGEM-T vector (Promega), and its sequence was confirmed (identified by sequence ID: 65 and 66 in Fig. 41). The hFLex / IZ / Trailex fusion gene was finally asexually propagated to the expression vector pGS in the same manner as the hFLex / Trailex fusion gene described in Example 7.1. The fusion protein was expressed and purified as described in Example 7.1. 3. Construction of hFLex / Fc / Trailex fusion protein. The extracellular region of the human Flt3 ligand plus the message peptide cDNA was obtained from a 550bp PCR amplified fragment derived from pGEM-T / hFlex. The hFLex PCR product contains Hindlll at the 5 'end, followed by a Kozak sequence to aid performance. Human IgG1 cDNA (hinge plus CH2 plus CH3) was amplified from pGEM-T / IgFc by PCR. We used the overlapping PCR method to fuse the FLex gene to the 5 'end of the human IgGl cDNA to generate the hFLex / Fc fusion gene. Human Trail (Trailex) extracellular functional site cDNA was amplified by PCR. 89753-60-60200427698 was obtained from pGEM-T / hTmil. The 3 'end of the Trailex PCR fragment contains an EcoRI position. Using the overlapping PCR method, the previously obtained hFLex / Fc fusion gene was fused to the 5f end of the Trailex gene. The final PCR product was purified and vegetatively propagated to the pGEM-T vector (Promega) for sequence determination (sequence identifiers: 67 and 68, shown in Figure 42). The FLex / Fc / Tmilex fusion gene fragment was then excised by digestion with Hindlll and EcoRI, and inserted into a pDR vector that was split with the same restriction enzyme. A schematic diagram of the hFLex / Fc / Trailex fusion gene is shown in Figure 43. The appropriate pDR-hFLex / Fc / Trailex expression vector was transfected into CHOdhfr_ cells using a Lipidamine 2000 reagent (Gibco BRL) according to the manufacturing instructions. in. Then, the transfected cells were selected in DMEM medium containing no GHT and gradually increasing the MTX content up to 1.0 // M. The resistant clones were selected and expanded for further analysis. The culture supernatant obtained from the cell asexual propagation line was analyzed for fusion protein production by sandwich ELISA using goat anti-human Trail as capture antibody and goat anti-human FL-HRP as detection antibody. Asexual lines that produce the highest amount of fusion protein are selected and grown in serum-free medium. The hFLex / Fc / Trailex fusion protein was then purified from serum-free culture supernatants by protein A affinity chromatography. In Examples 8-16, chSM / FL, SM / FL, huSM / FL, CD20 / FL, her2 / FL, Trail / FL, respectively represent FL / Fc / chSMFv, FL / Fc / huSMFv, FL / Fc / CD20Fv, FL / Fc / HER2Fv and hFLex / IZ / Trailex. Example 8 Characterization of chSM / FL (FL / Fc / chSMFv) and huSM / FL (FL / Fc / huSMFv) 89753 -61-200427698 Characterization of Bifunctional Fusion Proteins 1. SM / FL was applied to human cord blood CD34 (+) cells in Expansion effect in vitro · Human umbilical cord blood-derived CD34 + cell line uses immunomagnetic beads (Pharmacia) and is isolated according to the manufacturing instructions. The purity of CD34 + cells was analyzed by flow cytometry. Cultures were established in 0.4% agarose or 0.3% agar medium in the presence of 10% pre-screened heat-inactivated bovine fetal serum (FBS) (Hyclone, Logan, UT) to evaluate 〇] \ 4-CSF, IL-3, G-CSF, SCF or CSF-1 responding CFU-GM, CFU-G, CUR-M colonies were performed in the absence and presence of SM or SM fusion proteins. The cells were cultured at 37 ° C in 5% CO2, and the medium was replaced by half at the beginning of the culture week. The number of clones of CD34 + cells in each group was counted on day 14. The results (shown in Figure 44) show that SM / FL has the ability to stimulate CD34 + cell proliferation, similar to FL. 2. The effects of chSM / FL and huSM / FL on NK and DC cells in vivo. C57BL / 6 mice were purchased from Experimental Animal Center (Shanghai, China). FITC-conjugated anti-CD3, PE-conjugated anti-NK1.1 and FITC-conjugated anti-CDllc were commercially obtained (R & D or Sigma). C57BL / 6 mice were intraperitoneally A single injection of 10 micrograms of chSM / FL and huSM / FL or FL was received daily for 0, 3, 6, 8, 10, 12, 15, or 18 days. The rats were sacrificed 24 hours after the last injection. Bone marrow, spleen and liver were collected and a single cell suspension was prepared. Cells were stained with FITC-conjugated anti-CD3 and PE-conjugated anti-NK1.1 in two colors to identify NK cells. Cells were stained with FITC-conjugated anti-CD1 to identify DC cells. 89753 -62- 200427698 flow cell count analysis was performed to assess the percentage of NK and DC cells. The absolute numbers of NK and DC cells in each organ are shown in Figure 45. The results show that SM / FL bifunctional protein has the effect of causing proliferation in NK and DC cells in the spleen, liver and bone marrow equivalent to FL. The number of NK and DC cells peaked between 10 and 13 days, and this peak lasted for 3 or 4 days. This points to the considerable potential of SM / FL for the treatment of cancer. 3. Inhibitory effect of SM / FL bifunctional fusion protein on tumor cell growth. Cell line (Hepal-6, B16) was obtained from ATCC. The cell line Hepal-6 was transfected with the p230 gene to generate the Hepal-6 / 230 cell line. Cell B16 was transfected with the p230 gene to generate a B16p230 cell line. When measured by flow cell count analysis, the P230 line was highly expressed on the cell surface of the cell lines Hepal-6 / 230 and B16p230. Cells in the logarithmic growth phase (SMMU, B16p230, Hepal-6p230 or Raji) were digested with 0.05% trypsin and 0.02% EDTA, and then washed twice with PBS containing 1% FBS. Cells were resuspended in 1640 / DMEM plus 10% FCS and adjusted to 6 x 104 cells / ml. The cell suspension was added to a 96-well plate (100 μl / well) and cultured in serial dilutions of chSM / FL or huSM / FL at 37 ° C for 7 days in 7% CO 2. CellTiter96 aqueous non-radiocellular proliferation assay (Promega) was used to determine the proliferation of three tumor cell lines according to the manufacturer's instructions. The results are shown in Figure 46, and show that chSM / FL and huSM / FL lines effectively inhibit the growth of SMMU, B16p230, Hepal-6p230 tumor cells without inhibiting the growth of control cells (Raji cells). This indicates that the inhibitory effect of SM / FL is specific to these three tumor cells. 89753 -63- 200427698 4. In vitro antitumor activity of chSM / FL and huSM / FL · Cell lines (SK-BR-3, QYC) were obtained from the International Joint Cancer Society (Shanghai, China). The cell line (Hepal-6, B16) was obtained from ATCC. The cell line Hepal-6 was transfected with the p230 gene to produce the Hepal-6 / 230 cell line. Cell B16 was transfected with the p230 gene to generate a B16p230 cell line. When measured by flow cell count analysis, the P230 line was highly expressed on the cell surface of the cell lines Hepal-6 / 230 and B16p230. Cells in the logarithmic growth phase (Hepal-6, B16, Hepap230 or B16p230) were digested with 0.05% trypsin and 0.02% EDTA, and then washed twice with 10% FBS in PBS. Cells were resuspended in 1640 / DMEM plus 10% FCS and adjusted to 6 X 104 cells / ml. The cell suspension was added to a 96-well plate (100 μl / ^ well) and cultured in serial dilutions of chSM / FL or huSM / FL at 37 ° C for 7 days in 7% CO2. CellTiter96 aqueous non-radioactive cell proliferation assay (Promega) was used to determine the proliferation of tumor cell lines according to the manufacturing instructions. The results are shown in Figure 47, and show that chSM / FL and huSM / FL lines effectively inhibit the growth of Hepap230 and B16p230 tumor cells. The growth of Hepal-6 and B16 is not inhibited by the fusion protein. Example 9 (FL / Fc / HER2Fv), CO2Q / FL · (FL / FcCD20Fv) and Trail / FL (/ LF / ex / TZ / rm / tec) in vitro characterization. In this experiment, three types of assessment Bifunctional fusion proteins Her2 / FL, CD20 / FL and Trail / FL have tumor suppressive effects on tumor cells in vitro. The results confirmed that Her2 / FL, CD20 / FL, and Trail / FL have effective tumor suppressor activity, and are similar to Herceptin, rituximab, and Trail in some cases. 1. Inhibitory effect of Her2 / FL bifunctional fusion protein on tumor cell growth 89753 -64- 200427698 Use · The cell line SK-BR-3 was obtained from the International Joint Cancer Society (Shanghai, China). The cell lines BT-474, D2F2, and 4T1 were obtained from ATCC. The cell line D2F2 was transfected with the human her2 gene to produce the D2F2 / E2 cell line. The cell line 4T1 line was transfected with the her2 gene to produce a 4Tlher2 cell line. When measured by flow cell count analysis, the her2 antigenic line appears to a high degree on the cell surface of the cell lines D2F2 / E2 and 4Tlher2. 2. Cells in logarithmic growth stage (SK-BR-3, BT-474, D2F2, 4T1, D2F2 / E2 or 4Tlher2) were digested with 0.05% trypsin and 0.02% EDTA, and then washed with PBS containing 1% FBS for two Times. Cells were resuspended in 1640 / DMEM plus 10% FCS / and adjusted to 6 × 104 cells / ml. Add the cell suspension to a 96-well plate (100 μl / well) and serially dilute with her2 / FL fusion protein or the positive control group Herceptin at 37 ° C at 7% C02 was cultured for 7 days. The proliferative line of the tumor cell line was determined using CellTiter96 aqueous non-radioactive cell proliferation test (Promega) according to the manufacturing instructions. The ED50 value of the fusion protein or Herceptin was calculated using the four-parameter algorithm 7 = foreign sentence weight / QD7 + 10,000. The results are shown in Figure 48, and show that her2 / FL and Herceptin lines effectively inhibit the growth of SK-BR-3, BT-474, D2F2 / her2, and 4Tl / her2 tumor cells. The growth of D2F2 and 4Ή cells is not inhibited by fusion proteins or Herceptin. 3 The cytotoxicity of fine muscle fusion protein to tumor cells. The cell line SK-BR-3 was obtained from the International Joint Cancer Society (Shanghai, China). The cell lines π ·, Li] were obtained from ATCC. The cell line grade was transfected with the human plutonium gene to generate the D2F2 / E2 cell line. Cell line His line was stained with the human ㈣ gene car τ ′.. Field t ΉΤ1 stomach was determined by flow cell counting analysis, her2 89753 -65- 200427698 antigen line was highly expressed in cell line D2F2 / E2 and 4TlHer2 on the cell surface. Cells in logarithmic growth stage (SK-BR-3, BT-474, D2F2, 4A, D2F2 / E2 or 4TlHer2) were digested with 0.05% trypsin and 0.02% EDTA, and then washed twice with PBS containing 1% FBS. Cells were resuspended in 1640 / DMEM plus 10% FCS and adjusted to 6 X 104 cells / ml. The cell suspension was added to a 96-well plate (100 μl / well) and serially diluted with Her2 / FL fusion protein or a positive control group (Herceptin) at 37 ° C at 7 % C02 for 7 days. The cytotoxicity of Her2 / FL and Herceptin was measured using CytoTox 96 non-radioactive cytotoxicity test (Promega). According to the manufacturing instructions, the ED50 value of the fusion protein or Herceptin was calculated using a four parameter algorithm To get. The results are shown in Figure 49, and show that her2 / FL and Herceptin can effectively induce cytolysis of SK-BR-3, BT-474, D2F2 / E2, and 4Tlher2 tumor cells. Neither her2 / FL or Herceptin could cause lysis of D2F2 and 4T1 cells. 4. Cytotoxicity of CD20 / FL fusion protein to tumor cells. Cell line Raji was obtained from ATCC. Raji cells in logarithmic growth phase were washed twice with 10% FBS in PBS. Cells were resuspended in 1640 / DMEM plus 10% FCS and adjusted to 2 X 105 cells / ml. Add the cell suspension to a 96_ well plate (100 μl / well) and serially dilute with CD20 / FL fusion protein or positive control rituximab at 37 ° C at 7% Incubate at C02 for 7 days. The cytotoxicity of CD20 / FL and rituximab was measured using CytoTox 96 non-radioactive cytotoxicity test (Promega) according to the manufacturing instructions. The results are shown in Fig. 50, and it is shown that CD20 / FL and the Rituximab system effectively killed Raji tumor cells 89753 -66- 200427698. 5. Inhibitory effect of Trail / FL bifunctional fusion protein on tumor cell growth. Cell lines L929, MDA-MB-231 and U-138MG were obtained from ATCC. The cell line Renca is obtained from the Korea Cancer Institute. Cells in logarithmic growth phase (L929, MDA-MB-231 or Renca) were digested with 0.05% trypsin and 0.02% EDTA, and then washed twice with PBS containing 1% FBS. The cells were resuspended in 1640 / DMEM plus 10% FCS and adjusted to 5 X 105 cells / ml. Add the cell suspension to a 96-well plate (100 μl / well) and incubate with Trail / FL fusion protein or the serial dilution of the positive control Trail at 37 ° C in 7% C02 for 12 hours . The proliferation of tumor cells was measured using CellTiter96 aqueous non-radioactive cell proliferation assay (Promega) according to the manufacturing instructions. The results are shown in Figure 51 and show that Trail / FL inhibits the growth of L929, MDA-MB-231 and Renca tumor cells, similar to Trail. Neither Trail / FL nor Trail inhibited the growth of negative control cells U-138MG. This confirms that the inhibitory effects of Trail / FL and Trail are specific. 6. Cytotoxicity of Trail / FL fusion protein · M929 L929 and U-138MG cells were digested with 0.05% trypsin and 0.02% EDTA, and then washed twice with PBS containing 10% FBS. The cells were resuspended in 1640 / DMEM plus 10% FCS and adjusted to 5 X 105 cells / ml. Add the cell suspension to a 96-well plate (100 μl / well) and incubate with Trail / FL fusion protein or a positive dilution of the control trail in serial at 14 ° C at 37 ° C for 14 or 16 hours. Trail / FL and Trail cytotoxicity were measured using CytoTox 96 non-radioactive cytotoxicity test (Promega) according to the manufacturing instructions. The ED50 value of the fusion protein or Herceptin was calculated using the four-parameter algorithm 89753 -67- 200427698. The results shown in Figure 52 show that the Trail / FL and Trail lines effectively induced lysis of L929 cells. However, neither Trail / FL nor Trail could cause cytolysis of control U-138MG cells. Example 10 In vivo antitumor activity of chSM / FL and huSM / FL The proteins used in these experiments include: SM5-1 chimeric antibody (chSM); SM5-1 humanized antibody (huSM); chSM / FL double Functional fusion protein; huSM / FL bifunctional fusion protein; anti-CD3 chimeric antibody-FL fusion protein (chCD3 / FL); anti-CD3 humanized antibody-FL fusion protein (huCD3 / FL). Female C57BL / 6 mice were injected subcutaneously with B16, Hepal-6, B16p230 or hepap230 tumor cells. When the tumors reached a diameter of 0.5 cm, the mice were randomly divided into seven groups, each with ten mice. Six groups of mice were injected intravenously with chCD3 / FL, huCD3 / FL, chSM, huSM, chSM / FL, or huSM / FL at a dose of 4 mg / kg / week for 6 weeks. The group of mice injected intravenously with PBS was the negative control group. Tumors were found to degenerate after treatment. The experimental results (shown in Table 3) show that chSM, huSM, chSM / FL, and huSM / FL are effective in causing tumor regression of the p230 antigen. The FL fusion protein line significantly enhances the antitumor activity of chSM or huSM antibodies. Table 3. Anti-CD3 / FL fusion protein anti-SM5-1 antibody anti-SM5-1 / FL fusion protein PBS after treatment of tumor degenerative cell line Hepal-6 0/10 0 / 10 0/10 0/10 0/10 0/10 0/10 Hepal-6 / p230 0/10 0/10 7/10 8/10 10/10 10/10 0/10 B16 0/10 0/10 0/10 0/10 0/10 0/10 0/10 B16 / p230 0/10 0/10 8/10 7/10 10/10 10/10 0/10 89753 -68- 200427698 Example 11 Borrowed in vivo Specific tumor immune response caused by chSM / FL and huSM / FL The proteins used in these experiments include: SM5-1 chimeric antibody (chSM); SM5-1 humanized antibody (huSM); chSM / FL dual function Fusion protein; huSM / FL bifunctional fusion protein; anti-CD3 chimeric antibody-FL fusion protein (chCD3 / FL); and anti-CD3 humanized antibody-FL fusion protein (huCD3 / FL). Female C57BL / 6 mice were injected subcutaneously with B16p230 or hepap230 tumor cells. When the tumors reached a diameter of 0.5 cm, the mice were randomly divided into seven groups with 8 mice each. Six groups of mice were injected intravenously with chSM, huSM, chSM combined with FL, and F; huSM, chSM / FL, or huSM / FL combined with L at a dose of 4 mg / kg / week for 6 weeks. The group of mice injected intravenously with PBS was the negative control group. Tumors were found to degenerate after treatment. The experimental results (shown in Table 4) show that the administration of FL chSM (or huSM), compared to chSM (or huSM) alone, exhibited antitumor activity. The bifunctional fusion protein chSM (or huSM) showed the strongest antitumor activity in this study. Table 4. Antitumor activity of bifunctional fusion proteins _ Treatment of tumor regression The tumor neoplasms from the second challenge did not exist chSM 5/10, 5/10, 6/10 0/10 huSM 4/10, 5/10, 4/10 0/10 chSM + FL 6/10, 6/10, 8/10 6/10 huSM + FL 7/10, 5/10, 6/10 6/10 chSM / FL 10/10, 8/10 , 10/10 28/30 huSM / FL 10/10, 10/10, 10/10 30/30 In order to determine whether the tumor degradation caused by the fusion protein will cause the generation of an active antitumor immune response, mice (such as Those who received the fusion 89753-69-200427698 protein by intravenous method), using maternal tumor cells, such as B16p230 or hepap230 cells, were inoculated again and stimulated subcutaneously. Tumor regression was found after vaccination. The results (shown in Table 5) show that chSM or huSM do not cause an active antitumor immune response. However, chSM / FL and huSM / FL will blame the active antitumor immune response to resist the maternal tumor, and the tumor neoplasm from the second challenge of tumor cells will not exist. Table 5. Treatment of tumor neoplasms from the second challenge by the presence of an active anti-tumor immune response cell line with a bifunctional fusion protein. B16 Hepal-6 B16 / p230 chSM / FL 0/6 5/6 JB16 / p230 huSM / FL 0/6 5/5 Hepal-6 / p230 chSM / FL 5/5 0/5 Hepal-6 / p230 huSM / FL 5/5 1/5 To determine the specificity of the anti-tumor immune response found in the above experiments Therefore, mice with B16p230 tumors that have been treated with the fusion protein and have been degraded are stimulated with B16 or hepal-6 tumor cells. Mice bearing hepap230 tumors that had degenerated after treatment with the fusion protein were again challenged subcutaneously with Hepal-6 or B16. Tumors were found to degenerate after treatment. The results (shown in Table 5) show that B16 tumors were excluded in mice in which B16p230 tumor degradation had been induced, but hepal-6 tumors grew progressively in mice in which B16p230 tumor degradation had been induced. . In other experiments, hepal-6 tumors were ruled out in mice in which hepap230 tumor regression had been induced, but B16 tumors grew progressively. These results confirm that the antitumor immune response caused by the bifunctional fusion protein is specific for the tumors that were given during this challenge 89753 -70- 200427698. Example 12

The antitumor activity of Her2 / FL, CD20 / FL, Trail / FL fusion proteins in vivo is to study the in vivo antitumor activity of bifunctional fusion proteins constructed by antibodies or molecules that fuse FL to other cells that cause tumor cells to wither. Activity, so the following experiments were completed. The experimental results confirmed that the bifunctional fusion proteins constructed by FL fusion to anti-he: r2 mAb, anti-CD20 mAb, or Trail all inhibited tumor growth. The antitumor activity of L her2 / FL in βτ body, k fluoride% preparation bismuth service, and BT474 were obtained from ATCC. Male Balb / c hairless mice were obtained from the Experimental Animal Center (Shanghai, China). Balb / c hairless mice were injected subcutaneously with 5 × 106 BT-474 tumor cells. When tumors reached a diameter of 0.5 cm, mice were randomly divided into experimental and control groups, each with ten mice. Mice in the experimental group were injected with her2 / FL intravenously at a dose of 10 mg / kg / week for 6 consecutive weeks. Control mice were injected intravenously with PBS. Continuous tumor growth was observed in all animals over 6 weeks. Statistical analysis of differences was performed using the Student's t test. The results (shown in Figure 53) show that treatment with the her2 / FL fusion protein has a highly significant antitumor activity (ρ $ 0.038). 2. CD20 / FL 于 活 # 绔 之 # Grinding tumor was obtained from ATCC. Female Balb / c hairless mice were obtained from the Experimental Animal Center (Shanghai, China). Balb / c hairless mice were irradiated with 2GY once a week for 3 consecutive weeks. Then 89753 -71-200427698, irradiated hairless mice were injected subcutaneously with 2xl07 Raji tumor cells. When the tumor reached a diameter of 15 cm, the mice were randomly divided into experimental and control groups, each with ten mice. The experimental group of mice was injected with CD20 / FL intravenously, at a dose of 10 mg / kg / week, continuously. 6 weeks. Control group mice were injected intravenously with PBS. Continuous tumor growth was observed in all animals for 6 weeks. Statistical analysis of the differences was performed using the Student's t test. The results (shown in Figure 54) are shown Treated with CD20 / FL fusion protein, it has highly significant antitumor activity (p $ 0.03). 3. Trail / FL antitumor activity in vivo. Ruler v. V. General details, QYG system are obtained from International Joint Cancer Society (Shanghai, China). Female Balb / c hairless mice were obtained from the Experimental Animal Center (Shanghai, China). Balb / c hairless mice were injected subcutaneously with lxlO7 QYC tumor cells. When the tumor reached a diameter of 0.5 cm, The mice were randomly divided into experimental and control groups, each with ten mice. The experimental group of mice was injected intraperitoneally with Tmil / FL at a dose of 10 mg / kg / week for 6 consecutive weeks. Mice were injected intravenously with PBS. Continuous tumor growth was observed in all animals over a period of 6 weeks. Statistical analysis of the differences was performed using the Student's t test. The results (shown in Figure 55) show that the Trail / FL fusion protein Treatment with highly significant antitumor activity (ρ $ 0.039). Example 13 Specific tumor immune response caused by her2 / FL, CD20 / FL and Trail / FL 1. Specific tumor immune response caused by her2 / FL · ΒΆνοΙζ Ben Yuan, Zhi Lao 89753 -72- 200427698 The mouse breast cancer cell lines D2F2 and 4T1 are derived from ATCC. The cell line D2F2 / E2 is a cell line D2F2 transfected with the human her2 gene. The cell line 4Tlher2 is a her2 gene The transfected cell line 4T1. The her2 antigen line is highly expressed on the cell surfaces of the cell lines D2F2 / E2 and 4Tlher2. The D2F21E2 and 4Tlher2 tumor cell lines develop subcutaneous tumors in Balb / c mice. D2F2 / E2 and tumors Growth in mice was effectively inhibited by anti-her2 mAb. Female Balb / c mice were injected subcutaneously with D2F2, 4T1, D2F2 / E2 or 4Tlher2 tumor cells. When the tumor reached a diameter of 0.5 cm, Mice inoculated with tumor cells were randomly divided into five groups of 8 mice each. The rats were injected intravenously with FL, anti-her2 mAb, and anti-her2 mAb or huSM / FL in combination with FL at 4 mg / kg / week. At the dose, for 6 consecutive weeks, the group of mice injected intravenously with PBS was the control group. Continuous tumor growth was observed in all animals over 6 weeks. The experimental results (shown in Table 6) show that the bifunctional fusion protein her2 / FL has the ability to inhibit the growth of D2F2 / E2 or 4Tlher2, which is equivalent to anti-her2 mAb. Mice bearing D2F2 / E2 or 4Tlher2 tumors that had degenerated after treatment with fusion protein or mAb were again subcutaneously stimulated with maternal tumor cells. Continuous tumor growth was observed in all animals over 6 weeks. The results (shown in Table 6) show that anti-her2 mAb does not effectively induce an active immune response. However, her2 / FL defies an active immune response to fight maternal tumors. 89753 -73-200427698 Table 6. Inhibition of tumor growth by bifunctional fusion proteins. Number of animals with tumors treated. Tumor degradation cure rate after treatment (%). Number of animals with tumors after second challenge with tumor rate ( %) PBS 8 0 0 8 100 FL 16 4 25 14 87.5 anti-her2 mAb 16 13 81 16 100 anti-her2 mAb + FL 16 14 87 12 75 her / FL 24 21 87 2 8 The D2F2 / E2 mice were once again stimulated subcutaneously with .D2F2 or 4T1 tumor cells. Mice bearing 4Tlher2 tumors that had degenerated after treatment with fusion protein were again challenged with D2F2 or 4T1 tumor cells. Continuous tumor growth was observed in all animals over 6 weeks. The results (shown in Table 6) show that D2F2 tumors were excluded from the mouse in which D2F2 / E2 tumors had been degraded, whereas 4T1 tumors grew progressively. In another experiment, 4T1 tumors were excluded in mice in which 4Tlher2 tumor regression had been induced, whereas D2F2 tumor lines grew progressively. These results confirm that the antitumor immune response caused by the bifunctional fusion protein is tumor-specific. 2. Active tumor immune response by CD20 / FL. The cell line A20 was obtained from ATC C. The cell line A20 / CD20 line was produced via transfection of the D2F2 cell line with the human CD20 gene. When measured by flow cell count analysis, the CD20 antigenic line appears to a high degree on the cell surface of A20 / CD20 cells. The A20 / CD20 tumor cell line develops subcutaneous tumors in Balb / c mice. 89753 -74- 200427698 A20 / CD20 tumor growth in mice was effectively inhibited by anti-CD20 mAb treatment. Female Balb / c mice were injected subcutaneously with 2 × 10 06 A20 / CD20 tumor cells. When the tumors reached a diameter of 0.5 cm, the mice were randomly divided into arrays with 8 mice each. Mice were injected intravenously with FL, anti-CD20 mAb, and anti-CD20 mAb or CD20 / FL in combination with FL at a dose of 4 mg / kg / week for 6 consecutive weeks. Mice injected intravenously with PBS were the negative control group. Continuous tumor growth was observed in all animals over 6 weeks. The experimental results (shown in Table 7) show that the bifunctional fusion protein CD20 / FL has the ability to inhibit the growth of A20 / CD20 tumors, which is equivalent to anti-CD20 mAb treatment. Mice bearing the 'A20 / CD20 tumor that had been degraded by treatment with a fusion protein or mAb were again subcutaneously challenged with maternal tumor cells. Continuous tumor growth was observed in all animals over 6 weeks. The results (shown in Table 7) show that anti-CD20 mAb does not induce an active antitumor immune response. However, CD20 / FL will blame an active immune response to combat maternal tumors. Table 7. Number of animals with tumor treated with active anti-tumor immune response induced by CD20 / FL. Tumor regression cure rate after treatment (%) Number of animals with tumor after second challenge with tumor rate (%) PBS 8 0 0 8 100 FL 16 4 25.0 14 87.5 Anti-CD20 mAb 12 10 83.3 12 100 Anti-CD20 mAb + FL 14 12 85.7 10 71.4 CD20 / FL 20 18 90.0 2 10.0 89753 -75- 200427698 3. Caused by Trail / FL Active tumor immune response · Knowledge, kwzdi you, available from Korea Cancer Institute. Female Balb / c mice were injected subcutaneously with Renca tumor cells. When the tumors reached a diameter of 0.5 cm, the mice were randomly divided into arrays with 8 mice each. Mice were injected intravenously with FL, Trail, and Trail or Trail / FL in combination with FL at a dose of 4 mg / kg / week for 6 consecutive weeks. The group of mice injected intravenously with PBS was the control group. Continuous tumor growth was observed in all animals over 6 weeks. The experimental results (shown in Table 8) show that the bifunctional fusion protein Trail / FL has the ability to inhibit Renca tumor growth, which is equivalent to Trail. Mice bearing Renca tumors that degenerate after treatment with fusion protein or Tmil were again subcutaneously challenged with maternal tumor cells. Continuous tumor growth was observed in all animals over 6 weeks. The results (shown in Table 8) show that Trail does not effectively induce an active immune response. However, Trail / FL will spit out an active immune response to fight maternal tumors. Table 8. Number of animals with tumor treated with active anti-tumor immune response induced by Tmil / FL. Tumor regression cure rate after treatment (%) Number of animals with tumor after second challenge with tumor rate (%) PBS 8 0 0 8 100 FL 16 5 31.3 14 87.5 Anti-CD20 mAb 14 10 71.4 14 100 Anti-CD20 mAb + FL 14 12 85.7 10 71.4 CD20 / FL 18 17 94.4 2 11.1 In summary, my research has confirmed the dual function fusion protein Not only will it induce 89753 -76- 200427698 to cause tumor regression in vivo, but it will also induce a strong active antitumor immune response to resist subsequent maternal tumors. Example 14: Immunohistochemical analysis of tumors To further illustrate the mechanism of SM / FL and hSM / FL fusion proteins, immunohistochemistry of tumors was performed on mice treated with fusion proteins. In these experiments, most tumor cell lines were killed after administration of chSM / FL and hSMM / FL fusion proteins. The tumor line is surrounded by a wide range of infiltrates of DC, NK, or other lymphocytes, showing that chSM / FL and huSM / FL fusion proteins will cause DC and NK cells to aggregate in tumor tissues and mediate or help kill tumor cells * 1. Vaccination and tumor growth. Materials used in these experiments include the Lipidamine 2000 Transformation Kit (Invitrogen); HepaP230 tumor cell line. Hepa / P230 cells were digested with 0.05% trypsin and 0.02% EDTA and adjusted to 2.7 x 107 cells / ml. Hepa / P230 cells were inoculated subcutaneously into C57BL / 6 mice with 200 microliters of tumor cell suspension. When the tumor reached a diameter of 0.5 cm, the rats were injected intravenously with chSM / FL at a dose of 4 mg / kg / week for 3 consecutive weeks. Continuous tumor growth was observed in all animals. Immunohistochemical analysis of tumor samples was performed after treatment. 2. Ke Shou, group iron permitting points # (HE Attack β / Immunohistochemical analysis by HE staining, performed using standard methods. In short, the tumor samples were fixed in 10% formalin for 24 hours, And embedded in paraffin. Then, 4-micron thick sections were stained with hematoxylin and eosin. The results showed that FL alone did not significantly kill tumor cells. 89753 -77- 200427698 However, the killer cells found The degree will increase when SM5-1 chimeric or humanized Mb and FL are used together. At the same time, some infiltrates of lymphocytes containing DC, NK, T cells and B cells are found in and surround the tumor tissue It is worth noting that the SM / FL fusion protein will cause tumor cells to lyse in vivo and cause extensive infiltration of lymphocytes into the tumor mass, while the control fusion protein means (anti-CD3mAb / FL) No. This indicates that the SM / FL fusion protein has the ability to effectively cause DC, NK and other lymphocytes to aggregate at the tumor site in vivo. The results are shown in Table 9. Table 9: chSM / FL Guangdong huSM / FL Tumor immunity after administration of fusion protein Weaving chemical analysis results of tumor necrosis (50 X) NK DC TB anti-CD3 mAb / FL · + • + + + FL + ++ ++ ++ chSM +++ •--huSM ++++ +-_ chSM + FL +++ ++ ++ ++ + huSM + FL +++ ++ ++ +++ + chSM / FL ++++ ++++ ++++ ++++ ++++ huSM / FL ++++ ++++ ++++ ++++ ++++ 3 · The tumor recurrence after administration of her2 / FL, CD20 / FL or TraWFL fusion protein # In order to further explain the mechanism of other fusion proteins, the immunohistochemical analysis of tumors was performed as described above, which was excised from the treatment of her2 / FL, CD20 / FL or Trail / FL fusion proteins with D2F2 / E2, A20 / CD20 or Renca rats. The results are shown in Table 10. 89753 -78- 200427698 Table 10. Immunohistochemistry of tumors after administration of her2 / FL, CD20 / FL or Trail / FL fusion proteins. ^ Analysis and treatment of tumor necrosis results (50 X) NK DC TB anti-CD3 mAb / FL + + + + FL + ++ ++ ++ ++ anti-HER2 mAb +++---primary antibody CD20 mAb ++++ + one order TRAIL ++ + + + ++ + Anti Her2 + FL +++ ++ ++ ++ + Anti-CD20 + FL +++ ++ ++ +++ + TRAIL + FL ++ +++ +++ +++ ++ + Anti Her2 / FL ', ++++ +++++ ++++ ++++ ++++ Anti-CD20 / FL +++++ ++++ ++++ ++++ ++++ TRAIL / FL + +++ ++++ ++++ +++ ++++

The results showed that chSM / FL, huSM / FL, her2 / FL, CD20 / FL, and TRAIL / FL fusion proteins inhibit tumor cell growth by enhancing and activating reflection. The fusion protein causes NK and DC cells to aggregate at the tumor site, and DC, NK, and other lymphocyte lines exert their antitumor activity. Example 15 In vivo biodistribution of fused I white matter To study the specific binding of chSM / FL or huMM / FL to tumor cells, the biodistribution characteristics of the fusion protein were examined. Mice bearing B16p230 tumors were individually intravenously injected with 1251_ identified SM, chSM / FL, huSM, and huSM / FL. After 48 hours, the selected organs were immediately removed and radioactivity was measured. The results (shown in Figure 56) show the biodistribution of chSM / FL and hSM / FL fusion proteins 89753 -79- 200427698, similar to the desired mAb chSM or humanized mAb huSM. The fusion protein all retains the specificity of its parent mAb and is highly concentrated at the tumor site. The biodistribution of mAbs and fusion proteins depends on their specificity, which is an important factor for clinical applications. This specific tissue distribution will reduce the amount of drug needed to achieve the desired effect; and reduce harm to non-standard tissues. In vivo distribution characteristics of her2 / FL, CD20 / FL, and TRAIL / FL fusion proteins were also examined. Mice bearing 4Tl / her2, A20 / 20, and Renca tumors were injected intravenously with 1251 / her2 / FL, CD20 / FL, TRAIL / FL, and huSM / FL. After 48 hours, the selected organs were immediately removed and radioactivity was measured. The results of the study (shown in Figure 57) show that her2 / FL, CD20 / FL, and TRAIL / FL fusion protein lines are localized at tumor locations, similar to chSM / FL and hSM / FL. Example 16 Inherited immunotherapy using tumor-specific lymphocytes HepaP230 or B16p230 cells were digested with 0.05% trypsin and 0.02% EDTA and adjusted to 2.7 x 107 cells / ml. Hepal-6 / P230 or B16 / P230 cells were subcutaneously inoculated into 200 μl tumor cell suspensions into C57BL / 6 mice. When the tumor reached a diameter of 0.5 cm, mice were injected intravenously with chSM / FL at a dose of 4 mg / kg / week for 3 consecutive weeks. Continuous tumor growth was observed in all animals. Mice that had been treated with the fusion protein chSM / FL or huSM / FL and in which tumor hepap230 or B16p230 had deteriorated were sacrificed, and the spleen was collected. Spleen cells were isolated and adjusted to 1.0 x 10 cells / ml. Then, innocent mice were injected with 5.0 × 10 07 spleen cells obtained from the mice. Hepap230 or B16p230 tumor regression had occurred in the mice described below, and they were individually challenged with hepap230 or B16p230 89753 -80- 200427698 tumors. Continuous tumor growth was observed in all animals over 6 weeks. The results (shown in Table 11) show that mice using spleen cells obtained from mice treated with the fusion protein chSM / FL or huSM / FL and in which tumors hepap230 or B16p230 had degenerated spleen cells were excluded from maternal tumors. Obtained from the non-fusion protein combination, that is, chSM, huSM, FL, chSM combined with FL or huSM combined with FL, the metastasis of spleen cells in treated mice failed to cause tumor exclusion in recipient mice. These results indicate that metastatic lymphocytes initiate a specific anti-tumor immune response, and that this specific immune response relies on DC and NK cells. Table 11. Treatment of spleen cell donors with inherited immunotherapy using tumor-specific lymphocytes. Number of recipients. Mortality after transfusion. Hepap230 B16p230 Anti-CD3 mAb / FL 15 15/15 15/15 FL 15 9/15 10/15 chSM 15 12/15 14/15 huSM 15 13/15 14/15 chSM + FL 15 10/15 10/15 huSM + FL 15 10/15 10/15 SM / FL 15 0/15 1/15 hSM / FL 15 1 / 15 0/15 The results also show that the antitumor mechanism of chSM / FL and huSM / FL fusion proteins is based on a specific active tumor immune response. 4Tl / her2, A20 / 2O, and Renca cells were digested with 0.05% trypsin and 0.02% EDTA, and adjusted to 2.7 x 107 cells / ml. 4Tl / her2, A20 / 20 or Renca cells were subcutaneously inoculated into mice with 200 microliters of tumor cell suspension. When tumors reach 0.5 cm in diameter, mice are injected intravenously with her2 / FL, CD20 / FL or 89753 -81-200427698

Trail / FL at a dose of 4 mg / kg / week for 3 consecutive weeks. Continuous tumor growth was observed in all animals. Mice treated with the fusion protein her2 / FL, CD20 / FL or Trail / FL and in which tumors 4Ή / 1 ^ 2, A20 / 20 or Renca cells had degenerated were sacrificed, and the spleen was collected. Spleen cells were isolated and adjusted to 1.0 × 10 9 cells / mL. Then, innocent mice were injected with 5.0 x 107 spleen cells obtained from mice in which Tl / her2, A20 / 20, or Renca tumors had developed, followed by individual stimulation with 411/1 ^ 2, A20 / 20, or Renca tumors. Continuous tumor growth was observed in all animals over 6 weeks. The results (shown in No. 11) show that mice obtained from spleen cells treated with the fusion protein chSM / FL or huSM / FL and in which the spleen cells of the degenerate tumor hepap230 or B16p230 had developed were excluded from maternal tumors. The results (shown in Table 12) are consistent with chSM / FL and huSM / FL. It is shown that chSM / FL, huSM / FL, her2 / FL, CD20 / FL, and Trail / FL are activated by lymphocytes and mediate anti- Tumor activity. Table 12. Treatment of spleen cell donors with activated anti-tumor lymphocytes Number of recipients Mortality after transfusion * Anti-CD3 mAb / FL 15 15/15 FL 15 9/15 Anti-Her2 mAb 15 11/15 Anti-Her mAb + FL 15 9/15 HER2 / FL 15 4/15 Anti-CD20 mAb 15 13/15 Anti-CD20 mAb + FL 15 10/15 CD20 / FL 15 2/15 89753 -82- 200427698 TRAIL 15 8/15 TRAIL + FL 15 10/15 TRAIL / FL 15 5/15 * Cell lines: 4Tl / her2, A20 / 20 and Renca cell lines are used individually in Her2, CD20, and TRAIL related experiments. Many modifications and variations of the present invention can be implemented without departing from its spirit and scope, as those skilled in the art will understand. The specific specific embodiments described herein are presented by way of example only, and the present invention is intended to be limited in view of the scope of patents attached to the article, and the scope of equivalents accompanied by such scope of patents as titles ; And the invention is not limited to the specific embodiments that have been presented by way of example herein. The citation of the above-mentioned bulletin or document is not intended to recognize any of the foregoing as a related prior art, and the content or date of such bulletin or document does not constitute any recognition. The U.S. patents and other publications cited herein are hereby incorporated herein by reference. [Schematic description] Figure 1 shows the structure of (A) a tetravalent bispecific antibody and (B) a FLex / Fc / Fv bifunctional fusion protein. Figure 2 shows the nucleotide sequence (sequence identification code: 1) and amino acid sequence (sequence identification code: 2) of hFLex. SP, message peptide. Figure 3 shows the nucleotide sequence (sequence identification code: 3) and amino acid sequence (sequence identification code: 4) of FL / Fc. SP, message peptide. Figure 4 shows the nucleotide sequence of the linker (Gly4Sei03) (sequence identification code: 5) and amino acid sequence (sequence identification code: 6). Figure 5 shows the anti-p230 antibody (SM5-1) variable region gene The PCR products were split on an agarose gel on a 1% agar 89753 -83- 200427698. The figure shows mSM5-l heavy chain variable region, *: 7) and amino acid sequence (sequence sequence (sequence) Figure 7 shows the identification code, light chain variable = · 8) ,, and peptide.: 9) Sequence with amino acid (sequence identification code 3: ^. Reading sequence (sequence identification code Figure 8 shows deletion chimerism) (QiSM) money identification code: 11) and amino acid sequence (sequence identification code: 12). :: broad-sequence termination, translation stop codon. The shaded area indicates the insertion sequence., ...; • = 2M chimeric antibody The nuclear sequence of the light chain (sequence recognition sequence and amino group sequence (sequence identification code: 14)) osp, message peptide ^ #stop codon. I, Figure 10 shows the chimeric heavy chain expression vector Table W. It shows the region encoding the expression vector: HCMVpr0m, the human cytomegalovirus is an early promoter of each other; vH, the heavy-bond variable region base of huSM ; Ch, human ^ 1 chain foot region base JU. BGHpA, bovine growth hormone polygland # acidification message, SV40on, replicator of simian virus 40 early promoter and source; DHFR, dihydrofolate reductase gene; pUC source, the source of the copied plasmid;-? refers to the endolactamase gene. Figure 11 shows a diagram of the SM5-1 chimeric light chain expression vector. Shows the regions of the vector that encode different functions ... HCMVprom, Human cytomegalovirus main immediate early promoter; VL, light chain variable region gene of huSM; cL, human K chain constant foot region gene; BGHpA, bovine growth hormone polyadenylation message; SV40 0ri, replicating ape Viral 40 early promoters and sources; DHFR, dihydrofolate reductase gene; pUC source, replicated plasmid source; Amp refers to / 3-in 89753-84- 200427698 amidase gene. Figure 12 shows smma Antibody (huSM) heavy chain variable, #second order (sequence identification code: 15) and amino acid order (sequence 2 (nucleotide cis-peptide. ^ Sequence "code: 16). Offense, news =, show people Nucleic acid sequence (sequence recognition) of the variable region of light chain of antibody (huSM) Code: 17) Sequence with amino acid (Sequence recognition: Polypeptide. It is called A-picture. It shows the weight of SMS-1 humanized antibody (huSM).) 4. Identification code, and amino acid sequence (Sequence Recognition: Gan: sequence (sequence termination, translation termination codons. The shaded area indicates the insertion sequence) column: p, message skin; .㈣ show _ humanized antibody light chain nucleus # acid sequence • 21) and amino acid sequence (Sequence recognition ^ Translation stop codon. ) SP, message peptide; termination, transposition Figure 16 shows the sequence of fenamic acid cis amino acid of HuSMVH / Fc / FL (sequence identification code: 24). sp material sequence center J code: 23) and codon. The shaded area indicates the insertion sequence. Translocation termination Figure Π shows the core code of h_H / Fc / link / FL: work and amino acid sequence (sequence identification code: 26). Sequence (Sequence recognition FIG. 18 shows FL / Fc / huSMFv Xigu 4 Yu Wei, 'ν << ribobismuth sequence (sequence acid sequence (sequence identification code: 28). Ma. 27) and amines Figure 19 shows lAb and The dirty image of the FLex fusion gene product shows C_VH / Fc / FL's nuclear acid 4. Amino acid sequence (sequence identification code, tropic, message month too; 1: 1: 29) and codons. The shaded area indicates the inserted sequence The figure of the handle termination 89753 -85- 200427698 shows the nuclear acid code of VH / Fc / bond / fl: 31) and amino acid sequence (sequence identification code: 32). (Sequence recognition map ~, not FL / Fc / chSMFv "Nuclear: y: acid sequence (cis recognition sequence (sequence recognition code · 34). (33) and amine Fig. 23 shows 2B8 heavy chain variable Nucleic acid sequence of the region (with amino acid sequence (sequence identification code U, message I phase code: 35)) Figure 24 shows the nucleotide sequence of the light chain variable region. With amino acid sequence (sequence identification code: 38) .sp, m ”code 37) Figure 25 shows the cis-sine code of the heavy bond of the anti-Gamma chimeric antibody: 39) and the amino acid sequence (sequence identification code: 4). The sequence does not translate the stop codon. The shaded area indicates the intervening sequence. The termination, Figure 26 shows the anti-CD20 respectable human translocation> Tu Yi; ^ amino acid; 二 amino acid sequence (sequence recognition Base &amp; sequence (sequence identifier n, message translation stop codon. Figure 27) Nucleic acid sequence (sequence: 3) and amino acid sequence (sequence 3) Sequence identification code: 44).% Message skin; second-day sale termination codon. The shaded area indicates the insertion sequence., ... Figure 28 shows the can sequence of ⑽0VH / Fc / link / FL (code .45 ) And amino acid sequence (sequence identification code: 46). B々 Figure 29 shows the nuclear acid sequence of HVFe / ⑽Fv (sequence identification code amino acid sequence (sequence identification code ·· 48). The structure of the fusion gene product CD20Ab and Kongyin χχ / Ι ^ / Ι; ν. Figure 31 shows the nuclear acid sequence (cis: 49) and amino acid sequence (order (Identification code: 50). SP, message month too .... 89753 -86- 200427698 ·: nucleotide sequence (cis.) Of the variable region of the two light chain her2 light chain (sequence identification code: 52) .Science information peptide. "] Code '~ ^ &amp; heir2 Humanized antibody heavy chain core #Acid sequence (sequence code: 53) and amino acid sequence (sequence identification code: 54) The translation stop codon. The shaded area indicates the insertion sequence. 'Termination, Figure 34 shows the acid sequence ((, .55) and amino acid sequence (sequence) of the light chain of the anti-her2 humanized antibody. Cardiopeptide, terminated, showing the nuclear #acid sequence of he_muscle heavy chain (cis: 58) 〇sp? Ti4 ^; stop region. The shaded area indicates the insertion sequence. Ze, Figure 36 shows the material acid sequence (cis.59) and amino acid sequence (sequence identification code: 60) of W // Fe / chain / fl. 0 force code picture 37 head FL / Fc / her2Fv Nucleus: acid sequence (sequence identification code a based acid sequence (sequence identification code: 62). Sp, message peptide; termination, translation :,: code. The shaded area indicates the insertion sequence. &Quot; ^ Figure 38 The composition of her2 Ab and FUx fusion gene product certificate ~ The nucleotide sequence of 1 ^ LeWTrailex sequence (sequence identification code · sequence of the moon acid sequence (sequence identification code: 64)). sp, message peptide; stop codon. Figure 4. Structure of the hFLex / Trailex fusion gene of Trail and FLex. Figure 41. Nucleotide sequence of π hFlex / IZ / Trailex (sequence recognition aminobismuth sequence (sequence identification code: 66). Sp, message peptide; termination "ririi 2 '得 咩 止 89753 -87- 200427698 Figure 42 The nucleotide sequence (sequence identifier: 67) and amino acid sequence (sequence identifier: 68) of hFLex / Fc / Trailex are shown. SP, message peptide; stop, translation stop codon. Figure 43 shows the fusion of Trail and FLex Structure of the gene hFLex / Fc / Trailex. Figure 44 shows the expansion effect of SM / FL on human cord blood CD34 (+) cells. Figure 45 shows the effects of chSM / FL and huSM / FL on NK and DC cells in vivo. Figure 44 46A shows the inhibitory effect of chSM / FL fusion protein on different cell lines in vitro. Figure 46B shows the inhibitory effect of huSM / FL on different cell lines in vitro. Random 47A shows various FL fusion proteins for B16 melanoma cell proliferation in vivo Figure 47B shows the inhibition of various FL fusion proteins of Hepal-6 cell proliferation in vitro. Figure 47C shows the inhibition of various FL fusion proteins of B16 / p230 cell proliferation in vitro. Figure 47D shows H Inhibition of various FL fusion proteins of epal-6 / p230 cell proliferation in vitro. Figure 48 shows her2 / FL (A) herceptin (B) in vitro. Figure 49 shows her2 / FL. (A) Cytotoxicity in vitro with herceptin (B). Figure 50 shows the cytotoxicity of CD20 / FL in vitro. Figure 51 shows Trail / FL (A) and Trail (B) for different cells. Inhibition of 89753 -88-200427698 in vitro. Figure 52 shows the cytotoxicity of Trail / FL (A) and Trail (B) in vitro. Figure 53 shows the effect of her2 / FL on tumor growth in vivo. Figure 54 Shows the effect of CD20 / FL on tumor growth in vivo. Figure 55 shows the effect of Trail / FL on tumor growth in vivo. Figure 56 shows the biological distribution of SM / FL and huSM / FL. Figure 57 shows her2 / FL, CD20 / FL And Trail / FL biodistribution. 89- 89753

Claims (1)

200427698 Patent and application patent garden: L hybrid protein, this chimeric protein contains Flt3 ligand or its biologically active fragment and proteinaceous or peptide-based tumor killer. 2. The chimeric protein according to item 丨 of the application, wherein the tumoricidal agent causes the cell to wither. 3. The chimeric protein according to item 1 of the scope of the patent application, wherein the Flt3 ligand or a biological / tongue fragment thereof stimulates the proliferation of hematopoietic stem or primitive particle cells. 4. The chimeric protein according to item 1 of the scope of patent application, wherein the Flt3 ligand or its biologically active fragment stimulates the proliferation of cells selected from bone-like precursor cells, monocytes, and macrophages B cells, dendritic cells, and NK cells 05. The protein is synthesized according to item 1 of the patent application scope, wherein the Flt3 ligand or its biologically active fragment is a mammalian chaotic 3-ligand. 6. The chimeric protein according to item 1 of the application, wherein the mammalian Flt3 ligand or its biologically active fragment is a human Flt3 ligand. 7. The chimeric protein according to item 1 of the scope of patent application, wherein the Flt3 ligand or a biologically active fragment thereof is a soluble Flt3 ligand. 8 'Chimeric protein according to item 1 of the scope of the patent application, wherein the Flt3 ligand contains less than 100 amino acid residues, and the sequence identification code of this Flt3 ligand pair is the amine group proposed in 2. The acid sequence has at least 40% identity, in which the percent identity is determined on the amino acid sequence of the same size as the amino acid sequence proposed in Sequence 2, and the Flt3 coordination system essentially retains its biological activity . • According to the desired protein in the scope of the patent application, the Flt3 ligand 89753 200427698 is bound to the antibody, and the antibody system specifically binds to the sequence identification code: 2 the amino acid sequence proposed in F2, and Flt3 is coordinated The system essentially retains its biological activity. 10. The chimeric protein according to item 1 of the scope of patent application, wherein the Flt3 ligand comprises a sequence identification code: the amino acid sequence proposed in 2. 11. The chimeric protein according to item 1 of the scope of patent application, wherein the Flt3 ligand contains at least 80% of the amino acid sequence of 28 to 128 identical to the sequence identification code: 2. 12. The chimeric protein according to item 1 of the scope of the patent application, wherein the Flt3 ligand comprises a sequence identification code: 2 of the amino acids 28 to 128. 13. The chimeric protein according to item 1 of the scope of patent application, wherein the Flt3 ligand comprises an amino acid sequence selected from amino acid residues 28-160 including the sequence identification code: 2 and the sequence identification code: 2 of the Amino acid residues 28-182. 14. The chimeric protein according to item 1 of the application, wherein the tumoricidal agent is an antibody. 15. The chimeric protein according to item 14 of the patent application, wherein the anti-system is selected from the group consisting of intact antibodies, Fab fragments, Fab1 fragments, F (ab ') 2 fragments, FV fragments, diabody, single chain antibodies, and antibody fragments Many specific antibodies are formed. 16. The chimeric protein according to item 14 of the application, wherein the anti-system is selected from the group consisting of anti-p230 antibody, anti-CD29 antibody, anti-Her2 antibody, anti-Her3 antibody, anti-Her4 antibody, anti-EGFR antibody or Its biologically active fragment. 17. The chimeric protein according to item 14 of the application, wherein the antibody is a human or humanized antibody. 18. The chimeric protein according to item 1 of the scope of the patent application, wherein the tumoricidal agent 89753 200427698 is selected from the group consisting of Fas ligand, Δττ ,, iNr TRAIL or its biologically active extracellular functional site. 19. According to the scope of the patent application, proteins are synthesized after μ, in which the ligand system is located at the terminus of the chimeric protein. 20. The protein is synthesized after the first item of the scope of the patent application, in which the inhibitory ligand system is located at the C-terminus of the chimeric protein. According to 21 patents, the scope of patent application! The chimeric protein of the item, in which the self-relying body and the tumoricidal system are separated by a linker peptide. 22. According to the scope of application for patent No. 21 (Gly4Ser) 3. Brother 1 4 Loss-of-synthesis protein, of which the linking peptide is 23. Gen. Patent Application No. 1 of the Fanyuan. 24. Synthetic protein, which contains the sequence identification code. 24. Sequence identification code: 26. Other code: 30. Sequence identification. Code .3; Sequence J code: 28, Sequence recognition 〇1 [^. Ma. 2 Sequence recognition code: 34, Sequence power! Code. 44, sequence recognition /, catty 4 other code: 58, sequence ^ sequence identification code: 48, sequence identification code. 64 code. 60, sequence identification code: 62, sequence 1 M code, 64, sequence identification &quot; Sequence 4 amino acid sequence. The U sequence is presented in Code: 68. 24. An early-leaving nucleic acid comprising a nuclear acid sequence encoding a root chimeric protein. 25 of item 1 of the patent cadre · According to the item of the patent application fan garden:-, sequence center :, sequence identification code: 27, sequence identification code: criminal, sequence identification code: sequence identification code ^ sequence identification code · zl7,: 57, sequence identification code: 59, sequence 1, ^ sequence identification code Ma. 61, sequence identification code 89753 200427698: 63, sequence identification code acid sequence. 65 or sequence identification code · 67 nucleosides proposed in 67. 26 kinds of isolated nucleic acids, and 枋 # π g, /, $ $ $ according to the scope of the application for patents 24 "nucleon acid sequence complementary nuclear acid order. Gudi, 27 · —A vector containing a chimeric protein that encodes a gene called alpha target # 1, which encodes a nucleotide sequence of &lt; nucleotide sequence. 28. According to item 27 of the scope of the patent application, &lt; it further includes a performance modulation sequence, which can be operatively linked to a nucleic acid encoding a Flt3 ligand, and a proteinaceous or peptide-based tumoricidal agent. 29 · -recombinant cells' which contain a nucleic acid according to item 24 of the patent application. 30. Recombinant cells according to the scope of the patent claim ii, which are eukaryotic cells. 31. The recombinant cell according to item 3G of the patent application, which is a CH0, cos or NSO cell. 32. A method for manufacturing a chimeric protein, which comprises growing a recombinant cell containing a nucleic acid according to item 24 of the scope of the patent application, so that the encoded messenger protein is expressed by the cell, and recovering the expressed protein Aged protein. 33. The method of claim 32, further comprising isolating and / or purifying the recovered desired protein. 34 · —A product according to the method of claim 32 of the scope of patent application. 35. A pharmaceutical composition comprising an effective amount of a chimeric protein comprising a pit3 ligand and a proteinaceous or peptidic tumorigenic agent 'and a pharmaceutically acceptable carrier or excipient. 36. A kit comprising an effective amount of a chimeric protein comprising a Flt3 ligand and a proteinaceous or 89753 200427698 tumorimide, and a 7F device for administering the chimeric protein. 37. A method for treating neoplasms in mammals, the method comprising administering to a mammal in need or wanting such treatment an effective amount of a chimera comprising a Flt3 ligand and a proteinaceous or peptide-based tumorcide protein. 38. The method according to item 37 of the application, wherein the mammal is a human. 39. The method according to item 37 of the application, wherein the neoplasm is melanoma, breast cancer or hepatocellular carcinoma. 40. A combination comprising: a) an effective amount of a chimeric protein comprising a Flt3 ligand and a proteinaceous or peptidic tumor killer; and b) an effective amount of an antitumor agent. 41. The combination according to item 40 of the scope of patent application, wherein the anti-neoplastic agent is an agent for oral treatment of melanoma, breast cancer or hepatocellular carcinoma. 2_ A method for treating neoplasms in mammals, the method includes Or mammals that want such treatment administer an effective amount of a combination according to item 40 of the scope of the patent application. 43. A method for inducing the cavitation of vector cells by caspase j in a cell. This method includes administering an effective amount of a Flt3 ligand and a proteinaceous or peptidyl killer to a cell that requires or wants such induction Chimeric protein of a tumor agent 44. The method according to item 43 of the scope of patent application, wherein the cell is a mammal or a cell. The method according to the scope of the patent application, wherein the cells are milking animals 89753 200427698 neoplasm cells. 46. The method according to item 43 of the patent application, wherein the cell line is contained in a mammal. 47 —From &gt; .. A qualitative or response method that involves administering to a mammal in need or wanting such slander an effective amount of a vaccine according to item 47 of the scope of patent application. 49. A method for generating tumor-specific lymphocytes, the method comprising administering to a mammal an effective amount of a chimeric protein comprising a Flt3 ligand and a proteinaceous or peptide-based tumorcide to generate tumor-specific lymphocytes, and The mammal recovers the tumor-specific lymphocytes that have been produced. 89753 6-