CN1150030C - Recombinant human alpha-fetoprotein and its use - Google Patents

Abstract

Generally disclosed is a method for inhibiting the proliferation of a mammalian self-reactive immune cell, comprising administering to a mammal a therapeutically effective amount of recombinant human alpha-fetoprotein or an anti-proliferative fragment or analog thereof; a method for inhibiting a mammalian tumor, comprising Administration of therapeutically effective amount of recombinant human alpha-fetoprotein or its anti-tumor fragments or analogs to mammals; and cell culture method, including using culture medium containing recombinant human alpha-fetoprotein or its fragments or analogues.

Description

Recombinant human alpha-fetoprotein and its use

The present invention relates to expression and purification of cloned human alpha-fetoprotein; methods for treating autoimmune diseases; methods for cancer treatment and diagnosis; and cell growth and cell culture.

Alpha-fetoprotein (AFP) is a serum protein, which generally only has a high content in fetal blood. Higher alpha-fetoprotein levels in adult blood are associated with liver regeneration and certain cancers.

In summary, the present invention is characterized by substantially pure biologically active recombinant human alpha-fetoprotein comprising a sequence substantially identical to amino acids 1-389 of Figure 1 (Sequence 9) or a fragment thereof; Amino acid 198-590 or fragment thereof of 1 (SEQ ID NO: 10); Amino acid 198-389 or fragment thereof of FIG. 1 (SEQ ID NO: 7); Amino acid 390-590 of FIG. 1 (SEQ ID NO: 8); Amino acid 266 of FIG. 1 (SEQ ID NO: 11) -590 or fragments thereof.

In another related aspect, the invention features a method of producing biologically active recombinant human alpha-fetoprotein or fragments or analogs thereof using insect cells, the method comprising

a) providing a transformed insect cell (for example, Spodoptera frugiperda), which contains a recombinant DNA molecule encoding human alpha-fetoprotein or a fragment thereof or an analog thereof, which molecule and an expression control factor can be Operably linked, the control factor directs the expression of human alpha-fetoprotein or its fragment or analogue;

b) culturing the transformed cells; and

c) recovering biologically active human alpha-fetoprotein or its fragments or analogues.

In related aspects, the invention also features substantially pure human alpha-fetoprotein or fragments or analogs thereof produced by any of the methods disclosed herein, and substantially pure human alpha-fetoprotein or fragments thereof or analogs thereof produced by any of the expression systems disclosed herein. A therapeutic composition of alpha-fetoprotein (or a fragment or analog thereof).

In another aspect, the invention features a method of inhibiting proliferation of autoreactive immune cells in a mammal (e.g., a human patient) comprising administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein or an immune cell antiproliferative fragment thereof or analog. This approach is based on my discovery that non-glycosylated recombinant human alpha-fetoprotein produced in prokaryotes such as E. coli can be used to suppress autoreactive immune cells of mammalian origin. Such immune cells preferably comprise T cells or B cells; and the recombinant human alpha-fetoprotein (or immune cell antiproliferative fragment or analog thereof) used in this method is preferably produced in prokaryotic cells (such as Escherichia coli), And it is non-glycosylated.

In yet another aspect, the invention features a method of treating an autoimmune disease in a mammal (such as a human patient), comprising administering to the mammal a therapeutically effective dose of recombinant human alpha-fetoprotein or an immune cell antiproliferative fragment thereof or analog. The autoimmune disease is multiple sclerosis, rheumatoid arthritis, myasthenia gravis, insulin-dependent diabetes, or systemic lupus erythematosus. In other preferred embodiments, the autoimmune disease is acquired immunodeficiency syndrome or involves rejection of transplanted organs, tissues or cells. The recombinant human alpha-fetoprotein used in the method is preferably produced in prokaryotic cells such as E. coli and is non-glycosylated. In other preferred embodiments, the method further comprises administering to the mammal an effective amount of an immunosuppressant that is lower than the standard dose of such an immunosuppressant alone. The immunosuppressant is preferably cyclosporine, steroids, azathioprine, FK-506 or 15-deoxyspergualin. In another preferred embodiment, the method comprises administering to the mammal a tolerizing agent. The recombinant human alpha-fetoprotein used in the method is preferably produced in prokaryotic cells (eg, E. coli) and is non-glycosylated.

According to the present invention, administration of recombinant human alpha-fetoprotein ("rHuAFP") (or its fragments or analogues) may be an effective method for preventing, treating or improving autoimmune diseases in mammals. To illustrate this point, the inventors have demonstrated that recombinant HuAFP produced in a prokaryotic expression system effectively inhibits the proliferation of T cells in response to self-antigens, despite the fact that this rHuAFP is modified in a different manner than naturally occurring HuAFP. So far, the use of native HuAFP has been limited by its unavailability, which is obtained from limited sources of umbilical cord and umbilical cord serum through a tedious purification process. Since rHuAFP with biological activity can be prepared in large quantities by recombinant DNA technology, it is currently feasible to use rHuAFP to treat autoimmune diseases. The use of rHuAFP is particularly advantageous because no adverse side effects associated with human alpha-fetoprotein are known and larger doses are believed to be safe to administer.

In another aspect, the invention features compositions and methods for the prevention, treatment and diagnosis of tumors, particularly cancer. This aspect of the invention is based on the inventors' discovery that non-glycosylated recombinant human alpha-fetoprotein produced in prokaryotes such as E. Tumors, such as breast or prostate cancer, and other cancerous mammals resulting from malignant cell proliferations expressing receptors recognized by recombinant human alpha-fetoprotein.

In one aspect, the invention features a method of inhibiting tumors in a mammal, such as a human patient, comprising administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein or an antitumor fragment or analog thereof. The tumor is preferably a malignant tumor (such as breast tumor and prostate tumor); and the recombinant human alpha-fetoprotein is produced in prokaryotic cells (such as Escherichia coli) and is non-glycosylated. In a preferred embodiment, said tumor cells express a receptor recognized by recombinant human alpha-fetoprotein. The tumor is usually a carcinoma such as an adenocarcinoma or a sarcoma. In preferred embodiments, the tumor responds to the hyperplasia of hormones such as estrogens or androgens. Administration of recombinant human alpha-fetoprotein preferably inhibits the proliferation of mammalian tumor cells or kills these tumor cells. The method also includes administering a chemotherapeutic agent to the mammal.

In another aspect, the invention features a method of preventing tumor development in a mammal comprising administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein. Recombinant human alpha-fetoprotein is preferably produced in prokaryotic cells such as E. coli and is non-glycosylated.

In yet another aspect, the invention features a hybrid cytotoxin comprising recombinant human alpha-fetoprotein (or a fragment or analog thereof) linked to a cytotoxic agent. Examples of such cytotoxic agents include, but are not limited to, diphtheria toxin, Pseudomonas exotoxin A; ricin and other plant toxins, such as abrin, modeccin, Volkensin, parasitic toxin; Cholera toxin (produced by Vibrio cholerae); so-called Shiga-like toxin (produced by Escherichia coli and other Enterobacteriaceae); thermolabile enterotoxin of Salmonella; and thermolabile thermotoxin of Escherichia coli toxin. In other preferred embodiments, the cytokinetic agent is non-proteinaceous. Examples of such non-proteinaceous cytotoxic agents include, but are not limited to, anticancer agents, such as doxorubicin, and alpha-emitting radionuclides, such as astatine, and beta-emitting nuclides, such as yttrium. The cytotoxic agent of the hybrid cytotoxin is connected with recombinant human alpha-fetoprotein through peptide bond, and the hybrid toxin is produced by expressing the hybrid DNA molecule of genetic engineering. In other preferred embodiments, the cytotoxic agent of the hybrid cytotoxin is a protein; this cytotoxic agent is chemically conjugated to recombinant human alpha-fetoprotein.

In other aspects, the invention features a detectably labeled recombinant human alpha-fetoprotein or a detectably labeled fragment or analog thereof capable of binding human tumor cells. The molecule is preferably labeled with a radionuclide, such as technetium-99m, I-125, I-131 or indium. Other detectable labels include, but are not limited to, enzymes, fluorophores, or other components or compounds that emit a detectable signal (e.g., radioactivity, fluorescence, color), or emit a detectable signal upon exposure of the label to its substrate. The detected signal, or the detectable signal can be an epitope that can be recognized by an antibody (for example, an alpha-fetoprotein epitope or an epitope that is deliberately introduced into recombinant alpha-fetoprotein by engineering means, such as HA or myc epitopes). The molecule is preferably directed against a malignancy (eg, breast, prostate, or carcinoma) expressing a receptor recognized by recombinant human alpha-fetoprotein (or a fragment or analog thereof). Typically, this recombinant alpha-fetoprotein is produced in prokaryotic cells such as E. coli and is non-glycosylated.

Detectably labeled recombinant human alpha-fetoprotein (or fragments or analogs thereof) can be used for in vivo imaging of tumor cell-bearing sites in human patients. Generally, the method comprises: (a) providing a detectably labeled recombinant human alpha-fetoprotein molecule (or fragment or analog thereof); (b) administering the molecule to a patient; (c) allowing the labeled Molecules bind to the site, and unbound molecules are cleared from the site; and (d) obtaining an image of the site with tumor cells. The site is preferably the breast or prostate. In other preferred embodiments, the site includes, but is not limited to: liver tissue, lung tissue, spleen tissue, pancreas tissue, brain tissue, lymphoid tissue or bone marrow. The image is preferably obtained using dynamic gamma scintigraphy.

Detectably labeled recombinant human alpha-fetoprotein (or fragments or analogs thereof) can also be used in methods of diagnosing tumors in mammals such as human patients. The method comprises: (a) contacting a biological sample with detectably labeled recombinant human alpha-fetoprotein; and (b) detecting the label bound to the sample, when the detected label is above background levels, indicating that the patient has a tumor . The method preferably includes, prior to said contacting step, a biological sample comprising fixed and sectioned cells, and the label bound to the sample is bound to a portion of the cell membrane corresponding to the cells. In preferred embodiments, the biological sample is from the breast or prostate of a human patient.

Detectably labeled recombinant human alpha-fetoprotein (or fragments or analogs thereof) can also be used in methods for detecting tumors in living mammals in vivo. The method comprises: (a) administering a diagnostically effective amount of a detectably labeled recombinant human alpha-fetoprotein; and (b) detecting the presence of a detectably label bound to mammalian tissue, as indicated by an amount of label above background A tumor is present in the mammal.

In a preferred embodiment, the method involves a patient suspected of having breast cancer, and the tissue is breast tissue. In other preferred embodiments, the method involves a patient suspected of having prostate cancer and the tissue is prostate tissue. Detectably labeled recombinant human alpha-fetoprotein is preferably linked to a radionuclide (such as technetium-90), and the detection step is accomplished by radiographic imaging (such as dynamic gamma scintigraphy).

In another aspect, the invention features a kit for in vivo, in situ, or in vitro detection of a tumor or any cell expressing a receptor recognized by recombinant human alpha-fetoprotein (or a fragment or analog thereof). Typically, the kit contains a recombinant human alpha-fetoprotein that is recognized by the tumor, and this protein may be detectably labeled. If the recombinant human alpha-fetoprotein is unlabeled, the kit preferably contains a second protein with a detectable label (such as a radionuclide, such as technetium 90, I-125, I-131 or indium). reagent. When the detectable label is an enzyme, the kit also includes a substrate for the enzyme. The kit also includes a reagent for linking a detectable label to recombinant alpha-fetoprotein. In another embodiment, the kit for detecting tumors or any harmful cells expressing a receptor that can be recognized by recombinant human alpha-fetoprotein (or its fragment or analogue) comprises a A reagent that can specifically bind to recombinant human alpha-fetoprotein and a reagent that contains detectably labeled recombinant human alpha-fetoprotein that can be specifically bound by an anti-human alpha-fetoprotein antibody. The recombinant human alpha-fetoprotein of the kit is preferably produced in prokaryotic cells (Escherichia coli) and is non-glycosylated.

The use of recombinant human alpha-fetoprotein in the treatment and diagnosis of cancer has many advantages. For example, rHuAFP can be administered directly to the tumor site. Recombinant HuAFP can also be chemically defined and synthesized, and produced in large quantities using recombinant DNA techniques, for example, using the methods disclosed herein. In addition, unlike conventional cancer chemotherapy and radiotherapy, recombinant human alpha-fetoprotein causes minimal side effects such as nausea, vomiting, and neurotoxicity. Therefore, larger doses of rHuAFP can be safely administered.

The diagnostic method of the present invention is advantageous because it can diagnose tumors quickly and easily. For example, the use of rHuAFP as a diagnostic agent (e.g., radiographic imaging by scintigraphy) is particularly advantageous for real-time imaging of cancers for localization and staging of cancers (e.g., breast cancer) before surgery or within operations, after surgery It is also beneficial in the inspection. The application of this diagnostic method allows for the non-invasive determination of the presence, location or absence of tumors, which is beneficial in monitoring the disease in patients.

In another aspect, the invention features a cell culture medium comprising recombinant human alpha-fetoprotein or a cell-stimulating fragment or analog thereof. This aspect of the invention is based on the inventors' discovery that non-glycosylated recombinant human alpha-fetoprotein produced in prokaryotes such as E. coli is a cell proliferation agent, eg, promoting bone marrow growth in vitro. This recombinant human alpha-fetoprotein is preferably produced in prokaryotic cells (E. coli) and is non-glycosylated.

Accordingly, this aspect of the invention features a cell culture method comprising (a) providing a cell culture medium containing recombinant human alpha-fetoprotein; (b) providing a cell; (c) said The cells are cultured in a medium to allow proliferation and maintenance of the cells. The cells are preferably mammalian cells. Examples of such mammalian cells include myeloid cells (e.g., T cells, natural killer cells, lymphocytes), hybridomas, or genetically engineered cell lines. Examples of other cells include hematopoietic cells such as stem cells, blast cells, progenitor cells (e.g., erythroid progenitor cells such as burst colony forming units and colony forming units), myeloblasts, macrophages, monocytes, macrophages , lymphocytes, T-lymphocytes, B-lymphocytes, eosinophils, basophils, tissue mast cells, megakaryocytes [eg, see "Best Taylor's Physiological Basis for Medical Practice"( Best and Taylor's Physiological Basis of Medical Practice), by John B. West, Williams & Wilkins, Baltimore]. In other preferred embodiments, the method involves ex vivo cell culture.

In another aspect, the invention features a method of inhibiting myelotoxicity in a mammal, such as a human patient, comprising administering to the animal a therapeutically effective amount of recombinant human alpha-fetoprotein or a myelotoxicity inhibiting analog or fragment thereof . The recombinant human alpha-fetoprotein is preferably produced in prokaryotic cells (Escherichia coli) and is non-glycosylated.

In another aspect, the invention features a method of inhibiting myeloid cell proliferation inhibition in a mammal, the method comprising administering to the mammal an effective amount of recombinant alpha-fetoprotein or an anti-inhibitory fragment or analog thereof. Recombinant human alpha-fetoprotein is preferably produced in prokaryotic cells (eg, E. coli) and is non-glycosylated.

In another aspect, the invention features a method of promoting proliferation of bone marrow cells in a mammal comprising administering to the mammal an effective amount of recombinant human alpha-fetoprotein or a cell stimulating fragment or analog thereof. Recombinant human alpha-fetoprotein is preferably produced in prokaryotic cells such as E. coli and is non-glycosylated.

In yet another aspect, the invention features a method of preventing rejection of a bone marrow cell transplant in a mammal comprising administering to the mammal an effective amount of recombinant human alpha-fetoprotein or an anti-rejection fragment or analog thereof. Recombinant human alpha-fetoprotein is preferably produced in prokaryotic cells such as E. coli and is non-glycosylated. Administration of rHuAFP (or fragments or analogs thereof) according to the method of the present invention may be an effective method to promote and enhance cell growth in vitro, ex vivo or in vivo. In addition, administering the compounds of the present invention may also be an effective method for preventing, treating or ameliorating myelotoxemia in mammals.

It is advantageous to use rHuAFP (or a fragment or an analog thereof) as the main component of the tissue culture medium because of the advantage that there is little possibility of infection with pathogens.

"Human alpha-fetoprotein" refers to a human alpha-fetoprotein gene substantially identical to that described by Morinaga et al [Proc. Natl. Acad. Sci., USA 80:4604 (1983) A polypeptide that encodes the amino acid sequence of a protein. Methods for producing human alpha-fetoprotein in prokaryotic cells are disclosed in US Pat. No. 5,384,250 and are consistent with the methods disclosed herein.

"Expression control factor" refers to a nucleotide sequence that includes a recognition sequence for a factor that controls the expression of a protein coding sequence and is operably linked to that sequence. Thus, expression control factors typically include sequences that control transcription and translation, eg, promoters, ribosome binding sites, repressor binding sites, and activator binding sites.

"Substantially identical amino acid sequence" refers to a polypeptide having at least 80%, typically at least about 85%, more typically at least about 90%, often at least about 95%, more often at least about 97%. The length of the comparison sequences is usually at least about 16 amino acids, usually at least about 20 amino acids, more usually at least about 25 amino acids, typically at least about 30 amino acids, and preferably more than 35 amino acids.

Polypeptide homology is generally determined using sequence sequencing software (eg, Sequence Analysis Software Package, University of Saskatchewan Biotechnology Center, 1710, University Avenue, Madison, WI 53705). Protein analysis software pairs similar sequences by evaluating the degree of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions generally include substitutions within the following groups: glycine, propylamino; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine amino acids; lysine, arginine; and phenylalanine, tyrosine.

As used herein, "substantially pure" refers to a protein or polypeptide that has been separated from its naturally occurring components. Generally, a protein of interest is substantially pure when at least 60-75% of the total protein in a sample is the protein of interest. Minor variations or chemical modifications generally have the same polypeptide sequence. Substantially pure proteins generally contain greater than about 85-90% of such protein in a sample, and preferably have a purity of greater than 99%. Generally, purity is determined on a chromatographic column, on a polyacrylamide gel, or by HPLC analysis.

When a protein is separated from impurities with which it is naturally associated, the protein is substantially free of naturally associated components. Thus, a chemically synthesized protein or a protein produced in a cellular system other than the cell in which it is naturally produced will be substantially free of its naturally associated components. Thus, the term may be used to describe polypeptides and nucleic acids of eukaryotic origin that are synthesized in E. coli or other prokaryotic organisms.

The present invention provides substantially pure human alpha-fetoprotein. Various methods for isolating human alpha-fetoprotein (AFP) from biological materials can be devised based in part on the structural and functional properties of human alpha-fetoprotein. Alternatively, anti-AFP antibodies can be immobilized on a solid substrate to obtain a highly specific affinity for the purification of human AFP.

In addition to substantially full-length polypeptides, the present invention also provides biologically active recombinant fragments or analogs of human alpha-fetoprotein. For example, fragments that have ligand binding or immunosuppressive activity.

Natural or synthetic DNA fragments encoding human alpha-fetoprotein or desired fragments thereof will be incorporated into DNA constructs that can be introduced into and expressed in cell cultures. The DNA construct prepared for introduction into this host usually includes an origin of replication that can be utilized by the host cell, a DNA fragment encoding a desired portion of human alpha-fetoprotein, a transcriptional and A translation initiation regulatory sequence, and a transcriptional and translational termination regulatory sequence operably linked to the alpha-fetoprotein coding segment. The transcriptional regulatory sequence usually includes a heterologous promoter recognized by the host. Selection of a suitable promoter depends on the host, but promoters such as trp, tac, and phage, tRNA, and glycolytic enzymes can be used under suitable conditions (Sambrook et al., "Molecular Cloning: A Laboratory Guide." : Laboratory Manual), Cold Spring Harbor Press, Cold Spring Harbor, NY 1989). In some instances it may be desirable to include recognition sequences for the proper localization of factors capable of regulating transcription in the host cell (eg, the lac repressor of E. coli). Commercially available expression vectors can be used that include a replication system and transcriptional and translational regulatory sequences, as well as commonly used for insertion of a DNA fragment encoding the gene to be expressed.

The above various promoters, transcription and translation sequences are generally referred to as "expression control factors".

It is also possible to integrate a DNA fragment encoding all or part of human AFP into the host cell chromosome.

Vectors containing the DNA fragment of interest can be transferred into host cells by well known methods which vary with the type of cellular host (Sambrook et al., supra). The term "transformed cell" is also intended to include progeny of transformed cells.

Prokaryotic hosts that can be used for high-level expression of recombinant proteins include: Escherichia coli, Bacillus subtilis (Bacillus Subtilis), and various strains of Pseudomonas.

The method of the present invention provides a means to produce large quantities of biologically active human alpha-fetoprotein. AFP produced by the method of the present invention is biologically active despite the fact that such AFP has not been modified in the same manner as naturally occurring human AFP.

The expression "immune cell anti-proliferation" refers to the ability to inhibit the growth of unwanted immune cells (eg, autoreactive T cells as measured using the assays disclosed herein).

"Tumor" refers to any unwanted growth of a cell that has no physiological function. Typically, a tumor cell is released from its normal control of cell division, ie, a cell whose growth is not regulated by the usual biochemical and physical influences in the cellular environment. In most cases, the proliferation of tumor cells results in the formation of clones of cells, which can be benign or malignant. Examples of tumors include, but are not limited to, transformed and immortalized cells, tumors, and carcinomas, such as breast cell carcinoma and prostate cancer.

The term "therapeutically effective amount" refers to the amount of non-glycosylated recombinant human alpha-fetoprotein or its anti-tumor fragments or analogs that can inhibit the proliferation of tumors or can inhibit the proliferation of autoreactive immune cells or stimulate the proliferation of cells (such as bone marrow cells). dose.

The phrase "diagnostically effective amount" refers to a detectably labeled recombinant human alpha-fetoprotein or a detectably labeled fragment or analog thereof that can be detected in a target site in a mammal, such as a human patient. dosage.

The term "cell stimulation" means increasing cell proliferation, increasing cell division, promoting cell differentiation and/or development, or prolonging cell lifespan.

"Myelotoxicity suppression" means inhibition of bone marrow detachment.

Other features and advantages of the invention will emerge from the following description of preferred embodiments of the invention and from the claims.

The accompanying drawings are described first.

Attached picture

Figure 1 is the nucleotide sequence (SEQ ID NO: 4) and deduced amino acid sequence (SEQ ID NO: 5) of cDVA encoding human alpha-fetoprotein.

Fig. 2 is the 10% SDS-PAGE analysis (swimming lane A, molecular weight marker of rHuAFP fragment I (sequence 11); Swimming lane B, native human alpha-fetoprotein (AFP); Swimming lane C, unpurified rHuAFP; Swimming lane D, rHuAFP fragment I and lane E, rHuAFP (amino acids 1-590 of FIG. 1 , SEQ ID NO: 5)).

Figure 3 is a histogram showing the inhibitory effect of rHuAFP and domain fragments produced by E. coli on human "autologous mixed lymphocyte reaction (AMLR).

Figure 4 shows a series of graphs showing the purity and biochemical characteristics of rHuAFP produced by baculovirus and E. coli prepared by polyacrylamide gel electrophoresis and column chromatography. Figure 4A is a 10% non-denaturing alkaline polyacrylamide gel showing the purity of rHuAFP. Mouse amniotic proteins (transferrin, AFP, and albumin) are shown in lane 1, native HuAFP (lane 2), rHuAFP produced by baculovirus (lane 3), and rHuAFP produced by E. coli (lane 4). Figure 4B is a 10% sodium dodecyl sulfate polyacrylamide gel showing the purity of rHuAFP produced by baculovirus and E. coli expression systems. Molecular weight markers are shown in lane 1 and native HuAFP, rHuAFP produced by baculovirus and E. coli are shown in lanes 2, 3 and 4, respectively. Figure 4C is a series of FPLC chromatograms of native HuAFP, rHuAFP produced by baculovirus and E. coli eluted on Mono Q anion exchange column. Overlaid chromatograms represent native HuAFP (chromatogram 1), rHuAFP produced by baculovirus and E. coli (chromatograms 2 and 3, respectively). Figure 4D is a graph obtained by passing 50 μg native HuAFP and rHuAFP through a reversed Delta Pak C18 column (Waters) and eluting with a gradient of 0-100% acetonitrile in 0.1% "trifluoroacetic acid" (TFA). Serial HPLC chromatograms. Overlaid chromatograms represent native HuAFP (chromatogram 1) and rHuAFP produced by baculovirus and E. coli (chromatograms 2 and 3, respectively).

Figure 5 is a histogram showing that anti-native HuAFP antibodies produced by rHuAFP produced using baculovirus and E. coli expression systems prevent immunosuppression of AMLR. Immunosuppression by rHuAFP produced with baculovirus and E. coli expression systems was significant (P>0.002), as was the immunosuppression of AMLR mediated by rHuAFP by monoclonal anti-native HuAFP (aAFP) antibodies. Significant (P<0.03). AMLR cultures were established with or without protein and contained 2 x ¹⁰⁵ effector T cells, 2.5 x ¹⁰⁵ irradiated autologous non-T cells, harvested at 144 hours, and based on incorporation of autoreactivity ³ H-thymidine in T cells was measured for their own proliferation. The self-proliferation inhibitory effect of rHuAFP was carried out as follows: a mouse anti-human AFP monoclonal antibody was added to the AMLR culture at a dilution factor of 1/8 (125 μg/ml), and AMLR could be inhibited by 100 mg/ml rHuAFP produced by baculovirus. (hatched bar) and 100 [mu]g/ml rHuAFP produced by E. coli (open bar) inhibited. Control cultures consisted of AMLR in the presence of 1/8-fold anti-human AFP (aAFP) monoclonal antibody.

Figure 6 is a series of histograms (Figures 6A and 6B) showing the immunosuppressive effect mediated by rHuAFP when tested with human AMLR (Figure 6A) and "peripheral blood lymphocytes" (PBL) (Figure 6B). Figure 6A shows the results of an autologous mixed lymphocyte reaction (AMLR) prepared by culturing 250,000 T cells and an equivalent amount of autologous irradiated non-T lymphocytes. Recombinant HuAFP preparations derived from E. coli and baculovirus-expressed systems and albumin were both added at a concentration of 100 μg/ml at the beginning of the culture. Proliferative effects were determined by ³ H-thymidine incorporation at 144 hours. FIG. 6B shows the results of PBLs (2×10 ⁵ ) stimulated with 1 μg/ml Concanavalin A (ConA) and cultured in RPMI medium supplemented with 2 mg/ml albumin alone for 48 hours. Albumin and rHuAFP derived from Escherichia coli and baculovirus were added at a concentration of 100 μg/ml to start the culture. Proliferation effect was measured according to the incorporation of ³ H-thymidine during DNA synthesis. The measured SEM was less than 5% of the mean.

Figure 7 is a plasmid map of pVT-PlacZ.

Figure 8 is a series of graphs showing the inhibitory effect of rHuAFP on the kinetics of T cell activation (Figure 8A), and the dose-effect relationship of rHuAFP on autoproliferating T cells. Fig. 8A is a graph showing the proliferation effect of cells cultured for more than 4 days in the absence of rHuAFP () and in the presence of 100 μg/ml () rHuAFP. (·) represents the background proliferation of effector cell populations cultured alone. The recombinant HuAFP-mediated inhibition of AMLR was significant (P<0.01) within the above time. Figure 8B shows the inhibitory effect of rHuAFP at a dose of 6-100 μg/ml () on self-proliferating T cells at 144 hours. () indicates the control effect of the reaction in the absence of protein. The inhibitory effect of rHuAFP in the range of 12.5-100 μg/ml on autoreactive T cells was significant (P<0.005).

Figure 9 is a histogram showing the effect of rHuAFP on the post-confluent growth of estrogen-stimulated MCF-7 breast cancer cells.

Figure 10 is a graph showing the proliferation of mouse bone marrow in serum-free RPMI medium with or without 400 µg/ml rHuAFP and 5 µg/ml transferrin.

Expression of recombinant human alpha-fetoprotein

Construction of cDNA library

A cDNA library was constructed using size fractionated cDNA (0.5-3 kg) prepared from poly(A) ⁺ RNA isolated from hepatocytes (~3 g wet weight) of 4.5 month old human aborted fetuses. (Alternatively, fetal cDNA libraries are available from Clontech Laboratory, Inc., Palo Alto, CA.). Total RNA was prepared by the guanidinium thiocyanate method (Chirgwin et al., "Biochemistry" 18:5294, 1979) and purified by oligo(dT)-cellulose chromatography (Collaborative Research, Bedford, MA) ["Modern Molecular "Current Protocols in Molecular Biology" (Current Protocols in Molecular Biology), Ausubel et al., Wiley Interscience, New York 1989] Screening for mRNA. Utilize Librarian II cDNA synthesis kit (Invitrogen, San Diego, (A) to synthesize cDNA and carry out fractionation on 1% agarose gel. Extract the fragment of 0.5-3kb, and it is ligated with vector pTZ18-RB (Invitrogen) , and then use it to transform competent Escherichia coli DH1αF' (Invitrogen)). Colony lifts were performed using Colony/Plaque Screen filters (DuPont, Wilmington, DE), and the transferred bacterial colonies were lysed and denatured by incubation in a solution containing 0.5M NaOH, 1.5M NaCl for 10 minutes. Filters were washed in a solution containing 1.5M NaCl, 0.50M Tris-HCl (pH 7.6) for 5 minutes and air dried. The filters were then washed 5 times in chloroform, soaked in 0.3M NaCl to remove cell debris, and then air-dried. DNA was immobilized on nitrocellulose membranes by baking under vacuum at 80° for 2 hours. Place the baked filter membrane in a solution containing 6×SSC (1×SSC=150mM NaCl, 15mM sodium citrate [pH7.0]), 1×Denhardt’s solution (0.2g/l polyvinylpyrrolidone, 0.2g/l In a solution of "Bovine Serum Albumin" (BSA), 0.2 g/l Ficoll 400), 0.05% sodium pyrophosphate, 0.5% SDS, and 100 µg/ml E. coli DNA, prehybridization was performed at 37°C for 3 hours. Hybridize at 37°C for 18-24 hours in the same solution without SDS, containing 1-2×10 ⁶ cpm/ml by 5′-terminal phosphorylation “[Current Protocols in Molecular Biology” (Current Protocols in Molecular Biology) ), supra] with ³² P-labeled two oligonucleotides. The sequences of the oligonucleotides used to detect the library are: 5'-TGTCTGCAGGATGGGGAAAAA-3' (Sequence 1) and 5'-CATGAAATGACTCCAGTA-3' (Sequence 2), corresponding to 772-792 of the human AFP coding sequence respectively bits and 1405-1422 bits. The filters were washed twice with 6*SSC, 0.05% sodium pyrophosphate for 30 minutes at 37°C and then once in the same solution for 30 minutes at 48°C. Positive clones were identified by exposing KodakXAR film to dry filters for 24-48 hours in the presence of a Du Pont Cronex Lightning Plus intensifying screen. Positive clones were isolated, amplified, and subjected to Southern blot analysis [Current Protocols in Molecuar Biology, supra]. Briefly, purified DNA was hydrolyzed with appropriate restriction enzymes and the resulting fragments were separated on a 1% agarose gel. The DNA was then transferred to a nitrocellulose membrane. The hybridization conditions were as above, except that, in addition to the above two probes, a third ³² P-labeled oligonucleotide (5'-CATAGAAATGAATATGGA-3' (SEQ ID NO: 3), representing the human AFP coding fragment was also used 7-24 digits). Five positive clones were identified out of 3,000 colonies screened. One of these clones, pLHuAFP, was used in the construction described below.

Construction of full-length human AFP cDNA

The following 5 DNA fragments were used to prepare a construct containing a translation initiation codon followed by the human AFP coding sequence and a translation termination codon.

Fragment 1: Two non-phosphorylated oligonucleotides are annealed to form a double-stranded DNA molecule consisting of a 5′-end sticky EcoRI recognition site, followed by an ATG initiation codon, and human The first 60 base pairs (bp) of the AFP cDNA. Also included is the PstI restriction site at position 60 of the coding sequence (in this scheme, nucleotide 1 is the first nucleotide of the first codon (Thr) of the mature protein, corresponding to Morinaga nucleotide 102 of et al., supra). This fragment was ligated with pUC119 linearized with EcoRI and PstI (pUC19 having the intergenic region of M13 from HgiAI at position 5465 to AhaII at position 5941 inserted at the NdeI site of pUC19). The resulting DNA was amplified in E. coli NM522 (Pharmacia, Piscataway, NJ). The EcoRI-PstI insert was recovered by enzymatic digestion of the recombinant plasmid followed by electrophoretic separation on a 5% polyacrylamide gel and extraction from the gel.

Fragment 2: A 97 bp human AFP cDNA fragment (57-153) was obtained by digesting pLHuAFP with PstI and NsiI and gel purification as described above. This clone contains the complete coding fragment and 5' and 3' untranslated sequences of human AFP.

Fragment 3: A 224bp human AFP cDNA fragment (position 150-373) was obtained by digesting pLHuAFP with NsiI and AlwNI and purifying as above.

Fragment 4: A 1322bp human AFP cDNA fragment (position 371-1692) was obtained by digesting pLHuAFP with AlwNI and StyI and purifying as above.

Fragment 5: Two non-phosphorylated oligonucleotides were annealed to form a double-stranded DNA of 86 bp, which contained the human AFP sequence of the TAA stop codon of the AFP coding fragment from position 1693 to position 1773 of the StyI site, which This is followed by a cohesive BamHI site. This synthetic DNA was used without any further manipulation.

pBlue Script (StrataGene, La Jolla, CA) was thoroughly hydrolyzed with EcoRI and BamHI and added to the ligation mix containing the 5 purified fragments described above. The control ligation mix contained only linearized pBluescript. A portion of the above two ligation mixtures was used to transform competent E. coli DH52 (GIBCO/BRL, Grand Island, NY). Recombinant plasmids were isolated from several transformants and screened by in-depth restriction enzyme analysis and DNA sequencing. A recombinant plasmid was screened out and named pHuAFP. It was subsequently used to insert the human AFP gene into several expression vectors. pHuAFP includes a unique EcoRI-BamHI fragment, which not only includes the ATG start codon at the 5'-end and the TAA stop codon at the 3'-end, but also contains the complete coding sequence of human AFP.

AFP expression vector

High-level synthesis of human AFP in E. coli was successfully achieved in 3 different expression systems. The TRP system enables direct expression. The Rxl system produces a fusion protein containing 20 amino acids encoded by trpE and vector sequences. The MAL system expresses AFP fused to the malE gene product, a 42kd maltose binding protein.

TRP expression system: the 1186bp EcoRI-Bam HI AFP coding fragment of pHuAFP is cloned into the expression vector pTrP4 (Olsen et al., " Biotechnology Journal " (J.Biotechnol.) g: 179,1989), the clone is located in the trp promoter and a Modified downstream of the ribosome binding site.

Briefly, pHuAFP was digested with EcoRI and BamHI, and the ends were filled in with Klenow polymerase. The 1186bp AFP fragment was then gel purified. pTrp4 was digested with ClaI, its ends filled in with Klenow polymerase, and the linearized vector was gel purified. The AFP fragment and pTrp4 backbone of 1186bp were connected, and it was used to transform competent Escherichia coli, namely the following bacterial strains: DH5α, BL21 (F.W.Studier, Brookhaverc National Laboratory, Upton, NY), SG927 (American Type Culture Collection, Rockville, MD: Accession No. 39627), SG928 (ATCC, Accession No. 39628), and SG935 (ATCC, Accession No. 39623).

R×1 expression system: The human AFP cDNA was cloned into the expression vector pR×1 [Rimm et al., “Gene”, 75:323, 1989] close to the trp promoter, which is in the TrpE translation frame. Human AFP cDNA was excised from pHuAFP by digestion with EcoRI and BamHI and cloned into appropriately processed pR×I (BioRad Laboratories, Hercules, CA). The resulting plasmid construct designated pR×1/HuAFP was then used to transform the aforementioned E. coli strain and CAG456 (D.W. Cleveland, Johns Hepkins University, Baltimore, MD).

MAL expression system: AFP cDNA was inserted into the expression vector pMAL (New England Biolabs, Inc., Beverly, MA), placed under the control of the tac promoter and in the MalE translation frame. Briefly, pHuAFP was hydrolyzed with BamHI and the ends filled in with Klenow polymerase. The remainder of the human AFP cDNA plasmid DNA was released by EcoRI digestion followed by gel purification. The purified fragments were ligated into appropriately digested pMAL-C. The recombinant plasmid with the correct orientation - its name is pMAL/HuAFP - was used to transform Escherichia coli DH52, TBI (New England Biolabs) and SG935.

The AFP coding fragments used to construct the above three expression systems were sequenced, and it was found that they encoded the full-length AFP.

Expression of AFP in E. coli

Bacterial cultures were incubated at 30°C or 37°C with aeration. Overnight E. coli cultures were grown in LB medium supplemented with appropriate required antibiotics (tetracycline-HCl 50 μg/ml, ampicillin-Na 100 μg/ml).

TRP and R×1 expression systems: induction of the trp promoter under tryptophan starvation conditions. Induction was performed in MgCA medium prepared as follows: 1 g casamino acids (DifcoLaboratory, Detroit, MI), 6 g Na ₂ HPO ₄ , 3 g KH ₂ PO ₄ , 0.5 g NaCl, 1 g NH ₄ Cl were added to 1 liter of Milli- The pH was adjusted to 7.4 in Q water (Millipore Corporation, Bedford, MA), and the solution was autoclaved. The cooled medium contained 2 mM MgSO ₄ , 0.1 mM CaCl ₂ , and 0.2% glucose. Cultures grown overnight in MgCA supplemented with antibiotics were diluted 100-fold and cells were grown at 30°C to an A550 of 0.4, harvested by centrifugation, and stored as pellets at -20°C.

MAL expression system: The tac promoter was induced with the obligatory inducer isopropylthio-β-D-galactoside (IPTG). The overnight culture was diluted 100-fold with LB medium supplemented with antibiotics and the cells were grown at 37°C to an A550 of 0.4. IPTG was then added to a final concentration of 0.3 mM, and the bacteria were incubated for an additional 2 hours. Cells were harvested by centrifugation and the pellet was stored at -20°C.

Detection of AFP expression in Escherichia coli

Analytical studies were performed to determine the expression and behavior of recombinant AFP. Or suspend the cell pellet in SDS-lysis solution (0.16M Tris-HCl [pH6.8]), 4% W/VSDS, 0.2m dithiothreitol (DTT), 20% glycerol, 0.02 bromophenol blue) , boiled for 5 minutes, and used for SDS-PAGE analysis; or the cell pellet was suspended in 10mM Na ₂ HPO ₄ , 30mM NaCl, 0.25% Tween20, 10mM EDTA, 10mM ethylene glycol bis(ethyl-aminoethyl ether) tetraacetic acid (EGTA ) in a lysis buffer composed of 1 mg/ml lysozyme at 4°C for 30 minutes, and then sonicated for 3×1 minute in a pulsed manner at 50% power (“Sonics and Materials” ), Danbury, CT: model VC300 Sonifier). The lysate was centrifuged at 10,000g for 20 minutes, and the supernatant containing soluble protein was poured into another tube and frozen at -20°C until use. The pellet containing insoluble protein was resuspended in SDS-lysis buffer, boiled for 5 minutes, and stored at -20°C until use. Total protein released in SDS-lysis buffer as well as soluble and precipitated fractions were analyzed by SDS-PAGE and immunodetected after Western blot transfer. In the above studies, Coomassie blue-stained gels were routinely scanned with an image densitometer (BioRad, Model 620). The amount of recombinant AFP produced can thus be quantified as a percentage of total cellular protein.

Purification of AFP expressed in the TRP system

All manipulations were performed at 4°C unless otherwise stated. Each aliquot of frozen cell pellet obtained from 1 liter of culture was resuspended in 25 ml Lysis Buffer A [50 mM Tris-HCl [pH 7.5]], 20% sucrose, 100 μg/ml "phenylmethylsulfonyl fluoride" (PMSF) ] and incubate for 10 minutes. EDTA was added to a final concentration of 35 mM and the extract was left for an additional 10 min. The lysis buffer was further incubated for 30 minutes after addition of 25 ml (lysis buffer B (50 mM Tris-HCl [pH 7.5]), 2.5 mM EDTA, 0.2% TritonX-100). Centrifuge the cell lysate at 12,000g for 20 minutes, and wash the pellet containing recombinant AFP twice with 50ml washing buffer (50mM Tris-HCl [pH8.0], 10mM EDTA, 0.2% Triton X-100), after each washing Both were centrifuged as described above. The pellet was dissolved in 50 ml of denaturing buffer (0.1M _K2HPO4 [pH8.5]), 6M guanidine hydrochloride, 0.1M 2-mercaptoethanol), sonicated, and mixed for 4 hours on a Nutator (Clay Adams). The dissolved extract was diluted 50-fold with a solution containing 50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, and the recombinant AFP protein was refolded for 24 hours. This 50-fold dilution step is important because there appears to be microaccumulation of AFP prior to dilution. After dilution and reconcentration, AFP no longer accumulates. The above solution was concentrated 100-fold on a YM10 membrane using an Amicon filter unit and clarified through a Millex 0.22 μm membrane filter (Millipore). AFP was further purified at room temperature on a Mono Q column (Pharmacia) equilibrated in 20 mM Tris-HCl (pH 8.0), and bound protein. Eluted fractions were analyzed by SDS-PAGE, alkaline PAGE (APAGE) and Western blotting.

The general methods for expression and purification of polypeptides described above can also be used to produce and isolate useful human alpha-fetoprotein or analogs (as described below).

Polyacrylamide gel electrophoresis and Western immunoassay

Following the method of Hames et al. ("Gel Electrophoresis of Proteins: A Practical Approach", IRL Press, London, 1981), using a mini-Protean electrophoresis device (BioRad) in a discontinuous buffer system SDS-PAGE and basic PAGE were carried out. Recombinant human AFP was immunodetected after SDS-PAGE or APAGE by soaking the gel in transfer buffer (12.5 mM Tris-HCl, 96 mM glycine, 20% methanol [pH 8.2]) for 15 minutes. Each gel was then overlaid with a sheet of Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore) and sandwiched between two electrode grids of the mini-Protean transfer device (BioRad) so that the gels were in close proximity. cathode. The system was immersed in transfer buffer, and a current of 150 mA was applied for 2 hours. Unreacted sites on the Immobilon PVDF membrane were blocked in a solution containing 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 3% gelatin for 1 hour. Rabbit anti-human AFP antiserum and goat anti-rabbit antibody conjugated to alkaline phosphatase (BioRad) were used as primary and secondary antibodies, respectively. Alkaline phosphatase activity was detected with 5-bromo-4-chloro-3-indolyl phosphate and P-nitroblue tetrazolium (BioRad).

Quantification of AFP expression

Recombinant human AFP was quantified using the Human AFP "Enzyme-Linked Immunosorbent Assay" (ELISA) kit (Abbort Laboratories, Chicago, IK).

AFP production was determined by scanning silver-stained gels. When transforming SG935 cells with the plasmid encoding AFP using the "tryptophan" (Trp) expression system, AFP accounts for 2-5% of the total protein of E. coli cells (about 3-7mg AFP per liter of culture). As mentioned above, most of the AFP in the primary extract is insoluble. The above redissolution process can recover 50-60% stable, semi-purified, monomeric form of AFP (approximately 50 mg per 201 E. coli). The above product can be further purified to obtain 25 mg of purified monomeric AFP.

N-terminal analysis

Automated Edman degradation was performed with a Porton protein/peptide gas-phase microsequencer, and sequences were optimized with a custom-built integrated microwell HPLC. Protein sequence analysis was assisted by a selection program in the PC/Gene software package (Intelligenetics).

Cloning, expression and purification of HuAFP using a baculovirus expression system

Recombinant baculoviruses expressing HuAFP (or fragments or analogs thereof) are constructed according to standard methods known in the art (eg, see US Patent No. 4,745,051). This method generally involves two steps. The gene to be expressed, such as rHuAFP or its fragments or analogs (see below), is first cloned into a plasmid transfer vector downstream of a baculovirus promoter flanked by non-essential loci of baculovirus DNA, such as the polyhedrin gene. This plasmid is then introduced into insect cells along with circular wild-type genomic DNA, allowing homologous recombination to occur. Recombinant progeny are then screened, for example, by sequential plaque analysis to purify recombinant virus from the non-recombinant parental strain. In order to obtain sufficient virus for protein expression, virus amplification is usually necessary. The recombinant virus is plaque purified and its DNA structure confirmed by standard methods well known in the art.

The rHuAFP cDNA fragment was isolated from plasmid PI18 using EcoRI/BamHI and purified using Geneclean as described below. Cloning and expression of HuAFP cDNA in baculovirus was performed with plasmid pVT-PlacZ. The human AFP cDNA was cloned into the plasmid pVT-PlacZ (Fig. 7) in the reading frame whose 3'-terminus is the signal peptide sequence of melittin produced by insects. The baculovirus vector pVT-PlacZ was modified by replacing its multiple cloning site with the oligonucleotide 5′-GATCTAGAATTCGGATCCGGT-3′ (SEQ ID NO: 21) and its complementary fragments, including EcoRI and BamHI restriction site, said modification also includes reducing the number of non-AFP coding nucleotides located between the melittin signal peptide cleavage site and the AFP cDNA site inserted on the EcoRI endonuclease sequence. The insert was then ligated into the EcoRI and BamHI DNA sequences of the modified pVT-PlacZ vector.

Recombinant baculoviruses containing the rHuAFP coding sequence were prepared according to standard methods. Purified recombinant baculovirus containing the HuAFP coding sequence was prepared by co-transfection with the pVT-PlacZ transfer vector and wild-type baculovirus, followed by two rounds of plaque purification. Inoculate Sf9 insect cells at a density of 1×10 ⁶ cells/ml in a serum-free Grace medium contained in a 500 ml spinner flask, and infect with recombinant baculovirus at a multiplicity of infection of 5. The supernatant containing the secreted rHuAFP was harvested by centrifugation at 200xg, and the cells were removed. The medium containing rHuAFP was concentrated 10-20 fold by ultrafiltration with a YM30 Amicon membrane, dialyzed overnight against PBS, and injected onto a ConA lectin column (Pharmacia). Bound rHuAFP was eluted with 0.4 M of methyl α-D mannopyranoside and eluted from the MonoQ column during a 0-100% linear gradient of 1 M NaCl in 20 mM phosphate buffer, pH 8.0 purification. Recombinant HuAFP was identified using methods well known in the art.

The inventors found that rHuAFP produced by baculovirus accounts for about 20% of the total protein secreted by Sf9 insect cells into serum-free medium. This AFP was also monomeric by non-reducing basic PAGE. Most of the HuAFP produced by baculovirus was bound to immobilized ConA. In this way, more than 90% of contaminating proteins, which cannot bind to lectin particles, can be effectively removed. Final purification of the baculovirus-produced rAFP preparation by eluting the protein from Mono Q beads with 270-310 mM NaCl yielded a single peptide with an apparent molecular weight of approximately 68 KD. We obtain at least 1mg of purified protein per liter of grown culture.

The molecular weight of rHuAFP produced by baculovirus was similar to that of the natural human molecule (Fig. 4B). The above findings, together with the binding of baculovirus-produced rHuAFP to the ConA column and the observed non-binding of E. coli-produced rHuAFP to the ConA column, suggest that the baculovirus-produced rHuAFP is glycosylated. However, rHuAFP (BrAFP) produced by baculovirus is expected to be less glycosylated than the native molecule because it has been documented that Sf9 cells infected with recombinant baculovirus lack the capacity for complex glycosylation. ability, but this glycosylation is common in proteins produced by higher true organisms. The single bands on APAGE and SDS-PAGE (Figures 4A and 4B), as well as the single peaks on the FPLC and HPLC chromatograms shown in Figures 4C and 4D, respectively, confirmed the identity of the isolated rHuAFP produced by baculovirus. purity. N-terminal sequencing further confirmed the identity of purified rHuAFP. The N-terminal sequence of rHuAFP is: Asp-Leu-Gly-Phe-Met-Thr-Leu-His-Arg-Asn (SEQ ID NO: 22). Western blot analysis of serum-free supernatants from Sf9 cells infected with recombinant baculovirus detected a single and nonspecific anti-HuAFP Ab immunoreactive band, whereas in uninfected or infected with wild-type virus This band is absent in Sf9 cells.

Experiments were performed using methods known in the art to determine the biological activity of the baculovirus produced by HuAFP. For example, the immunosuppressive activity of 100 [mu]g/ml of baculovirus-produced HuAFP was determined based on its ability to inhibit human AMLR as described above. As shown in Figures 5 and 6A, rHuAFP produced by baculovirus was able to suppress the proliferative effect of autoreactive lymphocytes stimulated by autologous non-T cells at 144 hours. Adding the same amount of human serum albumin can not weaken the effect of lymphocyte proliferation.

To confirm that rHuAFP is a substrate of T cells determined to inhibit their own proliferation, rHuAFP-mediated inhibition of AMLR was blocked with a commercially available mouse anti-human AFP monoclonal antibody (MAb). As shown in Figure 5, anti-HuAFP completely prevented the inhibitory effect of rHuAFP produced by 100 μg/ml E. coli and baculovirus on the proliferation of autoreactive T cells. The addition of 100 μg/ml HSA could not attenuate the AMLR effect, and the presence of MAb alone in the reaction culture had no effect.

Additionally, we tested the biological activity of rHuAFP in inhibiting mitogen-induced proliferation of peripheral blood lymphocytes (PBL) in RPMI tissue culture medium supplemented with only 2 mg/ml purified human albumin (ICN, Mississanga, ON). As shown in Table II (below) and Figure 6B, 100 μg/ml rHuAFP inhibited ConA-stimulated PBLs, whereas the same concentration of albumin had no effect.

Other rHuAFPs can be produced from the baculovirus expression system described above using any of the methods disclosed herein according to standard methods [e.g., rHuAFP (amino acid 1 (THr)-590 (Val); sequence 5); domain I (amino acid 1 ( THr)-197(Ser); Sequence 6); Domain II (amino acids 198(Ser)-389(Ser); Sequence 7); Domain III (amino acids 390(Gln)-590(Val); Sequence 8); Domain I+II (amino acids 1 (THr)-389 (Ser); Sequence 9); domain II+III, (amino acids 198 (Ser)-590 (Val); Sequence 11 )].

In one embodiment, the vector pVT-PlacZ/HuAFP (amino acids 1-590) is constructed by inserting the cDNA encoding HuAFP into pVT-P10, which is an intermediate vector for constructing pVT-PLacZ [Richardson et al., (1992) "Engineering Glycoproteins for Secretion using the baculovirus expression System". See "Baculovirus and Recombinant Protein Production Processes", JM Viak, E.-J. Schlaeger and AR Bernard, Editions Roche, Basel, Switzerland, pp. 67-73]. The pVT-P10 vector was digested with BamHI, followed by incubation with mung bean nuclease (New England Biolabs, Mississ-auga, Ont.). Then the vector was further hydrolyzed with EcoRI downstream of the blunt-ended BamHI site, so as to facilitate the direct cloning of HuAFP cDNA. The rHuAFPcDNA encoding amino acids 1-590 was obtained by PCR amplification using the following oligonucleotide primers: (5'-AAAAAACTCGAGATACACTGCATAGAAATGAA-3'; Sequence 23), which contains an Xhol site (5'- AAAAAAGAATTCTTAAACTCCCAAAGCAGCACG-3'; SEQ ID NO: 24) and contains an EcoRI site, and plasmid p118 as template DNA contains the coding fragment of HuAFP. The PCR reaction is carried out according to standard methods, for example, in a reaction buffer containing 34 μL _HO , 10 μL 10× reaction buffer, 20 μL dNTP, 2 μl DNA template, 10 μL 10 pmol/μL 5′ primer, 10 μL 10 pmol/μL 3′ primer, 1 μL glycerol, 10 μL dimethyl in a reaction mixture of sulfoxide (DMSO) and 1 μL Pfu polymerase. The annealing, extension and denaturation temperatures were also carried out according to standard conditions, for example, at 50°C, 72°C and 94°C, respectively, and Gene Amp PCR System 9600 (PerKing Elmer Cetus) was used for 30 cycles. DNA from PCR reactions was purified using the Genaclean kit (Bio II Inc., LaJolla, CA). The rHuAFP fragment was first digested with Xhol and then treated with mung bean nuclease. The HuAFP cDNA was then digested with EcoRI to facilitate its direct cloning into pVT-P10.

The rHuAFP cDNA generated by PCR was ligated into the blunt-ended 5'BamHI site and 3'EcoRI site. The membrane fusion hemagglutinin protein of measles virus was synthesized from the vector pJVNheI by digestion with the restriction enzyme BamHI [Vialard et al., (1990) "Synthesis of the membrane fusion hemagglutinin protein of measles virus using a novel baculovirus vector with the alpha-galactosidase gene" Fusion and hemagglutinin proteins of Measles (Virus, using a novel baculovirus vector containing the β-galactosidase gene), "Journal of Virology" (J.Virology) 64: 37-50] in its 3' end contains a protein derived from SV40 The β-galactoside group of the polyadenylation site, and then insert it into a compatible BgIII, resulting in the final structure: pVT-PLacZ/HuAFP (containing amino acids 1-590). This structure is then used to express rHuAFP(1-590).

Fragments and Analogs

The present invention includes biologically active rHuAFP fragments. Fragments of rHuAFP having biological activity have at least one of the following activities: (a) direct a specific interaction with a target cell, e.g., bind to a cell expressing a receptor recognizable by rHuAFP (e.g., cancer cells such as MCF-7 or bone marrow cell membranes); (b) terminate, attenuate or inhibit the growth of tumor or autoreactive immune cells (eg, bind to cell surface receptors and produce an anti-proliferative signal); stimulate, enhance, Extend or otherwise affect the proliferation of cells such as myeloid cells (eg, bind a proliferative or stimulatory signal on a cell surface receptor); or prevent or inhibit or prevent immunopathological antibody responses. The ability of a rHuAFP fragment or analog to bind a receptor recognized by rHuAFP can be determined using any standard binding assay known in the art. Biological activity of such fragments and analogs is assayed using methods well known in the art, such as those disclosed herein.

Typically, rHuAFP fragments are produced using the polypeptide expression and purification techniques described above. For example, a suitable rHuAFP fragment can be produced by transforming a suitable host bacterial cell with a portion of a HuAFP-encoding cDNA fragment bound to a suitable expression vector, such as the cDNA described above. Alternatively, the fragments described above can be generated using standard PCR techniques and cloned into expression vectors (supra). Fragments can also be produced by chemical synthesis (e.g., using the methods disclosed in "Solid Phase Peptide Synthesis," Second Edition, 1984, The Pierce Chemical Co., Rockford, II). Thus, once the rHuAFP fragments are expressed, they can be isolated using various chromatographic and/or immunological methods known in the art. Cells containing rHuAFP can be lysed and fractionated using standard methods prior to affinity chromatography. Candidate rHuAFP fragments are tested for their ability to exhibit alpha-fetoprotein biological activity using methods known to those skilled in the art (eg, using methods disclosed herein).

As described above, rHuAFP fragments can also be expressed as fusion proteins, produced in E. coli as a fusion with maltose binding protein. Using the maltose-binding protein fusion and purification system (NewEngland Biolabs, Beverly, MA), the cloned human cDNA sequence can be inserted downstream of the gene encoding maltose-binding protein (malE) and located in the reading frame of the gene, and then can be superseded. Quantitative expression of malE fusion protein. When the human cDNA sequence lacks common restriction sites, PCR method can be used to introduce restriction sites of the adaptable vector at the 5' and 3' ends of the cDNA fragment, so as to insert the cDNA fragment into the vector. Following expression of the fusion protein, purification can be performed by affinity chromatography. For example, the fusion protein can be purified by taking advantage of the ability of the maltose binding protein portion of the fusion protein to bind amylose immobilized on a column.

To facilitate protein purification, the plasmid pMalE contains a Factor Xa cleavage site upstream of the site where the cDNA is inserted into the vector. Thus, the above purified fusion protein can then be cleaved with Factor Xa to separate the maltose binding protein from the human recombinant cDNA gene product. This cleavage product can be subjected to further chromatography to purify rHuAFP from maltose binding protein. Alternatively, rHuAFP fragments can be expressed as fusion proteins that contain polyhistidine fragments. This alpha-fetoprotein fusion protein can then be isolated by binding the polyhistidine fragment to an affinity column having a nickel moiety that binds the polyhistidine fragment with high affinity. The fusion protein can be eluted by changing the pH in the affinity column. rHuAFP can be released from the polyhistidine sequence present in the resulting fusion protein by cleavage of the fusion protein with specific proteases.

Recombinant HuAFP fragment expression products (e.g., produced by any of the prokaryotic systems described above) can be assayed immunologically, such as by Western blot of recombinant cell extracts, immunoprecipitation analysis, or immunofluorescence (e.g., using the method disclosed in Ausubel et al., "Current Protocols In Molecular Biology", Greene Publishing Associates and Wiley Interscience (Jhon Wiley & Sons), New York, 1994).

If desired, the purified gene product or fragments thereof can be used to prepare anti-human recombinants by well-known methods (see Coligan et al., "Current Protocols in Immunology" (Current Protocols in Immunology) 1992, Greene Publishing Associates and Wiley-Interscience). Polyclonal or monoclonal antibodies to alpha-fetoprotein. To prepare monoclonal antibodies, mice can be immunized with recombinant protein, antibody-secreting B cells isolated, and immortalized with nonsecreting myeloma cell fusion donors. Hybridomas are then screened for production of antibodies specific to recombinant human alpha-fetoprotein (or fragments or analogs thereof) and cloned to obtain a homogeneous population of cells capable of producing monoclonal antibodies.

As used herein, the term "fragment" when used to refer to a rHuAFP polypeptide preferably has at least 20 contiguous amino acids, preferably at least 50 contiguous amino acids, more preferably at least about 100 contiguous amino acids , most preferably at least about 200-400 or more contiguous amino acids. rHuAFP fragments can be prepared by methods known to those skilled in the art, e.g., by proteolytic cleavage or expression of recombinant peptides, or by ordinary protein processing (e.g., removal of amino acids irrelevant to biological activity from nascent polypeptides) preparation.

Interesting recombinant HuAFP fragments include, but are not limited to domain I (amino acid 1 (Thr)-197 (Ser), see Figure 1, sequence 6); domain II (amino acid 198 (Ser)-389 (Ser), see Figure 1, sequence 7), domain III (amino acid 390 (Gln)-590 (Val), see Figure 1, sequence); domain I+II (amino acid 1 (Thr)-389 (Ser); see Figure 1, Sequence 9); domain II+III (amino acid 198 (Ser)-590 (Val), see Fig. 1, sequence 10), and rHuAFP fragment I (amino acid 266 (Met)-590 (Val), see Fig. 1 , sequence 11). The activity of the fragments is assayed experimentally, using conventional techniques and assays, eg, as disclosed herein.

The invention also includes analogs of full length rHuAFP or fragments thereof. An analog can differ from rHuAFP by amino acid sequence differences or modifications that do not affect its sequence (eg, post-translational modifications), or both. The homology of the analogues of the present invention to all or part of the amino acid sequence of rHuAFP is generally at least 80%, more preferably 85%, most preferably 90% or even 99%. Modification (often without altering its primary sequence) involves in vivo or in vitro chemical derivatization of the polypeptide, for example, acetylation or carboxylation; such modification may be performed during polypeptide synthesis or processing, or following treatment with a separate modifying enzyme . Analogs can also differ from naturally occurring rHuAFP by primary sequence changes, e.g., substitution of one amino acid for another with similar properties (e.g., valine for glycine, arginine for lysine etc.) or carry out the substitution, deletion or insertion of one or several non-conservative amino acids without losing the biological activity of the polypeptide. These include natural and induced genetic variants [e.g. random mutagenesis by irradiation or methyl ethanesulfonate treatment or by the method disclosed by Sambrook et al. ("Molecular Cloning: A Laboratory Manual", vol. 2nd ed.Cold Spring Harbor Press, 1989, or Ausubel et al., supra)] for site-specific mutagenesis, also including cyclized peptide molecules and analogs containing residues other than L-amino acids, e.g., D-amino acids or non- Naturally occurring or synthetic amino acids, eg, beta or gamma amino acids, or L-amino acids with unnatural side chains (see, eg, Noren et al., Science 244:182, 1989). For example, Ellman et al. (see Science 255: 197, 1992) disclose methods for the site-specific incorporation of unnatural amino acids into the protein backbone of proteins. Also included are chemically synthesized polypeptides or peptides with modified peptide bonds (eg, non-peptide bonds as disclosed in US Patent 4,897,445 and US Patent 5,059,653) or modified side chains to achieve the desired pharmaceutical properties described herein. Useful mutants and analogs are identified using routine methods, such as those disclosed herein.

Recombinant HuAFP as an immunosuppressant

Immunosuppressive effects of rHuAFP (or fragments or analogs thereof) are assayed for in vivo or in vitro immunomodulatory activity by any standard method. As described below, the art provides several animal systems for in vivo testing the immunosuppressive characteristics of rHuAFP (or fragments or analogs thereof) on autoimmune diseases, eg, non-obese diabetic (NOD) mice. In addition, there are a variety of in vitro systems available for testing the immunosuppressive properties of rHuAFP, for example, one such in vitro assay measures the inhibition of autoantigen-induced T cell proliferation in an autologous mixed lymphocyte reaction (AMLR) .

The following examples demonstrate that non-glycosylated rHuAFP and rHuAFP fragments can inhibit the autoproliferation of T cells in response to self-antigens. These examples are intended to illustrate, not limit, the invention.

Example

Materials and methods

Gel electrophoresis, western blotting, and purification

The purity and identity of rHuAFP were determined by sodium dodecylsulfonate polyacrylamide gel electrophoresis (SDS-PAGE) and native alkaline PAGE (APAGE) using standard methods. The gels were then analyzed either by Coomassie blue staining or by electrophoretically transferring the separated polypeptides to Immobilon membranes (Millipore, Mississauga, ON) for immunoblot analysis. Recombinant HuAFP non-specific rabbit anti-native HuAFP polyclonal antibody complexes were identified with alkaline phosphatase-conjugated goat anti-rabbit IgG, and immunoassays were detected with BCIP/NBT chromogenic solution (BioRad Laboratories Mississange, ON) according to the manufacturer's instructions. reaction zone.

Column chromatography was performed according to standard methods.

Polymerase chain reaction (PCR) of rHuAFP fragment

A plasmid construct encoding human alpha-fetoprotein fragments was prepared using PCR techniques well known to those skilled in the art of molecular biology techniques, using oligonucleotide primers designed to amplify the specific portion of the gene of human alpha-fetoprotein [for example, see "PCR "PCR Technology", H.A.Erlich, StochtonPress, New York, 1989; "PCR Protocols: A Guide to Methods and Applications", M.A.Innis, David H.Gelfand, JohnJ.Sninsky , and Thomas J. White, Academic Press, Inc., New York, 1990, and Ausubel et al., supra].

Prepare the following 6 kinds of rHuAFP fragments, and measure their biological activity (for example, using the method disclosed herein):

Domain I amino acid 1 (Thr)-197 (Ser), (Figure 1, sequence 6)

Domain II amino acid 198(Ser)-389(Ser), (Figure 1, sequence 7)

Domain III amino acid 390(Gln)-590(Val), (Figure 1, sequence 8)

Domain I+II amino acid 1(THr)-389(Ser), (Figure 1, sequence 9)

Domain II+III amino acid 198(Ser)-590(Val), (Figure 1, sequence 10)

rHuAFP Fragment I Amino Acid 266(Met)-590(Val), (Figure 1, Sequence 11)

The amino acid sequence was deduced from the indicated sequence of human alpha-fetoprotein (1(Thr)-590(Val); sequence 5 in Figure 1). Fragments of rHuAFP designated Domain I, Domain II, Domain III, Domain I+II, Domain II+III, and rHuAFP Fragment I were synthesized using standard PCR reaction conditions in a 100 μL reaction mixture containing 34 μL _H2O , 10 μL 10× reaction buffer, 20 μL 1 mMdNTP, 2 μL DNA template (HuAFP cloned on pI18), appropriate amount of 5′ and 3′ oligonucleotide primers (10 μL 10 pmol/μL 5′ primer, 10 μL 10 pmol/ 3' primer), 1 μL glycerol, 10 μL dimethylsulfoxide (DMSO), and 1 μL Pfu polymerase (Stratagene, LaJolla, CA). The primers used for PCR amplification were:

Domain I25 5'-AAAAAAAGGTACCACACTGCATAGAAATGAA-3' (sequence: 14)

Domain I3 5'-AAAAAAAGGATCCTTAGCTTTCTCTTAATTCTTT-3' (sequence: 15)

Domain II5 5'-AAAAAAATCGATATGAGCTTGTTAAATCAACAT-3' (sequence: 16)

Domain II3 5'-AAAAAAAGGATCCTTAGCTCTCCTGGATGTATTT-3' (sequence: 16)

Domain III5 5'-AAAAAAATCGATATGCAAGCATTGGCAAAGCGA-3' (sequence: 16)

Domain III3 5'-AAAAAAAGGATCCTTAAACTCCCAAAGCAGCACG-3' (sequence: 17)

5' rHuAFP fragment I 5'-AAAAAAATCGATATGTCCTACATATGTTCTCAA-3' (sequence: 18)

Therefore, the primer pairs DomI25 and DomI3, DomII5 and DomII3, DomIII5 and DomIII3, 5' rHuAFP Fragment I and DomIII3, DomI25 and DomII3, and DomII5 and DomIII3 can be used to extract domain I, domain II, domain III, cDNA sequences of rHuAFP fragment I, domain I+II, and domain II+III. The annealing, extension, and denaturation temperatures were 50°C, 72°C, and 94°C, respectively, and 30 cycles were performed. PCR products were purified using standard methods. Purified PCR products encoding domain I and domain I+II were individually digested with KpnI and BamHI and cloned into KpnI/BamHI-treated pTrp4, respectively. Purified PCR products encoding domain II, domain III, domain II+III, and rHuAFP fragment I were digested with Bsp106I and BamHI, respectively, and cloned into Bsp106I/BmHI-treated pTrp4, respectively. Each plasmid construct was then transformed into competent E. coli cells. Since the expression product begins with an amino acid sequence encoded by the translation initiation signal methionine, it is expected that this signal will be removed, or in any case not affect the biological activity of the final expression product.

Autologous mixed lymphocyte reaction (AMLR)

Human peripheral blood mononuclear cells (PBMC) were extracted and fractionated into non-T cell populations and AMLRs using standard methods. Effector T cells were isolated by passing 1.5×10 ⁸ PMBC over a commercially available Ig-anti-Ig affinity column (Biotek Laboratories), followed by 2×10 ⁵ effector cells with autologous ¹³⁷ Cs-irradiated ( 2500 rad) non-T-stimulatory cells were cultured together. The medium used was supplemented with 20 mM HEPES (Gibco), 5×10 ⁻⁵ M2-mercaptoethanol (BDH, Montreal, QC), 4 mM L-glutamine (Gibco), 100 μ/ml penicillin (Gibco) and 100 μg/ml For RPMI-1640 of streptosulfatin, add 10% autologous fresh human serum from effector T cell donors when used in AMLR. Various concentrations of purified rHuAFP, human serum albumin (HSA), anti-HuAFP monoclonal antibody Clone#164 were added at the beginning of the culture (final concentration in culture was 125 μg/ml) (Leinco Technologies St.Louis, MO) . The AMLR cultures were grown at 37 °C in 95 air and 5% CO _for 4–7 days. DNA synthesis was measured at the indicated intervals by 6 h pulses with 1 ^μCi3H -thymidine (specific activity 56-80 Ci/mmole, ICN). Cultures were harvested on a multi-sample collector (Skatron, Sterling, VA) and ³ H-TdR incorporation was measured on a Packard 2500 TR liquid scintillation counter. Results are presented as mean cpm ± standard error of the mean of 3 or 4 replicate cultures.

Peripheral blood lymphocyte (PBL) assay

Human peripheral blood lymphocytes (PBL) were isolated from red blood cells by density centrifugation on Ficoll-Hypaque (Sigma, St. Louis, MO) from heparinized blood from normal humans diluted 1:1 with PBS. They were washed at least 3 times with PBS, and cell viability was identified by trypan blue exclusion. Human PBL (2.5×10 ⁵ cells) were cultured by standard methods. Results are expressed as mean Cpm thymidine incorporation ± SEM of 3 replicate cultures.

result

expression and purification

Resolving a single band on Coomassie blue-stained APAGE and SDS-PAGE shown in Figures 1A-1B confirmed the purity of the isolated rHuAFP expressed in E. coli. Soluble monomeric rHuAFP derived from Escherichia coli was obtained from the HuAFP-containing protein fraction eluted by universal Q-Sepharose chromatography. By FPLC Mono-Q anion exchange, use 220-230mM NaCl to recover about 1mg of pure rHuAFP from each liter of bacterial culture, which is a single uniform peak, and its migration on SDS-PAGE is about 65KD (1B), On SDS-PAGE, the molecular weight of recombinant HuAFP was lower than that of native HuAFP, because the prokaryotic expression system lacks the enzymatic machinery required for protein glycosylation. Rechromatography of pure rHuAFP samples on FPLC and HPLC gave a single peak as shown in Figures 1C and 1D, confirming the purity of this rHuAFP. In addition, the N-terminal sequencing data were consistent with the expected N-terminal amino acid sequence of rHuAFP.

Alternatively, Escherichia coli containing an expression plasmid encoding rHuAFP was cultured as described above. The characteristics of purified rHuAFP Fragment I are shown in Figure 2 (lane D). N-terminal amino acid sequence analysis shows that the amino acid sequence that rHuAFP fragment I has is: Ser ₂₆₇ -Tyr-Ile-Cys-Ser-Gln-Gln-Asp-Thr ₂₇₅ (sequence 13), this sequence and the expected rHuAFP fragment I The N-terminal amino acid sequence (see Figure 1, sequence 11) is identical, wherein the initial methionine is excised inside the cell.

Inhibition of Autologous Mixed Lymphocyte Reaction (AMLR)

The immunosuppressive activity of rHuAFP was determined based on its ability to inhibit human autologous mixed lymphocyte reaction (AMLR). As shown in Figure 8A, rHuAFP inhibition of the proliferative effect of autoreactive lymphocytes stimulated by autologous non-T cells was determined by measuring autoproliferation over a period of 4-7 days. As shown in Fig. 8B, the results obtained from dose-response studies performed at the T cell self-proliferation peak confirmed that the addition of rHuAFP at the beginning of the culture inhibited AMLR in a dose-dependent manner. In addition, parallel activity studies confirmed that the suppressive activity of rHuAFP on human autoreactive T cells was not due to nonspecific cytotoxic effects.

To further confirm that rHuAFP is the inhibitor responsible for the suppression of autoproliferating T cells, rHuAFP-mediated suppression of AMLR was blocked with a commercially available mouse anti-human AFP monoclonal antibody (MAb). As shown in Figure 5, anti-HuAFP MAb could completely prevent the inhibitory effect of 100 μg/ml on the proliferation of autoreactive T cells. Addition of 100 μg/ml HSA did not disrupt the AMLR effect, nor did MAb alone in the reaction cultures.

Recombinant polypeptides produced in prokaryotic expression systems run the risk of being contaminated with host cell lipopolysaccharide (LPS) when extracted from cells. It has been confirmed that a small amount of LPS can antagonize the biological activity of cytokines, thereby destroying the immune response of macrophages. Therefore, the effect of endotoxin on various rHuAFP preparations was determined by performing AMLR experiments with recombinant protein depleted of endotoxin by passage through Detoxi-gel (pierce) and untreated rHuAFP. The above experiments show that both formulations have the same level of immunosuppressive activity.

As shown in Figures 8A and 8B, the results of this study also indicated that rHuAFP could inhibit the proliferation of autoreactive T cells with the same potency as glycosylated rHuAFP, and had an inhibitory effect on self-proliferating T cells in vitro, when When the concentration of rHuAFP was in the range of 5μg/ml-100μg/ml, it had obvious inhibitory effect. In addition, as shown in Table 1 and Figure 3, rHuAFP Fragment I was able to inhibit the proliferative effect of autoreactive lymphocytes stimulated by autologous non-T cells at 144 hours. Furthermore, as shown in Figure 3, domains I and III are also able to inhibit AMLR.

Table I Reaction scheme Thymidine incorporation (CPM) T cell 7118±964 AMLR 83103±6480 AMLR+rHuAFP Fragment I (100μg/ml) 29692±2963

Inhibition of Peripheral Blood Lymphocyte Response (PBL)

The immunosuppressive activity of 100 μg/ml rHuAFP Fragment I, and rHuAFP produced by E. coli and baculovirus (as described below) were also tested for the ability to suppress human peripheral blood lymphocyte responses (PBL). As shown in Table II, rHuAFP and Fragment I (produced as described above) produced by E. coli and baculovirus were confirmed to inhibit the proliferation effect of ConA-stimulated human peripheral blood lymphocytes.

Table II 3H-thymidine incorporation (CPM±SE) protein free 102,353±5,566 91,502±4,333 99,700±4,464 100 μg/ml human albumin 89,860±5,800 82,924±11,085 94,123±1,633 100 μg/ml E. coli rHuAFP 33,641±3,893 - - 100 μg/ml baculovirus rHuAFP - 31,331±6,303 - 100 μg/ml Fragment I - - 39,019±161

autoimmune disease

As noted above, autoimmune diseases are characterized by loss of tolerance to self-antigens, causing immune system cells such as T or B cells (or both) to react with self-tissue antigens. Autoimmune diseases involve all organ systems, although some organs are more affected than others. Examples of tissues affected by autoimmune symptoms include the white matter of the brain and spinal cord in patients with multiple sclerosis; the lining of joints in patients with rheumatoid arthritis; and the insulin-secreting beta islet cells on the pancreas in patients with insulin-dependent diabetes. Other forms of autoimmune disease can destroy the connections between nerves and muscles in people with myasthenia gravis or the kidneys and other organs in people with systemic lupus erythematosus. Examples of other autoimmune diseases include, but are not limited to Addison's disease, Crohn's disease, Grave's disease, psoriasis, scleroderma, and ulcerative colitis.

The art provides a variety of transgenic and non-transgenic experimental animal systems for testing the treatment of human diseases associated with autoimmune diseases [see, for example, Panl, W.E., Fundamental Immunology), 2nd ed, Raven Press, New York, 1989; and Kandel et al., Principles of Neural Science, 3rd ed., Appleton and Lange, Novwalk, CT, 1991; and Current Protocols In Immunology, Coligan , J.E., Kruisbeek, A.M., Margulies, D.H. Sherach E.M., and Strober, Green Publishing Associates (John Wiley & Sons). New York 1992]. The above experimental results confirm the immunosuppressive activity of non-glycosylated rHuAFP, and it is reasonable to think that taking this rHuAFP (or its fragments or analogs) produced in the prokaryotic system can treat other autoimmune diseases. Therefore, the present invention provides the use of rHuAFP (or its fragments or analogues) for treating (ie preventing, or suppressing, or improving, or promoting remission) all autoimmune diseases.

The following are examples of animal systems that can be used to determine the efficacy of recombinant human alpha-fetoprotein or immune cell antiproliferative fragments or analogs thereof in the treatment of autoimmune diseases. These examples are used to illustrate the present invention, not to limit the present invention.

multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease involving scattered parts of the white matter of the central nervous system. In MS, myelin basic protein and proteolipid proteins are the main targets of autoimmune responses involving T lymphocytes, among other immune system components. Loss of the myelin sheath in nerve cells (demyelination), leading to neurological symptoms and eventually coma or paralysis.

Experimental autoimmune encephalomyelitis (EAE) is the primary model used in the art to test and measure the effects of therapeutic agents for the treatment of MS. EAE is an inflammatory autoimmune demyelinating disease induced in experimental animals by immunization against the central nervous system. When animals (such as mice, rats, intestinal mice, rabbits, monkeys, etc.) are injected with a left agent such as Freund's complete adjuvant plus myelin basic protein or proteolipid protein, EAE can be induced, which can be induced in Pathologically similar to MS [see, eg, Alvord et al., "Experimental Allergie Enceph-alomyelitis—A Useful Model for Multiple Sclerosis," Liss, New York, 1984; Swanberg, "Methods in Enzyme" (Meth. Enzymol.) 162:413, 1988; and McCarron et al.) J. Immunol., 147:3296, 1991].

To test rHuAFP or a fragment or analog thereof, EAE is induced in a suitable experimental animal, for example, a mouse or a rabbit by methods known in the art. To evaluate the immunosuppressive effect of a compound on EAE, ie its ability to prevent or ameliorate EAE, the compound is administered daily at an appropriate dose by standard methods such as intravenous or intraenteric injection. Generally, dosing is initiated before the induction of EAE and/or after clinical symptoms of EAE appear. Control pairs receive a placebo, such as human serum albumin, which is administered in a manner similar to that of rHuAFP or related molecules. The effect of the test molecule on EAE is determined according to any standard method. For example, EAE-induced animals were monitored daily for weight loss and muscle paralysis. Histological examination of brain and spinal cord tissue, if desired [e.g., by any standard histochemical or immunohistochemical method, see e.g. Ausubel et al., Current Protocols In Molecular Biology, Greene Publishing Associates (John Wiley & Son, New York, 1994; Bancroft and Stevens, Theory and Practice of Histochemical Techniques, Churchill Livingstone, 1982] and microscopic examination of tissue samples looking for evidence of EAE , such as evidence of perivascular infiltration. Comparative studies between treated and control animals are used to determine the relative efficacy of test molecules in preventing or ameliorating EAE. Molecules that prevent or ameliorate (alleviate, or inhibit, or alleviate, or attenuate) the symptoms of EAE are considered useful in the present invention.

Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic disease that inflames the synovial membrane of many joints, causing damage to cartilage and bone. RA is associated with human lymphoid antigen (HLA)-DR4 and is considered an autoimmune disease involving T cells, see, eg, Sewell et al., The Lancet 341:283,1993. RA results from the interaction of synoviocytes with various cytokines (and their soluble products) that infiltrate the synovial lining of the joints from the circulatory system. The series of biological events that take place culminates in a damaging effect that invades and erodes the collagen and cartilage matrix of the joint.

Several AR animal models, such as the MRL-lpr/lpr mouse, are well known in the art, and these models produce arthritis symptoms that mimic human disease (see, eg, Fundamental Immunology supra). Alternatively, autoimmune collagen arthritis (ACA) and adjuvant arthritis (AA) can be induced in suitable animals using standard methods.

To determine the immunosuppressive effect of rHuAFP or its fragments or analogs on RA, i.e. the ability of the compound to prevent or ameliorate RA, the test molecule was administered to MRL-lpr/lpr mice using standard methods such as intravenous or intraperitoneal injection , administered daily at an appropriate dose. Generally, dosing is initiated prior to the onset of RA and/or after the onset of clinical symptoms of RA. Control animals receive a placebo, eg, human serum albumin, which is administered in a manner similar to that for rHuAFP or related molecules. The effect of the test molecule on RA is monitored using standard methods. For example, by daily monitoring of the analysis of the cellular components of synovial joints. If desired, synovial joints can be examined histologically (e.g., by any standard histochemical or immunohistochemical method, see, for example, Ausubel et al., supra; Bancroft and Stevens, supra) and microscopic examination of tissue samples, To look for evidence of RA, for example, evidence of erosion of the collagen and cartilage matrix in the joints. Comparative studies between treated and control animals are used to determine the relative efficacy of test molecules in preventing or ameliorating RA. Test molecules that prevent or ameliorate (reduce, or inhibit, or alleviate, or promote attenuation of) the symptoms of RA are considered useful in the present invention.

myasthenia gravis

Myasthenia gravis (MG) is a neuromuscular transmission disorder in which autoantibodies are produced against acetylcholine receptors at the neuromuscular junction. Antibodies attack the linker, causing weakness and paralysis. It is twice as common in women as in men and usually occurs around age 30. The main feature of this disease is muscle weakness. Clinical signs include ptosis and double vision. MG is associated with hyperthyroidism.

Experimental autoimmune MG (EAMG), whose symptoms resemble the basic features of the human disease, has been studied in a variety of animals including rabbits, monkeys, Lewis rats, and inbred strains of mice. [See eg: "Principles of Neutral Science", supra]. A single injection of acetylcholine receptors with an adjuvant, for example purified from the electrical tube of the eel (Torpedo californica), causes acute weakness within 8-12 days, followed by chronic weakness after 30 days or so. Responses to eel receptors are T cell-determining. The C57BL/6 strain (H-2 ^B ) is a high effector of the Torpedo receptor and is highly susceptible to EAMG.

To test for RhuAFP or fragments or analogs thereof, EAMG is induced in suitable experimental animals such as C57BL/6 strain (H- ^2B ) mice by methods well known in the art. To examine the immunosuppressive effect of a compound on EAMG, ie the ability to prevent or ameliorate EAMG, the compound is administered by standard methods, for example, daily in appropriate doses in the form of intravenous or intraperitoneal injection. Generally, dosing is initiated prior to the induction of EAMG and/or after the onset of clinical symptoms of EAMG. Control animals receive a placebo, such as human serum albumin, which is administered in a similar manner as rHuAFP or a related molecule. The effect of test molecules on EAMG is monitored by standard methods. For example, nerve stimulation analysis can be performed on electromyographic analysis of EAMG-induced animals (eg, using the method disclosed by Pachner et al., Ann. Neurol. 11:48, 1982). If desired, tissue samples can be subjected to histological examination (e.g., by any standard histochemical or immunohistochemical method, see, e.g., Ausubel et al., supra; Bancroft and Stevens, supra) and microscopic examination of tissue samples to determine Evidence of EAMG was found, for example, evidence of monocyte infiltration and/or localization of autoantibodies to acetylcholine receptors at the neuromuscular junction. Comparative studies between treated and control animals are used to determine the relative efficacy of test molecules in preventing or ameliorating EAMG. Molecules that prevent or ameliorate (alleviate, or inhibit, or alleviate, or facilitate attenuation of) the symptoms of EAMG are considered useful in the present invention.

insulin dependent diabetes

Diabetes is a disease of glucose metabolism. Insulin-dependent diabetes mellitus (IDDM), also known as type I diabetes, is an autoimmune disease characterized by T-cell-mediated destruction of pancreatic β-cells on Lang's island, accompanied by a variety of autopeptides The resulting immune response leads to hyperglycemia and other pathological symptoms. IDDM patients rely on exogenous insulin to maintain normal glucose metabolism. Individuals at risk of developing IDDM can be identified prior to the onset of hyperglycemia by abnormalities in insulin resistance, islet cells, glutamate carboxylase, and other autoproteins (see, for example, Baekkeskov et al., J. Clin. .lnvest.) 79:926, 1987; Dean et al., "Diabetologia" (Diabetologia) 29:339, 1986; Rossini et al., "Annu.Rev.Immunol.) 3:289, 1985; Srikanta et al. , "New England Journal of Medicine" (N. Engl. J. Med.) 308:322, 1983). In general, the type of autoantibody can predict the risk of eventual disease development and/or morbidity (see, eg, Keller et al., The Lancet 341:927, 1993).

Examples of animal models that spontaneously develop human-like disease include Bio-Breeding (BB) rats and non-obese diabetic (NOD) mice. Diabetes can also be induced experimentally with streptozotocin.

BB rats can spontaneously develop a disease similar to IDDM, with insulitis (infiltration of islets by monocytes) and resistance to self cells and insulin (see, for example, Baekkeskov et al., J. Clin. Invest. 79 : 926, 1987; Rossini et al., supra; Nakhooda et al.) Diabetes 26: 100, 1977; Dean et al., Clin. Exp. Immunol. 69: 308, 1987).

NOD mice typically develop insulitis at 5-8 weeks of age, with 70% of females and 40% of males becoming diabetic by 7 months of age. Transfer of T cells from diabetic mice into naive non-diabetic NOD mice induces diabetes within 2-3 weeks (see, for example, Bendelac et al., J. Exp. Med. 166:823, 1987 ). NOD mice usually die within 1-2 months after developing diabetes unless they are treated with insulin.

Diabetes can be induced chemically by multiple low-dose injections of streptozotocin, which is toxic to pancreatic beta cells and can cause severe insulitis and diabetes (see, for example, Kikutani et al., "Advances in Immunology" ( Adv. Immunol.) 51:285, 1992).

Therefore, various animal models simulating human IDDM are provided in the art, and these animal models can be used for testing and testing methods for preventing or improving diabetes involving rHuAFP (or its fragments or analogs).

In order to determine the immunosuppressive effect of rHuAFP or its fragments or analogues on the pathogenesis of diabetic mice, that is, the ability of the compound to treat or prevent insulitis and diabetes, a suitable dose was administered daily at an appropriate dose by a standard method such as intravenous or intraperitoneal injection. The test animals are administered the appropriate test compound. Generally, administration is performed before the onset of insulitis and diabetes and/or after the onset of clinical symptoms of diabetes. Control animals receive a placebo, such as human serum albumin, which is administered in a similar manner as rHuAFP or a related molecule. The effect of test molecules on insulitis and diabetes is monitored using standard methods. For example, weight loss, ketone body formation, and blood glucose concentrations are monitored daily. If desired, islet cells can be examined histologically (e.g., by any standard histochemical or immunochemical method, see, for example, Ausubel et al., supra; Bancroft and Stevens, supra) and microscopic examination of tissue samples to look for Insulitis and evidence of beta-cell damage. Comparative studies between treated and control animals can be used to determine the relative effectiveness of test molecules in preventing or ameliorating the symptoms of diabetes. Molecules capable of preventing or ameliorating (relieving, or inhibiting, or alleviating, or promoting attenuation) the symptoms of diabetes, such as IDDM, are considered useful in the present invention.

systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a serious systemic autoimmune disease. About 90% of patients with this disease are young women. This pronounced female advantage does not exist before puberty and after menopause. The disease usually begins in early adulthood with a characteristic rash on the patient's cheekbones and forehead. Hair loss is common, as can severe kidney damage, arthritis, fluid buildup around the heart, and inflammation of the lining of the lungs. Blood vessels in the brain also become inflamed in nearly half of patients, leading to paralysis and seizures. The disease, like other autoimmune diseases, fluctuates: long periods of quiescence in good health can be abruptly terminated and inexplicably relapsed. A large number of different autoantibodies are known in SLE, eg, against DNA, RNA, and histones (see, eg, Fundamental Immunolgy, supra).

Several animal models of human SLE are known in the art, e.g., inbred mouse strains including NIB mice and their _F1 hybrids, MRL mice, and BXSB mice (see, e.g., Bielschowsky et al., "University of Otago Proc. Univ. Otago Med.Sch. 37:9, 1959; Braverman et al., J.Invest.Derm. 50:483, 1968; Howie et al., "Immune "Adv. Immunol.) 9:215, 1968; "Genetic Control of Autoimmune Disease" (Genetic Control of Autoimmune Disease), Rose, M. Bigazzi, PE, and Warner, NL, Elsevier, Amsterdam, 1979; and Current Protocols In Immunology, supra). For example, the NZB×NZW F ₁ mouse is an excellent human model of SLE, and female mice develop high levels of anti-double-stranded and anti-ssDNA autoantibodies, other antinuclear antibodies, and kidney disease; usually at about 8 months Death occurs (eg, see Theofilopoulos et al., "Advances in Immunology" Adv. Immunol.) 37:269, 1985).

In order to determine the immunosuppressive effect of rHuAFP or its fragments or analogs on SLE, that is, the ability of the compound rHuAFP to prevent or improve SLE, a suitable drug such as NZB×NZW F ₁ Animal administration of test compounds to mice. Generally, administration is initiated prior to the onset of SLE and/or after the onset of clinical symptoms of SLE. Control animals receive a placebo, eg, human serum albumin, which is administered in a manner similar to that for rHuAFP or a related molecule. The effect of the test compound on SLE is monitored by standard methods. For example, the analysis of autoantibodies, such as anti-DNA antibodies, can be monitored. If desired, renal tissue can be examined histologically (e.g., by any standard histochemical or immunohistochemical method, see, for example, Ausubel et al., supra; Bancroft and Stevens, supra) and microscopic examination of tissue samples to look for Evidence of SLE, eg, evidence of lupus nephritis. Comparative studies between treated and control animals can be used to determine the relative effectiveness of test compounds in preventing or ameliorating SLE. Molecules that prevent or ameliorate (attenuate, or inhibit, or alleviate, or promote alleviation of) the symptoms of SLE are considered useful in the present invention.

therapeutic administration

As mentioned above, recombinant alpha-fetoprotein, such as rHuAFP (or its fragments or analogs) can effectively inhibit the proliferation of autoimmune cells, therefore, can be used to prevent or improve autoimmune diseases, including, but not limited to multiple sclerosis, Rheumatoid arthritis, diabetes mellitus, systemic lupus erythematosus, and myasthenia gravis. Therefore, recombinant human alpha-fetoprotein (or its fragments or analogs) can be formulated by known methods to prepare pharmaceutically acceptable compositions.

Recombinant alpha-fetoprotein, such as rHuAFP (or fragments or analogs thereof), is preferably administered to patients with autoimmune disease in an amount effective to prevent or ameliorate the symptoms of the disease. Generally, the dose of 0.1ng/kg body weight-10g/kg body weight is appropriate. Administration can be carried out on a daily basis, if desired. Because there are no known deleterious side effects associated with recombinant human alpha-fetoprotein, higher doses are considered safe to administer. For example, a human patient may be treated with a therapeutically effective amount of rHuAFP (or a fragment or analog thereof) contained in a physiologically acceptable carrier. Suitable carriers and their formulations are disclosed in Remington's Pharmaceutical Sciences by E.W. Martin. The dosage of rHuAFP varies depending on the way of administration, the age and body weight of the patient, the type of disease, and the body size of the susceptible or suffering patient. For example, preferred modes of administration include subcutaneous, intravenous, intramuscular, or intradermal injections, which provide continuous maintenance of drug levels in the patient. In other preferred modes of administration, rHuAFP may be administered to a patient by injection or implantation in a sustained release formulation, for example, in a slowly dissociated polymeric or crystalline form; prior to such sustained administration, rHuAFP may be administered with The initial administration is carried out in the more usual manner (as described above). Alternatively, rHuAFP can be administered with an infusion pump (eg, an external or implantable infusion pump) to allow precise control over the rate of drug release, or to implant rHuAFP into the nasal passages in a manner similar to that used to facilitate insulin inhalation. As an alternative to nasal mucosal absorption, rHuAFP can be infused into the lungs as an aerosol precipitate of powder or as a solution.

In addition, the method of the present invention can also be used in the form of combination therapy, wherein rHuAFP is administered simultaneously or sequentially with a therapeutic agent such as a common or specific resistance agent, for example, the therapeutic agent is an anti-idiotypic agent (such as a monoclonal antibody) or Treatment Vaccines or oral agents (such as insulin, collagen, or myelin basic protein) or cytokines (such as I1-15) or interferon (alpha-interferon) or immunosuppressants. The immunosuppressant is preferably administered at an effective dose which is lower than when the immunosuppressant is used alone. Preferred immunosuppressants are cyclosporine, FK-506, steroids, azathioprine, or 15-deoxyspergualin.

Treatment generally begins when an autoimmune disease is diagnosed or suspected and is generally repeated daily. Administration of rHuAFP before onset can prevent or prevent the occurrence (or development or deterioration) of autoimmune diseases. The effectiveness of therapeutic or prophylactic regimens can be tested, if desired, by monitoring or diagnosing the patient's autoimmune disease.

Use of recombinant human AFP for treatment or diagnosis of cancer

cytotoxic agent

Hybrid cytotoxins of rHuAFP are prepared by conjugating full-length rHuAFP or a fragment or analog thereof to any number of known toxic agents using conventional techniques. These toxins can be used to inhibit tumorigenesis (as described below). A useful cytotoxin is preferably one that is appreciably cytotoxic only when present inside the cell, and is substantially excluded from particular cells lacking the targeting domain. As noted above, peptide toxins meet the above criteria and can be readily incorporated into hybrid molecules. Mixed cytotoxins (ie, cytotoxins consisting of all or part of two or more toxins) can also be used if desired. Several useful toxins are described in more detail below.

Toxin molecules for use in the methods of the invention are preferably toxins, such as peptide toxins, which are only significantly cytotoxic when present in cells. Of course, in this case the toxin molecule must be able to enter the cell bearing the target receptor. This ability depends on the nature of the molecule and the nature of the cell's receptors. For example, a cellular receptor that can naturally take up a ligand appears to provide a means for molecules including toxins to enter cells bearing the receptor. As described below, peptide toxins useful in the methods of the invention are fused to the binding domain of rHuAFP (or a fragment or analog thereof) by creating a molecule encoding a hybrid protein.

Many polypeptide toxins have a common eukaryotic receptor binding domain; in this case, the toxin must be modified to prevent intoxication of non-receptor bearing cells. Any such modifications must be made in a form that retains the cytotoxic function of the molecule [see "US Department of Health and Human Service, US Serial No. 401.412"]. Potentially useful toxins include, but are not limited to: cholera toxin, ricin, O-Shiga-like toxins (SLT-I, SLT-II, SLTII _v ), LT toxin, _C3 toxin, Shiga toxin, pertussis toxin , tetanus toxin, pseudomonas exotoxin, saporin, modeccin and gelanin.

The cytotoxic portion of some of the molecules useful in the present invention may, if desired, be provided by mixed toxin molecules. A mixed toxin molecule is a molecule derived from two different polypeptide toxins. Typically, a polypeptide toxin has an enzymatically active domain and a transport domain, in addition to the domain responsible for general eukaryotic cell binding, as described above. The binding and translocation domains are necessary for cellular recognition and toxin entry, respectively. Once the toxin molecule enters the cell, the cytotoxic activity is determined by the enzymatically active domain.

Natural proteins known to have a translocation domain include diphtheria toxin, Pseudomonas exotoxin A and possibly other peptide toxins. The transport domains of diphtheria toxin and Pseudomonas exotoxin A have been well characterized [eg, see Hoch et al., Proc. Natl. Acad. Sci. USA 82:1692 , 1985; Colombatti et al., "J.Biol.Chem." (J.Biol.Chem.) 261; 3030, 1986; and Deleers et al., "Communication of the Federation of European Biochemical Societies" (FEBS Lett.) 160:82, 1983], Also, Hwang et al. ("Cell" (Cell) 48: 129, 1987) and Gray et al. ("Proc. Methods determine the presence and location of such domains on other molecules.

For example, a useful rHuAFP/mixed toxin hybrid molecule is obtained by combining the enzymatically active A subunit of E. coli Shiga-like toxin (see, for example, Calderwood et al., "Proc. Natl. Acad. Sci.USA) 84:4364, 1987) is formed by fusion with the transport domain of diphtheria toxin (amino acid residues 202-460) and rHuAFP. The rHuAFP part of this 3-part hybrid enables the molecule to specifically bind cells bearing receptors recognized by rHuAFP, while the diphtheria toxin transport part functions to insert the enzymatically active A subunit of Shiga-like toxin into target cells Inside. The enzymatically active portion of the Shiga-like toxin acts on the protein synthesis machinery of the cell like diphtheria toxin to prevent protein synthesis, thereby killing the cell.

The functional moieties of the hybrid cytotoxins of the present invention are linked together by non-covalent bonds or covalent bonds or both. Non-covalent interactions can be ionic, hydrophobic, or hydrophilic, and involve leucine-zipper interactions or antibody-protein G interactions (see, for example, Denick et al., Nature )359:752, 1992). An example of a covalent linkage is a disulfide bond.

Hybrid cytotoxins are made by chemically conjugating rHuAFP (or fragments or analogs) with any number of known toxic moieties, such as those described above. The reaction is carried out using standard techniques well known to those skilled in the art. A common method of conjugating a protein and a protein toxin (including, for example, bacterial toxins such as diphtheria toxin or Pseudomonas exotoxin A, or plant toxins such as ricin) is via a disulfide bond (e.g., see Chang et al., "J. Biol. Chem.) 252:1515, 1977) or by heterobifunctional molecules (e.g., see Cawley et al., "Cells" (Cell), 22:563, 1980). See Stevens et al., US Patent No. 4,894,227.

Alternatively, the hybrid cytotoxins are obtained using techniques known to those of ordinary skill in the art [see, for example, Murphy, U.S. Patent No. 4,675,382, and Chadhary et al., Proc. Natl. Acad. Sci. .USA) 84:4538,1987] expressing hybrid DNA encoding rHuAFP (or its fragments or analogs) and toxin (or its toxic part) produced by engineering methods. For example, recombinant fusion proteins of rHuAFP and cytotoxic agents can be prepared by methods known in the art (eg, see Murphy, supra, and Huston et al., Meth. Enzymol. 203:46, 1991). If the hybrid cytotoxin is produced by expressing a fusion gene, a peptide bond acts as the link between the cytotoxic agent and the target ligand. A method that can be used to conjugate a protein or polypeptide and a protein toxin employs the polymer, monomethoxypolyethylene glycol (mPEG) [as described by Maiti et al., "International Journal of Cancer Supplement" (Int. J . Cancer Suppl.) 3:17, 1988].

After synthesis of the hybrid cytotoxin, if desired, it is affinity purified using standard methods using antibodies raised against the target portion of the molecule, eg, human alpha-fetoprotein. Similarly, hybrid cytotoxin molecules can also be purified by standard immunological techniques using antibodies against cytotoxic agents. The resulting hybrid cytotoxins are then formulated for use against unwanted cells, such as cancer cells, by methods standard in the pharmaceutical art.

Molecules of the invention can be screened for their ability to reduce the viability of cells bearing a target receptor using assays known in the art, such as those disclosed herein.

Since the hybrid cytotoxins of the present invention are potent cytotoxic agents for cells bearing receptors recognizable by rHuAFP, rHuAFP can be used in the treatment of diseases involving deleterious alpha-fetoprotein receptor-positive cells, such as cancer cells.

diagnostic agent

The recombinant rHuAFP or its fragments or analogs can be combined with a detectable label to make a preparation that can be used in vivo, in situ or in vitro to detect and localize tumors. Methods for attaching such labels to proteins are well known in the art. For example, a detectable label can be attached chemically, but when the label is a polypeptide, it can also be attached using genetic engineering techniques.

Detectable labels are generally selected from a variety of such labels known in the art, but typically radioisotopes, fluorophores, enzymes (e.g., horseradish peroxidase), or other moieties or compounds capable of emitting a detectable signal (e.g. radioactive, fluorescent, colour) or a moiety or compound that emits a detectable signal upon contact of the label with its substrate. Various detectable label/substrate pairs (e.g., horseradish peroxidase/diaminobenzidine, avidin/streptavidin, luciferase/luciferin, β-galactosidase/X -gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside) and methods for labeling proteins for this detection purpose are well known in the art. This can be measured as follows Uses of such preparations: For example, a tumor cell line such as MCF-7 is implanted into a host such as a mouse, and whether the preparation of the present invention has detectably labeled the tumors arising from incoming cells. This preparation can also be used to detect the presence of any deleterious alpha-fetoprotein receptor-bearing cells, for example, by Western blot analysis or by histochemical staining of tissue samples by known methods .

Recombinant HuAFP as an anticancer agent

Anticancer agents of the present invention (such as rHuAFP or fragments or analogs thereof; or hybrid cytotoxins of rHuAFP) can be used to inhibit tumors, such as breast or prostate cancer. It will be understood by those skilled in the art that any method for measuring the effect of anticancer agents in vitro and in vivo can be used in the present invention. For example, mice or rats bearing prostate cancer (eg, a tumor xenograft of a LNCaP androgen receptor positive human prostate cancer cell line) are monitored for reduction in tumor growth following administration of a test compound. In one example, a human tumor cell line grown in culture [e.g., such as MCF-7 (ATCC HTB 22), T-47D (ATCC HTB 133), MDA-MB-231 (ATCC HTB 26), BT-20(ATCC HTB 19), NIH: OV-CAR-3(ATCCHTB 161), Ln Cap.FGC(ATCC CRL 1740) and Du-145(ATCC HTB 81) were released from monolayer cells, Diluted into a single cell suspension and solidified into a pellet by centrifugation, then treated with 15 μl of fibrinogen (50 mg/ml) and 10 μl of thrombin (50 units/ml) at 37° C. for 30 minutes. The fibrin mass containing the tumor was then cut into small pieces approximately 1.5 mm in diameter. Each tumor mass was then implanted under the renal capsule of the mouse using standard methods. If desired, mice can be immunosuppressed with routine daily subcutaneous (S.C.) injections of 60 mg/kg cyclosporine A (Sandimmune IV) starting immediately before tumor implantation. Mice were supplemented with estrogen and androgen, if desired, using standard methods, such as implantation of siliconized rubber tubes containing estradiol or injections of testosterone propionate. Typically, hormone supplementation begins the day of tumor implantation. Typically, administration of the test molecule is initiated prior to and/or following tumor implantation. Control animals receive a placebo, such as human serum albumin or a diluent, which is administered in a manner similar to that for rHuAFP or a related molecule. The effect of the test molecule on tumor growth is monitored by any standard method. For example, tumor growth is monitored by weekly tumor size measurements by laparotomy, using a dissecting microscope equipped with an ocular micrometer. Molecules that have been experimentally shown to prevent or attenuate or inhibit the growth of such transplanted tumors are considered useful in the present invention.

Toxicity of test compounds to cells bearing receptors recognized by rHuAFP can be determined in vitro by any standard method. For example, cultured cancer cell lines, such as the MCF-7 estrogen receptor-positive human breast cancer cell line, are maintained in Dulbecco's Modified Eagle's Medium (DMEM) in plastic tissue culture flasks (Costar), DMEM Contains penicillin (100U/ml), streptomycin (100μg/ml), 5% fetal bovine serum, insulin (10ng/ml), L-glutamine (2mM) and non-essential amino acids (1%). Inoculated in complete medium on 96-well V-bottom plates (Linbro-Flow Laboratoris, Mclean, VA) at a concentration of 1×10 ⁵ /well. Putative toxins were added at various concentrations (10 ⁻¹² M-10 ⁻⁶ M) and the cultures were incubated at 37° C. in 5% CO ₂ gas for 18 hours. After incubation, the plates were centrifuged at 170×g for 5 minutes, the medium was removed and replaced with 100 μl of leucine-free medium (MEM, Gibco) containing 8 μCi/ml ( ³ H-leucine; New England Nuclear, Boston, MA). After an additional 90 minutes of incubation at 37°C, the plates were centrifuged at 170 xg for 5 minutes, the medium was removed, and the cells were harvested on glass fiber filters using a cell harvester (Skatron, Sterling, VA). Filters were washed, dried, and counted using standard methods. Cells cultured with medium alone were used as controls. Test compounds that attenuate or arrest or inhibit cell growth as an indication of toxicity compared to untreated control cells are assayed and are considered useful in the present invention.

Methods for determining whether a test compound confers protection against tumorigenesis (breast or prostate cancer) generally involve the use of an animal known to produce tumors (e.g., the transgenic mice disclosed in U.S. Patent No. 4,736,866 ). Appropriate animals are treated with the test compound according to standard procedures and a reduction in the incidence of tumor morbidity detected compared to untreated control animals is indicative of protection.

As described below, the inventors have discovered that aglycosylated rHuAFP produced in a prokaryotic expression system is effective in treating cancer. For example, rHuAFP has been found to be a potent inhibitor of breast cancer growing in vitro.

The experimental examples disclosed below demonstrate the efficacy of rHuAFP as an anticancer agent. These experimental examples are for illustrating the present invention, not limiting the present invention.

Example

Materials and methods

Culture Media and Tumor Cells

Dulbecco's Modified Eagle's Medium (DMEM), RPMI1640, fetal calf serum, glutamine, non-essential amino acids and penicillin-streptoxin mixture were purchased from GIBCO (BRL). Donor bovine serum was purchased from Hyclone, Logan, UT, and porcine insulin was purchased from Squibb, Inc., Princeton, NJ.

The MCF-7 estrogen-receptor-positive human breast cancer cell line was provided by Dr. Alberto C. Baldi, Institute of Experimental Biology and Medicine, Buenos Aires, Argentina. Mother cultures were maintained in plastic tissue culture flasks (Costar) in DMEM containing penicillin (100 u/ml), streptomycin (100 μg/ml), 5% fetal calf serum, insulin (10 ng/ml) , L-glutamine (2mM) and non-essential amino acids (1%).

Estrogen-stimulated growth of MCF-7 cells after confluency in culture.

The assay is based on the finding that MCF-7 cells grown in estrogen-containing media go through the confluent phase and accumulate into foci; however, in the absence of estrogen, when cell-cell contacts are formed in culture Cell proliferation ceases and foci cannot be formed (eg, see Gierthy et al., Breast Cancer Res. 2 Treat. 12:227, 1988). 1×10 ⁷ MCF-7 breast cancer cells were seeded into 16 mm wells of 24-well tissue culture plates. The medium is phenol red-free DMEM supplemented with 5% donor bovine serum (prescreened for detectable estrogen deficiency), L-glutamine (2 mM), non-essential amino acids (1x, GIBCO), insulin (10 ng /ml), penicillin-streptomycin (1x, GIBCO) and estradiol diluted to a final concentration of 1.8×10 ^-9 M. The cultures were replenished with medium at 24 hours and then every 4 days with 2 ml of medium containing rHuAFP and human serum albumin each time to give a final protein concentration of 100 μg/ml per well. Cells reached confluency within 5 days, with numerous foci appearing within 10 days in estrogen-only wells. Cells were fixed with buffered formalin and stained with 1% Rhodamine B. Stained foci were quantified using an Artek 870 Macro-Micro-Automated Colomy Counter. Data presented are the number of mean clusters per treatment group.

result

Anti-MCF-7 breast cancer cell activity of rHuAFP

The results shown in Figure 9 indicate that rHuAFP can inhibit the growth of estrogen-stimulated MCF-7 breast cancer cells after confluence in vitro. Control experiments with human albumin or no protein had no effect on the formation of MCF-7 foci. The above data indicate that rHuAFP has a direct inhibitory effect on the growth of cancer cell cultures.

therapeutic administration

As mentioned above, rHuAFP is effective in suppressing tumors, eg, breast cell carcinoma. Accordingly, the compounds of the present invention can be formulated into pharmaceutically acceptable compositions by known methods. Human patients can be treated with a therapeutically effective amount of an rHuAFP anticancer agent contained in a physiologically acceptable carrier. Suitable carriers and their formulations are disclosed in Remington's "Pharmaceutical Sciences" by E.W. Martin. The dose of the anticancer agent varies depending on the mode of administration, the age and body weight of the patient, and the type of disease. Generally, the amount will be in the range of that used in other agents used in the treatment of cancer, however, in some instances lower amounts will be required because of the greater specificity of the compound. For example, as described below, rHuAFP is administered systemically at a dose that inhibits the proliferation of malignant cells, typically in the range of 0.1 ng to 10 g/kg body weight.

In addition, the methods of the present invention may also employ combination therapy, wherein rHuAFP and chemotherapeutic agents are administered simultaneously or sequentially. Typically, the chemotherapeutic agent is administered by standard methods or, alternatively, the chemotherapeutic agent is administered at a lower dose than that of the chemotherapeutic agent alone. Examples of chemotherapeutic agents include, but are not limited to, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, hexamethylolmelamine, phosphorous sulfide, busulfan , nitrosourea nitrogen mustard, Luo nitrogen mustard, racemustine, streptozotocin, dacarbazine, methotrexate, fluorouracil, cytarabine, mercaptopurine, thioguanine, pentastatin, Changchun Flower alkali, vincristine, etoposide, podophyllotoxin, actinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, cisplatin, Mitoxantrone, Hydroxyurea, Procarbazine Hydrochloride, O-Chlorobenzene-p-Chlorobenzenedichloroethane, Aminophenetidone, Prednisone, Hydroxyprogesterone, Diethylstilbestrol, Tamoxifen, Fluoxetine amides, or gonadotropin-releasing hormone analogs.

Treatment generally begins when a tumor is diagnosed or suspected, and is usually repeated daily. Daily administration of rHuAFP also prevented tumorigenesis. Evaluating the effectiveness of therapeutic or protective regimens, if desired, by monitoring or diagnosing cancer in patients.

In addition, the compounds of the present invention may also be used in the treatment of mammals to kill any unwanted alpha-fetoprotein receptor-bearing cells associated with pathological conditions. The methods of the invention can also be used to treat non-human mammals, eg, livestock or livestock. As described below, the anticancer agent of the present invention can be administered systemically or locally.

systemic drug delivery

The compounds of the present invention, when used as anticancer agents, can be administered systemically, for example, formulated in a pharmaceutically acceptable buffer such as physiological saline. Preferred methods of administration include, for example, subcutaneous, intravenous, intraperitoneal, intramuscular or intradermal injection, whereby drug levels are continuously maintained in the patient. In other preferred methods of administration, the compound may be delivered to the patient by injection of a sustained release formulation, for example, in a slowly dissociated polymeric or crystalline form; such sustained administration may be preceded by the more common Methods (eg, as described above) begin dosing. Alternatively, the compounds may be administered using an infusion pump to precisely control the rate of drug release, or placed into the nasal passages in a manner similar to that used to facilitate insulin absorption. As an alternative to nasal mucosal absorption, the compounds of the invention can be delivered to the lungs as aerosol deposits or solutions of powders.

Topical administration

The anticancer agents of the present invention can also be administered topically to treat cancer. Since the effect of a desired therapeutic agent is often dependent on the size of the tissue involved, eg, the size of a tumor, it is particularly desirable to deliver the drug in a manner that results in a high local concentration near the tumor.

Recombinant HuAFP as a diagnostic agent

Recombinant HuAFP (or fragments or analogs thereof) linked to a detectable label can be used for diagnostic purposes when detecting, monitoring or analyzing the presence of a tumor (eg, breast or prostate cancer).

For example, human patients can be studied in vivo with detectably labeled rHuAFP (Tc-99m-labeled rHuAFP) to determine the presence of tumors. Typically, detectably labeled rHuAFP is administered intravenously and visualized with a scanner, eg, radioactive scintigraphy, by methods known to those skilled in the art.

In another embodiment, rHuAFP (or fragments or analogs thereof) linked to a detection label can be used to detect tumor or any cell with a receptor recognizable by rHuAFP in a tissue sample such as a biopsy or body fluid. exist. Following the above assays, tissue samples, biopsies or body fluid samples taken from the patient, especially lymph, blood, serum, or urine should be tested for the presence of such cells. Thus, the subcellular localization or presence of receptors recognizable by rHuAFP can be determined using fractionated cells by any standard biochemical or histochemical method (see, for example, Ausubel et al., supra; Bancroft and Stevens, "Theory and Practice of Histological Techniques" ( Theory and Practice of Histological Techniques), Churdill Livingstone, 1982) for in situ or in vitro assays. Suitable control samples for analysis include tissue samples or body fluids taken from individuals who do not have cells bearing alpha-fetoprotein (negative control), or samples containing a known, predetermined number of alpha-fetoprotein receptors (positive control). ).

Diagnostic tests can be performed by any standard method in solution or on solid (insoluble) supports (eg, polystyrene, nitrocellulose, or beads) or on tissue samples prepared for histological examination. For example, to determine whether a patient from whom a test sample is taken has cells with receptors that can be recognized by rHuAFP, the level of detectably labeled rHuAFP binding in the test sample is compared to that in the negative control sample and/or the positive control sample. Combine levels for comparison. When the level of binding in the test sample is higher than the level of binding in the negative control sample, or at least equal to the level of binding in the positive control sample, it is indicated that the patient has cells carrying alpha-fetoprotein receptor.

Materials for use in diagnostic assays according to the methods of the present invention may be provided in the form of kits with instructions for use. Typically, the kit will consist in part of rHuAFP (or a fragment or analog thereof). Such a kit may also include another reagent, for example, a detection label to be used to label rHuAFP (or a fragment or analog thereof). For example, the kits described above can be used for in vitro detection of the presence of tumors in human tissue samples, or for in vivo detection purposes.

The experimental paradigm described below demonstrates the effectiveness of rHuAFP in diagnosing tumors.

Example

Materials and methods

animal

MCF-7 human breast cancer cells implanted in the lateral breast of CB-17 SCID mice were grown to a size of 1 cm in diameter (approximately 5 grams) under estrogen-stimulated conditions using methods known in the art.

technetium label

Tc-labeled recombinant alpha-fetoprotein was prepared from AFP samples mixed with 0.5 ml of 0.9% NaCl injection (Baxter Healthcare Corporation, Deerfield, IL). The above solution was added to UltraTagRBC Reaction Vial (Mallinckrodt Medical Inc., St. Louis, Mo 63134 Lot No. 0683040) containing stannous chloride dihydrate, sodium citrate dihydrate in lyophilized form stored under argon substances, and anhydrous glucose. The contents of the above vials were mixed by gentle swirling and incubated at room temperature for 5 minutes. A volume of 0.8-1.2 Gbq Technetium 99mTc Sodium Pertechnetate Injection (99mTc Generator Mallincrolet Medical, Inc. St. Louis, MO) in a volume of 1-2 ml was added at the end of the incubation. The contents of the above vials were mixed by gentle swirling and incubated for 15 minutes. Formulation samples were analyzed at 0, 3 and 6 hours after preparation. Thin layer chromatography of the preparation in 0.9% NaCl by ITLC-SG (Gelman Instrument Co., Ann Arbor, MI) showed that 95-99% of 99Tc was bound to recombinant alpha-fetoprotein.

imaging

The experimental animals were sedated with Medafane. A 24 gauge, 3/4 inch catheter (Surflo IV catheter, Terumo Medical) was then secured at the lateral tail pulse. The animals were then further anesthetized by slow intravenous infusion of 20-25 mg/kg body weight pentobarbital. Anesthesia was maintained by additional injections of 5 mg pentobarbital as needed to restrain their movements.

Isotope biodistribution data were collected with an Elscint Dymax 409 gamma camera. These data were subsequently analyzed by computer (Siemans Gammasonics Microdelta). Animals were imaged three times and placed on polyethylene sheets in a dorsal-lying position. To avoid movement of the animal during imaging, the animal may be restrained if necessary, with its limbs taped to the above board so that breathing is not restricted. The acquired 60-min motion images were used to determine the biodistribution of the marker protein. Typically, 12 sequential 5 min images were acquired with a low energy general purpose calibration and 1.5 hardware upscaling into a computer matrix with 128 x 128 pixels. Research animals are typically injected with 37MBq of Tc-99m-labeled protein.

result

Tracer biodistribution and kinetics

Following administration of 37 MBq (approximately 4-6 μg Tc-99m recombinant human alpha-fetoprotein) from the tail vein, the kinetics of tracer biodistribution were determined during the first 1 hour and 24 hours post-injection. Tissue uptake kinetics were determined as 60% of injected activity/100 Region of Interest (ROI, Region of Interest) pixels (%IA). Rapidly eliminated from the kidneys within the first hour, moderately localized to the liver, with little apparent activity in other organs. At the first hour, tumor uptake was (mean ± SEM: 1.9 ± 0.3% IA), while the tumor to heart (T/H) site ratio was 0.84 ± 0.23. By 24 hours, tumor uptake was (0.8+/-0.1% IA) and ratios of T/A and tumor to background (T/B upper chest) fractions were 1.43±0.41 and 2.66±0.54, respectively. A comparative study of 99mTc-labeled rHuAFP and 99mTc-labeled human serum albumin (used as a non-specific protein control) repeated on the same animals showed that for 99mTc-labeled rHuAFP, at 1 hour and 24 hours The T/B image ROI activity ratios at 24 hours were 2.7 and 5.8, and the activity of 99mTc-labeled rHuAFP was 40% higher than that of Tc-99m human serum albumin at 24 hours. The above results indicated that rHuAFP can be labeled with Tc-99m, and that the labeled preparation has low non-specific tissue absorption and can be rapidly cleared from the blood by the kidneys. Localization in human breast cancer xenografts was rapid initially and accelerated over time due to specific tumor uptake. These results suggest that rHuAFP labeled with Tc-99m can be used as a diagnostic agent for breast cancer.

diagnostic use

As described above, rHuAFP (or a fragment or analog thereof) linked to a detectable label can be used for diagnostic purposes, for the detection, monitoring or analysis of tumors such as breast cell carcinoma. Thus, patients presenting with symptoms typical of cancer, such as symptoms of breast or prostate cancer, or with a medical history indicating a predisposition to such cancer, can be tested by the methods of the invention. Other patients eligible for this test include those with a family history of breast or prostate cancer. Patients who are receiving drugs or have been exposed to toxins involved in cancer induction should also be tested.

The diagnostic methods of the invention employing detectably labeled rHuAFP (or fragments or analogs thereof) can be used to detect the presence of cancer either before or after the onset of clinical symptoms associated with cancer.

The method of the invention facilitates the diagnosis of tumors prior to and concurrent with clinical symptoms such as palpable masses. For example, the subject methods of the present invention can be used to diagnose breast cancer before clinical symptoms appear. Additionally, the methods of the present invention allow physicians to accurately diagnose tumors such as breast or prostate cancer.

The diagnostic imaging method of the present invention can be performed with a diagnostically effective amount of rHuAFP diagnostic agent contained in a physiologically acceptable carrier. For example, suitable carriers and their formulations are disclosed in "Remington's Pharmaceutical Sciences" by E.W. Martin. The amount of diagnostic agent to be administered depends on the manner of administration, the age and weight of the patient, the type of disease, the extent of the disease, and the size of the patient suffering from the disease. Generally, however, the amount used is within the range of other agents used in the diagnosis of cancer, although lower amounts may be required in some cases due to higher compound specificity. For example, detectably labeled rHuAFP is administered intravenously to such patients in such a dose that the tumor can be visualized, for example, radioactively by scintigraphy. Typically, dosages will be in the range of 0.1 ng-10 g/kg body weight.

All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in equal weight as if each individual publication or patent application were specifically and individually indicated to be incorporated as part of the present invention. reference.

Cell culture medium of the present invention

The present invention also provides a culture medium containing rHuAFP (or its fragment or analog) for cell culture. Although the culture medium of the present invention generally does not need to use serum (for example, fetal calf serum, bovine serum, horse serum, normal mouse serum, human serum, pig serum, rabbit serum, etc.), because the rHuAFP is intended to be used to replace or supplement The use of serum, but those skilled in the art will understand that serum can be added if desired. Formulation components are generally prepared by methods well known in the art. Therefore, any standard medium, such as RMPI-1630 medium, CMRL medium, Dulbecco's modified Eagle's medium (D-MEM), Fischer's medium, Iscove's modified Dulbecco's medium, McCoy's medium, 's medium, minimal essential medium, NCTC medium, etc. can be formulated with rHuAFP (or its fragments or analogs) at desired effective concentrations. If necessary, medium supplements such as salt solution (such as Hank's balanced salt solution or Earle's balanced salt solution), antibiotics, nucleic acids, amino acids, carbohydrates and vitamins are added by known methods. If desired, growth factors, colony stimulating factors, cytokines, etc. can also be added to the culture medium by standard methods. For example, the medium of the present invention may contain any of the following substances and rHuAFP (or fragments or analogs thereof) alone or in combination: erythropoietin, granulocyte/macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating Factor (G-CSF), macrophage colony-stimulating factor (M-CSF), interleukins (such as IL-1, IL-2, IL-3, IL-4, IL-5, etc.), insulin growth factor (IGF) , transferrin, albumin, and stem cell growth factor (SCF). The culture medium of the present invention can be used for culturing various eukaryotic cells, such as mammalian cells, yeast cells, amphibian cells and insect cells. The medium of the present invention can also be used for culturing any tissue or organ. The medium can also be used in a variety of culture conditions and for a variety of biological purposes. Examples of such culture conditions include, but are not limited to, bioreactors (such as continuous or hollow fiber bioreactors), cell suspension cultures, semi-solid cultures, and long-term cell suspension cultures. The medium of the present invention can also be used for industrial purposes, for example, culturing hybridoma cells, genetically engineered mammalian cells, tissues or organs.

Recombinant human alpha-fetoprotein as a cell proliferation agent

The cell growth promoting effect of rHuAFP (or fragments or analogs thereof) is determined using any assay method used for in vitro and in vivo analysis of cell proliferation. As described below, animal systems are available in the art for testing the cell growth promoting or enhancing properties of rHuAFP (or fragments or analogs thereof) in vivo. Additionally, there are a variety of in vitro systems available for testing the growth promoting or growth enhancing properties of rHuAFP (or fragments or analogs thereof).

Any cell that proliferates in response to rHuAFP (or a fragment or analog thereof) can be identified using standard methods known in the art. For example, cell proliferation, such as bone marrow cells, can be monitored by culturing the cells in liquid media containing the test compound, which is artificially added to serum-free or serum-based media, alone or in combination with other growth factors. Alternatively, the bone marrow cells can be cultured on a semi-solid matrix of dilute agar or methylcellulose, while the test compound is artificially added to the serum-free or serum-reduced medium alone or in combination with other growth factors. Progeny of isolated precursor cells proliferate in response to rHuAFP or a fragment or analog thereof, held together in identifiable colonies in said semi-solid matrix. For example, one myeloid cell can form a clone consisting of multiple myeloid cells, such as NK cells. This system provides a convenient method to analyze whether a cell responds to rHuAFP (or a fragment or analog thereof) alone or in combination with other growth factors.

Subpopulations of expanded cells are identified and isolated, if desired, according to standard methods. For example, cells can be analyzed using a fluorescence activated cell sorter (FACS). The method generally involves labeling the cells with an antibody conjugated to a fluorochrome and separating the labeled cells from the unlabeled cells on a FACS, such as a FACScan (Becton Dickson). Thus, virtually any cell can be identified and isolated by assaying for the presence of cell surface antigens (see, eg, Shah et al., J. Immunol. 140:1861, 1988). When a cell population is obtained, it is subjected to biochemical analysis, or it is used as the original population for another cell culture, so that the effect of the cells can be determined under the conditions determined by the culture conditions.

In one embodiment, the effect of rHuAFP (or a fragment or analog thereof) on the growth of human bone marrow cells was examined as follows. Typically, human bone marrow samples are obtained using standard methods after licensing. For example, bone marrow is obtained from the iliac crest of a healthy donor, and the marrow cells are diluted in phosphate-buffered saline at room temperature. Cells are then washed and grown in a suitable growth medium. For example, cultures can be established by inoculating bone marrow cells in 20-30 ml of McCoy's medium containing 50 U/ml penicillin, 50 U/ml streptomycin and 2 mM L-glutamine. The cultures are grown in the presence or absence of other growth factors alone or in combination, such as transferrin or GM-CSF. The cultures were then incubated at 37°C for the required time in a humidified atmosphere containing 5% _CO2 , 5% _O2 and 90% _N2 . Cell proliferation assays were performed using standard methods. For example, replicate samples cultured with and without test compound are analyzed by pulsing cells with 1-2 μCi of ³ HTdR. After a certain incubation time, the cultures were harvested onto glass fiber filters and the ^{incorporated3H} was measured by liquid scintillation. Comparative studies of treated cells and control cells, eg, cells cultured in the presence of rHuAFP versus cells cultured in the absence of rHuAFP, are used to determine the relative potency of the test molecule in stimulating cell proliferation. Molecules that stimulate cell proliferation are considered useful in the present invention.

To determine the proliferative effects of rHuAFP (or fragments or analogs thereof), e.g., the in vivo hematopoiesis of test compounds, sublethally irradiated mice (or Mice treated with immunosuppressants such as cyclosporin or FK-506, or with chemotherapeutic agents such as 5-fluorouracil or cyclophosphamide, or any other method known in the art to deplete the bone marrow) and Normal mice are dosed with the test molecule. Typically, administration of the test compound to the treated mice is initiated before and/or after treatment of the animal with, for example, sublethal radiation, immunotherapy or chemotherapy. Control animals receive a placebo, such as human serum albumin or a diluent, which is administered in a similar manner to rHuAFP or a related molecule. The effect of the test molecule on hematopoiesis is monitored using standard techniques. For example, the peripheral blood and spleen of treated animals and control animals are analyzed for white blood cell counts. Qualitative and quantitative analysis of bone marrow such as lymphocyte lineage or myeloid lineage or any other type of cells can also be determined and analyzed using conventional methods. Data from treated and control animals are compared and used to determine the relative potency of the test molecule in promoting cell proliferation, eg, production of myeloid cells, mature B lymphocytes, thymocytes, or peripheral T lymphocytes. Test molecules that stimulate cell proliferation are considered useful in the present invention.

The following examples demonstrate the ability of aglycosylated rHuAFP to stimulate bone marrow growth in vitro. This example is used to illustrate the present invention, but not to limit the scope of the present invention.

Example

Materials and methods

animal

Adult male and female CBA/J mice were obtained from the Jackson Laboratory (Bar Harbor, Maine). All mice were housed and maintained in our animal housing facility. Animals used in this study were 12-20 weeks old.

Cultures

Tibias and femurs of CBA/J mice were rinsed with modified Dulbecco's phosphate-buffered saline (PBS), and bone marrow cells were collected using sterile syringes and 25-gauge needles. Obtain a homogeneous single-cell suspension by repeatedly passing the cell mixture through a Pasteur pipette. All cells were washed twice by centrifugation in PBS at 250xg for 10 minutes, and their viability was measured by trypan blue dye exclusion. Cell viability of 95% or higher was recorded in all experiments. Cells are then adjusted to the desired concentration prior to use. Bone marrow cells (250,000) were cultured in 96-well round bottom microtiter plates (Flow Laboratories, Mississauga, Ontario, Canada). The medium was serum-free RPMI plus 4 mM L-glutamine, 20 mM Hepes, 100 u/ml penicillin, 100 μg/ml streptomycin (GIBCO Laboratories, Burlington, Ontario, Canada), 5 μg/ml transferrin and 5×10 ^-5 B - Mercaptoethanol (Eastman Chemicals Co., Rochester NY). Cells were cultured without rHuAFP or with rHuAFP at a concentration of 400 μg/ml, respectively. The total volume of all cultures was 0.2 ml. Cells were grown at 37°C in an atmosphere of 95% CO2 with 5% _CO2 . Six hours before harvest, cultures were pulsed with 1 μCi of tritiated thymidine (NEN, specific activity 77.1 Ci/mmol). Cells were then harvested on glass fiber mats (Flow Labs) using a multi-sample harvester (Skatron, Flow Labs). Incorporation of water-insoluble tritiated thymidine was determined by standard liquid scintillation techniques using an LKB 1215 RackbetaII.

result

Effect of rHuAFP on bone marrow proliferation in serum-free medium

The effect of purified rHuAFP on cultured murine bone marrow was determined in serum-free medium. In this embodiment, 2.5 x ¹⁰⁵ viable cells obtained from CBA/J mice were cultured for 72 hours in serum-free RPMI without or with transferrin at a final concentration of 400 μg/ml and a final concentration of 5 μg/ml . The data presented in Figure 10 demonstrate that bone marrow cells mount a significant proliferative response in the presence of aglycosylated rHuAFP; the stimulation index (SI) was 35. This proliferative effect was not observed in bone marrow cells cultured in the absence of rHuAFP.

treat

As noted above, rHuAFP is effective in promoting cell proliferation and, therefore, can be used in therapies involving the promotion of cell proliferation, such as myeloid cell proliferation, and in the prevention of immunosuppressive therapy, radiotherapy or chemotherapy, or other agents known to suppress the immune system and suppress bone marrow Treatments that produce side effects of therapies that lead to myelotoxicity. Therefore, rHuAFP (or fragments or analogs thereof) is used to treat erythroblast progenitor or stem cell deficiencies or related diseases. Recombinant HuAFP (or fragments or analogs thereof) can also be used in the treatment of cancer and other pathological conditions leading to bone marrow toxicity, treatment by radiation or drug effects, including, for example, leukopenia, bacterial and viral infections, anemia, B cell or T cell Defects, including defects in immune cells or erythroblasts from autologous or nonautologous bone marrow transplantation. Recombinant HuAFP (or fragments or analogs thereof) can also be used to stimulate the development of megakaryocytes and natural killer cells in vitro or in vivo.

The media, compositions and methods of the invention are also useful in the treatment of cancer treated by bone marrow transplantation (BMT), which involves removing bone marrow from a patient and maintaining the cells in ex vivo culture while simultaneously administering radiation therapy to the patient Or chemotherapy, and reimplanting these cells into the patient after treatment ends to restore the patient's bone marrow. Thus, rHuAFP can be used in BMT as a means of reconstituting bone marrow in ex vivo cell culture medium and for promoting proliferation of bone marrow cells in vivo. Recombinant HuAFP (or fragments or analogs thereof) may also be used in other cell therapies, such as cell expansion and/or gene therapy, therapies requiring ex vivo cell cultures. Recombinant HuAFP (or fragments or analogs thereof) can also be used to prevent rejection of autologous or allogeneic bone marrow transplantation.

therapeutic administration

Recombinant HuAFP (or its fragments or analogs) can be formulated into pharmaceutically acceptable compositions by known methods. Recombinant human alpha-fetoprotein, such as rHuAFP (or fragments or analogs thereof) is preferably administered to a patient in an amount effective to prevent or ameliorate symptoms of myelotoxicity. Generally, a dose of 0.1 ng/kg-10 g/kg body weight is sufficient. For example, a human patient can be treated with a therapeutically effective amount of rHuAFP (or a fragment or analog thereof) contained in a physiologically acceptable carrier. For example, suitable carriers and their formulations are disclosed in Remington's Pharmaceutical Sciences by E.W. Martin. The amount of rHuAFP to be used depends on the mode of administration, the age and weight of the patient, the type of disease, and the size of the patient predisposed or suffering from the disease. For example, preferred methods of administration include oral administration, subcutaneous, intravenous, intraperitoneal, intramuscular, transdermal or intradermal injection, whereby drug levels in the patient are continuously maintained. In other preferred methods of administration, rHuAFP may be administered to the patient by injection or implantation of a sustained release formulation such as a slowly dissociated polymerized crystalline form; prior to such sustained administration, the more common method ( As above) start medication. Alternatively, rHuAFP can be administered with an external or implantable infusion pump to precisely control the rate of drug release, or to place rHuAFP into the nasal passages in a manner similar to that used to facilitate insulin absorption. As an alternative to nasal mucosal absorption, rHuAFP can be delivered to the lungs as a powder in aerosol precipitation or as a solution.

The therapeutic methods and compositions of the present invention also include co-administration of other human growth factors. Examples of cytokines or hematopoietins used for this purpose include, but are not limited to, such as interleukin (IL-1), GM-CSF, G-CSF, M-CSF, tumor necrosis factor (TNF), transferrin and Factor of erythropoietin. Growth factors such as B-cell growth factor, B-cell differentiation factor, or eosinophil differentiation factor can also be used for administration with rHuAFP (or a fragment or analog thereof). The above dosages may be adjusted to compensate for the above mentioned other ingredients in the therapeutic composition. The progress of treated patients can be monitored by conventional methods.

Generally, treatment begins when myelotoxicity is diagnosed or suspected and is repeated regularly or daily to ameliorate or prevent the development or worsening of symptoms. Administration of rHuAFP before onset also prevents or prevents the development of myelotoxicity. The effectiveness of treatment or prophylaxis can be measured, if desired, by monitoring or diagnosing myelotoxicity in patients.

The methods of the invention can also be used to treat non-human mammals, eg, livestock or livestock.

Other implementations

In other embodiments, the invention encompasses the use of rHuAFP (or a fragment or analog thereof) for the prevention or treatment of acquired immune deficiency syndrome (AIDS). In order to determine the immunosuppressive effect of rHuAFP or its fragments or analogs on AIDS, i.e. the ability of the compound to prevent or ameliorate the autoimmune effects of AIDS, it is administered daily to suitable animals at the above-mentioned suitable doses by standard methods such as intravenous or intraperitoneal injection. A test compound is administered (eg, to a human patient). Generally, administration is initiated prior to the onset of AIDS and/or after the onset of clinical symptoms of AIDS. Control animals receive a placebo, such as human serum albumin, which is administered in a similar manner as rHuAFP or a related molecule. The effect of test compounds on AIDS is monitored by standard methods. For example, assays for the inhibition, prevention or amelioration of helper T cell destruction by test compounds can be monitored. Studies comparing treated and control animals can be used to determine the relative efficacy of test compounds in preventing or ameliorating AIDS. Molecules that prevent or ameliorate (attenuate, or inhibit, or alleviate, or promote alleviation of) the symptoms of AIDS are considered useful in the present invention.

The present invention also includes the use of rHuAFP (or fragments or analogs thereof) in a therapeutically effective amount for inhibiting mammalian organs (such as heart, liver, lung, pancreas and kidney), tissues (such as skin, bone marrow, dura mater, bone, Rejection of implanted collagen, implanted bioreactors) or transplantation of cells (eg, beta islet cells, stem cells, hematopoietic cells, lymphocytes, neuroendocrine cells, or adrenal cells). The transplanted organ, tissue or cells may be derived from any source, for example, such biological material may be isotype, isotype, autologous, synthetic, artificial or genetically engineered. For example, this approach may be employed when the patient is the recipient of an allogeneic transplant, such as a heart or kidney from another species.

In one embodiment, the immunosuppressive effect of rHuAFP on clinical transplantation, i.e., the ability of rHuAFP to prevent or ameliorate transplant rejection (e.g., hyperacute rejection, acute rejection, and chronic rejection) is administered using, for example, intravenous injection or intraperitoneal injection. The standard method is to do the assay by administering rHuAFP daily to NIH minipigs at the appropriate dose. Typically, administration of rHuAFP is initiated prior to transplantation, such as kidney transplantation, and/or following the transplantation process. Control animals receive a placebo, such as human serum albumin, administered in a similar manner to rHuAFP. The effect of rHuAFP on graft rejection was monitored using standard methods. One manifestation of the rejection process is impairment of the function of the transplanted organ, for example, the analysis of urine output can be monitored. If desired, renal tissue can be examined histologically (e.g., by any standard histochemical or immunohistochemical method, see, for example, Ausubel et al., supra; Bancroft and Stevens, supra), and tissue samples obtained by biopsy Microscopic examination to look for evidence of graft rejection, such as the presence of chronic interstitial fibrosis, vascular thrombosis, or abnormal lymphatic infiltration. Comparative studies of treated and control animals can be used to determine the relative efficacy of rHuAFP in preventing or ameliorating suppression of rejection. Recombinant HuAFP (or fragments or analogs thereof) capable of preventing or improving (attenuating, or inhibiting, or alleviating, or promoting alleviating) the symptoms of transplant rejection are considered to be useful in the present invention.

All publications, manufacturer's specifications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in the same manner as if each publication or patent application were specifically and individually indicated to be incorporated by reference. The reference in this article is the same.

Sequence Listing

(1) General Information:

(i) Applicant: Murgita, Robert A.

(ii) Title of invention: Expression and purification of cloned human alpha-fetoprotein

(iii) Number of sequences: 20

(iv) Correspondence address:

(A) To: Fish and Richardson P.C.

(B) Street: 225 Franklin Street, Suite 3100

(C) City: Boston

(D) Continent: MA

(E) Country: USA

(F) edited by: 02110-2804

(V) In computer readable form:

(A) Media type: floppy disk

(B) Computer: IBM PC compatible

(C) Operating system: PC-DOS/MS-DOS

(D) Software: Patent In Release #1.0, #1.30

(vi) Current application information

(A) Application number: PCT/US96/---

(B) Filing date: January 24, 1996

(C) Classification:

(vii) Prior application materials:

(A) Application No.: 08/377,317

(B) Application date: January 24, 1995

(C) Classification:

(A) Application No.: 08/377,311

(B) Application date: January 24, 1995

(C) Classification:

(A) Application No.: 08/377,309

(B) Application date: January 24, 1995

(C) Classification:

(A) Application No.: 08/377,316

(B) Application date: January 24, 1995

(C) Classification:

(A) Application No.: 08/505,012

(B) Filing date: July 21, 1995

(C) Classification:

(viii) Lawyer/Agent Information:

(A) Name: Clark, Paul T.

(B) Registration number: 30,162

(C) Data/Document No.: 06727/003001

(ix) Correspondence information:

(A) Tel: (617)542-5070

(B) Fax: (617)542-8906

(C)Tel: 200154

(2) Sequence 1 data:

(i) Sequence features:

(A) Length: 21 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 1

TGTCTGCAGG ATGGGGAAAA A

(2) Sequence 2 data

(i) Sequence features:

(A) Length: 18 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 2

CATGAAATGA CTCCAGTA

(2) Sequence 3 data

(i) Sequence features:

(A) Length: 18 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 3

CATAGAAATG AATATGGA

(2) Sequence 4 information:

(i) Sequence features:

(A) Length: 2022 bases

(B) type: nucleic acid

(C) chain type: not involved

(D) Topology: linear

(ii) Molecular type: protein

(xi) Sequence description: Sequence 4

ATATTGTGCT TCCACCACTG CCAATAACAA AATAACTAGC AACCATGAAG TGGGTGGAAT 60

CAATTTTTTT AATTTTCCTA CTAAATTTTA CTGAATCCAG AACACTGCAT AGAAATGAAT 120

ATGGAATAGC TTCCATATTG GATTCTTACC AATGTACTGC AGAGATAAGT TTAGCTGACC 180

TGGCTACCAT ATTTTTTGCC CAGTTTGTTC AAGAAGCCAC TTACAAGGAA GTAAGCAAAA 240

TGGTGAAAGA TGCATTGACT GCAATTGAGA AACCCACTGG AGATGAACAG TCTTCAGGGT 300

GTTTAGAAAA CCAGCTACCT GCCTTTCTGG AAGAAGTTTG CCATGAGAAA GAAATTTTGG 360

AGAAGTACGG ACATTCAGAC TGCTGCAGCC AAAGTGAAGA GGGAAGACAT AACTGTTTTC 420

TTGCACACAA AAAGCCCACT GCAGCATGGA TCCCACTTTTT CCAAGTTTCCA GAACCTGTCA 480

CAAGCTGTGA AGCATATGAA GAAGACAGGG AGACATTCAT GAACAAATTC ATTTATGAGA 540

TAGCAAGAAG GCATCCCTTC CTGTATGCAC CTACAATTCT TCTTTCGGCT GCTGGGTATG 600

AGAAAATAAT TCCATCTTGC TGCAAAGCTG AAAATGCAGT TGAATGCTTC CAAACAAAGG 660

CAGCAACAGT TACAAAAGAA TTAAGAGAAA GCAGCTTGTT AAATCAACAT GCATGTCCAG 720

TAATGAAAAA TTTTGGGACC CGAACTTTCC AAGCCATAAC TGTTACTAAAA CTGAGTCAGA 780

AGTTTACCAA AGTTAATTTT ACTGAAATCC AGAAACTAGT CCTGGATGTG GCCCATGTAC 840

ATGAGCACTG TTGCAGAGCA GATGTGCTGG ATTGTCTGCA GGATGGGGAA AAAATCATGT 900

CCTACATATG TTCTCAACAA GACACTCTGT CAAACAAAAT AACAGAATGC TGCAAACTGA 960

CCACGCTGGA ACGTGGTCAA TGTATAATTC ATGCAGAAAA TGATGAAAAA CCTGAAGGTC 1020

TATCTCCAAA TCTTAAACAGG TTTTTAGGAG ATAGAGATTT TAACCAATTT TCTTCAGGGG 1080

AAAAAAATAT CTTCTTGGCA AGTTTTGTTC ATGAATATTC AAGAAGACAT CCTCAGCTTG 1140

CTGTCTCAGT AATTCTAAGA GTTGCTAAAG GATACCAGGA GTTATTGGAG AAGTGTTTCC 1200

AGACTGAAAA CCCTCTTGAA TGCCAAGATA AAGGAGAAGA AGAATTACAG AAATACATCC 1260

AGGAGAGCCA AGCATTGGCA AAGCGAAGCT GCGGCCTCTT CCAGAAACTA GGAGAATATT 1320

ACTTACAAAA TGAGTTTCTC GTTGCTTACA CAAAGAAAGC CCCCCAGCTG ACCTCGTCGG 1380

AGCTGATGGC CATCACCAGA AAAATGGCAG CCACAGCAGC CACTTGTTGC CAACTCAGTG 1440

AGGACAAACT ATTGGCCTGT GGCGAGGGAG CGGCTGACAT TATTATCGGA CACTTATGTA 1500

TCAGACATGA AATGACTCCA GTAAACCCTG GTGTTGGCCA GTGCTGCACT TCTTCATATG 1560

CCAACAGGAG GCCATGCTTC AGCAGCTTGG TGGTGGATGA AACATATGTC CCTCCTGCAT 1620

TCTCTGATGA CAAGTTCATT TTCCATAAGG ATCTGTGCCA AGCTCAGGGT GTAGCGCTGC 1680

AAAGGATGAA GCAAGAGTTT CTCATTAACC TTGTGAAGCA AAAGCCACAA ATAACAGAGG 1740

AACAACTTGA GGCTCTCATT GCAGATTTCT CAGGCCTGTT GGAGAAATGC TGCCAAGGCC 1800

AGGAACAGGA AGTCTGCTTT GCTGAAGAGG GACAAAAACT GATTTCAAAA ACTGGTGCTG 1860

CTTTGGGAGT TTAAATTACT TCAGGGGAAG AGAAGACAAA ACGAGTCTTT CATTCGGTGT 1920

GAACTTTTTCT CTTTAATTTT AACTGATTTA ACACTTTTTG TGAATTAATG ATAAAGACTT 1980

TTATGTGAGA TTTCCTTATC ACAGAAATAA AATATCTCCA AA 2022

(2) Sequence 5 data

(i) Sequence features:

(A) Length: 590 amino acids

(B) type: amino acid

(C) chain type: not involved

(D) Topology: linear

(ii) Molecular type: protein

(xi) Sequence description: Sequence 5

Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr

1 5 10 15

Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe

20 25 30

Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val

35 40 45

Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser

50 55 60

Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys

65 70 75 80

His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser

85 90 95

Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro

100 105 110

Thr Ala Ala Trp Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser

115 120 125

Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile

130 135 140

Tyr Glu Ile Ala Arg Arg His Pro Phe Lsu Tyr Ala Pro Thr Ile Leu

145 150 155 160

Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pro Ser Cys Cys Lys Ala

165 170 175

Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys

180 185 190

Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Pro Val Met

195 200 205

Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu

210 215 220

Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln Lys Leu Val

225 230 235 240

Leu Asp Val Ala His Val His Glu His Cys Cys Arg Ala Asp Val Leu

245 250 255

Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln

260 265 270

Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr

275 280 285

Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp Glu Lye Pro

290 295 300

Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe

305 310 315 320

Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val

325 330 335

His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser Val Ile Leu

340 345 350

Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr

355 360 365

Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys

370 375 380

Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Phe

385 390 395 400

Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Glu Phe Leu Val Ala Tyr

405 410 415

Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Ser Glu Leu Met Ala Ile Thr

420 425 430

Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp

435 440 445

Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His

450 455 460

Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly Val Gly Gln

465 470 475 480

Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Leu

485 490 495

Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys Phe

500 505 510

Ile Phe His Lys Asp Lsu Cys Gln Ala Gln Gly Val Ala Leu Gln Arg

515 520 525

Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys Pro Gln Ile

530 535 540

Thr Glu Glu Gln Leu Glu Ala Leu Ile Ala Asp Phe Ser Gly Leu Leu

545 550 555 560

Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe Ala Glu Glu

565 570 575

Gly Gln Lys Leu Ile Ser Lys Thr Gly Ala Ala Leu Gly Val

580 585 590(2) Sequence 6 information:

(i) Sequential features:

(A) Length: 197 amino acids

(B) type: amino acid

(C) Chain type: not involved

(D) Topology: linear

(ii) Molecule type: protein

Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr

1 5 10 15

Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe

20 25 30

Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val

35 40 45

Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser

50 55 60

Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys

65 70 75 80

His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ssr Asp Cys Cys Ser

85 90 95

Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro

100 105 110

Thr Ala Ala Trp Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser

115 120 125

Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile

130 135 140

Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu

145 150 155 160

Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pro Ser Cys Cys Lys Ala

165 170 175

Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys

180 185 190

Glu Leu Arg Glu Ser

195

(2) Sequence 7 data:

(i) Sequence features:

(A) Length: 192 amino acids

(B) type: amino acid

(C) chain type: not involved

(D) Topology: linear

(ii) Molecular type: protein

(xi) Sequence description: Sequence 7

Ser Leu Leu Asn Gln His Ala Cys Pro Val Met Lys Asn Phe Gly Thr

1 5 10 15

Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu Ser Gln Lys Phe Thr

20 25 30

Lys Val Asn Phe Thr Glu Ile Gln Lys Leu Val Leu Asp Val Ala His

35 40 45

Val His Glu His Cys Cys Arg Ala Asp Val Leu Asp Cys Leu Gln Asp

50 55 60

Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln Gln Asp Thr Leu Ser

65 70 75 80

Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr Leu Glu Arg Gly Gln

85 90 95

Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro Glu Gly Leu Ser Pro

100 105 110

Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe Asn Gln Phe Ser Ser

115 120 125

Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val His Glu Tyr Ser Arg

130 135 140

Arg His Pro Gln Leu Ala Val Ser Val Ile Leu Arg Val Ala Lys Gly

145 150 155 160

Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr Glu Asn Pro Leu Glu

165 170 175

Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys Tyr Ile Gln Glu Ser

180 185 190

(2) Sequence 8 data:

(i) Sequence features:

(A) Length: 201 amino acids

(B) type: amino acid

(C) chain type: not involved

(D) Topology: linear

(ii) Molecular type: protein

(xi) Sequence description: Sequence 8

Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Phe Gln Lys Leu Gly Glu

1 5 10 15

Tyr Tyr Leu Gln Asn Glu Phe Leu Val Ala Tyr Thr Lys Lys Ala Pro

20 25 30

Gln Leu Thr Ser Ser Ser Glu Leu Met Ala Ile Thr Arg Lys Met Ala Ala

35 40 45

Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp Lys Leu Leu Ala Cys

50 55 60

Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His Leu Cys Ile Arg His

65 70 75 80

Glu Met Thr Pro Val Asn Pro Gly Val Gly Gln Cys Cys Thr Ser Ser

85 90 95

Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Leu Val Val Asp Glu Thr

100 105 110

Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys Phe Ile Phe His Lys Asp

115 120 125

Leu Cys Gln Ala Gln Gly Val Ala Leu Gln Arg Met Lys Gln Glu Phe

130 135 140

Leu Ile Ash Leu Val Lys Gln Lye Pro Gln Ile Thr Glu Glu Gln Leu

145 150 155 160

Glu Ala Leu Ile Ala Asp Phe Ser Gly Leu Leu Glu Lys Cys Cys Gln

165 170 175

Gly Gln Glu Gln Glu Val Cys Phe Ala Glu Glu Gly Gln Lys Leu Ile

180 185 190

Ser Lys Thr Gly Ala Ala Leu Gly Val

195 200

(2) Sequence 9 data:

(i) Sequence features:

(A) Length: 389 amino acids

(B) type: amino acid

(C) chain type: not involved

(D) Topology: linear

(ii) Molecule type: protein

(xi) Sequence description: Sequence 9

Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr

1 5 10 15

Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phs Phe

20 25 30

Ala Gln Phe Val Gln Glu Ale Thr Tyr Lys Glu Val Ser Lys Met Vel

35 40 45

Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser

50 55 60

Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys

65 70 75 80

His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser

85 90 95

Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro

100 105 110

Thr Ala Ala Trp Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser

115 120 125

Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile

130 135 140

Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu

145 150 155 160

Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pro Ser Cys Cys Lys Ala

165 170 175

Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys

180 185 190

Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Pro Val Met

195 200 205

Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu

210 215 220

Ser Gln Lys Phe Thr Lys Val Aan Phe Thr Glu Ile Gln Lys Leu Val

225 230 235 240

Leu Asp Val Ala His Val His Glu His Cys Cys Arg Ala Asp Val Leu

245 250 255

Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln

260 265 270

Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr

275 280 285

Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro

290 295 300

Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe

305 310 315 320

Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val

325 330 335

His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser Val Ile Leu

340 345 350

Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr

355 360 365

Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys

370 375 380

Tyr Ile Gln Glu Ser

385

(2) Sequence 10 data:

(i) Sequence features:

(A) Length: 393 amino acids

(B) type: amino acid

(C) Chain type: not involved

(D) Topology: linear

(ii) Molecular type: protein

(xi) Sequence description: Sequence 10

Ser Leu Leu Asn Gln His Ala Cys Pro Val Met Lys Asn Phe Gly Thr

1 5 10 15

Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu Ser Gln Lys Phe Thr

20 25 30

Lys Val Asn Phe Thr Glu Ile Gln Lys Leu Val Leu Asp Val Ala His

35 40 45

Val His Glu His Cys Cys Arg Ala Asp Val Leu Asp Cys Leu Gln Asp

50 55 60

Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln Gln Asp Thr Leu Ser

65 70 75 80

Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr Leu Glu Arg Gly Gln

85 90 95

Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro Glu Gly Leu Ser Pro

100 105 110

Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe Asn Gln Phe Ser Ser

115 120 125

Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val His Glu Tyr Ser Arg

130 135 140

Arg His Pro Gln Leu Ala Val Ser Val Ile Leu Arg Val Ala Lys Gly

145 150 155 160

Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr Glu Asn Pro Leu Glu

165 170 175

Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys Tyr Ile Gln Glu Ser

180 185 190

Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Phe Gln Lys Leu Gly Glu

195 200 205

Tyr Tyr Leu Gln Asn Glu Phe Leu Val Ala Tyr Thr Lys Lys Ala Pro

210 215 220

Gln Leu Thr Ser Ser Ser Glu Leu Met Ala Ile Thr Arg Lys Met Ala Ala

225 230 235 240

Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp Lys Leu Leu Ala Cys

245 250 255

Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His Leu Cys Ile Arg His

260 265 270

Glu Met Thr Pro Val Asn Pro Gly Val Gly Gln Cys Cys Thr Ser Ser

275 280 285

Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Leu Val Val Asp Glu Thr

290 295 300

Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys Phe Ile Phe His Lys Asp

305 310 315 320

Leu Cys Gln Ala Gln Gly Val Ala Leu Gln Arg Met Lys Gln Glu Phe

325 330 335

Leu Ile Asn Leu Val Lys Gln Lys Pro Gln Ile Thr Glu Glu Gln Leu

340 345 350

Glu Ala Leu Ile Ala Asp Phe Ser Gly Leu Leu Glu Lys Cys Cys Gln

355 360 365

Gly Gln Glu Gln Glu Val Cys Phe Ala Glu Glu Gly Gln Lys Leu Ile

370 375 380

Ser Lys Thr Gly Ala Ala Leu Gly Val

385 390

(2) Sequence 11 data:

(i) Sequence features:

(A) Length: 325 amino acids

(B) type: amino acid

(C) chain type: not involved

(D) Topology: linear

(ii) Molecular type: protein

(xi) Sequence description: Sequence 11

Met Ser Tyr Ile Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr

1 5 10 15

Glu Cys Cys Lys Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His

20 25 30

Ala Glu Asn Asp Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg

35 40 45

Phe Leu Gly Asp Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn

50 55 60

Ile Phe Leu Ala Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln

65 70 75 80

Leu Ala Val Ser Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu

85 90 95

Leu Glu Lys Cys Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys

100 105 110

Gly Glu Glu Glu Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala

115 120 125

Lys Arg Ser Cys Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln

130 135 140

Asn Glu Phe Leu Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser

145 150 155 160

Ser Glu Leu Met Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr

165 170 175

Cys Cys Gln Leu Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala

180 185 190

Ala Asp Ile Ile Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro

195 200 205

Val Asn Pro Gly Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg

210 215 220

Arg Pro Cys Phe Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro

225 230 235 240

Ala Phe Ser Asp Asp Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala

245 250 255

Gln Gly Val Ala Leu Gln Arg Met Lys Gln Glu Phe Leu Ile Asn Leu

260 265 270

Val Lys Gln Lys Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Leu Ile

275 280 285

Ala Asp Phe Ser Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln

290 295 300

Glu Val Cys Phe Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Gly

305 310 315 320

Ala Ala Leu Gly Val

325

(2) Sequence 12 data:

(i) Sequence features:

(A) Length: 324 amino acids

(B) type: amino acid

(C) chain type: not involved

(D) Topology: protein

(ii) Molecular type: protein

(xi) Sequence description: Sequence 12

Ser Tyr Ile Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu

1 5 10 15

Cys Cys Lys Leu Thr Thr Lsu Glu Arg Gly Gln Cys Ile Ile His Ala

20 25 30

Glu Asn Asp Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe

35 40 45

Leu Gly Asp Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile

50 55 60

Phe Leu Ala Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln Leu

65 70 75 80

Ala Val Ser Va1 Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu

85 90 95

Glu Lys Cys Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly

100 105 110

Glu Glu Glu Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys

115 120 125

Arg Ser Cys Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Aan

130 135 140

Glu Phe Leu val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser

145 150 155 160

Glu Leu Met Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys

165 170 175

Cys Gln Leu Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala

180 185 190

Asp Ile Ile Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro Val

195 200 205

Asn Pro Gly Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg

210 215 220

Pro Cys Phe Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala

225 230 235 240

Phe Ser Asp Asp Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gln

245 250 255

Gly Val Ala Leu Gln Arg Met Lys Gln Glu Phe Leu Ile Asn Leu Val

260 265 270

Lys Gln Lys Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Leu Ile Ala

275 280 285

Asp Phe Ser Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu

290 295 300

Val Cys Phe Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Gly Ala

305 310 315 320

Ala Leu Gly Val

(2) Sequence 13 data:

(i) Sequence features:

(A) Length: 9 amino acids

(B) type: amino acid

(C) chain type: not involved

(D) Topology: linear

(ii) Molecular type: protein

(xi) Sequence description: Sequence 13

Ser Tyr Ile Cys Ser Gln Gln Asp Thr

1 5

(2) Sequence 14 data:

(i) Sequence features:

(A) Length: 30 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 14

AAAAAAGGTA CCACACTGCA TAGAAATGAA

(2) Sequence 15 data:

(i) Sequence features:

(A) Length: 33 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 15

AAAAAAGGAT CCTTAGCTTT CTCTTAATTC TTT

(2) Sequence 16 data:

(i) Sequence features:

(A) Length: 33 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 16

AAAAATCG ATATGAGCTT GTTAAATCAA CAT

(2) Sequence 17 data:

(i) Sequence features:

(A) Length: 33 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 17

AAAAAAGGAT CCTTAGCTCT CCTGGATGTA TTT

(2) Sequence 18 data:

(i) Sequence features:

(A) Length: 33 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 18

AAAAAAATCG ATATGCAAGC ATTGGCAAAG CGA

(2) Sequence 19 data:

(i) Sequence features:

(A) Length: 33 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 19

AAAAAAGGAT CCTTAAACTC CCAAAGCAGC ACG

(2) Sequence 20 data:

(i) Sequence features:

(A) Length: 33 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 20

AAAAAAATCG ATATGTCCTA CATATGTTCT CAA

(2) Sequence 21 data:

(i) Sequence features:

(A) Length: 21 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 21

GATCTAGAAT TCGGATCCGG T

(2) Sequence 22 data:

(i) Sequence features:

(A) Length: 10 amino acids

(B) type: amino acid

(C) chain type: not involved

(D) Topology: linear

(ii) Molecular type: protein

(xi) Sequence description: Sequence 22

Asp Leu Glu Phe Met Thr Leu His Arg Asn

1 5 10

(2) Sequence 23 data:

(i) Sequence features:

(A) Length: 32 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 23

AAAAAACTCG AGATACACTG CATAGAAATG AA

(2) Sequence 24 information:

(i) Sequence features:

(A) Length: 33 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(xi) Sequence description: Sequence 24

AAAAAAGAAT TCTTAAACTC CCAAAGCAGC ACG

Claims (26)

1. Use of recombinant human alpha-fetoprotein or its fragments comprising domain I, domain II, domain III, domain I+II, domain II+III, or fragment I in the preparation of medicines, the medicine is used At: a) inhibiting the proliferation of autoreactive immune cells in a mammal; b) treating an autoimmune disease in a mammal; c) inhibiting tumors in a mammal; d) preventing the development of tumors in mammals; e) inhibiting myelotoxicity in mammals; f) inhibiting the inhibitory effect on the proliferation of mammalian bone marrow cells; g) promoting mammalian bone marrow cell proliferation; or h) Prevention of transplant rejection of bone marrow cells in a mammal. 2. The use of claim 1, wherein the mammal is a human patient. 3. The use of claim 1, wherein the recombinant alpha-fetoprotein or fragments thereof are produced by prokaryotic cells and are not glycosylated. 4. The use of claim 3, wherein the prokaryotic cell is Escherichia coli. 5. The use of claim 1, wherein the immune cells comprise T cells. 6. The use of claim 1, wherein the immune cells comprise B cells. 7. The use of claim 1, wherein the medicament is for inhibiting the proliferation of autoreactive immune cells, and further comprises an effective dose of an immunosuppressant, said dose being lower than the standard dose of said immunosuppressant alone. 8. The use according to claim 7, wherein the immunosuppressant is cyclosporine. 9. The use of claim 7, wherein the immunosuppressant is a steroid, azathioprine, FK-506 or 15-deoxyspergualin. 10. The use of claim 1, wherein said medicament further comprises a tolerizing agent. 11. The use of claim 1, wherein the autoimmune disease is multiple sclerosis. 12. The use of claim 1, wherein the autoimmune disease is rheumatoid arthritis. 13. The use of claim 1, wherein the autoimmune disease is myasthenia gravis. 14. The use of claim 1, wherein the autoimmune disease is insulin-dependent diabetes. 15. The use of claim 1, wherein the autoimmune disease is systemic lupus erythematosus. 16. The use of claim 1, wherein the tumor is a malignant tumor. 17. The use of claim 16, wherein the malignancy is breast cancer. 18. The use of claim 16, wherein the malignancy is prostate cancer. 19. The use of claim 1, wherein the tumor is a carcinoma. 20. The use of claim 1, wherein the tumor is an adenocarcinoma. 21. The use of claim 1, wherein said tumor is a sarcoma. 22. The use of claim 1, wherein said tumor proliferates in response to estrogen. 23. The use of claim 1, wherein said medicament inhibits cell proliferation of said tumor in said mammal. 24. The use of claim 1, wherein said medicament kills cells of said tumor in said mammal. 25. The use of claim 1, wherein the medicament is for tumor suppression and further comprises an effective dose of a chemotherapeutic agent which is lower than the standard dose when the chemotherapeutic agent is used alone. 26. The use of claim 1, wherein cells of said tumor express a receptor recognized by said recombinant human alpha-fetoprotein or a fragment or analog thereof.

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